COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD HX64140148 QP535.P1 F74 A review of the lite Cduttibia Uni bertfttp firfemtr? Htbrarg Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons (for the Medical Heritage Library project) http://www.archive.org/details/reviewofliteratuOOforb OHIO AGRICULTURAL EXPERIMENT STATION TECHNICAL SERIES, BULLETIN No. 5 A REVIEW OF THE LITERATURE OF PHOSPHORUS COMPOUNDS IN ANIMAL METABOLISM By E. B. FORBES AND M. HELEN KEITH WOOSTER, OHIO, U. S. A., MARCH, 1914 OHIO AGRICULTURAL EXPERIMENT STATION GOVERNING BOARD THE AGRICULTURAL COMMISSION OF OHIO Columbus A. P. Sandles, President S. E. Strode C G. Williams H. C. Price B. P. Gayman, Secretary STATION STAFF Charles E. Thorne, M. S. A., Director DEPARTMENTAL ORGANIZATION ADMINISTRATION The Director, Chiej William H. Kramer, Bursar D. W. Galehouse, Assistant Dora Ellis, Mailing Clerk Glenn Hall, Engineer W. K. Greenbank. Librarian W. J. Holmes, Printer AGRONOMY C. G. Williams, Chiefs F. A. Welton, B. S., Associate William Holmes, Farm Manager C. A. Patton, Assistant C. A. Gearhart, B. S. , Assistant E. C. Morr, Office Assistant C. H. Lebold, Asst. Foreman ANIMAL HUSBANDRY B. E. Carmichael, M. S., Chief J. W. Hammond, M. S., Associate Geo. R. Eastwood, B. S., Assistant Don C. Mote, M. S., Assistant W. J. Buss, Assistant Anthony Russ, Herdsman E. C. Schwan, Shepherd (Carpenter) BOTANY A. D. Selby, B. S., Chiej True Houser, B. S., Assistant (Germantown) P. K. Mathis, Office Assistant D. C. Babcock, A. B-, Assistant Richard Walton, B. S., Assistant Alfred S. Orcutt, M. S., Assistant CHEMISTRY J. W. Ames, M. S., Chief Geo. E. Boltz, B. S., Assistant J. A. Stenius, B. S., Assistant C J. Schollenberger, Assistant Miss Mabel Corbould, Assistant CLIMATOLOGY J. Warren Smith, C/2/V/"(Columbus) 2 C. A. Patton, Observer COOPERATION The Director, Chief W L. Elser, B. S., Executive Assistant^ C. W. Montgomery, Assistant H. P. Miller, B. S., Comity Agent (Ravenna) . P. L. Allen-, A. B., County Agent (Burton,) W. M. Cook, A. B., County Agent (Xenia) M. O. Bugby, B. S. County Agent (Warren) A. L. Higgins, B. S., County Agent (Das'ton) P. N. Meeker, County Agent (Hamilton) M. C. Thomas, County Agent (Troy) C. Ellis Bundy, County Agent (Paulding) DAIRYING C. C. Hayden, M. S., Chief A. E. Perkins, M. S., Assistant T. R. Middaugh, Office Assistant ENTOMOLOGY H. A.Gossard, M. S-, Chief J. S. Houser, M. S. A., Associate W. H. Goodwin, M. S., Assistant R. D. Whitmarsh, M. S. Assistant J. L. King, Assistant FORESTRY Edmund Secrest, B. S., Chief J. J. Crumley, Ph. D., Assistant A. E. Taylor, B. S., Assistant J. W. Calland, B. S., Assistant D. E. Snyder, Office Assistant HORTICULTURE W. J. Green, Vice Director, Chief F. H. Ballou, Assistant, (Newark) E. J. Riggs, B. S., Assistant, (Columbus) Paul Thayer, B. S., Assistant C. W. Ellenwood, Office Assistant Ora Flack, Foreman of Orchards W. E. Bontrager, Foreman of Grounds C. G. Laper, Foreman of Greenhouses NUTRITION E. B. Forbes, Ph. D., Chief M. Helen Keith, A. M., Assistant F. M. Beegle, B. S., Assistant L. E. Morgan, M. S., Assistant Charles M. Fritz, B. S., Assistant The Director, Chief C. G. Williams, Associate in soil fertility investigations J. W. Ames, M. S., Associate in soil chemistry George N. Coffey, Ph. D., Associate in charge of soil survey E. R. Allen, Ph. D., Associate in soil biology H. Foley Tuttle, M. S., Assistant A. Bonazzi, B. Agr., Assistant DIVISION OF EXPERIMENT FARMS District Experiment Farms Northeastern Test-Farm, Strongsville. Edward Mohn, Resident Manager Southwestern Test-Farm, Germantown. Henry M. Wachter, Resident Manager Southeastern Test-Farm, Carpenter. H. D. Lewis, Resident Manager Lewis Schultz, Horticultural Foreman Northwestern Test-Farm, Findlay. John A. Sutton, Resident Manager County Experiment Farms Miami County Experiment Farm. Troy M. C. Thomas, Agent m Charge Paulding- County Experiment Farm, Paulding C. Ellis Bundy, Agent in Charge Harry Ray, Foreman Clermont County Experiment Farm, Owensville Victor Herron, Agent in Charge Howard Elliott, Foreman Hamilton County Experiment Farm, Mt. Healthy Victor Herron, Agent in Charge ^With leave of absence. 2 In cooperation with Weather Service, U. S. Department of Agriculture. s In cooperation with Bureau of Plant Industry, U. S. Department of Agriculture. PREFACE. This review of the literature of phosphorus metabolism was undertaken as a part of a general program of experimentation in this field which is being conducted by the Department of Nutrition of this institution. In our selection of material we have in mind the bearing of this work on practical human nutrition and animal husbandry, and have sought to adapt it to the requirements of the college graduate who has an especial interest in nutrition. A certain over-emphasis of the subject of this discussion must inevitably result from the restriction of its scope to a consideration of the compounds of but a single element, but we have sought throughout the discussion to maintain correct perspective by indi- cating the connections of our subject with those matters with which it stands in natural relationship. Since the compounds of phosphorus have to do, in prominent ways, with all of the life processes of animals, our subject, though narrow in form, might easily be construed to cover the whole field of nutrition. It has been necessary, therefore, in the conduct of this study, arbitrarily to limit its scope in order to render its com- pletion in any way a practical possibility, and we have been obliged entirely to ignore some important divisions of the subject for no better reason than that life is short. The thoroughness with which we have treated the several sec- tions of this discussion varies much. Our treatment of the field of pathology has been especially incomplete. Here it was our idea to include only such matters as we considered of interest to the student of nutrition in general, and we have not gone into a discus- sion of diseases, as such, in a way to serve the requirements of the pathologist. We have made no careful search of the literature of pathology for materials on phosphorus metabolism. The whole subject of phosphorus poisoning has been omitted, this matter being considered as quite apart, in reality, from phosphorus metabolism in the usual sense — and the literature is enormous. The relation of phosphatids, especially lecithin, to the action of alkaloids and other drugs, as well as antitoxins, precipitins, hemolysins, bacteriolysins, agglutinins and opsonins is of a special nature, having to do with the maintenance of .immunity rather than with phosphorus metabolism in the usual sense, and is not included in this discussion. 4 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Clinical reports are submitted in great brevity and usually without comment. They certainly have a value, but it is impos- sible to say, in a given case, whether this value be great or small. Some of the reasons for this are as follows : The first interest of the medical profession is the cure of the patient and not the acquirement of experimental evidence ; it is usually impossible to provide proper experimental controls or checks; even in hospitals the environment of the patient does not always constitute a proper basis for exact experimentation; the rights of the patient prevent the full development and realization of such unfavorable results of treatment as might follow under strict- ly experimental conditions; and the imagination of the patient un- doubtedly cuts a figure in symptoms and results which is not char- acteristic of experiments on animals with less capacity to think. The chemistry of phosphorus compounds, and the phosphorus compounds of animal bodies and products, and of foods and drugs have been briefly considered with the idea of making clear the bear- ing of the matter on normal phosphorus metabolism, in which field we have considered all of the material which has come to our atten- tion. The selection of articles for inclusion in this review was made by the use of indices, supplemented by a page-to-page search of the journals considered as most likely to contain the desired material. The main sources of the articles included are as indicated in the fol- lowing list, though many references were also taken from scattered sources. American Journal of Physiology 1 (1898) to 32, (Dec. 1913) Archiv fur die gesammte Physiologie 1 (1868) to 156, 442 (Mar. 1914) Biochemical Journal 1 (1906) to 7, (Dec. 1913) Biochemische Zeitschrift 1 (1906) to 59 (Feb. 1914) Chemical Abstracts 1 (1907) to 8, 1220 (Mar. 20, 1914) Die Ernahrung der landwirtschaftlichen Nutztiere, vierte Auflage; O. Ilell- ner 1907. Ergebnisse der Physiologie 1 to 13 (1913) Experiment Station Record (1899) to 29, 899 (Feb. 1914) Handbuch der Biochemie des Menschen unci der Tiere, Carl Oppenheimer, 1908-1913. International Catalog of Scientific Literature. Q. Physiology 1 to 8 (1911) Jahresbericht fiber die Fortschritte der Thierchemie 1 (1871) to 41 (1911) Journal fur Landwirtschaft 49 (1901) to 61 (1913) Journal of the American Medical Association 34 (1900) to 61 (Dec, 1913) Journal of Physiology 26 (Dec. 1900) to 47, No. 6 (Feb. 1914) Metabolism and Practical Medicine, Vols. I, II, III, Carl von Noorden, 1907. Physiologie und Pathologie des Mineralstoffwechsels, Albert Albu and Carl Neuberg, 1906. Review of American Chemical Research 6 (1900) to 12 (1906)— last issue. Revue de la Societe d' Hygiene Alimentaire 1 (1904) to 7 (1909) entire. The Journal of Biological Chemistry 1 (1905-06) to 17, 304 (Mar. 1914) PHOSPHORUS METABOLISM 5 The Journal Of the American Chemical Society 1 (1879) to 36, 616 (Mar. 1914) Vierteljahreschrift der Nahrungs- Chemie 9 (1894) to 12 (1897) last issue. Zeitschrift fur Biologie 39 (N. F. 21) to 63 (N. F. 45), Heft 9 (Mar. 1914) Zeitschrift fur Kinderheilkunde. Originate 1 (1910-11) to 10, Hefte 1-4 (Feb. 1914) Zeitschrift fur Nahrungs Untersuchung 1 (1898) to 10 (1905) Zeitschrift fur physiologische Chemie 1 (1877) to 89, 324 (Feb. 1914) Zeitschrift fur Untersuchung der Nahrungs- und Genussmittel to 27, 360 (Feb. 1914) In general we have considered only original articles. Most of the study of the literature has been pursued by a uniform method of preparation of abstracts including (1) the complete reference, (2) the object of the investigation, (3) the methods employed, (4) the nature of the data recorded, (5) numerical results and (6) the author's conclusions in full. Gross misconceptions have been generally ignored, in consider- ation of the extreme brevity with which we have been obliged to treat much good material. We have not gone into a discussion of analytical methods, though they have been considered, and much work has been thrown out because of the use of methods unsuited to the purpose. It is true, however, that much work, known to have been accomplished by faulty methods, has been retained, often because the methods have been fairly satisfactory for comparative purposes, and again, frequently because better methods have not been discovered or have not come into general use. In very few cases, for instance, do the differential estimations of the groups of organic phosphorus com- pounds rest upon critically established analytical procedures,' and even so simple a problem as the determination of inorganic phos- phorus in plant and animal substances is still a problem,- fairly well settled for some substances, it is true, but by no means for all from which we report determinations. Candor requires the observation that much of the work con- sidered is without great value, but until supplanted by results of higher grade we are obliged to give it space. This applies espec- ially to metabolism data, the experiments being, as a rule, much too short to afford safe bases for conclusions ; and elaborate discussions of urinary conditions, feces data not being included, are far too common. , Numerical data have been largely retabulated, and very com- monly they have been recalculated. If found inconsistent they have usually been thrown out, but have sometimes been corrected, with the author's- approval. 6 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In addition to data on phosphorus metabolism, we have also commonly taken from the general studies the data on calcium and nitrogen, since these elements are more prominently involved than others with the metabolism of compounds of phosphorus. We are glad to acknowledge here the assistance in abstracting received from B. M. Hendrix, Dr. H. W. Houghton, E. C. Lathrop, Grace Mac Leod, Dr. Martha A. Phelps, Dr. J. B. Rieger, Florence C. Sargent, Marion E. Sparks and Dr. G. M. Tucker. The abstracting has been done in the following institutions : Ohio Agricultural Experiment Station, University of Illinois, Li- brary of Congress, Surgeon General's Library, Library of the De- partment of Agriculture, New York Academy of Medicine, New York Public Library, Columbia University, Massachusetts Institute of Technology, Boston Medical Library, Harvard Medical Library,- Boston Public Library, Harvard University, Boston Natural His- tory Museum, Connecticut Agricultural Experiment Station, Yale University, Cleveland Medical Library and Western Reserve Med- ical College. PHOSPHORUS METABOLISM TABLE OF CONTENTS Part I Chemistry of Organic /Compounds of Phosphorus Page 1 . Nucleoproteins and Their Derivatives 13 2 . Phosphoproteins 32 3 . Phosphocarnic Acid 49 4. Phytin 51 5 . Phosphatids 58 Part II The Phosphorus of Foods 1 . General Discussion 76 2 . Organic and Inorganic Phosphorus in Foods 80 3 . Phytin in Foods 81 4 . Lecithins in Foods 84 5 . Nuclein Phosphorus in Foods 90 6. Pyrophosphoric Acid in Foods 91 7 . Proprietary Preparations 92 8 . Effects of Water on the Phosphorus of Forage Plants 93 9. Effects of Fertilizers on the Composition of Foods 95 10 . Addition of Phosphates to Silage 101 11. References to Extensive Presentations of Total Phosphorus Determinations in Foods ...,.: 103 Part III The Phosphorus of Animal Bodies and Products 1 . General Studies, Grown Men and Animals 104 2 . Fetuses and New-Born Young 108 3 . Bones, Teeth, Marrow, Cartilage Ill 4. Muscle : 120 5. Feathers 126 6. Brain, Nerves, Cerebrospinal Fluid 127 7. Liver 136 8. Spleen 139 9 . Pancreas 140 10. . Kidney 141 11 . Suprarenal Capsules 143 12. Thymus 143 13 . Thyroid 144 14 . Lungs 145 15 . Hypophysis 146 16 . Digestive Mucosa 146 17. Genitalia 146 18. Blood 149 19. Milk 153 20. Eggs 169 21 . Digestive Secretions 172 22. Chyle 177 23. Lymph 177 8 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Part rV Normal Phosphorus Metabolism Page 1 . Maintenance of Neutrality 178 2. The Absorption and Elimination of Compounds of Phosphorus General Consideration 181 The Influence of Phosphates on Digestion 186 Autolysis of Compounds of Phosphorus 187 Method of Urinary Elimination of Phosphates 188 Organic Phosphorus in the Urine 190 Phosphorus Compounds in the Feces 194 3. Phosphorus Excretion as Affected by Various Conditions Acids and Acid Salts 198 Sodium and Potassium 200 Calcium and Magnesium 203 Diet and Species 207 Summary 211 4. Relative Metabolism of Phosphorus and Other Elements 213 5. Metabolism Experiments With Inorganic Phosphates 221 6. Metabolism of Nucleoproteins and Nucleic Acids 229 General Discussion , 229 Digestion Studies with Enzymes from the Alimentary Tract 231 Effects of Bacteria in the Gastrointestinal Tract 234 Ferments of Individual Organs 235 Animal Experiments on the Digestion of Nucleoproteins and Nucleic Acids 237 Accompaniments of the Ingestion or Injection of Nuclear Material 245 Significance of Nucleoproteins and Nucleic Acids as Active Agents in Specific Body Processes 255 Therapeutic and Prophylactic Use of Nucleins and Nucleic Acids ; 256 Summary 258 7. Metabolism of Casein Production of Casein in Milk Glands 260 Digestion of Casein 261 Animal Experiments With Casein 272 Summary 274 8. Metabolism of the Compounds of Glycerophosphoric Acid The Function of Lecithin in Intermediary Metabolism. . . .276 The Influence of Lecithin in Digestion 282 Summary 286 The Digestion of Lecithin and Glycerophosphates 286 Summary 289 Balance Experiments With Lecithin and Other Compounds of Glycerophosphoric Acid 290 Summary 295 Growth and Composition of Animals as Affected by Com- pounds of Glycerophosphoric Acid 296 Summary 303 9. Metabolism of Phosphocarnic Acid 303 PHOSPHORUS METABOLISM 9 Page 10. Metabolism of Phytin Feeding Experiments with Men and Animals 305 Clinical Experiments with Phytin 315 Summary 317 11. The Nutritive Values of Organic and Inorganic Phosphorus Metabolism Experiments with Men and Animals 318 Miscellaneous Experiments , 318 Organic Phosphorus Synthesis in Fasting Salmon . . . .354 Advantage of Complexity of Organization in Food Phosphorus . 355 Feeding Experiments with Rats and Mice on Rations Composed of Simple Purified Nutrients 357 Summary 364 12. Common Foods in Relation to Phosphorus Metabolism Miscellaneous Experiments with Men and Animals 366 The Phosphorus Content of Milk as Affected by Foods .... 379 Diet and the Phosphates of the Bones 384 13. Phosphorus Requirements of Animals General Discussion 397 Phosphorus Excretion During Fast 399 Normal Phosphorus Requirements of Adult Human Beings 403 Phosphorus Requirements of Infants 414 " Cattle 419 " Horses 422 " Swine 424 " Sheep 425 " Dogs 426 " Rats 428 14. Phosphorus Metabolism as Affected by Various Conditions Altitude 429 Amount of Food 430 Fast .438 Incubation 446 Infancy 449 Mental Work 461 Nerve Stimulation 463 Physical Exercise 464 Pregnancy 470 Sleep 472 Thirst and Water Drinking 473 Time of Day 474 Part V Phosphorus Metabolism in Disease 1 . Acid Intoxication 476 2. Acromegaly 479 3 . Alcoholism 483 4. Arthritis 484 5. Beriberi 484 10 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Page 6. Blood in Relation to Pathological Phosphorus Metabolism Chlorosis and Anaemia 492 Leukaemia 493 Blood Dissolution 496 Phosphorus of the Blood as Affected by Anaemia 496 Chlorosis 496 Leukaemia 497 Various Diseases 497 Nuclein Therapy in Anaemia 502 7. Calcareous Degeneration 503 8. Cancer 503 9 . Caries 504 10 . Castration and Ovariotomy 505 11. Composition of the Body in Disease 506 12. Diabetes Mellitus 510 13 . Endoarteriitis 512 14 . Fatty Degeneration 512 15. Fever ! 514 16 . Glycerophosphate Therapy 520 17. Gout 522 18 . Hemorrhage 526 19 . Hookworm 527 20. Icterus 527 21 . Lecithin Therapy 528 22 . Malnutrition of the Bones 534 23. Mental and Other Nervous Disorders Nerve Degeneration 536 Epilepsy 540 Paralysis 541 Insanity 543 Administration of Phosphorus Compounds and Other Drugs in Mental and Other Nervous Diseases 545 24. Nephritis 547 25. Nuclein Therapy 549 26. Oedema 549 27. Organic Phosphorus-Containing Compounds and Preparations, Effects on Phosphorus Metabolism 549 28 . Osteomalacia 552 29 . Parathyroidectomy 559 30. Phosphaturia 561 31 . Phytin Therapy 566 32. Rachitis 566 33 . Thyroid Glands in Relation to Phosphorus Metabolism 577 34 . Toxins and Antitoxins 582 35 . Tuberculosis 583 INTRODUCTION As agricultural scientists our interest in the mineral elements lies in that larger intermediary metabolism between the soil and the sea which begins with the weathering of the rocks, includes the whole of plant and animal metabolism, and ends with the formation of new rocks. Throughout this vast sweep of chemical change the mineral elements occupy a unique and dominating position, entering in es- sential ways into every process, and exerting an influence in metab- olism entirely out of proportion to the amounts in which they are involved. In a large and general way life may be regarded as a coordin- ated system of responses to electrical stimulation. The ions, and es- pecially the inorganic ions, are the bearers of this electricity, and it is because of this fact that they are able to play a leading role in the direction of the whole process of metabolism. Among the several inorganic elements involved in animal life phosphorus is of especial interest. No other one enters into such a diversity of compounds and plays an important part in so many functions. Structurally, it is important as a constituent of every cell nucleus and so of all cellular structures ; it is also prominent in the skeleton, in milk, in sexual elements, glandular tissue and the nervous system. Functionally, it is involved in all cell multiplica- tion, in the activation and control of enzyme actions, in the main- tenance of neutrality in the organism, in the conduct of nerve stim- uli, and through its relation to osmotic pressure, surface tension and imbibation of water by colloids it has to do with the movement of liquids, with the maintenance of proper liquid contents of the tissues, with cell movements and with absorption and secretion. Throughout the intricacies of these processes — in considering the relations of the animal to its food — let it be our point of view that inheritance has furnished the plans, the details and specifica- tions which are to govern the whole course of metabolism; that food builds the structure and maintains its processes, in so far as made possible by the nature and amounts of its constituents ; that variability in the composition and functions of the animal body, and excess of capacity in its structures constitute a provision of safety, a means of adaptive response to changes in dietary conditions ; that time lends to these adaptations such permanency, in the individual, as to constitute specific effects of foods on the life and structure of 12 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 the animal ; that these specific effects of foods are, in general, due rather to their limitations than to stimulation of .supernormal func- tion ; that the nature and possible extent of these effects have been separately determined for each species by the particular conditions, and the variability of conditions of life to which, through the ages, they have become adapted, and that in relation to practical animal nutrition our interests are in the highest states of function rather than in irreducible physiological minima, since the whole range of success and profit lies close, and ever closer, to maximum possibili- ties. The following discussion attempts no complete picture or con- tinuous account of phosphorus metabolism, but seeks merely to ar- range in natural and useful sequence the fragmentary material of which it is composed. BULLETIN OF THE Ohio Agricultural Experiment Station Number 5 March, 1914 PART I CHEMISTRY OF ORGANIC COMPOUNDS OF PHOSPHORUS NUCLEOPROTEINS AND THEIR DERIVATIVES Place in the Scheme of Chemical Compounds. The facts as to the method of growth of the individual animal lead us almost certainly to the conclusion that the transmission of characters from generation to generation, and the determination of the nature of all cellular growth has as its material basis the chemical structure of the nucleoproteins of the cell. In comparison with the almost inconceivable complexity of composition implied by this idea our knowledge of the chemistry of the nucleoproteins is especially superficial and our methods of study most violent. This field must remain for all time in large part unknown and unknowable, and it is only with the grossest facts as to composition, as made manifest by the destruction-of the compounds and the study of the wreckage products that we are here concerned. Nucleoproteins are those compound proteins which yield protein and nucleic acid on cleavage. Any of the simple proteins may occur in this union; and the number of nucleoproteins is practically unlimited. Of those which are found in natural products the protein has been identified in but very few cases. Under the action of weak acids or of gastric juice the cleavage of nucleoproteins does not, in most cases, produce a nucleic acid directly ; but instead, a protein compound of nucleic acid containing less of protein and, consequently, a larger proportion of phosphorus. It is customary to call these simpler protein compounds of nucleic acids by the name nucleins, and to let the name nucleoprotein apply to the naturally occurring substances which are combinations of nucleins with further protein. Nucleoprotein as defined above includes both of these groups. (13) 14 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The nucleins are decomposed by caustic alkali into protein and nucleic acids. All nucleic acids are rich in phosphorus, and on cleavage yield phosphoric acid, purin bases, a carbohydrate (or car- bohydrate derivative) and usually pyrimidin bases. The paranucleins, or pseudonucleins, are derived from the phos- phoproteins (nucleoalbumins) in the same way that nucleins are de- rived from nucleoproteins, and in certain cases paranucleic acids have been obtained, corresponding to nucleic acids. A. Kossel (1891a), proposed the name paranuclein, and noted that para- nucleins differ from true nucleins in not yielding purin. Giertz (1899) says that they may be definitely distinguished from true nucleins in that they readily dissolve in water made slightly alkaline with baryta, and that they are decomposed by excess of baryta with the splitting off of phosphoric acid. Some have reported the cleavage of nucleoprotein directly, with the separation of nucleic acid, and either protein, or its peptones and amino acids (Altmann, 1889; A. Kossel, 1893; Umber, 1901). The reader is referred to the following articles for reviews of the work on this subject reported up to the dates of the several papers: — Nolf (1898), Steudel (1907a), Schaumann (1910), Brugsch and Schittenhelm (1910) and Brahm (1913). Occurrence of Nucleoproteins . Nucleoproteins occur chiefly in the cell nuclei ; but they also often occur in the cytoplasm, and are found in the blood serum and other fluids. They have been obtained most abundantly from those products and organs which are rich in nuclei. The nuclein first discovered was that formed from pus by peptic digestion. This discovery was the work of Miescher. Plosz (1871 and Ibid., p. 441), also obtained a similar substance from the blood corpuscles of a bird and of a snake. Miescher's more extensive study, to which we owe consid- erable of our knowledge of these compounds, was made upon sperm and spermatozoa, chiefly from salmon. The substance which Mies- cher (1878, 1896, 1897) called protein-free nuclein was named nucleic acid by Altmann (1899) on account of its acid properties. The nucleic acids to which the greatest amount of attention has been given are those of the thymus, of spermatozoa and of yeast ; and in addition to these we are fairly familiar with those of the pancreas, adrenals, spleen, testes and fish eggs. The only plant nucleic acid besides that present in yeast, which has been much in- vestigated, is that which T. B. Osborne and his associates have iso- lated from wheat embryo, and have named tritico-nucleic acid (Os- borne and Campbell, 1900a ; Osborne and Harris, 1902 ; Wheeler and Johnson, 1903; Mendel, Underhill and White, 1903; Osborne and Heyl, 1908; Levene and LaForge, 1910). PHOSPHORUS METABOLISM 15 Other early discussions and reports of nucleins besides those of Miescher and of Plosz are those of J. W. Miiller (1873), of Piccard (1874) on salmon sperm, of von Jaksch (1876) on nucleins from human brain, and of Klinkenberg (1882) on some nucleins from various seeds used as fodders. As long ago as 1847 Justus Liebig isolated from flesh an acid to which he gave the name inosinic acid, and which he analyzed, but he did not find the phosphorus. In 1878 Schiitzenberger (1878) named decomposition products of brewer's yeast, which were such as are now recognized as those of nucleoproteins. PROPERTIES OF NUCLEOPROTEINS The nucleoproteins, after isolation and purification, are loose, white powders, insoluble in water, but soluble in alkali solutions by union with the alkali. From such solutions they are precipitated by acids ; excess of mineral acids dissolves them, but acetic acid less readily. They are acid in character. The percentage composition of nucleoproteins is very variable and the determinations doubtful; but all contain phosphorus, per- haps from 0.5 to 3.0 percent. That obtained by Liebermeister (1906) from the blood serum of the horse was very low in phos- phorus, only about 0.08 percent. It has been commonly stated that the nucleoproteins contain iron in organic combination; but the amounts found vary greatly, and the nature of its relation to the molecule, if it be a part of the nucleoprotein molecule, is not known (see S. S. Zaleski, 1886 ; Ham- marsten, 1894 ; Scaff idi, 1908 ; Salkowski, 1909a, 1909b ; Capezzuoli, 1909a). Salkowski (1909b) finds iron present, but very loosely bound. Ascoli (1899) reports it in plasminic acid, showing rela- tions which he thinks indicate a metaphosphoric combination; but Sauerland (1910) fails to find iron in the free nucleic acids from pancreas and spermatozoa heads, and doubts its existence in the nucleoproteins. The cleavage products are characteristic constituents. The only attempts which we have noticed, to make complete determina- tions of all cleavage products are those of Wohlgemuth (1903, 1904a, 1904b, 1905). The nucleoproteins give the color reactions of proteins. So far as examined, they are all dextrorotatory (Gam- gee and Jones, 1903a, 1903b, 1903c). On peptic digestion nucleoproteins yield true nucleins. Weak acids cause decomposition, and the solutions of the alkali combina- tions are decomposed even by heating with water. 16 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 A list of references to nucleoprotein investigations is given below : NUCLEOPROTEIN STUDIES ARRANGED WITH REFERENCE TO THE SOURCE OF THE NUCLEOPROTEIN From Thymus: Bang, 1900a, 1900b, 1903 and 1904; Gamgee and Jones; 1903a, 1903b, 1903c; Halliburton, 1895; Halliburton and Brodie, 1894; Huis- kamp, 1901a, 1901b, 1903; A. Kossel, 1900; Lilienfeld, 1894; Malengreau, 1900, 1901; Steudel, 1913b. From Liver: Goubau, 1911; Halliburton, 1892, 1895; Salkowski, 1909a, 1909b; Scaffidi, 1908, 1909a; Wohlgemuth, 1903, 1904a, 1904b, 1905. From Pancreas: Gamgee and Jones, 1903a, 1903b, 1903c; Hammarsten, 1893, 1894; Jones and Whipple, 1902; Knopf, 1914; Levene, 1904; Levene and Stookey, 1904; Umber, 1900, 1901. From Suprarenals: Gamgee and Jones, 1903a, 1903b, 1903c; Jones and Whipple, 1902. From Brain: Halliburton, 1895; von Jaksch, 1876; Levene, 1899. From Blood Corpuscles: Bang, 1903, 1904; Halliburton, 1895; Halliburton and Friend, 1889; A. Kossel, 1881, 1882; Plosz, 1871. From Blood Serum: Liebermeister, 1906; Pekelharing, 1895. From Leucocytes: Bang, 1903, 1904; Lilienfeld, 1894. From Yeast: A. Kossel, 1879, 1880, 1881. From Other Sources: Spleen; Capezzuoli, 1909a; Goubau, 1911; Sato, 1909. Thyroid; Oswald, Ad., 1899. Kidney; Goubau, 1911; Halliburton, 1892, 1895. Mammary glands; R. Odenius, 1899. Human Placenta; Bottazzi, 1903; Cocchi, 1901. Testes; Goubau, 1911. Spermatozoa; Steudel, 1911a, 1911b, 1913a. Lymph glands; Bang, 1903, 1904. Muscle; Pekelharing, 1896. Marrow; Halli- burton, 1895. Pus; Goubau, 1911; A. Kossel, 1881. Egg- yolk; A. Kossel, 1885, 1886. Several nucleins; Klinkenberg, 1882. Barley sprouts; Petit, 1893. Several seeds; Vorbrodt, 1910. PROPERTIES OF NUCLEIC ACIDS Nucleic acids are white, amorphous, acid powders, insoluble in water, but soluble in ammoniacal or alkaline water. Since all of the phosphorus of the nucleoprotein molecule is in the nucleic acid fraction, the percentage here is high, perhaps 8.0-10.0 percent. The nucleic acids precipitate proteins from solution. The affinity for protein is perhaps their most significant property. Nucleins stand between nucleoproteins and nucleic acids in properties and composition, corresponding with their intermediate position in production. Feulgen (1912, 1913a,) has prepared, analyzed and studied the properties of compounds of nucleic acid with dyes, the composition of which he thinks is such as to indicate the formation of tetrabasic salts, and he suggests that perhaps nucleoproteins are such saltlike combinations of nucleic acids with the basic proteins. Feulgen says that Bang's work on lymph glands gives support to this con- ception, and throws doubt on the existence of nucleins. PHOSPHORUS METABOLISM CLEAVAGE PRODUCTS OF NUCLEOPROTEINS 17 Outline of the Processes: Hydrolytic cleavages produce from nucleoproteins, simple proteins, carbohydrates, phosphoric acid, and purin and pyrimidin bases, with the intermediate formation of nu- cleins and nucleic acids, as may be represented by the following scheme : Nucleoproteins Proteins Proteins Carbohydrates Pentoses Hexoses Unidentified Nucleins Nucleic acids Phosphoric acid Purin bases Pyrimidin bases Adenin Thymin Guanin Cytosin Hypoxanthin Uracil Xanthin The carbohydrates may appear as formic or levulinic acid. Ox- idative processes transform hypoxanthin to xanthin, xanthin to uric acid, and sometimes further oxidize the uric acid (probably allan- toin is formed in such a way, and perhaps oxalic acid and urea) . PUKIN BASES Four purins have been obtained, namely,- guanin, adenin, hypo- xanthin and xanthin. These are spoken of also as the nuclein bases, or alloxur bases. The relations of these to one another and to simple purin are indicated by the following structural formulae : 'Nr^CH HN— CO N=C— NH 2 II II II HC 2 5 C— HN 7 H.N— C * C— HN HC C-HN 'CH 3 N— *C— N* Purin CH N— C— N Guanin CH N— C— N Adenin HN— CO I I HC C— HN \ CH N— C— N Hypoxanthin // HN— CO OC C— HN HN— C— N Xanthin S CH 18 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Guanin and adenin contain the amino group (NH 2 ) while hy- poxanthin and xanthin are oxygen derivatives. The figures placed beside members of the purin ring are used to designate the points at which substitutions are made in making up the molecule of the derivatives, and they may be used in designating these derivatives ; thus guanin is 2-amino-6-oxypurin, adenin is 6-aminopurin, hypo- xanthin is 6-oxypurin and xanthin is 2, 6-dioxypurin. By removal of the amino group guanin becomes hypoxanthin, by removal of amino and introduction of oxygen adenin becomes xanthin, hypoxan- thin by oxidation becomes xanthin, and by further oxidation this becomes uric acid, the structural formula of which follows : HN— CO I I OC C — HN X II II CO HN— C — HN Uric acid (2, 6, 8 — trioxypurin) The importance of these purin bases as decomposition products of cell-nuclei and of nuclein was shown by the researches of Al- brecht Kossel, and made clear in his repeated discussions. He dis- covered and named adenin (Kossel, A., 1886), and we have already mentioned the fact that he marked the distinction between nucleins which yield purin bases on cleavage, and those which do not, by naming the latter paranucleins. The processes transforming one of these purins to another and the enzymes causing such trans- formations have received special attention from Walter Jones. Jones has pointed out that, since guanin and adenin may so readily be changed into the other bases, it is very probable that, at least in some of the cases, the hypoxanthin or xanthin found among the decomposition products was of such origin, and was not present in the nucleic acid as such. By use of his methods, which control changes of that kind, he was able to prove that the nucleic acids of thymus, spleen and pancreas contain no other purin bases but guanin and adenin, and he concluded that the nucleic acids from these three sources, at least, are identical. (Jones and Austrian, 1907; Jones, 1908; Jones and Whipple, 1902). We recommend to the reader his clarifying discussion (1908) "On the Identity of the Nucleic Acids of the Thymus, Spleen and Pancreas," in which he attempts to interpret and adjust the apparently contradictory findings of various authors as to the identity of nucleic acids and of their decomposition products. PHOSPHORUS METABOLISM 19 Guanylic acid contains but one purin base, namely guanin ; and inosinic acid, likewise only one, hypoxanthin. PYRIMIDIN BASES Three pyrimidin bases have been obtained from nucleic acids,- cytosin, uracil and thymin. The relations of these to one another and to pyrimidin are expressed in the graphic formulae as follows : 1 N= 6 CH HN— CNH 2 HN— CO HN— CO II I II II II *HC 5 CH OC CH OC CH OC C— CH 3 II II II I II II II 3 N— 4 CH N=CH HN— CH HN— CH Pyrimidin Cytosin, Uracil, Thymin, 6-amino-2-oxy- 2, 6-dioxy- 5-methyl- pyrimidin pyrimidin uracil The ring nucleus of these compounds is simpler than that of the purins, but might readily be made from that in case of decompo- sition, as may be seen by inspection of the graphic formulae. Each of these compounds was first prepared in Kossel's labora- tory (A. Kossel and A. Neumann, 1893, 1894 ; A. Kossel, 1894 ; As- coli, 1900b), and has been further studied there as well as else- where. All three of these bases are in some of the nucleic acids, though thymin and cytosin are most common. In some cases where uracil has been obtained it was probably a secondary product derived from cytosin. By some it has been held, also, that all of the pyrimidins are secondary products derived from purins (Burian, 1907b) ; but that idea seems to be satisfactorily disproved, at least as to its uni- versal application, by the work of Steudel (1905b, 1907e), and by T. B. Osborne and Heyl (1908) with regard to tritico-nucleic acid. This last acid (from wheat embryo) and yeast nucleic acid contain only cytosin and uracil. Wheeler and Johnson (1903) showed the cytosin from this acid and that from spleen nucleic acid to be identi- cal. Only the simplest of the nucleic acids, guanylic and inosinic acids, are without pyrimidin bases, so far as is known. The expres- sion thymonucleic acids is sometimes used as a group name for such as contain thymin, and that seems to be practically all of those of animal origin. CARBOHYDRATES Among the cleavage products of all true nucleic acids or nu- cleins are carbohydrates or their derivatives ; but the specific identi- fication of the carbohydrate contained in the nucleic acid molecule 20 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 has not generally been made. Kossel and many others have found both a pentose and levulinic acid, which latter is a hexose deriva- tive. All the thymonucleic acids (from animal organs) give evi- dence of some kind of hexose (they yield levulinic acid as a hexose derivative) ; while the vegetable nucleic acids (yeast nucleic acid and triticonucleic acid from wheat embryos) and guanylic and in- osinic acids contain a pentose. Boos (1909), however, in reinves- tigating the question of yeast nucleic acid finds indication that it contains no pentose. When pentose as well as hexose has been ob- tained from the acids derived from animal organs (Levene 1903a; Wohlgemuth, 1903, 1905) it may have been due to an admixture of guanylic acid. F. Bauer (1907) considers that the pentose of inosin- ic acid is racemic arabinose, while Neuberg and Brahn (1907) and Haiser and Wenzel (1909) and Wohlgemuth make it a laevo-xylose, and Levene feels that he disproved both of these ideas, and proved that the pentose in every case is a dextro-ribose (Levene and Ja- cobs, 1909b, 1909c). INTERMEDIARY PRODUCTS By partial cleavage several workers have obtained various de- composition products from nucleic acids which are thought to be of especial interest as throwing light on the makeup of the more complex compound. Frequently the purin bases split off first, but not with sufficient ease to indicate that they were in salt-like combination. Alsberg (1904) isolated what he called heminucleic acid, containing only one- half of the purin bases, and Kossel and Neumann (A. Kossel, 1894 ; A. Kossel and A. Neumann, 1896) obtained an acid free from all the purin bases, without loss of phosphoric acid, by boiling thymus nucleic acid with water. This second acid was called thymic acid, and it has received considerable attention, owing to the suggestion that it might bear the same relation to paranucleins that the nucleic acids bear to true nucleins. Milroy (1896) proved that thymic acid is not identical with paranucleic acid by showing that synthetic compounds of thymic acid with proteins, though much like the nat- ural paranucleins, do not give the same acid on cleavage. Steudel and Brigl (1911) obtained the same, or a similar thymic acid, by nitric acid oxidation of the thymus nucleic acid. A. Kossel (1894), A. Neumann (1898, 18.99), and Kostytschew (1903) found three other modifications of nucleic acid from the thymus gland which they called a-nucleic acid, /^-nucleic acid and PHOSPHORUS METABOLISM 21 nucleothyminic acid, these differing from one another in the amount of nuciein bases present, and showing certain differences in properties. Alsberg (1904) also obtained, on more energetic cleavage, an acid free from all its purin bases and from phosphorus, which he called nucleotin, and which corresponds with Schmiedeberg's (1900) idea that the ground substance of nucleic acids is a nucleotin-phos- phoric acid which is combined in some way with purin bases. The thymic acid of Kossel and Neumann and the thymin-glucophosphor- ic acid of Levene and Mandel (1908a) would be such nucleotin- phosphoric acids. Under the influence of enzyme action or nitric acid oxidation Steudel (1908a)- obtained, from nucleic acid, products in which the sugar was in organic union with phosphoric acid after the purin bases were removed. This investigation of Steudel's also made evident that the purins were closely bound to the carbohydrate sec- tion of the nucleic acid; and the same relationship is indicated by the isolation and the synthesis of compounds of the carbohydrates with purin and pyrimidin bases which have been made by Levene and his associates. Levene, however, has not succeeded in forming them by such cleavage as that of Steudel mentioned above. The union with the carbohydrate, at least in the case qf purin bases, is said to be glucoside-like, and the compounds are called nu- cleosides. Jones (1911b) showed that enzyme cleavage sometimes forms such bodies by splitting off phosphoric acid from nucleic acids. Under special conditions Jones (1912) and Jones and Richards (1914) have obtained a simple nucleic acid, guanylic acid, from the complex nucleic acids of pancreas and of yeast. Considering all of these intermediary products, it seems evi- dent that the purins are more easily separated by cleavage than the pyrimidin bases, that the purin cleavage is gradual, that the phos- phoric acid may be as easily separated as at least a part of the pur- in bases, and that the carbohydrates never split off so as to leave the purin and pyrimidin bases united with phosphoric acid. By mild acid hydrolysis phosphoric acid is more readily split off from purin than from pyrimidin union. MONONUCLEIC ACIDS It is convenient to distinguish between nucleic acids containing only one base, which may be called mononucleic acids, or simple nu- cleic acids, and those which contain more bases, and which may be 22 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 called polynucleic acids, or complex nucleic acids. The latter are the ordinary nucleic acids, and may be looked on as combinations of the simple acids (usually four such). Levene calls the two groups, whether natural or artificial, mononucleotides and poly- nucleotides. Jones (1912) and Jones and Richards (1914) have succeeded in cleaving a polynucleotide, yeast nucleic acid, so as to obtain a mononucleotide, guanylic acid, from it; and the work of Feulgen (1913b) and of Knopf (1914) suggests that Hammarsten's method of preparing guanylic acid from the pancreas may involve a like process. INOSINIC ACID An acid yielding on hydrolysis only phosphoric acid, hypoxan- thin and a pentose, and named inosinic acid, is found in meat ex-_ tracts. It was isolated by Liebig (1847), but the formula then given does not include phosphorus. It is agreed that this is a simple acid containing but one molecule of each component. It is, therefore, what Levene calls a mononucleotide. The empirical formula is C 10 H 13 N 4 PO 8 , but several structural formulae have been suggest- ed. The work of Levene and Jacobs seems to have proved that the linking is such as to include the pentoside inosin, made up of hy- poxanthin and the pentose. Similar formulae are given by Levene and Jacobs and by Haiser and Wenzel; one which the former au- thors suggest is given below, the purin being bound to the sugar in the glucoside manner, and the phosphoric acid in ester manner: H H H H OC— NH HO \ 0=P— O— O— i CIL— C— C— C— C -i N— C CH / HO HO OH O — / HC ^ N-C— N Phosphoric acid Pentose Hypoxanthin (See Haiser, 1895; F. Bauer, 1907; Neuberg and Brahn, 1907; Levene and Jacobs, 1908, 1909a, 1909b, 1911a; Haiser and Wenzel, 1909.) GUANYLIC ACID Bang discovered in the pancreas of the ox a nucleic acid yielding no base but guanin, and he named it guanylic acid. He at first thought glycerin was present, but that idea was not confirmed. The carbohydrate found was shown to be a pentose, and the relation of PHOSPHORUS METABOLISM 23 N:P, that of 5/.1. In his latest reports he gives the formula C 44 H 65 N 20 P 4 O 34 , and speaks of the molecule as made up of 4 guanin, 4 pentose and 4 phosphoric acid groups, together with some unidenti- fied substance not containing nitrogen or phosphorus. (Bang, 1898, 1901a, 1901b; Bang and Raaschon, 1903; Bang, 1908, 1910a, 1910b.) Steudel and Brigl (1910), however, and Levene and Jacobs (1909c, 1909e, 1912c) are inclined to give to guanylic acid a formula and structure more like that of inosinic acid. According to them the empirical formula is C 10 H 14 N 5 O s P. Levene and Jacobs (1912c) find some reason to think the structure is not identical with that of inosinic acid, and give the structural formula as follows: OH Phosphoric acid P — OH I O o H H - H— O-CH,— C— C— C— CH OH o-J // CH \ N— C— N N— C C— NH, OC— NH Pentose Guanin The phosphoric acid is cleaved from guanylic acid more readily than from inosinic. Guanylic acid has been found at least in the pancreas, the spleen, the liver and milk glands. (Steudel 1907d ; von Fiirth and Jerusalem, 1907, 1908 ; Jones and Rowntree, 1908 ; Levene and Man- del, 1908b; R. Odenius, 1899.) Jones (1912) has produced it from yeast nucleic acid. See also Jones and Richards (1914). The suggestions of an adenylic acid, corresponding to guanylic, have apparently been made on insufficient evidence (A. Kossel and A. Neumann, 1894; Bang, 1904). POLYNUCLEIC ACIDS General Similarities. The numerous nucleic acids of animal origin containing both purin and pyrimidin bases, which have been investigated, show striking similarities, and very likely many of them are identical. It is supposed that as a rule they consist of phosphoric acid, adenin, guanin, 2 (or 3) pyrimidin bases, and a hexose, the definite nature of which is not determined. The plant 24 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 polynucleic acids which have been studied contain pentose in place of hexose. Levene and LaForge (Levene, 1909b; Levene and LaForge, 1910) find the pentose of both yeast nucleic acid and triticonucleic acid to be d-ribose, and that these acids yield the same products on partial hydrolysis. Therefore they think that these acids are identical. It is customary to think of these complex acids as made up on the basis of four phosphorus atoms to the mole- cule. For convenience, a general list of references to studies of these nucleic acids is given here, grouped with reference to their source. NUCLEIC ACID STUDIES ARRANGED WITH REFERENCE TO THE SOURCE OF THE NUCLEIC ACID From Thymus: Altmann, 1889; de la Blanchardiere, 1913; Burian, 1904a, 1904b, 1907a; Herlant, 1900; Iwanoff, 1903; Jones, 1908; Jones and Austrian, 1907a; Kossel, A., 1894; Kossel, A., and A. Neumann, 1893, 1894, 1896; Kosty- tschew, 1903; Levene and Mandel, 1908a; Levene and Jacobs, 1912a, 1912b; Neumann, Albert, 1898,1899; Steudel, 1904, 1905a, 1905b, 1907b, 1907c, 1907d, 1908a, 1908b, 1912, 1913b; Tschernorutzky, Helene, 1912b. From Spermatozoa or Sperm: Alsberg, 1904; Altmann, 1889; Herlant, 1900; Inouye, 1904, 1906; Kossel, A., 1896; Levene and Mandel, 1906e; Miescher, 1878, 1896, 1897; Noll, 1898; Sauerland, 1910; Schmiedeberg, 1900; Steudel, 1906a, 1906b, 1907b, 1907c, 1907d, 1911a. From Spleen: Bang, 1903, 1904; Inouye, 1904; Jones 1908; Levene, 1901b, 1903a, 1903b, 1904, 1905; Levene and Mandel, 1906a. From Pancreas: Feulgen, 1913b; Jones, 1908; Levene, 1901b, 1903a, 1903b, 1903d, 1904; Levene and Jacobs, 1909c, 1909e; Sauerland, 1910; Steudel, 1907f; von Fiirth and Jerusalem, 1907. From Testes: Inouye, 1904; Levene, 1903f, 1904. From Fish Roe: Levene, 1901b; Levene and Mandel, 1906b; J. A. Mandel and Levene, 1906b; Tschernorutzky, Helene, 1912a. From Yeast: Altmann, 1889; Ascoli, 1899, 1900b; de la Blanchardiere, 1913; Boos, 1906, 1909; Burian, 1904a, 1904b, 1907a; Herlant, 1900; Jones, 1912; Jones and Richards, 1914; Kowalevsky, 1910'; Levene, 1901b, 1903d, 1909b; Le- vene and Jacobs, 1909c, 1909f, 1909g, 1910, 1911b; Levene and La Forge, 1912; Liebermann, 1888b, 1889, 1890; Liebermann and Bitto, 1893; Tschernorutzky, Helene, 1912b. From Wheat Embryo: Levene and La Forge, 1910; Osborne, T. B., and Campbell, 1900a; Osborne, T. B., and Harris, 1902; Osborne, T. B., and Heyl, 1908; Wheeler and Johnson, 1903. From Brain: Levene, 1899, 1903c, 1903f, 1904. From Liver: Levene, 1903e; Levene and Jacobs, 1909c. From Kidney: J. A. Mandel and Levene, 1906a. From Intestinal Tissue: Araki, 1903b; Inouye and Kotake, 1905. From Mammary Glands: Basch, 1898; Lobisch, 1906; J. A. Mandel and Levene, 1905. From Human Placenta: Kikkoji, 1907b. From Leucocytes: Ascoli, 1900a; A. Kossel, 1893. General Work: Levene, 1903c, 1910; Levene and Medigreceanu, 1911b, 1911c, 1911d; Schmiedeberg, 1907. Elementary Composition. Since these compounds are in them- selves so complex, and, moreover, always exist, whether in the body or in the food, in conjunction with undetermined proportions of PHOSPHORUS METABOLISM 25 other compounds, and since it is quite doubtful whether they have ever been examined either in a pure condition or in their natural state, it seems that the percentage content of phosphorus or other constituents has no especial significance other than as contributing to a knowledge of the formulae, and then only as an aid to the study of the structure as indicated by chemical reactions and cleavage products. The question of the percentage composition of the proteins which are. united to the nucleic acids is practically untouched. On account of such interest as they may possess, a few empirical form- ulae of nucleic acids are presented. They are determined in part by analysis and in part by probable structure. FORMULAE OF A FEW NUCLEIC ACIDS Nucleic acid Investigator Date and reference Empirical formula Schmiedebergf Steudel Levene Kowalevsky Osborne and Harris 1900 1912 1909b 1910 1902 C40H56N14P4O26 C43H61N15P4034 C38H49N15P4029 C29H42N13P3023 Triticonucleic acid, (from wheat embryo) C41H61N16P403I Structure. Two extensive investigations which have led to somewhat definite pictures of the structure of these nucleic acids are those of Steudel and of Levene and his collaborators. With regard to the principal thymus nucleic acid (and Steudel thinks that from herring sperm is identical with this) these authorities are agreed that the molecule contains the four bases guanin, adenin, thymin and cytosin in unimolecular relation, and each bound to a hexose molecule which in turn is bound to phosphoric acid, there being four phosphoric acid molecules ; also that the linking of the hexose with the purin bases (and perhaps that with pyrimidin bases) is glucoside-like, but the union with pyrimidins is much less easily broken than that with purins. Steudel's picture involves a more condensed form of phosphoric acid than Levene's. Levene, applying the name mononucleotide to the individual complexes of a single base, carbohydrate and phosphoric acid, which he considers quite analogous to inosinic and guanylic acids (see the graphic formulae given) , looks upon these acids as polynucleotides made up of four mononucleotides. Jones (1912) has brought about a cleav- age of yeast nucleic acid with the production of the mononucleotide, guanylic acid. Steudel's formula does not attempt to represent the linking within the hexose, nor in the base-hexoside. 26 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Steudel's (1912) formula for thymus acid is: (OH) 2 = P— C„H u N 5 O d (Guanin-hexoside) /\ O O \/ P — C 10 H 14 N 3 O 6 (Cytosin-hexoside) /\ O O \/ P— C n H 15 N 2 7 (Thymin-hexoside) /\ O O \/ (OH) 2 = P— C u H 14 N 5 5 (Adenin-hexoside) In Levene and Jacobs's formula (p. 27) the two pyrimidin-nu- cleotides are represented as linked to each other by their sugar, while the phosphoric acids of the purin-nucleotides serve to link the purin-nucleotides with the sugar (not the phosphoric acid) of the pyrimidin-nucleotides. These conceptions correspond with the in- termediary products obtained.* From yeast nucleic acid Levene and Jacobs (1909f, 1909g, 1910, 1911b) find the phosphoric acid more readily cleaved and the purins less readily than from thymus nucleic acid, and the structural form- ula suggested is : OH / 0=P-C 5 H 8 4 C 5 H 4 N 5 \ O / 0=P-C 5 H 8 4 C 5 H 4 N 5 O / 0=P— C 5 H 8 4 - C 4 H 4 N 3 O / 0=P-C 5 H 8 4 C 4 H 3 N 2 8 \ OH PHOSPHORUS METABOLISM 27 Levene and Jacob's (1912b) formula for thymus nucleic acid is: O — H. H I I H 2 C — C — C — C — C — C — Guanin I ! I I I I ■ i OH H OH OH H O I HO— P=0 OH , / p=o o o OH O H I H H 2 C-C— C — C — ( 1 1 i :— c- i - Thymin 1 1 1 H H OH ( i H H H OH 1 1 1 H j 1 1 1 H 2 C — C— C— C — ( :— c- - Cytosin o H H — o— o OH / PrO \ HO— P=0 OH O | OH HOH OH H H 9 C — C — C— C — C — C — Adenin H H- H O Levene's ideas as to the structure and cleavage of nucleic acids, together with the distribution of cleaving enzymes in the animal body were clearly summarized by Levene and Medigreceanu (1911d) . See Ferments of Individual Organs. Helene Tschernorutzky is making a study of the glucoside re- lation in these acids by attempting cleavage by means of enzymes which are known to have the power to split glucosides. So far the 28 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 evidence is unfavorable, for the work reported in August, 1912 (Tschernorutzky, H., 1912b) resulted in a separation of inorganic phosphate as well as purin bases, indicating a more thoroughgoing cleavage, a nuclease action. Burian (1904a, 1904b, 1907a) believes the purin bases to be linked to the remainder of the nucleic acid at the 7-place of the purin ring. Levene thinks that the pyrimidins are linked at either the 8- or the 4- place. (Levene and LaForge, 1912). ENZYMES CONCERNED IN NUCLEIC ACID CLEAVAGE The name nuclease has been used to signify an enzyme which brings about complete disruption of the nuclein molecule. It was first applied by Iwanoff (1903). Evidence of such an enzyme (or enzymes) has repeatedly been found in aqueous extracts of animal organs (Jones, 1904a, 1904b; Levene and Medigreceanu, 1911b, 1911d) ; but it is probable that the process is due to several enzymes acting in succession. Jones distinguishes four enzymes having the power to deamin- ize, two of which, guanase (Jones and Partridge, 1904) and adenase (Jones and Winternitz, 1905), act directly on the purin bases (guanin and adenin, respectively), and two on the glucosides of these bases (guanosin-deaminase converting guanosin to xanthosin, and adenosin-deaminase converting adenosin to. inosin, the hypo- xanthin glucoside) (Jones, 1911b). Accordingly he recognizes two general paths by which the nucleic acid may be transformed for the formation of uric acid; (1) a nuclease may cleave off the phos- phoric acid and the carbohydrate, leaving the purin (and the pyri- midin) bases, the purin bases then being transformed by the de- aminizing ferments guanase and adenase into xanthin and hypoxan- thin, respectively, the hypoxanthin being oxidizable to xanthin, and xanthin to uric acid through the agency of xanthooxidase; or (2) the nucleic acid may first be broken down by a cleavage of phos- phoric acid only, by which guanosin and adenosin are left, and they may be deaminized at once into xanthosin and inosin, and then have their carbohydrates cleaved off, with the formation of xanthin and hypoxanthin ; or the carbohydrates may break off before deaminiza- tion, which would transform them to guanin and adenin, upon which guanase and adenase act as in the other line of cleavage ; in either case the xanthooxidase completes the process. The following dia- gramatic scheme taken from Amberg and Jones (1911b) may help to make this clear. The arrows indicate possible changes, for each of which a distinct ferment might be required. The numbered ones have been proved to be present in one or more organs or fluids. PHOSPHORUS METABOLISM 29 DIAGRAMATIC SCHEME OF POSSIBLE CLEAVAGES OF NUCLEIC ACID AND THE TRANSFORMATIONS RESULTING IN THE FORMATION OF URIC ACID Nucleic acid, shown as dinucleoside OH OH I / 0=P— 0-C 5 H 8 3 -C 5 HN 4 \ \ O NH 2 / O = P-O • C 5 H g 3 • C 5 H 2 N 4 (NH 2 ) I OH OH OH / / C 5 H 2 N 4 C 5 HN 4 • C 5 H 9 4 C J H,N t (NH,)-C ! H,0 1 C 5 H 3 N 4 (NH 2 ) \ \ NH, NH 2 Xanthosin OH / Inosin __ C 5 HN 4 • C 5 H 9 4 C 5 H 2 N 4 (OH)-C 5 H 9 4 \ OH Xanthin <- C 5 H 2 N 4 — OH \ OH -> Hypoxanthin C 8 H 3 N 4 (OH) Uric acid OH / C 5 HN— OH \ OH 30 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Enzymes represented: 1. Phosphonuclease — cleaving off phosphoric acid. 2. Purin nuclease — cleaving off purins. 3. Guanosin deamidase — removing amino group (NH 2 ) of guanosin. 4. Adenosin deamidase — removing amino group (NH 2 ) of adenosin. 5. Adenase — removing amino group of adenin. 6. Guanase — removing amino group of guanin. 7. Xanthosin hydrolase — hydrolyzing xanthosin, removing carbohydrate. 8. Inosin hydrolase — hydrolyzing inosin, removing carbohy- drate. 9. Xanthooxidase — oxidizing hypoxanthin or xanthin to uric acid. By the terminology of Levene and Medigreceanu (1911d) such nucleic acids as the above would be called "dinucleotides," the simplest of polynucleotides, and the first enzymatic process (brought about by "nucleinases") is the cleavage of polynucleotides to simple nucleotides. The enzymes cleaving these nucleotides, such as Jones calls phosphonucleinase, Levene and Medigreceanu call nucleotidases, and the glucosides resulting they call "nucleosides." The enzymes causing the cleavage of nucleosides are named "nucleo- sidases." STUDIES ON ARTIFICIAL NUCLEIN SYNTHESIS Nucleins have not been made artificially. A number of workers have, however, succeeded in bringing about synthesis of organic compounds of phosphoric acid which seem to resemble in their make-up fractions of the nuclein molecule, and certain of them have been called nucleins. None of these, so far as we have learned, are unions of the phosphoric acid with both carbohydrate and nitrogen- ous components, and therefore they have not even all the constitu- ents of the nucleic acids. The processes which have been observed may be looked on as partial syntheses of nucleins, for phosphoric acid unions have been made with carbohydrates on the one hand, and with simple proteins on the other. Apparently direct union of simple proteins with phosphoric acid radicals occurred in the studies of Pohl (1889), of Liebermann (1888b), of Fuld (1902a), and perhaps of Malfatti (1892, 1893). In all of these cases metaphosphoric acid or its salt was used. Bech- hold (1901) reports a similar compound of orthophosphoric acid with albumin formed by use of phosphorus oxychloride. Such PHOSPHORUS METABOLISM 31 compounds as these were, at the time they were made, looked upon as synthetic nucleins (or paranucleins) . Giertz (1899) definitely proved that in such a compound as Liebermann's the phosphoric acid does not bear the same relation to the albumin that it does in the natural paranuclein from casein. We now know that even the para- nucleins are not made up simply of phosphoric acid and albumin. Nucleic acids readily precipitate proteins from solution, and, according to the investigation of Milroy (1896), there are then formed firm chemical compounds which somewhat resemble nu- cleins. Those formed from thymic acid and protein were not identi- cal with natural paranuclein. By methods similar to that of Bechhold, but carried out at low temperatures, Neuberg and his associates (Neuberg and Pollak, 1910a, 1910b, 1910c, 1910d ; Neuberg and Kretschmer, 1911) have succeeded in forming phosphate compounds with the carbohydrates saccharose, glucose, fructose, maltose, lactose and galactose, with glycerin and also with the phosphorus-free proteins lactalbumin and serum globulin. These phosphorized proteins are said to resemble casein in elementary composition and in behavior, in so far, at least, that the first cleavage produced in tryptic and in peptic digestion seems to correspond to the digestion of casein as described by Sal- kowski (1899, 1901). The authors consider the phosphoric acid to have entered into the protein molecule by uniting with the amino- or imino- group of the protein to form a substituted phosphaminic acid. Carbohydrate-phosphoric acid esters have frequently been pro- duced in recent years in connection with yeast fermentation of sug- ars, the union apparently being effected by a ferment present in the yeast. These complexes act as catalytic agents in accelerating the fermentation by yeast or yeast-juice, and it is thought that their formation and subsequent hydrolysis furnish the mechanism by which the well-known favorable influence of phosphates in such fer- mentation is exerted. Von Lebedew and Griaznoff (1912) give a definite picture of the process as they conceive of it. The extensive work of Young and Harden, of von Lebedew, and of Euler and his associates is especially to be noted. (See Wroblewski, 1901 ; Iwanoff, 1907; A. Harden and Young, Proc. Chem. Soc. 21 (1905), 189; A. Harden and Young, 1906, 1908a, 1908b, 1909, 1910, 1911a, 1911b; Young, 1907, 1909, 1911 ; A. Harden, 1910 ; A. Harden and Norris, 1910; von Lebedew, 1910a, 1910b, 1911a, 1911b, 1911c; Euler and Fodor, 1911; Euler and Lundeqvist, 1911; Euler and Kullberg, 1911a, 1911b; Euler and Ohlsen, 1911, 1912; Harden and Young, 1912 ; Euler, 1912a, 1912b, 1913 ; Euler and Backstrom, 1912 ; 32 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Euler and Funke, 1912; Euler and Johansson, 1912a, 1912b, von Lebedew, and Griaznoff, 1912.) Young identified his compound as a diphosphoric acid, ester of the hexose fruc- tose, and others confirm this finding of a diphosphoric ester. Euler has given much attention to the enzyme involved, which he names "phosphatese." Euler and Johansson (1912b) find that neither glycerol, inositol, mannitol nor alanine forms such an ester with phosphates, nor do glucose, fructose and mannose till after they have been altered in some way by an enzyme or by dilute alkali. Von Lebedew and Griaznoff find that dioxyacetone forms an ester with phosphate but glycerin aldehyde does not. According to their conception the yeast fermentation of glucose first cleaves the sugar into these two compounds and then the latter ferments direct- ly; the former by way of the intermediate phosphate ester forma- tion. Langheld (1910, 1911, 1912) has synthesized first meta- and from them orthophosphoric acid esters from the alcohols. Among his products is a monoester of fructose which he thinks may be identical with the hexose compound of Young and of von Lebedew. These synthetic investigations will probably give valuable evi- dence, though indirect, as to the structure of the natural organic phosphorus compounds and as to the anabolic processes which may go on in the animal body. THE PHOSPHORUS OF NUCLEOPROTEINS Probably all of the phosphorus of the nucleoproteins is con- tained in the nucleic acid fraction, and as has been shown, it is there present in a highly oxidized state and apparently ready formed as phosphoric acid, more or less dehydrated, or else as esters of ortho- phosphoric acid. If, therefore, the natural substances, the nucleo- proteins, are completely digested, they yield phosphoric acid; and if only partially digested, the phosphorus is left in the form of sub- stances, nucleins or nucleic acids, which may or may not be of direct use in the body. The question of the extent to which digestive cleavage does take place will be considered in the discussion of met- abolism of nucleoproteins. CHEMISTRY OF PHOSPHOPROTEINS CASEIN Our treatment of the literature on the chemistry of casein is by no means exhaustive. Our earliest note is of a discussion by Braconnot in 1830. Since that date there have been probably a thousand articles on this subject. For general reviews see Fuld (1902b) andRaudnitz (1903). PHOSPHORUS METABOLISM 33 NATURE OF THE COMPOUND Casein is a phosphoprotein. The recommendations made in 1908 by the joint committee of the American Physiological Society and the Society of Biological Chemists place phosphoproteins among the conjugated proteins and define them as "Compounds of the pro- tein molecule with some, as yet unidentified, phosphorus-containing substance other than a nucleic acid or lecithins," adding "they are possibly esters of some phosphoric acid or acids and protein." The earlier view which looked on them as simple proteins and used the names nucleoalbumin or phosphoglobulin is thus discarded. Plimmer (1913b), judging from the comparative instability toward alkali hydrolysis, thinks that the phosphoproteins are not esters, and that the phosphorus is probably combined with one of the amino-acids. Phosphoproteins differ from nucleoproteins in not yielding purin bases on cleavage, and therefore not containing true nucleic acids. Incomplete peptic digestion does, however, leave a phos- phorized body resembling the nucleins, which are derived in the same way from nucleoproteins. The residues from phosphoproteins are called para- or pseudonucleins. All the attempts that have been made to isolate a paranucleic acid from paranuclein have failed in that the product obtained still contained protein. Levene and Als- berg (1901) reported such an acid from the paranuclein of ovovitel- lin, as also did Salkowski (1901), Reh (1908) and M. Dietrich (1909) from the paranuclein of casein. Salkowski examined his preparation in the form of an iron compound, Reh as a uranyl com- pound, and Dietrich separated four calcium preparations. Reh thought his compound corresponded with a uranyl ammonium phos- phate in which the ammonium was replaced by an albumose com- plex bound in a manner more or less ester-like but not like a salt, and he calls it a polypeptid phosphoric acid. The formula given for the uranyl ester is C 29 H 5C N s P 2 U 2 0, 4 . Dietrich speaks of his com- pound as a phosphorus-containing casein peptone. OCCURRENCE OF CASEIN Casein occurs only in the milk of mammals. It forms the main part of the curd of milk. (For table of casein contents of the milk of different species consult index.) The amount in cow's milk is usually given as not far from 3 percent, while that of human milk is, by most observers, put as low as 1 percent, or even lower. In this respect the milks of the goat, pig and elephant seem to be nearly like that of the cow, those of the ass and mare nearly like that of woman, while those of the buffalo, dog, cat, ewe, and guinea pig have higher casein content than that of the cow. 34 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In milk the casein is in suspension in combination with calcium and perhaps calcium phosphate. According to Lindet (1913a, 1913b) there are two caseins in milk which may be distinguished by their action on polarized light. The solubility of both in the milk is favored by the presence of phosphates and citrates. ELEMENTAEY ANALYSIS AND EMPIRICAL FORMULA The table below shows the elementary analysis of casein from cow's milk as reported by different . authors and the second table similar analyses of casein from the milk of different species. ELEMENTARY ANALYSIS OF CASEIN FROM COW'S MILK— Percent Author quoted Date and refer- ence C H N S P Makris 1876 53.622 7.422 14.20 Hammar- sten 1883, 1885a 52.96 7.05 15.65 0.758 0.847 22.78 Mean results from many substances. Chitten- den and Painter 1885 0.88 0.85 Seven samples Beohamp 1893 0.752 Mean of four determinations Von Szontagh 1893 0.87 Hemp el (Leh- m arm's 1894 54.00 7.04 15.6 0.771 0.847 Computed to ash-free basis work ) Wr6blew- ski 1894a, 1894b 53.00 7.00 15.70 0.80 0.85 25.65 Osborne, Thomas 1902 0.86 Storch 1902 53.00 6.4 15.7 0.80 0.85 22.65 Quoted by Ellen- berger 1902 53.07 7.13 15.64 0.76 0.8 Mann, Chem- istry of Pro- teids Burow 1905 52.825 7.095 15.64 0.725 0.808 22.906 Taken through Tangl, 1908 Tang] 1908 52.69 6.81 15.65 0.832 0.877 23.14 Kikkoji 1909 0.86 + Van Slyke and Bosworth 1912, 1913a 53.50 7.13 15.80 0.72 0.71 22.08 Ash only 0.06 percent PHOSPHORUS METABOLISM 35 ELEMENTARY. ANALYSIS OF CASEIN FROM DIFFERENT KINDS OF MILK— Percent Source of milk Author quoted reference C H N S P 53.00 7.00 15.7 0.'8 0.86 1876 52.353 7.266 14.650 ( 1894a, 1 1894b 52.24 7.325 14.97 1.117 0.679 j 1894a, ( 1894b 52.41 7.28 15.03 1.11 0.651 1908 52.82 7.04 14.47 0.78 0.26 1910 52.51 7.07 14.61 0.731 0.243 1908 52.88 7.81 15.78 0.833 0.773 1902 54. 7.0 14.4 0.84 1.04 1908 52.57 7.01 16.28 0.588 1.057 1908 52.92 7.05 15.71 0.717 0.809 1905 52.805 7.02 15.64 0.718 0.815 1908 52.90 6.86 15.48 0.700 0.760 1905 52.866 7.045 15.595 0.733 0.810 1908 52.36 7.09 16.44 0.528 0.871 Cow Woman Buffalo Ass Ass Sheep Goat Goat Rabbit Mare Summarized from previous table Makris Wroblewski 1 Wrohlewski 2 Bergrell and Lang-stein 3 . . . Langstein and Edelstein 4 . Tangl Storch Tangl Tang-K Burow 5 , Tang-1 Burow 5 Tangl 22.8 23.66 21.925 23.32 22.495 22.794 23.002 23.300 22.952 22.705 (1) Mean of eight analyses on five preparations. (2) Mean of six analyses on three whitest preparations. (3) Mean of two. Hot water washing may have removed some S and P. (4) Mean values for five preparations. (5) Taken from Tangl, 1908. From Hammarsten's analytical data Richmond (1901) com- puted the "probable approximate formula" for casein as separated from cow's milk by Hammarsten's method, C 162 H 258 N 41 SP0 52 , and for the salt separable by the porcelain mter,C 162 H 255 N 41 SP0 52 CaNa.I/2 (Ca 3 P 2 8 ). The formula given by T. Osborne (1902) is C 708 H 1130 N 180 S 4 P 4 O 224 , indicating a more condensed molecule. Raudnitz (1903) quotes formulae of the simple protein from Knop, C 64 H 100 N 16 2 and from Millon- Commaile, C 10S H 97 N 14 O 29 . PHYSICAL AND CHEMICAL PROPERTIES OF CASEIN Casein is a white, amorphous powder, practically insoluble in water. It is an acid and as such readily dissolves in solutions of the hydroxides or the carbonates of alkalis or alkaline earths by forming soluble salts. These salt formations have been much in- vestigated. (See Soldner, 1888; Bechamp, 1893; Courant, 1891; de Jager, 1897 ; Osborne, W. A., 1901-02 ; Long, 1906a ; Robertson, 1906-07, 1908; VanSlyke and Bosworth, 1912, 1913b, 1913c, 1913d; Bosworth and VanSlyke, 1913). L. L. VanSlyke and A. W. Bos- worth (VanSlyke and Bosworth, 1912, 1913c; Bosworth, 1913) de- cide that it has 8 valences toward metals, and that its molecular weight is 8888. The specific rotation of alkali solutions of casein has been investigated by Long (1905,1906a). . 36 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Being a protein, casein also forms union with acids, and ion- protein compounds with salt ions. Long (1907b) discusses the combining power toward acids. The alkali or alkaline earth solu- tions of casein are precipitated by a very little acid, and are soluble in excess of the acid (in most cases). Increase of temperature favors the precipitation by acids. The presence of certain salts is said to favor the solubility of casein and its salts, but sodium chloride, magnesium sulphate and some others "salt out" casein from solution. (See Biedert, 1887; Schroter, 1887; Storch,1897, 1902 ; Schmidt-Nielsen, 1906; Robertson, 1906-07.) According to Dakin and Dudley (1913a, 1913b), dilute alkalis acting on casein at low temperatures racemize the casein by tauto- meric changes within the protein complex. Such racemized casein, and racemized caseose resulting from it by hydrolysis, were found to escape digestion and absorption when given either per os or sub- cutaneously to a dog. The most characteristic property of casein is its coagulation with rennet in the presence of lime salts. Hydrolysis by boiling acids, or under the influence of enzymes, cleaves casein as it does other proteins. Buxton and Rake (1911) discuss the various types of coagulation in connection with their study of the flocking out of colloids in general. COAGULATION BY ACIDS AND BY HEAT Pure casein salt solutions and fresh milk do not coagulate on boiling, but in the presence of free acid coagulation may take place below the boiling temperature. The coagulum formed in the case of milk includes fat and calcium phosphate. The slight pellicle which coats over milk when it is warmed is of the same composition. The usually accepted explanation of the precipitation of casein by acids is that the casein is held in solution by chemical union with a base (lime in the case of milk) , that added acid removes the base, allowing the insoluble casein to precipitate, and that excess of acid unites with casein forming a compound which is more or less readily soluble. Probably what actually occurs is not quite so simple; it may be rather that there are salts of different proportions of base formed in the presence of different proportions of acid, and that some of these salts are soluble and some are insoluble. L. L. and D. D. VanSlyke (1906, 1907) showed by conductivity methods that in some cases, at least, the taking up of acid is a process of ad- sorption; while Sammis and Hart (Sammis, 1907a, 1907b; Sammis and Hart, 1909) have shown that the amounts of the different PHOSPHORUS METABOLISM 37 acids required for coagulation are not chemically equivalent, and that the time required is affected by the presence of other substan- ces as well as by the temperature and the concentration. All of these observations emphasize complication in the process. The papers of VanSlyke and Hart (1902, 1905a, 1905c) should be mentioned in this connection. Other studies of VanSlyke and Hart consider the reaction with acids particularly in relation to cottage cheese (1904) and to the so-called ''mottling" of butter (1905b). In the coagulation it may be that, in addition to such chemical changes as have been described, there is also concerned a chemico- physical aggregation of molecules as suggested by T. Brailsford Robertson (1908) in the article, "On the Influence of Temperature upon the Solubility of Casein in Alkaline Solutions." In this con- nection the nature and complexity of casein as a protein should be borne in mind. The protein molecule always shows both acid and basic qualities — is amphoteric — indicating that it yields both hydro- gen (H + ) and hydroxyl (OH - ) ions on electrolytic dissociations such as is conceived to take place spontaneously in any water solution. The molecule may be represented by the formula HXOH, in which X represents a carbon nucleus of variable complexity. Any two such molecules may unite, and, with the elimination of water, form a molecule of the type HXXOH, according to the equation : HX(OH+H)XOH=HXXOH+H 2 0. Proteins generally are made up by the union of simple bodies of this type. According to Robertson's conception, in a solution of a protein or its salt the molecules of the protein unite with each other to a certain extent, in this way forming polymers. The reaction is re- versible, and the point of equilibrium between the compound and its polymeric modification varies under the influence of whatever con- dition affects the concentration of the protein ions. Addition of water, or of acid, alkali or salt, or the application of heat has such an effect, and consequently alters the relative number of heavier molecule-complexes. Robertson's experiments give evidence that one of the effects of increase of temperature on a solution of casein is a shifting of the equilibrium in the direction of the higher complexes. He explains coagulation as being a result of these molecular aggre- gates becoming so large as to assume the properties of matter in mass and to become practically an unstable suspension and then a precipitate. 38 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 COAGULATION OF CASEIN BY RENNET For general discussions see Fuld (1902b) and Kastle and Rob- erts (1909). The Enzyme. Coagulation is brought about by an enzyme called "rennin" or "chymosin," which is usually obtained from the water infusion of the fourth stomach of the calf or sheep. As was first noted by Bang (1900c), the corresponding enzyme obtained from the gastric juice of the pig, of man, and of some other animals differs somewhat from chymosin, and hence is designated "para- chymosin." It has been shown that this parachymosin action is to be attributed to pepsin, the same enzyme which has the proteolytic action in these juices, and that proteolytic enzymes generally have coagulating power under suitable conditions. Several workers hold also that the distinction between chymosin and parachymosin is apparent, not real, and that chymosin also is identical with pepsin. Many articles have appeared, mainly in Hoppe-Seyler's Zeitschrift f iir physiologische Chemie, on the two sides of this controversy. For a recent discussion of evidence for the identity, see Van Dam (1912) ; and for counterargument and evidence, see Rakoczy (1913). Rakoczy interprets his experiments, and harmonizes those of others, by the theory that there is in the stomach of young animals of certain species (notably the calf) an independent coagulating enzyme accompanying the pepsin, though the pepsin also has co- agulating action of its own together with its proteolytic action, but this special enzyme is lacking in the adults of the same species and in the young of other species. He notes also that in the very young of some species both enzymes are entirely lacking and coagulation is due only to the acid of the stomach. Enzymes (or their zymogens) having rennin-like action are found in several organs of the higher animals and in blood (see Ed- munds, 1896), and they are said to occur even in invertebrates, and to be widely diffused in the plant world. That of the pancreatic gland has been most studied. (See Roberts, 1879, 1881; Edkins, 1891; Harris and Gow, 1892; Halliburton and Brodie, 1896). The Coagulum and Conditions for its Formation. Rennet coag- ulation differs from coagulation by acids, and cannot be looked upon as a simple removal of the base from a caseinate. The presence of soluble calcium salts (or other alkaline earth salts) seems to be es- sential, and the precipitate formed is not casein or a casein salt, but a salt of a slightly different nucleoalbumin called "paracasein." Many writers, following Halliburton, call this modification produced PHOSPHORUS METABOLISM 39 by rennin the "casein," and that from which it is derived, "casein- ogen." Foster and a few others have used the term "tyrein" for the rennet clot. A number of investigations have been made concerning the conditions essential or favorable to formation of the coagulum, es- pecially with regard to the effects of the degree of acidity and of conditions affecting the amount of calcium present,* either as free soluble salt or bound to the casein. (Hammarsten, 1877 ; Schaf- f er, 1887 ; Soldner, 1888 ; Arthus and Pages, 1891 ; Courant, 1891 ; Leze and Hilsont, 1894 ; de Jager, 1897 ; Locke, 1897 ; VanSlyke and Hart, 1902, 1905a, 1905c; VanDam, 1909a; Van Slyke and Bosworth, 1912; Bosworth, 1913; Schryver, 1913.) Soluble salts of calcium, barium and strontium favor or hasten coagulation, while salts of ammonium, sodium and potassium retard or prevent coagulation. The bulk of the coagulum from milk is a calcium paracaseinate, but it carries down with it calcium phosphate and fat, both of which bodies have been helped to remain in their state of suspension in milk by the presence of the casein salt. Lindet (1912a, 1912b) has concluded that about one-half of the phosphorus contained in the rennet curd is in the form of phosphate of lime (probably tricalcic) , the other half being organically combined phosphoric acid. Hammarsten's Interpretation of the Process of Rennet Coagu- lation. According to Hammarsten (1877, 1896), whose view has been commonly held, the distinctive effect of the ferment is not pre- cipitation, but is the transformation of casein into paracasein. This is evidenced by the fact that if rennet be allowed to act on solutions free from lime salts no precipitate occurs ; but there is an invisible alteration of the casein, for now, even if the ferment be destroyed by boiling the solution, addition of lime salts will cause immediate coagulation. (See also Spiro, 1906.) Hence the process of rennet coagulation is a two-phase process ; the first phase is the transform- ation of casein by rennin, the second is the visible coagulation caused by lime salts. Furthermore, if the purest casein and the purest rennin were used, Hammarsten always found after coagulation that' the filtrate contained very small amounts of a protein. This protein he desig- nated as the "whey protein." In accordance with these observations, Hammarsten (1911) ex- plains the rennin action "as a cleavage process, in which the chief mass of the casein, sometimes more than 90 percent, is split off as paracasein, a body closely related to casein, and in the presence of sufficient amounts of lime salts the paracasein-lime precipitates out while the proteose-like substance (whey-protein) remains in solu- tion." 40 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 By continued action of rennin on paracasein a further trans- formation has been found in several cases (Petry, 1906; VanHer- werden, 1907; VanDam, 1909b), but perhaps due to a contamination of the rennin with pepsin, or to the identity of these two enzymes. The action which forms paracasein and whey protein takes place in a short time (Hammarsten, 1896; Schmidt-Nielsen, 1906). The composition and solubilities of paracasein have received consider- able attention. (See Loevenhart, 1904; Kikkoji, 1909; VanSlyke and Bosworth, 1912.) It is more readily digested by pepsin-hydro- chloric acid than is casein (Hosl, 1910). Duclaux (1884c) and Loevenhart (1904) and others do not accept Hammarsten's theory; but to most workers it seems prob- able, at least, that the action of the rennin is to cause a cleavage of casein v/ith formation of paracasein. However, the chemical and physical differences observed between casein and paracasein appear to be so slight that Loevenhart and some others think that they are only physical, perhaps differences in the size of the colloid or solu- tion aggregates. Loevenhart conceives of a large part of the work of the rennet (or of the acid, in acid and heat coagulation) as being a freeing of the calcium to make it available for precipitation. Some think that the aggregates of paracasein are larger than those of casein, but there is more evidence of their being smaller, which idea corresponds with the findings of Bosworth, though he looks upon the change as a true cleavage. Bang's Description of the Precipitation. Bang (1911b) studied the progress of the coagulation process by means of interruptions at definite intervals. His observations confirm the idea that rennin causes the formation of paracasein, and that the calcium salt serves only for the precipitation of the paracasein; the rennin has to do also with the mobilizing of lime salts. According to Bang, before coagulation occurs paracaseins with constantly greater affinity for calcium phosphate are produced. These take up increasing amounts of calcium phosphate, until finally the combination formed can no longer remain in solution. Bosworth's View of the Rennin Action. By a very recent work of L. L. VanSlyke and A. W. Bosworth (VanSlyke and Bosworth, 1912, 1913a, 1913b, 1913c; Bosworth and VanSlyke, 1913) in which ash-free casein and paracasein were compared as to their element- ary composition, and as to the salts they form with bases, and the properties of these salts, it is indicated that the two compounds are alike in percentage composition and in combining equivalent, the paracasein molecule being one half of the casein molecule. More- over, Bosworth (1913) has shown that, if the rennin cleavage be PHOSPHORUS METABOLISM 41 carried out under conditions which avoid autohydrolysis, no other protein is formed ; also that, if the calcium casemate present be one containing four equivalents of calcium, the paracaseinate does not precipitate, save in the presence of a soluble calcium salt, while, if the calcium casemate be one of two equivalents of base, rennin does cause immediate coagulation. Bosworth concludes that the rennin action is a cleavage (probably hydrolytic) of a molecule of caseinate into two molecules of paracaseinate, the coagulation being a second- ary effect due to a change in solubilities, dicalcium paracaseinate being soluble in pure water but not in water containing more than a trace of calcium salt, and the monocalcium caseinate being insolu- ble in water. The alkali paracaseinates, as well as caseinates, are soluble. This explanation seems to promise to harmonize the ob- servations with regard to acidity and the effects of the presence of soluble salts. The investigations of these authors and of Hart with regard to the changes which the paracasein, the calcium and the phospho- rus undergo during the ripening of cheese (VanSlyke and Hart,1902, 1905a, 1905c; VanSlyke and Bosworth, 1907, 1913d; Bosworth, 1907) contributed toward this interpretation. Mellanby (1912) holds an altogether different idea. He rep- resents the coagulation of milk as due to an absorption of a proteo- lytic enzyme (rennin is considered to be identical with pepsin) by the casein, and the precipitation of this complex by calcium ions, or other such ions, the calcium being supposed to act by reason of its electric charge without entering into chemical union. Schryver (1913) has a theory that the action of the enzyme is to free the surface of the complex colloidal molecules from adsorbed compounds which interfere with aggregation. HYDROLYTIC CLEAVAGE OF CASEIN Since casein is a protein, it undergoes hydrolysis when boiled with mineral acids or alkalis, or when warmed with the digestive proteolytic enzymes. Under the influence of any of these agents the elements of the water molecule enter into the molecule of the protein, and the protein molecule splits into simpler units. Aside from the phosphoric acid, the nature of the intermediate and final products, as far as known, is the same for casein as for other pro- teins. The processes are complex and but partially understood. The latest complete analysis of casein which we have seen is that of T. B. Osborne and H. H. Guest (1911). From their own de- terminations and from all data previously published they collected a table of the highest values which had been found up to that time 42 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 by methods which were considered reliable. It is to be noted that there is still about a third of the molecule unaccounted for, owing to faults in the methods or difficulties in manipulation. We give their table below together with the only other such analyses we have found which are at all extensive. (Abderhalden, 1905; Osborne and Guest, 1911 ; Abderhalden and Schittenhelm, 1906b ; Abderhal- den and Langstein, 1910. See also Abderhalden and Funk, 1907, and those referred to in the table.) HYDROLYTIC CLEAVAGE PRODUCTS OF CASEIN— Percent Casein from jow's milk From goat's milk From human milk Cleavage products Formulae Tahle of Ab- derhalden, Table of Os- borne and Analysis by Ab- derhalden and Schittenhelm, 1906b ' Analysis by Ab- derhalden and - 1905 Guest, 1911 Langstein, 1910 Glycocoll C-,H B N0 2 0.00 0.00 Alanine C 3 H 7 N0 2 0.9 1.50 1 1.5 1.2 Valine C 5 H u N0 2 1.0 * 7.20 x 1.3 Leucine CoHisNOa 10.5 9.35 2 7.4 8.8 Proline C5H e N0 2 3.1 f 6.70 3 4.62 2.85 Phenylalanine CHnNOs 3.2 3.20 4 2.75 2.8 Glutaminic CbHbNO* 10.7 15.55 1 11.25 10.95 acid Aspartic acid C4H7NO4 1.2 1.39 1 1.1 1.0 Cystine C e H 12 4 N 2 S 2 0.065 5 1 Serine C 3 H 7 N0 3 0.23 6 0.50 8 Tyrosine CoHuNOs 4.5 4.50 7 4.95 4.58 Oxyproline C 5 H 3 N0 3 0.25§ 8 0.23 8 Histidine C<,H 9 N 3 2 2.59 9 2.50 10 Arginine C 8 H 14 N 4 2 4.84 9 3.81 10 Lysine C 6 H 14 N 2 2 5.80 9 5.95 10 Tryptophane CxiH^NaOo 1.5 1.50 i Diaminotri- oxydodecanic acidj Cn>H 26 N 2 5 0.75 u 0.75 "■ Present Ammonia NH 3 1.61 u Sulphur s 0.76 13 Phosphorus P 0.85 13 Sum 51.13 67.85 (*) Amino-valerianic acid. (f) Pyrrolidine-carboxylic acid. (§) Oxy- a -pyrrolidine- carboxylic acid. (}) Sometimes called "caseinic acid." Authors to whom credit is given: (1) Osborne and Guest. (2) Levene and VanSlyke, original has "leucine+isoleucine." (3) VanSlyke. (4) Abderhalden. (5) K. A. H. Morner. (6) Fischer, original has 0.5 percent. (7) Reach. (8) Fischer, original has 0.23 percent. (9) E. Hart. (10) Osborne, Leavenworth and Brautlecht. (11) Fischer and Abderhalden. (12) Osborne and Harris. (13) Hammarsten. Folin and Denis (1912) have recently reported the determina- tion of tyrosine by a new colorimetric method. They find 6.5 per- cent in casein. E. Fischer and Abderhalden (1903) by enzyme di- gestions found a-pyrrolidine carbonic acid. PHOSPHORUS METABOLISM 43 With regard to the relation of phosphorus to the casein mole- cule, Osborne and Guest say in their discussion: "The presence of phosphorus in casein raises the question as to whether it is a con- stituent of the protein molecule or of some non-protein group united with a protein group as is a haematin with globin in haemoglobin, or nucleic acid with protein in the nucleins. Such data as are avail- able indicate that the union is other than a salt-like combination of a phosphorus-containing acid with a protein base." Calculations from the amount of the casein and of its nitrogen unaccounted for by the above analyses "give no evidence that casein differs in con- stitution to any marked degree from other proteins which contain no phosphorus. It might be supposed that the phosphorus of casein was a part of some organic radical If this is, in fact, so the organic radical must be one which contains nitrogen in ap- proximately the same proportion as the mono-amino-acids." DIFFERENCES IN THE CASEIN FROM DIFFERENT KINDS OF MILK Consistency of the Coagulum. There is a marked difference in the appearance and the digestibility of the coagulum formed by acids or rennet in cow's milk and human milk. The milk of the mare, and the ass (Langgaard, 1875; Ellenberger, 1899; Storch, 1902; Zaitschek, 1904; von Szontagh, 1905) are said to resemble human milk in this respect and, according to Arthus and Pages (1891), the milk of the dog also belongs in this class. The cow's milk gives a much tougher and more compact coagulum, in coarser flakes, than the small, jelly-like flakes from human milk. This difference, however, may not be due to any chemical difference in the casein itself. Certain observations indicate that it might be due to the greater proportionate amount of fat in the human milk (Hempel, 1894), or of salts in cow's milk (Dogiel, 1885), or to the acidity of cow's milk (Courant, 1891). Soxhlet (1893) says that dilution of cow's milk reduces the size of the flakes. Biedert and Schroter (Biedert, 1887 ; Schroter, 1887) notice great differences in the amount precipitable with MgS0 4 . Ass's milk is not easily co- agulated by acids and its casein not easily salted out by salts (Storch, 1902), and the coagulum is fine. Completeness of Peptic Digestion. Early investigators report that peptic digestion of cow's casein leaves an insoluble, undigested nuclein (pseudonuclein) but casein from human milk does not (von Szontagh, 1892, 1894; Willdenow, 1893; von Moraczewski, 1895a; Alexander, 1898) ; but later work seems to prove that this differ- ence is merely one of the ease of digestion of the two pseudonu- cleins. (Wroblewski, 1894a, 1894b ; Sebelien, 1894, 1895 ; Salkow- ski, 1893a, 1893b, 1896a; Salkowski and Hahn, 1894-5; Kobrak, 44 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1900; Rotondi, 1902). Salkowski says that the casein will be com- pletely digested if the ratio between casein and digesting liquid is 1:500 and the casein has previously been dissolved so that the in- fluence of hard, dried particles of casein is excluded. About 1 per- cent remains undissolved if the ratio of casein to solvent is 1 :250. Zaitschek and von Szontagh (Zaitschek, 1904; von Szontagh, 1905) found woman's, ass's and mare's milk completely digested in 72 hours at 38°C, while only 8, 14 and 15 percent, respectively, of the casein of cow's, buffalo's and goat's milk is rendered soluble under the same conditions. Long (1906a, 1907a) finds the caseins of goat's and cow's milk much alike, but the former has a somewhat higher equivalent weight than the latter, and it is more slowly acted on by pepsin-hydrochloric acid, and leaves more pseudonuclein. Elementary Composition. By reference to the table on p. 35 it will be seen that there is not much difference in the elementary com- position of caseins from various sources except as to the phosphorus content. Probably the analyses of Langstein and Edelstein (1910) are the best we have on human casein. Then, as compared with about 0.85 percent phosphorus in cow's casein, we find only about 0.24 percent in human casein and as high as 1.04 percent in ass's casein. Products of Complete Hydrolysis. The analyses of Abderhal- den and his associates (see p. 42) show, so far as they go, no marked differences between the cleavage products of the caseins from cow's, goat's and woman's milk, considering the lack of close agreement of all such determinations. On the other hand, they by no means ex- clude the possibility of significant differences of this kind, as ac- counting for the variations in elementary composition and in chem- ical behavior. Other Comparisons. In the other ways in which caseins have been compared — as to solubilities, reactions with chemicals, repeat- ed solution and reprecipitation, pancreatic digestion and nutritive value — there seems to be enough similarity observed to indicate that all the caseins are alike in general nature, but unlike in some chemi- cal respects which are not yet determined. J. Bauer and St. Engel (1911) concluded that the caseins from cow and human milk are alike. THE PHOSPHORUS OF CASEIN What is to be said as to the form in which phosphorus is present in casein ? The quotation from Osborne and Guest shows that practi- cally nothing is known as to the nature of that within the protein itself, not even whether it be present as a phosphoric acid radical PHOSPHORUS METABOLISM 45 or as a nitrogenous, organic complex. Aside from the phosphorus within the protein, casein is also practically always intimately as- sociated with calcium phosphate. Perhaps the synthetic work of Neuberg and Pollak (1910b, 1910d) throws some light on the nature of the combination of phos- phorus in the phosphoprotein molecule, as well as that in the nuclein molecule. At least, they have succeeded in bringing about a union of simple proteins (lactalbumin and blood globulin) with the phos- phorus of phosphorus oxychloride. A substance obtained in this way had an elementary composition much like that of casein (but with somewhat higher phosphorus content) and yielded to pancre- atic and peptic digestion in just about the same way as casein. The authors looked upon this compound as a substituted phosphaminic acid. With the simple amino acids or their esters they were able also to cause organic union of the phosphorus, but the organic phos- phorus compounds were not isolated. OTHER PHOSPHOPROTEINS OVOVITELLIN Ovovitellin is the lecithalbumin found in the yolk of hen's eggs. It is a combination of protein with lecithin, the lecithin not being removable by ether. According to^ T. B. Osborne and Campbell (1900b) the body which has been studied under this name is a mix- ture of various vitellin-lecithin combinations, containing from 15 to 30 percent of lecithin. The protein, moreover, when freed from lecithin still contains phosphorus. Osborne and Campbell give the composition of this protein (which they call "nucleovitellin") : C=51.24; H=7.16; N=16.38; S=1.04; P=0. 94; 0=23.24 percent. Hammarsten (1911) tells us that Gross (Zur Kenntniss des Ovovi- tellin, Inaug. Diss., Strassburg, 1899) gave the analysis of vitellin prepared by (NH 4 ) 2 S0 4 precipitation as follows : C=48.01 ; H=6.35 ; N=14.91— 16.97; P=0.32— 0.35; S=0.88 percent; and we have from T. B. Osborne (1902) the formula C 671 H 1112 N 182 S 5 P 4 0,. 7 , with 0.82 percent P. Plimmer (1908) found 0.65-1.14 percent P, with a mean of 0.99 percent in five preparations examined. Plimmer found with the vitellin in egg-yolk another protein having a lower phosphorus con- tent (0.10-0.85 percent P, with a mean of 0.35 percent in five prepar- ations), which he suggests may be vitellin without its phosphorus- containing portions. He gave the new substance the name "live- tin." Abderhalden and Hunter (1906) and T. B. Osborne and D. B. Jones (1909) report the following cleavage products: 46 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 CLEAVAGE PROUCTS OF VITELLIN OF HEN'S EGG— Percent Acid Glycocoll Alanine Amino-valerianic acid (Valine) Leucine Aspartic acid Glutaminie acid Phenylalanine , Proline Serine Tyrosine Cystine Histidine Arginine Lysine • Ammonia , Tryptophane Phosphorus , Total Abderhalden and Hunter (1906) 1.1 Present 2.4 11.0 0.5 12.2 2.8 3.3 1 i.e Not determined Osborne and Jones (1909) Not complete 0.00 0.75 1.87 9.87 2.13 12.95 2.54 4.18 3 3.37 Not determined 1.90 7.46 4.81 1.25 Present 0.94 Folin and Denis (1912) found in ovovitellin 5.2 percent tyrosin. The study of Osborne and Campbell already cited includes ex- amination of the paranuclein resulting- from peptic digestion. By re- peated digestions they obtained substances containing 3.29, 2.52 and 4.19 percent P. They interpret their analyses as showing that this paranuclein and the paranucleoprotein from which it was derived "are both compounds of one and the same proteid body [which is free from phosphorus, and which these authors call "vitelline"] with a phosphoric acid, possiblyH 3 P0 4 , H s P 2 9 , or some simple organo-phosphoric acid." For the paranucleic acid prepared by Lev- ene and Alsberg (1901) the phosphorus content is given as 9.88 percent P. Bunge (1885a) made a study of the paranuclein derived from peptic digestion of the egg-yolk as a whole. In this body he found 5.19 percent P, Hugounenq and Morel (1905a, 1905b) later finding 8.7 per cent P. This body Bunge looked upon as that from which haemoglobin is made for the young organism, and he gave it the name "haematogen." The significance of the iron content is emphasized. Finally, it is evident that much more study may well be given to the constitution and metabolic possibilities of ovovitellin, which is so significant for the development of the young bird, and import- ant as a food for man. Phosphorus is present in a lecithin portion PHOSPHORUS METABOLISM 47 and in a protein p.ortion, and probably the two are chemically com- bined. In the lecithin fraction it is glycerylphosphate, and in the protein fraction some undetermined radical, very likely a simple phosphoric acid or a phospho-organic acid. ICHTHULIN The compound in the eggs of fish corresponding to the ovovi- tellin of hen's eggs has been given the name "ichthulin." The two were early studied together and their similarities in chemical nature as well as biological significance were pointed out. (See Gobley 1850a ; Valenciennes and Fremy, 1854 ; Diaconow, 1867a ; G. Walter, 1891; Levene, 1901a; Hammarsten, 1905a; Plimmer and Scott, 1908; Gobley quotes other authors as far back as 1817.) Such a 'body has been isolated and studied from the eggs of carp, sturgeon, cod and perch. Levene (1901a) formed the paranuclein by peptic digestion, and reports the analysis of the paranucleic acid, which corresponds with that of the product from ovovitellin. This acid (not entirely free from protein) showed 10.34 percent P. We give below Levene's table of the analyses of ichthulin made by different workers, and add values taken from Hammarsten's (1905a) later report. ELEMENTARY ANALYSIS OF ICHTHULIN FROM FISH EGGS— Percent Author Kind of fish C H N S P Pe - Fremy Salmon 52.5- 53.3 8.82 15.2 1.00 0.6 22.7 Gobley Carp 52.6 7.74 15.5 0.90 0.37 23.24 Walter Carp 53.52 7.6 15.63 0.41 0.43 0.10 22.19 Levene Cod 52.44 7.45 15.96 0.92 0.65 22.58 Hammarsten Perch 14.81 1.111 0.743 OTHER PHOSPHOPROTEINS OF ANIMAL ORIGIN Hammarsten (1885b) reports obtaining a nucleoalbumin from the albuminous gland of a snail. This body is spoken of as "helicoproteid from the name of the snail (Helix pomatia) . Lieber- mann (1891a, 1891b, 1893a, 1893b) isolated what he called lecithal- bumins from the mucous membrane of the stomach (hog), from kidney (sheep), liver (lamb), lungs and spleen. He called especial attention to the observations that these substances are strongly acid, and readily form union with alkalis or other basic bodies, that they are rendered still more acid by C0 2 ; then by soda solutions are 48 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 rendered strongly alkaline, and afterward can again be made acid by C0 2 . He suggests that in the stomach walls they may thus serve in the production of hydrochloric acid from sodium chloride by taking up the sodium temporarily and gradually giving it up to the C0 2 of the blood. He also found that the alkaline solutions of Na,HP0 4 , or of sodium urate carrying a slight excess of soda, when passed through these lecithalbumins as a filter, show a strongly acid nitrate, and leave a strongly alkaline residue on the filter. This he suggests may indicate the process by which an acid urine may result in the kidney from filtration from alkaline blood. Lonnberg (1890) looking for mucin in the cortical and medul- lary substance of the kidney and the mucous membrane of the urin- ary bladder, decided that the bodies he isolated were not true mucins, but were nucleoalbumins. Malengreau (1900) found two nucleoalbumins in the thymus. Plimmer and Kaja (1909) ; also Plimmer and Scott (1908) re- port phosphoproteins in the pancreas and pancreatic juice of the dog, in the salivary glands of sheep and in the eggs in the ovary of the frog. They did not find them in the testes of the ox or of codfish, nor in thymus. PHOSPHOPROTEINS OF VEGETABLE ORIGIN From time to time proteins containing phosphorus have been isolated from plant bodies, and some of these so closely resemble the animal phosphoproteins in their properties that Liebig gave them the name of vegetable casein; this name, however, was generally given up when, later, Weyl showed that none of the substances most resembling casein are present in the natural bodies, but that there are there such as may be called plant vitellins — phyto-vitellins. All of the investigations of these compounds are thrown somewhat in doubt because of the presence of considerable amounts of salts such as potassium, calcium, or magnesium phosphates, or of alkalis in combination with organic acids, which are very difficult of removal. Hammarsten (1911, p. 105) saj^s: "It is not clear whether the phos- phorized plant proteids contain their phosphorus as impurities or whether they are the same as the animal phosphoproteins." The vegetable vitellins that have been most thoroughly inves- tigated are, according to Gustav Mann, (Chemistry of the Proteids, 1906 pp. 374, 375; 23 references on this subject), the gluten-casein of wheat ; similar substances from rye, maize, spelt and barley ; leg- umins from peas, vetches, beans, lentils, etc. (T. B. Osborne says that these are globulins) ; conglutins (so named by Ritthausen) of lupines, almonds, nuts, etc. Several of these have been analyzed by Ritthausen and by Osborne. PHOSPHORUS METABOLISM 49 We have not found report of any recent work on any of the compounds of this group. PHOSPHOCARNIC ACID Phosphocarnic acid is a complex phospho-nitrogenous com- pound the nature of which seems never to have been fully deter- mined, the substance never having been isolated free, but only in its iron compound, carniferrine, or mixed with decomposition prod- ucts. It was first described by Siegfried in 1894 (see Siegfried 1894, 1895, 1896) who obtained it at first from prepared meat ex- tracts and later directly from muscle. It is perhaps the most im- portant organic phosphorus compound among the meat extractives. The properties make it well fitted to serve as a carrier of phosphoric acid, iron, lime and magnesia in the body fluids, since it readily unites with the basic elements and forms with them compounds soluble in either neutral, weakly acid or weakly alkaline solutions. It probably has a similar significance in milk, of which also it is a constant constituent. The cleavage products which Siegfried obtained from phospho- carnic acid are carnic acid (which seems to be identical with, or closely related to, the antipeptone of Kuhne) , phosphoric acid, car- bon dioxide, a carbohydrate group which reduces Fehling's solution, succinic acid and paralactic acid. (See also T. R. Kruger, 1896.) Since carnic acid is a peptone, phosphocarnic acid differs from nucleins primarily in that on hydrolytic cleavage it yields a peptone direct instead of a protein. For such compounds Siegfried pro- posed the name "nucleon," and he designated phosphocarnic acid as muscle-nucleon." The nucleon obtained from milk showed nearly the same com- position as that from muscle, but yielded decomposition products of slightly different properties ; the lactic acid obtained was not para- lactic but was fermentation lactic acid, and in place of carnic acid there was obtained a modification designated as orylic acid. T. R. Kriiger (1899) states that milk nucleon is not precipitated by ammonium sulphate, while the muscle nucleon is so precipi- tated, and that such precipitation alters the proportion of N to P. Siegfried (1899) pointed out at the same time that the precipitates which he and others were obtaining from muscle were not uniform in the ratio of nitrogen to phosphorus, which might be due to the ex- istence in muscle of nucleons of different composition. That would not be the only possible explanation of this inconsistency, however, for it is possible that differences in the conditions of precipitation 50 OHIO EXPERIMENT STATION: TECHNICAL BTJL. 5 brought down a mixture of substances, or even caused some slight decomposition of the nucleon. In Siegfried's analyses of carnifer- rine from muscle reported in 1896 the nitrogen content ranged from 5.45 to 6.03 percent, the mean of six determinations being 5.65 per- cent. The phosphorus content found in four cases ranged from 1.84 to 2.59 percent (P), the mean being 2.12 percent. Using the mean value for nitrogen, and relating it to the lowest and highest values for phosphorus, gives the ratio N :P as 3.07 and 2.18 respect- ively. The ratio N:P found at this time in a preparation from muscle of a new born calf was as 1:1, and Kriiger had found a ratio of 1 :1 during his work with muscle from a steer. Macleod (1899), in the same laboratory, had precipitated carniferrine from the ex- tracts of muscle from a number of dogs, and had found that they varied in this respect. The 1 analyses he gives of nine such extracts show these values for N:P,— 4.3, 1.6, 1.8, 1.5, 1.7, 1.5, 1.3, 2.0, 3-.7. This all gives sufficient evidence that we have not a uniform product, and that if phosphocarnic acid is a chemical unit it under- goes decomposition in the processes used in isolation. The animal substances in which phosphocarnic acid has been found (or, rather, from the water extracts of which its iron complex has been precipitated after removal of proteins and phosphates) are as follows: By Siegfried (1896) : Liebig's and Kemmerich's meat extract, beef muscle, dog's liver and heart, and cow's milk. By Balke and Ide (1896) : Kemmerich's meat extract, the heart, liver and kidney of horse and of dog. By Wittmaack (1897) : The milk of cow, woman and goat. By Panella : The striated muscle fibre of dogs and rabbits (Pan- ella, 1902a, 1903b) ; brain of dog, rabbit and calf (Ditto, 1902b, 1903a) ; blood of dog, rabbit and calf (Ditto, 1902c, 1903c) ; both white and red muscle of rabbit (Ditto, 1903e, 1903f ) ; testicular sub- stance of ass and horse (Ditto, 1903d) ; both white and gray matter of brain (Ditto, 1903g, 1903h) ; testicular substance of horse (Ditto, 1903i, 1904a) ; non-striated muscle, in larger amount than in striat- ed (Ditto, 1904b) ; spleen of cattle, horses, sheep, swine and dogs (Ditto, 1904c) ; the amount in the brain of the dog falls off during fast (Ditto, 1906a). In 1906 Panella (1906b) published a paper which seems to throw doubt on the numerical data of previous work by showing that the values found for muscle are too high if less than 20 gm. of substance are taken as the sample. Using 50gm. or more as sample PHOSPHORUS METABOLISM 51 yielded nearly constant results of about 0.01 percent nucleon in fresh muscle, or 0.02-0.03 percent in dry. These values are much lower than those which were earlier reported. PHYTIN Introduction. The literature of the chemical study of phytin has recently been reviewed by A. R. Rose (1912b) with especial reference to its significance in plant economy. This review is pub- lished in the Biochemical Bulletin, and is the most complete discus- sion to which we can refer the reader. Phytin is the name generally applied to the alkali and alkaline earth salts of an organic phosphoric acid which is found ex- tensively in the vegetable world, and which by most investigators is supposed to contain inosite combined in some way with phosphor- ic acid. It appears now probable that the natural products are not all identical, and distinction must be made between the salts of phytic acid and of other phyto-phosphoric acids. Rose calls the acid "inosite-phosphoric acid." Discovery. Rose tells us that the aleurone grains, in which this compound is found in seeds, were first discovered microscopi- cally by Hartig in 1854, and that the particular P-bearing spheroid- al bodies in these grains were isolated by Pfeffer in 1872, and named by him "globoid." Chemical study began with Palladin in 1893, who obtained it from Senapis niger. He showed the compound to be rich in phosphorus, and to contain magnesium and calcium but no nitrogen ; also that it was non-reducing and yielded no reducing sub- stance on acid hydrolysis. Palladin's work was followed up by Schulze and Winterstein (1896) and Winterstein (1897) who identi- fied the body with the globoid of Pfeffer. The name "inosite-phos- phoric acid" was proposed by these investigators in accordance with their finding that the magnesium salt on being digested for 30 hours with cone, hydrochloric acid at 130-140° yielded inosite and phosphoric acid. Posternak studied the substance extensively (Posternak, 1900, 1903a, 1903b, 1903c, 1903d, 1905 ; and Bull. Soc. chim. 33 (1904), 116). He considered that the inosite was not pre- existent in the molecule, but that the compound was an anhydro- oxymethylene-diphosphoric acid, and he gave it the name "phytin," which has been retained. Occurrence. Phytin is found most frequently in the aleurone of seeds or in other parts of plants which serve for storage, as some roots and tubers. Of investigations as to the amount of phosphorus present in this form we mention Schulze and Winterstein (1896), 52 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Posternak (1903a, 1903b), Hart and Andrews (1903), Patten and Hart (1904), Suzuki and Yoshimura (1907), Suzuki, Yoshimura and Takaishi (1907), Vorbrodt (1910), Rising (1910), Hart and Totting- ham (1910), and Bernardini (1912). ANALYTICAL DATA ON THE OCCURRENCE OF ORGANIC PHOSPHATE PHOSPHORUS, "PHYTIN"— Percent Substance analyzed Phosphorus in the form of organic phosphate Fraction of total phos- phorus in this form Black mustard (Si/tapis nigra) (1) Red fir Spruce seed {Picea cxcctsa) (2) Pine seed (Pinus ccmbra) (2) Hempseed (cortex removed) Hempseed ( Cannabis sativa) (2) Sunflower seed (cortex removed) Pea Pea, yellow White kidney bean Bean, brown Lentils Lentils {Lens escuZentd) (2) Windsor bean {.Viciaf aba minor) (2) Wheat bran Wheat bran (3) Wheat grain (4) Wheat grain (Triticum sativum) (2) Graham flour Rice bran (3) Rice seed (4) Rice flour Rice, seed (5) Rice embryo (5) Barley bran \Hordeum xmlgarc) (3) Barles", grain (4) Barley, grain (6) (5) Barley bran (6) (5) Barley grain minus bran (C) (5) Barley, grain (Hordeum distich.) (2) Barley, grain (Hordeum distich.) (2) R3 - e flour Fye, grain (Sicale ccreale) (2) ... Millet (Panicumfrumcntaceum) (3) Sesame seeds (SesamUm indicum) (3) Castor bean, seed (Ricinus communis) (3) Rape seed (Brassicanapus oleifera) (2) ... Oil cake of rape (Brassica napus) (3) . . Rape seed (Brassica napus) (4) Radish root juice (7) Turnip root juice (7) Apple, juice (7) Pear, juice (7) Corn, grain (4) (5) Corn, bran (4) (5) Corn, germ (4) (5) Com, endosperm (4) (5) Corn, grain (Zea mays) (2) Oat, grain (4) (5) Oat, hull (4) (5) . . . Oat, grain minus hull (4) (5) Schulze, Winterstein Posternak Vorbrodt Vorbrodt Posternak Vorbrodt Posternak Posternak Rising Posternak Rising Posternak Vorbrodt Vorbrodt Patten, Hart Suzuki, Yoshimura Suzuki, Yoshimura, Takaishi Vorbrodt Rising Suzuki, Yoshimura Suzuki, Yoshimura, Takaishi Rising Bernardini Bernardini Suzuki, Yoshimura Suzuki, Yoshimura, Takaishi Hart, Tottingham Hart, Tottingham Hart, Tottingham Vorbrodt Vorbrodt Rising Vorbrodt , Suzuki, Yoshimura Suzuki, Yoshimura Suzuki, Yoshimura Vtrbrodt Suzuki, Yoshimura Suzuki, Yoshimura, Takaishi Suzuki, Yoshimura Suzuki, Yoshimura Suzuki, Yoshimura Suzuki, Yoshimura Hart, Tottingham Hart, Tottingham Hart, Tottingham Hart, Tottingham Vorbrodt Hart, Tottingham Hart, Tottingham Hart, Tottingham 0.33 0.60 0.15 0.07 1.33 0.11 0.72 0.26 0.11 0.42 0.29 0.25 0.03 0.02 0.92 0.58 6.' 136 0.08 1.68 o.'ii 0.436 5.14 0.24 6.'i9 0.15 0.17 0.17 0.12 0.09 0.12 0.34 0.13 0.11 0.20 0.53 0.13 0.00 0.13 0.15 0.17 0.18 0.09 0.22 91.46 21.65 14.39 91.44 15.00 86.26 70.80 19.00 81.60 52.00 82.60 9.29 4.39 68.10 52.00 55.50 29.90 29.00 74.17 41.64 69.00 45.90 82.90 44.00 56.55 38.00 68.20 29.80 36.40 34.30 25.00 28.90 44.97 16.24 41.61 38.05 44.46 62.11 15.13 15.06 48.14 46.15 44.80 0.00 34.20 35.70 48.90 43.90 21.90 53.70 piler. (1) (2) Computed. Percent of fat-free seeds. NaCl extract, precipitated hot. Organic phosphorus soluble in 1.0 percent acetic acid; computed from P 2 B by com- (3) Percent of dry substance obtained by absolute alcohol precipitation from 0.2 per- cent HC1 extract. (4) Organic phosphorus soluble in 0.2 percent HC1. (5) Percent of total computed by compiler. (6) Phosphorus soluble in 0.2 percent HC1 (considering that the inorganic fraction of this is small enough to be ignored). (7) Total organic phosphorus of expressed juice; grams per 100 c.c. PHOSPHORUS METABOLISM 53 To quote finely from Posternak (1903a) : Phytin is stored as reserve material in all grains, tubers, rhi- zomes and bulbs, where it is destined for the development of the em- bryo. Other bodies from which it has been extracted besides those reported in this paper are — rape, lupine, wheat, corn, potatoes, dahl- ia bulbs, carrots and even onions. In the grains, where there is very little mineral phosphate, it forms at least 70-90 percent of the total phosphorus. Anderson (1912b) was unable to find in wheat bran any of the characteristic salts of phytic acid. The purified barium salts of the compounds obtained corre- sponded to the formulae: C 25 H 55 54 P 9 Ba 5 and C 20 H 45 O 49 P 9 Ba 5 . The only acid that could be isolated was C 20 H 35 O 49 P 9 , and all of the barium salts obtained could be changed into salts of this acid by processes which liberate reducing substances. Hence it was concluded that this acid is the only organic-phosphoric acid present, and that wheat bran does not contain phytin. Rather (1912, 1913a, 1913b) has investigated a corresponding compound obtained from cottonseed meal. It was his conclusion that this and the wheat bran compound are identical, and that the free acid from both is represented by the formula C 12 H 41 P 9 Q 42 . An- derson (1914a), however, thinks that Rather's silver salt prepara- tions are not chemically pure, and from his own investigations (1912c,- 1914a) believes that the organic phosphoric acid in cotton- seed meal must be inosite hexaphosphoric acid, C 6 H 18 24 P 6 , or some multiple of the same. Anderson gives the same formula for com- pounds obtainable from oats (1914b), from corn (1914c), and, final- ly, from commercial phytin (1914d). Salts of Phytic Acid. Phytic acid forms neutral salts, acid salts, double salts and acid double salts of the alkalis, alkaline earths and heavy metals. The solubility of these salts decreases in the order above named. The magnesium compounds are more soluble than the calcium, and the calcium more soluble than the barium or strontium. They are all more soluble in cold than in hot water, and heating often precipitates them. Several such salts have been isolated from plant bodies; and Anderson (1911) has added largely to the number by artificial production. Elementary analysis of these salts throws light on the basicity of the acid and the relative number of C and P atoms in the molecule. Rose gives us a table compiled from the various elementary analyses reported in the lit- erature. Rose's conclusion is that the relative number of atoms in the molecule' corresponds with the ratio 6C:6P; and — "It seems 54 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 probable that the molecular weight when accurately determined will be reported as 714 or will differ from this by the molecular weight of three molecules of water. The molecule seems to contain twelve hydrogen atoms readily separated in ionization, six of which are ex- ceedingly reactive ; the remaining hydrogen atoms gradually dimin- ish in activity by twos, the last four being slow to enter into an ex- change with bases. The more readily formed salts are therefore those corresponding to an octavalent acid and the other common ones are in six and tenvalent combinations." Synthesis of Phytic Acid. Anderson speaks of a paper by Con- tardi, which we have not seen (Atti R. Accad. dei Lincei, Roma [5] 19 1 23), in which the author reports having prepared the hexa- phosphoric acid ester of inosite by heating inosite in an excess of phosphoric acid in a stream of carbon dioxide at 160-1 65 °C. The free ester obtained from this preparation was said to be identical with phytic acid. Carre, however, (Bull. Soc. chim. de France [4] 9, 195) repeating these experiments, found that the products de- scribed by Contardi were merely mixtures of free phosphoric acid with the alcohol, together with their decomposition products mixed with monobarium phosphate. Anderson has made repeated ef- forts to synthesize phytic acid, without accomplishing it; nor was he able to synthesize a hexa-phosphoric acid ester of inosite. A tetra-orthophosphoric acid ester, a di-pyrophosphoric acid ester of inosite and a di-inosite tri-pyrophosphoric acid ester were obtained in pure form, and analyzed. (Anderson, 1912a.) Of them Anderson says: "These compounds are in physical and chemical properties very similar to phytic acid. They form analogous acid salts Which in appearance and solubility seem almost identical with salts of phytic acid. Whether esters, such as above, are found in nature is at present unknown. It is, however, not impossible that a part of the organically bound phosphorus existing in plants may be present in such, or similar forms." Constitution. Winterstein (1897, 1908), repeatedly obtaining a cleavage of the compound into inosite and phosphoric acid, be- lieved it to be a conjugated inosite-phosphoric acid. Posternak (1903a, 1903d), however, was of the opinion that, although inosite is obtained in such cleavage, the grouping present in the phytin is that of a simpler compound, that of the alcoholic isomer of formal- dehyde, CH'OH, and that when this is set free from the acid poly- merization occurs, forming the inosite by union of six such groups. He interpreted phytin as being the first product in the organization PHOSPHORUS METABOLISM 55 of inorganic phosphorus taking place under the influence of chlor- ophyll in direct sunlight. He called it anhydro-oxymethylene-di- phosphoric acid and proposed the empirical formula, C 2 H s P 2 9 , and the structure indicated by the following : H / CH— OPO(OH) 2 / O \ CH— 0-PO(OH) 2 \ H The analyses of Patten and Hart (1904) were said to support this formula. Suzuki, Yoshimura and Takaishi (1907), because of enzymatic splitting off of inosite, decided that inosite was originally present in the phytin, and that the substance must be inosite hexaphosphoric acid. They constructed the following formula to represent their view. HO OH O— P— O— HC— CH— 0— P— / \ HO | | OH HO || OH \ / O— P— 0— HC— CH— 0— P— HO^ || X OH HO OH V / 0— P— 0— HC— CH— O— P— O . . / \ HO OH The discovery by Neuberg (1908) that both inosite and phytin yield furfurol when distilled with phosphorus pentoxide and phos- phoric acid, respectively, led him to believe that the inosite ring ex- ists already formed in the phytin, and he suggested the following structural formula : 56 OHIO EXPEEIMENT STATION: TECHNICAL BUL. § HO OH HO\ H H /OH HO-^-P— O— C — C— 0— P— OH / \ || \ / HO— P— O— CH HC— O— P— OH HO/ | | \^0H HO HC — CH OH I I o o HO I | OH \ / HO— P P —OH / \/\ HO OH Levene, (1909a) (to quote from Rose) "working- with a prepar- ation from hempseed, was led to believe that the 'phytin' of this gram contained in its molecule phosphate, inosite and a carbohy- drate of the pentose group. His work was criticized by Neuberg (1909), who claimed that there were impurities in the preparation. In view of the known intimate association of the phytin with pro- tein and carbohydrate in the aleurone grain, and the possible occur- rence of a chemical combination of both phyto-phosphate and carbo- hydrate with protein, it is conceivable that Levene had a product holding pentose as an integral part and not as an impurity, though in view of all the available evidence Neuberg's criticism seems at the present time somewhat justifiable." Starkenstein (1910), who thinks that inosite bears some defi- nite relation to the phenomena of growth of animals as well as of plants, believes that the acid to which phytin corresponds is not a simple ester but a complex pyro-phosphoric acid compound, and that the acid salts usually resulting from reaction with divalent metals are to be explained by the union of the divalent atoms with hydrox- yls of adjacent phosphoric-acid residues. His interpretation would give the ratios of C:P:OH as 6:6:12, which is supported by most of the analyses. Titration with uranium acetate shows only one half of the true phosphoric acid value, because only one half of the free hydrogens are readily reactive. The analyses of Levene, Vorbrodt (1910) and Rising (1910) do not support any of these formulae as to the relative amounts of car- bon and phosphorus ; and we understand that those of Plimmer and Page do not (Chem. Abs.). PHOSPHORUS METABOLISM 57 Anderson (1914d) has succeeded in obtaining from commercial phytin a crystallizable barium salt, and the acid from the same, analysis of which corresponds with the formula C 2 H 6 8 P 2 or C 6 H 18 24 P 6 . No direct evidence has been gained as to the molecular mag- nitude, but the second formula is thought to be the more probable. This differs from Neuberg's formula for phytic acid by three mole- cules of water, and is identical with inosite hexaphosphate. Ander- son, therefore, believes the phytic acid to be either inosite hexa- phosphate or an isomer of the same. He finds, as did Starkenstein, that only one half of the acid hydroxyls are particularly reactive. Both the salts and the free acid when kept at ordinary temperatures decompose slowly, with liberation of inorganic phosphate, the acid decomposing much faster than the salt. Inosite was not found among the products of this spontaneous decomposition. Possible relations between phytin and lecithin (phosphatids) on the one hand and nucleic acids on the other have been pointed outbylljin (1906), Parrozzani (1909) and Rising (1910). Phytin-Splitting Enzyme. Suzuki, Yoshimura and Takaishi (1907) isolated an enzyme from rice and wheat bran which splits phytin into phosphoric acid and inosite, and which is probably wide- spread in the plant world. They named the new enzyme "phytase." A part of Vorbrodt's (1910) study was with regard to enzy- matic decomposition of the organic phosphorus compounds of bar- ley and corn. He concluded that : "1. The organic phosphorus compounds of barley and corn grains, both those soluble in 1% acetic acid and those insoluble, can be decomposed under the influence of enzymes, in which process mineral phosphoric acid is split off. "3. Temperature exerts a decided influence on the progress of the decomposition of the organic phosphorus compounds; a little above. 0° there is no decomposition of the soluble organic phosphorus compounds of barley, the optimum is about 28°C, above 48° the progress of decomposition is very slow. "4. The enzymes which split the organic phosphorus com- pounds are already present in the barley grain, but in the corn grain the amount is very insignificant ; they develop in large amount during germination, when they are present more abundantly in the germ and the scutellum than in the rest of the seed. McCollum and Hart (1908) found evidence of such a phytin- splitting enzyme in the liver and the blood of the calf, but not in the muscle or kidney. Jegorow (1912) doubts the existence of phytase, since under the conditions of the work by Suzuki phosphoric acid splits off with- out the enzyme preparation. He finds phytin quite unstable. Ino- site and phosphoric acid result from boiling with water. 58 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 PHOSPHATIDS INTBODUCTION Our present knowledge of the phosphatids has been attained largely through the systematic study of the composition of the brain. Of these studies the most significant as supplying a basis for the classification of phosphatids (originating the name "phos- phatid") was that of Ludwig W. Thudichum (1901). Thudichum's earlier work (1875) and that of Gutnikov (1896-7) should also be mentioned. Other more recent analyses and general discussions of the brain which include classifications of the phosphatids are those of Waldemar Koch (1904), of Coriat (1905) and of Frankel (1909a). The basis of classification which Thudichum introduced has proved to be very satisfactory and has continued in use with but slight modifications. Since the phosphatids resemble the fats in their solubilities, and in some other properties, it has been found convenient to discuss these groups together with cholesterins and cerebrosides under the comprehensive name of "lipoids," or "fat- like substances." Ivar Bang defines lipoids as, "compounds which are soluble in organic solvents such as ether, alcohol, chloroform and benzol." Bang's book, published in 1911, is freely used in the preparation of this article and his classification is followed. Rosenheim (1909) presented certain "Proposals for the Nomenclature of the Lipoids," in which several names are discarded, and the group divisions are fitted to those which are retained. The classification does not ma- terially differ from that of Bang. The phosphatids are in every way the most important lip- oids. They are of great physiological interest, both as food con- stituents of specific importance and as cell constituents with signif- icant functions. Apparently they are primary constituents of cells, that is, are found in all cells, and are necessary for the life of the cell. They are interesting in their chemical relations and are as difficult to obtain pure as are the proteins. The name "phosphatid" was proposed by Thudichum to signify that we have here bodies similar to phosphates though not just like them. Phosphoric acid was considered to be the central group of the molecule, with which are combined an alcohol, fatty acids and a nitrogenous base. The alcohol is at least nearly always glycerin, and the base is mainly choline. If there be one such phosphoric acid molecule only, the compound is a monophosphatid ; two such, united, may form a diphosphatid. Further, there may be one or PHOSPHORUS METABOLISM 59 two, perhaps three, possibly more nitrogenous groups making mon- amino, diamino, etc., compounds. The phosphatids are classified on this basis. Thus, if on elementary analysis a substance of this class is shown to have the relative number of atoms of nitrogen and phosphorus such that N:P=2:1, it is placed in the diamino-mono- phosphatid group. These names are still used although the pres- ent conception makes glycerin more centrally significant in the mole- cule than Thudichum thought it to be. Bang's classification makes use of a difference pointed out by Frankel (1909b) by which the phosphatids which contain an un- saturated fatty acid form one class, and those which contain only saturated fatty acid radicals form another. The former, in distinc- tion from the latter, are semisolid or viscous ; they do not crystal- lize; they oxidize in the air, they are easily decomposed and they readily react with other substances. They are for these reasons particularly difficult to obtain pure, and doubtless most of the prep- arations that have been examined have been mixtures. Accord- ing to Erlandsen, the cadmium chloride method of preparation causes some decomposition. The following is Bang's classification including the best-known phosphatids : BANG'S CLASSIFICATION OF PHOSPHATIDS A. Unsaturated Phosphatids I. Monamino-monophosphatids, (N:P, 1:1) Lecithin, cephalin and myelin. II. Monamino-diphosphatids, (N:P, 1:2) Cuorin, liver phosphatid and monamino-diphosphatid from egg-yolk. III. Triamino-diphosphatids, (N:P, 3:2) B. Saturated Phosphatids . I. Diamino-monophosphatids, (N:P, 2:1) Sphingomyelin and diamino-phosphatids from muscle, from egg-yolk and from horse pancreas. II. Triamino-monophosphatids, (N:P, 3:1) Neottin and carnaubon. III. Protagon. C. Insufficiently Characterized Phosphatids D. Plant Phosphatids 60 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 A. UNSATURATED PHOSPHATIDS 1. MONAMINO-MONOPHOSPHATIDS LECITHIN, OR LECITHINS Occurrence. The best known of the phosphatids and the one which is apparently most widely distributed in the animal kingdom is lecithin. However, it is now recognized that not all of the sub- stances which have been reported as lecithin should be given this name. It is only within quite recent years that sufficiently dis- criminating work has been done to differentiate lecithin from cer- tain other phosphatids. It is possible that it is a primary con- stituent of animal cells, but it is not impossible that it is sometimes lacking and its place taken by another phosphatid. Apparently it is not present in any vegetable substance. Its identity is little to be questioned, however, as found in heart muscle (Erlandsen, 1906, 1907 ; MacLean, 1908a) , muscle of the extremities (Erlandsen, 1906, 1907), egg-yolk (Stern and Thierf elder, 1907 ; Erlandsen, 1907; Mac- Lean, 1909c) and liver (Baskoff, 1908). MacLean (1912b), using special purification processes, proves the principal phosphatid of kidneys and of muscle to be at least lec- ithin-like in its nitrogen and phosphorus content. The discovery of lecithin was made by Gobley, who found it in egg-yolk in 1846, in the brain of hen, sheep and man in 1847, in the eggs and milt of carp in 1850, in the blood of ox and man in 1851, and in bile in 1856. He more fully studied that in human brain in 1877. (Gobley, 1846, 1847, 1850a, 1850b, 1851, 1856, 1877.) Constitution. Gobley recognized the existence in lecithin of glycerophosphoric acid, of fatty acid, and of nitrogen in an NH 3 group. The nitrogenous group was thought by Liebreich (1865) to be neurine, but Strecker (1868) showed it to contain oxygen and to be choline. It is now recognized as being mainly choline. C 8 H — OH N=(CH 3 ) 3 \ OH (See also Bergell, 1900, 1901.) MacLean (1908a, 1909a, 1909c) has shown that not all of the nitrogen in lecithin is present as choline, and, moreover, that leci- thins from different sources do not- agree in the proportion of choline they contain. Very careful examination of lecithin from heart PHOSPHORUS METABOLISM 61 muscle showed not more than 42.6 percent Of the nitrogen to be present as choline, while in commercial lecithin 80 percent and in that from egg-yolk 66 percent of the nitrogen was present in this form. MacLean was led to feel that some of the nitrogen must be in the form of amino acid. Trier (1912, 1913b) has isolated from the hydrolytic products of egg lecithin, as well as from related compounds of plant origin, a compound of amino-ethyl-alcohol which he thinks may be the mother substance of choline, and which he names "colamin." The structure of the lecithin molecule was early studied by Diaconow (1867b, 1868a, 1868b), by Strecker (1868) and by Gilson (1888). Diaconow held that the choline is bound with the phos- phoric acid in salt-like union, while Strecker and Gilson believed that it is ester-like. The constitution suggested by Strecker is practi- cally that which is now generally accepted. OH CH 2 — O— P— O— C 2 H 4 I II \ . CHOR O (CH 3 ) 3 =N I / CH 8 OR OH In this formula R stands for a fatty acid radical. Two of these radicals are supposed to take the place of the hydrogen in two of the OH radicals of the tribasic alcohol, glycerin, and the phos- phoric acid that of the other. This formula is supported by the observations on optical activity as made by Ulpiani (1901) and by Willstatter and Ludecke (1904), but not by the recent study of Mal- engreau and Prigent (1912) on the rate of cleavage of the different radicals in acid hydrolysis. According to these observations, the splitting off of choline and of the fatty acids takes place at the same time, and much more rapidly than the phosphoric acid is freed from its glycerophosphoric acid combination. Hence it is thought that the linkings of the phosphoric acid with other residues (gly- ceryl and choline) are not of the same nature, as they would be according to the above formula. Of the phosphatid components the fatty acids are of most val- ue in distinguishing one phosphatid from another. Thudichum thought that oleic acid is characteristic of lecithins, and that it is associated with some other, usually palmitic or margaric acid. Hen- riques and Hansen (1903) showed that there must be some acid present more unsaturated than is oleic acid. Cousin (1903) and Erlandsen (1906, 1907) both speak of linoleic acid as a phosphatid 62 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 constituent. Attempts to identify the acid by its iodine number have brought results which cannot at present be reconciled. Of course oxidation, which so easily takes place, rapidly reduces the iodine number. Bang's conclusion, after considering all the evi- dence, is that there are in lecithin two fatty acids, one of which is stearic and the other an as yet undetermined unsaturated acid. The saturated acid may be palmitic (Serono and Palozzi, 1911). These disagreements and uncertainties with regard to the con- stituents of lecithin are doubtless due in part to the fact that the pure compound has not been obtained, since lecithin so readily oxi- dizes, and is so difficult to free from other phosphatids, fats, etc. It is quite doubtful if the substances examined were individual com- pounds. Bang says that elementary analysis has shown Merck's preparation to be quite impure and unsuited to scientific work ; and that the preparation of the proprietary product known as "agfa-leci- thin" by Bergell's method, which makes use of the CdCl 2 precipita- tion, has been shown by Erlandsen to cause a serious decomposition. Hence conclusions which have been drawn on the basis of work with these products are of little value. 1 Bang considers that no suitable method of preparation has been described except that of Erlandsen. It cannot be said, finally, whether there is but one lecithin or whether there are several lecithins isolated from animal products by the same process. Elementary Analysis. The elementary analyses and empirical formulae reported by different workers are nearly alike, although not identical. A few are quoted here : ELEMENTARY ANALYSES OF LECITHINS— Percent Author Thudichum Koch Erlandsen MacLean Erlandsen Stern and Thierfelder Mac Lean Baskoff Date, refer- ence Source of lecithin C II N P 4.00 » Brain 66.75 18.67 1.81 1902b Brain 64.03 10.4 1.8 3.79 1906, 1907 1908a Muscle of heart Muscle of heart 66.29 66.27 10.17 10.32 1.87 1.85 3.95 3.975 1907 Muscle of thigh 65.96 10.20 1.82 3.93 1907 Egg-yolk 64.63 10.96 2.08 3.97 1909a Egg-yolk 64.18 10.6 1.876 3.95 1908 Liver 64.64 10.71 1.95 4.00 Formula C 13 H &1 NP0 8 ( 1 ) C 43 H 8 oNP0 9 CsHsoNPOs ( J ) Quoted through Bang (1911). (1) Altchul's (1912) assertion that "Agfa-lecithin" is not made by Bergell's method, but by a method worked out by himself, which gives a pure product, is answered by Bang (1912) by saying that he based his statement as to the method used on the authority of the "Agfa" company. PHOSPHORUS METABOLISM 63 Properties. Lecithin is a white, yellow, or orange-colored sub- stance, usually amorphous, and of a waxy consistency when dry. Some have obtained it in crystalline form, the crystals being thin flakes. It is very hygroscopic, and in the air it takes up moisture, and softens to a semifluid mass. It also readily takes up oxygen from the air, and in doing so grows darker in color. Unlike the fats, lec- ithin becomes wet and sinks in water. Gradually it takes up water and swells to a gelatinous mass, and finally spreads to an opaque, colloidal solution. Lecithin is precipitated from this colloidal so- lution by certain acids and salts, and precipitation is prevented by other salts. It is thought that the tendency of phosphatids in gen- eral to pass into the colloidal state, and their properties as colloids, may be significant in their functioning within the cells. The pre- cipitating power seems to be due to the individual ions and depend- ent on their concentration. It is a reversible reaction, and the total effect is a summation of the effects of the individual ions present. Quantitative studies of such precipitations of lecithin and other lipoids have been made by W. Koch (1902a, 1903, 1907a, 1909b, 1910b), Koch and MacLean (1910), Koch and Mostrom (1910), Koch and Pike (1910), Koch and Williams (1910), Porges and Neubauer (1907), Long (1908), Long and Gephart (1908a) and Feinschmidt (1912). See also the physicochemical studies of Handovsky and Wagner (1911) concerning the reactions of such emulsions with in- organic salts and with proteins. . Lecithin is easily dissolved by alcohol, either hot or cold, and is precipitated from this solution by water. It is soluble also in ether, chloroform, carbon bisulphide, benzene and some other or- ganic solvents, but nearly insoluble in cold acetone and in methyl acetate. The power of lecithin (and other lipoids) in such organic solvents, to take up organic substances, has recently been investi- gated by Loewe (1912a, 1912b, 1912c, 1912d) and is attributed to adsorption. R. Cohn (1913) thinks that on longer action the ad- sorption becomes so intensive that probably a chemical adsorption results. According to Ulpiani (1901), and to Willstatter and Liidecke (1904), lecithin shows by its optical activity that it has an asym- metric molecule. Paul Mayer (1905,1906b) separated the racemic form under the influence of an enzyme (lipase) into dextro- and laevo-lecithin, which then behaved differently toward the lipase, the laevo-lecithin remaining unchanged while the dextro-lecithin broke down into fatty acids and dextro-glycerophosphoric acid. 64 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Lecithin is readily saponified by alkalis and especially by baryta, yielding fatty acids, glycerophosphoric acid and choline. The cleavage with dilute acids is quite slow. Casanova's (1911) observations indicate that the first step in the cleavage resulting from the taking up of water in the presence of oxygen is a separa- tion of choline and glycerophosphoric acid esters of the fatty acids, further taking up of water being required for the cleavage of these esters. Malengreau and Prigent (1912) found that only about 70 percent of the choline and fatty acids was split off in two hours by 0.1N sulphuric acid, that after 13% hours they were nearly all split off, but the cleavage of the glycerophosphoric acid was much more slow, and was still incomplete after 72 hours. Lecithin is cleaved by digestive ferments. Compounds. Lecithin shows more power to enter into com- bination with other substances than do most organic compounds. These combinations are of different kinds; sometimes additions in molecular relation, forming well-characterized compounds, and sometimes in other combinations of which the nature is not known, which may be chemical complexes or may be adsorption products. Thudichum suggests that it may be of considerable physiological significance that the phosphatids generally react with so many kinds of substances and are thereby changed, and that these reactions' are so dependent on the concentrations of the reacting substances. Combinations of lecithin with inorganic acids, with some bases, and with both inorganic and organic salts have been studied. These seem to be usually chemical and perhaps mainly addition products, though it has been shown in some cases that partial decomposition accompanies the taking up of salt. Bang gives the following summary of the classes of organic compounds with which lecithin combinations have been made; (1) glucosides, such as phlorizin, salicin and amygdalin, (2) alkaloids, such as chlorides of morphine and nicotine and sulphate of strych- nine, (3) toxins, as cobra poison and bee poison, (4) cholesterin, (5) enzymes, (6) dyes, (7) carbohydrates, (8) proteins. These substan- ces have not been very well studied as yet and only the last two need be considered here. Compounds of Lecithin and Carbohydrate. Apparent combina- tions of lecithin and carbohydrate have been prepared. For ex- ample, if an alcoholic solution of lecithin and glucose be evaporated to dryness, a substance is obtained which shows different solubilities from either component, which is taken as indication that a chemical union has taken place. Similar substances are said to have been PHOSPHORUS METABOLISM 65 isolated from organs and from blood. These preparations give glu- cose on decomposition, also the cleavage products of lecithin; but as they are not constant in composition, nor permanent in their solu- tions, or under repeated evaporation, it seems probable that no chemical union exists but that we have here simply an example of the power lecithin shows to change by its presence the solubility of other compounds. These artificial preparations have been spoken of as identical with Drechsel's jecorin, but that idea must immedi- ately be given up when it is shown that jecorin always contains sul- phur and sodium. However, jecorin may be also a similar loose combination, or mixture of sugar with a phosphatid or its decompo- sition products. Jecorin was first found by Drechsel (1886), and has usually been obtained by the use of his method. Drechsel isolated it from the liver of a horse, and the following year Baldi (1887) found it in the liver of the rabbit and the dog, in beef spleen, horse blood, horse muscle and human brain. He said it was present in largest amount in liver and next in spleen. Since then it has repeatedly been isolated from liver (P. Manasse, 1895; Meinertz, 1905; Siegfried and Mark, 1905 ; P.Mayer, 1906a ; Mark, 1907 ; Waldvogel and Tintemann, 1906 ; Baskoff, 1908, 1909a, 1909b) and from blood (Henriques, 1897; Bing, 1899; P. Mayer, 1906a), and is also reported from adrenals (P. Manasse, 1895), spleen (Waldvogel and Tintemann, 1906) and bone marrow (Otolsky, 1906). Both smooth and striated muscles showed a very little jecorin in Erlandsen's (1906,1907) analyses. The nature of jecorin is not established. All the preparations examined contained, in acTdition to phosphorus and other constitu- ents of lecithin, sulphur and sodium and a reducing substance which has been identified as glucose. Bing (1899) looked upon it as a compound of lecithin and glucose, and it has been investigated as such. However, it cannot be simply such a cohipound, on ac- count of the sulphur and the sodium content, as has been said, and it is doubtful if the lecithin is chemically united with the glucose. It may be that the union of the reducing substance with the lecithin- like fraction or fractions takes place at the time of their simultan- eous precipitation. At least, it seems probable that the various substances that have been obtained are not identical, for they differ in elementary composition, and contradictory properties have been reported. Baskoff (1908), after a review of the subject, and careful ex- perimental investigation of Drechsel's jecorin purified in several ways, and a consideration of other products separated during the 66 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 processes, concludes that "Drechsel's jecorin may be characterized as a lecithin-like complex with about 14 percent glucose, which also always contains sulphur and an inorganic substance," and that there are other jecorin-like bodies not identical with it. The close sim- ilarity in chemical composition of his several purified products argues against such an indefinite mixture of substances as Paul Mayer, Meinertz, Siegfried and Mark, and A. Mayer and Terroine have suggested. (References as previously given and A. Mayer and Terroine, 1907.) Baskoff finds the relation of N :P=2 :1, corresponding to a diam- ino-monophosphatid. Others have found different ratios. Lecithin-Protein Compounds, or Lecitho-Proteins. The vitellin isolated from egg-yolk contains considerable lecithin (or other phos- phatid) from which it is freed with great difficulty, and there is some question as to whether it should not be looked on as a phos- phatid-globulin. However, there seems to be sufficient evidence that not all of the phosphorus is present as phosphatid but that we have here a nucleoalbumin, still containing phosphorus after all phosphatid is removed. Hoppe-Seyler and others consider that the original vitellin is chemically combined with lecithin and that the phosphatid-free vitellin is therefore to be looked on as a denatured compound. According to T. B. Osborne and Campbell (1900b), the so-called ovovitellin is a mixture of several vitellin-lecithin combina- tions containing 15-30 percent of lecithin. The chemical union is not proved, nor is the identity of the lecithin present, which may be the diamino-phosphatid which Erlandsen found in egg-yolk. Other lecithin-protein compounds have been discussed under the name of lecithalbumin. Liebermann (1891a, 1891b, 1893a, 1893b) isolated what he called lecithalbumins from the mucous membrane of the stomach (hog), from kidney (sheep), liver (lamb), lungs and spleen. The methods used in preparing and handling these substances were not sufficiently discriminating to prove their nature. Several observers think that the phosphatids of the serum and of chyle are in combination with globulin. According to Erlandsen's interpretation of his extractions from muscle, the lecithin found there is entirely free or in easily cleavable condition, while the diamino-phosphatids are in some complex union with proteins or other substances so that they cannot be extracted until the proteins have been precipitated with alcohol. Micro- scopical observations indicate that the phosphatids are in some kind of union with other cell constituents. PHOSPHORUS METABOLISM 67 Bing (1901) discusses the solubilities of a variety of lecithin compounds. For an extended general review of literature on leci- thin see Merck (1912). CEPHALIN Cephalin was first studied as isolated from brain by Thudichum (Thudichum, 1875; Thudichum and Kingzett, 1876), and has since been reported by W. Koch and his associates as found in various tis- sues and organs, and in milk and yeast, by Stern and Thierfelder in egg-yolk, and by Cousin, Falk and Frankel and associates in brain and nerves. (W. Koch, 1902b, 1903, 1904, 1906; W. Koch and Goodson, 1906; W. Koch and Woods, 1905; W. Koch and Mann, 1907; Stern and Thierfelder, 1907; Cousin, 1906; Falk, 1908; Frankel and Neubauer, 1909 ; Frankel and Dimitz, 1909.) Erland- sen did not find it in either kind of muscle. According to Thudi- chum, it is the principal phosphatid in the brain, and Falk's analysis of peripheral nerves shows considerably more cephalin than lecithin. It is doubtful if cephalin has ever been obtained in the pure state. Koch reports that "agfa" lecithin is two-thirds cephalin. Koch, however, at a later date, abandoned the method which served as the basis for the quantitative separation of lecithin and cephalin. (Jour. Am. Chem. Soc. 31, 1909, p. 1349.) Cephalin is a highly unsaturated monamino-monophosphatid. It is autooxidizable. The nitrogenous base is probably not choline, but a base of lower CH 3 content (W. Koch, 1902b ; Frankel and Neu- bauer, 1909). Baumann (1913) reports that this base exists in cephalin as a primary amino group, and Renall (1913) recognized in cattle-brain cephalin an amino ethyl alcohol, which he con- siders a characteristic constituent of cephalin. The base is united with glycerophosphoric and fatty acids, the latter, according to Cousin (1906) containing saturated acids, al- most entirely stearic, and unsaturated acids of the linoleic series. Cousin worked with the brain of cattle. Frankel and Dimitz (1909) think that the cephalin of brain is a mixture of palmityl-cephalin and stearyl-cephalin, with more of the former. Parnas (1913) states that all of the fatty acid cleaved from cephalin by baryta is stearic acid. Falk (1908) says that the cephalin of the peripheral nerves is not the same as that of the brain. Thudichum gave the name cephalic acid to the unsaturated acid, which he considered the distinguishing component of cephalins, 68 OHIO EXPERIMENT STATION! TECHNICAL BUL. 5 and which through its marked variability in composition imparts variability to cephalins. He also found a second base. Some in- vestigators have found inorganic base elements, as Ca or K, which seemed to belong to the cephalins. The solubilities of the prepar- ations reported by different workers do not agree. Some of the elementary analyses reported are as follows: ELEMENTARY ANALYSES OF CEPHALIN— Percent Author Date and Reference Source of Cephalin C H N P Formula Koch 1901 1902b 1909 1908? 1907 Brain Brain Brain Nerve Egg-yolk 60.00 59.5 62.05 57.0 59.68 9.38 9.8 9.85 9.1 9.74 1.68 1.75 1.69 1.94 1.57 4.27 3.83 3.45 4.4 3.64 C 42 H 79 NFOi3 C42H82NPO13 Frankel and Neubauer . Falk * Stern and Thierfelder C 1 ) These figures are taken through Bang (1911a). Bang gives as the probable formula, C 42 H 80 NPO 9 ; Hammarsten gives C 42 H 82 NP0 13 . If there are several cephalins, the formulas and percentage compositions vary accordingly. OTHER MONOPHOSPHATIDS Other monophosphatids which may be mentioned here are the cephalin variations, the paramyelin (C 38 H 75 NP0 9 ) and the myelins (C 40 H 75 NPO 10 ) of Thudichum, a monophosphatid found by Erland- sen in the acetone solution from heart muscle, and vesalthin ob- tained by Pari (Frankel and Pari, 1909 ; Frankel, Linnert and Pari, 1909) as the CdCl 2 salt, C 32 H 63 NP0 9 .CdCl 2 , from the corresponding extract from pancreas. II MONAMINO-DIPHOSPHATIDS According to Erlandsen, diphosphatids are regularly found in large amount in heart muscle, and only in small amount in the stri- ated muscle. One or two have been found elsewhere in the animal body, but none have been fully studied. They have not been found in the brain. CUORIN For cuorin, found in heart muscle, Erlandsen gives the formula C 71 H 125 NP 2 21 , which implies a much larger molecule than that of lecithin. It is made up of glycerophosphoric acid, three fatty acids and a nitrogen-containing base that is not choline. The fatty acids are mainly highly unsaturated and are thought to belong to the lino- leic or the linolenic acid series. Cuorin is even more autooxidizable PHOSPHORUS METABOLISM 69 than lecithin, and the properties are much altered by such oxida- tion. MacLean (1912a, 1912b) finds cuorin in horse kidney and muscle. OTHER MONAMINO-DIPHOSPHATIDS Other monamino-diphosphatids similar to cuorin, but not iden- tical with it, have been obtained by Baskoff (1908) from horse liver, and by MacLean (1908b, 1909b) from egg-yolk. Elementary analy- ses of the three substances are given here : ELEMENTARY ANALYSES OF MONAMINO-DIPHOSPHATIDS— Percent Author Date and Refer- ence Source C H N P O S N:P Erlandsen Baskoff . . MacLean . 1906, 1907 1908 1909b Heart Liver Egg-yolk 61.63 61.12 59.12 9.03 8.95 9.44 1.015 1.23 0.812 4.46 4.00 3.59 23.86 (24.04) 27.048 None 0.6 1:1.99 1:1.47 1:2 Baskoff called his compound "heparphosphatid." It contained sulphur. The N-component was thought to be other than choline, but was undetermined. It had a lower fatty acid content than leci- thin. It did not react with sugar. MacLean found his compound in about equal amount with cephalin in egg-yolk. Ill TRIAMINO-DIPHOSPHATIDS Triamino-diphosphatids have been obtained only by Frankel and his co-workers as CdCl 2 compounds (Frankel and Nogueira, 1909a, 1909b; Frankel and Linnert, 1910). The methods used are unreliable. These compounds were obtained from kidney and from brain. Only that from the brain, which is called "sahidin" has been studied in detail. From hydrolysis it appears to be a compound of choline with glycerophosphoric acid and fatty acids, the latter being of both saturated and unsaturated type. It is quite conceivable that the compound under examination was a monamino-monophos- phatid of the lecithin type containing a nitrogenous substance as an impurity. B. SATURATED PHOSPHATIDS I. DIAMINO-MONOPHOSPHATIDS This class of compounds appear to be primary constituents of cells, but they have not been studied sufficiently to furnish definite conceptions as to their constitution or their relations to one another. 70 OHIO EXPERIMENT STATION: TECHNICAL BUL, 5 SPHINGOMYELIN Thudichum, and Rosenheim and Tebb (Rosenheim and Tebb, 1907, 1908a, 1908b, 1908c, 1909a, 1909c, 1910) have felt that they showed the so-called "protagon" of the brain to be mainly a mixture of a diamino-monophosphatid, which Thudichum named sphingo- myelin, and cerebrosides. Rosenheim and Tebb found the same characteristics in the substance isolated from the cortex of the ad- renals, but with a smaller amount of sphingomyelin. Thudichum gives the formula C 52 H 104 N 2 PO 9 +H 2 O for sphingomyelin. Rosen- heim and Tebb (1908c) report an elementary analysis with 3.46 percent P, from which Bang computes the formula C 49 H lo4 N 2 PO 10 . If the work of these two sets of observations is correct, sphing- omyelin is, with carnaubon only, an exception among the phos- phatids in that it contains no glycerin, and therefore is not a glycerophosphoric acid compound. It does show phosphoric acid, fatty acids (perhaps only one, which Thudichum iden- tified as an isomer of ordinary stearic acid) and choline (or neurine) and a crystalline alcohol. Rosenheim and Tebb (1908b) made a study of the peculiar property of sphingomyelin by which it separates from its pyridine solution, or a pyridine solution of pro- tagon, in doubly refracting spheroid crystals which are strongly laevorotatory, the dextrorotation of the original protagon solution thus being gradually lessened and finally changed to laevorotation. Since this substance is probably widely distributed in cellular tis- sues, it is desirable that it should receive much further study. OTHER DIAMINO-MONOPHOSPHATIDS Other diamino-monophosphatids are the apomyelin and amido- myelin of Thudichum, a phosphatid isolated from liver by Baskoff (1908) which showed the ratio P:N=1 :2.55, one taken from kidney by Nogueira for which the formula C 34 H 72 N 2 PO 10 is given, and two others the properties of which are reported as resembling those of sphingomyelin. Of these, that of Stern and Thierf elder (1907), was obtained from egg-yolk, and the elementary analysis showed 3.22 percent P and a ratio P:N— 1:1.9. One of the compounds which Erlandsen obtained as a CdCl 2 combination, but was not able to isolate free, seemed to have the formula C 40 H 75 N 2 PO 12 . It was ob- tained from both types of muscle, though apparently it was not present in the free state but in protein combination. Cleavage in- dicated but one molecule of fatty acid and probably two basic radicals. The fatty acid was an oxyacid. Otherwise it much re- sembled lecithin. PHOSPHORUS METABOLISM 71 Burow (1910) finds three iron-containing phosphatids in the spleen of cattle and of man. Only one of these was obtained in suf- ficient amount for analysis and that proved to be a diamino-mono- phosphatid. The name "ferroid" was proposed to show its iron- lipoid character. Baskoff's (1908) analyses of purified jecorin indicate that that compound may be a diamino-monophosphatid (see p. 65). II. TRIAMINO-MONOPHOSPHATIDS The triamino-monophosphatids which have been reported are the neottin of egg-yolk, found by Frankel and Bolaffio (1908), and the carnaubon of kidney, found by Dunham and Jacobson (1910). NEOTTIN Neottin is a saturated compound, optically inactive. It is thought to contain choline and three saturated fatty acids, stearic, probably palmitic and perhaps cerebronic. CARNAUBON Carnaubon differs decidedly from all other animal phosphatids that have been examined, except jecorin, in that it contains a sugar (galactose or aminogalactose) which is but incompletely split off in simple hydrolysis and therefore must form an essential part of the phosphatid molecule. Such compounds are common in plants. No glycerin is found in carnaubon, and Dunham and Jacobson suggest that the structure may be similar to that of lecithin, but with, the sugar in the place of glycerin. Three fatty acids were found, carnaubic, stearic and palmitic, with phosphoric acid and choline. Assuming that carnaubon is made up of aminogalactose, these three fatty acids and one phosphoric acid in combination with two choline groups, the formula C 74 H 150 N 3 PO 13 is proposed. MacLean (1912a, 1912-13) isolated from horse kidney a sub- stance having all the properties of this compound of Dunham and Jacobson, but it was a diamino-monophosphatid. He thinks it prob- able that carnaubon is not a tri-, but a diamino-monophosphatid and that the methods used by Dunham and Jacobson were inefficient to obtain a pure substance. By water extractions MacLean (1912a, 1912b) separates water-soluble substances which contain but little phosphorus, and are not of the nature of ordinary phosphatids, but are easily confused with them in the processes ordinarily used for isolation of phosphatids. As these substances have a high nitrogen content, it may be that several of the substances hitherto described as containing large percentages of nitrogen are really mixtures of 72 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 simpler bodies with these. He recommends a purification by a pro- cess of emulsification with water and precipitation with acetone. III. PROTAGON For a long time the name "protagon" was in use for a substance isolated from brain and thought to be the principal, perhaps the only, phosphatid of brain. It was found also in many other parts of the body. That these preparations were crystalline and fairly constant in composition even after recrystallization justified the belief that protagon was a chemical unit ; later work, however, has shown that such a conception is untenable. Since the first finding and naming of protagon by Liebreich in 1865 it has received much attention. A list of references is given below. The question of protagon being the main or only constituent of the brain was satisfactorily answered by Frizzell's analyses, report- ed by Chittenden, and especially by the abundant evidence from Thudichum's work. Thudichum also showed that it is a mixture, and that it does not contain lecithin. Thudichum's work, however, seems not to have met with due recognition until after his death. Further evidence against the chemical entity of protagon has been produced mainly by Gies and his associates, and by Rosenheim and Tebb. They have shown that it is made up of phosphorus-rich and phosphorus-poor components mixed in variable proportions. Kossel and Freytag had already recognized the presence of cerebrosides, which are phosphorus-free compounds consisting of the sugar galac- tose, fatty acids and a nitrogen complex. Thierf elder and his as- sociates have separated and studied cerebron, another phosphorus- free compound. The phosphorus compound was identified by Thudichum and by Rosenheim and Tebb, as sphingomyelin, but it is perhaps not a single phosphatid. Sulphur has usually been found in protagon, and apparently in organic combination, and Koch thought that he had evidence of a complex containing an ethereal sulphuric acid combined with a phosphatid and a cerebroside, as one of the substances present in the mixture. Frankel concludes that protagon is a mixture of members of the group which he designates "galacto-phospho-sulphatids," or combinations of phosphatids and sulphatids with galactose. It is not to be looked upon as a chemical unit. References on protagon: Liebreich, 1865; Diaconow, 1867a; Gamgee and Blankenhorn, 1879a, 1879b, 1879c, 1880; Baumstark, 1885; A. Kossel, 1891b; A. Kossel and Freytag, 1893; Ruppel, 1895; Chittenden, 1897 ; Noll, 1899 ; Worner and Thierf elder, 1900 ; Kita- PHOSPHORUS METABOLISM 73 gawa and Thierf elder, 1906 ; Ulpiani and Lelli, 1902 ; Barbieri, 1905 ; Lesem and Gies*, 1903 ; Cramer, 1904 ; Orgler, 1904 ; Posner and Gies, 1905; Lochhead and Cramer, 1907; Gies, 1907; Steel and Gies, 1907b ; Wilson and Cramer, 1908 ; Cohen and Gies, 1908 ; Rosenheim and Tebb, 1907, 1908a, 1908b, 1908c, 1909a, 1909c, 1910; W. Koch, 1907b, 1910a, 1912 ; Frankel, 1908. D. PLANT PHOSPHATIDS Apparently phosphatids are as widely distributed (and there- fore probably essential) in plant cells as in animal cells, and are as varied in their make-up, though the work of differentiation and classification has not yet been carried even as far as it has for those of animal origin. Nearly all of the investigations we have found reported have been made by E. Schulze and E. Winterstein and their associates (Schulze and Steiger, 1889 ; Schulze and Likiernik, 1891a, 1891b; Schulze and Frankfurt, 1894; Schulze, 1895, 1897, 1907, 1908a, 1908b; Schulze and Winterstein, 1903; Hiestand, 1906; Win- terstein and Hiestand, 1906, 1908 ; Winterstein and Stegmann, 1909a, 1909b; Winterstein and K. Smolenski, 1909; K. Smolenski, 1909; Schulze and Pfenninger, 1911; Trier, 1911, 1913a, 1913b, 1913c, 1913d). A few other articles should be mentioned, especially that of Njegovan (1911) ; also those of Wintgen and Keller (1906) ; Parrozzani (1909); Vorbrodt (1910) and Bernardini (1912). It was at first supposed that all the phosphorus-containing fat- like substances obtained were identical with those of animal tissues, and, as for the latter, so for these, the name lecithin was used, and the cleavage products somewhat supported the idea ; but it was later shown that there are several different phosphatids here and that the true lecithins are not found at all in plants. The phosphatids of the two kingdoms are essentially different, in that vegetable phos- phatids nearly or quite always contain a sugar, apparently firmly bound and in constant stoichiometric relation with the rest of the molecule, such as is not the case in animal phosphatids, with the probable exception of jecorin, and perhaps carnaubon. The amount of carbohydrate found has been widely different in different prepar- ations, suggesting that a part, at least, of it is present as a phos- phatid-sugar combination such as the lecithin-sugars mentioned above. The plant phosphatids contain, together with the sugar, glycerophosphoric acid, fatty acids and choline, and sometimes other nitrogenous residues, either basic or amino acid. 74 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 ARTIFICIAL SYNTHESIS OF PHOSPHATIDS Glycerophosphates have been made by several investigators, and they have found extensive use as drugs. See Adrian and Trillet (1897, 1898), Lumiere, A. L., Lumiere and F. Perrin (1901), Carre (1904), Willstatter and Liidecke (1904), Power and Tutin (1905), Tutin and Hann (1906), Neuberg and Kretschmer (1911), Langheld (1910, 1911, 1912), DuBois (1914), and also a re- view of literature by Merck (1911). The question of the identity of the synthetic products with the glycerophosphate of lecithin was considered by Willstatter and Liidecke, Tutin and Hann, and Power and Tutin, with evidence that they are not identical, and that the difference is in the structure of the molecule. Power and Tutin say that, in the substances handled, the mono-ester, C 3 H 5 (OH) 2 O.PO (OH) 2 , has often been contaminated A with the di-ester, C 3 H 5 OH ^POCOH). Tutin and Hann con- clude that both the synthetic and the natural products are mixtures of the asymmetric or a-acid, CH 2 O.P0 3 H 2 , CH.OH CH 2 .OH and the symmetrical or /?-acid, CH 2 .OH CH.O.P0 3 H 2 CH 2 .OH DuBois (1914) discusses these and other possible glycerophos- phates, and gives in detail the properties of such as are known. Con- sidering the evidence brought forth by others in comparing the nat- ural and synthetical products leads him to the "conclusion that the synthetical glycerophosphates produced at low temperatures (100- 110°), and the natural glycerophosphates obtained from lecithin are almost identical, and in all probability, a mixture of the a- and /?- isomerides, in which the a-isomeride predominates." According to Francois and Boismenu (1913) commercial cal- cium glycerophosphate may be a mixture of salts of 5 forms of the acid, namely the tri-ether, and the a- and /?- forms of both di- and mono-ethers. The calcium salt of the tri-ether, however, is so diffi- cultly soluble that it is not usually permitted by the manufacturer to be present in the commercial article. PHOSPHORUS METABOLISM 75 THE PHOSPHORUS OF PHOSPHATIDS In the phosphatids, and in no other natural products, so far as known, phosphorus is present as glycerophosphoric acid, a glycerin ester of orthophosphoric acid. The most ready cleavage sets free this glycerophosphoric acid, and further cleavage produces free ortho- phosphoric acid. This relation seems to be unquestioned. See Imbert and Belugon (1897), with regard to the reactions of glycerophosphoric and phosphoric acids with bases, and Malengreau and Prigent (1911) with regard to the hydrolysis of glycerophos- phoric acid. For general discussions on the phosphatids see Thudichum (1901), Hiestand (1906), Bang (1909), Frankel (1908, 1909a), MacLean (1909d), Bang (1911a) and Merck (1911, 1912). 76 OHIO EXPERIMENT STATION: TECHNICAL BUL. 6 PART II. THE PHOSPHORUS COMPOUNDS OF FOODS GENERAL DISCUSSION A statement of the phosphorus content of a foodstuff is of slight value apart from a consideration of other nutrient constitu- ents, but we can not, within the scope of this article, go into the matter of general composition of foods. In our selection of material for presentation in this section, therefore, we have sought to include especially data showing the connection of the phosphorus content of foods with other matters of interest, particularly as related to gen- eral type of food products, or to conditions of growth, preparation or manufacture ; and also such material as is available showing the differential estimation of the various groups or kinds of phosphorus compounds in foodstuffs. For data on total phosphorus of foods see the works cited on p. 103. Analyses of meats, milks, and eggs will be found in the section or Phosphorus of Animal Bodies and Products, and are not included here except as they appear in miscellaneous tables. From the following tables of Forbes, Beegle and Mensching (1913) we note that among the milling products of wheat the phos- phorus is contained principally in the byproducts which are used as foods for live-stock. The white flour is that portion of the grain of wheat which contains the least phosphorus. Wheat gluten, such as is used as a diabetic flour is also poor in phosphorus. The bran contains more phosphorus than any other part of the kernel. For further details regarding the phosphorus constituents of wheat see Osborne and his associates, p. 91, Rengniez, p. 78, Rising, p. 79, Ames and Boltz, p. 96-98, and Swanson, p. 80, of this work; also Girard (1884), Girard and Lindet (1903), Balland (1903), Surmont and Dehon (1903, 1904) and Fauvel (1907) ; also references on p. 103 to general works on food analysis and nutri- tion. PHOSPHOEUS METABOLISM 77 Corn contains less phosphorus than do oats and wheat, the pearl hominy and bolted corn meal used by human beings containing less phosphorus than the whole grain. Gluten feed contains more phosphorus than does the whole corn, and distiller's grains about the same as corn. • Kafir corn contains less phosphorus than does Indian corn, while polished rice is very poor in phosphorus, about the same as pearl hominy. Rice polish, however, which is fed to live-stock, is exceedingly rich in phosphorus. On a dry basis the fruits are very low in phosphorus ; such veg- etables as onions, cabbage, beets, and potatoes contain much more. Among various kinds of roughage, leguminous forage contains considerable phosphorus, as also does blue-grass. Timothy hay, corn stover and wheat straw contain much less. The phosphorus content of all sorts of roughage is much affected by the soil. Leguminous seeds are characteristically rich in phosphorus, much richer than the leaves and stems of the same species, while the oil meals are still richer, cottonseed meal excelling linseed oil meal in this regard. MINERAL ELEMENTS OF CEREAL PRODUCTS— Parts per 100 of Dry Substance (Forbes, Beegle and Mensching, 1913) Potas- sium Sodium Cal- cium Magne- sium Sul- phur Chlor- ine Phos- phorus Inor- ganic phos- phorus Organ- ic phos- phorus Wheat - .590 .058' .156 1.464 1.147 .323 .007 .425 .396 .192 .410 .153 .272 .014 .045 .185 .219 .460 .288 .040 1.279 .035 .127 .583 .223 .186 .788 .031 .733 .030 .113 .000 .000 .461 .154 .077 .278 1.458 .184 .066 .032 .124 .056 .022 .038 .139 .108 .078 .085 .134 .014 .015 .030 .005 .268 .047 .142 .169 .159 .112 .013 .009 .030 .142 .019 .004 .590 .430 .372 .049 .324 .126 .122 .088 .036 .239 .054 .195 . .172 .194 .130 .142 .028 .741 .224 .168 .198 .297 .263 .355 1.000 .285 .171 .122 .124 .182 .636 .509 .408 .419 .864 .214 .186 .114 .189 .095 .081 .958 1.000 .029 .077 .055 .156 .073 .070 .052 .052 .098 .065 .028 .062 .389 .077 .117 .040 .151 .425 .102 .135 1.233 .984 1.147 .220 .928 .303 .264 .156 .111 .589 .314 .458 .503 .746 .434 .271 .104 1.684 .038 .017 .043 .034 .069 .037 .098 .028 .019 .031 .019 .106 .056 .018 .162 .471 .059 .012 .003 .028 .387 .085 White bread Wheat bran . . Wheat middlings . .* . . . .092 1.199 .915 Wheat gluten Red dog flour .183 , .830 .275 .245 .125 Pearl hominy .092 .483 Distiller's grains, corn.. Distiller's grains, rye . . .258 .440 .341 .275 .375 .259 .101 1.656 The animal products are, on a dry basis, rich in phosphorus, even whey containing notable amounts, while, of course, such foods as tankage, which contain some bone, are much richer than any oth- er foods in phosphorus. Milk and eggs do not differ greatly in phos- phorus content. Both are rich in phosphorus, and contain much more of this element than does meat. 78 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 MINERAL ELEMENTS OF FRUITS, VEGETABLES AND ROUGHAGE— Parts per 100 of Dry Substance (Forbes, Beegle and Mensching, 1913) Potas- sium Sodium Cal- cium Magne- sium Sul- phur Chlor- ine Phos- phorus Inor- ganic phos- phorus Organ- ic phos- phorus .802 1.347 1.291 .667 1.442 2.484 1.208 1.547 3.870 .347 1.840 1.774 .873 .832 .613 1.338 1.847 1.405 .842 .132 .066 .045 .240 .115 .097 .028 .061 .175 .714 .185 .067 .145 .722 .489 .345 .099 .065 .141 .237 .135 .027 .076 .037 .084 .261 .590 .084 .027 .131 .729 1.236 1.378 2.029 1.130 .192 .326 .507 .336 .217 .780 .033 .056 .129 .086 .136 .209 .215 .331 .358 .283 .292 .692 1.096 .400 .111 .262 .092 .240 .063 .570 .044 .066 .021 .087 .601 .901 .117 .141 .224 .138 .190 .259 .352 .298 .162 .159 .187 .334 .159 2.090 .037 .050 .421 .285 .183 .243 .069 .055 1.380 .048 .259 .084 .167 .161 .199 1.230 .308 .234 .209 .040 .064 .110 .119* .077 .323 .262 .186 .270 .260 .069 .183 .237 .283 .238 .123 .173 .102 .242 .038 .024 .033 .098 .089 .037 .210 .136 .138 .130 .174 .006 .080 .121 .152 .122 .052 .072 .039 .142 .015 .003 .031 .012 .030 Date .040 .113 .126 .048 .140 .086 .063 .103 .116 .131 ' .116 .071 .101 .063 .100 .023 .021 MINERAL ELEMENTS OF LEGUMINOUS SEEDS, NITROGENOUS CON- CENTRATES AND ANIMAL PRODUCTS— Parts per 100 of Dry Substance (Forbes, Beegle and Mensching, 1913) Soy beans , Navy beans . , Cowpeas Peanuts Linseed oil meal Cottonseed meal Milk, skim Whey , Mutton Eggs Tankage "Banner" bone flour Blood, swine "Black albumen" . Potas- sium 2.095 1.390 1.636 .061 1.224 1.811 1.272 2.762 .624 .206 .601 .065 1.040 .027 Sodium .380 .086 .189 .563 .282 .283 .488 .459 .214 .389 1.830 .091 1.370 1.247 Cal- cium Magne- sium .230 .235 .117 .068 .403 .291 1.336 .721 .006 .250 3.242 23.990 .031 .039 .244 .206 .243 .180 .544 .599 .146 .138 .062 .059 .159 1.160 .028 .011 Sul- phur .444 .224 .280 .254 .455 .536 .357 .139 .607 .762 .669 .647 .820 Chlor- ine .025 .047 .047 .024 .095 .042 .953 1.948 .235 .621 2.687 1.200 1.550 Phos- phorus .649 .429 .532 .399 .786 1.479 .979 .640 .474 .856 1.789 14.940 .280 .122 Inor- ganic phos- phorus .017 .088 .023 .049 .078 .551 .402 .230 trace 14.940 .076 .037 Organ- ic phos- phorus .632 .341 .509 .350 1.401 .428 .238 .244 .856 .204 .085 From the work of Vorbrodt (1910) we quote the table on the following page setting forth a separation of general groups of phos- phorus compounds. Rengniez (1911) reports most of the phosphorus of flour to be in the forms of phytin and nuclein. The highest phosphorus con- tent was found in flour made from the germs. Wheat germ flour showed 2.9 percent total phosphoric acid, of which 2.17 percent was in the form of phytin and 0.70 percent in the form of nuclein. PHOSPHORUS METABOLISM 79 CONTENT OF DIFFERENT FORMS OF PHOSPHORIC ACID IN SEEDS Vorbrodt, (1910) Seeds of Indian corn (Zea Mays) . . . Wheat (Triticum sativum) Barley (Hordeum distich.)! "II Rye (Secale cereale) Lentil (Lens esculenta) II Windsor bean ( Vicia faba minor) Rape (Brassica napus olei- fera) Hemp ( Cannabis sativa) . . Spruce (Picea excelsa) Pine (Pinus cembrd) Total P2O5 Percent 0.814 1.04 1.07 0.83 0.98 0.70 1.07 1.23 1.74 1.57 1.07 Mineral P2O5 Percent Percent of seed of total 0.050 0.134 0.15 0.127 0.302 0.134 0.081 0.102 0.213 0.138 0.079 6.14 12.88 14.02 15.30 30.82 19.14 7.57 8.29 12.24 8.70 7.38 Soluble organic P2O5 Percent of seed 0.398 0.311 0.389 0.285 0.283 0.065 0.047 0.468 0.261 0.340 0.154 Percent of total 29.90 36.36 34.34 28.88 9.29 4.39 38.05 15.00 21.65 14.39 Protein P2O5 Percent of seed 0.319 0.567 0.531 0.388 0.382 0.435 0.894 0.66 1.266 1.092 0.837 Percent of total 39.18 54.51 49.63 46.75 38.98 61.71 83.56 53.66 72.76 69.56 78.22 Lecithin P2O5 Percent of seed 0.047 0.028 0.030 0.013 0.069 0.048 0.029 0.053 Percent of total 5.77 2.69 1.33 9.86 4.49 1.85 4.95 General phosphorus separations are also quoted from the work of Rising (1910). PHOSPHORUS COMPOUNDS IN PEAS, BEANS AND FLOURS Rising (1910) Phos. of nu- Total Phosphorus of Phosphorus of Phosphorus clein sub- stance and phosphopro- tein Mois- ture phosphorus phosphatids phosphates of phytin Sum of Per- Per- Per- Per- Per- of solids other Per- cent Per- cent Per- cent Per- cent cent found deter- cent of total cent of total cent of cent of directly mina- tions P P total P total P Yellow peas 13.7 0.579 0.565 0.0586 10.1 0.019 3.3 0.110 19 0.378 65.1 Brown beans.. 11.8 0.560 0.5167 0.0397 7. 0.019 3.4 0.290 52 0.168 30. Rye flour 11.85 0.313 0.3278 0.0073 2.8 0.090 2.8 1 0.0885 25 0.142 45.3 13.5 0.1665 0.-1891 0.0067 4. 0.0116 7. 0.1144 69 0.0564 33. Graham flour. . 12.4 0.287 0.2400 0.0058 2. 0.0381 13.2 0.0819 29 0.1142 39. ( 1 ) Apparently a misprint for 28. Hooper (1911) states that unmilled rice contains about 0.65 percent P 2 5 , while polished rice contains 0.38 percent. The por- tion of the rice which is removed by the milling process contains 3.36 percent P 2 5 . He reports Indian wheat to contain 0.69 percent P 2 5 , and flour from the same 0.21 percent P 2 5 . Aron and Hocson (1911a) found in husked rice 0.7-0.8 percent P 2 5 ; undermilled rice, 0.45-0.6 percent; and overmilled rice, 0.15- 0.35 percent. Bernardini (1912) gives the distribution of phosphorus in rice as follows : 80 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 DISTRIBUTION OF PHOSPHORUS IN RICE (Bernardini, 1912) P2O5 contained in In embryo, percent In whole seed, percent Total 6.20 0.04 0.22 5.14 0.04 0.76 0.950 0.003 0.018 Phytin 0.436 0.502 Adler (1912a, 1912b) investigated the nature of the phospho- rus compounds of brewing products. Eighty percent of the phos- phorus of beer is inorganic; 20 percent organic. Of the organic phosphorus a part is combined with carbohydrates, and a part with proteins. Lecithin and phytin are not present. Of the phos- phates, half are united with alkalis and half with alkaline earths. Wort contains both organic and inorganic phosphorus, and in about the same proportion as in beer. Malt contains alkali and alkaline earth phosphates, as well as phytin. Malt sprouts also contain al- kali and alkaline earth phosphates, in greater amounts than either the barley or the malt. Swanson (1912) reported investigations with regard to the re- lations between the percentages of acidity and percentages of ash, amino compounds, and total and water-soluble phosphorus of wheat flours, the work being part of a general study on the relation be- tween chemical composition and baking qualities of flour. The total phosphorus varies from 0.093 percent (in second middlings) to 0.373 percent (in ship-duster flour), the amount in wheat being given as 0.482 percent; the water-soluble phosphorus at 25° varies from 0.017 percent (first and second middlings) to 0.114 percent (fifth break) ; and the water-soluble at 40°, from 0.023 percent (first middlings) to 0.191 percent (ship-duster flour), that of wheat being 0.218 percent. ORGANIC AND INORGANIC PHOSPHORUS IN FOODS Methods for the estimation of inorganic phosphorus as distinct from organic phosphorus in foods are not yet sufficiently well estab- lished to warrant the placing of emphasis upon minor differences in results obtained. One of us (E. B. F.) in collaboration with Leh- mann, Collison and Whittier published methods for this estimation in both plant and animal tissues (Ohio Agr. Exp. Sta. Bui. 215), and results obtained by these methods are submitted in the tables on pp. 77 and 78. In later studies of these methods the one used for PHOSPHORUS METABOLISM 81 animal tissues has promised well, while the outcome of further work with the method for vegetable products seems problematical. Cer- tain general conclusions, however, we feel it safe to draw from these data. Thus, all of the parts of plants contain inorganic as well as organic phosphorus. The proportion of the total phosphorus which is inorganic is, in general, much higher in leaves, stems, fruits and roots than in seeds. Among the cereal products the proportion of inorganic phosphorus in the total is much higher in brewer's grains and malt sprouts than in others. Organic and inorganic phosphorus in meat and milk are some- what nearly equally divided. Inorganic phosphorus predominates in whey, while eggs are practically free from inorganic phosphorus. Since the whole matter of inorganic phosphorus estimation in vegetable substances is in an unsettled state we have made no effort to present all the data available. Below are references to a few of the investigations which report inorganic phosphorus estimations in vegetable substances : Zaleski (1902, 1907) ; .Schlagdenhauff en and Reeb (1902) ; Hart and Andrews (1903) ; Schulze and Castoro (1904) ; Suzuki and Yoshimura (1907) ; Suzuki, Yoshimura and Takaishi (1907) ; Stut- zer (1908) ; Heubner and Reeb (1908) ; Forbes, Lehmann, Collison and Whittier (1910) ; Forbes, Whittier and Collison (1910) ; Hart and Tottingham (1910) ; Hartwell and Quantz (1910) ; Rising (1910) ; Vorbrodt (1910) ; Hartwell and Hammett (1911) ; J. W. Ames and Boltz (1912) ; Hartwell (1913). PHYTIN IN FOODS Posternak (1903a, 1903b, 1905) found phytin in the aleurone grains of red fir, hemp and sunflower, in peas, lentils, white kidney beans, rape, lupine, wheat, corn, potatoes, dahlia-bulbs, carrots and even onions, and says that in grains, where there is little mineral phosphate, it forms at least 70-90 percent of the total phosphorus. He looks upon it as a storage of reserve material for the develop- ment of the embryo. The following table is taken from the re- ports of 1903. , PHYTIN AND LECITHIN PHOSPHORUS IN FOODS (Posternak, 1903)"*" Red fir Hempseed (cortex removed) Sunflower seed (cortex removed) Peas Lentils White kidney beans Total phos- phorus, per- cent Phosphorus of phytin, per- cent 0.656 1.460 0.830 0.367 0.299 0.512 Phosphorus of I Phosphorus of phytin, in per-| lecithin, in cent of total percent of to- phosphorus tal phosphorus 0.600 1.330 0.723 0.260 0.247 0.418 91.46 91.44 86.26 70.80 82.60 81.60 1.1 3.1 1.8 6.2 6.7 6.0 82 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Gilbert and Posternak (1905) say that in a mixed diet the main part of the phosphorus is in the form of phytin. It is now well-known that phytin as estimated by Gilbert and Posternak includes compounds other than phytin. Suzuki, Yoshimura and Takaishi (Suzuki and Yoshimura, 1907; Suzuki, Yoshimura and Takaishi, 1907), from their studies of the distribution of phytin, report the following determinations of the forms of phosphorus in bran, seeds, seedlings and a few other sub- stances. PARTITION OF THE PHOSPHORUS COMPOUNDS OF FOODS (Suzuki, Yoshimura and Takaishi) — Percent Material examined Rice bran Wheat bran Seeds of sesame (Sesamum indicutn) Seeds of castor bean (Rtc inns communis) Oil cake, rape (Brassica napus) Barley bran {Hordeum ■vulgar e) Millet bran (Panicum frumeiitaceum) Rice seed seedling's Rape seed seedling's Barley seed seedlings Wheat seed seedlings Steamed bone meal (air- dry) Fresh bones of young cock (1 percent HC1 used) . . Total phos- phorus Phosphorus in lecithin In dry sub- stance 2.27 1.114 0.772 0.261 1.195 0.541 0.765 10.077 9.186 In dry sub- stance 0.02 0.010 0.030 0.013 0.034 0.010 0.026 0.009 Percent of total 0.86 0.81 3.91 5.13 1.85 3.40 1.47 4.10 6.95 9.83 4.14 8.05 3.32 6.97 0.09 Phosphorus sol- uble in 0.2 per- cent HC1 Of that soluble in 0.2 percent HC1 In dry sub- stance Percent of total 1.92 0.638 0.144 0.110 0.592 0.327 0.363 10.066 7.168 84.48 57.24 18.61 42.29 49.52 60.44 47.45 44.28 76.00 64.51 78.18 58.62 73.34 56.56 74.26 99.89 78.03 Inorganic In dry sub- stance 0.13 0.050 0.089 trace 8.919 6.514 Percent of total 5.89 4.49 16.45 trace 25.22 trace 70.02 trace 65.98 trace 63.27 88.51 70.91 Organic In dry sub- stance 1.68 0.579 0.125 0.109 0.532 0.238 0.344 0.267 0.167 Percent of total 74.17 52.00 16.24 41.61 44.46 44.00 44.97 41.64 42.52 62.11 2.88 56.55 6.89 55.50 10.72. 2.65 1.82 The authors conclude : "1. The greater part of the phosphorus of plant seeds consists of the organic compound soluble in water and dilute mineral acids which has been obtained by Schulze, Palladin, Winterstein, Poster- nak, Patten and others, and named 'Anhydro-oxy-methylen-diphos- phoric acid' or 'Phytin/ "From rice bran we have isolated about 8 percent and from wheat bran about 2 percent phytin. "2. But in roots, bulbs and fruits inorganically combined phosphoric acid predominates. "3. The occurrence of phytin in bones is doubtful. "4. During the germination of plant seeds, either in the light or in the dark, the inorganically combined phosphoric acid increases notably. PHOSPHORUS METABOLISM 83 "Also if one grinds rice or wheat bran or different seeds and lets them stand suspended in water for some days, phosphoric acid forms in considerable quantity at the expense of phytin. "5. An enzyme was isolated from rice and wheat bran that splits phytin into phosphoric acid and inosite. It is apparently a new enzyme and seems to be widely distributed in the plant world." Hart and Tottingham (1910) proved the presence of such a compound also in the grains of corn, oats and barley, and distributed throughout the entire seed, in these cereals, more than in wheat. Below is their description of the separation of parts of the seeds. "In the wheat kernel phytic acid as a salt exists largely in the outer aleurone layers and consequently is found in very large pro- portion in wheat bran. In order to determine whether similar dis- tribution obtained in the corn kernel this seed was mechanically div- ided into three parts — the outer layer or corn bran (pericarp) , the germ, and the starch and gluten cells (endosperm) ." The determin- ations below are stated on an air-dry basis. Total P P soluble in 0.2 percent Percent -r, HC1 * Percent Entire seed 0.29 0.13 Corn bran 0.13 0.00 Corn germ 0.38 0.13 Corn gluten 0.42 0.15 "It is clear from these data that in the maize grain, phytin is not localized in the outer layers In this instance there ap- pears to be none in the outer skin or seed coats, while there is, on the contrary, more or less uniform distribution throughout the en- tire seed." The oat "seed was mechanically separated into the hull or bran layers (pericarp) and kernel. The former is fibrous and forms a considerable part of the grain. The latter consists of the aleurone layer and starch cells (endosperm) and the embryo." _ , , _ P soluble in 0.2 percent Total P jjOl Percent Percent Entire seed 0.41 0.18 Oat kernel 0.41 0.22 Oat hull 0.41 0.09 "It is apparent from the data that while the seed coats carry a total amount of phosphorus comparable with the other parts of the grain, the proportion of phytin in the outer layers is relatively small. On the other hand it constitutes 50 percent of the total phosphorus bearing bodies of the remaining parts of the seed." 84 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Of barley "the separation of the entire seed into the outer fibrous coats (pericarp), here designated as bran, and into the aleurone layer, starch cells and embryo (kernel) was carried out mechanically." Total P P soluble in 0.2 percent HCi Percent Percent Entire seed 0.50 0.19 Bran, or hull 0. 22 0.15 Kernel, or germ 0.57 0.17 "These results make it manifest that phytin is not concentrated in the outer seed coats, although it does constitute a very large pro- portion of the total phosphorus existing there. Phytin appears to be distributed throughout the entire seed." Vorbrodt (1910), after studying the distribution of the kinds of phosphorus compounds in maize seed, says : "In corn seeds almost nine-tenths of the total phosphoric acid and almost all of that of the soluble organic compounds is in the germ and the scutellum. In the germ and scutellum there are large amounts of nucleopro- teins, while the rest of the seed contains but little of them." See also Rising p. 79, and Rengniez p. 78, Bernardini p. 79-80. DISTRIBUTION OF LECITHIN IN PLANTS AND IN MISCELLANEOUS FOODS The data of the table below are taken from Schulze and Steiger (1889), Schulze and Frankfurt (1894), and Schulze (1897, 1908b). Phosphorus determinations were made on the ether-absolute alco- hol extract and these values were interpreted as lecithin (3.84 per- cent P). The values, therefore, must not be taken as the absolute content, but they show, rather, the relative amounts of phosphorus in such a combination. Where determinations have been found to differ in the reports of different years, that given latest has been taken, and the dates are recorded in all cases. PHOSPHORUS METABOLISM 85 PHOSPHATID PHOSPHORUS IN VEGETABLE SUBSTANCES— Percent, Dry Basis Substance Seeds of Date i Phosphorus in ether-al- cohol ex- tract 2ther-alco- lol soluble phosphorus computed to lecithin Total phos- phorus Phosphatid phospho- rus in per- cent of total 1908 1897 1889 1889 1897 1908 1897 1894 1897 1908 1894 1908 1897 a 1908 1894 " " 0.082 0.084 0.031 0.063 0.049 0.035 0.022 0.017 0.021 0.009 0.011 0.011 0.026 0.026 0.038 0.033 0.059 0.020 0.021 0.019 0.006 0.004 0.009 0.0x4 0.001 0.007 0.009 0.025 0.006 0.020 0.028 0.024 0.016 0.039 0.012 0.073 2.14 2.19 1.09 0.81 1.64 1.05 1.27 0.90 1.03 0.43 0.47 0.25 0.57 0.53 0.73 0.85 0.44 0.55 0.25 0.29 0.30 0.67 0.67 0.49 0.27 0.11 0.99 0.86 1.55 0.54 0.56 0.50 0.15 0.10 0.25 0.37 0.04 0.19 0.22 0.69 0.17 0.54 0.77 0.65 0.45 0.86 0.32 1.94 1.53 1.32 2.10 1.14 1.16 2.60 5.5 3.7 1.0 1.0 Horsechestnut (Aesculus hippocastamim) 3.2 1.3 " II " III " " " II " II From the work of Stoklasa (1896a, 1896b) we quote the follow- ing summary of conclusions as to the distribution of lecithins : "1. Roots. Annual plants contain very little lecithin in their roots, the maximum being 0.3 percent ; at completion of growth this falls to 0.1 percent. Perennials or biennials contain a larger quan- tity at the close of the season, this serving as a reserve for use in building new cells. "2. Stalk. Stems contain 0.3 to 0.4 percent lecithin : after the fruit ripens this decreases rapidly, being at most 0.1 percent then for annuals. 8Q OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 "3. Leaves. A considerable quantity of lecithin is continually present in the leaves ; it begins to disappear after fertilization and during fruit formation, if at the same time the leaves begin to turn yellow. Pure leaf substance is the richest part of the plant in leci- thin, except the anthers and pollen grains at the time of blooming. Leaves contain up to 40 percent of their total phosphorus as lecithin. Since I have observed that the removal and destruction of lecithin and chlorophyl-coloring matter run parallel, and since I consider it most probable that this coloring matter contains a quantity of phosphorus corresponding to that of lecithin, I am of the opinion that chlorophyl itself is a lecithin. "4. Flowers. The petals, it appears, contain the most lecithin at the stage of fullest development of the buds ; after fertilization the lecithin decreases. The male organs, pollen filaments, anthers and pollen grains, are the parts richest in lecithin. The pollen . grains, containing almost 6 percent of lecithin, are the richest of all. This corresponds to the fact that the sperm of higher animals is es- pecially rich in lecithin." From the work of Stellwaag (1890) we quote the following es- timations of lecithin in the ether and benzene extracts of miscel- laneous vegetable foods. LECITHIN AND PHOSPHORUS IN ETHER EXTRACT AND BENZENE EXTRACT OF FODDERS— Percent (Stellwaag, 1890) Fat of Hay Peas Vetch Tick bean .... Lupine blossom Buckwheat Soja bean .... Cabbage Poppyseed cake Cottonseed cake Potatoes Barley Ether Extract Lecithin Phosphorus Traces 27.37 22.94 21.29 4.59 1.88 1.26 6.99 13.27 4.35 3.07 4.25 Traces 1.049 0.881 0.818 0.172 0.072 0.066 0.268 0.40 0.166 0.117 0.163 Benzene Extract Lecithin 6.95 7.65 4.11 2.53 1.57 3.27 6.24 1.52 2.37 Phosphorus 0.247 0.26 0.14 0.083 0.063 0.123 0.222 0.058 0.09i Von Bitto (1894) prefers a methyl alcohol extraction (20 ex- tractions) to the method of Schulze and Steiger, for phosphatid de- termination, and reports the following amounts found in that way : PHOSPHORUS METABOLISM 87 LECITHIN PHQSPHORUS AND LECITHIN IN VARIOUS SEEDS— Percent Dry Basis P Lecithin 0.0687 0.0621 0.0746 0.0750 0.0222 0.0256 0.0227 0.0185 1.788 1.618 Lupinus luteus (yellow lupine) . . . 1.933 1.955 0.578 0.667 0.592 0.482 Schulze (1895) supports his method in comparison with that of von Bitto. Th. Dietrich (1902) reports 1.48 to 1.89 percent of lecithin in the dry substance of brewer's grains. Stoklasa (1895) found in sugar beet leaves 1.12 percent, and in the root 0.43 percent lecithin, on the dry basis. W. Koch (1905a) reported lecithin estimations on a number of common foods, as below: LECITHIN IN SOME COMMON FOODS (W. Koch, 1905) 1 lb. calf brain . . 1 lb. shad roe .... 12 eggs 1 lb. calf liver . . . 1 lb. sweetbreads . 1 lb. lamb fries . . 1 lb. meat (beef) . 1 lb. peas or beans 1 lb. salmon 1 lb. bread 1 lb. vegetables . . 1 pint human milk 1 pint bovine milk 1 lb. mushrooms . . Gm. lecithin 20-25 18-20 12-16 12-15 12-15 10-12 5-7 5-7 5-6 0.5-1 0.3-0.5 0.4 0.3 0.2 Heubner and Reeb (1908) made determinations of the amount of phosphorus in the forms of its different compounds in a number of common foods, and their table is given below. Heubner (1911) says later that the method used here is inexact for the estimation of organic phosphorus, for instance, phytic acid. 88 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Q O O ft <5 H Woo w OS 5- S s H * P n as H « COM i— i " ft I— I o w o M ri , d » o ° COt-OsaoONWCOlNCO u-3o Nino^cccoooHO 2 ft M oooooooooo oooooooooo ra £,D 5 1 -HMMlO^t-t-Nt-tSI HMOMOTfCilHINH O ft rHOOOOcNOOOO ,£] VI VI odddddodoo VI a u 3 -ft a o 3 ft °3 OiCOOt--d(OTilCDOMO MOOOONOOOO . ooooooooco H 2 ft oooooooooo CTtOiO00t- "5 r^iOCNO •lOT-HiOOlt^ OOOO .(OHOOO dddd 'ddddd o » v^^- 05 « P4 -91 — ' O 4^ r3 ft w THiOtMLOOmOOOtM^II o rfNOOONNOlMH rt O vx OOOOOOOOOO 03 02 O A PS % ft Ed *>> § 3 in mmoHofico^t-© H i-IOOOOOOOOO "O ft °3 OOOOOOOOOO CD VI Oi rf rt <] M T3 PS O iH CO rH Cl 00 O • • d •2 © 03 OCOCDr-tOOOOCOCOCOCO doicsidc^odcOTiicsiod CNrHrHt-OOairHiHiH bO M a1 E- r bi tz: CDS ' £ A PHOSPHORUS METABOLISM 89 Vageler (1909) finds^ as do others, that seeds contain but a small amount of inorganic phosphorus ; also that the largest amount of phosphatid is to be found in a plant at the time of its fullest veg- etation and while the seeds are forming; when autumn comes and the seeds have ripened and leaves are withering, the easily soluble phosphorus is low; also that the phosphorus content of a normal, healthy plant is greater than that of an ill-nourished plant. Vageler also questions whether it may not be that the low values that ob- servers have found for the phosphorus content of the ether-alcohol extract of dried vegetable products is due to a partial breaking up of phosphatid during the drying process. ANALYSES OF FRESH AND DRIED VEGETABLE TISSUE FOR WATER AND PHOSPHATID PHOSPHORUS (Vageler, 1909)— Percent Fresh material Dried rr aterial Substance examined Water Phosphatid phosphorus Water Phosphatid phosphorus 94.93 89.74 90.165 85.55 14.74 17.26 14.94 0.1487 0.0376 0.2934 0.0780 0.0169 0.0496 0.0203 10.37 9.596 7.218 4.898 12.98 8.03 11.66 0.0731 0.0353 0.0578 0.584 Hay 0.0091 Lupine seeds. . . > 0.0398 0.0189 Winterstein and Smolenski (1909) find that the phosphatid ob- tained from wheat flour is a mixture of at least three phosphatids with cholesterin and other substances; and K. Smolenski (1909) isolated two phosphatids from wheat germ. LeClerc and Wahl (1909) report finding considerable loss of phosphoric acid and of other ash constituents in the change from barley to malt. At the same time there was an increase of alco- hol-ether soluble phosphorus, and probably a part of the other forms of organic phosphorus are transformed to phosphatids, while con- siderable, as others have found, becomes inorganic. Of the 130 barleys analyzed the general average of total P,0 5 is 1.04 percent, and of lecithin 0.52 percent in the water-free .substance; and of' 43 malts the general averages are 1.03 percent total phosphoric acid and 0.75 percent lecithin. The average percent of alterations during malting are computed to a loss of 12.7 percent of the total phosphorus and a gain of 44.3 percent of the lecithin. Fraps and Rather (1912) have investigated the composition of the ether extract of several kinds of hay, straw and other roughage. The phosphorus content of the ether extract may be considered roughly to indicate the phosphatid content of the products. From 90 OHIO EXPEEIMENT STATION: TECHNICAL BUL. 5 these figures it would appear that the phosphatid content of these species of roughage is low, as indeed all roughage is, and also that peanut hay is distinctly richer in ether-soluble phosphorus than the other kinds of hay studied. Below are figures from this study. NITROGEN AND PHOSPHORIC ACID OF THE ETHER EXTRACT OF HAYS AND FODDERS (Fraps and Rather, 1912) Feed Alfalfa hay Bermuda hay Buffalo grass hay Burr clover Corn shucks Cowpea hay Guam grass Johnson grass hay . . . Johnson grass hay . . . Kafir fodder Millet Oat hay Para grass hay Peanut hay Rice straw, Japan . . . . Rice straw, Honduras Sorghum Vetch hay Total ether extract Percent 1.26 1.55 1.28 2.72 0.61 3.15 1.78 1.29 1.38 1.99 1.53 2.12 0.86 8.17 1.47 1.24 1.47 1.59 Percent of extract Nitrogen 0.23 0.38 0.33 0.44 0.20 0.56 0.26 0.34 0.39 0.19 0.33 0.23 0.07 0.26 0.18 0.23 0.42 Phosphoric acid 0.06 0.08 0.06 0.10 0.19 0.13 0.14 0.09 0.15 0.10 0.09 0.05 0.25 0.18 0.02 0.06 0.09 0.06 See also Posternak, p. 81, Rising, p. 79, Suzuki, Yoshimura and Takaishi, p. 82, Vorbrodt, p. 79, and Bernardini, p. 79-80. DISTRIBUTION OF NUCLEIN PHOSPHORUS IN FOODS There are no well-established methods for the routine estimation of nuclein phosphorus in miscellaneous foods. Nuclein phosphorus has been estimated, however, by various more or less satisfactory methods, and for the results of some of these determin- ations we would cite the work of Heubner and Reeb (1908), Jebbink (1910) (60 articles of human diet) and Jordan, Hart and Patten (1906). In Klinkenberg's (1882) study of nucleins he gives us the fol- lowing as the content of nuclein phosphorus in seed cakes com- pared with meat residues and with yeast. NUCLEIN PHOSPHORUS OF SEED CAKES AND MEAT RESIDUES Klinkenberg (1882) Poppy seed cake Peanut cake Rape seed cake Cotton seed cake Palm cake Residue from meat extract preparation I. . Residue from meat extract preparation II. Yeast Nuclein P Percent 0.0707 0.0360 0.0676 0.0630 0.0344 0.0307 0.0530 0.198 PHOSPHORUS METABOLISM 91 About 3.5' percent of wheat germ is tritico-nucleic acid, which, was first studied by T. B. Osborne and Campbell (1900a), and later by T. B. Osborne and Karris (1902) and Levene and LaForge (1910). This acid is different in its makeup from the animal nucleic acids. Osborne and Campbell isolated the acid itself and also obtained combinations of it with leucosin, a globulin and two proteoses, as well as showing that some of it was left in combination with other insoluble substances. With regard to the combinations in the wheat embryo, however, it is said finally : "That the wheat embryo in fact contained the same nucleic acid compounds as we have obtained from the extracts is highly improbable. All that we can conclude is that the embryo contains the different pro- tein substances described, together with nucleic acid, and that these may unite to form a number of different compounds according to the conditions which prevail at any given time." Funatsu (1907) also estimated nuclein phosphorus in oil cakes. The following data are submitted : DETERMINATIONS OF THE PHOSPHORUS PARTITION IN PRESS CAKES (Funatsu, 1907) Total P2O5 Lecithin P 2 O s Nuclein P2O5 P0O5 soluble in. dilute HC1 Percent Percent of total Percent Percent of total Percent Percent of total 1.38 2.25 2.82 0.17 0.12 0.20 12.4 5.0 7.0 0.23 0.30 0.26 16.5 13.2 9.0 0.98 1.80 2.37 71.0 81.7 84.0 See also Rising p. 79, Rengniez p. 78 and Bernardini p. 79-80. PYROPHOSPHORIC ACID IN VEGETABLE SUBSTANCES The question of the presence of pyrophosphoric and metaphos- phoric acids in cottonseed meal, first raised by Hardin (1892), has called forth investigation, especially because Crawford (1910) thought that he had evidence that the poisonous effects sometimes observed as a result of feeding cottonseed meal to cattle and hogs is due to the presence in the meal of a salt, either organic or inor- ganic, of pyrophosphoric acid. The evidence of the presence of these acids is in qualitative reactions in extracts of cottonseed meal, which resemble those of the acids in question, and in toxic effects of the meal, or extracts from it, which are similar to those of the acids. More recently, however, both Rather (1912) and Anderson (1912c) have independently shown that all of the reactions in ques- tion are given by organic phosphorus compounds, salts of which 92 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 they have isolated from cottonseed meal. ' Both find the acids of the salts isolated to be inosite phosphoric acid (Rather 1913b, An- derson 1914a). It is not markedly toxic in small doses.Withers and Ray (1913) have shown that the only fraction of cottonseed meal which is toxic to rabbits in the amounts used in feeding is that which is not soluble in water or dissolved by digestion with pepsin and pancreatin (one day each), and that if this fraction contains any pyrophosphoric acid it is a non-toxic amount. It is to be con- cluded that probably there is no pyrophosphoric acid in this meal; also that the principal phosphorus compound present is organic, and of the nature of phytin. PROPRIETARY PREPARATIONS Phosphorus compounds as constituents of medicinal prepara- tions have occupied a prominence out of proportion to their useful- ness in this relation. While they are of undoubted value as simple nutrients, the idea of their possession of a stimulating function by reason of direct contribution to the nervous tissues, or by reason of other specific effects, has been very greatly overworked. It is true, however, that there is at hand evidence that certain of the organic compounds of phosphorus, lecithin especially, may, in certain path- ological conditions, have a curative value; but even in these cases there is need of further experimental work to differentiate between the effects of these compounds in the uncombined state, and the ef- fects of the related compounds as they exist in natural foods. We put no emphasis, therefore, on the usefulness of proprietary medic- inal preparations as affected by their phosphorus compounds, but merely enumerate those which have come to our attention, with brief notes as to their general character. Springer (1894, 1902) prepared a cereal decoction which he recommends to be used with the diet, especially in cases of retarded growth. It is prepared by boiling a mixture of wheat, barley, oats, rye, maize and bran in water and straining out the residues. Mouneyrat (1902a, 1902b) prepared what he called "Histo- genol," a combination of methyl arsenate of sodium and the nucleic acid from herring milt. Among casein preparations are "Plasmon" (E. Bloch, 1900 ; Poda and Prausnitz, 1900; Micko, 1900) and "Nutrose," impure sodium salts of casein, "Eucasein," an ammonium salt described by Salkow- ski (1896b), "Sanose," 80 percent casein and 20 percent albumose (Schreiber and Waldvogel, 1897), and "Sanatogen," sodium-casein glycerophosphate (Snowman, 1905; Gumpert, 1905). PHOSPHORUS METABOLISM 93 Special glycerophosphate and lecithin preparations are dis- cussed by Frey (1906), especially Baraba's "Nervinol." Laves (1900) describes "Roborat," a cereal preparation said to contain an abundance of lecithin, and Heim (1904) describes "Bio- son" as a protein-iron-lecithin compound containing- 1.27 percent lecithin. Schroder (1905, 1906) describes "Bioplastin," a lecithin prepar- ation, "Histogenol," and "Caudol," a dry malt extract containing phosphates. "Fersan," prepared from the blood of cattle, contains iron and phosphorus organically combined (Kornauth and Czadek, 1900 ; Kor- nauth, 1901). "Protylin" is a synthetic paranuclein containing about 2.7 per- cent phosphorus (Kornfeld, 1904; Laguesse, 1905; Fjodoroff, 1907). EFFECTS OF WATER ON THE COMPOSITION OF FORAGE PLANTS Kellner, Kohler and Barnstein (1894, 1895) give data which show that both hay and straw were low in the phosphoric acid con- tent of the ash after the dry summer of 1893; and the following figures from von Seelhorst, -Georgs and Fahrenholtz (1900) show a like effect in clover and grass, with moisture controlled by irrigation. PHOSPHORIC ACID CONTENT OF CLOVER AND HAY AS AFFECTED BY THE AMOUNT OF MOISTURE SUPPLIED (Von Seelhorst et al., 1900) Percent Phosphoric Acid Water Plot manured Plot not manured 0.456 0.536 0.540 0.725 0.597 0.666 0.428 0.553 0.523 0.660 0.644 0.650 Clover Much 91 Little Meadow fescue »» Forbes, Whittier and Collison (1910) showed in experiments with oats, grown with various amounts of water, that there is a re- lation between the phosphorus content of the plant and the water available during growth. The numerical data are below : 94 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 ANALYSES OF OAT PLANTS GROWN WITH DIFFERENT AMOUNTS OF WATER— Dry Matter Basis— Percent Lot Total water ap- plied to each pot C. C. Moisture in plant Percent Ash in dry sub- stance Percent Phosphorus in dry substance Percent Calcium in dry substance Percent Planted Oct. 7, 1908; cut Nov. 20, 19C 1 1675 86.11 16.77 .884 .526 2 2420 88.51 15.38 .898 .451 3 3165 88.20 15.12 .966 .461 4 3910 87.77 14.72 .979 .515 5 4655 88.06 13.96 .892 .453 Planted Dec. 13, 1909; cut Feb. 26, 1910 6 1800 83.78 15.17 .647 .440 7 3100 84.57 15.17 .666 .409 8 4400 86.79 15.65 .805 .498 9 5700 87.28 16.02 .787 .463 10 7000 87.75 16.20 .855 .463 Planted Dec. 13, 1909; cut Mar. 7, 1910 11 2400 79.68 15.37 .684 .461 12 4000 82.44 16.19 .768 .504 13 5600 85.94 16.20 .801 .495 14 7200 85.18 16.18 .818 .488 15 8800 84.76 15.98 .809 .480 Planted Apr. 4, 1910; cut May 11, 1910 16 900 88.97 18.47 1.12 .542 17 1425 89.59 18.68 1.16 .539 18 2000 89.95 18.54 1.20 .554 19 2625 90.50 18.20 1.22 .493 20 3300 90.73 18.05 1.27 .551 A similar relationship between water and phosphorus was shown by these same authors in forage plants grown in arid regions with and without irrigation. The data are below : EFFECTS OF IRRIGATION ON MINERAL CONSTITUENTS OF GRASSES Dry Matter Basis — Percent No. Ash Nitrogen Calcium Potas- sium Phos- phorus Source of sample 1 8.62 2.32 1.67 1.80 .175 Alfalfa; Fallon, Nev.; not irrigated for five years; water table 8 ft. below sur- face of field. 2 10.34 2.50 1.27 2.38 .220 Alfalfa; Fallon, Nev.; irrigated frequently. 3 16.38 .845 .342 .99 .099 Blue-joint (Elymus) ; Fallon, Nev.; sample from dry soil. 4 9.47 766 .250 1.06 .133 Blue-joint (EZy?nus); Fallon, Nev.; sample from land continuously wet by seepage from irrigation ditch. 5 9.78 .895 .410 .92 092 Indian bunch-grass, (JErt'ocoma cuspidatd) Fallon, Nev.; sample from dry soil; annual rain-fall less than three inches. 6 16.10 .734 .462 .67 .103 Indian bunch-grass, (Eri'ocoma cuspidata) Fallon, Nev. ; sample from land con- stantly wet by seepage from irrigation ditch. 7 12.11 1.89 1.00 2.14 .142 Bermuda grass. Tuma, Ariz. Not irrigated. 8 11.46 1.39 .709 1.55 .243 Bermuda grass. Yuma, Ariz. Irrigated frequently. PHOSPHOEUS METABOLISM 95 EFFECTS OF FERTILIZERS ON THE COMPOSITION OF FOODS Since some of the phosphorus compounds of plants are readily soluble in the water which falls upon them as rain, and since we do not know that the salts absorbed by the fertilized plants are the same and therefore have the same solubility as those absorbed by the unfertilized plants, we have no assurance that samples collected from plants which have been grown out-of-doors fairly represent the effects of the fertilizers on the phosphorus compounds of the plants. In the interpretation of numerical data on this matter, therefore, it would seem wise to make liberal allowance for experi- mental error. It seems altogether probable that the apparent in- consistencies in results which have been obtained are due in part to the factors above suggested. From the mass of material available we select but a small portion. From the work of Chavan (1908) we quote the following fig- ures showing direct effects of fertilizers on the composition of grass. INFLUENCE OF PHOSPHORUS AND POTASSIUM IN FERTILIZERS ON THE COMPOSITION OF GRASSES (Chavan, 1908) Percent — Dry Basis Fertilizer applied Protein P2O5 K 2 CaO Ash P 10.29 8.90 11.06 10.25 0.25 0.56 0.24 0.57 1.41 1.80 2.56 2.61 1.52 1.20 1.10 0.89 6.47 6.05 K 6.82 P, K 7.21 Parrozzani (1908) states that increasing the inorganic phos- phorus available to maize increases the lecithin and phytin phos- phorus, but does not affect nuclein phosphorus nor total nitrogen, though protein nitrogen increases slightly and amid-nitrogen de- creases correspondingly. Forbes, Whittier and Collison (1910) made analyses of 24 samples of Kentucky blue-grass taken in 1910 from a variety of types of soil in Ohio and Kentucky, the conditions of taking the samples and the age of the plants being as nearly alike as possible. The range of phosphorus content is from 0.164 to 0.403 percent P in the dry matter. Samples taken the two previous years were all within the same range. The inorganic phosphorus in 17 of the samples ranged from 0.064 to 0. 267 percent, and the organic, from 0.064 to 0.154 percent of the dry matter. Blue-grass from experi- ment plots differently fertilized gave results as in the table below. 96 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Potassium chloride appears to have increased the organic phospho- rus of the grass. The effects of the fertilizers are generally direct and consistent. ANALYSES OF BLUE-GRASS DIFFERENTLY FERTILIZED Forbes, et al. (1910) Percent of Dry Matter No. Ash Nitro- gen Cal- cium Potas- sium Phos- phorus Inor- ganic phos- phorus Organ- ic phos- phorus Source of Sample 1 2 3 4 ' 5 5.48 5.08 5.47 5.01 6.16 1.49 1.52 1.50 1.46 1.48 .230 .247 .222 .311 .250 1.83 1.58 1.86 1.63 1.82 .277 .235 .265 .236 .264 .176 .135 1.38 .130 .161 .101 .100 127 .106 .103 Wooster, O. Station lawn. Fertilized with sodium phosphate. Wooster, O. Station lawn. No fertilizer. Wooster, O. Station lawn. Fertilized with potassium chloride. Wooster, O. Station lawn. Fertilized with lime (calcium oxide). Fertilized with sodium phosphate, 'po- tassium chloride and lime (calcium oxide). Lewoniewska (1911) reports observations on the phosphorus content of oats (grain) as affected by the soil. Protein and lecithin phosphorus varied little, but both phytin and phosphates varied much. With variation in the phosphorus of the soil the relation of total phosphoric acid to nitrogen varied between 100:50 and 100 :32, while the relation of phosphorus compounds soluble in 1 per- cent acetic acid varied between 100:20 and 100:6. From the reports of Ames and his associates (J. W. Ames, 1910; J. W. Ames, Boltz and Stenius, 1912) with regard to the effects of fertilizers on the phosphorus content of wheat we quote several tables : EFFECT OF SEASON AND PLANT FOOD SUPPLIES ON THE PHOS- PHORUS AND NITROGEN CONTENT Phosphorus content of wheat grain and straw— Percent • 1904 1907 1908 Maximum variation due to season Grain Straw Grain Straw Grain Straw Grain Straw .395 .366 .029 .072 .105 -.033 .3839 .3053 .0786 .0563 .0548 .0015 .3545 .3234 .0311 .0364 .0469 -.0105 .0405 .0607 .0356 .0581 PHOSFHORUS METABOLISM 97 AVERAGE PERCENTAGE OF PHOSPHORUS IN THE WHEAT GRAIN AT WOOSTER Treatment Phosphorus in grain Percent .3143 .3080 .3690 .3332 .3615 AVERAGE PERCENTAGE OF PHOSPHORUS IN THE WHEAT GRAIN AT STRONGSVILLE Tear and cross dressing Plot treatment 1908 1909 Floats Lime Floats Lime .4059 .4168 .4449 .4572 .4287 .3270 .2837 .4122 .3643 .4173 .4646 .4182 .4689 .4671 .4736 .2947 .2775 Phosphorus, without nitrogen or potassium . . Phosphorus, potassium and nitrogen .4116 .3845 .4522 From the extended studies from which the above figures are taken Ames concludes in part as follows : "The composition of the wheat crop grown on the unfertilized plots of two soils, containing different amounts of phosphorus, po- tassium and nitrogen, is in accordance with the composition of these soils. "The proportion of phosphorus, potassium and nitrogen in the wheat plant is increased by the addition of these elements to the soil. "Although the extent of variation due to seasonal conditions is greater than that produced by changes in the composition of the soil, the variations due to soil treatment are relatively the same for the different seasons. "Phosphorus applied to soil, showing a deficiency of this ele- ment as measured by crop yields, increases the amount of phos- phorus in the grain. Associated with this increased accumulation of phosphorus there is an increased quantity of potassium and a decreased amount of nitrogen. 98 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 "The addition of lime to the soil increases the amount of phos- phorus assimilated by the wheat plant. With this increase in the phosphorus content there are the same variations in the nitrogen and potassium as are produced by the addition of phosphorus. "The composition of the wheat crop from plots on the same soil treated with five different carriers of phosphorus, namely: acid phosphate, bone meal, dissolved bone black, basic slag, and barn- yard manure, shows that the phosphorus content of the wheat plant has been increased to the greatest extent by manure. "The application of untreated rock phosphate to a soil well supplied with nitrogen and potassium, increases the phosphorus con- tent of the wheat plant to a marked extent. "The percentage of nitrogen in the wheat plant varies with the supply at its disposal, and is also influenced to a considerable extent by the supply of phosphorus." "A comparison of the composition of the wheat plant grown on the same soil, under different conditions of fertilization, gives a better indication of the available supply of nitrogen, phosphorus and potassium in the soil than can be obtained from the analysis of the soil itself." From a study by J. W. Ames and Boltz (1912) of the effects of fertilizers on the composition of alfalfa we quote the table on the fol- lowing page. From the study from which these figures were taken, Ames and Boltz conclude in part as follows : "The phosphorus supply of the soil, as increased by the addition of acid phosphate, is reflected by the phosphorus content of the crop, which follows the same order as the yields obtained. "When the fertilizer used contained both phosphorus and nitro- gen, the increase in the amount of phosphorus over that found in the crop from unfertilized soil is not as great as where phosphorus without nitrogen was applied. "From 43 to 63 percent of the total phosphorus present in the alfalfa plant is combined as organic phosphorus. There is in each instance, for every condition of fertilization, a greater proportion of the total phosphorus present as organic phosphorus in the second cutting, while the amount of total phosphorus is always larger in the first cutting. The water-soluble phosphorus, which includes prac- tically all the inorganic phosphorus and a considerable portion of that in organic combination, stands in the same order as the total phosphorus and is present in greater amounts in the crop of the first cutting. The quantities of pepsin-insoluble phosphorus which is combined with nitrogen as a highly insoluble compound, and would be of doubtful value from a nutrition standpoint, amounts to about 20 percent of the total phosphorus." PHOSPHORUS METABOLISM 99 PHOSPHORUS COMPOUNDS OF ALFALFA HAY AS AFFECTED BY COMPOSITION OF FERTILIZER (Ames and Boltz, 1912) Fertilizing elemen per acre ts Lime Forms of Phosphorus Total Organ- ic Inor- ganic Organ- ic Percenl of total Water- soluble Percent of total Phos- phorus Potas- sium Nit per r o- acre n in- soluble First cutting First cutting Lbs. Lbs.. Lbs Lbs. 2,500 Percent .2382 .2734 Percent .1180 .1267 Percent .1201 .1466 Percent 49.54 46.35 Percent 80.32 80.22 Percent .0560 .0559 Second cutting . . Second cutting. . Unfertilized . . 2,500 5,000 .1885 .2106 .1190 .1270 .0694 .0836 63.13 60.31 73.24 65.71 .0455 .0519 First cutting First cutting 45 45 2,500 5,000 .3415 .3128 .1494 .1513 .1921 .1615 43.75 48.37 83.50 86.48 .0947 .0656 Second cutting. . Second cutting . . 45 45 2,500 5,000 .2507 .2447 .1183 .1529 .1324 .0919 47.19 62.49 79.47 68.22 .0550 .0480 First cutting First cutting .... 45 45 25 25 2,500 5,000 .3116 .2913 .1473 .1295 .1643 .1618 47.28 44.46 85.48 83.92 .0619 .0598 Second cutting. . Second cutting. . 45 45 25 25 2,500 5,000 .2674 .2184 .1402 .1265 .1272 .0919 52.43 57.92 73.80 77.50 .0473 .0529 First cutting. . . . First cutting .... 45 45 25 25 3 3 L2 2,500 2 5,000 .2914 .2388 .1407 .1169 .1507 .1219 48.28 48.96 78.53 73.17 .0640 .0515 Second cutting. . Second cutting . . 45 45 25 25 ] 2 2,500 2 5,000 .2340 .2316 .1329 .1191 .1011 .1125 56.80 51.43 70.58 77.50 .0445 .0529 First cutting. . . . First cutting .... 45 45 2 2,500 2 5,000 .2603 .2758 .1158 .1243 .1445 .1515 44.49 45.07 85.87 .0689 .0508 Second cutting. . Second cutting. . 45 45 2 2,500 2 5,000 .2423 .2316 .1456 .1344 .0967 .0972 _ 60.10 58.03 69.40 79.46 .0506 .0522 *First cutting. . . First cutting. . . 24 24 56 56 7 7 2 2,500 2 5,000 .2472 .2698 .1096 .1248 .1376 .1450 44.34 46.26 .0662 .0522 *JSecond cutting. Second cutting. 24 24 56 56 7 7 2 2,500 2 5,000 .2184 .2232 .1174 .144C .1010 .0786 53.75 64.79 ::::: .0410 .0543 *From 16,000 pounds of stable manure. Vuaflart (1911, 1912) reports analyses from which it appears that the phosphoric acid variations in flours, from year to year, do not correspond with those of wheats of the same year. He found also that the flour was not as much enriched in phosphoric acid by increased superphosphate treatment as was the grain. PHOSPHORIC ACID OF WHEAT AND FLOUR Parts per 100 Parts of Nitrogen (Vuaflart, 1912) Wheats Flours Maximum Minimum Mean Maximum Minimum Mean 1908 1909 1910 1911 59.8 52.3 56.7 58.1 49.8 42.8 37.4 44.2 54.5 47.5 47.4 49.9 18.1 17.9 16.7 22.1 14.1 12.1 11.3 12.8 15.3 14.4 13.7 17.9 100 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Swanson (1913) shows that the mineral constituents of flour are much more prominently influenced by the presence or absence of bran than by variations in the ash content of the wheat, the phos- phorus content of the flour varying with its grade. Forbes, Beegle and Mensching (1913) also studied the effects of fertilizers on the phosphorus of the wheat grain. The wheats rep- resented by the following analyses are from the 1911 crop of the 5-year rotation series of the Department of Soils of the Ohio Agr. Exp. Station. MINERAL ELEMENTS OF WHEATS VARIOUSLY FERTILIZED— Parts per 100 of Dry Substance Description 1 Unfertilized 2 Phosphorus 3 Potassium 4 Unfertilized 5 Nitrogen 6 Nitrogen ; phosphorus 7 Unfertilized 8 Phosphorus; potassium 9 Nitrogen ; potassium . . 10 Unfertilized 11 Nitrogen; phosphorus; potassium 12 Nitrogen; phosphorus; potassium 13 Unfertilized 1 Inor- Ash Potas- So- Cal- Magne- Sul- Chlor- Phos- ganic dium cium sium phur ine phorus phos- phorus 1.87 .523 .122 .051 .153 .243 .089 .403 .0229 1.82 .561 .127 .055 .149 .224 .073 .406 .0219 1.73 .497 .135 .047 .152 .237 .112 .37 .0192 1.71 .442 .144 .049 .150 .256 .100 .357 .0181 1.73 .467 .147 .055 .154 .253 .096 .349 .0202 1.75 .467 .139 .057 .149 .243 .085 .358 .0186 1.80 .473 .113 .056 .152 .248 .102 .35.6 .0208 1.72 .443 .157 .044 .145 .213 .087 .372 .0179 1.61 .465 .136 .043 .139 .258 .100 .337 .0185 1.67 .459 .128 .052 .144 .245 .091 .340 .0179 1.78 .449 .154 .046 .150 .228 .080 .395 .0207 1.77 .456 .128 .048 .149 .228 .079 .388 .0176 1.82 .451 .168 .053 .149 .241 .090 .359 .0170 Organ- ic phos- phorus .380 .384 .351 .339 .329 .339 .335 .354 .318 .322 .374 .370 .342 According to Hartwell (1913) the phosphorus content of the purple-top, flat, or strap-leaf turnip (Brassica rapa, L.) is most markedly influenced by the amount of available phosphorus in the soil, so much so that he recommends its use as a means of determin- ing the relative amount of available phosphorus in different soils. Of the range of variation in the specific tests reported he says : — "The percentages of phosphorus in dry matter varied from .27 in turnip roots grown on an extremely deficient soil, to 1.82 in turnips from a soil so abundantly supplied with phosphorus that further manuring even depressed the yield." The inorganic phosphorus (Hartwell and Hammett, 1911) was found to be influenced much more than the total phosphorus. Previously (Hartwell and Quantz, 1910) it had been found that about 70 percent of the phosphorus of this turnip is extractable with 0.2 percent HC1, and mostly pre- cipitable from this extract by molybdate and magnesia mixtures; apparently then, it is present largely as inorganic phosphorus. PHOSPHOKUS METABOLISM 101 Summary. - All things considered, we regard the observed ef- fects of soils, fertilizers, and climate upon the phosphorus content of foods, in so far as they are derived from grains, to be without im- portant practical significance. These variations, as a rule, are not large ; the food products of greatest value contain but small propor- tions of the total phosphorus ; and neither the farmer nor anyone else is dependent for cereal foods upon the products of any particu- lar farm. With reference to roughage, however, the case is different; roughage can not be economically transported ; it is so bulky. Prac- tically speaking, the farmer is largely dependent on the roughage of his own farm, especially so for green forage. The variations in the phosphorus content of the roughage are large, and these varia- tions as affected by soil, fertilizer and climate constitute factors of first-class practical importance to the breeder of live stock, espec- ially as affecting the growth of bone. ADDITION OF PHOSPHATES TO SILAGE An unusual use of a phosphate is reported by Sani (1912). In the proportion of 300 gm. CaH 2 P0 4 per quintal of green clover this salt preserved the foodstuff in a silo with much less chemical change and loss than occurred in untreated silage. The temperature of the treated clover did not rise so high as in the untreated silage, and after removal from the silo it was found to be immune to mold. The flowers retained almost their natural color. In 11 months the treated fodder lost 13.7 percent of its weight and the untreated fod- der 18.68 percent. Forbes and Fritz (1914) studied the effects of the ensilage process upon the solubility of rock phosphate (floats). The phos- phate was added to the green corn at the time it was put into the silo. The table on the following page sets forth the results. The increase in total phosphorus during the ensilage process shows that there was a loss of 8.2 percent of dry substance from the untreated corn and 3.5 percent from the phosphated corn. The increase in water-soluble phosphorus in the untreated corn was not quite equal (7.4 percent) to the arithmetical increase due to the loss of dry matter. During the ensilage of this untreated corn there was a loss of citrate-soluble phosphorus, in the residue from water extraction, which signifies a process of reversion to less soluble forms. The one significant increase during the ensilage of the untreat- ed corn was in the inorganic phosphorus soluble in 0.2 percent HC1. 102 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 This was much more than enough to account for arithmetical in- crease from loss of dry substance. PHOSPHORUS IN SILAGE CORN WITH AND WITHOUT ADDED FLOATS, AND IN SILAGE MADE FROM THE SAME Percent Water-Free Basis Product Total P Water-soluble P Citrate-soluble P • Inorganic P soluble in 0.2% HC1 Total water- sol. -+- citrate- sol. P Untreated green sil- 0.200 0.203 0.218 0.207 0.231 0.228 0.214 0.224 0.374 0.371 0.367 0.371 0.405 0.387 0.361 0.384 0.151 0.150 0.147 0.149 0.159 0.161 0.161 0.160 0.135 0.138 0.135 0.136 0.158 0.157 0.155 0.157 0.020 0.021 0.021 0.021 0.009 0.007 0.009 0.008 0.055 0.064 0.065 0.061 0.061 0.061 0.059 0.060 0.086 0.082 0.082 0.083 0.112 0.110 0.112 0.111 0.156 0.181 0.178 0.172 0.234 0.243 0.235 0.237 Silage from untreat- o.iess G-reen silage corn plus floats (250:1) Silage from treated 0.197 oiii In the phosphated corn there was a loss of water-soluble phos- phorus simply through the addition of the floats, that is, the water- soluble phosphorus in the fresh corn was 0.149 percent and in the phosphated corn 0.136 percent, which probably signifies a combina- tion of water-soluble phosphorus of the corn with bases in the floats. This probably took place during the partial drying at 50° C, though perhaps to some extent during the subsequent storage of the sample for nearly a year before the analyses were made. The water-soluble phosphorus in the phosphated silage was not higher than in the untreated silage. The excess of water-soluble phosphorus in the phosphated silage over the amount in the unen- siled, phosphated corn was more than enough to account for the loss in dry matter, but was not as great in amount as in the silage from the untreated corn, again suggesting reversion. The citrate-soluble phosphorus in the treated fodder and in the silage from the same was naturally higher than in the untreated corn and silage, since a part of the phosphorus of the floats was citrate-soluble. There was no increase in citrate-soluble phospho- rus, however, in the residue from the water extraction, during the ensilage of the phosphated corn. PHOSPHORUS METABOLISM 103 The very considerable increase in inorganic phosphorus soluble in 0.2 percent HC1 during the ensilage of the phosphated corn gives us the most significant figure of the test. In the treated corn fod- der the inorganic phosphorus soluble in 0.2 percent HC1 was 46.4 percent of the total, while in the silage from the same was 61.7 per- cent of the total. It is also of interest that the phosphated silage contained more than twice as much inorganic phosphorus soluble in 0.2 percent HC1 as the treated silage. The total phosphorus of the floats was 12.666 percent, the water-soluble phosphorus 0.0129 percent and the phosphorus soluble in 0.2 percent HC1 8.721 percent, all on a water-free basis. Conclusion. The ensilage of corn will render soluble in 0.2 per- cent HC1 such an amount of the phosphorus of floats, added to corn, as to constitute a practical consideration in the feeding of livestock. REFERENCES TO EXTENSIVE PBESENTATIONS OF TOTAL PHOSPHORUS CONTENT OF FOODS Wolff (1871, 1880), Konig (1903, 1904, 1910), Albu and Neuberg (1906), Schaumann (1910), Sherman (1911). 104 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 PART III THE PHOSPHORUS OF ANIMAL BODIES AND PRODUCTS GENERAL STUDIES THE PHOSPHORUS OF GROWN MEN AND ANIMALS Gilbert and Posternak (1905), in their general discussion of phosphorus therapy from the standpoint of metabolism, sum up the total phosphorus of the human body as being about 30-40 gm. P 2 5 at birth, and 1600 gm. at middle life. This 1600 gm. is made up of about 1400 in the skeleton, 130 in the muscles, 12 in the brain and nerves, 10 in the liver, 6 in the lungs, about 4 in the blood, etc. Gilbert and Posternak generalize with regard to the phosphorus distribution in meats in the following way. Meat contains 0.467 percent P 2 5 , and of this 0.274 percent (or 60 percent of the total) is made up of organic compounds. Lecithin 0.060 Phosphoearnic acid 0.039 Soluble in water but not precipitated by lime 0.039 Nuclein 0.008 Organic phosphorus insoluble in water (other than nuclein) 0.128 The rest is soluble in water and is precipitable by lime, and hence is looked upon as probably inorganic phosphate. Beaunis has compiled ash analyses of various animal tissues and products, as in the following table, from which one may observe the relative prominence of phosphoric and other oxides in these parts. Approximately half of the ash of bone, muscle, brain and liver is phosphoric anhydrid. In other parts the proportion of phosphorus is much less. Since all of the other elements reported, except sulphur, constitute larger percentages of their oxides than does phosphorus of its oxide, this method of statement exaggerates the actual phosphorus content of these ashes, as compared with the other elements. PHOSPHORUS METABOLISM 105 H U P Q O ►J I— I *< o H ui j -a ■« ° a "B O o I— I- H «! I— I o (J xn P M O B h a • !~»00**IO .COOCOCOCO . .1003COCO ■ r~oo-s*iocM •CDTfli— IOCS MCOCMC COCO .TTICOt^O c— co • TtHF-oo*— i dto 'Hoood i-ICM CU.-I • oco •oicN CO .CO^lO-rH CM 'COCOr-ICO CD >-H oo -wmr^coia t)! •dco'ddd ■ OM •odo ^3 d J •r 1 TO OS to r— 10 CO oo co co . .-^fcnf-CM co oo m co io io ^n t^ Oi CO CM CM O O ■OOHTf •OOCMO oo .coinoor~co . .nooo 00 •050SCMCMCM CM -CMCMCMOO HTflCM •i-HCnJth'o t-a .inr— COCMI>- OO «i— (Oit^r- iCO 00 -TjIr-I^Hr • CMCOt-h •Oi-Ir-i • COOOOOiCO-*! .O-* • COCO-^I-^CMIO -^HIO •ajror-^o'r-^o • t-^cM ■ 1OC002 05CM • cdr-Hi— It-HCO • i— IMHO^OO .00CM • mcMcocMc^m • ■— ios ■^*incdocM04 oo' •* .OiCMCOCO t— ■CO'"*!— CM ^ 'O^OH OS -locscnio »0 'CO-^CTlTti •rHCO -lO •a 3 'E-S o u s a .3 « ° C C/3 3 C 106 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 From Wolff's "Farm Foods," Eng. ed., we quote the following table, from the work of Lawes and Gilbert. The two most im- portant factors determining the percentage of phosphorus in animals is their relative development of skeleton, the tissue richest in phosphorus, and of fat, the tissue poorest in phosphorus. Of the three common meat animals, cattle contain the highest percentage of phosphorus, and swine the lowest percentage of the same. MINERAL SUBSTANCES OF THE BODIES OF OXEN, SHEEP, AND PIGS Lawes and Gilbert — Percent, Live Weight . Ox Fat calf Sheep Pat lamb Pig Half- fat Pat Thin: Half- fat Pat Very- fat Thin Fat Phosphoric Acid.. . . 1.839 2.111 0.085 0.205 0.146 0.040 0.038 0.087 0.059 0.013 1.551 1.792 0.061 0.176 0.126 0.024 0.033 0.071 0.055 0.006 1.535 1.646 0.079 0.206 0.148 0.021 0.041 0.047 0.063 0.005 1.118 1.321 0.056 0.173 0.120 0.037 0.052 0.037 0.072 0.021 1.199 1.350 0.052 0.168 0.104 0.042 0.035 0.053 0.051 0.020 1.040 1.184 0.048 0.148 0.097 0.034 0.031 0.041 0.044 0.026 1.108 1.240 0.055 0.158 0.129 0.030 0.028 0.049 0.066 0.016 1.126 1.281 0.052 0.166 0.103 0.026 0.039 0.043 0.053 0.012 1.066 1.079 0.053 0.196 0.110 0.022 0.053 0.021 0.056 0.005 0.654 0.636 0.032 0.138 0.073 0.013 Sulphuric Acid.. .. Carbonic Acid. - • • 0.029 0.021 0.043 0.003 1 Francis and Trowbridge (1910) have reported phosphorus de- terminations on the different parts of the carcass of eight beef ani- mals of different ages and in various conditions. Their figures on four animals representing extremes of age or condition are quoted on the following page. Moisture, fat, ash and total phosphorus were determined. The emaciated and the very fat steers contained, in most of the parts, less phosphorus in the dry, fat-free tissue than did the fat calf and the aged cow, the two former perhaps being in more abnormal states of nutrition. Nerking (1908b) has estimated lecithin in many animal tissues. For data see next page. The high content of nerve tissue in lecithin is notable, the glandular organs contain considerable amounts, and there is evidence that under certain conditions the bone marrow may serve as a storage depot for this important nutrient. PHOSPHORUS METABOLISM 107 PHOSPHORUS IN THE DIFFERENT PARTS OF THE CARCASS OF BEEF ANIMALS (Francis and Trowbridge, 1910) Percent Steer; 1 yr. 10 mos.; emaciated; submaintenance for 11 mos. 1 Steer; 11 mos.; fat Steer; 4 yr. 6 mos. ; very fat Cow; 7 yr. 6 mos.; fat Fresh Moisture and fat free Fresh Moisture and fat free Fresh Moisture and fat free Fresh Moisture and fat free 0.019 0.039 0.137 0J59 0.323 0.183 0.333 0.067 0.109 0.163 0.184 0.173 0.179 0.142 0.168 0.170 0.174 0.116 0.101 0.801 1.037 2.305 1.356 1.299 0.479 0.815 0.762 0.887 0.882 0.844 0.637 0.804 0.824 0.799 0.055 0.072 lo.'isi 0.193 0.347 0.020 0.034 0.164 0.191 0.143 0.158 0.125 0.149 0.163 0.167 0.266 0.225 i.'isi -j 1.204 1.339 1.470 1.072 0.818 0.922 0.968 0.883 0.798 0.821 0.964 0.869 0.021 0.048 0.076 0.117 0.425 0.175 0.307 0.016 0.012 0.142 0.146 0.093 0.098 0.064 0.082 0.123 0.102 0.149 1.756 0.887 2.453 0.953 1.179 1.045 0.710 0.759 0.842 0.892 0.872 0.732 0.804 0.799 0.030 0.072 0.103 0.164 0.354 0.140 0.339 0.016 0.020 0.176 0.182 0.126 0.141 0.125 0.149 0.158 0.153 0.153 0.222 1 772 1.071 2.565 Digestive and excretory 1.115 1.323 Offal fat Shin, shank, head and tail . . 0.899 983 1 176 1.168 1.018 Rib 1.112 923 Composite of leans and fats • • 1.085 ( x ) "The condition of the skeleton of this steer was remarkable; the marrow having practically disappeared, being replaced with a watery malodorous liquid with none of the properties of normal marrow and totally lacking in greasy or fatty appearances." LECITHIN DETERMINATIONS IN ANIMAL TISSUES— (Nerking, 1908) Percent, Dry Basis Organs, etc IV Rabbit V Rabbit Mean of IV and V Rabbit VI Cat VII Hedgehog Lungs Heart Brain Spinal cord . . . Kidneys Spleen Eyes. Stomach Liver Intestine Gall Blood.. Muscle Bones Pelt Bone marrow . Testes Adrenals Whole animal 5.99 5.618 12.23 33.62 4.30 3.27 3.05 2.67 4.22 0.48 Trace 0.914 2.68 0.283 0.568 4.531 5.93 6.108 12.593 36.75 5 73 5.20 1.32 3.94 3.42 0.777 0.'813 2.49 0.260 0.392 0.89 1 3.39 5.54 5.96 5.863 12.41 35.19 5.02 4.24 2.19 3.31 3.82 0.629 6! 864 2.59 0.272 0.480 2.711 6.10 4.55 13.74 26.20 6.26 0.39 4.99 5.36 4.28 10.49 22.31 18.19 8.55 6.56 6\37 5.23 1.508 Trace 3.71 0.871 0.585 41.7 l 11.27 92.00 0.382 2 0.4478 2 0.82142 Other rabbits 1 n m 1 These determinations were made on fresh substance. ■ Percent of live weight. Lecithin, percent of live weight 0.3626 0.4025 0.7995 108 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 THE PHOSPHORUS OF FETUSES AND NEW-BORN YOUNG Analyses of the entire bodies of animals dying at birth, or very soon after, have been discussed chiefly in connection with Bunge's suggestion that the composition of the ash of new-born animals gen- erally corresponds with that of the ash of milk, perhaps peculiarly with that of the milk of the same species (Bunge, 1874). Our table shows the ash analyses of Bunge (1874, 1886, 1889) and of Abderhalden (1899c) on the young of several animals, and those of other investigators on new-born human infants. (Giacosa, 1895; deLange, 1897, 1900; Michel, 1899, 1900; Hugounenq, 1899b, 1900; Soldner, 1902.) In this direction the work of Soldner, in conjunction with W. Camerer, Jun., is more extensive than that of others, and their latest results expressed as total grams in the body may be of interest, as given at the foot of the table. (Our figures here are taken from Camerer and Soldner, 1903.) PHOSPHORUS METABOLISM 109 fc pel O CO P5 PS £ o W W H SP fo -sd O a nrt P P o o PS PQ > W JH PS PU h- 1 H Q fc W W H PS W o w Pu H W fe PS O GQ co <1 fc CO O M 1— 1 H ^ S k; ^ S -COOiCO00CO i-Ht— IrHiHi— irH»-4e4tHCO . . .K3MH • • -mm ..... ■ ' -o'o • • • iococnj^^hooouocmco Ni-HQOlOOTflr-HOOOt- CN1 >-i CM CM i-I H cm 00 ■* COt^t--0-# HO .IOCOCOCOOCOOO com 'Hnoohho o'o •oo'ooooo COt-HOIOOOOCM'— <-HCM COTf*TJ1tncOCOTJ-I 'oir-IcNCNiojoioi IOI^CTiC5CMTt(^*^<0303 01C»HOONf-t^»01 00 00 „CJS OS OO 00 00 00 00 l>.COCMCOi— f-^HCMT^lOCO r^coi^-cMcoosi— icoco-^i »o CO id "* cd~* t^ t^ 00 OS IO»— <~H00CO^*COT*ICMOi COCOOCMCOOiCMOOTjft^. t^t^00*U0I>^T-HO03C3i— H COCOCOCOCO-'SI'^COCO^ OiOi 05COCO-#-*CM-* COCO CO 00 CM CM CO F- CM ooooooo Ot^Oi-ICOOiCMr-IO»Tt( THCOCMmTfl^-llOCOOO-Vl T-iOOll CMQ3O0C00CM OiOOCO^CNOO r-J 00 i-i © 00 HO TK 1T5 CT> CM TflCOTflTKCOCOCOCOCMCO COO .- ood ■oo'r-irsododocd Or« .OCOtSf. 105CM05 CM CO CMOOOf— 1TJHTJ1© co' C-i o' © 00 i-i oo W03020CM*J d d d d d rt ri ri 7i rt aaaaag 3 3 3 3 3rt"5 g'o'a WWWMM«Ofiqci ho ho-9 a * * O fr) «H 5 ft 2 rtf TH I 8 .a rt -^ w 110 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Michel (1899, 1900) gives two reports of the same analyses of human embryos and fetuses of different stages, and of a new-born infant. We quote the table which gives these analyses on the basis of 100 gm. of fresh substance. These data reflect the rapid deposit of phosphorus which occurs late in fetal life. COMPOSITION OF HUMAN FETUSES AND A NEW-BORN INFANT Michel (1899) Body weight Grams Percents of fresh substance fetus Months Water Nitrogen Protein (calcu- lated) Ash CaO MgO P2O5 CI Fat 2.5 3.4 5 5 6 7 Pull term 17.80 125.80 445 448 672 1024 3335 93.82 89.95 87.80 86.95 85.02 84.73 69.16 0.685 1.100 1.322 1.390 1.644 1.563 2.179 4.39 7.05 8.46 8.90 10.50 10.04 13.96 L729 1.948 2.485 2.512 2.487 3.37.3 6!465 0.597 0.790 0.850 0.804 1.393 0.027 0.0258 0.0314 0.0328 0.0307 0.0405 0.489 0.643 0.842 0.833 0.788 1.282 6.'240 0.289 0.193 6 '.397 0.888 0.798 1.210 1.823 11.750 Brubacher (1890), in his study of rachitis, gives the following mineral determinations on the entire bodies of two undernourished human fetuses. BRUBACHER'S ANALYSES OF HUMAN FETUSES Percents of Fresh Substance Age of fetus Weeks Water Fat Ash CaO MgO P2O5 Si0 2 Fe 2 3 28 36 80.75 75.28 3.95 8.42 3.00 3.12 1.04 1.13 0.04 0.04 1.09 1.15 0.01 0.01 0.01 0.01 Siwertzow (1909) presents the following data on the lecithin content of the organs of human fetuses and children. He concludes that of the brain, liver, heart and muscles, the brain contains more lecithin than all these other organs put together, the liver coming next, and then the heart, the muscles having least of all. The lec- ithin content of each of the organs gradually increases in the fetus to a maximum at the time of birth. During the first months of extrauterine life the lecithin remains constant or even decreases, and then rises again, reaching a new, permanent maximum at the second year. The facts established seem to the author to show that in the new-born child there is a store of lecithin, as has been shown for iron, and that this is gradually used up during the first four months. PHOSPHORUS METABOLISM 111 LECITHIN CONTENT OF ORGANS OF THE HUMAN FETUS AND OF YOUNG CHILDREN (Siwertzow, 1903) Percent, Dry Substance Age Number ex- amined Brain Liver Heart Muscle 6 months 2 9.39 0.25 0.00 1.30 8 1 11.95 2.82 2.22 1.32 9 2 12.86 3.80 3.55 1.38 10 " 4 16.21 4.90 4.51 2.18 Child . 1 month 4 15.66 3.05 2.40 1.54 2 months 2 15.35 3.20 2.39 1.44 3 2 14.85 2.74 2.44 1.65 4 2 16.40 3.37 2.13 1.58 6 " 1 17.67 4.83 2.59 1.83 10 1 21.59 5.77 3.93 2.40 2 years 1 22.78 8.15 7.52 3.86 3 yrs. 5 mos. 1 23.15 7.58 7.23 4.34 THE PHOSPHORUS OF INFANTS' BODIES AFTER DISEASE Steinitz (1904) made analyses of the bodies of infants that had suffered serious nutritional disturbances and found them not to dif- fer much, save in fat, from the new-born infants reported by Cam- erer and Soldner. We give the ash analyses, together with those of Steinitz and Weigert (1904, 1905) on a one-year-old child that had died of tuberculosis with rickets. ASH ANALYSIS OF BODIES OF INFANTS— Percent of Ash Case I II III IV K 2 Na 2 CaO MgO Fe 2 3 P2O5 CI 8.3 8.7 37.8 1.1 1.0 39.8 6.5 7.2 7.7 38.1 1.2 1.2 38.7 6.1 6.85 7.8 37.8 1.32 0.94 36.3 5.7 6.88 9.03 35.85 0.99 0.425 34.7 7.38 Cause of death Nutritional disturbance Tuberculosis Cases I, II and III, Steinitz (1904) Case IV, Steinitz and Weigert (1904) THE PHOSPHORUS OF BONES, TEETH, MARROW AND CARTILAGE EEFEEENCES TO OLD WOEK Probably the most extensive of the early work on this subject was that of von Bibra (1844) who published a book of 430 pages on chemical studies made by himself and several others on the bones and teeth of men and other vertebrates. Other more or less com- plete analyses are those of Frerichs (1842), Nasse (1842), Stark (1845), Fremy (1855), von Recklinghausen (1858), Edwards (1860), Zalesky (1866), Papillon (1870, 1870-71, 1873), Volkmann (1873), Mallet (1874), Hofmeister (1873), and Siedamgrotsky and Hofmeister (1879). From Fremy's work we quote the table on the following page. Schrodt (1876) presents analyses of the individual bones of a normal, healthy dog. The inorganic matter of the water-free bones varied mostly between 60 and 70 percent, but this figure for the sternum was as low as 49.57 percent, and in the third neck vertebra as high as 72.98 percent. In the ash the C0 2 varied between 112 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 4.61 and 9.06 percent, the CaO between 50.40 and 53.28 percent, the MgO between 0.78 and 1.08 percent and the P 2 5 between 39.08 and 40.47 percent. The ratio of calcium carbonate to calcium phos- phate is said to be, in compact bones, 1:6.4, and in spongy bones 1:5.7. It is noted that the ash constituents, with the exception of carbonic acid, occur in almost the same quantities in relation to one another in all the bones. ANALYSES OF BONE OF VARIOUS ANIMALS (Fremy, 1855)— Percent Animal Bone Woman Man Egyptian iaummi Dog Lion, young . . . Sea-lion Rabbit Elephant, India Rhinoceros, Java Calf, 5 mos. . . Cow, old Ox Bull Lamb Sheep Goat Sperm whale . . Eagle Owl . Fowl Turkey Heron Teal Sea turtle Crocodile Carp Eel Ray Cod .'.[\\\'.'.\'. Skull Femur Femur Humerus Femur Ash Carapace Bony skin Cartilage Spines 64.1 64.2 65.0 62.1 64.7 63.1 66.3 66.8 65.3 69.1 71.3 70.4 69.3 67.7 70.0 68.0 62.9 70.5 71.3 68.2 67.7 70.6 73.5 64.0 64.6 61.4 57.0 30.0 65.3 61.3 Calcium phos- phate 57.8 56.9 58.7 59.0 60.0 53.9 58.7 62.2 60.0 61.2 62.5 61.4 59.8 60.7 62.9 58.3 51.9 60.6 61.6 64.4 63.8 62.5 68.4 56.0 58.3 58.1 56.1 27.7 64.4 55.1 Magnesium phosphate 1.7 1.3 1.7 1.2 1.5 1.5 1.1 1.2 2.3 1.2 2.7 1.7 1.5 1.5 1.3 1.2 0.5 1.7 1.5 1.1 1.2 1.5 1.3 1.2 trace 1.1 trace 1.3 Calcium car- bonate 10.9 10.2 5.9 6.1 6.3 9.3 6.3 5.6 5.2 8.4 7.9 8.6 8.4 8.1 7.7 8.4 10.6 8.4 8.8 5.6 5.6 10.2 5.6 10.7 9.7 4.7 2.2 4.3 1.3 • 7.0 COMPOSITION OF THE BONES OF A GANDER (Hiller, 1885) Bone Inorganic matter of fat- and water-free bones Percent Percent of ash Carbonic acid Lime Magnesia Phos- phoric acid 58.48 56.64 54.99 47.94 62.67 60.61 56.72 57.11 59.45 58.91 67.16 65.91 61.75 61.13 62.50 61.54 55.71 4.67 5.30 4.31 4.03 5.20 5.36 4.00 5.67 4.34 4.48 4.93 5.49 4.48 4.92 5.47 5.25 5.35 51.83 51.61 52.16 51.52 51.57 52.04 52.42 51.86 52.88 52.18 52.50 52.11 52.39 51.30 51.79 51.66 51.34 0.68 0.77 0.83 0.92 0.85 0.67 0.75 0.80 0.67 0.81 0.81 0.84 0.84 0.76 0.86 0.85 0.75 38.90 2 38.83 3 39.08 4 38.86 5 39.54 g 38.63 7 38.11 8 Ribs 38.19 9 39 45 10 38.48 11 38.75 12 13 Right and left radius and ulna 38.57 39.28 14 38.97 15 16 38.50 38.99 17 39.53 Hiller (1885) made a similar study (though with less separa- tion of the individual bones) of the bones of a two-and-a-half -year- old gander in normal, average condition. The conclusions were the PHOSPHORUS METABOLISM 113 same as those of Schrodt, except that the bone showing the highest content of inorganic matter was the humerus, and the ratio of cal- cium carbonate to calcium phosphate varied, independently of the compactness or sponginess of the substance, between 1 :6 and 1 :8. ANALYSIS OF MINERAL MATTER AS OBTAINED BY REMOVAL OF ORGANIC MATTER " WITH GLYCERIN AND POTASSIUM HYDRATE Gabriel (1894) preferred obtaining the mineral matter of bones and teeth by removing the organic matter by heating the powdered bone with alkaline glycerin at 200°C. The substance obtained in this way retains water and carbonic acid which are driven off at the high temperature of ashing. Gabriel says that the mineral matter of bones contains water in two forms, water of crystallization, and water of constitution, the latter not being driven off by heat alone but only by heating with silicic acid. COMPARISON OF THE MINERAL MATTER OF THE TEETH OF CATTLE AS PREPARED BY ASHING AND BY THE GLYCERIN METHOD Gabriel (1894)— Percent of Ash Treatment CaO Mg-O K2O Na20 H20of crystal- ization P2O5 CO2 CI 1 Organic matter removed by 50.68 50.76 53.67 50.59 1.52 1.52 1.56 1.47 0.23 0.20 0.25 0.24 0.97 1.16 1.13 1.07 2.27 2.21 2.21 38.78 38.88 41.55 39.13 4.16 4.09 0.59 4.09 0.05 2 Organic matter removed by fr.05 3 4 Ashed in the usual way (3) computed to H2O and CO2 0.10 0.09 ANALYSES OF "GLYCERIN-ASHES" OF BONES OF DIFFERENT SPECIES (Gabriel, 1894)— Percent of Ash H 2 of Animal Bone CaO MgO K 2 Na 2 crystal- lisation P a 6 co 2 CI Teeth 50.76 1.52 0.20 1.16 2.21 38.88 4.09 0.05 Cattle Tooth, enamel 51.98 0.53 0.20 1.10 1.80 39.70 3.23 0.21 Cattle Tooth, dentine 50.36 1.83 0.14 0.80 2.90 38.60 3.97 0.03 Humerus 51.31 0.77 0.32 1.04 2.46 36.65 5.86 0.01 Cattle Femur 51.28 1.05 0.18 1.09 2.33 37.46 5.06 0.04 All bones 51.01 1.27 0.19 1.11 3.05 38.19 4.11 0.06 INFLUENCE OF AGE ON THE COMPOSITION OF BONES The changes in the appearance of bones as an effect of age are so characteristic that anyone who is acquainted with beef recognizes at once the soft vascular bones of young cattle and the white, flinty bones of old animals. Cattle from regions where they are not mar- keted at an early age are sometimes distinguished from cattle raised in more intensive farming regions by referring to the former as "hard-boned" cattle. 114 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Such data as we have on the changes in composition are all old, but probably they give a fair interpretation of the facts. They are from Weiske and from Wildt (Weiske, 1872a, 1889; Wildt, 1872). With both the birds and the rabbits which Weiske exam- ined the water content of the bones decreased as the animals in- creased in age, and in the dry bones the mineral content increased and the organic content decreased. The fat content of the water- free bones increased in the rabbit, while in the birds it is said to be larger in the young than in the old fowl. With regard to the com- position of the ash, we have computed averages from Weiske's rab- bit study. LIME, MAGNESIA, AND PHOSPHORIC ACID CONTENT OF THE ASH OF RABBIT BONES AT DIFFERENT AGES (Weiske, 1872) Percent Age CaO MgO P 2 6 51.24 53.45 53.68 54.06 1.60 1.22 1.23 1.07 42.39 42.47 41.53 Calcium increased, magnesium decreased and phosphorus re- mained nearly constant in the ash. The same relations held in Weiske's data on bird bones. Weiske (1889) analyzed the bones of fowls of various ages, all receiving grain food. These results show with increasing age an increase of all the mineral constituents in the dry, fat-free bone. In the ash, there were increased percentages of CaO and C0 2 and de- crease in MgO. COMPOSITION OF THE BONES OF FOWLS AS AFFECTED BY AGE Weiske (1889) Percent, Dry, Fat-free Bone Age Mineral matter P2O5 CaO MgO Just hatched 25.29 32.77 37.40 42.59 44.92 42.27 46.57 ' 40.12 48.71 57.14 54.20 57.05 9.31 13.45 14.84 17.19 17.79 16.43 17.48 14.70 18.25 22.25 19.98 21.73 10.57 15.35 17.68 20.42 21.97 20.68 23.32 19.99 24.47 28.98 27.86 28.99 1 week 0.36 2 weeks 0.47 4 weeks 0.54 0.63 0.62 0.49 24 weeks 0.49 0.33 0.40 0.61 0.43 0.58 ■ MM Wildt (1872) reported analyses of the ash of the bones of rab- bits of various ages. The following table is from this work. The inorganic substance increased with age as also did the calcium and the carbonic acid, while magnesium and phosphorus diminished. PHOSPHORUS METABOLISM 115 ANALYSES OF THE ASH OF THE BONES OF RABBITS OF DIFFERENT AGES (Wildt, 1872) Inorganic sub- stance x Percent Percent of ash Inorganic sub- Age of rabbits Carbonic acid CaO MgO Phosphor- ic acid bone Percent 8 months 53.39 50.82 55.18 58.94 65.63 67.68 68.72 70.26 71.77 74.24 72.90 73.65 3.65 3.84 3.99 4.00 4.52 4.69 4.92 4.94 5.54 5.71 5.81 5.66 52.17 52.16 52.10 51.91 52.10 52.49 52.60 52.64 52.78 52.61 52.76 52.84 1.38 1.36 1.26 1.22 1.09 1.01 1.02 1.05 0.93 0.91 0.93 0.83 42.05 42.13 42.19 42.20 41.64 41.03 40.80 40.80 40.05 40.04 39.78 39.80 15.56 17.23 18.62 23.39 30.13 30.90 37.17 41.80 39.22 44.39 41.68 45.00 (*) Apparently reckoned on residue from water-extraction of fat-free bone. See also Graff enberger (1891). COMPOSITION OF THE TEETH Hoppe-Seyler (1862) submits a table of analyses (p. 117) of the enamel of the teeth of several species of animals. General sim- ilarity is evident. Kiihns (1895) compares human teeth at different ages. The more notable differences are in the calcium and magnesium of the enamel, the former increasing and the latter decreasing with age, and the increase with age of the phosphorus of the dentine. (P. 116.) Bertz (1899) says that in teeth the bases were found to exceed the acids ; that, contrary to the findings of others, no alkalis or car- bonates were found ; that there is no chlorine in enamel ; that it is doubtful if there is a trace of chlorine in true dentine; and that dentine is found to contain twice as much magnesia and half as much fluorine as enamel. (See table on next page.) St. Bondzynski and Gonka (1907) found that the organic sub- stance of calf teeth, entirely freed from mineral matter, contained 0.37 percent phosphorus. The ground-substance of bone (femur of horse) similarly treated showed no organically combined phos- phorus. For other analyses of teeth see Gabriel, p. 113, Gassmann (1908) and C. Cohn (1889), under Caries, and also Weiske (1891b, 1892, 1894, 1895c, 1896, 1897), Graffenberger (1891), Carnot (1892) and Aeby (1873c). 116 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 o CO Q O (— i tt q in rH °° «s| w i_i OB fifl > «H o "3 o o r-i <& Ph s H "*. '. d ; o 0Q 00 t- • CO • d ] o P4 rjl m . N • d * CO o oil oo o • ri '. o C3 o © CO • r-l . oo' • CO * '3 03 U) U o lO • r-l • 0> '. Total dry substance Percent r-l • r-l • 00 . d * o •a i "o P* Ph OIH CO t- ot- rid OS r-l CJCO do cam coio t-Ol do (MtJI lOTtl C0 5O Olt- rtoi eo'cd Wd PHOSPHORUS METABOLISM 117 COMPOSITION OF THE ENAMEL OF TEETH Hoppe-Seyler (1862) Percent of Inorganic Matter Animal Man Man Man Hog Hog Rhinoceros . Elephant . . , Mastodon . . . Paleotherium Horse Dog Age New-born New-born New-born Young Adult Fossil Fossil Fossil Fossil P 2 5 CaO MgO CI 30.53 41.42 0.72 Trace 35.69 44.91 0.79 0.15 36.61 45.03 0.50 t 39.06 48.67 0.74 0.30 40.59 51.57 0.91 0.40 40.57 51.23 0.75 0.42 38.85 49.71 0.92 0.28 39.62 52.82 0.30 0.38 40.20 52.39 0.59 0.37 40.22 51.10 0.56 0.43 43.63 51.46 2.27 0.51 Fe 2 3 0.34 Trace 0.48 0.47 1.30 0.29 0.17 0.40 Trace PHOSPHORUS COMPOUNDS OF BONE The Inorganic Phosphate. From all of these analyses it is quite evident that lime and phosphoric acid are the principal constituents of the mineral part of bones, the two together forming fully 90 percent of the ash. Carbonic acid and magnesia are also certainly present, and there is no doubt that these are united as some form of calcium phosphate, magnesium phosphate and calcium carbonate. There are also small quantities of sodium, potassium, chlorine and fluorine. Papillon (1870, 1870-71, 1873) claims to have introduced strontium, magnesium and aluminum, and H. Stoelzner (1908) strontium, into bones in place of a small part of the calcium. Attempts have been made to assign definite formulae to the calcium phosphate and to a combination of phosphate and carbonate of calcium ; but, so far as we know, none of these have been on any other basis than the percentage composition. Other chemical and physico-chemical evidence .should be brought out before confidence is placed in any formula claiming to represent chemical union of the elements in the usual sense. The articles in which we have noted that attention was given to this subject are: Berzelius, 1816; von Gohren, 1865; Aeby, 1872a, 1872b, 1873a, 1874a, 1874b; Wibel, 1874; Schrodt, 1876; Levy, Moritz, 1894; Gabriel, 1894 ; Gassmann, 1910, 1913. Gabriel's conclusion, after his studies on the mineral constitu- ents of bones and teeth, the organic matter being removed by the glycerin method, which avoids high temperatures, he expresses in this way: "The composition and properties of bone and tooth-ash 118 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 have their simplest expression in the formula (Ca 3 (P0 4 ) 2 -(-Ca 5 HP 3 13 +Aq), in which 2 — 3 percent of the lime is replaced by magnesia, potash and soda, and 4 — 6 percent of the phosphoric acid by car- bonic acid, chlorine and fluorine." Gassmann favors the formula OP0 3 Ca )Ca C0 3 , \ OPO a Ca (which he attributes to A. Werner) on account of having- found the ratio of the three constituents constant in this proportion in normal and in rachitic bones, and from having isolated from an ignited mix- ture of ground teeth and calcium chloride a compound corresponding in its chemical analysis to the analogous chloride. To account for the increased proportion of Mg in rachitic bones Gassmann suggests that this does not belong to a salt-forming body. Lecithin and Phosphorus Determinations on Bone Marrow. A part of the fat of bone marrow is phosphorized, and on the older un- derstanding, which called all the phosphatids lecithin, and computed the amount of lecithin from the percent of phosphorus found in the alcohol-ether extract, lecithin determinations on bone marrow are reported. Otolsky (1906) and Bernazky (1908) have studied in par- ticular the composition of the lecithin of bone marrow. From these articles we have seen only abstracts. One kilogram of bone marrow yielded by Otolsky's process 1.3-1.5 gm. lecithin, containing 3.25 per- cent phosphorus, and yielding choline, glycerophosphoric acid and fatty acids on cleavage. Bernazky considered the distribution of phosphorus in the organs of the horse, and made a special study of the lecithin of bone marrow. He says that the red bone marrow of the horse contains twice as much lecithin as the white bone marrow, and that no dif- ferences were observed in either the amount or the composition of lecithin in the bone marrow of horses immunized against diphtheria, scarlet fever and the yellow staphylococcus. Hutchison and MacLeod (1901-2) studied the red marrow from the ribs of the horse. They report that the fat, lecithin and choles- terin together constituted 17.9 percent of the marrow, the soluble salts 2.34 percent and the insoluble salts 0.66 percent. Among the soluble salts was included 0.48 percent P,0 5 . A nucleoproteid was found. Nerking (1908a) found, of lecithin, in red bone marrow, 0.1576, 0.1469, and 0.3006 percent, and in yellow bone marrow, 0.1636 and 0.2046 percent. PHOSPHORUS METABOLISM 119 Glikin (1907, 1908a, 1908b, 1909a) made a number of Fe 2 3 » P 2 6 and lecithin determinations on the fat of the bone marrow of different kinds of animals at different ages which seem to support the view that the marrow of very young animals is richer in lecithin than that of older animals, and that it is highest in the young of those species which are most helpless at birth. PHOSPHORUS, IRON AND LECITHIN DETERMINATIONS ON THE FAT OF BONE MARROW OF SEVERAL SPECIES OF ANIMALS AT DIFFERENT AGES (Glikin, 1907) Percent Animal Cattle. Cattle. Cattle. Cattle., Calf.... Calf.... Calf.... Calf.... Horse. . Horse • • Horse . . Horse.. Horse. . Horse.. Swine. Swine . Swine. Swine . Pigs.. Pigs . . Pigs 1 .. Pigs 2 .. Pigs. . Sheep. Sheep. Sheep. Sheep . Dog... Dog-... Dog-... Dog-... Dog-... Dogr... Man 3 . . Man 4 .. Man 5 .. Man 6 . Man 7 .. Age Older Older Older Older Younger Younger Younger Younger 18 yrs. Man Child 8 . Child9. ChildW. Child". Cat Rabbit. Guineapig New-born Bird Young 10 yrs. 6-7 yrs. 7-8 yrs. 2 yrs. Foal Older Older Older Young 20 hrs. 24hrs. 6 wks. 8 wks. 8 wks. Older Older Younger Younger Older Older Older 5 wks. Stillborn 10 wks. 88 yrs. 70 yrs. 70 yrs. 61 yrs. 56 yrs. 34 yrs. 2 yrs. 16 mos. 13.5 mos. 7 mos. New-born New-born P20fi 0.1152 0.2309 0.2378 0.2010 0.2515 0.3193 0.3198 0.5950 0.0787 0.0963 0.1055 0.1749 0.1709 0.3741 0.1842 0.2092 0.2105 0,1992 0.4564 2.6538 2.7413 4.1555 2.5834 2.4800 0.1529 0.2253 0.3254 0.5732 0.3235 0.1777 0.3081 1.6418 3.3170 0.8376 0.1613 0.2433 0.2052 0.1947 0.1783 0.2905 1.1770 2.1930 2.5730 5.3839 2.919 2.306 1.520 2.25 Fe203 0.0243 0.0266 0.0283 0.0223 0.1105 0.1085 0.1254 0.0254 0.0379 0.0975 0750 0.0486 6.' 0396 0.0264 0.0145 1.29 1.02 0.2960 0.1460 0.0138 0.0191 0.0249 0.0839 0.1672 0.0542 0.0489 0.4437 4.35! 0.3214 0.0354 0.1157 0.0668 0.0693 0.0807 0.0552 1.025 0.2237 6.'8049 Lecithin 1.31 2.62 2.70 2.28 2.86 3.63 3.64 6.76 0.89 1.09 1.20 1.98 1.94 4.25 2.09 2.38 2.39 2.96 5.18 30.16 31.16 47.23 29.36 28.19 1.73 2.56 3.70 6.51 3.67 2.02 3.50 18.66 37.70 9.52 1.83 2.76 2.33 2.21 2.02 3.30 13.38 24.93 29.24 61.19 33.18 26.21 17.34 25.63 bottle nursed 2 Badly nourished 8 Lung emphysema ^Stomach, liver, kidney cancer 5 Pleurisy 6 Influenza, inflammation Kidney inflammation, enlargement of the heart 8 Pleurisy 9 Bronchopneumonia 10 Lung emphysema "Pneumonia Bolle (1910a, 1910b) submits many lecithin estimations on the marrow of several species of animals, and says that his findings agree with those of Glikin in that lecithin seems to be always pres- ent in the fat of bone marrow, the amount decreasing with advanc- ing age, and that in paralysis there is an impoverishment in this particular. The tables did not seem to the abstractor to show clear 120 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 evidence of decrease with age, at least they did not exhibit anything 1 like a regular gradation of lecithin according to age. The method of lecithin estimation is open to considerable improvement. Suzuki and Yoshimura (1907) found lecithin in bones, and also, an acid-soluble organic phosphorus compound. For other lecithin figures on bone marrow see Glikin 1909a, un- der Phosphorus Metabolism in Paralysis. Phosphorus Compounds in Cartilage. Grandis and Copello (1902) state that the epiphyseal cartilage of the femur and tibia of the calf, compared with the articular cartilage, contains decidedly more phosphorus, a part of which is held in organic combination. PHOSPHORUS COMPOUNDS OF MUSCLES The studies on the amounts of phosphorus in muscles, and on the forms in which it is present, have a two-fold significance, (1) as related to the phosphorus requirement of the organism, and (2) as bearing on the value of meats as foods. Muscles contain phospho- rus in the forms of nucleoproteins, phosphatids, nucleon (phospho- carnic acid) and inorganic phosphates. STUDIES OF THE AMOUNT AND DISTRIBUTION OF PHOSPHORUS Katz's (1896) ash analyses on muscle are often quoted because his was the first extensive study of the kind, and it covers many species of animals. (See table on following page.) More recent studies are those of Grindley and associates, from the latest of which, by L. D. Hall and Emmett (1912), we quote the following : PHOSPHORUS IN THE BONELESS FLESH OF DIFFERENT CUTS OF BEEF (Hall and Emmett, 1912) Percent Soluble phosphorus Insoluble Wholesale cuts Inorganic Organic Total soluble Total 0.093 0.094 0.085 0.091 0.085 0.092 0.072 0.062 0.065 0.050 0.048 0.032 0.033 0.024 0.015 0.013 0.016 0.017 0.022 0.019 0.015 0.006 0.125 0.127 0.109 0.106 0.098 0.108 0.089 0.084 0.087 0.065 0.054 0.059 0.046 0.052 0.052 0.050 0.033 0.053 0.051 0.043 0.039 0.023 184 Clod Chuck 0.173 0.161 158 148 0.141 142 135 Rib 0.130 0.104 0.077 Plate Flank See also Grindley and Emmett (1905), Emmett and Grindley (1906, 1909a, 1909b). For materials on the phosphorus compounds of the muscle of swine, as affected by foods, see Hart, McCollum and Fuller (1909) under Nutr. Val. Org. and Inorg. P., and Forbes (1909) under Comm. Foods in Rel. to P. Met. PHOSPHORUS METABOLISM 121 GO p o K O w CO W •J O CO H H < w a HHHHHHNHOHOOO oooooooo'ooooo CD-stHcqcncocoMcorocor— oio c— icococoi-ico CNJ CN «j >0 lO CD CO lO OO iH O CO 03 r-r~r~r^t~t~ir~r~coaooc5or- §ftOOQ«OOMfcWBd( 122 •OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 A. Kossel (1882) reported the following estimations of total and nuclein phosphorus in the muscle of cattle and hens. TOTAL AND NUCLEIN PHOSPHORUS IN MUSCLE Kossel (1882) Percent Species H 3 P0 4 in fresh organ Nuclein HsPOi Nuclein H 3 P0 4 in total H 3 P0 4 Embryonic muscle Cattle Cattle Hen, fasted ' ' well fed 0.462 0.610 0.838 0.663 0.1490 0.0923 0.0542 0.0442 32.2 15.1 6.5 6.7 Embryo 28 cm. long The distribution of phosphorus in the muscle of salmon in nor- mal condition is reported by D. Noel Paton and his associates (Paton, 1898; Paton et al. 1897-98) as follows: DISTRIBUTION OF PHOSPHORUS IN MUSCLES OF SALMON Paton, et al. (1897-98) Percent Fish number Type of muscle Ether extract "Lecithin" W ater extract. "Phosphates" Residue, "Nucleins and pseudonucleins" Total 14 14 76 76 Thick Thin* Thick Thin 0.042 0.046 0.060 0.060 0.131 0.094 0.095 0.119 0.056 0.041 0.055 0.063 0.228 0.181 0.210 0.242 C 1 ) The "thin" muscle is said to comprise about % of the entire body. Milroy's (1908) determinations on the muscle of herring show wide and irregular variations from month to month, ranging from 0.45 to 0.82 percent P 2 5 in fresh muscle. Urano (1907) has made complete mineral analyses of the mus- cular tissue of frogs, and also of the press juice from the same, the blood plasma, and the muscle extraction residue. The salts of the blood and lymph in the muscle were removed by treatment with isotonic (6 percent) cane sugar solution; after which the muscle juice was expressed by a pressure of 1000 atmospheres. This press juice was dried, and extracted with hot water, and the extract and residue analyzed separately. Blood plasma was prepared by centri- f uging. The phosphorus figures are as •stated below : PHOSPHORUS IN MUSCLE OF FROG— Percent P0 4 Sample Juice Ash Muscle A 0.378 0.633 0.368 0.422 0.311 0.529 0.531 0.109 0.432 0.468 69.399 64.9 76.9 48.0 63.8 60.849 60.236 16.732 52.558 55.469 0.2268 B 0.3861 0.2245 0.2532 C 0.2084 0.529 0.531 D 0.109 0.432 0.468 PHOSPHORUS METABOLISM 123 Richet (1900) analyzed beef muscle serum. A kilogram of the serum contained 8.9 gm. ash, of which 3.15 gm. was P 2 5 . The form in which the phosphorus was present in the serum was not de- termined. Whitfield (1894) made a special examination to determine whether the myosin of muscle is a nucleoalbumin and whether there is any nucleoalbumin in muscle, and decided that myosin is not a nucleoalbumin and that muscle contains no nucleoalbumin. Pekel- haring (1896), however, isolated from the muscles of rabbit, dog and ox substances which he identified as nucleoprotein. Martin Muller's (1897) nucleon determinations in human muscle vary from 0.1123 to 0.2174 percent, in the muscle of adults, and from 0.0000 to 0.0570 in the muscle of new-born infants. Panella (1902a, 1903b, 1903e, 1903f, 1904b, 1906b) decided that phospho- carnic acid is a constant, normal constituent of the striated muscles of dogs and rabbits, being found in both the red and the white muscles of rabbits ; but his paper of 1906 throws doubt on the quan- titative value of all earlier determinations, as they may have been made on too small samples. The amounts reported at that time are 0.01 percent of fresh and 0.02-0.03 percent of dry muscle. Bajmakov (1904) (through Biochem. Centralbl.) reports an- alyses of the muscles of calves (5) and of children (9) of different ages, which show that the dry substance, ash, phosphorus and iron content of muscle, as well as the total protein and "organized pro- tein," increase with advancing age. The iron is said to increase at a more rapid rate than the phosphorus. J MacLean (1912b) has made a careful purification and examin- ation of the phosphatids of horse muscle, and decides that the chief one present is a lecithin, the proportion of N:P being as 1:1. Cavazzani (1904b) found the phosphocarnic acid content of oysters to vary with the stages in the life cycle from 0.1942 to 0.5978 percent with a mean value of 0.3725. COMPARISONS OF STRIATED AND NON-STRIATED MUSCLES There seem to be differences in chemical composition between the striated, or voluntary, and the non-striated, or involuntary muscles; and the heart muscle (myocardium), which is striated but involuntary, differs somewhat from both. The reports as to these differences are not closely concordant, but we give such as there are. Saiki (1908) was apparently the first to make determinations on the mineral constituents of non-striated mammalian muscle. He used the same analytical methods as Katz. Saiki is alone in finding the sodium content higher than the potassium in non-striated muscles, so far as we know, though others do find somewhat more of sodium and more of chlorine than in striated muscles. 124 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 MINERAL ANALYSES OF NON-STRIATED MUSCLES OF PIG Saiki (1908) Source Water Percent Percent, dry, fat-free substance K Na Fe Ca Mg CI S P Stomach I Stomach II Urinary bladder I Urinary bladder II 81.4 80.8 80.6 79.6 0.415 0.332 0.342 0.204 1.549 1.095 1.058 1.045 0.028 0.036 0.042 0.049 0.111 0.117 0.205 0.187 0.026 0.023 0.013 0.012 0.922 0.808 0.816 0.786 0.833 0.444 0.445 0.424 0.373 0.422 0.381 0.428 0.416 0.437 Average 80.6 0.323 1.187 0.039 0.155 0.019 0.416 Edw. B. Meigs and L. A. Ryan (Meigs and Ryan, 1912; Ryan and Meigs, 1912) report finding in the water-extract of the smooth muscle of the stomach of the frog (Rana catesbiana) 0.0958 and 0.0919 parts phosphorus per 100 parts of muscle; in the alcohol ex- tract 0.0356 and 0.0260 parts, and in the residue 0.0146 and 0.0105 parts, making in the whole muscle 0.1460 and 0.1284 parts phos- phorus. They conclude that "1. The fibres of this tissue are not surrounded by semi-permeable membrane. 2. Most of the water of the smooth muscle fibres is held by the colloids of the living tissue as organic water. 3. Most of the potassium, phosphorus, sulphur, and magnesium, which appear in the ash of smooth muscle, are pres- ent in the living tissue in a non-diffusible form." A large portion of the phosphorus is thought to be in the form of lipoid. The fol- lowing are their ash analyses of striated and smooth muscles of the frog: MINERAL CONSTITUENTS OF SMOOTH AND STRIATED FROG MUSCLE Meigs and Ryan (1912) Percent, Fresh Basis K Na Fe Ca Mg P CI S H 2 0.3483 0.3518 0.0572 0.0500 0.0536 0.0099 0.0094 0.0096 0.0007 0.0007 0.0335 0.0228 0.0281 0.0042 0.0042 0.0042 0.0288 0.0313 0.0300 0.0132 0.0126 0.0129 0.1554 0.1541 0.1547 0.1460 0.1284 0.0650 0.0674 0.1492 0.1322 79.99 79.74 0.3500 0.3063 0.3437 0.3250 0.0662 0.1407 79.87 0.0648 0.0804 0.1191 0.1200 0.1724 0.1501 0.1612 82.61 82.01 0.0726 0.0007 0.1372 0.1195 82.30 According to Vincent's (1902) comparison of the proteins of smooth and striated muscles, the smooth contain 6-8 times as much nucleoprotein as the striated, and the heart muscle has an inter- mediate place in this regard. Panella (1904b) found more nucleon in non-striated than in striated muscles. Balke and Ide (1896) found in the heart of the horse 0.116 and 0.105 percent, and in that of the dog 0.253 percent of nucleon. PHOSPHORUS METABOLISM 125 Krehl (1893) found that the lecithin content of the healthy- heart muscle is about constant at 4.2 to 4.6 percent, while in differ- ent maladies it varies from 1.1 to 6.3 percent without reference to the state of nutrition. Rubow (1905) studied the composition of the heart and of or- dinary striated muscle as affected by various conditions. From this investigation we quote the following data which show heart muscle to be richer in lecithin than is skeletal striated muscle. LECITHIN CONTENT OF HEART AND ORDINARY STRIATED MUSCLE Percent of Muscle Subject and condition Muscle No. of subjects Ether extract Lecithin Fat Dog, starved 19-22 days Dog, well fed Dog, starved 19-22 days Heart Heart Striated Striated Heart Back Heart Shoulder 9 2 2 1 1 1 1 12.23 11.18 10.07 6.07 18.71 15.03 17.62 14.54 8.01 7.49 5.08 3.41 7.54 2.50 8.02 4.15 4.21 3.69 5.99 2.66 10.17 12.53 9.60 10.39 Erlandsen (1906, 1907) made an exhaustive study of the phos- phatids of muscle from the heart and from the upper thigh of the ox, distinguishing different kinds of phosphatids. Both forms of muscle contained lecithin, a little of a protagon- or jecorin-like sub- stance, a new monamino-diphosphatid, "cuorin," and a diamino- monophosphatid. Phosphorus was also found as phosphocarnic acid, inorganic salts and in some nitrogenous relation not identified. The heart muscle was the richer in phosphatids in general, and es- pecially in the two new phosphatids. A high content of cuorin seems to be characteristic of heart muscle. It contains less of phos- phocarnic acid than the thigh muscle. The phosphatids were thought to be present in both types of muscle partly in a free state and part- ly combined with protein. From the values given in the table below, Costantino (1912) draws the conclusions: "1. There is no measurable variation between striated and non-striated muscle in respect to total phosphorus. Heart muscle tissue shows, however, a higher percentage of phosphorus. "2. There is an important difference in respect to plain and striated muscle content of inorganic phosphorus. Plain muscle contains about the same amounts of organic and inorganic phospho- rus ; striated muscle contains a much higher percentage of inorganic than organic phosphorus. Heart muscle shows the same relation as plain muscle. 126 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 "3. Both the plain and heart muscle tissue show higher values of organic than phosphatid phosphorus. Phosphatid phosphorus is shown to be about y% as much as organic phosphorus in plain muscle, and only 1/3 as much in case uterus." of muscle fibres of cow's PHOSPHORUS OF STRIATED AND NON-STRIATED MUSCLE OF CATTLE (Costantino, 1912) Percents of dried muscle Percents of total phosphorus Organi- Type Total Inor- cally Phos- Organ- Inor- Organ- Phos- Organ- phos- phorus ganic com- phatid ic, not ganic phatid ic, not phos- bined phos- phos- phos- phorus phos- phos- phorus phos- phorus phatid phorus phorus phatid phorus 9 0.7575 0.6126 0.1449 0.1242 0.0207 81.50 18.50 16.39 2.11 ? 0.6498 0.5287 0.1211 0.1059 0.0152 81.36 18.64 16.29 2.35 Heart 1.0814 0.4327 0.6487 40.01 59.99 Heart 1.0430 0.3951 0.6479 0.4407 0.2072 37.88 62.12 42.25 19.87 Non-striated Retractor penis 0.5670 0.2313 0.3357 40.79 59.21 Retractor penis 0.5917 0.2839 0.3078 0.1439 0.1639 47.97 52.03 24.33 27.70 Stomach 0.6010 0.3148 0.2862 0.1518 0.1344 52.38 47.62 25.25 21.371 Stomach 0.5751 Uterus 0.8449 0.3353 0.5096 0.1644 0.3452 39.68 60.32 19.46 40.86 Uterus 0.7329 0.2142 0.5187 0.2081 0.3106 29.22 70.78 28.39 42.39 ( x ) The compilers compute this to be 22.36. MacLean (1913) found heart muscle to contain lecithin, cuorin and a trace of diamino-monophosphatid which resembles the sub- stance described by Stern and Thierf elder and by Thudichum, An analogous compound has been obtained from kidneys by Dunham and Jacobson and by MacLean. For the distribution of phosphorus, potassium and chlorine within the muscle fiber see Menten (1909). PHOSPHORUS IN FEATHERS In connection with Weiske's study (1889) of the effects of age on the composition of the bodies of birds, analyses were made of the feathers. From the following data it appears that as a growing fowl increases in age the phosphorus of the feathers decreases in percentage during the first 18 weeks. At the 18th week the feath- ers contain a lower percentage of mineral matter generally than at any other time during the first year, and this is also the low point for percentage content of calcium, sulphur and phosphorus. PHOSPHORUS METABOLISM ANALYSES OF FEATHERS OF BIRDS Weiske (1889) Percent of Dry, Fat-free Feathers 127 Bird Age, ■weeks Mineral matter CaO MgO S0 2 P 2 5 Feed 2.04 0.31 0.140 0.11 0.190 Corn 1 1.98 0.16 0.051 0.23 0.190 Corn 2 2.19 0.45 0.047 0.48 0.160 Corn 4 2.09 0.40 0.120 0.27 0.150 Corn 8 1.51 0.15 0.044 0.25 0.140 Corn 12 1.50 0.19 0.050 0.14 0.140 Corn 18 0.88 0.15 0.022 0.052 0.060 Corn 24 1.80 0.40 0.049 0.059 0.117 Corn 34 1.78 0.62 0.024 0.10 0.075 Corn 44 2.11 0.87 0.011 0.22 0.067 Corn 52 1.82 0.82 0.031 0.15 0.062 Corn 52 1.89 0.80 0.041 0.13 0.070 Corn 0.40 0.050 0.016 0.037 0.074 Meat 0.62 0.092 0.006 0.147 0.133 Meat THE PHOSPHORUS OF BRAIN, NERVES AND CEREBROSPINAL FLUID HISTORICAL REVIEW TO THE TIME OF THUDICHUM None of the general analyses of brain before those of Thudi- chum need receive especial attention. Thudichum mentions one by Hensing (1719) when the phosphorus of the brain was dis- covered. Fourcroy's (1793) work was quite extensive and Vau- quelin's (1811) was significant enough to have led to its translation from the French into both English and German, and to be reviewed at some length in Thudichum's "Die chemische Konstitution des Gehirns." Couerbe's (1834) analyses were followed by Fremy (1841, 1842). We find early ash analyses by Breed (1851b) and by Geoghegan (1877-78). VonBibra (1853, 1854a) made phosphorus determinations on the fat obtained from the brains of fifteen kinds of animals, and of men, and showed that this phosphorus is a con- stituent of the fat itself. The next year (vonBibra, 1854b) he reports phosphorus in the spinal cord and nerves of several species, and concludes that in brain and spinal marrow the phospho- rus content depends on the fats containing phosphorus, and in nerves principally on the cerebric acid. Liebreich's (1865) analysis is significant for its discovery of protagon, which, however, is now known not to be a single compound. After Gobley and others made the various studies of "lecithin," Gobley (1877) reported a general analysis of brain. The later one of Gutnikov (1896-97) should also be mentioned. Some of these old reports make interesting reading. Couerbe (1834), finding a lower phosphorus content in the fat of an idiot's brain and a higher in that of the insane than normal, concludes: "Phosphorus is the exciting principle of the nervous system," and Note: For general discussions of the subject of the chemical composition of the brain and nerves the reader is referred to Thudichum (1901), Halliburton (1901b, 1905> Coriat (1905) and Frankel (1909a). 128 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 "Absence of phosphorus from the brain reduces man to the level of the brute, while a great excess irritates the nervous system, caus- ing mental alienation." THE WOEK OF THUDICHUM At the time of his report in 1875 Thudichum (1875) had anal- yzed a thousand brains, mostly normal brains from human subjects, and had found and identified many of the compounds which he af- terwards described and classified more fully. "Die chemische Kon- stitution des Gehirns" appeared in 1901 and seems to have greatly stimulated and influenced later work on brain chemistry in general and especially on the chemistry of lipoids. The classification of compounds found is given below; also analytical determinations of the principal constituents of the white and the gray matter : THUDICHUM'S CLASSIFICATION OF THE BRAIN CONSTITUENTS A. GEOUP OF PHOSPHOEUS-CONTAINING CONSTITUENTS OE PHOSPHATIDS Subgroup of the monophosphatids containing one nitrogen. N:P— 1:1. Lecithin Cephalin Paramyelin Myelin Subgroup of monophosphatids containing two nitrogens. N:P=2:1 Amidomyelin Amidocephalin Sphingomyelin Apomyelin Subgroup of diphosphatids containing two nitrogens. N:P=2:2. Assurin Subgroup of nitrogen-containing phosphatid-sulphatids Subgroup of nitrogen-free monophosphatids Lipophosphoric acid Butophosphoric acid B. GEOUP OF NITEOGEN-CONTAINING PHOSPHOEUS-FEEE CONSTITUENTS ' Subgroup of cerebrosids or cerebrogalactosids Phrenosin Kerasin Subgroup of cerebrin acids Cerebrinic acid Sphaerocerebrin Subgroup of cerebrosulphatids Subgroup of amidolipotids or nitrogen-containing fats Bregenin Krinosin Subgroup of alkaloids Hypoxanthin Gladiolin Tenysin Subgroup of amino acids and amids Leucin and its homologues Tyrosin Urea C. GEOUP OF CONSTITUENTS WHICH CONTAIN ONLY THEEE ELEMENTS Subgroup of nitrogen-free alcohols Cholesterin Phrenosterin (?) Subgroup of carbohydrates Inosite Glycogen (?) PHOSPHORUS METABOLISM 129 Subgroup" of nitrogen-free organic acids Formic acid Sarcolactic acid Succinic acid Oxyglyceric acid (?) D GROUP OF ORGANOPLASTY OR ALBUMINOUS SUBSTANCES Subgroup of nitrogen-containing sulphatid-phosphatids Neuroplastin Gangliocytin, cytophosphatid- or nuclein-substances Subgroup of nitrogen-containing sulphatids Albumen Collagen E. GROUP OF INORGANIC PRINCIPLES, ACIDS, BASES AND SALTS These are found in part in the water- extract, in part in combination with many of the before-named constituents. Sulphuric acid Hydrochloric acid and chlorine in chlorides Phosphoric acid Carbonic acid Potassium Sodium Ammonium Calcium Magnesium Copper Iron Manganese Aluminum, silicic acid, fluorine SUMMARY OF ANALYSES OF BRAIN In compounds forming bases with products obtained; or in compounds with phosphoric acid, and then combined, as phosphates, with these products; or in combination with min- eral acids as free mineral salts in the fluids and extracts. Gray matter Percent White matter Percent Ether-extract with cephalins, lecithins 85.270 7.608 1.950 0.424 0.500 0.193 0.102 0.017 0.025 0.092 70.230 8.630 11 497 Cerebrosids, cerebrin acids and myelin. . 6.910 1.403 0.2171 0.0456 0.1717 OTHER STUDIES OF THE COMPOUNDS PRESENT IN BRAIN Waldemar Koch and William H. Goodson (1906) give analyses of two fractions of the gray matter, the prefrontal and the motor areas, which do not differ materially in the normal brain, and of the white medullated matter of the corpus callosum, which differs some- what from them, particularly in the amount of cerebrins. The sciatic nerve differs from the corpus callosum in having considerably more protein and less water, which may be due to a greater amount of connective tissue. The table below gives the analyses together with some for the study of the effects of degeneration of brain and nerve tissue. W. Koch's (1904) analysis of epileptic brain is given in the chapter on metabolism in disease. 130 OHIO EXPERIMENT STATION: TECHNICAL BTJL. 5 ANALYSES OF NORMAL AND DEGENERATED BRAIN AND NERVE TISSUE (Koch and Goodson, 1906) Percent Brain (Human) Sciatic . nerve (Human) Prefrontal area Motor area Corpus callo- sum Spinal cord (Dog) Normal Degener- ated Normal Degener- ated Normal Degener- ated Total solids 17.5 15.2 18.4 17.4 30.0 35.8 31.5 28.4 Percent of solids Simple proteids Nucleoproteids Lecithins Cephalins Cerebrins Sulphur compound.. Extractives Inorganic salts 25.8 23.8 14.8 8.9 7.1 5.9 11.0 7.0 19.1 37.7 12.3 8.5 7.7 5.6 10.8 6.6 26.6 21.0 12.4 11.0 8.6 5.9 10.6 6.0 12.1 35.3 15.0 15.0 12.0 6.0 10.0 6.0 16.7 11.4 14.5 7.6 17.7 7.3 5.8 2.7 12.6 34.9 7.1 7.8 7.2 10.0 5.0 3.6 18.1 6.7 26.4 15.9 5.3 3.7 2.2 14.9 12.8 26.3 15.1 6.4 3.4 2.0 Other single determinations of phosphorus compounds reported by different workers are collected in the following table : SOME DETERMINATIONS OF BRAIN OR NERVE CONSTITUENTS Compound and source Author Date Moist substance Percent Dry substance Percent Phosphocarnic acid Panella 1903g . 1903h 0.2050 0.9037 Panella Panella Panella 1903h 1903h 1903h 0.2142 0.2245 0.2837 1.1803 1.0044 1.3273 Panella 1903h 0.3259 Brain, calf, gray matter Panella 1903h 0.1917 Panella 1903h 1903h 0.2871 0.3502 1.1841 1.6937 Panella 1903h 0.3520 1.5820 Panella 1903h 0.3316 1.7240 Lecithin Chevalier 1886 14.80 Embryo, 62 cm. (cattle) Raske 1886 0.610 6.6331 Embryo, 68 cm. (cattle) Raske 1886 0.315 3.4923 Petrow- sky 1873 3.1714 17.2402 White matter of brain Petrow- sky 1873 3.1347 9.9045 Burow 1900 3.954 Nuclein Geog- hegan 1 1877-78 0.1390 0.1624 0.1340 0.1368 1 Percents computed by compilers. Special studies of cephalin are reported by Thudichum and Kingzett (1876), W.Koch (1902b, 1903), Cousin (1906), Falk (1908), and by Frankel, Neubauer and Dimitz (Frankel and Neu- bauer, 1909 ; Frankel and Dimitz, 1909) . Falk says that the ceph- alin of the peripheral nerves is not the same as that of brain. PHOSPHORUS METABOLISM 131 He reports on the chemical composition of peripheral nerves. Frankel (1908, 1909b) describes several brain lipoids, and Argiris (1908) reports quantitative estimations of phosphorus compounds on brains of birds and fish. Scott (1899) discusses the Nissl granules of nerve cells, which are of nucleoprotein nature and are said to consist of chromatin which has diffused from the nucleus into the cytoplasm. Levene (1903c) gives further discussion of these chromatins, saying that they are compounds of nucleic acid with one or more proteins, or with protein and carbohydrate. He reviews the work on the sub- ject, considering the question of the identity or difference of nucleic acids from different sources. The nucleoprotein which Levene (1899) isolated from brain and analyzed was said to differ from other nucleoproteins in its low phosphorus content, and in having a considerably higher amount of proteins bound to its nuclein. Halli- burton's (1894) paper on the proteins of brain we have not seen. The so-called "protagon," which was once looked upon as the most distinctive component of brain tissue, has been proved to be a mixture, and we have given the history of its study in our chapter on the chemistry of phosphatids. Noll (1899) made a number of determinations of protagon in medullary tissues, and all of the early analyses include it as an item. Rosenheim and Tebb (1909c) find that sphingomyelin, containing 4 percent phosphorus, is the most important component of the so-called protagon'. W. Koch (1907b, 1910a) thought he had evidence of a combin- ation which might be called phosphatid-cerebrosid-sulphatid. Bar- bieri (1909) describes cerebroin, containing 0.7 percent phosphorus, found chiefly in the gray matter of the brain (ox). It yielded neither glycerin nor sugar. Bethe (1902) reports a cerebrinic phos- phoric acid of about 1 percent phosphorus content found in the brain of the horse. Jolly (1898) looked for evidence of phosphorus in the organic portions of brain in some other form than such as yields orthophosphoric acid on simple ashing, but did not find it. Masuda (1910) reports total phosphorus figures for the three divisions of the brain as in the following table : TOTAL PHOSPHORUS CONTENT OF THE THREE PARTS OF THE BRAIN Masuda (1910) Percent P Cerebrum Cerebellum Mid-brain 0.1838 0.3324 0.2545 0.2813 . 0.2743 0.1593 0.2829 0.2760 0.2971 0.2824 0.2638 0.3437 0.4002 0.3736 0.3480 132 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 For total phosphorus determinations on the brain of horse see Baumstark (1885) and Chittenden (1897), pig, — Hart, McCollum and Fuller (1909), sheep, — Kutanin (1910), and pigeon, — Funk (1912a). For partial separations of the groups of phosphorus compounds in the brain of the horse see Baumstark (1885), and the same for sheep, Chittenden (1897). DIFFERENTIAL ESTIMATIONS OF PHOSPHORUS COMPOUNDS OF BRAIN UNDER SPECIAL CONDITIONS In their report of "A Chemical Study of the Brain in Healthy and Diseased Conditions, with Especial Reference to Dementia Praecox," W. Koch and Mann (1909) give the following as the parti- tion of phosphorus among the various classes of compounds. We have brought together all of those which are spoken of as normal and grouped the pathological cases as do the authors. The values are percents of the total phosphorus. PARTITION OF PHOSPHORUS AMONG THE BRAIN COMPOUNDS Koch and Mann (1909) 6-weeks child 1 2-yr. child, cortex 2-yr. child, corpus callosum 2-yr. child, whole by computation . 12 yr. child, cortex 12-yr. child, corpus callosum 12-yr. child, whole by computation . 16-yr., Case 20 24-yr.,Casel9 43-yr., Case 70 49-yr., Case 18 Dementia praecox, Case 28 Dementia praecox, Case 29 Dementia praecox, Case 41 Dementia praecox, Case 42 General paralysis, Case 22 General paralysis, Case 23 General paralysis, Case 24 General paralysis, Case 40 Melancholia, Case 25 Melancholia, Case 26 Brain of a dog • Total P Percent of solids Part 1.50 1.46 1.45 1.45 Whole brain 1.72 1.48 1.45 1*50 1.50 Percent of total P Protein P Part Whole brain 5 4.7 3.7 6 3.9 3.8 5.1 3.6 4.7 5.9 4.8 4.6 4.6 4.2 4.1 4.5 Lipoid P Part Whole brain 54 67 72 70.3 73.1 75 71.8 73.7 73.0 73.6 70.8 69.3 70.6 68.2 69.9 69.2 74.7 70. Extractive P Part 22 Whole brain 41 27 23 25.1 23.2 19 24.2 22.6 21.9 22.9 24.4 24.7 24.7 26.9 25.6 26.6 21.2 25.5 (!) Birth premature; brain underdeveloped. The authors' conclusions with regard to the phosphorus are : — 1. With growth of the brain there is an increase in the lipoid phos- phorus and a decrease in the extractive phosphorus. 2. The phosphorus agreement between the brain of man and dog is quite close. 3. Comparison of brains from cases in which the causes of PHOSPHORUS METABOLISM 133 death were of an entirely different character showed no variations of importance.- 4. The brains from cases of dementia praecox showed no marked change in the amount and distribution of phos- phorus as compared with the normal. 5. The results from brains of cases of general paralysis show that the destructive changes in this disease affect the brain generally and not one constituent in particular. There is, however, a tendency for the lipoid phosphorus to be decreased, indicating a greater destruction of the phosphatids. CHANGES OF THE BRAIN WITH GROWTH Koch and Mann (1907) give the following comparison of the chemical composition of three human brains at different ages, and draw conclusions as quoted : COMPARISON OF BRAINS OF DIFFERENT AGES— Percents of Solids 6- weeks child Whole 2-years old 19-year s old Gray White Whole . Gray White Whole 46.6 48.4 31.9 40.1 47.1 27.1 37.1 12.0 10.0 5.9 8.0 9.5 3.9 6.7 Ash 8.3 5.8 3.2 4.5 5.9 2.4 4.1 Lecithins and cepha- 24.2 24.7 26.3 25.5 23.7 31.0 27.3 6.9 8.6 17.2 12.9 8.8 16.6 12.7 Lipoid S as SO* . . . 0.1 0.1 0.5 0.3 0.1 0.5 0.3 Cholesterin (by dif- 1.9 2.4 15.0 8.7 4.9 18.5 11.7 88.78 84.49 76.45 80.47 83.17 69.67 76.42 Total sulphur .... 0.52 0.53 0.63 0.58 0.46 0.50 0.48 Total P 1.72 1.50 1.64 1.48 1.45 1.45 1.45 DISTRIBUTION OF SULPHUR— Percent of Total Sulphur Protein S . Lipoid S . . Neutral S . Inorganic S 62 63 55 59 73 51 6 6 27 17 7 36 26 22 13 17 12 6 6 9 5 7 8 7 62 22 9 7 DISTRIBUTION OF PHOSPHORUS— Percent of Total Phosphorus Protein P Lipoid P Water-soluble P 5 6 6 6 5 5 54 62 72 67 63 81 41 32 22 . 27 32 15 5 72 23 "In the above table is to be observed with the growth of the brain : "A decrease in moisture, proteins, extractives, and ash, a change usually found in growing tissues. "An increase in cerebrins, lipoid sulphur and cholesterin, in other words the substances which predominate in the white matter. "The relative increase in the lecithin and cephalin is not so ap- parent, as at an early age the brain is supplied with practically the same proportion of lecithin and cephalin as the adult ; in fact, it is the richest tissue in these constituents, the adrenal coming next with 12 percent and the liver with 10 percent. 134 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 "In the distribution of phosphorus and sulphur there is to be observed a tendency of the water-soluble forms to become converted into more complex water-insoluble forms, principally lipoids, which increase much more in actual amount than the above increase in rel- ative amount might indicate. The variation in both the protein sulphur and phosphorus is less than might be expected." M. Cohn (1907) gives the following determinations on the brains of children. No changes were noted as characteristic of the diseases. ANALYSES OF THE BRAINS OF CHILDREN M. Cohn (1907) Age Sex Diagnosis Wt. of brain H2O Percent of dry substance No. Grams Percent N P Ca 1 Fetus, 25 cm. 56 91.1 9.44 0.0631 2 1 day 495 89.30 9.69 1.69 0.0519 3 VA mos. M Intestinal catarrh 402 87.80 9.47 1.64 4 3K mos. F Naval sepsis 547 86.17 9.76 1.58 0.0231 5 7 mos. F Pneumonia 656 83.65 8.95 1.68 0.0285 6 8 mos. M Post pneumonia empyema . 824 83.35 9.09 1.58 0.0263 7 11 mos. F Pneumonia, measles 846 83.93 8.92 1.56 8 VA yrs. F Pneumonia, measles 801 82.34 9.35 1.62 0.0237 9 2H yrs. F Pneumonia, measles 994 82.15 8.58 1.55 0.0197 10 Zte yrs. M Scarlatina 1247 81.57 8.05 1.51 11 4 yrs. M Diphtheria 1304 80.33 7.97 1.50 0.0191 12 6K yrs. F Scarlatina 1280 80.01 8.15 1.51 0.0181 13 20 yrs. F Erysipelas, nephritis 1270 77.52 7.57 1.50 0.0169 14 6% mos. M Tetanus 822? 84.40 9.72 1.71 0.0282 15 10 M mos. M Atrophy, tetanus 750 84.31 9.15 1.72 0.0225 Dhere and Maurice (1909) made analyses of the peripheral nerves of 19 dogs betwen the ages of 4 weeks and 8 years, from which they report the following results, and conclude that the phos- phorus content of nerves (on the dry basis) diminishes with in- crease of age, but the diminution does not occur equally among the different groups of phosphorus compounds. ANALYSES OF PERIPHERAL NERVES OF DOGS OF DIFFERENT AGES Dhere and Maurice (1909) Ages No. Percents of dry substance Percents of total P Group Total P Lipoid P Nuclein P Inorg'anic P Lipoid P Nuclein P . Inorganic P I II III 4 wks. — 4 mos. 6 — 13 mos. 2-8 yrs. 9 6 4 0.990 0.717 0.605 0.430 0.335 0.285 0.072 0.061 0.056 0.488 0.321 0.264 43.43 ( 46.72 -, (gain of 1 7.6/o) 47.11 \ (gain of \ 8.5*) 7 27 I 8.51 < (gain of ( 17.0*) 9.26 < (gain I 27. 4*) 49.30 I 44.77 i (loss of 1 9.2*) 43.63 \ (loss of I 11.5*) See also Kutanin (1910) PHOSPHORUS METABOLISM 135 J. Smith and W. Mair .(1912) give the following report of a study of the lipoid content of the brains of dogs of different ages, — a Utter of eight pups and their mother. They find that, while the to- tal lipoids and the cerebrosids show a marked and constant increase with age, the percentage of phosphorus and phosphatids show a maximum at 3 weeks, and thereafter a continuous decrease, al- though the absolute amount present increases constantly. Atten- tion is called to the fact that while the milk contains only minute traces of these lipoids, in the brain alone at the age of 3 to 6 weeks there is a daily addition of .045 gm. phosphatid, .007 gm. cerebrosid, and .015 gm. cholesterin. This is taken as evidence of a synthesis of the compounds in the body. LIPOID CONTENT OF DOGS' BRAINS AT DIFFERENT AGES Smith and Mair (1912) Age and subject 3 days, 3 dogs 3 weeks, 3 dogrs 6 weeks, 1 dog 12 weeks, 1 dog Adult, 1 dog Human adult 1.38 23 5.40 25 8.90 34 15.0 38 19.25 47 Percentage Composition of Chloroform Extract Cholesterin . Cerebrosid . . Phosphorus . Phosphatid . Other lipoids 18.3 16.8 18.5 17.1 21.8 1.5 3.5 6.8 11.5 21.4 2.10 2.42 2.27 2.11 1.76 52. 60. 57. 52. 44. 28. 20. 28. 19. 13. 21.8 10.7 1.58 39.5 28. Amounts Found in the Brain — Grams Phosphatid . Cerebrosid . . Cholesterin . Other lipoids Total extract 0.16 0.80 1.70 2.90 4.0 0.005 0.046 0.20 0.67 1.95 0.058 0.22 0.55 1.00 2.0 0.10 0.28 0.55 1.20 1.17 0.32 1.34 3.00 5.77 9.12 Messing (1912) studied the mineral constituents of normal and pathological brains. The P 2 5 content increases with age and the weight of the brain, until about the 60th year, after which it de- creases. Arteriosclerosis causes a decrease of the phosphorus, and an increase of both calcium and S0 3 . Mathilde Koch (1913) has made similar determinations to those of Koch and Mann on brains from pig fetuses of 50, 100 and 200 mm. length, and from albino rats at birth, and in the adult state, carrying on work started by Waldemar Koch. She finds that chemically, anatomically and physiologically, the brain of the albino rat is at about the same stage at birth that the pig fetus is at 50 or 100 mm., and represents as young nervous material as can conveniently be an- alyzed at present. The phosphatids there comprise 15.2 percent of the solids, and the total phosphorus 1.87 percent. Of the phos- phorus 13.3 percent is in protein, 33.1 percent in lipoid and 53.55 136 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 percent in water-soluble form. From examinations of rat brains at 1, 10, 20, 40, 120 and 210 days (Koch, W., and M. L., 1913) it is learned, among other things, that in the period of the most rapid growth and of the beginning of medullation, from the 10th to the 20th day, there is most rapid development of phosphatids. Phos- phatids are found in both cells and sheaths. PHOSPHORUS IN THE LIVER Phosphorus Content. Szymkiewicz (1891) made sulphur and phosphorus determinations on the ash of some 80 samples of the livers of cattle of various ages, from the 13-cm. fetus to the cow of 13 years. In the cases of the smaller fetuses a mixture of 2-6 livers was used. There were no characteristic variations in the phosphorus content of the livers of the fetuses (minimum, 1 .52 and maximum, 1.98 percent), though there were irregular individual variations. As a rule the phosphorus content after birth is ap- preciably lower than before birth, although among the 24 there were some individual cases in which the phosphorus content is higher than the lowest found in the fetus. Among the 24 there is no evi- dence of any characteristic effect due to advancing age or to sex. The mean of the values on calves, oxen and cows is 1.39 percent P. Kriiger (1895) obtained results practically like those of Szymkiewicz except that his averages for adult animals are some- what lower than for calves. In the livers from human subjects the phosphorus was considerably higher in the new-born infants than in the adults. AVERAGE PHOSPHORUS (P) CONTENT OF LIVERS OF CATTLE OF DIFFERENT AGES (Friedrich Kriiger, 1895) Percent Dry Basis Fetuses, length in centimeters Calves Cows Oxen 30-40 1.74 40-50 1.73 50-60 1.73 60-70 1,65 70-80 1.69 / 80-90 90-100 1.72 1.46 1.29 1.30 Weiske's (1886) experiment testing the effects on lambs of feeding hay which had been treated with dilute sulphuric acid showed the phosphorus content of the livers of the 3 lambs on such feed and on normal feed to range between 0.450 and 0. 485 percent P, with the mean value of 0.468 percent. Paton (1895-6), in discussing the relation of the liver to the metabolism of fat, submits the following lecithin determinations on the livers of animals under known conditions of feeding. The amount of lecithin in the liver appeared to be somewhat nearly con- stant, its considerable percentage variations being due in large part to the storage or removal of fat, as determined by the general state of nutrition of the animaL PHOSPHORUS METABOLISM 137 CONTENT OF LECITHIN IN LIVER OF VARIOUS ANIMALS UNDER DIFFERENT CONDITIONS OF FEEDING— Paton (1895-96) Lecithin Animal Percent of liver Percent of solids of liver Percent of ether extract of liver Feed Rabbit 2.60 2.70 2.05 1.90 2.57 3.04 3.31 1.72 1.42 2.24 11.4 11.9 9.1 10.3 11.7 5.2 9.8 8.9 56.1 58.4 51.3 36.4 50.0 60.0 70.0 11.0 3.53 34.2 Bran and oats Rabbit Rabbit Cat Kitten Kitten Fast of 48 hours Kitten " " 59 " Kitten Kitten Rabbit, very fat. . . Turnips Balthazard (1901a) investigated the lecithin content of the liver in a number of cases of different diseases or artificially pro- duced pathological conditions in animals. He found the lecithin high in all the pathological cases examined; thus it was increased by infection in tuberculosis and diphtheria, by intoxication with a mineral poison, phosphorus; by bacterial poison — typhus toxin, or by autointoxication, as in inanition and uremia. Balthazard con- cluded that a large part of the hepatic lecithin comes from the de- struction of leucocytes of the circulating blood. The livers of 4 pigs to which Hart, McCollum and Fuller (1909) had given different amounts and different kinds of phosphorus in the diet showed a phosphorus content of from 1.27 to 1.43 percent P in the air-dry substance, with an average of 1.35 percent. Forbes (1909) found the phosphorus of the livers of pigs to vary between 0.291 and 0.367 percent, and the phosphorus in the ash between 24.18 and 28.23 percent, as affected by the food received. The bile and liver of cattle were analyzed by Daniel-Brunet and Rolland (1911b) and the phosphate content of liver showed a range from 2.90 to 3.48 parts P 2 5 per 1000 parts fresh substance (1.26- 1.52 parts P). For phosphorus estimations in the liver in various pathological states see Robin (1911). Partition of the Phosphorus in Liver. Plosz (1873) identified nuclein in liver, and A. Kossel (1882) submitted the following fig- ures for nuclein phosphorus in liver. TOTAL PHOSPHORUS AND NUCLEIN PHOSPHORUS IN LTVER Kossel (1882)— Percent Species H3PO4 in fresh organ Nuclein H3PO1 Nuclein H3PO4 in total H 3 POi Dog 0.846 1.267 1.077 0.834 0.444 0.390 0.511 0.264 52.5 Cattle 30.8 Hen, -well fed. . . . 47.4 31.7 138 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Drechsel (1886) showed that jecorin is found in liver. Paul Manasse (1895) isolated from the liver of the horse a phosphatid which yielded grape sugar on cleavage and hence was supposed to be jecorin. Waldvogel and Tintemann (1906) also report jecorin in the liver. Baskoff (1908) found in addition to lecithin and jecorin another phosphatid which he named heparphosphatid. Ken- naway and Leathes (1909) found the jecorin fraction of the lipoids from the liver of the pig higher in phosphorus than that from the liver of the dog or the goat. The compounds which Liebermann (1893a) studied and called lecithalbumins were found in most abundance in liver and lungs. Phosphocarnic acid is reported by Siegfried (1896) and by Balke and Ide (1896). Balke and Ide give the amount as 0.183 percent in the liver of the dog and 0.015 percent in that of the horse. Levene and Mandel (1908b) say that among the nucleic acids is guanylic acid, and others describe a ferruginous nucleoprotein, or mixture of such nucleoproteins, to which the name "ferratin" is given. Masing (1911b) published a series of analyses which indicate a percentage decrease in amount of nucleic acid in the rabbit and in rabbit livers with advancing stages of development, both before and after birth. NUCLEIN-PHOSPHORUS IN RABBIT EMBRYOS AND RABBIT LIVERS Masing (1911) Stage of development 18 embryos 0.5—1.5 cm. long from 1st half of gestation; total N about 21 mg 2 embryos averaging 21.5 gm. in weight; about the beginning of the 4th week of gestation 1 embryo at same stage as the last; considerably less N 2 embryos somewhat older 5 livers from stage somewhat later than the other livers 4 embryos (5 livers) about 1-2 days before birth. 2 embryos, mature Young just after birth (small breed; 4 livers) 11 days old animal (skin and stomach contents removed) 2 livers of 11 days old animals 2 livers of 22 days old animals Liver of a full-grown animal Ditto Average Nuclein P Average weight of of whole weight of animal animal for 0.35 gm. N liver Grams Milli- grams Grams 20.3 21.5 17.8 2 22.5 17. 28. 14.7 28 1.8 36 13. 2.6 43 12. 33 11.7 2.3 57 11.9 72 4.0 210 9.5 1800 65. Nuclein P of liver for 0.35 gm. N. Milli- grams 22.8 20.4 18.0 17. 16. 12 11.5 10. PHOSPHORUS METABOLISM 139 Scaffidi (1908) made nucleoprotein determinations on the livers of rabbits in 'connection with his study of the iron distribution in the liver after feeding an iron paranucleinate called "triferrin," This paranucleinate did not affect either the nucleoprotein content of the liver or the phosphorus content of the nucleoprotein, as it did ap- parently the iron. The amounts of nucleoprotein found in the livers of the several rabbits range from 0.3595 to 1.2334 percent of the liver, and the phosphorus content of the nucleoprotein from 2.51 to 2.83 percent, while the iron content of the nucleoprotein in the con- trol animals ranged from 0.18 to 0.44 percent, averaging 0.30 per- cent, and that of the experiment animals from 0.34 to 1.10 percent, averaging 0.67 percent. PHOSPHORUS IN SPLEEN Phosphorus Content. Kriiger (1895) examined spleens and livers of cattle fetuses, calves and adults . The phosphorus content of the spleen was found to be highest when the fetus was 30-60 cm. in length, and lowest in the adult animal. In the adults, livers and spleens gave about the same figure. AVERAGE PHOSPHORUS (P) CONTENT OF THE SPLEEN OF CATTLE OF DIFFERENT AGES (Kriiger, 1895)— Percent Dry Basis Fetuses, length in centimeters Calves Cows Oxen 30-40 2.38 40-50 2.43 50-60 2.39 60-70 2.13 70-80 1.94 80-90 1.70 90-100 1.48 1 .82 1.26 1.37 Dhere and Maurice (1910) find a steady decrease of phospho- rus in the spleen of dogs with advancing age. INFLUENCE OF AGE ON THE QUANTITY AND PARTITION OF PHOS- PHORUS IN THE SPLEEN OF DOGS Dhere and Maurice (1910) Num- ber of dogs Phosphorus, percent of fresh substance Phosphorus, percent of dry- substance Percent of total phosphorus Age Lipoid Nucleic Inorganic Few hours to 15 days 21 9 6 4 0.35 0.34 0.29 0.25 1.70 1.61 1.32 1.09 13.55 16 75 8.67 7 85 77 78 4 weeks to 4 months 75 40 6 months to 15 months 16.91 6.57 21 17 ii ns 76.52 67 78 Partition of the Phosphorus of Spleen. A. Kossel (1882) sub- mitted the following figures for total and nuclein phosphorus in spleen. 140 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 TOTAL AND NUCLEIN PHOSPHORUS IN SPLEEN— (Kossel, 1882) Percent Species H 3 P0 4 in fresh organ Nuclein H3PO4 Nuclein H3PO4 in total H3PO4 Cattle 1.162 0.837 1.005 0.867 0.593 0.636 0.676 74.6 70.9 63.3 67.3 J- Duplicates Liebermann (1893a) found considerable of lecithalbumin in spleen. Gourlay (1894) isolated from spleen a substance he called nucleoalbumin. Phosphocarnic acid, phosphatids and nucleoproteins are found. Besides the jecorin (Waldvogel and Tintemann, 1906), which is free from iron, Burow (1910) found three iron-containing phosphatids in the spleens of cattle and of men. The presence of iron in the lipoids of spleen is considered significant in connection with the view that the chief physiological function of the spleen, aside from leucocyte formation, may be iron metabolism. Capez- zuoli (1909a) found considerable iron in the nucleoprotein. Ac- cording to Sato (1909), the iron of the nucleoprotein is present in two forms, one more stable than the other. Sato (1909) obtained 0.709, 0.406, 0.481, and 0.498 percent nu- cleoprotein from the spleens of cattle. Corper (1912) reports that the spleen of the dog contains 6-7 percent lecithin; about 0.27-0.52 percent water-soluble phosphorus and about 0.39 percent insoluble phosphorus. _1_ Panella (1904c) made water and nucleon determinations on the fresh spleen of cattle, horses, sheep, swine and dogs, and found it. present in all in amounts varying somewhat with the species. The nucleon content of the spleen of cattle, sheep and dogs seems to de- crease with increasing years. The values found are as follows : WATER AND NUCLEON CONTENT OF FRESH SPLEEN— Percent Animal Water Nucleon Fresh subs. Dry subs. Cattle ' 78.88 78.64 77.27 78.92 78.79 0.31 0.36 0.40 0.57 0.67 1.46 1.17 1.76 2.70 3.16 PHOSPHORUS IN THE PANCREAS Phosphorus Content. Liining (1899) reports the phosphoric acid of the ash of the pancreas of two aged women as 46.99 and 45.29 percent, respectively. See also Gossmann (1898) and Juchler (1912). PHOSPHORUS METABOLISM 141 Juchler (4913) found phosphorus in the pancreas of adults in amounts varying within narrow limits, apparently mainly in organic condition. The percentage of total P 2 5 in the fresh pancreas at various ages is as follows : New-born 2.36 ; 7 mos.-5 years 1.03 ; 20 years-48 years 0.55 ; 50 or more years 0.42 ; adults in general 0.48. Partition of the Phosphorus. Frankel, Linnert and Pari (Frankel and Pari, 1909; Frankel, Linnert and Pari, 1909) have made a special study of a phosphatid found in the pancreas of cattle. Plimmer and Kaja (1909) show that a prominent feature in the phosphorus changes in the pancreas, accompanying the secretion of pancreatic juice, is the destruction of phosphoprotein, which de- creases from about 2.5 percent of the total phosphorus to less than half of one percent of the total phosphorus. The total phosphorus of the pancreatic juice is very small in amount. Its total protein phosphorus is phosphoprotein, and inorganic phosphorus is also present. It contains no lecithin. Below are the analytical data on this matter. PHOSPHORUS (P 2 5 ) CONSTITUENTS OF PANCREAS AND PANCRE- ATIC JUICE— Percent Condition of Phosphorus Dog's pancreas, normal Dog's pancreas after action of secretin Pancreatic juice of dog I II III IV I II III I II III Ether soluble (lecithin) Water soluble (nucleic acid-(- inorganic) . . Protein (nucleo- protein -(-phos- phoprotein) Phosphoprotein . 8.6 37.6 16.4 53.8 2.5 28.5 15.5 7.1 56.0 2.6 30.6 17.4 7.8 52.0 1.6 28.6 14.4 6.4 57.0 1.9 31.1 16.2 7.3 52.7 0.4 28.0 19.8 6.9 52.2 0.3 31.0 16.2 6.7 52.8 0.0 0.0 56.3 present 43.7 43.7 0.0 86.3 trace 13.7 13.7 0.0 59.8 trace 36.7 40.2 A. Kossel (1882) reports the total phosphorus (H 3 P0 4 ) of the pancreas of cattle as 1.257 and 1.215 percent, the nuclein phospho- rus 0.580 and 0.606 percent, and the percent of nuclein phosphorus in the total phosphorus as 46.1 and 49.9 percent. These pairs of figures are duplicate determinations. PHOSPHORUS IN KIDNEY Phosphorus Content. From Gossmann (1898) we compute the percentages of phosphoric acid in the kidneys of man and steer, as follows : man 0.107 percent ; steer 0.401 percent. Forbes (1909) found in the kidneys of swine phosphorus vary- ing between 0.205 and 0.318 percent and in the ash 18.47-27.90 per- cent. 142 OHIO EXPERIMENT STATION: TECHNICAL BTJL. 5 Partition of the Phosphorus. A. Kossel (1882) found in bovine kidney 0.7584 percent H 3 P0 4 , 0.285 percent nuclein H 3 P0 4 and 37.6 percent nuclein P in the total P. Lonnberg (1890) reports finding nucleoalbumins in the cortical substance and the medullary substance of kidney, and in the mucous membrane of the urinary bladder. Liebermann (1891b, 1893a, 1893b) found in the parenchyma of the kidney a compound which he called lecithalbumin, and which, after removal, had such properties as to suggest that in the tissues it might be the agent by which the kidney excretes acid urine from alkaline blood. The substance in the kidneys which others had called protagon Panzer (1906) states to be a cholesterol compound not containing phosphorus. Balke and Ide (1896) obtained 0.043 percent phosphocarnic acid from the kid- ney of the horse and 0.205 percent from that of the dog. Lusena (1903) gives the lecithin content of the kidneys of rabbits as aver- aging 2.086 percent for the normal organs and 1.856 for organs af- fected by experimental fatty degeneration. Rubow (1905) reported the following lecithin phosphorus figures for the kidneys of dogs in various conditions. LECITHIN AND FAT CONTENT OF THE KIDNEYS OF DOGS Rubow (1905) Percent, Fresh Basis Condition of dog Kidney Ether extract Lecithin Fat j Right 1 Left j Right 1 Left j Right extirpated 1 Left J Left extirpated 1 Right j Left extirpated 1 Right j Left extirpated 1 Right 13.81 13.63 15.21 14.99 19.46 21.28 14.06 14.97 15.04 15.74 13.61 13.13 15.65 15.79 12.49 15.33 6.60 6.86 8.66 8.63 7.57 7.95 7.87 7.39 8.55 8.32 7.89 7.90 12.86 14.42 5.40 6.34 7.47 Phosphorus-poisoned, about 3 mg. per kg. body weight, injected.. Phosphorus-poisoned, about 3 mg. per kg. body weight, injected.. Under chloroform for 5 hrs. two 7.79 5.74 5.74 7.10 7.47 4.60 Under chloroform for 5 hrs. two 7.43 Dunham (1903-4, 1904-05, 1905-06, 1908) and Dunham and Jacobson (1910) isolated and studied a peculiar phosphatid found in the kidneys of cattle, which they call "carnaubon." Frankel and Nogueira (1909a, 1909b) describe three unsaturated phosphatids. MacLean (1912a, 1912b, 1912-13) has made painstaking examina- tions of the phosphatids of the kidney of the horse and finds the chief one to be a lecithin, or at least a phosphatid having the N:P relation 1:1. He finds also cuorin and a diamino-monophosphatid which he considers as probably the carnaubon of Dunham and Ja- cobson freed from a nitrogenous impurity present in their product. PHOSPHORUS METABOLISM 143 PHOSPHORUS IN SUPRARENAL CAPSULES Paul Mariasse (1895) isolated from the adrenals of the horse ,and of cattle a sugar-yielding phosphatid supposed to be jecorin. Orgler (1904) reports a substance similar to the so-called protagon of the brain (0.6 percent of the substance) , and Rosenheim and Tebb (1909b) found in the adrenals a small amount of the sphingomyelin such as they consider a prominent constituent of the protagon of brain. Bernard, Bigart and Labbe (1903a, 1903b) identified as lecithin, or a mixture of lecithins, the distinctive fat occurring in the spong- iocytes of the suprarenal capsules and increasing during the func- tioning of the organs. It is looked upon as a secretion of these cells. The phosphorized fat was found to be in the horse 45.3 per- cent, in the sheep 48.8 percent, in the rabbit 52.7 percent and in man (one case) 13.1 percent of the total fat of the organ. The total amount found was in the horse 6.77 percent of the gland and in man 2.08 percent. The following are Alexander's (1892) determinations of the lecithin and the nuclein phosphoric acid. LECITHIN AND NUCLEIN PHOSPHORIC ACID DETERMINATIONS ON THE ADRENALS OF THE HORSE (Carl Alexander, 1892) Age of Lecithin Nuclein phosphoric acid horse Percent of fresh substance Percent of dry substance H 3 POt, percent of fresh substance H3PO4, percent of dry substance 16 years. . 3 years . . 2 years . . 2 years . . 4.2973 2.81383 2.573 3.4502 26.018 16.6114 16.071 21.964 0.7781 4.592 PHOSPHORUS IN THE THYMUS Lilienfeld (1894) reports a general chemical examination of leucocytes from the thymus of calves. He found a nucleoprotein, protagon, inosite, monopotassium ^ phosphate, lecithin and nucleo- histone. The total phosphorus is' given as 3.01 percent of the dry substance, the lecithin as 7.51 percent and the nuclein as 68.78 per- cent. Huiskamp (1901a) says that 69.4 percent of the proteins of the thymus are in nucleohistone and 18.7 percent in nucleoprotein, leav- ing only 11.9 percent of other proteins. Malengreau (1900, 1901) states that there are two nucleoalbumins and two histones in the thymus. Herlitzka and Borrino (1902, 1902b, 1903) try to distin- guish the parts played by the nucleohi stones and the nucleoproteins of the thymus, the liver and the kidney. 144 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The nucleohistone of the thymus has been given considerable attention. (See Lilienf eld, 1894, 1895 ; Bang, 1900a, 1900b ; Kossel, A., 1900 ; Malengreau, 1901 ; Huiskamp, 1901a, 1901b, 1903; Herlitz- ka and Borrino, 1902a, 1902b, 1903). A nucleohistone is a nucleo- protein in which the nucleic acid is united with one of the simple proteins, a histone. The result is a nucleoprotein differing from most of the members of this group through the content of a larger proportion of nucleic acid and relatively little protein. According to Goubau (1911) it is characteristic of little-differentiated cells. There seems to be present in the thymus at least one nucleoprotein of this type and one of the type containing relatively less nucleic acid. It will be observed that a very large proportion of the proteins of this gland are united with phosphorus in one or the other of these ways. Goubau finds nucleohistone much more abundant in thymus than in any other mammalian organs, and says that this nucleohis- tone of the thymus is of a different type from that of the other or- gans ; it is insoluble in physiological salt solution. It is not found in human or horse serum nor in ascitic fluid containing bile. It is prob- able that the histones and nucleohistones are fixed in such organs as the thymus and the spleen, and hence cannot be carried away in body fluids. PHOSPHORUS IN THE THYROID According to Gourlay (1894), the only protein of the thyroid is a nucleoalbumin which, from microchemical examination, he con- cluded is derived, at least in part, from the colloid matter of the acini. Baldoni (1899-1900) gives these phosphorus and nucleoprotein determinations on thyroids of different animals. ANALYSES OF THYROID GLANDS— (Baldoni, 1900) Percent Species Water Ash P2O5 in ash P2O5 in fresh substance* Nucleoprotein in dry substance* Nucleoprotein in fresh substance 68.09 74.71 69.79 70.81 73.30 73.79 0.92 0.74 1.00 1.03 0.92 0.98 12.59 23.24 12.48 13.59 14.66 16.31 0.116 0.172 0.125 0.140 0.135 0.160 5.55 3.49 4.15 3.43 3.69 4.45 1.771 Cattle (Tuscan).. Cattle (Maremma) Buffalo 0.883 1.254 1.001 0.985 1.166 * Computed by F. & K. Markotum (1895) describes from the thyroid of cattle a phos- phorus-containing substance "thyreonucleoalbumin," which is said to have the following composition: C 51.46, N 15.56, H 6.94, P 0.32, S 1.5 and O 24.12. Markotum says that the alteration of the nerv- ous system by the thyroid is supposed to be through this compound. PHOSPHORUS METABOLISM 145 Oswald (1899) finds that the active agent of the thyroid gland as used in pathological cases, the so-called thyroid gland colloid, is a mixture of two compounds. The iodine-containing body, thyreo- globulin, which was shown to have the characteristic influence on nitrogen metabolism when the gland was administered to dogs, is free from phosphorus; but it is associated in the colloid with a small amount of nucleoprotein containing 0.16 percent phosphorus. Fenger (1913), in connection with an extensive study of the iodine content of the thyroid glands of animals in both the adult and the fetal stage, has found with regard to cattle thaf'enlarged glands in general, both fetal and adult, contain less total iodine and much more total phosphorus than normal thyroids/' and that "normal fetal glands are relatively larger and contain more iodine and phos- phorus per unit of body weight than thyroids from fully mature an- imals." In a later paper Fenger (1914) reported studies on the com- position of the thyroid gland in cattle as affected by sex and preg- nancy. Among his conclusions are the following : "Castrated males contain less thyroid tissue than either un- castrated males or females, but the iodine content per unit of body weight is about half-way between the uncastrated male and female animals. "The phosphorus content of the thyroid gland seems to be fair- ly uniform in all four cases and should only be considered as indi- cation of normal physiological activity." From the analytical data we select the following: PHOSPHORUS AND IODINE CONTENT OF THYROID GLAND OP CATTLE Phosphorus in fresh gland Mg. Iodine in fresh gland Mg. Fresh gland tissue per 100 lbs. body weight Grams Phosphorus per 100 lbs. body weight Mg. Iodine per 100 lbs. body weight Mg. Bulls Pregnant cows Non-pregnant cows and heifers . . 30.66 23.97 21.08 20.52 19.38 19.73 18.50 16.98 2.14 1.92 2.44 2.44 2.19 2.00 2.11 2.22 1.38 1.64 1.85 1.84 PHOSPHORUS IN THE LUNGS By Sieber and Dzierzgowski (1909) the mixed lung tissue of several horses was found to contain 76.6 percent water, and of the dry substance 1.107 percent was phosphorus; which equals 0.259 percent in the fresh substance. 146 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The compounds which Liebermann (1893a) studied and called lecithalbumins were found abundant in lungs. According to Sieber (1909b), the phosphatid extract of lungs (horse) includes lecithin and jecorin in the approximate proportion 6:1. A cholesterol-like substance was also isolated. There is a discussion by Zoja (1894) on the lecithin of the alveolar cells of the lungs and the semeiological significance of the myelin drops of the sputum. PHOSPHORUS IN THE HYPOPHYSIS According to the analyses of Malcolm (1904), the pituitary gland contains in its "glandular portion" 0.72 percent P and in its "nervous portion" 0.8 percent. PHOSPHORUS IN THE DIGESTIVE MUCOSA After peptic digestion of the mucous membrane of the stomach, Liebermann (1891a) obtained a lecithin-albumin complex which he thought existed as such in the walls of the stomach and there func- tioned significantly in the control of stomach acidity. Borri (1906) studied the phosphorus compounds of the mucous membrane of the intestine (dog) , distinguishing nucleoprotein, phosphorized fats, lecithalbumin and perhaps some other phos- phorized organic body as well as inorganic phosphate. He says that there are no nucleoalbumins present. The phosphate content of a water extract of the membrane was distinctly less after fast. The ingestion of oleic acid after a three-days' fast increased the phos- phate content of the extract, which is interpreted as supporting the idea that glycerin to unite with the fatty acid was obtained from the lecithalbumin of the membrane. Araki (1903b) isolated nucleic acid from the mucous membrane of the small intestine to the extent of 0.66 percent. PHOSPHORUS IN THE GENITALIA Paton and his associates (Paton, 1898; Paton, et al., 1897-98), in their extensive study of the metabolism of salmon in fresh water, gave considerable attention to the forms and amounts of phosphorus present in the genitalia and muscles of salmon at the time of leaving the sea in May, and again in October after the long fast in fresh water, during which time the reproductive organs and products mature. The muscles were shown to have the phosphorus most largely present as inorganic phosphates, while the ovaries had greater amounts of lecithin and of phosphoprotein (ichthulin) , and the testes had more than a half of it as nuclein. The muscles lose phosphorus while the genitalia gain it. The following are given as representative figures showing the distribution of phosphorus in each of the three classes of tissue. PHOSPHORUS METABOLISM 147 DISTRIBUTION OF PHOSPHORUS IN THE MUSCLES AND GENITALIA OF SALMON (Paton, 1897-98)— Percent Tissue No. of fish In ether extract (lecithin) In water extract (inorganic phosphates In residue (nucleins or pseudo- nucleins) Total Muscle, thick . . Muscle, thin. . . Muscle, thick.. Muscle, thin. . . 14 14 76 76 14 76 53 68 0.0416 0.0460 0.060 0.060 0.114 0.150 0.063 0.060 0.131 0.094 0.095 0.119 . 0.057 0.075 0.068 0.040 0.056 0.041 0.055 0.063 0.114 0.189 0.161 0.178 0.228 0.181 0.210 0.242 1.285 0.414 0.292 0.278 Estuary fish The "thin" muscle is said to make up about K of the body Estuary fish taken later in the year Testis Testis Milroy (1908) has since made a similar study of herring. The percentage content of phosphorus (P 2 5 ) in ovaries, and the weights of phosphorus per average fish found in these organs are averaged for the months of the year as follows : PHOSPHORUS (P2O5) IN GENITALIA OF THE HERRING— (Milroy, 1908) 3 £ a fao Condition 3 3 a OOP til OQ OP . > a PIS ctf £ ft Pen is +> £> a; focc Ovaries P2O5, percent 077 1.23 0.71 0.93 0.91 1.03 1.19 0.93 0.91 P2O5, per fish 0.003 0.011 0.01 0.05 0.26 0.48 0.34 0.16 0.016 Testes P2O5, percent P2O5, per fish 0.86 0.20 2.10 1.21 Plimmer and his associates (Plimmer and Scott, 1908 ; Plimmer and Kaya, 1909), in their study of the distribution of phosphopro- teins in animal products, found them chiefly in milk, egg-yolk and the ova of fish ; but they were present also, in smaller proportions, in the pancreas and salivary glands. They do not appear in the thymus nor in the testes of the bull or the codfish. Determinations were made also of the proportionate amounts of the different kinds of phosphorus compounds in the testes of codfish and in the eggs of the frog. DISTRIBUTION OF PHOSPHORUS IN CERTAIN TISSUES Plimmer and Kaya (1909)— Percent of Total P 2 5 Water sol- Protein Ether uble (nucle- (nucleo-pro- Substance Animal soluble ic acid and Inorganic tein and Phospho- examined (lecithin) inorganic) phospho- protein) protein Codfish 17.8 22.6 20.5 59.6 , . Codfish 8.9 31.9 20.4 59.2 Eggs, ovarian .... Frog 26.2 4.3 69.5 61.9 Eggs, soon after being laid .... Frog 19.1 13.0 Trace 67.9 41.1 Eggs, soon after being laid .... Frog 21.4 9.6 Trace 69.0 40.9 148 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Panella (1903d, 1903i, 1904a) found, of phosphocarnic acid, 1.1733 percent in the dry testicular substance of the ass, and 0.871 percent in that of the horse. Konig and Grossfeld (1913a), in considering the food value of fish sperm mention the high phosphorus content (4.46 percent of the ash-free, dry substance of herring sperm and 3.74 percent of that of carp sperm), and the high content of lecithin (20.7 per- cent of the fat of herring sperm and 20.2 percent of that of carp sperm). In a second article (1913b) they report the phosphorus content of the ichthulin and albumin and the lecithin content of the fat of the roe of several species of fish. The dry, ash-free ichthulin from herring roe contains 1.56 percent and the albumin 1.05 percent phosphorus, while the lecithin content of the fat of herring roe was reported as 43.61 percent. Rosenbloom (1913a) reports determinations of the percent- ages of lipins, phospholipins, neutral fat, fatty acids and cholesterol in the ovary and corpus luteum of the cow in the non-pregnant state, and in the pregnant state. The results indicate that there is prac- tically no increase in the above substances during pregnancy in the cow. The phospholipins amount to 3.94 percent of the dry sub- stance in the non-pregnant ovary and 3.48 and 3.63 percent at two stages of pregnancy ; those of the non-pregnant corpus luteum were 14.10 percent, and of the pregnant, 14.90 and 14.87 percent of the dry substance. Bienenfeld (1912), Ballerini (1912) and Sakaki (1912, 1913a, b, c and d) have reported special lipoid studies on the placenta. Sakaki isolated two phosphatids, sphingomyelin and jecorin. Spermatozoa heads, being mainly nucleus, are composed largely of nucleic acid, and this is, in the case of several fish at least, com- bined with protamin or histone in a compound more simple than most nucleoproteins of the animal body or its products. This relation has been especially studied by Miescher (1878, 1896, 1897) and by Mathews (1897). The heads are apparently free from lipoids, but the tails contain considerable amounts, and largely as a phosphatid which is reported as lecithin. Gobley (1850b) also found the leci- thin here. Mathews reports that jecorin is not present. The ash of spermatozoa is said to be mainly (three quarters) potassium phosphate. The seminal fluid contains nucleon, and its mineral matter is chiefly calcium phosphate and sodium chloride. PHOSPHORUS METABOLISM 149 Percival-(1902) made phosphorus studies on a number of ani- mal organs. The total phosphorus figures on the oxidized tissue are as follows, being given as percent of P 2 3 in the fresh substance, and arranged in order of decreasing amount of phosphorus : Thymus 1.223; heart (sheep) 1.011; pancreas (sheep) 0.749; lung (sheep) 0.745 ; brain (sheep) 0.635 ; spleen (beef) 0.570 ; liver (beef) 0.561 ; testicles of calf 0.517; muscles (beef) 0.506; testicles of bull 0.470; kidney (sheep) 0.454; ovary (cow) 0.429; udder (cow) 0.414; thy- roids (sheep) 0.369. For the phosphorus content of the ether extract of many gland- ular organs see Gerard and Verhaeghe (1911). PHOSPHORUS OF THE BLOOD Kinds of Phosphorus Compounds Present. The blood was, naturally, early recognized as an important part of the animal or- ganism the chemical composition of which might be of great signi- ficance. In some cases the blood was ashed, and determinations of mineral constituents made on the ash ; but in other cases the indi- vidual organic constituents were noted. Ash analyses are not very enlightening, and quantitative determinations of the individual com- pounds cannot be interpreted with definiteness unless the corpuscles and serum be examined separately. Phosphorus is present as inorganic salts, chiefly potassium phosphate and calcium phosphate, and in organic combination as phosphatids and nucleoproteins or nucleins, and, according to Pan- ella, as phosphocarnic acid. In 1851 Gobley (1851) found lecithin in blood. "Protagon" studies were reported by both Herrmann (1866) and Hoppe-Seyler (1866a). One of the first findings of nucleoprotein was in blood corpuscles (Plosz, 1871). Pribram (1871) made phosphorus and calcium determinations which distinguished between the amounts precipitable directly from the defibrinated and centrifuged blood serum and that obtained in precipitable form after ashing, or, as it may be interpreted, between the calcium and phosphorus present in inorganic form and the total present. The indication was that none of the calcium was in organic union, while more than two-thirds of the phosphorus was in such a form. Fokker (1873) reported find- ing calcium acid phosphate bound to protein in the blood serum of the ox. Nucleoprotein is found in the blood plasma and the serum as well as in the corpuscles ; of leucocytes it forms the chief constitu- ent. (See Plosz, 1871; A. Kossel, 1881, 1893; Halliburton and Friend, 1889 ; Halliburton, 1895 ; Lilienf eld, 1894; Pekelharing, 1895; Bang, 1903, 1904; and Liebermeister, 1906). 150 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 . The amount of phosphocarnic acid was studied by Panella (1902c). He found an average of 0.3631 percent in the blood of dogs, 0.282 percent in the blood of rabbits and 0.0674 percent in the blood of calves. The most frequently quoted early analyses of the blood of dif- ferent species, including human subjects in different conditions, are those of Becquerel and Rodier (1844), Jiidell (1868), von Bunge (1876), Jarisch (1877) and Miroczkowski (1878). We have selected from the data of a number of authors items showing the amounts in the blood, of the different phosphorus com- pounds, and of a few other constituents which may be considered in connection with the same. Some of these are given in connection with studies on the changes brought about in pathological condi- tions, and are included in the tables with such pathological data. The following table of Abderhalden's is quite unusual with regard to the number of species considered and the completeness and care with which the analyses were made. Abderhalden has tabulated these analyses to show the amounts of the individual constituents contained in 1000 parts by weight of blood, in 1000 parts of serum and in 1000 parts of corpuscles. He calls attention to the similarity of composition of the serum from the different animals, and the great similarities of composition of the blood of two animals of the same species. It is also very evi- dent that the bloods of the three kinds of ruminants (cattle, sheep and goat) agree much more closely with each other than with that of the carnivora (dog and cat) or with that of the horse, pig and rabbit. "The ruminants show a considerably lower phosphoric acid content than the carnivora or the horse, pig and rabbit. Perhaps in the ruminants there is an organic acid present with the phospho- ric acid." PHOSPHORUS METABOLISM 151 =? 4 2 of.co co on en on If* OS CO if. CD CO 00 M • COM oo if. -3 co os os ; Ol-q 00C0CO COOSMCOO MMCO . M MCO M ©©©• ©O©© • • MO- 00C0O1M toono- enco-q o • CO OS- -aonooco i^mm; if.cocnco ; ; coo; OS OSOl COlfs. If.. -3OSO100 • . COM ©CO©©; ©CO© ppt-ippp- m if* b b • m bo b co b m b bo b • oioto-a; ostoM to >f. to to to os ; if. co os oo. o.tf-M coo-^ooco OsGQ C3 -*3 2 **£ o tog 2 M o ©3 to as o oo 3 f co M CO > >TJ 8 M ^ M s» LJ Hi! W 8 f 3 o 00 O ^§ *2 !» fO _ HH M 02 © CO O »fl o 9 u_i | — i 3 H © GO o _ &- O tr* co 152 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 _ & g- a m m i p ! p£ , b-S <= 2 _ P*£2.2 B\S S » ° » o°9 £,s° B o '" g So "S o P'.—.p'tr' hi i-s O. . ffl «■£- p 5 p 2 5 tO Ol © • if- tO © © Ol © O ■ tD tO Ol tO tO © Ol : tO 00 tf»- M O -1 • O ID O to to to • CO © en O1C0. O 0(000 ©10© OOM©©©- HWOO. M©© ©-a tooo • -cioo© O^OOHl -q if>.-q to -q Oi to Ol © ©©.. ©©©© tooio- Oi to-q o tO-q©- lf>-CO© to GO Ul ©. Ol © © CO MM- tO tO OOO 00 00 00 CO tO^- 1>0(© too. ©to©© ©to© HOiOO- H(DO © M tO Qo; -qHM CO©-q©I tO -3 © }H 00 © 00 -J Ol • ©Hi ©Oloo©©©- CO© O'O 00 MOO o©o. ©o©o to©coto MM. O CO tO tO tO M • 00 M 00 © too- toOOiCn ©ol to o too©- © too MlHO©- MOO ©OOCOOO" -qoo (H OOOO: MOO 00 ©©©Ol tDtH to w OHOO00 P* © M 00 V] tO ' ©©©• ©op© to © O • Ol to bo © otopp. ptoo !_i t- © o : m bo © oioi to■ CO 10 Ol © £- • ©© © Ol rf^ © -q M * ©CO if»--4>f*©© ; ©to bo © MtOCO co©o to ©©If**"- M tO- © ©: Ol©. © pMOO; ©t-3© © M -1 © © ■ MOO CO MO! to en* tooo© CO tO tO M tO I CO Ol © © O0C0MCO -J CO If. "ooppp. ©ih P- © GO M Ol © ' © tO OM©MOltO- -5C0 f*HcooH : CO © ©OO- ©M- MMbS to tOOl 1^- © • M © • O Ol CO CO OlCO. o O- © to- O00 CO 00 ©to- en ti' co ©; Ol lf» -3 toco! © tt-- i— -'• O ©top©; pt-SO m to b © • M © © ^ iH©to©; oito© 01 C" M S © • to if". tO C0Ol©CO00©. MM © O Cn M © Ol ' Ol if* cotOMCo-a : «5P -»9 2 .JO °" »d o Mg p. On too'n ©cc oo2 cog 03 e o t°g P" wg 2 ,«2 -*P pp cog «S? fr- >■ Hi GO H CO O *J a H CLfH re o s-O S.O ^^ 2 O co <1 •"0O 3d oH o G0 tdO o GO o o o f o H O PHOSPHORUS METABOLISM 153 Weiske (1886) analyzed the blood of lambs in connection with a study of the effects of ingestion of acids. From this work the fol- lowing data are quoted. . INORGANIC ELEMENTS OF LAMB'S BLOOD Weiske (1886) Percent, Dry Basis Animal P N K2O Na20 CaO MgO Feed, 6 months 0.150 0.150 0.130 15.255 14.960 15.090 0.415 0.430 0.305 2.220 2.445 0.395 0.066 0.074 0.065 0.029 Trace Trace Normal hay, barley- Hay treated with di- lute H2SO4, barley Killed at beginning 1 of the experiment Lamb III For comparison with the data on blood of mammalia we quote the following figures for constituents of the blood of birds, from the work of Weiske. DETERMINATIONS ON THE BLOOD OF BIRDS Weiske (1889) Percent, Dry Basis Bird Mineral matter N CaO Mg-O P2O5 5.99 6.14 5.57 5.52 5.85 5.27 14.84 15.19 15.22 15.37 15.11 15.98 0.12 0.08 0.06 0.07 0.10 0.04 0.08 0.06 0.06 0.07 0.07 0.09 1.69 1.71 1.82 1.55 1.79 1.77 Fowl Fowl Fowl Bergell (1898b) gives the following values of the phosphorus content of the blood ash of cattle : Blood of calf, 8.36, 6.73, 7.83 per- cent P 2 5 ; of cow, 6.21 percent; of ox, 4.17, 5.06, 4.64, 5.66 per- cent. He thinks there is a significance in the ratio of serum phos- phorus to erythrocyte phosphorus. Burger and Beumer (1913b) found lecithin in the blood cells of man and sheep in minute amounts only. The phosphatids consist chiefly of sphingomyelin, with some cephalin, an ether-soluble diam- inomonophosphatid, and a water-soluble phosphatid. PHOSPHORUS COMPOUNDS IN MILK GENERAL COMPOSITION It is the idea of Duclaux (1884a, 1884b, 1884c, 1893, 1894) that in milk both casein and calcium phosphate exist partly in suspen- sion and partly in solution, the casein being, in fact, an equilibrium Note:_ For a general discussion of the constituents, properties and changes of composi- tion of milk the reader is referred to the review and summary of literature by Raudnita (1903); also to Basch (1903) and Kastle and Roberts (1909). 154 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 of the three conditions solid, colloidal and dissolved. The phos- phates in suspension are thought to be those of calcium, of magne- sium and of iron or aluminum, the calcium phosphate in suspension being about twice the amount of that in solution. Vaudin (1894a, 1894b, 1895, 1897b) finds alkali citrates in milk, which, aided by the presence of lactose, he considers essential for holding the calcium phosphate in solution. Richmond (1901) looked upon casein as being a compound containing sodium, calcium and phosphorus united in the milk with Ca 3 (P0 4 ) 2 , the salts of the milk including also both mono- and di- basic phosphates. Richmond considers that of the 0.75 percent of ash in milk 0.27 percent is salts combined with casein, the remaining 0.48 percent being salts in solution in the milk plasma. Soldner (1888) thought that the calcium united with casein is not a phosphate but the oxide only. Barille (1909a, 1909b), from microscopical and chemical exam- inations of milk, has put forth the idea that in it there are crystal- lizable compounds of the phosphates with carbonic acid which he calls "carbonophosphates," and which he considers beneficial for the assimilation of milk. Pasteurization, he states, precipitates and decomposes these carbonophosphates almost completely and hence is injurious. Sterilization with ultraviolet rays leaves the carbono- phosphates intact, and is recommended. The data may be of in- terest. CARBONOPHOSPHATES IN MILK AS AFFECTED BY STERILIZATION Barille (1909)— Grams Sample No. Phosphoric acid per liter, as P2O5 Carbonic acid per liter Milk analyzed Insoluble phosphates Soluble phosphates Carbono- phosphates In free state and as car- bonophos- phates As carbon- ates and dissociated carbonates From Department at 1 2 3 4 5 5+ 6 6+ 7 7+ 0.812 0.728 0.767 0.750 0.521 0.690 0.666 0.882 0.660 0.710 0.677 0.519 0.711 0.640 0.540 0.630 0.530 0.598 0.350 0.370 0.214 0.045 0.011 0.050 0.372 0.079 0.372 none 0.210 0.190 0.052 0.046 0.043 0.064 0.078 0.068 0.132 0.075 0.091 0.080 0.136 Commercial pasteurized ( Prom dairy at Paris Same pasteurized by us. .. From dairy near Paris Drawn in our presence Same pasteurized by us. . . From dairy in Paris 0.076 0.072 0.077 0.140 0.065 0.173 0.042 0.148 Same submitted to ultra- 0.107 PHOSPHORUS METABOLISM 155 Aside from inorganic phosphates and casein, in whatever com- binations they may be present, there are other phosphorus com- pounds reported ; a phosphatid, usually considered to be a lecithin, a nucleon similar to the phosphocarnic acid of muscle, and perhaps an- other phosphorized protein, the opalisin of Wroblewski (1894a, 1894b, 1898). He found opalisin present in abundance in human milk, in less quantity in mare's milk, and in very small amount in cow's milk. The relative amounts of the constituents in milk are not constant, as is shown by the data we submit, and the milks of dif- ferent species of mammalia differ. Naturally, a part, but not all, of the variations appearing in the tables are due to the use of differ- ent methods of determination. Schmidt-Mulheim (1883) compares the first and last portions drawn at milking. The last is always considerably richer in fat, but the fat-free serum does not vary much in composition. The table of Ellenberger, Seeliger and Klimmer (1902) compar- ing the composition of the milk of woman, cow and ass will serve to show the general composition of milk, and some of the variations with species. Ellenberger's special study was on the milk of the ass. (See also Ellenberger, 1899, 1902.) COMPOSITION OF ASS'S, WOMAN'S AND COW'S MILK Ellenberger, Seeliger and Klimmer (1902) — Percent Ass's milk Woman's milk Cow's milk Dry matter .... Albumin and globulin .... Fat 91.23 8.77 1.50 0.94 0.53 0.1 1.15 0.02 6.0 0.4 86.4 13.6 1.6 1.0 0.5 0.12 4.8 0.06 6.6 0.25 88.0 12.0 3.3 3.0 0.3 0.06 3.5 Lecithin Milk sugar .... Salts 0.05 4.5 75 We quote below the averages of Katayama's (1908) extensive analyses of the milk of different pure breeds of cattle, and of the milk of unimproved native cattle from different countries. (The averaging has been done in the main by the compilers). 156 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE COMPOSITION OF MILK OF VARIOUS BREEDS OF CATTLE Katayama (1908) — Percent Num- ber Dry sub- Pat Nitrogen Ash Lime Phosphor- Breed of sam- ples stance CaO ic acid P a O s Silesian red .... 3 13.39 4.63 0.580 0.705 0.152 0.216 Red-brown East 2 14.14 5.65 0.677 0.842 0.193 0.222 Scheinf elder 3 13.03 4.57 0.519 0.703 0.152 0.212 Voigtlander .... 3 12.39 4.19 0.543 0.778 0.162 0.209 Simmenthal .... 3 13.94 4.66 0.625 0.781 0.196 0.238 Black-spotted East Friesian 3 12.03 3.22 0.605 0.784 0.193 0.247 3 12.86 4.32 0.545 0.738 0.166 0.236 Wilstermarscher 3 12.37 3.87 0.524 0.699 0.155 0.200 Swiso . , 3 12.45 3.98 0.574 0.739 0.164 0.219 East Prussian 6 11.12 3.16 0.449 0.713 0.154 0.188 Average of these improved breeds .. 12.55 4.08 0.547 0.742 0.167 0.216 10.76 2.02 0.429 0.666 0.143 0.180 14.95 6.36 3.36 0.682 0.863 0.227 0.273 3 12.28 0.510 0.747 0.185 0.227 2 13.23 4.94 0.489 0.654 0.170 0.200 2 10.44 0.75 0.516 0.732 0.200 0.280 Dutch East Africa 6 12.40 4.08 0.680 0.708 0.176 0.225 Buffalo 1 9.69 4.37 0.336 0.467 0.150 0.153 Average of native 12.04 3.60 0.567 0.695 0.178 0.225 9.69 0.70 0.336 0.467 0.150 0.153 13.60 5.89 0.649 0.795 0.204 0.296 DISTRIBUTION OF PHOSPHORUS IN MILK As to the distribution of phosphorus among the constituents of milk we do not find close agreement in the evidence. Raudnitz (1903) gives the following figures for phosphorus in various conditions, which must be looked upon as approximations. They differ considerably from other estimations which we quote. PARTITION OF PHOSPHORUS OF MILK— (Raudnitz, 1903) Total P2O5 Percent Casein P2O5 Lecithin P2O5 Percent Preformed phosphoric acid Authors mentioned Species Percent Percent of total Percent Percent of total 0.24 0.047 0.135 0.0581 0.012 0.0117 about 21 25 8.2 0.005 0.006 0.005 0.18 0.034 rest about 79 72 nearly 90 Baginsky ers Schlossmann (1905b) compared different milks, and the follow- ing values show a contrast between human and bovine milk in the amount of phosphorus present in inorganic form. PHOSPHORUS METABOLISM 157 PARTITION OF PHOSPHORUS IN MILK— (Schlossmann, 1905b) Total P2O5 Gm. per liter Casein P2O5 Lecithin P2O5 Nucleon P2O5 Rest, as inorganic Species Gm. per liter Percent of total Gm. per liter Percent of total Gm. per liter Percent of total Percent of total Cow ,1.65 0.455 0.58 0.16 35 35 0.091 0.16 5.5 35 0.09 0.153 5.5 33 54 Marre (1911) finds nearly all of the lecithin of cow's milk in buttermilk. He states that pasteurization of milk decomposes the lecithin. Forbes, Beegle and Mensching (1913) found 56 percent of the phosphorus of skimmed cow's milk to be inorganic. The partition of phosphorus as given by Gilbert and Posternak (1903, 1905) is as follows : GRAMS P 2 5 PER LITER OF MILK Species Total In casein In lecithin In nucleon Total organic Woman .... 1.81 0.43-0.47 0.580 0.132 0.091 0.153 0.087 0.171 0.758 0.456 Stoklasa (1897) gives the average lecithin P 2 5 of cow's milk as 0.091 gm. per liter, or 5 percent of the total, and that of woman's milk 0.153 gm. per liter, or 35 percent of the total, which agrees with Schlossmann. Jensen's (1904, 1905a, 1905b, 1905-6) details, reported from 15 milk analyses, distinguishing between the phos- phorus of casein and that of insoluble or soluble salts, make the casein phosphorus about 25 percent of the total in cow's milk. Values reported by Paton, Dunlop and Aitchison (1899-1900) indicate that in goat's milk the phosphorus of casein and lecithin is about 29 percent of the total ; that of nucleon is about 16 percent, and the inorganic about 55 percent of the total. Sikes (1906) made about three hundred estimations of phos- phorus, and a somewhat smaller number of determinations of cal- cium, on human milk. The following are his conclusions : Phosphorus: 1. The average amount of P 2 5 in human milk during the first fortnight after the commencement of lactation is 0.0297 percent, the variations being between 0.0140 and 0.0522 per- cent. 158 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 2. The percentage of the non-proteid P 2 5 averages 0.0169, and of the proteid P 2 5 0.0124, for the total number of cases. For primipara and for multipara the numbers are 0.0160 and 0.0129, and 0.0183 and 0.0116, respectively. 3. The ratio of the proteid P 2 5 to the total P 2 5 averages 42.3 percent, but it varies between 14.7 and 77 percent. In primip- ara the average is 44.6 and in multipara 38.8 percent. 4. The chart for primipara shows that the total amount of P 2 5 increases up to the ninth day, while that for multipara shows a maximum a little earlier. After this in both the P 2 5 falls to a lower level. 5. The variations mentioned are chiefly due to the non-proteid PA. 6. The proteid P 2 O g does not vary much on successive days. This uniformity is rather more noticeable in multipara than in primipara. 7. If one assumes that the average amount of proteid in human milk is 2 percent, the numbers mentioned will give the av- erage amount of P 2 5 in it as 0.62 percent. For primipara the number will be 0.64 and for multipara 0.58 percent. Calcium. 1. The average amount of calcium in human milk in the first fortnight after delivery is 0.0301 percent. 2. In primipara the average is slightly lower than in multip- ara. 3. The major part of the calcium is combined with the proteid. The ratio of the proteid calcium to the total calcium is, on the aver- age, 84 to 100. 4. The variations of the calcium from the average stated are small. 5. The curve giving the percentages on successive days after delivery is more uniform than the similar curve of the percentages of P 2 D in the same samples. 6. When both the calcium and the proteid were estimated on the same samples, the calcium was found to average 1.06 percent of the total proteid. Njegovan (1913) investigated the lecithin content of milk by a carefully considered method, and concluded that the existence of lecithin in milk is doubtful. MINERAL CONSTITUENTS OF MILK We have brought together from various sources a number of individual or average determinations of the mineral constituents of the milks of different species, grouping them as to species. No at- tempt was made to consider the methods of analysis. PHOSPHORUS METABOLISM 159 1 ercent. Percent of ash. Mean of 2 d by compilers. er and Soldner, 1900 and 1902,1903. lactation period, tation period. 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Mean of 8. Some fi last drawn. n of 2. uding- traces of globulin. n of 2. Quoted by Schlossmann, ted by Schlossmann. rage up to time of doubling- weig rage after doubling- weigfht of yoi h salts in combination, n 3rd to 116th day of lactation. 3 0} >> w to 4) f V} o X •M ■a o X o a ' ent proportions ol hay and he er lOOcc. er lOOcc uted from 26. ige of many analyses. a; computed b 's tables. 's tables, compilers, by compilers, of 6 monthly o CO 15; 2 method: d from Konig d from Konig ad from 40 by 35; computed 100 cc. Mean n ■s .■as 3 ° » (4 X u V a is e^e-S a ft ft ft a n V ri ra viraO I? P ±> a <§ a s e^i 3 > >r s ff O S w§0) <«!£& s a a a <« n - 200020 .s "55 to CD in 50 i-it~o,H en en cm t~i— ii>-oj en cm Tft men CM rt CO CM CO COOOTflTti coo iA to u o XI -p O O to •d a M Buffalo Elephant Gamoose Cat Rabbit Guinea pig Guinea pig M^ 5 2 C3 ho O 5 CO Tf( 'o 4-> CO r-{CO CM en S P. CO O) C3SIO -■ CM cog 3 co cm"cm CM tS"" CM rH ?cn co XI CO iar-5 ■*•* gTf! co- co ,— ICN1 to 00 CO -f Tff l> to CnOOt- CO 00 CD < .5 A 1 ft f c o s ■d c — -c ftc ^1 • o • is : .."a ea C <« - gs,. §JsS W coy 1 bderhalden bderhalden bderhalden.... bderhalden llenberger. See grer and Klimm Drdas and de S to is o o 2 rt £ I I on Szontagh on Szontagh stertag-andZun anSlyke and Bo worth ;ach — extremes iach — mean igel and Frehn 1 5 > s e c fc as <<«(£ WW M E- t>t>WC!> ^MWSI 162 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Rogers (1909) gives the casein content of buttermilk as 2.4, of kefir 2.56-2.59 and of kumiss 0.77-0.85 percent. Engel and Frehn (1910), computing the casein content of human milk from the nitrogen loss of the milk on precipitation with acetic acid, report the examination of 31 samples from several wet nurses, at different times in the lactation period, and of four samples of mixed milk, from which they conclude : "The relative casein value of human milk undergoes wide variations, even with the same individ- ual, for which no laws could be recognized. The factors which have empirically been taken to be of great influence for the compo- sition of human milk, like the time of lactation, individuality, the phase of the emptying of the breast, or the length of intervals be- tween nursing, play no recognizable part in determining the casein content." PHOSPHATID CONTENT OF MILK The content of phosphatid in milk varies with the time of lac- tation, and is reduced by heating, as well as by skimming. Fetzer (1910) has noticed that the milk from animals suffering from mas- titis contains less lecithin than that from healthy animals. It was further noted that where a diminution in the lecithin content took place there was a corresponding decrease in the fat content. Very few of the studies have distinguished lecithin in milk from other phosphatids possibly present. W. Koch (1906) thought he identified both lecithin and cephalin in milk. Wrampelmeyer (1892) reports that 100 gm. of butter contains 0.0451 gm. P 2 5 , indicating 0.017 percent lecithin. PHOSPHATID OF MILK OF DIFFERENT SPECIES AS REPORTED BY VARIOUS AUTHORS— Reported as Percent Lecithin Date Species Author Cow Human Ass Dog Sheep Goat Mare Notes 1897 1900 1902 J-1908-j 1909b 1909 0.090- 0.113 0.054 0.05 0.0364- 0.1163 0.0765 0.0629 07) 0.0449 0.170- 0.186 0.058 0.06 0.0240- 0.0799 0.1329 0.0499 (10) 0.024' 0.0058- 0.0393 0.0165 (6) 0.17 0.0509- 0.1672 0.0833 (4) 0.0349- 0.0753 0.0488 (ID 0.0073- 0.0174 0.0109 (8) Gm. per 100 ccmilk Nerking and Extremes Average and no. of Kida analyses Mean of 4 NUCLEON CONTENT OF MILK The nucleon content of the milk of woman, the goat and the ass are shown by the following figures to be somewhat nearly the same, while cow's milk seems to contain only half as much of this constit- uent. PHOSPHORUS METABOLISM" 163 NUCLEON CONTENT OF MILK OF DIFFERENT SPECIES AS REPORTED BY VARIOUS AUTHORS— Computed as Carnic Acid, Percent or Grams per 100 c. c. Milk Date Species Author Cow Human Ass Goat Notes 1896 1896 1897 1897 1902 1905, 1908 0.0583 0.0502 0.0566 0.124 0.1315 0.120 0.1 0.110 by compilers. Gm. per 100 cc. Decreases on standing. Ellenberger, Seeligrer andKlimmer Average for year. Mean of 3 computed by the compilers. Blumenthal (1896) found that the 0.5021 gm. carnic acid (measure of phosphocarnic acid) in a liter of cow's milk decreased to 0.2430 gm. while the milk stood at room temperature for eight days. This is taken as indicating that phosphocarnic acid is a source of the decomposition products of milk. SOME COMPARISONS OF WHOLE MILK, SKIMMED MILK AND CREAM A considerable part of the lecithin of milk and a smaller pro- portion of the casein are removed with the cream ; hence skimmed milk is richer in casein and much poorer in lecithin than whole milk. Bordas and de Raczkowski (1902b), however, claim that the lecithin is not wholly removed in the cream, while Glikin (1909b) does not agree with them, and says that if the cream is completely separated by centrifuging the lecithin is also completely removed. Following are Bordas and deRaczkowski's analyses of milk, 3.200 liters of which gave 2.800 liters of skimmed milk and 0.370 liters of cream. PHOSPHORUS COMPOUNDS IN WHOLE MILK, SKIMMED MILK AND CREAM— Grams per 100 Whole milk Skimmed milk Cream 2.88 0.176 0.0044 0.0124 0.058 3.24 0.184 0.0013 0.0037 0.018 1.15 0.096 0.0252 0.0691 0.334 The figures below are from d'Hont (1890), who obtained 12 liters of skimmed milk and 3.5 liters of cream from 15.5 liters of whole milk. 164 OHIO EXPERIMENT STATION: TECHNICAL BUL. 6 ANALYSES OF WHOLE MILK, SKIMMED MILK AND CREAM Percent Whole milk Skimmed milk Cream Ash 5.05 4.70 3.50 0.79 0.22 0.226 0.025 5.05 3.62 0.788 0.214 0.22 21.95 3.32 3.02* 0.585 0.155 0.17 * Prom Kjeldahl nitrogen determination. Dornic and Daire (1910) report that buttermilk contains more lecithin than whole cow's milk, and more even than woman's milk. CHANGES OF PHOSPHORUS COMPOUNDS AS A RESULT OF HEATING 0E STANDING Baginsky (1883) found that heating milk to 120°, and de- hydrating as in Scherff' s method of preservation, did not decrease the ratio of phosphorus in casein to phosphorus in the whey. Bordas and de Raczkowski (1903) noted that milk heated to various temperatures over the free flame, on the water bath, or in the autoclave loses through decomposition a certain amount of its lecithin. Diffloth (1904) also studied the effects of heating on milk. He found that a like effect is produced by simply allowing the milk to stand. Comparisons were made of the phosphorus changes due to standing and to heating as in sterilization or pas- teurization. Total phosphorus was determined on the solids taken up by acetic acid, soluble inorganic phosphorus on the clear liquid left after complete coagulation, soluble organic on the same (this is interpreted as lecithin) , and the insoluble phosphorus by difference. According to these figures, and the author's interpretation of them, lecithin decomposition takes place even when the milk stands at or- dinary temperature, but heating causes a much greater change. The duration of heating seems to have a greater influence than the de- gree of heat ; but with the same length of time of heating the dim- inution of assimilable phosphates (formation of insoluble phos- phates and decomposition of lecithin) increases as the temperature increases. (See table on next page.) Kida (1909) reports the following percentages of reduction in the lecithin content of cow's milk as a result of heating for thirty minutes at different temperatures: 80°, 7.52 percent; 80°, 11.39 per- cent; 95°, 25.27 percent; 100°, 21.22 percent. PHOSPHORUS METABOLISM 165 Grosser (1913) finds that boiling milk 5-30 minutes does not al- ter the freezing point or the phosphorus content of that portion of the milk which passes through a Bechold filter — colloids are held back. PHOSPHORUS PARTITION IN COW'S MILK AS AFFECTED BY STAND- ING AT 15° C. AND AT HIGHER TEMPERATURES Diffloth (1904)— Grams P 2 O s per Liter of Milk Time of standing 1 Temper- ature Total phos- phorus Soluble inorganic Soluble organic Insoluble Increase in insol- uble Decrease in leci- thin 15°C 60° 60° 95° 95° 110° 4.58 4.58 4.58 4.58 4.58 4.58 4.58 4.58 1.92 1.92 1.96 1.85 1.81 1.82 1.79 1.75 2.12 2.08 2.01 1.90 1.60 1.50 1.47 1.38 0.54 0.58 0.61 0.83 i 1.17 1.26 1.32 1.45 o'.64 0.07 0.29 0.63 0.72 0.78 0.91 0.04 0.11 0.22 0.52 0.62 0.65 0.74 CHANGES IN MILK WITH PROGRESS OF LACTATION Cow's Milk. Houdet (1894) made a chemical study of colos- trum of the cow. From his conclusions we quote the following: "The composition of colostrum remains about the same from the time the liquid appears until after the birth of the young. In the days following the birth marked changes occur at once." Among the changes noted is this, "The calcium phosphate and other salts, abundant at first in both soluble and insoluble condition, diminish gradually until about the fourth or fifth day, when they show about the same content as in normal milk." In the main, the observations of Trunz (1903) correspond with this last statement of Houdet, though perhaps there was longer time in getting back to normal, (6-7 days for one cow). Trunz made complete analyses of the milk of two cows at frequent inter- vals (30 samples for each) throughout lactation. We have com- puted a few summary statements from the phosphorus data. For further detail the original article should be consulted. PHOSPHORUS OF MILK AS AFFECTED BY PERIOD OF LACTATION Trunz (1903)— Grams per 1000 Day of highest phosphorus content First 4 days Average of later values Cow No. Total P2O5 P2O5 in salts Number of samples Total P2O5 P2O5 in Mini- mum Maxi- mum Mini- mum Maxi- mum salts 655 674 Day of calving 1 Day after calving- 2.227 2.137 3.284 3.144 1.534 1.564 2.564 2.557 25 19 2.015 2.075 1.529 1.622 166 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 According to the statements of Kort (1899), based on a number of investigations, the content of cow's milk in mineral matter, par- ticularly in phosphoric acid and calcium, decreases more or less reg- ularly as the time of birth of the calf draws near, even in spite of decreased flow of milk ; the colostrum is rich in ash ; the percentage of phosphoric acid and calcium in the milk is lowest when lactation is at full height, though the absolute quantity is then the greatest. Schrodt and Hansen (1885) determined the ash composition of the milk of the same cows (10) at seven dates between January and September, the times of calving having been October 15 to Jan- uary 1. The cows were in stall from January to May, and in pas- ture from June to September. The phosphoric anhydride data, in percent of the ash, are as follows: January 1, 23.11; March 30, 23.11 ; May 20, 24.61 ; June 16, 22.41 ; July 26, 23.59 ; August 30, 26.51 ; and September 26, 25.41. Bordas and de Raczkowski (1902a) report the following figures, and say that both the phosphoric acid and the lecithin constantly fall off in amount after the time of calving, animals of the same breed, and fed in the same way, showing the milk of the first month to be highest in lecithin. PHOSPHORUS CONSTITUENTS IN MILK OF COWS AT DIFFERENT STAGES OF LACTATION (Bordas and de Raczkowski, 1902)— Percent Breed Jersey Norman- dy Date of calving Cow with calf July 4 July 1 June 12 May 13 Not with calf Casein content Total phosphoric Other organic phos- As glycerophos- 3.86 0.218 0.0049 0.0139 0.0654 3.17 0.204 0.0068 0.0198 0.0909 2.90 0.200 0.0044 0.0124 0.0582 2.89 0.164 0.0033 0.0100 0.0472 3.04 0.168 . 0.0033 0.0100 0.0472 , 2.30 0.148 0.0041 0.0116 0.0545 2.61 0.156 0.0031 0.0093 0.0436 Note: The date of taking the samples is not given; samples probably taken all on the same date. The general question of the influence of the stage of lactation on the composition and properties of milk has been studied by Eckles and Shaw (1913). They analyzed samples taken throughout the year from 11 cows of four breeds which were kept on a uniform ration of alfalfa and grain, and from 5 other Jersey cows not kept on uniform rations. The casein seldom went beyond the limits of 80 to 82 percent of the total protein, and averaged 81,4 percent. Both total protein and casein were abnormally high following PHOSPHOEUS METABOLISM 167 parturition, then declined continuously till the third or fourth week, when the minimum was reached. They then remained fairly con- stant until near the end of the lactation period, when they rose rap- idly and reached the maximum at the end of the period. Human Milk. Schlossmann (1905b) gives the following values for the total phosphorus in woman's milk at different times, indicat- ing no characteristic change with duration of lactation. 12 21 213 191 (right breast) 191 (left breast) 191 (right breast) 191 (left breast) Grams per P 2 Os per liter. . 0.65 0.56 0.52 0.37 0.35 0.51 0.34 Engel and Frehn's opinion that the wide variations in casein content observed in human milk are not due to the stage of lactation has been mentioned above (see p. 162). Edlefsen (1901) gives a table of the gross analysis of human milk at different periods. Case- in appears regularly to decrease with advance in period of lactation. ANALYSES OF HUMAN MILK AT DIFFERENT PERIODS OF LACTATION Edlefsen, (1901)— Percent Day of lacta- tion Total protein Casein Albumin Tat Sugar 3 12 48 103 116 2.695 1.875 1.00 0.843 0.835 1.810 1.160 0.44 0.375 0.310 0.885 0.715 0.560 0.468 0.525 3.225 3.035 3.64 3.415 4.105 3.59 5.15 7.06 5.835 5.95 Valenti (1905, 1908) found that the content of nucleon in hu- man milk is greatest during the first and second months of lacta- tion, generally decreasing from the third to the sixth month, in- creasing again during the seventh month, and then remaining con- stant until the twelfth month. RELATIONS OF THE COMPOSITION OF MILK TO THAT OF THE YOUNG VonBunge (1874), in connection with studies of the alkalis of milk and other articles of diet and of the young organism, intro- duced the idea which became known as Bunge's law that the compo- sition of the ash of milk (with the exception of the iron) corre- sponds closely with that of the ash of the total organism of suck- lings. VonBunge (1886, 1889) and his pupils Abderhalden (1899b, 1899c) and Proescher continued to investigate the question, considering first whether the ash of blood and blood serum also have 168 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 the same composition, and whether there are characteristic differ- ences between the species in this respect running parallel in the milk and the body of the young. Pages (1894) criticized the supporters of Bunge's law on the ground that it was too sweeping, and that it was applied to all ash constituents, not distinguishing those which are essential for the growth of the bodies. We have not seen Pages's thesis. A little later Proescher (1898) and Abderhalden (1899a, 1899d) compared the composition of the milk of each spe- cies in relation to the time required by the young of that species to double their live-weight. The numerical support for the idea of such a relation is best illustrated by the table below, taken from Abderhalden (1899d, p. 594). Protein, ash, lime and phosphoric acid in the milk of this series of animals all increase in the same order as does the rate at which the young double their weight. RELATION OF THE COMPOSITION OF THE MILK OF DIFFERENT SPECIES TO THE TIME REQUIRED FOR THE YOUNG OF THE SAME SPECIES TO DOUBLE IN WEIGHT Abderhalden (1899) Species Time required for the new-born animals to double the body weight Days 100 parts by weight of milk contain Protein Ash Lime Phosphoric acid 180 60 47 22 15 14 m 9 6 1.6 2.0 3.5 3.67 4.88 5.21 7.00 7.44 10.38 0.2 0.4 0.7 0.7713 0.8406 0.8071 1.02 1.3282 2.4998 0.0328 0.124 0.160 0.1974 0.2453 0.2489 6.' 4545 0.8914 0.0473 0.131 Cattle 197 Goat 2840 2928 3078 Cat Dog- 0.5078 Rabbit 0.9967 With regard to human milk, and the body of the human infant, investigation has yielded results which do not support the theory, or which at least show that the parallelism is less close here than for other animals. See the work of Cornelia deLange (1897, 1900), of Hugounenq (1899b, 1900), and of Camerer and Soldner (W. Cam- erer, Jun. 1900, 1902a; W. Camerer, Jun. and Soldner, 1900, 1903; Soldner, 1902). Another suggestion has been made by Burow (1900) to the ef- fect that there may be a significant relation between the lecithin content of the milk of different species and the brain weight of the suckling young of the same species. For cattle, dogs and men it was found to hold that the greater the relative brain weight, the higher is the lecithin content of the milk, reckoned in percent of the protein. "Here also nature has matched the needs of the sucklings with the composition of the milk." PHOSPHORUS METABOLISM 169 COMPARISON OF THE RELATIVE LECITHIN CONTENT OF MILK AND THE RELATIVE BRAIN WEIGHT OF THE YOUNG OF DIFFERENT SPECIES— (Burow, 1900) Mean composition of milk Species Lecithin Percent Protein Percent Ash Percent Lecithin: pro- tein=X:100 Brain weight: body ■weight Cattle 0.051 0.17 0.058 3.84 8.05 1.90 0.69 1.00 0.24 X=1.40 2.11 3.05 1:370 (mean of 4, 7-8 wks. old.) STUDIES OF THE PHOSPHORUS OF EGGS General Composition and Phosphorus Content. All of the types of phosphorus compounds found in eggs, with the exception of nu- clein, were noted by Gobley (1846, 1847) in his researches on hen's eggs and on the eggs of carp (1850a). The compounds are nuclein, phosphorized fat, nucleoalbumin and traces of inorganic phosphate. Liebermann (1888a) made chemical examinations of different portions of the yolk and of the white. He also followed the special chemistry of the embryo through to the maturity of the chick. He found nuclein and lecithin in the germ; but no phosphorus in the ether extract of the yolk ; he concluded, therefore, that the lecithin present is not free, but is in chemical combination, perhaps with vi- tellin, as previously suggested by Hoppe-Seyler. Extraction with water, or with dilute acid failed to reveal any phosphate directly recognizable without ashing. According to Kaas (1906), the amount of phosphorus in the white is increased by remaining in contact with the yolk. T. B. Os- borne and Campbell (1900c) report 0.122 percent phosphorus in the ovalbumin of egg white. Carl Voit (1877a) gives the average phosphoric acid content of the white of one egg as 0.007043 gm., which would be 0.197 percent, and that of the yolk as 0.20386 gm., which would be 2.57 percent. Lebbin (1900, 1901) found an average of 0.22 percent P 2 5 in the whites of hen's eggs and 1.43 percent in the yolks, or 0.636 percent in the whole edible part. Forbes, Beegle and Mensching (1913) found only traces of inorganic phosphorus in the edible portion of the hen's egg. According to Hammarsten (1911, p. 600, 604) Poleck and Weber found 1000 parts of the ash of the white of the egg 276.6- 284.5 parts potash, 235.6-329.3 soda, 17.4-29 lime, 17-31.7 magnesia, 4.4-5.5 iron oxide, 238.4-285.6 chlorine, 31.6-48.3 phosphoric acid (P 2 5 ), 13.2-26.3 sulphuric acid, 2.8-20.4 silicic acid and 96.7-116 170 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 parts carbon dioxide. In 1000 parts of the ash of the yolk were found 51.2-65.7 parts soda, 80.5-89.3 potash, 122.1-132.8 lime, 20.7-21.1 magnesia, 11.90-14.5 iron oxide, 638.1-667.0 phosphoric acid, and 5.5-14.0 parts silicic acid. Carpiaux (1908) gives the makeup of the ash of a fresh egg as being 17.37 percent K 2 0, 22.87 percent Na 2 0, 10.91 percent CaO, 1.14 percent MgO, 0.39 percent Fe 2 3 , 37.62 percent P 2 5 , 0.32 per- cent S0 3 and 8.98 percent CI. Malcolm (1902) has given us a comparison of the chemical composition of the yolks of eggs laid by different hens of the same breed, and on the same diet ; also of the different eggs laid by the same hen on a constant diet. His tables are given in part below. The conclusions are: 1. The percentage of lecithin in egg-yolk varies considerably. 2. The percentages of protein, fat and phos- phorus in the yolks of eggs from the same hen are in very close agreement, while there are very considerable differences in eggs from a number of hens, even where the breed is the same. ANALYSES OF EGG- YOLKS— (Malcolm, 1902) Percent Egg- No. Fat Nitrogen P2O5 Ether extract Chloroform extract Percent P2O5 content Percent P2O5 content Laid by hens all of one breed, and fed J on maize and barley ) Laid by one hen. . -j 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 27.786 29.453 28.501 28.662 31.887 28.956 29.114 30.080 30.' 124 29.565 3L043 31.575 30.431 30.120 36! 265 3.004 2.620 2.648 2.674 2.676 3.123 2.877 2.674 2.605 2.822 2.846 2.762 2.575 2.552 2.607 2.754 2.797 2.747 1.489 L356 1.527 1.396 1.517 1.511 1.569 i.*47i 1.654 1.543 1.392 1.425 1.441 1.477 L506 28.662 28.956 29.114 30.080 0.1408 0.2120 0.1884 0.1838 31.418 31.423 32.659 33.100 0.2085 0.2419 0.1930 0.2036 THE NXTCLEOALBUMINS OF EGGS The nucleoalbumin of hen's eggs, ovovitellin, seems to be pres- ent in chemical combination with lecithin, and, as isolated and pre- pared, it usually contains lecithin. According to T. B. Osborne and Campbell (1900b), it is extracted as a mixture of several protein- lecithin compounds which may be called lecithin-nucleo-vitellins. Plimmer (1908) reports another protein containing less phosphorus than vitellin. The corresponding compound from fish eggs, called ichthulin, was especially studied by Valenciennes and Fremy (1854) in their general consideration of the composition of salmon eggs; but G. Walter (1891) gave it a more particular study, as he obtained PHOSPHORUS METABOLISM 171 it from the eggs of carp, and he decided that vitellin and ichthulin may be considered identical. Hammarsten (1905a) isolated a nu- cleoalbumin from the eggs of perch which yielded a much larger proportion of pseudonuclein, on pepsin digestion, than Walter had found from the ichthulin of carp eggs. NUCLEIC ACID 01* FISH EGGS J. A. Mandel and Levene (1906b) obtained a nucleic acid from unfertilized fish eggs which in its chemical makeup resembled nu- cleic acids of plant origin more than those from animal substances generally. Helene Tschernorutzky (1912a) found indication of about 1.2 gm. nucleic acid in 100 gm. of dry, ripe, unfertilized her- ring eggs which had been extracted with alcohol and ether. The phosphorus content of the substances not removed by alcohol and ether was not all accounted for by this nucleic acid. THE PHOSPHATIDS OF EGGS A large part of the study of phosphatids, under the name of lecithin, has been made on that obtained from eggs. (See Diaco- now, 1867a, 1867b, 1868b; Strecker, 1868; Bergell, 1900; Cousin, 1903 ; Henriques and Hansen, 1903 ; Laves, 1903a, 1903b ; Wintgen and Keller, 1906; Erlandsen, 1907; Stern and Thierf elder, 1907; Frankel and Bolaffio, 1908; MacLean, 1908b, 1909b, 1909c; Serono and Palozzi, 1911; Riedel, 1912; Trier, 1912, 1913b.) Armand Manasse's (1906) determinations of the amount of lecithin obtainable from egg-yolk by different treatments show re- sults nearly enough alike (range, 8.856-9.96 percent) to give con- siderable support to the average figure, 9.41 percent. Serono and Palozzi (1911) give 11.05 and 12.09 as the minimum and maximum percents of lecithin in fresh yolk. Tornani (1909) says that the amount of lecithin, and the relation between cholesterol and leci- thin in egg-yolk is quite variable, that this relation changes as the eggs are kept, and is different in fertilized and unfertilized eggs. The amount found by Mesernitzy (1907) in fresh eggs was 15.35 percent of the dry substance, and the amount fell to about one-half of that by 20 days' incubation. Glikin (1908a) thinks that the eggs of those species of birds which are least capable of independent existence when hatched contain a larger supply of lecithin than oth- ers. Distinctions have been made of several different phosphatids in egg-yolk. Laves (1903a, 1903b) states that there are several leci- thins present, and that they are largely free, but partly in combina- tion with protein and perhaps partly with cerebrin. 172 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Henriques and Hansen (1903) found that changes in the char- acter of the fat of the food did not lead to much alteration in the io- dine number of the lecithin, though it did in that of the egg fat. The lecithin, then, seemed to retain its constancy of composition inde- pendent of the food fat. Barbieri (1910, 1912) made an examina- tion of the extracts of egg-yolk usually said to contain lecithin, and came to the conclusion that the yolk contains no lecithin, either free or combined, that is, that there was no choline and no phosphorus united to glycerol. The phosphorus was said to be dialyzable, and all or in part in the form of soluble phosphates. We have not seen confirmation of this conclusion. Stern and Thi erf elder (1907) found three distinct phosphatids in egg-yolk: the first, orange red, was thought to be lecithin; the second, bright yellow, resembled cephalin ; and the third, white, was apparently a diamino-monophosphatid. MacLean (1908b, 1909b, 1909c) reports a monamino-diphosphatid, and Frankel and Bolaffio (1908), a triamino-monophosphatid, neottin. Eppler (1913) separated two phosphatids from egg yolk. They differ in solubility in alcohol, and in the proportion of nitrogenous base (choline) which is present. OTHER PHOSPHORUS-CONTAINING COMPOUNDS REPORTED PRESENT IN EGGS The substance to which von Bunge (1885a) gave the name haematogen, and which he and Hugounenq and Morel (1905a, 1905b) examined with especial view to its being of the nature of an embryonic haemoglobin was found to be a pseudonuclein which may have been derived from the vitellin. The mucoid present in egg, ovomucoid, has been supposed by some to contain phosphorus (see Milesi, 1898), but if carefully pre- pared there is not more than a trace of phosphorus (Langstein, 1903). PHOSPHORUS IN THE DIGESTIVE SECRETIONS GASTRIC JUICE In the gastric juice which Schoumow-Simanowsky (1893-94) took by stomach fistulae from healthy dogs, phosphoric acid was. found to the extent of 0.00398 and 0.0036 percent. Pekelharing (1902) believes pepsin to be free from phosphorus, and that the phosphorus which others have found in their prepara- tions was present as a contamination of lecithin or perhaps a phos- phorus-containing mucus. Pekelharing mentions Nencki and Sieber as believing that there is a nucleoprotein in the gastric juice. PANCREATIC JUICE Pancreatic juice, caused to flow from dogs by the introduction of secretin, is reported by Plimmer and Kaya (1909) as showing the following distribution of phosphorus compounds. PHOSPHORUS METABOLISM 173 DISTRIBUTION OF PHOSPHORUS IN DOGS' PANCREATIC JUICE Plimmer and Kaya (1909) Percent of Total P s 5 Dog Ether-sol- uble P 2 5 (lecithin) Water-soluble P2O5 (nucleic acid and inorganic) Inorganic P2O5 Protein P 2 5 (nucleoprotein and phosphoprotein ) Phosphopro- tein P2OS I II III 56.3 86.3 59.8 Present Trace Trace 43.7 13.7 36.7 43.7 13.7 40.2 Frouin and Gerard (1912) determined the phosphorus in the pancreatic juice of the cow and the dog. Pancreatic juice was ob- tained from dogs having temporary fistulae by the injection of se- cretin. From 7 animals 1700 c.c. of the juice was collected in 6-7 hours. It contained 0.006 gm. P per liter. From cows with per- manent fistulae the pancreatic juice contained 0.0089 gm. P per liter; the juice as collected a month later by catheter in the pancre- atic duct contained 0.003 gm. P per liter. BILE The phosphorus-containing constituents of bile are phosphatids and a very little mineral phosphate. The mucus of the liver secre- tion is said to be either nucleoalbumin or nucleoprotein (Paijkull, 1888). The inorganic constituents of bile are the chlorides of so- dium and potassium and small amounts of the phosphates of cal- cium, magnesium and iron. Edlefsen (1880) taking the old form- ula for haemoglobin, which gives it a phosphorus content, worked out a reaction by which the haemoglobin of the red corpuscles dis- integrating in the liver may furnish a part of the phosphorus of bile, a part going to the urine. The phosphatids in general are, of course, usually spoken of, in all but the more recent writings, as lecithin, but it is shown (Ham- marsten) that there are in bile phosphatids of different solubility and different relation of N:P. Hammarsten found evidence of a jecorin-like phosphatid, in certain cases at least. The first finding of lecithin in bile was apparently that of Gobley (1856). It was then detected by the presence of oleic acid and margaric acid in some kind of combination not a fat. Quantitative determinations of the lecithin have commonly been simply by computation from the phosphorus found in the ether extract. Hammarsten (1901, 1902, 1904, 1905b) made a study of the bile of different species of animals, especially those of polar regions. He notes that the largest amount of lecithin is in the bile of the polar bear which uses a diet unusually rich in fat, and thinks it may be Nature's provision for the digestion of the fat. In the table the animals are arranged in order of decreasing phosphatid content 174 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 of the bile. The bile of the liver and that of the gall bladder are said not to be alike in composition, the lecithin being considerably higher in the bladder bile. PHOSPHORUS FOUND IN THE ETHER EXTRACT OF THE BILE OF VAR- IOUS ANIMALS— (Hammarsten, 1905) Animal Phosphorus in ether extract Percent P Lecithin computed from the same Percent Dog 0.911-1.14 0.048-1.17 0.100-0.611 0.768 0.502 0.420 0.334 0.332 0.289 0.272 0.191 0.181 0.168 0.162 0.043 0.033 Present but too littl 23.12-28.96 1.23-29.75 2.54-15.5 19.50 Hog 12.74 10.67 8.47 8.43 7.35 7.04 Cattle 4.86 4.60 Seal 4.27 4.10 1.08 Codfish 0.81 3 to be determined Daniel-Brunet and Rolland (1911b) reported certain chemical analyses of the bile and the liver of cattle. The following table shows the range of the values found for compounds of phosphorus. PHOSPHATES, NUCLEOPROTEINS AND LIPOIDS OF BILE OF CATTLE Parts per 1000 Phosphates P2O5 Nucleo- proteins Lipoids Total Cholesterins Lecithins and Min. Max. Min. Max. neutral soaps Min. Max. Min. Max. Min. Max. Bile 1.31 1.58 1.15 2.25 1.100 2.130 0.410 0.813 0.690 1.317 In another article (1911a) these authors say that, with cattle, neither sex nor castration was found to make any difference as to the quantity of the mineral elements, the glycogen or the nitrogen- ous compounds in the bile; but they did affect the lipoid content, bulls showing the most lipoids, cows less, and steers still less. Rosenbloom (1912, 1913b) reports the following analysis of human bile taken from a biliary fistula, as parts per 1000; total solids 29.8, bile salts 10.1, mucin and pigments 4.86, cholesterol 2.61, fat 6.85, soaps 2.6, lecithin 6.42, inorganic matter 9.2, fatty acids 1.2. See also Brand (1902), Yeo and Herroun (1884-5), Baginsky and Sommerfeld (1895), Pruszynski and Siemienski (1906), Bonan- ni (1906), and Long and Gephart (1908b). PHOSPHORUS METABOLISM 175 SALIVA The saliva contains phosphates. With regard to the quantita- tive relations of the mineral constituents of the saliva, the following data are at hand : Hammerbacher (1881) made an organic and inorganic analysis of mixed human saliva in which the inorganic salts formed 2.205 parts per 1000. The ash analysis was as shown below: Percent K 2 45.714 Na 2 9.593 CaO (with trace of iron oxide) 5.011 MgO 0.155 so 3 6.380 PA 18.848 CI 18.352 104.053 Oxygen equivalent of chlorine 4.135 99.918 By computation from the ash analysis, it was concluded that the ash of the saliva may have been made up of the following com- pounds : Compound Percent KC1 38.006 K 2 S0 4 13.908 K 3 P0 4 21.278 Na 3 P0 4 16.917 Ca 3 (P0 4 ) 2 9.246 Mg 3 (P0 4 ) 2 0.338 99.693 Excess CI 0.282 99.975 This aggregates 90.109 percent alkali salts and 9.584 percent alkaline earth phosphates. Hammerbacher says that Enderlin found 92.367 percent alkali salts and 5.509 percent earth phosphates with a trace of iron phosphates. 0.82 176 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Schafer's "Text-Book of Physiology," Vol. I, p. 348 quotes from Jacubowitsch, through Hermann's "Handbuch," V (2), p. 14, the following analyses of the ash from mixed saliva, expressed as parts per 1000 parts of saliva. Human Dog Total solids 1.82 6.79 Phosphoric acid 0.51 Soda 0.43 Lime 0.03 Magnesia 0.01 \ Alkaline chlorides 0.84 5.82 Schafer also quotes (p. 347) from Herter, through Hoppe-Sey- ler, "Physiol. Chem.," II, p. 191, the following as the salts found in submaxillary saliva, expressed as parts per 1000 of saliva: K 2 S0 4 0.209 KC1 0.940 NaCl 1.546 Na 2 C0 3 0.902 CaC0 3 0.150 Ca 3 (POJ 2 0.113 F. N. Schulz gives, in Oppenheimer's "Handbuch der Biochemie des Menschen und der Tiere," III, p. 29, the following ash analyses from Jacobi (Diss., Wurzburg, 1896) : Percent Percent I II CI 14.46 13.68 K 35.88 35.69 Na 32.91 21.61 PA 10.98 17.70 Sulphuric acid Trace ' 7.1 Ca 2.19 3.96 Mg 0.47 0.69 Barille (1911) considers that dental tartar is derived from the saliva through the precipitation of its salt as tricalcic phosphate and calcium carbonate as a result of the loss of carbon dioxide from the carbonophosphate in the saliva. Barille found in the inorganic part of tartar 70 percent of the phosphate and 8 percent of the car- bonate of calcium. Roger (1908) believes that the presence of phosphates is neces- sary for the amylolytic action of saliva. He showed that the sugar- forming power of saliva may be destroyed by precipitation of the phosphates with uranium acetate, and restored by addition of so- dium phosphate. PHOSPHORUS METABOLISM 177 PHOSPHORUS PRESENT IN CHYLE Hamill (1906-07) collected chyle through a fistula in the thigh of a human subject, and examined it for various constituents. The lecithin averaged 4.204 gm. per 100 gm. of ether extract. Lipase and amylase were both present. Lecithin given by mouth during the time of observation produced a rise in the ether-soluble phos- phorus of the chyle. PHOSPHORUS PRESENT IN LYMPH From the few phosphorus estimations on lymph which have come to our attention we are unable to conclude as to its usual content. Odenius and Lang (1874) found 13 parts per 1000 of soluble P 2 5 ; E. Ludwig (quoted by von Zeynek, 1895) found 15 parts total P 2 3 per 1000; von Zeynek (1895) found 0.095 parts soluble, and 0.214 parts insoluble P 2 5 per 1000 c. c, while Zaribnicky (1912) found in the lymph of the horse 0.089 parts total P 2 5 per 1000. The preceding figures were from analyses of human lymph. See also Dahnhardt (1866). 178 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 PART IV NORMAL PHOSPHORUS METABOLISM MAINTENANCE OF NEUTRALITY The approximate constancy of reaction of the body fluids, which is one of the conditions essential to life, is maintained at neu- trality (actually very slight alkalinity) by a group of counteracting agencies prominent among which are the phosphates. The need for such an adjustment is due to the facts that the predominating chemical reaction of the body is the oxidation of car- bon compounds to carbonic acid, and that sulphuric and phosphoric acids are prominent among the normal products of protein katabo- lism. This control of reaction in the body fluids is accomplished by phosphates, carbonic acid and carbonates, aided by the amphoteric proteins, and sustained by the excretory functions of the lungs for carbonic acid, and of the kidneys and intestine for phosphates. We shall consider in detail only Henderson's theory of the pe- culiar effectiveness of carbonic and phosphoric acids and their salts in this adjustment. (Henderson, L. J., 1906, 1098a, 1908b, 1908c, 1909, 1911, 1913 ; Henderson and Black, 1907, 1908 ; Fitz, Alsberg and Henderson, 1907.) Henderson's explanation of this phenom- enon is based on physico-chemical considerations and ion concen- tration measurements. The substances considered are the two pairs of compounds, carbonic acid and bicarbonate, and the mono- and di-phosphate. The processes concerned may be represented by the two equations, M,HP0 4 +HA^MA+MH 2 P0 4 MHC0 3 +HA?±MA+H 2 C0 3 where M stands for any basic radical ; A, for any acid radical. The indications are that in the fluids of the body the carbonic acid is present as carbonic acid (H 2 C0 3 ) or sodium bicarbonate (NaHC0 3 ), and all of the phosphoric acid as mono-sodium phosphate (NaH 2 P0 4 ) or di-sodium phosphate (Na 2 HP0 4 ). Now, mixtures of these compounds in solution possess greater power than any other known salts for balancing each other, and any acid or base that may be added, so that the solution remains neutral. While a pure solution PHOSPHORUS METABOLISM 179 of NaH 2 P0 4 is very weakly acid, and one of Na 2 HP0 4 is very weak- ly alkaline, one of a mixture of the two adjusts itself by ionization so that it is neutral ; and addition of an acid to such a solution does not give it even the slight acidity of mono-sodium phosphate till enough acid has been added to convert all of the di-sodium salt to mono-sodium salt. Bases produce an opposite effect, but will not give the mixture even the faint alkaline reaction of di-sodium phos- phate till all has been converted to that form. The case is similar for carbonic acid and bicarbonates, and when all four compounds are present in a solution, the base dis- tributes among these components so that the relative amounts of each are constant for any given temperature. By mathematical deductions Henderson shows that the strength of an acid theoretically best fitted to preserve neutrality in a pure solution is such that its ionization constant is near IX 10" 7 , which is the ionization constant of distilled water; or, more exactly, "that its ionization constant, divided by the degree of ionization of its salt, is precisely equal to the hydrogen ion concentration in pure water." The ionization constant of H 2 C0 3 is 3X10" T > and that of the H 2 P0 4 ~ ion is 2X10" 7 * Hence both of these are theoretically, as they are found to be practically, nearly ideal neutralizing agents near the neutral point. The condition to be maintained, however, is not exactly the neu- trality of distilled water, and at the alkalinity required very slight variations in hydrogen ion concentration are accompanied by great- er variations in sodium bicarbonate content than in the ratio of mono- and di-sodium phosphate. Hence the carbonate is more effi- cient than the phosphate, proportionately, in maintaining alkalinity. The concentration of bicarbonate is then from ten to twenty times the concentration of carbonic acid, and neutrality is attained only when the concentration of bicarbonate has fallen nearly to that of the carbonic acid. This efficiency of the bicarbonate-carbonic acid equilibrium is further increased by the maintenance of a nearly con- stant supply of carbonic acid through its removal by the lungs. The phosphates, however, have certain other advantages. At the most critical point, that is, when the bicarbonate is nearly used up and the system is very near the neutral point, the phosphates are most effective. Furthermore, they are present, especially in proto- plasm, probably in much greater amounts than the carbonate, and are correspondingly of greater service. In addition, the easy dif- f usibility of acid phosphates and their removal by the excretory or- gans must greatly enhance their practical efficiency. 180 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 With regard to the magnitude of the effect of these two agents together in the protoplasm, Henderson says: "Only when an amount of acid equal to about three-fourths of the total carbonic acid plus the total phosphoric acid of the protoplasm has been added to the protoplasm can there be a real beginning of acidity." This mechanism of equilibrium in the tissues and fluids very greatly reduces the alkali requirement of the body; but it must be sustained, as it is, in fact, by special retention mechanisms at the points of excretion, by means of which the alkalis are returned to the blood after the excretion by lungs and kidneys of acids with which these bases were combined. In so far as the alkalis are united with carbonic acid they are entirely saved to the body, as the carbonic acid leaves the body in the gaseous state through respiration. The amount of alkali in use in this way is said to be about 1.5 gm. at any moment, or 400 gm. per day. Alkalis are, however, removed from the body in the urine of omnivora and carnivora, chiefly as phosphates and sulphates. At the kidney there are two provisions, probably about equal in their effect, for sparing alkali. First, the fixed alkalis are replaced by ammonia, in so far as it is available. This is all clear gain of base, because the ammonia comes from nitrogenous compounds which are nearly neutral, and if completely oxidized becomes neutral urea. The other factor which saves alkalis to the body is the physico-chemical process by which acid urine is separated from the alkaline blood. Though the change of reaction is small, the amount of base involved may be considerable in the presence of phosphates and other salts and acids having the ability to undergo wide variation in the amount of base which they hold with but slight variation in hydro- gen ion concentration. As we have already noted, phosphates are peculiarly effective in this way near the neutral point. Carbonic acid and uric acid have the same property, and, at hydrogen ion con- centrations such as are reached in acidosis, the /?-oxybutyric acid and its salts, which are then present, are also effective in the same way. With regard to the relative magnitude of the parts played by the proteins, the bicarbonates and the phosphates in maintaining neutrality nothing definite can be asserted, nor can it be said that there are not other unrecognized agencies involved. Conditions within the body cannot be exactly duplicated outside, especially as to the influence of colloids; but as far as true solutions are con- cerned quantitative measurements can be made with pure substan- PHOSPHOEUS METABOLISM 181 ces which give some idea of the possible degree of effectiveness of the different agents; and the final word in the discussion between T. Brailsford Robertson and Lawrence J. Henderson (Robertson, 1909b, 1910a; Henderson, L. J., 1908a, 1908c, 1909) seems to be that, so far as the blood is concerned, in passing from the reaction of normal blood to that of blood in advanced acid intoxication, the proteins of the blood are about one-fifth as efficient as the bicar- bonates in maintaining its neutrality. In the tissues or tissue- fluids the proteins very likely play a larger part. Henderson attrib- utes a relatively greater effectiveness to bicarbonate than to phos- phate except at points very near to neutrality. Michaelis (1913) gives the average H + ion concentration of hu- man venous blood, in normal resting condition, as 2.75 X10" 8 at 20-21° and 4.46X10" 8 at 37.5° ; and the greatest variations from this mean, found under such conditions, amounted to -{-12 and — 11.7 percent of the mean. A difference of ± 6 percent is attributable to uncertainties of the method of determination, so that the variations observed are but very slightly beyond the limits of error of work. THE ABSORPTION AND ELIMINATION OF COMPOUNDS OF PHOSPHORUS GENERAL CONSIDERATIONS After the ingestion of ordinary food, with its variety of phos- phorus compounds, organic and inorganic, there begins at once the same process of simplification and absorption that is general for other groups of its nutrient components. The inorganic phosphates are, of course, absorbed as such. The organic phosphorus com- pounds are absorbed in part without change, in part after partial cleavage, and in part after complete separation into their simplest groups, with the liberation of phosphorus as inorganic phosphoric acid, the method differing with the nature of the nutrient. The phosphoric acid split off from organic compounds during digestion behaves just as does any other inorganic phosphoric acid introduced as such (Oeri, 1909). The larger part, though by no means all of the food phosphorus, therefore, is absorbed as inorganic phosphate. For details as to the digestion of the several groups of phosphorus compounds see the special discussions of these groups. Such inorganic phosphates as are insoluble in water are most of them readily soluble in the hydrochloric acid of the gastric juice. They are then absorbed, mainly in the small intestine, which is usu- ally faintly acid in the upper portions (Raudnitz, 1893 ; and Wuertz, 1912). 182 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 It is usually said to be true that absorption of phosphates takes place only in the acid portion of the small intestine, but von Wendt and Zuckmayer, as noted below, suggest means by which phos- phates may be absorbed from the alkaline portions of the ali- mentary tract. Since the phosphorus of the food is mostly in completely oxi- dized forms there can be no significant change in the state of oxi- dation during metabolism. There is evidence of some capacity, how- ever, for animals to oxidize the less completely oxidized compounds, as is observed by Heffter (1903), who states that in the healthy or- ganism phosphorous acid is completely oxidized. He states, also, that pyrophosphates and hypophosphites are excreted unchanged, while metaphosphates are changed to the ortho- form. The circumstances which determine whether phosphates shall be eliminated through the urine or the feces are first, the reaction of the contents of the alimentary tract, as determined especially by hydrochloric acid and carbonates; second, the nature and relative amounts of the other mineral elements, especially the bases, present in the digestive tract, as affecting the solubility of the phosphates ; third, after the absorption of the phosphates from the alimentary tract, all of those circumstances which control the secretion of phos- phates into the digestive tract and the reabsorption from the same ; fourth, the many circumstances which affect the reaction of the blood serum and the amounts and proportions of the other salts present, as affecting the form and therefore the solubility Of the phosphates, the more readily soluble tending to go out through the urine; and fifth, the species of animal involved. There may be a considerable variation as to path of outgo without apparent change of conditions; thus the experiments of Kochmann (1911) (see Inn. of Amount of Food on P. Met.) show that on the same amounts of the same kind of food there may be a change of urine phosphorus from 27 to 34.9 percent of the total outgo, and of feces phosphorus from 65.1 to 73 percent of the total outgo, without apparent cause. This whole problem, then, is almost hopelessly complicated, though we are able to record a considerable number of harmonious observations as to the bearing of certain individual factors. The general course of the minerals during their passage through the alimentary tract has been traced by Wildt (1874, 1879), who made a study of digestion in sheep by analyses of the contents of the different parts of the alimentary tract. By means of quanti- tative estimations of the silica and also the absorbable constituents of the food, in each part of the tract, it was possible to trace absorp- tion, to recognize both the organic and inorganic constituents of PHOSPHORUS METABOLISM 183 each of the digestive secretions, and later to tell where, when, and to what extent these constituents were again absorbed and returned to the blood stream. The amount of phosphorus secreted into the alimentary tract was, in each of the three sheep studied, greater than the total amount in the food. Secretion of phosphorus greatly exceeded ab- sorption in the first and second stomachs, but was less than absorp- tion in the third stomach ; changed again sO that secretion exceeded absorption in the abomasum and duodenum, and once more so that absorption exceeded secretion in the remainder of the tract. The principal secretion of phosphorus was in the saliva and in the secretion of the abomasum, and its largest absorption was from the small intestine. Calcium and magnesium, on the other hand, were secreted principally into the small intestine, and were absorbed most actively from the first and second stomachs and the colon. The following tables set forth the facts as Wildt determined them as to the absorption and secretion of phosphorus and the min- eral bases. ABSORPTION AND SECRETION OF PHOSPHORUS, CHLORINE AND THE MINERAL BASES IN THE ALIMENTARY TRACT OF THREE SHEEP— Grams - First and second stomachs Third stomach Abomasum and duodenum Small intestine Caecum Colon Large intestine Entire Last part Sheep I K "II III —2.525 +1.993 —2.104 -2.232 -4.811 -2.480 +1.665 +5.218 +9.683 +3.746 -4.714 -6.955 —7.436 —3.321 —2.073 -0.391 —1.607 -0.903 -0.186 0.525 -1.042 +2.178 +0.761 -0.912 Sheep I Na ' II III +14.794 +15.673 +13.006 -10.004 -10.167 -6.389 +2.983 +7.167 +20.715 +15.871 +7.060 -1.687 —14.837 -12.566 —17.728 -3.527 -1.746 -2.967 -3.560 -3.086 -3.740 -3.179 -1.703 -1.935 Sheep I Ca " II III -0.729 —0.236 -1.002 +0.163 +0.214 +0.278 -1.284 -0.803 +0.536 +1.415 +0.118 -0.328 +0.653 +0.398 +0.754 -1.023 -0.686 -0.585 +0.555 +0.107 +0.050 -0.424 -0.386 +0.198 Sheep I Mg" " II " III -0.675 -0.472 —0.724 -0.316 -0.128 -0.189 +0.109 —0.129 +0.596 +0.719 +0.557 -0.199 -0.175 —0.235 +0.328 -0.479 -0.274 -0.278 +0.022 -0.289 +0.089 +0.207 +0.311 -0.010 Sheep I P2O5 ' ' II III +7.359 +9.064 +5.416 -2.068 —2.861 -1.091 +0.155 +2.818 +8.939 +3.470 -2.164 -8.701 -7.348 —5.175 —2.440 -0.523 -0.642 -0.437 -0.707 -0.290 -0.416 +0.980 -0.353 -0.556 Sheep I CI "11 " III —1.284 +1.821 —1.538 +0.925 —1.425 —1.012 +12.835 +18? +27.422 —1.687 —10.619? -16.032 —9.558 -7.167 -9.584 -1.209 -0.150 -1.389 -1.491 -1.141 -0.828 -2.174 ( — )==more absorbed than secreted. ( + )=more secreted than absorbed. SECRETION DURING TWENTY-FOUR HOURS IN VARIOUS PARTS OF THE ALIMENTARY CANAL— Grams Mouth (Saliva) Abomasum Small Intestine Total DailyFood K Mg P s O B '.... 15.010 7.969 4.443 7.291 17.240 2.584 9.270 0.601 4.443 31.571 0.601 17.240 10.553 9.078 1.103 3.267 3.162 0.966 184 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 One point which a study of these tables makes clear is the utter absurdity of the usual statements as to digestibility of min- eral nutrients, and also of many statements as to coefficients of ab- sorption of inorganic salts. The mineral salts, including phosphates, reach the general cir- culation almost wholly through the portal system. The final dis- position of the phosphates under ordinary conditions of nutrition may then involve retention in the body, or elimination, either in urine or feces. There are also the various losses of phosphorus in- volved in reproduction and lactation. It would seem from the work of Taylor (1911) that there is no cutaneous elimination of phos- phates. Taylor studied the cutaneous elimination of nitrogen, sul- phur and phosphorus with two laboratory assistants in periods of 28 and 45 days. The average daily cutaneous eliminations were as follows : Substance Subject J. Subject D. Grams Grama S 0.028 0.015 P 0.003 0.002 N 0.190 , 0.160 The author concluded that the phosphorus figure stands for desquamation only, and that there is not a real cutaneous elimina- tion of phosphorus in any form. In discussing urinary and feces excretion of calcium salts, VonWendt (1905) suggests that the acidity of the urine is respon- sible for its content of calcium salts, while the calcium salts which are excreted into the intestine are rendered more or less insoluble by the alkalinity of the intestinal juices, their reabsorption being there- by restricted. Phosphates are supposed to be excreted into the in- testine as mono- and di-salts. Here the di-salt decomposes into mono- and tri- salts, and the latter, further, with the taking up of the elements of water, into basic phosphate and acid. Since, of these phosphates, onlythe mono-salt can be considered as relatively easily soluble in the weakly alkaline intestinal juice, their reabsorp- tion takes place principally in this form, though to some extent also as the di-salt. From the relation of Ca:P in the feces vonWendt concludes that the calcium is present as the trimetallic phosphate. Zuckmayer (1912) found that "tricalcol," an alkali-soluble col- loidal calcium preparation, was absorbed from the small intestine of a rabbit and a dog, while the tricalcium phosphate, directly intro- duced, was not usually absorbed. Zuckmayer states that in nor- mal digestion calcium (calcium phosphate) is withdrawn from food PHOSPHORUS METABOLISM 185 substances by the acid of the stomach, with the formation of acid calcium phosphate and calcium chloride, and probably also phos- phoric acid in the presence of protein cleavage products. He also suggests that in the small intestine, as soon as there is sufficient al- kali present, the dissolved calcium phosphates are again changed to tricalcic phosphate, which becomes absorbable through the agency of the colloidal protein cleavage products. In studying the conditions governing calcium and phosphorus metabolism in infants Bliihdorn (1912-13) experimented with cal- cium and phosphorus compounds added to feces extracts in vitro. If to 8 c.c. of distilled water were added 1 c.c. each of N/50 Na 2 HP0 4 and N/100 Ca Cl 2 a slight but distinct precipitate of Ca, (P0 4 ) 2 appeared. By similar addition of these salt solutions to water-extracts of feces it was determined that if Ca salts occur with phosphates in the intestine, in weakly acid or alkaline reaction, in- soluble Ca 3 (P0 4 ) 2 results, and in this way Ca and P may be with- drawn from absorption. A strong acid reaction prevented the pre- cipitation of Ca 3 (P0 4 ) 2 . The colloids of the feces extracts appeared to play no decisive role in these processes. Plimmer (1913a) studied the action, on phosphorus compounds, of enzymes obtained from pancreas, liver, intestine, castor beans, yeast and wheat bran. The phosphorized nutrients studied includ- ed glycerophosphoric acid, phytic acid, nucleic acid, casein and others. The most active tissue in the hydrolysis of the organic phosphorus compounds was thought to be the intestinal mucous membrane. Phytic acid was attacked readily only by an enzyme in the bran extract. Casein was the only one of these compounds which was hydrolyzed by the pancreas extract. From the activity of the intestinal mucosa in the cleavage of organic phosphorus com- pounds Plimmer concludes that they are absorbed only in the hy- drolyzed condition, the phosphorus as inorganic phosphate. The lipoclastic enzymes were shown not to be active in the hydrolysis of organic phosphorus compounds. While Plimmer's conclusion as to the absorption of phosphorus compounds only in a state of hydrolytic cleavage is doubtless true of the bulk of the phosphorus absorbed from organic compounds, his evidence is not of such nature as to controvert successfully the es- tablished facts as to the absorption of a part of the organic phos- phorus of the food in unsplit or incompletely hydrolyzed forms. 186 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 THE INFLUENCE OF PHOSPHATES ON DIGESTION The nature of the influence of phosphates in digestion was shown by Berg and Gies (1906-7), who studied the effects of ions on catalysis, with especial reference to peptolysis and tryptolysis. The H + ion is the favorable factor in peptolysis, and the OH" ion in tryptolysis ; the associated ions, or molecules, or both, exerting deterrent influences in variable degrees. Variations in swelling ef- fects on fibrin, in general, correspond to these digestive differences. Finzi (1903) determined that intravenous injection of neutral sodium phosphate caused increased secretion of saliva, and in- creased phosphate content of the saliva. Ferrata and Moruzzi (1907) determined that a phosphorus-con- taining food causes an increase in the lecithin content of the intes- tinal mucous membrane of the dog, while the nucleoproteids were increased after the taking of food of any kind. Roger (1908) found that the amylolytic power of the saliva de- creased with the addition of uranium acetate, and disappeared with the complete precipitation of the salivary phosphates. The ad- dition of sodium phosphate then caused the amylolytic action to re- appear. Conclusions were sustained by adequate numerical data. Giacosa and Dezani (1909) found phosphates in the press-juice from pig stomach, but also prepared from it a phosphorus-free di- gestive enzyme. A. Loeb (1910) showed with a dog that the urinary phospho- rus was reduced, temporarily, by the taking of food, apparently be- cause of its utilization in the preparation of the digestive juices. Lisbonne (1911) reports that phosphates do not activate salt- free amylases of the saliva and pancreatic secretion as do chlorides. W. Lob (1911) demonstrated that phosphates stand in an im- portant relation to the utilization of sugar. Glycolysis, which is favored by hydroxyl ions, is accelerated by the presence of phos- phate ions; with constant hydroxyl ion concentration, the acceler- ation rising with the absolute amount of phosphate present. Leci- thin or glycerophosphates hinder phosphate glycolysis. The activating effect of primary and secondary phosphates on various proteases is compared by Fernbach and Schoen (1911). The above notes show that phosphates stand in such a signifi- cant relation to the digestion and utilization of food that their ex- cretion into the alimentary tract must be regarded as of vital con- sequence. The possible significance of phosphates in relation to enzymatic processes in animal bodies is indicated by a series of studies by Wroblewski, Harden, Young, and Euler and associates, on the rela- tion of phosphates to the enzymes of yeast. PHOSPHORUS METABOLISM 187 Wroblewski.(1901) demonstrated the favorable effect of a certain concentration of phosphate on the action of zymase from yeast press-juice. Harden and Young (1906, 1908a, 1908b, 1909, 1910, 1911a, 1911b) also found that phosphate accelerates the fermenta- tion of sugar by yeast juice, the phosphate becoming non-precip- itable by magnesia mixture. Young (1907, 1909, 1911) shows that the organic phosphorus compound above mentioned is a hexose phos- phate. The method of action of the enzyme involved, phosphatese, is discussed in a series of papers by Euler and Kullberg (1911), Euler and Ohlsen (1911), Euler and Lundeqvist (1911), Euler and Ohlsen (1912), Euler (1912a) and Euler and Backstrom (1912). Other studies on the influence of phosphates on the enzymes of yeast are those of Euler and Beth af Ugglas (1911). AUTOLYSIS OF COMPOUNDS OF PHOSPHORUS The intermediary metabolism of phosphorus is accomplished through the agency of enzymes. Given the particular con- ditions necessary for their activity, they are able to bring about the various changes in the state of organization of matter which are es- sential to the maintenance of life. Our state of knowledge of these compounds is especially incomplete, and we make no effort to pre- sent a full discussion of the subject. We merely offer a few notes on investigations demonstrating the existence of such compounds, and the nature of the cleavage which they may induce; and we make the suggestion that reversibility of direction is a not infre- quent characteristic of enzyme action, some at least of the autolytic enzymes undoubtedly possessing, with change of conditions, capac- ity for synthesis in the same field of activity. Evidence of the ac- tivity of autolytic enzymes has been accumulated by a great number of workers, among whom are those whose names follow, these hav- ing given attention to compounds of phosphorus. Kutscher and Lohmann (1903) found no autolytic decomposi- tion of the lecithin of the brain of the ox, but Coriat (1904a) found an enzyme in brain tissue capable of splitting choline from lecithin. This enzyme acts only in neutral or slightly alkaline media, and the yield of choline is greater in the latter than in the former. Satta (1907, 1908) determined that both lecithin and nuclein phosphorus in ox liver, pancreas and thymus split off inorganic phosphorus in autolysis, the nuclein more rapidly than the lecithin. Below are data from this study : 188 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 THE AUTOLYSIS OF ORGANIC PHOSPHORUS COMPOUNDS Satta (1907, 1908) Animal Total phos- phorus as P2O5 Percent Percents of total phosphorus Organ A lcohol-ether-sol- uble (Lecithin, jecorin, etc.) Water- and acetic acid-soluble (Inorganic) Freed by ashing: (Nuclein) of autolysis Days Ox Ox Ox Ox Ox 2.9 2.8 2.7 2.65 2.89 30.60 28.2 25.20 23.60 21.61 31.79 51.8 62.95 66.46 69.9 37.61 20.0 11.85 9.94 8.49 2 7 12 21 Ox Ox Ox 3.87 3.23 3.47 41.69 34.20 11.47 22.71 60.50 84.73 35.60 5.30 3.80 i 5 Calf Calf Calf Calf Calf 3.9 4.0 3.8 3.8 3.8 10.95 9.56 9.26 9.1 9.0 20.05 24.27 43.72 51.75 59.46 69.00 66.17 ,47.02 39.15 31.54 1 3 10 30 Lymph, sarcoma of intestine.... Man 1.85 19.66 39.59 40.75 Satta and Fasiani (1910) determined that the presence of lip- oids increased the autolysis of liver, as measured by the amount of nitrogen passing into solution in a given time. The action seemed to be most pronounced when the liver of a starved animal was used for the autolysis. The action was not proportional to the amount of lipoids present. Simon (1911) demonstrated active autolysis in brain tissue, which affected not only the phosphorus compounds soluble in alco- hol and ether, but also those which are insoluble, the proportion of the former to the latter, represented by the inorganic phosphorus split off from organic compounds, being as 61 to 39. Grund (1912a, 1912b, 1913) determined that in degenerating muscle, following the division of its nerves, there occurs a large in- crease of fat, and of water in proportion to fat-free substance ; but in the dry, fat-free substance the total nitrogen, total phosphorus and phosphatid phosphorus remain constant. On the other hand, the ratio of protein phosphorus to total phosphorus greatly increas- es, as also the ratio of protein phosphorus to protein nitrogen. Thus the protein phosphorus maintains its integrity, while phosphorus- free proteins dissolve. Degeneration as caused by hunger is ac- companied by the katabolism of all the protein components. For a discussion of autolysis of the several groups of organic phosphorus compounds see the discussions of the metabolism of the same. METHOD OF URINARY ELIMINATION OF PHOSPHATES In view of the importance of the function of the kidneys in freeing the system from certain classes of katabolites, and thus assisting in the maintenance of that constancy of conditions in the PHOSPHORUS METABOLISM 189 blood which is. essential to the vital functions, it is a matter of in- terest to know the method of formation of urine and the agencies through which this is accomplished. M. Maly (1876) shows that acid constituents diffuse and di- alyze more rapidly than alkali from such mixtures as are found in the blood, this depending on the greater mobility of ionized hydro- gen, and of more complex substances which contain hydrogen, than of corresponding basic substances. Thus acid phosphates are sep- arated from alkaline blood. This theory receives support from the investigation of Fitz, Alsberg and Henderson (1907), who demonstrated by hydrochloric acid feeding to rabbits that the resultant shifting of the salt equi- librium toward the acid side increased the urinary elimination of phosphates. In harmony also with this idea is the observation of Teissier (1877) of inverse relations in the excretion of glucose and phos- phates, the significance of which probably lies in an accentuation of the state of acidosis during diminished glucose outgo. Liebermann (1893b) found that alkaline solutions passed through a filter of lecithalbumin become acid. He therefore ad- vanced the theory (with experiments) that acid urine may arise in such a way, by the acid bodies in the kidney cells removing base from alkaline urates as the urine passes, provided there is not too much alkali. Frey (1911) also investigated the nature of the process by which phosphorus is separated from the blood in the kidneys. By intravenous injection of Na 2 HP0 4 , and phosphorus determinations on the blood serum and urine, he found that the urine contained from 10 to 60 or 70 times the concentration of phosphorus that ex- ists in blood serum ; thus it appears that the process of separation of phosphorus from the blood in the kidneys is more than one of filtration. Folin (1905) in discussing the laws governing the compo- sition of the urine maintains the incorrectness of the usual view (see, for instance, G. Zuelzer, 1905 and Ott, 1886) that the phos- phates of normal acid human urine are present to the extent of about 60 percent in the diacid form, and states that the phosphates of clear acid urine are all monobasic, the acidity of such urines be- ing ordinarily greater than the acidity of all the phosphates, the ex- cess being due to free organic acids. It is Folin's idea that the precipitate with barium chloride which is ordinarily considered to include the diacid-phosphates in reality contains phosphorus only as an impurity in the abundant precipitate of barium sulphate. 190 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 According to Hammarsten (1911), p. 784, phosphorus may be found in urinary sediments as tricalcic and trimagnesium phos- phates in alkaline urines, as dicalcium phosphate in neutral or faint- ly acid urine, and as ammonium magnesium phosphate in urines which have become ammoniacal through alkaline fermentation, or in amphoteric urines in the presence of a sufficient quantity of am- monium salts. ORGANIC PHOSPHORUS IN THE URINE From an early date it has been recognized that a portion of the phosphorus of the urine is present in organic combination. Ronalds (1846) considered that he found organic phosphorus in the urine, though he states that his methods were unsatisfactory. Sotnitschewsky (1880) determined glycerophosphoric acid as a common constituent of human urine, though the amount present is sometimes very small. W. Zuelzer (1881a) found that the urine of a man 22-25 years old contained 1-2 mg. of phosphorus (stated as P 2 5 ) combined with glycerin. In fever no more was found, but much more was found after chloroform anaesthesia, subcutaneous injection of morphine, after the crisis of pneumonia, and in erysipelas. The maximum amount found by Zuelzer for 24 hours is 35 mg. P 2 5 . Lepine and Eymonnet (1882) examined the urine of more than 100 subjects. Normal human urine they found to contain about. 0.150 gm. of glycerophosphoric acid per liter, being about 0.15 to 0.3 of one percent of the amount of the nitrogen. With fatty degeneration of the liver in tuberculosis the value of the glycero- phosphoric acid, in percent of nitrogen, was found to be 1.0-1.8, or about 6 or 7 times the normal. In a later article (Lepine, Eymonnet and Aubert, 1884) they re- port increased proportions of unoxidized to total phosphoric acid in the urine in apoplexy, epilepsy and delirium tremens, and in a dog after subcutaneous injection of hydrochlorate of morphine, and also in a dog after the ingestion of potassium bromide. They state that in pernicious anaemia it may be present in four times the normal amount, without an increase in the total phosphorus. An excess above normal occurs also in some cases of icterus, typhoid fever and acute pneumonia ; while in cases of meningitis the unoxidized phos- phorus was present in less than the normal relation to the nitro- gen of the urine. Biilow (1894) found, with dogs, that the phosphoric acid esters of the urine did not vary greatly with the introduction, either per os or subcutaneously, of glycerophosphates, these salts, in so far as PHOSPHOEUS METABOLISM 191 they were excreted in the urine, being mostly decomposed to phos- phates. His figures are as follows : URINARY EXCRETION OF PHOSPHORIC ACID ESTERS BY DOGS Days H 3 P0 4 as ether-phosphoric acid 5 0.00619 Normal conditions 1 0.01085 3 gm. calcium glycerophosphate-)- 2 gin, calcium carbonate per os 1 0.01191 3 gm. sodium glycerophosphate, suhcutane- ously 1 0.00626 Next day 1 0.00573 3 gm. salol 5 0.00626 Next day Rockwood (1895) showed, by the method of Siegfried, that normal urine contains both carnic and phosphocarnic acids. He found, as did Siegfried, two compounds in the iron precipitate, one "carniferrin," the iron compound of phosphocarnic acid, soluble in alkalis, and the other a basic iron compound of carnic acid. Ceconi (1896) states that the organic phosphorus of the urine equals 11-28 mg. per day, the amount varying with the volume; that values greater than 20 mg. are found only in pathological condi- tions ; and that the organic phosphorus of the urine is not affected by variations in the food. Oertel (1898-9) found, in a study of the urinary phosphorus of 7 healthy men, an average total phosphorus content of 2.0 gm. P 2 5 , and about 0.05 gm. P 2 5 as organic phosphorus. The highest amount of organic phosphorus for one day was 0.120, and the low- est 0.03 gm. P 2 5 . The amount was not influenced by work, but differed much with individuals. Keller (1900a) determined that the organic phosphorus of the urine of infants was greater in amount after feeding cow's milk than after feeding human milk, though it was a smaller percentage of the total urinary phosphorus, since the total phosphorus from the cow's milk greatly exceeded that resulting from woman's milk. It varied in amount from less than one to about 10 percent of the to- tal urinary phosphorus. In a fasting experiment on himself Keller found in a four-day test that the amount increased daily, thus, 0.017, 0.029, 0.034, and 0.057 gm. P,0 5 . These increased amounts constituted increased percentages of the total urinary phosphorus, though this also in- creased from day to day. The significance of organic phosphorus of the urine was not es- tablished. It did not vary regularly with the phosphorus given in the food, nor with absorbed phosphorus, nor with nitrogen metabol- ized. It was not influenced by Na,HP0 4 ingested. On a similar diet normal children excreted more organic phosphorus than did 192 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 children suffering from such disturbance as would reduce powers of oxidation. The amount eliminated was not altered by feeding with a very low phosphorus diet. Lepine (1901) states that in fatty degeneration of the liver its high lecithin content is accompanied by an abnormal glycerophos- phate acid content of the urine, which may reach a value as high as twice the usual amount. He suggests that an abundance of in- completely oxidized phosphorus in urine may be an indication of a fatty condition of the liver. Mandel and Oertel (1902) observed no change in the organic phosphorus of the urine by food rich in organic phosphorus. Y. Henderson and Edwards (1903) compared phosphorus fig- ures on the urine by direct titration and after fusion. From the correspondence of results they conclude that no organic phosphorus was present. K. Bornstein (1905) considered the origin of the organic phos- phorus of the urine, in metabolism experiments on himself. Over- feeding with protein did not increase the organic phosphorus of the urine. The author did not determine the source of this organic phosphorus but was of the opinion that it was of endogenous origin. Symmers (1904-5a, 1904-5b) has investigated the elimination of organic phosphorus in the urine in various pathological condi- tions. Symmers calls attention to the idea, which has prevailed un- til recently, that the urinary excretion of organic phosphorus, eith- er in health or in disease, is negligible, and cites 8 earlier investi- gations of the subject. Symmers states that exercise and diet do not affect the urinary excretion of organic phosphorus. Analyses of the urine of 20 cases, embracing 9 diseases, are given. The or- ganic phosphorus varies from a small part to about nine-tenths of the total phosphorus. Symmers cautions, therefore, against the use of inorganic phosphorus determinations as indices of metab- olism. He finds the excretion of organic phosphorus to some ex- tent rhythmical,periods of excessive secretion alternating with what may be either retention or diminished production. Symmers finds organic phosphorus elimination pronounced in lymphatic leukaemia ; and also in nervous diseases of degenerative type, to such extent as could not be derived from the destruction of nervous tissue. In conclusion Symmers says that abnormal increase of organic phosphorus may be explained either as an increase in the production of phosphorized endogenous metabolic products, or as an expression of lessened oxidation, with the organic phosphorus compounds as the end-products. PHOSPHORUS METABOLISM 193 Symmers determined total phosphorus by titration with urani- um nitrate, after fusion; and inorganic phosphate also by uranium nitrate titration, but without fusion. Organic phosphorus was reckoned by difference. Franchini (1907, 1908a) found glycerophosphoric acid quite variable in amount in the urine of fasting rabbits. When lecithin was fed,the average glycerophosphoric acid content of the urine was increased, but there was no uniform effect noticeable. No choline or formic acid was found as a decomposition product from lecithin. Plimmer, Dick and Lieb (1909-10) found the inorganic phospho- rus of the urine to constitute 90-100 percent of the whole, its amount depending especially on the intake of P 2 5 . Organic phos- phorus excretion they found very irregular and not dependent on the diet. They concluded, therefore, that it must be of endogenous origin. Mathison (1910) determined organic phosphorus in the urine of 5 healthy persons on 3-6 consecutive days, and in the urine of one person as affected by exercise and by the ingestion of glycerophos- phoric acid and sodium glycerophosphate. Organic phosphorus was found normally present, usually in amounts greater than 0.1 gm. P 2 5 per day, though occasionally it fell below this figure, and in one case reached 0.3 gm. The proportion of the total phosphorus pres- ent in organic combination varied considerably from day to day but averaged about 6 percent. The organic phosphorus outgo was not affected by ingestion of glycerophosphoric acid. The organic phosphorus of urine was found readily dialysable and not precipitable by reagents which pre- cipitate protein. Kondo (1910) studied the effect of organic phosphorus of the food on the organic phosphorus of the urine. With a dog weighing 8 kg. the urinary organic phosphorus varied from 0.0086 to 0.0227 gm. P 2 5 per day, while the total urinary phosphorus varied from 0.3941-1.6436 gm. P 2 5 daily. The variations in organic phospho- rus, then, were slight in absolute quantity. The proportion of or- ganic to inorganic phosphorus was less (1.2-1.6 percent) on days of excessive phosphorus ingestion, in the shape of brain, casein and thymus, than on intermediate days (2.2-3.5 percent), when the phosphorus was present in horse flesh. Bogdanow (1911) found the organic phosphorus of the urine to rise significantly immediately after the fall in temperature in croupous pneumonia. 194 OHIO EXPERIMENT STATION: TECHNICAL BUL. 6 To summarize the evidence: Urine may contain organic phos- phorus as glycerophosphoric acid, and as phosphocarnic acid. This fraction is variable in amount from a very small to a large part of the whole, and is too large a factor to ignore in any quantitative work. This organic phosphorus increases after fast (Keller), chloroform anaesthesia, morphine injection, and in many pathologi- cal conditions. Its significance is not known. Its amount seems slightly to increase after the ingestion of glycerophosphates, but there is certainly no marked or constant effect of the constituents of the food, either phosphorus-containing or otherwise, or of the amount of phosphorus absorbed, or of the nitrogen metabolism, on this excretion. It appears to be to some extent an individual char- acteristic, though temporary physiological states seem to be the dominant factors in its quantitative variation. PHOSPHORUS COMPOUNDS IN THE FECES The phosphorus compounds of the feces are of mixed origin, being derived in part each from food residues, intestinal excretions, digestive secretions, intestinal epithelium, and bacterial products, the relative proportion of the whole which is contributed by each of these factors varying much with the nature of the diet and kind of animal, the feces being the main vehicle of outgo for metabolized as well as unmetabolized phosphorus in herbivora, while among carnivora and omnivora the main outgo of metabolized phosphorus is through the urine. Since there is no evidence of a direct rela- tionship between food and feces phosphorus compounds, compara- tively little attention has been given the determination of the ident- ity of the compounds in the feces. Inorganic salts occur in abund- ance both as intestinal secretions and as food residues after more or less reapportionment of bases during the digestive process. The following analyses of the ash of meconium are quoted from F. Miiller (1884). Analyses I, II, III and IV are of human mecon- ium and were quoted by Miiller from Zweifel. ASH ANALYSES OF MECONIUM— Percent of Ash I II III Horse Human Human Human Human 0.30 0.67 0.80 0.87 1.36 2.60 0.86 18.76 8.00 31.80 5.70 5.09 2.65 4.32 3.60 4.00 7.23 10.21 10.66 7.80 5.40 3.20 38.42 47.05 22.30 23.00 39.50 21.92 24.42 K 6.00 Na 24.20 ... 8.40 3.78 2.53 8.68 IV Human Insol. in HC1 Fe 2 3 CaO MgO P 2 O s SOa Alkalis CI 0.80 9.50 7.92 8.58 81.90 Ns 15.93 K7.09 8.90 PHOSPHORUS METABOLISM 195 Feces from long fasting were shown to resemble meconium. Flesh feces also resemble meconium, and seem to consist largely of excretion products of the intestinal canal rather than of food resi- dues. Muller also states that the time of passage of the contents of the alimentary tract affects its composition ; thus, if the movement be rapid one finds unaltered bile salts and abundant alkali salts, but if the feces remain in the large intestine for a week or more almost no alkali compounds appear in the feces as passed. Micko (1900), studying feces phosphorus with adult human be- ings, concludes that the organic phosphorus of the feces does not come for the most part from the food, but that some true nuclein is present in the feces from an ordinary mixed diet and also in the feces from diets of flesh and plasmon. In a later paper (1900) Micko, Muller, Poda and Prausnitz con- clude that human feces are derived in large part, with but few ex- ceptions, from intestinal secretions, and (Muller) that cow's milk used by either infants or adults leaves no phosphorus-rich casein residue in the feces. Schilling (1901a) states that when the food contains only a small amount of phosphorus the feces contain little or no crystalline tricalcium phosphate. Von Oefele (1901) states that in diabetes and syncopic neurosis the relative scarcity of triple phosphate crystals in the feces can be explained as due to coexistent phosphaturia. The presence of triple phosphates in the feces was found to have no especial diagnos- tic value. A number of workers have determined lecithin in the feces. Among these, Deucher (1898) determined lecithin in the feces in cases of occlusion of the pancreatic duct; P. Muller (1900) found lec- ithin, calculated as distearyl-lecithirt, in milk feces, to the extent of 1.13-10.1 percent of the ether extract, and considered it as derived largely from the food. Long (1906b) and Johnston (L. & J., 1906, 1907) have studied the phosphorus of the feces fat. Ether-soluble phosphorus com- pounds which they speak of as lecithins, are found in amounts of from one to several percent, but they suggest that these may be but remotely related to simple distearyl lecithin. They consider that this fraction may come in part each from food, intestinal epithelium, bacteria and bile residues. Commenting on the usual belief that lecithin can not escape digestion, since it is easily split by the pancreatic secretion, they suggest that, while this may be true for the largest part of the food lecithins, it may not be equally true of the lecithins of the bile under certain conditions. 196 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Franchini (1907, 1908a) determined lecithin in the feces of rab- bits. There was no uniform increase produced by the feeding of lecithin during fast, but the average lecithin content of the feces was greater. Peritz (1908-9b) reports lecithin in the feces in cases of tabes and paralysis as varying between 0.1311 and 7.221 gm. per day, there being no evidence of relationship between these amounts and the content of the food in lecithin. A. Bornstein (1909) found 0.02 gm. lecithin per day in the feces from a diet of crackers and milk. In connection with a phosphorus metabolism study Rogozinski (1910) made separations of the groups of phosphorus compounds in the feces of a man and of two dogs. The rations and feces phos- phorus data were as indicated below. PHOSPHORUS COMPOUNDS IN HUMAN AND IN DOG FECES Percent of Total P O Period (5 days) Day Diet Lecithin Phytin Inorganic Protein SERIES I DOG Meat, rice, pork fat Meat, rice, pork fat, phytin Meat, rice, pork fat Meat, rice, lecithin Meat, rice, pork fat Meat, rice, pork fat, Na phosphate Meat, rice, pork fat SERIES II DOG Meat, rice, pork fat Meat, rice, pork fat, phytin Meat, rice, pork fat Meat, rice, lecithin Meat, rice, pork fat Meat, rice, pork fat, Na phosphate Meat, rice, pork fat SERIES ni MAN Mixed diet Mixed diet Mixed diet Mixed diet Mixed diet . Mixed diet, phytin Mixed diet, phytin Mixed diet, phytin Mixed diet, phytin Mixed diet, phytin Mixed diet Mixed diet Mixed diet Mixed diet Mixed diet 1.82 6.34 51.00 0.45 69.62 14.25 1.46 4.04 47.52 2.19 6.18 48.69 1.20 10.27 39.74 1.16 8.92 50.43 1.29 6.84 40.15 40.84 15.68 46.98 42.94 48.79 39.49 51.72 1.68 26.16 42.76 0.36 62.42 14.71 1.71 18.72 40.68 5.06 13.53 40.21 2.11 20.47 39.94 1.40 22.84 43.54 1.92 21.61 43.13 29.40 22.51 38.89 41.20 37.48 32.22 33.34 6 7 8 9 10 11 12 13 14 15 20.46 3.15 43.82 20.18 4.17 46.59 18.19 3.01 50.51 21.88 0.00 44.87 19.49 1.58 44.80 17.24 2.66 14.96 2.45 16.95 1.49 24.05 0.00 19.52 0.00 16.90 20.45 21.56 14.80 20.47 0.00 3.43 2.24 0.00 0.44 45.67 50.77 52.88 52.87 54.38 53.97 50.69 49.93 55.47 47.38 32.57 29.06 28.29 33.25 34.13 34.43 31.82 28.68 23.08 26.10 29.13 25.43 26.27 29.73 81.71 PHOSPHORUS METABOLISM 197 The group of compounds designated "phytin" is that fraction soluble in acidified water; protein phosphorus is considered to be that portion of the organic phosphorus which is insoluble in acidi- fied water. In dog feces inorganic and protein phosphorus predominate, though there is also considerable phytin phosphorus, especially when phytin is fed. The feeding of lecithin also increased the feces lecithin. In human feces there is a very much larger proportion of leci- thin phosphorus than in dog feces. The phytin is much more com- pletely absorbed by the human being than by the dog, and the feces phytin is not increased in human beings by the ingestion of phytin. This difference in the utilization of phytin is in harmony with the natural differences of the diets of the two species. The dog has no need for capacity to digest phytin, since there is no phytin in its natural food. Stutzer (1908) found, in feeding experiments with sheep on hay, that the feces contained metabolic organic phosphorus com- pounds insoluble in acid gastric juice in quantities greater than would have been expected from the analysis of the food. Emmett (1909) determined phosphorus on the ether extract of pig feces, and found 0.39-0.74 percent P 2 5 in this extract. Lipschiitz (1910b) determined that the phosphorus content of the feces of young dogs on mother's milk, cow's milk, meat diet, and phosphorus-poor diet varied between 0.87 percent with mother's milk and 0.60 with meat diet, while the starvation feces contained about the same (0.63 percent) phosphorus content. The phospho- rus intake with cow's milk was 0.201 gm. per kg. live weight ; with phosphorus-poor diet 0.023-0.031 gm., and on meat diet 0.095 gm., from which it would appear that the phosphorus content of dog feces under these conditions originated, not in the food, but in the secretions of the digestive tract. Lipschiitz quotes figures for the phosphorus content of feces as follows : Blauberg— infants 6-7 days old, 0.26-0.49 percent ; Tiger- stedt — man on low-phosphorus diet, 0.9 percent ; Miiller — man, fast- ing, 1.8-2.2 percent, and dog, grown, fasting, 1.91 percent; and Munk — dog, grown, fasting, 3.65 percent, from which he concludes that the feces arising from the digestive tract of the growing organ- ism is notably poorer in phosphorus than that of adults. Fraps and Rather (1912) have investigated the composition and digestibility of the ether extract of a variety of hays and fod- ders, and of certain constituents, among others phosphoric acid, of 198 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 the ether extract. Since the ether extract of the feces is to a con- siderable extent a bile residue, and not directly related to the food, it seems to us that their statements as to the relative digestibility of the lecithin and other constituents of the ether extract are quite without warrant. VonWendt (1905) and Zuckmayer (1912) both concluded that phosphorus is in the feces largely in the form of the trimetallic phosphates. See p. 184. Summary. The phosphorus compounds of the feces are not well-known, but have been found to include nuclein phosphorus, in- organic phosphates in a variety of forms, lecithin-like compounds and other organic metabolic products, the nature of which has not been investigated. Studies of the feces in fasting, and during feed- ing on meat and milk, lead to the belief that a considerable part of the feces phosphorus, especially the lecithin-like compounds, have an origin other than in the food. To relate the feces phosphorus com- pounds as a whole directly to those of the food, as in computation of digestibility, is without justification. PHOSPHORUS EXCRETION AS AFFECTED BY ACIDS, SALTS, GENERAL CHARACTER OF DIET, AND SPECIES OF SUBJECT ACIDS One of the most potent influences affecting the elimination of phosphates is the ingestion of acids and acid salts. Under this influence calcium and phosphorus, being associated in the body, are affected at the same time, and thus are excreted in combination; in- creasing acidity of the urine being associated with increased excre- tion of both calcium and phosphorus. Schetelig (1880) studied urinary calcium under different condi- tions with a normal subject and 24 cases of a number of diseases. Incidentally phosphorus was considered. The ingestion of hydro- chloric acid caused an increase of both calcium and phosphorus in the urine. Ott (1886) reported the acid and neutral phosphates of human urine as being in the relation 60:40. He determined that cal- cium is present in urine in greater amount than the solubility of its acid and neutral phosphates in water would explain, and that the solubility of CaHP0 4 is increased by the presence of acid phosphates of the alkalis, gypsum, ammonium chloride and magnesium sul- phate. Neutral alkali phosphates decrease the solubility of tricalcio phosphate. Rudel (1893a), in experiments on dogs and infants, proved that ingestion of hydrochloric acid, calcium acetate, or chalk, would in- crease the urinary calcium, while sodium phosphate was without PHOSPHORUS METABOLISM 199 such effect, and 'calcium phosphate served markedly to reduce the urinary calcium. Subcutaneous injection of calcium acetate caused increased urinary calcium as also did prolonged peristalsis from tinct. opii. Rudel (1893b) showed with a nine-months-old infant that the introduction of sodium phosphate into the food would decrease the calcium to 41.76-61.5 percent of the normal. A notable increase in the urinary calcium followed the administration of dilute hydro- chloric acid. In harmony with these observations is that of Gerhardt and Schlesinger (1899) that the administration of sodium bicarbonate markedly reduced the urinary calcium and correspondingly in- creased the feces calcium. Similarly Proskauer (1910) found an evident increase of calcium and a smaller increase of magnesium in infant's blood in severe digestive disturbance. In harmony with this is the observation of Allers and Bondi (1907) of an increase of calcium in the blood in experimental hydrochloric acid poisoning, from 0.069 gm. to 0.159 gm. in 1000 gm. of blood. W. Camerer, Jr. (1902b), from a study of conditions affecting ammonia excretion, submits data on the relative amounts of total phosphorus, and phosphorus in acid salts, in the urine, as affected by age, sex, exercise, nature of the food, and the ingestion of acid and alkaline compounds. The figures are as follows: AVERAGE DAILY TOTAL PHOSPHORUS, AND PHOSPHORUS IN ACID SALTS, IN URINE— Grams ^^ m ■ Grown men on mixed diet Youths Children Men Sp. gr. 1.016 Women Sp. gr. 1.011 Animal food Mixed diet plus 4-5 gm. HCl Mixed Resting Exer- cise Sp. gr. 1.0165 Sp. gr. 1.0225 diet plus 8 gm. NaHCO Total P2O5 P2O5 in acid 2.89 1.70 1.80 1.01 1.80 0.82 0.70 0.38 1.08 0.64 2.07 1.31 1.56 1.14 3.5 1.55 1.79 1.08 2.635 70 Here we note a much higher proportion of acid salts resulting from the ingestion of HCl than from NaHC0 3 . Folin and Shaffer (1902) found that ingestion of hydrochloric acid increased the outgo of nitrogen, sulphur and phosphorus, with- out marked change in the proportions between these elements. VonTabora (1905) studied the phosphorus content of 122 sam- ples of gastric juice from normal and pathological subjects. The phosphorus content of the stomach, with normal or greater acidity, is negligible, following a phosphorus-free test meal, the maximum amounts being about 0.005 gm. P 2 5 in 10 c.c. of filtered juice. The calcium content was about 0.002 gm. in 100 c.c. 200 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The author concluded that the phosphate content of gastric juice is indirectly proportional to, and dependent on, the degree of acidity, in one and the same individual. In the presence of hydro- chloric acid all phosphate was considered to be present in the diacid form ; while in anacid conditions the monacid form predominated. Fitz, Alsberg and Henderson (1907) studied urinary phospho- rus elimination in rabbits as affected by the ingestion of hydrochlo- ric acid. The introduction of acid into the circulation increased, in four rabbits, the outgo of phosphorus in the urine. This increase was followed by a decrease, and then after further introduction of acid, by a premortal rise in phosphorus outgo. This increase in phosphorus elimination, under the influence of acid poisoning, is explained by the conversion of di- to mono-phos- phate in the blood, for the neutralization of the acid, and the res- toration of the normal proportions of phosphorus in these forms by the elimination of the excess of mono-phosphate. Granstrom (1908) found that feeding H 3 P0 4 to rabbits in- creased the feces calcium, and the phosphorus of both urine and feces. Adler (1909) reports a study of the effects of hydrochloric acid and calcium phosphate administration on the excretion of calcium and phosphorus by human beings, but the brevity of the collection periods renders results of doubtful significance. Wuertz (1912) found that the addition of HC1 to the diet of rabbits did not affect the distribution of phosphorus between urine and feces, the acid being so rapidly absorbed that its ingestion did not increase the length of the acid portion of the alimentary tract, but that the addition of calcium carbonate increased the alkalinity of the intestine, and, therefore, greatly increased the proportion of the phosphorus excreted in the feces. PHOSPHORUS EXCRETION AS AFFECTED BY SALTS OF SODIUM AND POTASSIUM Sick (1857) found that the ingestion of sodium phosphate in- creased urinary phosphorus by more than the added amount, with a decrease of earth phosphates, and increase of alkali phosphates. C. Ph. Falck (1872) determined, by intravenous injection of so- dium phosphate into dogs, that this salt was promptly eliminated by the kidneys, the principal part within a few hours. Doses of 5.4 to 10.2 gm. by intravenous injection (the volume of the solution not stated) caused vomiting. Bunge (1873) found that potassium phosphate, as well as the citrate and sulphate, has the effect, when ingested by human beings, to cause an increase in the elimination of sodium salts. PHOSPHORUS METABOLISM 201 Bertram (1878) found that in man potassium citrate decreases urinary excretion of phosphorus but little, while the calcium excre- tion is greatly diminished. A further decrease of the phosphoric acid takes place if in addition to potassium citrate, calcium carbon- ate is also given. In herbivora Bertram found that K 2 HP0 4 if ad- ded, to the usual diet led to the appearance of phosphorus in the urine. P. A. E. Wagner (1892) demonstrated the diuretic effect of di- sodium phosphate by injecting it in solution into the jugular vein of rabbits. The urine was then collected at 15-minute intervals. The maximum excretion of urine was during the second 15-minute inter- val. Gerhardt and Schlesinger (1899) found in diabetes that inges- tion of sodium carbonate reduced urinary phosphorus excretion ; but this was probably by virtue of its alkalinity alone, and not by virtue of any specific action of sodium, or to the solubility of its com- pounds. Desgrez and Guende (1906) studied the influence of phosphoric acid, monosodium phosphate and trisodium phosphate on the metab- olism of the guinea pig. Twenty-four male animals were used in four series of six each. The basal ration was consumed ad libitum, the amounts consumed being said to be about alike. The only phosphorus metabolism data are figures representing urinary phos- phorus outgo. The urinary nitrogen was increased by all of the phosphorus compounds, and in order of their acidity, that is, the neutral phosphate caused the least increase, the acid phosphate the next, and the phosphoric acid the most. For a computation by Raoult's formula of the molecular weight of the average molecule in the urine in these experiments see Des- grez and Posen (1907). According to Loewi (see von Noorden, 1907, vol. 3, p. 1079, 1080; German ed. vol. 2, p. 685, 686) von Bunge found that urinary phosphorus excretion is not influenced by sodium salts, but that po- tassium salts cause a considerable fall in urinary phosphorus. Loewi states that Bertram cleared up the question as to the method of action of potassium in this connection, by the observation (previous- ly mentioned) that the exhibition of potassium citrate (er- roneously translated "calcium citrate," Eng. ed.) considerably de- creases not only the phosphoric acid but also the calcium in the urine (in one case within three days from 0.5 to 0.28 gram), an action which is not shared by sodium (Beckmann) . The cause of the dif- ferent action of potassium and sodium on calcium excretion in the urine is, according to Loewi, who credits the observation to Bert- ram, that the calcium phosphates found in the body fluids are insol- 202 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 uble in potassium carbonate, and hence can not be excreted in the urine, while they are, however, soluble in the corresponding sodium compounds. Loewi states that the excretion of magnesium is not affected by either potassium (Bertram) or sodium (Beckmann). Hart, McCollum and Humphrey (1909), in their study of phytin metabolism with a dairy cow, found that the potassium of the feces varies with the phosphorus intake. With high phosphorus intake the potassium is in part deflected from urine to feces, while with a low phosphorus intake the potassium is eliminated mostly in the urine. Von Hoesslin (1909), in experiments with dogs, studied the effects of sodium chloride, and other salts, on metabolism. He con- cluded that the addition of sodium chloride to the food increased the outgo of phosphorus, especially in the urine. The excretion of ter- tiary sodium phosphate by the kidneys is increased by water, and still more by the sodium chloride intake. The phosphorus outgo, especially in the urine, is also increased by overheating. Phos- phates added to the diet caused diuresis, and an increase in the per- centage content of the urine in phosphorus. Oeri (1909) studied phosphorus metabolism as affected by in- gestion of disodium phosphate. The basal ration was composed of normal foods in usual combinations. The subjects of the experi- ments were a woman, aged 35 years, weight 55 kg., and the author himself, aged 25 years, weight 93 kg. The numerical data follow. AVERAGE DAILY PHOSPHORUS BALANCES OF MATURE MAN AND WOMAN AS AFFECTED BY INGESTION OF DISODIUM PHOSPHATE— Grams Periods and days Intake P2O5 Urine P2O5 Feces P2O5 Total excreted P2O5 Urine, percent P2O5 Feces, percent P2O5 Bal- ance P2O5 Diet Fore-period 1-6 Phosphate fed 7 After-period 8-12 Phosphate fed 13 After-period 14 6.45 7.46 5.45 7.48 5.45 2.24 2.62 2.02 2.64 3.15 2.50 3.90 3.07 3.62 2.83 4.74 6.52 5.09 6.26 5.98 47.3 40.2 39.7 42.2 52.7 52.7 59.8 61.3 57.8 47.3 +0.71 +0.96 +0.36 +1.22 -0.53 Milk, veal, bouillon, bread, potatoes, apples, sago, butter, coffee, jelly. Same plus disodium phos- phate. Same without phosphate. Same with phosphate. Same without phosphate. Fore-period 1-9 Phosphate fed 10 After-period 11-17 6.54 8.57 6.54 3.62 3.64 3.39 3.31 4.06 3.10 6.93 7.70 6.49 52.3 47.0 52.3 47.7 53.0 47.7 -0.39 +0.87 +0.06 Milk, veal, bouillon, bread, potatoes apples, pea soup, butter, coffee, beer, jelly. Same plus disodium phos- phate. Same without phosphate. The sodium phosphate was without marked or consistent effectj on the partition of phosphorus between urine and feces. PHOSPHORUS METABOLISM 203 EFFECTS OP ■CALCIUM AND MAGNESIUM ON PHOSPHORUS ELIMINATION Calcium Carbonate. Riesell (1868) found that the ingestion (by himself) of large amounts of calcium carbonate (10 gm. with each meal and some with the drink ) decreased the urinary phos- phorus to about one-half the normal, but during four days of such chalk treatment the urinary phosphorus rose nearly to the nor- mal, apparently through the absorption of the calcium phosphate formed in the intestine. Under the influence of the chalk, the al- kali phosphates of the urine were largely replaced by alkaline earth phosphates. Schetelig (1880) did not observe a decrease of urinary phospho- rus from taking calcium carbonate for 2 days in 3-gram doses. The amount was perhaps insufficient. E. Lehmann (1882, 1894) determined that the alkaline-earth carbonates, when ingested, have the effect quantitatively to reduce the urinary phosphorus. Riidel (1893a) shows that the administration of calcium car- bonate may cause some increase in the urinary calcium of children, but not by any means in proportion to the intake. Strauss (1896) studied the effect of calcium carbonate inges- tion on the elimination of phosphorus, uric acid and purin bases in man. Ingestion of calcium carbonate caused a decrease in urinary phosphorus, which did not disappear immediately after the with- drawal of the carbonate from the food, but only after about three days. The decrease in urinary phosphorus is due to decrease prin- cipally in the amount of monosodium phosphate, but partially to de- crease in disodium phosphate, thus decreasing the urinary acidity but never causing alkalinity. Uric acid and purin bases were not affected. Urinary calcium was slightly increased. Herxheimer (1897) tested the effects of ingestion of calcium carbonate baked into bread in the amount of 5 percent of the same. The carbonate had the effect to decrease the urinary phosphorus quite markedly, and also to increase the feces phosphorus, though to a less extent, the calcium apparently increasing the retention of phosphorus, though phosphorus in the food was not determined. There was in the urine a greater reduction of monophosphate than of diphosphate, which produced in the latter a great relative in- crease. Herxheimer states that 18 grams of carbonate baked in the bread was more effective than 30 grams as a powder. Volhard (1904) found that great quantities of calcium carbon- ate in the food did not seriously affect digestion or phosphorus metabolism. 204 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 G. Zuelzer (1905) states that calcium affects the urinary phos- phorus by uniting with this element in the intestine and passing off in this form in the feces. With a lime-poor diet the feces are poor and the urine is rich in phosphorus. On the basis of work by Zerner and . Ritter, and others, vonNoorden and Dapper (vonNoorden 1907, III, 946) state that even after the ingestion of considerable quantities of calcium car- bonate the urine remains slightly acid, since the calcium is excreted almost entirely by way of the intestine, in which it combines with phosphoric acid, thus restricting its absorption. Thus the total phosphorus of the urine is decreased, and, as the reaction of the urine approaches alkalinity, the proportion of disodium to mono- sodium phosphates is modified in favor of the former. This de- crease of phosphates involves an absolute and a relative decrease of monosodium phosphate, a substance which directly promotes the precipitation of uric acid ; while the relative excess of disodium phos- phate, which is capable of dissolving uric acid, is left free to exert a greater effect. Kochmann and Petsch (1911) found, in metabolism experi- ments with dogs, that increasing the lime of the diet, the phospho- rus remaining constant, increased the feces phosphorus and de- creased the urine phosphorus. When a calcium equilibrium had been established and protein, carbohydrates and fat were added to the ration, the calcium intake remaining constant, the calcium equi- librium was disturbed, and lime was lost from the bones. Bertram (1878) and Renvall (1904) also found that calcium carbonate in the diet served to deflect phosphorus from urine to feces. Various Salts of Calcium and Magnesium. Tereg and Arnold (1883) reported results of experiments with dogs in which they compared the effects .on metabolism of the ingestion of calcium car- bonate and primary, secondary and tertiary phosphates as supple- ments to uniform basal rations. The authors interpret the negative nitrogen balance in the fourth period as being due to the influence of the primary calcium phosphate in causing increased katabolism of body pro- tein, and cite in support of this idea the coincident loss of calcium and phosphorus. We would suggest that, comparing the calcium and phosphorus balances of Periods II, III and IV, the smaller the proportion of cal- cium to phosphorus in the salt supplement the less is the storage, or the greater the loss, of both calcium and phosphorus, suggesting that in these rations the lack of proportion between these constit- PHOSPHORUS METABOLISM 205 uents restricts the usefulness of both. The great increase of phos- phorus retention caused by the ingestion of lime in the fifth period shows that in these rations the lime content limited the deposition of phosphorus. AVERAGE DAILY NITROGEN, CALCIUM AND PHOSPHORUS BALANCES WITH A DOG ON NORMAL RATIONS SUPPLEMENTED BY INORGANIC SALTS— Grams N CaO P2O5 Period and date Body weight, initial and final Food Urine Food Urine Food Urine P2O5: N in urine Rations Feces Feces Feces Balance Balance Balance I 32000 22.68 17.76 4.31 0.780 0.0455 1.465 5.040 3.087 3.003 600 gm. dog biscuit Nov. 12-15 32900 +0.61 -0.7305 -1.049 17:100 II 22.68 5.140 8.710 600 gm. dog- biscuit; 17.45 0.1077 3.539 10 g-m. Ca3 (P04)2 4.90 4.960 4.728 Nov. 18-21 constant +0.33 +0.0701 +0.443 17:100 III 22.68 4.688 9.350 600 firm, dog- biscuit; 17.30 0.053 4.496 10 gm. CaHP04 5.10 4.557 4.810 Nov. 29-Dec 2 constant +0.28 +0.078 +0.044 32:100 IV 32900 22.68 1.905 9.255 600 g-m. dog- biscuit; 18.75 0.0779 5.895 7.5 gm. CaH4(P04>2 4.83 1.860 3.853 Dec 5-12 32600 -0.90 -0.0354 -0.493 31:100 V 22.68 6.380 0.158 4.310 5.040 1.336 2.088 600 gm. dog biscuit; 10 gm. chalk Dec 19-22 +1.914 +1.616 VI 33500 22.58 0.144 2.802 600 gm. horse flesh; 16.45 0.020 2.532 100 gm. horse fat 3.90 0.100 0.237 Dec 28-31 33520 +0.19 +0.022 +0.033 19:100 VII 33520 22.58 4.052 0.029 7.112 3.402 600 gm. horse flesh; 100 gm. horse fat; 10 gm. CaHP04 Jan. 7-10 33520 VIII 33520 22.58 0.894 0.061 0.740 5.612 4.572 0.909 600 gm. horse flesh; 100 gm. horse fat; Jan. 13-16 33430 +0.093 +0.131 5 gm. CaH4(P04>2 Tereg and Arnold compute that in Period IV the phosphorus loss could not all be accounted for as coming from the loss in flesh, a portion apparently coming from the bones. That all three of these phosphates were absorbed is not to be questioned, though the authors note that the calcium and phosphorus apparently ab- sorbed, that is, missing from the feces, are not in proportional amounts. Either these salts were decomposed in the intestine, and 206 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 the parts absorbed independently of each other, or, after absorption, they were decomposed in the blood, and the parts eliminated in un- like portions through the intestines and the kidneys. Of the primary salt (Period IV) nearly twice as much calcium and phosphorus were eliminated in the urine as of the secondary salt. In periods VI, VII and VIII where the basal ration was meat, the primary salt caused a much greater increase of the calcium and phosphorus of the urine than did the secondary salt, and in in- jection experiments the authors noted similar increase of the cal- cium and phosphorus of the urine. It is their opinion therefore that the primary salt is absorbed as such from the intestine. Steel and Gies (1907a) determined the effects of the addition of bone ash to a diet of prepared meat, cracker meal, lard and water, on the urinary excretion of calcium and phosphorus by dogs. It was desired to use bone ash in the ration to give bulk and body to the feces. Bone ash seems not to alter the urinary elimination of cal- cium, but a decrease of urinary phosphorus from 0.460 to 0.244 gm. P 2 5 in 21 days, during the feeding of bone ash, shows that the use of bone ash for the purpose intended would be justifi- able only in such experimental work as would be unaffected by a de- crease of phosphorus absorption or change in path of outgo. Lothrop (1909) investigated the effect of bone ash in the diet on the metabolism of the dog. The ingestion of bone ash decreased the urinary phosphorus. Hart, McCollum and Humphrey (1909) found, in their study of phytin metabolism with a milch cow, that calcium, magnesium and phosphorus were excreted principally in the feces, and that a low phosphorus intake served to increase the urinary elimination of cal- cium. R. Berg (1910a) conducted balance experiments on himself, using a basal ration to which he added, in different periods, various organic and inorganic phosphorus compounds. Tricalcic phosphate he found excreted largely as such in the feces. The dicalcic phos- phate was excreted in part as tricalcic phosphate and magnesium ammonium phosphate. Hypophosphites were almost all excreted unchanged. Berg considers that the best source of calcium is green vegetables prepared so as to retain their salts. PHOSPHORUS METABOLISM 207 De Jager (1910) submits the following data showing the effects of calcium salts on urinary phosphorus in man. URINARY PHOSPHORUS AS AFFECTED BY CALCIUM SALTS— Grams Days Periods Ave. Daily P a O s 4 Preliminary .2.462 10 4 gm. Ca-sulphate daily 2.344 16 Interval 2.489 5 7.5 gm. Ca-lactate daily 2.087 18 Interval 2.199 10 6 gm. Ca-sulphate daily 1.767 14 After period 1.999 The slight decrease of urinary phosphorus produced by the cal- cium sulphate is doubtless due to the relative insolubility of this salt. The lactate, with its weak, oxidizable acid radical, yields much more of its calcium free for combination and excretion with phosphorus in the feces. A. R. Rose (1912a), in his phytin metabolism study with a cow, found that the ingestion of calcium and phosphorus as the phytate deflected a part of both the calcium and magnesium from urine to feces, the calcium added as the phytate also being eliminated by the feces. With decreasing phosphorus in the food there was an in- crease of urinary calcium. Gregersen (1911) showed with rats that calcium and magnes- ium tend to deflect phosphorus excretion into the feces, either when the phosphorus excreted is contained in the food, or when it is met- abolized phosphorus from the body. Gregersen also found that this effect was not affected by such acid-saturating salts as sodium bicarbonate. In experiments with swine, Forbes, Beegle, Fritz and Mensching (1914) found that in common foods magnesium tends to deflect the phosphorus excretion from urine to feces, and excess- ive phosphorus content of the ration seems to limit the ab- sorption of magnesium. With an average daily intake of 2.17 gm. magnesium and 5.40 gm. phosphorus there was storage of magne- sium, but with an intake of 9.28 gm. magnesium and 20.71 gm. phosphorus there was loss of magnesium, apparently combined with phosphorus, through the feces. EFFECTS OF DIET AND SPECIES ON PHOSPHORUS ELIMINATION The bearing of species, especially as determining prevailing diet, on the paths of elimination of phosphorus is marked. The herbivora normally consume a diet which is comparatively poor in phosphorus and rich in alkalis and alkaline earths. The omnivora and carnivora, on the other hand,, live on a diet which is so much richer in phosphorus and poorer in mineral bases that a different and much more extensive acid-neutralizing capacity has been 208 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 evolved. Thus, the herbivora excrete their mineral acids in com- bination with fixed alkalis, while the omnivora and carnivora are able to excrete the same largely combined with ammonia. During the suckling period, however, the excretion of phosphates is much the same in both classes of animals. A few of the many papers showing the different methods of elimination of phosphorus in herb- ivora and other animals are mentioned in brief. Bischoff (1867) found that in dogs phosphorus is excreted to the extent of about 12-thirteenths in the urine, mostly combined with the alkalis, while the remainder leaves the body in the feces combined principally with calcium, iron and magnesium. Bertram (1878) concluded that the absence of phosphorus from the urine of herbivora was due to the richness of the food in vege- table alkali compounds and calcium salts, alkaline liquids containing carbonates having no solvent power for calcium phosphate, though they do dissolve magnesium phosphate. F. Miiller (1884) concluded from analyses of meconium and of feces from diets of meat that the dog excretes its lime mostly through the feces, while magnesium, and to a greater extent still, phosphorus, is excreted chiefly through the urine. The content of the feces in the alkalis varies indirectly with the length of stay in the intestine. Weiske (1872b) analyzed the urine of two goats, one receiving milk alone, ad libitum, and the other green clover and turnip leaves. The ash analyses are as follows : Vegetable diet Milk diet K2O 34.91 42.83 Na20 22.48 14.05 CaO 0.77 „ 0. 98 MgrO 3.28 0.61 Fe203 trace trace CO2 10.40 none Si02 „ 0.59 none S03 16.89 3.02 P2O5 trace 22.22 CI 13.35 20.67 102.67 104.38 O out for CI 3.01 4.66 99.67 99.72 On a milk diet the. goat excretes much phosphorus in the urine; on this vegetable diet no phosphorus was excreted in the urine. Jordan (1885-6) found in the urine of a sheep nearly half of the total potassium outgo, but no phosphorus. E. Wolff (1886) reports determinations of the phosphorus of clover hay, and of the feces resulting from its being fed to horses. The feces phosphorus equalled the food phosphorus. With a ration of oats, straw and meadow hay the feces phosphorus was 98.9 per- cent of the food phosphorus. PHOSPHORUS METABOLISM 209 Grundzach (1892) determined the ash constituents of normal human feces, and found in the ash 29.25 percent CaO and 13.76 per- cent P 2 5 . Paton, Dunlop and Aitchison (1899-1900) made a general study of the paths of elimination of phosphorus compounds by animals, and their principal findings are as follows : In dogs, on a vegetable diet, a large proportion of the phospho- rus of the food is not excreted in the urine. In dogs, on a diet of dog's biscuit and milk, a large proportion of the phosphorus injected subcutaneously as sodium phosphate is not excreted in the urine (no food or feces data) . During lactation there is a diminished urinary excretion of phosphorus by the dog, and a diminished feces excretion of phos- phorus by the goat. In the goat none of the phosphoric acid injected subcutaneous- ly as sodium phosphate, or formed in the body, or contained in the food, is excreted in the urine. The administration of calcium glycerophosphate by the mouth causes no increased excretion of phosphorus in the urine of the dog, or in the urine or milk of the goat. Rumpf and Schumm (1900) made urinary phosphorus esti- mations in a human metabolism experiment with a vegetable diet. The subject, weighing 62.5 kg. at the beginning of the 8-day experi- ment, gained 1.7 kg. during this time on a ration supplying 3431 calories and 73.88 gm. protein daily. The urinary nitrogen was 6.91 gin., and urinary phosphorus (P 2 5 ) 1.38-2.43 gm. per day. Bergmann (1901) studied phosphorus elimination in the dog and the sheep. Sodium phosphate injected subcutaneously into a dog receiving a ration of meat and bread was eliminated in the urine. The same result was obtained when, in addition to the phos- phate injection, calcium carbonate was added to the diet. With a wether, on a diet of hay and oats, sodium phosphate was injected subcutaneously, and all of it was excreted by the intestine, the urinary phosphorus remaining unweighable. Glycerophosphoric acid, likewise, when subcutaneously inject- ed, was excreted by the dog in the urine, and by the wether in the feces, as inorganic phosphoric acid. Bergmann also notes the well- known fact that, while dogs normally excrete P mostly in the urine, if Ca is abundant in the food much P is excreted with it in the feces. Tangl (1901) found in metabolism experiments with horses, on hay and oats, that more than half of the calcium, about three- fourths of the magnesium, and nearly all of the phosphorus of the 210 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 food is eliminated in the feces. Tangl concluded that a horse was able to obtain its calcium needs from the same low-calcium fodder which with cattle may produce malnutrition of the bones. See also W. Camerer, Sr. (1904) ; composition of human urine as affected by animal and vegetable diet, disease, etc. Gouin and Andouard (1907) conducted experiments showing that a calf receiving milk alone excreted 83.33 percent of the food phosphorus in the urine. An addition of powdered bone to the food did not alter the percent of the total phosphorus excreted in the urine. Another calf received 55.6 gm. P 2 5 in milk and vegetable foods, and excreted 13.74 percent of this amount in the urine; when 29.22 gm. more P 2 5 was added in the form of powdered bone the amount, of phosphoric acid in the urine was increased by 2.36 gm., and the percent of the total phosphoric acid of the food found in the urine: was reduced to 11.79. A third calf received no milk in the food. The urine contained 1.15 gm. phosphoric acid, which was 7.85 percent of the total ex- cretion. When 33.5 gm. P 2 5 was added to the diet in the form of bone, the quantity in the urine increased to 6.35 gm., and the per- cent of the total excretion to 20.92. Another calf passed no phosphorus in the urine. Powdered bone was added to the diet in the amount of 18.31 gm. daily for 7 weeks. The storage of phosphoric acid was 17.5 gm. per kilogram, of increase in live weight. These data show that bone phosphate may be retained by- calves, and that the vegetable foods used tended to deflect the ex- cretion of the absorbed phosphorus of the bone meal from urine to feces. The authors (G. and A.) referred these results to differen- ces in digestibility of the phosphorus in the different forms. Magnus-Levy (vonNoorden 1907, I, p. 41) speaks as follows regarding the effects of species and diet on phosphate elimination : "The complete removal of the phosphoric acid from the urine of man and of dogs, as in the case of herbivora, cannot be effected. Acid sodium phosphate, when subcutaneously injected into sheep, is excreted in the feces, and in dogs appears almost entirely in the urine, even when the intestine is overladen with calcium salts (Bergmann) . "In dogs the separation of phosphoric acid by the kidneys is not affected by the addition of alkali if the food is not rich in calcium (Beckmann) ; the phosphoric acid salts of the sodium and cal- cium of the food then pass out almost entirely by way of: PHOSPHORUS METABOLISM 211 the kidneys (Markuse, Leipziger, Zadik) . It is only when an ex- cess of calcium and alkali are together present in the food that the phosphoric acid usually excreted by the kidneys is conducted from and to the intestine." Gouin and Andouard (1908) attempted to answer the question as to why adult cattle do not secrete phosphorus in the urine. In the light of current understanding, however, their conclusions as to kidney resistance to phosphates are no longer significant. Oeri (1909) found that with mature human beings the excre- tion of calcium and phosphorus by the intestine is increased (1) by diets, which, like milk, contain considerable inorganic phosphorus and calcium, (2) by a diet rich in phosphorus, when calcium is separately administered, and (3) by a calcium-rich diet, when inorganic phosphorus is superposed, that is, whenever phosphorus and calcium are associated within the body. Stutzer (1910) submits analyses of the urine and feces of the common farm animals, showing the content of mineral elements in these excreta. The cows used were animals giving 12-15 liters of milk on medium rations of green and dry fodder. The sheep received hay only. To the pigs were fed potatoes with green fodder, but no milk. The specific gravity of the urine was as follows : Sheep 1.038 ; horse, 1.035; cow, 1.037; pigs, 1.020. There was much similarity in the methods of elimination of the several mineral elements by these animals. The influence of species on the paths of excretion could only be determined by feeding the animals to be compared all on the same ration. ANALYSES OF FRESH URINE AND FECES— Parts per 1000 Urine Fe ces Sheep Horse Cow ' Pig Sheep Horse Cow Pig Water 903 926 923 966 680 750 835 800 Organic material . 70 47 57 23 295 230 150 160 Nitrogen (a) Total amount 15.8 15.2 15.0 6.4 6.2 5.6 5.9 6 (b) Easily soluble 15.8 15.2 15.0 6.4 0.5 0.5 0.6 0.8 Phosphoric acid (a) Total amount 1.3 0.05 1.5 1.6 3.0 3.0 2.8 6 (b) Easily soluble 1.3 0.05 1.5 1.6 5 18.5 16.5 15.5 .8.0- 1,7 3.3 1.4 5 1.8 3.2 0.3 0.1 4.0 2.3 2.4 0.5 Magnesium 2.5 2.4 0.1 0.8 2.4 1.0 1.8 2 Sulphuric acid . . . - 1.0 1.6 0.3 2.7 1.4 0.5 1.2 6 3.8 3.0 1.0 1.0 1.0 0.1 0.1 0.1 Summary. The ingestion of acids or acid salts, or acid-form- ation in the body, increases urinary calcium and phosphorus, and in- creases the proportion of acid phosphates to total phosphates in the urine. 212 OHIO EXPEKIMENT STATION: TECHNICAL BUL. 5 The alkaline earths decrease urinary phosphorus elimination by uniting with phosphorus in the intestine to form difficultly sol- uble salts, thus hindering phosphorus absorption. Calcium carbonate causes a considerable replacement of alkali phosphates by alkaline earth phosphates in the urine. The de- crease of alkali phosphates is more largely mono- than di-sodium phosphate. Sodium phosphate is promptly eliminated by the kidneys and has a diuretic effect. Sodium chloride also has some tendency to increase the urinary phosphorus. Sodium carbonate may reduce urinary phosphorus excretion when this is due in part to acidosis. Potassium phosphate, as well as other potassium salts, increas- es the excretion of sodium salts. Potassium phosphate added to the usual diet of herbivora, leads to the appearance of phosphorus in the urine. In man, potassium citrate causes a fall of calcium phosphate in the urine by virtue of the insolubility of calcium phos- phates in potassium carbonate. Sodium salts have not this effect. Calcium phosphates affect phosphorus outgo in the urine in ac- cord with their acidity; the more acid the salt, the greater is the urinary excretion of calcium and phosphorus. The ingestion of bone ash decreases urinary phosphorus, probably through hindering phosphorus absorption. Calcium lactate, like the carbonate, may decrease urinary phos- phorus. Calcium sulphate appears to decrease urinary phosphorus to a very slight degree. The phosphorus of the diet of carnivora is eliminated principal- ly in combination with alkalis in the urine. The remainder is eliminated in combination with iron, magnesium and calcium in the feces. Carnivora excrete their magnesium more largely than their calcium in the urine. Sodium phosphate, injected subcutaneously in the dog, is ex- creted in the urine even if calcium carbonate be added in quantity to the food. Calcium added to the food of carnivora increases the feces phos- phorus ; and on a vegetable diet carnivora excrete a large part of the phosphorus in the feces. Herbivora, in accord with the high calcium content of the diet, excrete almost all of their phosphorus in the feces, the urine usually being practically free from phosphorus. Subcutaneous injections of sodium phosphate are excreted in the feces. During starvation, or on an animal diet, such for instance as milk, herbivora excrete much calcium and phosphorus in the urine. PHOSPHORUS METABOLISM 213 During lactation there is, with carnivora, a decrease of urinary- phosphorus ; with herbivora there is a decrease of feces phosphorus. Human beings excrete their phosphorus according to the same laws as carnivora and herbivora, the path of outgo varying with the diet. The prevalence of meat and cereals, which have acid ash, in ordinary mixed diets, deflects the larger part of the phosphorus into the urine. Vegetable diet or calcium carbonate increases the feces phosphorus, but the complete deflection of phosphorus from the urine to the feces has not been accomplished. On a milk diet three-fourths of the phosphorus is excreted in the urine by infants, and nearly the whole of it by calves. The alkaline earths deflect metabolic phosphorus as well as food phosphorus into the feces, and fasting feces contain considerable amounts of calcium and magnesium phosphate. The balance of magnesium may be changed from positive to negative in swine by excessive ingestion of phosphorus in common foods, the loss occurring through the feces. RELATIVE METABOLISM OF PHOSPHORUS AND OTHER ELEMENTS There is, without doubt, a certain interdependence of all of the constituents of the body, in their metabolism ; for instance, if nitro- gen is lacking in the ration the metabolism of all other elements which are used with nitrogen will be affected in some measure, the degree depending on the extent and duration of the nitrogen de- ficiency. The less the available supply of any nutrient, compared with the demand for the same, the more definitely does it become the lim- iting factor in production, and the more closely does its metabolism measure that of the body as a whole. Any element, therefore, might become the limiting factor in metabolism ; thus, on a ration which is practically iron-free, the total growth might vary directly with the iron content of the ration. In view of their distribution and relationships in the tissues it might be supposed that nitrogen, sulphur, calcium and phosphorus would ordinarily be found somewhat closely interdependent in met- abolism; and since phosphorus, as a universal cell constituent, is concerned in all growth and production, it would be assumed that this element would be found quantitatively related especially to those other elements whose metabolism is dominant during the par- ticular kind of growth or production under observation ; thus for the maintenance and repair of the mature animal, nutrients are used in 214 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 certain rather definite proportions, one to another; during normal growth the proportions would be somewhat different, and it is quite conceivable that pregnancy, lactation, egg-laying and wool-growing should each have some special requirements for nutrients which would affect their relative quantitative metabolism; further, con- ditions may be favorable for the growth of bone, without providing liberally for other increase. In each of these phases of normal metabolism phosphorus is involved, and its quantitative relations to other elements have been the subject of study and record, the ob- ject being, ordinarily, the determination of the states and conditions of nutrition as made manifest by the quantitative and qualitative estimation of the constituents which have been concerned. A meth- od of frequent use is, for instance, the estimation of relative metab- olism of bone and soft parts by the relation of the details of the in- come and outgo to the composition of these tissues. We have as the several factors of such considerations first, two possible sources of the constituents involved; the nutrients of the food, and products of tissue katabolism; second, two possible methods of disposition of these constituents, namely, retention and outgo. Usually our observations are confined to the determina- tion of final or net balance, the relative amounts of the constituents retained or lost indicating the identity of the parts involved. Some limitations of the value of this method of study are that we do not know that all organs are katabolized en masse, nor that this process is not to any extent partial or selective; and further, we know very little of the resynthesis of katabolized nutrients. Participation of products of tissue katabolism in synthetic or reten- tion processes may sometimes be inferred from the outgo of accom- panying products which are not retained ; thus purin nitrogen elim- ination may signify the katabolism of nucleins under circumstances where the subsequent retention of the accompanying phosphorus and non-purin nitrogen would mask this process. In spite of the uncertainties attending these attempts to judge of the origin of katabolized nutrients from the composition of the excreta, the method has its uses, though in times past it has been overworked. The supplementary capacities of the intestine as a means of elimination of katabolized constituents require that the feces as well as the urine be involved in such studies. Conclusions resting on urine analyses alone must be considered as open to question. Bischoff (1867) concluded that nitrogen and phosphorus ex- cretion rise and fall together, except in fasting ; the phosphorus ex- cretion then being relatively greater, the increase being due, PHOSPHORUS METABOLISM 215 according to Bischoff , to a loss of phosphates from the plasma, with- out corresponding protein metabolism. This extra phosphorus out- go in fast is now often ascribed to bone katabolism. J. Forster (1873b) published urinary nitrogen and phosphorus data from a man on a meat diet which show, in considerable meas- ure, at least a temporary independence of nitrogen and phosphorus metabolism. The figures are given below. EXCRETION OF NITROGEN AND PHOSPHORUS IN THE URINE OF A HEALTHY ADULT ON A MEAT DIET DURING TWENTY-FOUR HOURS— Grams Hours during 1 the day N , P2O5 10 A. M.— 1 P. M 2.74 3.51 3.36 3.36 2.52 2.56 0.76 2 P. M.— 5 P. M 0.62 6 P. M.— 9 P. M. 0.42 10 P. M.— 1 A. 1VL 0.41 2 A. M.— 5 A. M 0.32 6 A. M.— 9 A. M 0.29 A healthy adult ingested at 9 A. M. 500 gm. of finely chopped flesh, containing 18.04 gm. N and 48.3 gm. fat, after having fasted since the preceding noonday meal. He then drank water but took no other food for 24 hours. During this time he excreted nitrogen just about equal to the total present in the 500 grams of meat; and of phosphorus, we compute, 3 times as much as was in the meat. These facts, taken in connection with the rates of excretion as shown by the above figures, exhibit the truth that there is no close relation between urinary nitrogen and phosphorus excretion. The large phosphorus excretion and the steady decline in the rate of elimination show the influence of the previous diet. W. Zuelzer (1876) published extensive papers on the propor- tions of nitrogen and phosphorus in the urine of human beings and of dogs in various states of nutrition. In the absence of food and feces data the significance of the conclusions is limited, but certain notes of interest are recorded. With a dog, the P 2 5 of the urine from a fat-free flesh diet was 12.8 percent, during fast 8.7 percent, while receiving potatoes 27.1 percent, and while receiving rye bread 25.3 percent of the amount of the nitrogen. The chief meal of the day exerts an influence on the nitrogen and phosphorus of the urine of the hours immediately following, while this influence diminishes with lapse of time thereafter. In a table of ratios of phosphorus to nitrogen in the urine at different ages, from infancy to old age, it is shown that the propor- tion of phosphorus to nitrogen is greatest in infancy, and decreases 216 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 rapidly during the period of growth. There appears to be little change during the middle years of life, but there is evidence of a slight increase of phosphorus in proportion to nitrogen in old age. These figures must depend largely on the prevailing diet at the var- ious ages. Zuelzer was of the opinion that increased phosphorus excretion signified loss from the nervous system, an idea now long since abandoned. C. Voit (1881) submitted the following figures for the propor- tions of nitrogen and phosphorus in dog urine under different con- ditions : N:P 2 5 on meat diet 8.1:1 Bischoff N :P 2 5 during fast 6.4 :1 N:P 2 5 , diet of N-poor bread 3.8:1 Feder (1881) attempted to determine a normal ratio of P, S and N in the urine during fast, and feeding on various diets. This he found it impossible to do. He also showed that the hourly vari- ations in the phosphorus outgo, and in the proportion of nitrogen to phosphorus in the outgo, could not be due directly to nerve katabol- ism, a fact which becomes obvious when we consider the amounts of these variations in connection with the phosphorus content of the nervous system. W. Zuelzer (1881a) determined the proportion of N:P 2 5 in the muscular system as 100:15, in nerve substance as 100: 45 and in blood as 100 :4. He made much of the idea (1881b) of determining the source of the nitrogen and phosphorus of the urine by their proportionate amounts, but ignored the feces. Zuelzer (1881a) stated that a large relative amount of phosphorus as compared with nitrogen in the urine, or a high content of glycerophosphoric acid, indicates nerve katabolism. Politis (1884) considered the proportions of nitrogen and phos- phorus in the urine of dogs receiving brain in the food, and also the hourly variation, during two days, of the nitrogen and phospho- rus of the urine. He reached the conclusion that urinary nitrogen and phosphorus data alone were insufficient to demonstrate the breaking down of brain substance. The hourly curves for nitrogen and phosphorus elimination during the feeding of brain were much the same. Kolpakcha (1888) studied the source of the nitrogen of the urine in 8 extensive balance experiments with dogs, by comparison .of the relative amounts of the various constituents of the food with the relative amounts of these same constituents in the tissues of the animal, and in the excreta. The author gives formulae for de- termining mathematically the proportion of food-, circulating- and tissue-protein katabolized. PHOSPHOEUS METABOLISM 217 Among the conclusions reached by the author are the follow- ing: In fasting, two kinds of protein are broken down, (1) "stored protein," or protein stored in the body, after excessive feeding, that has not had time to become part of the tissue protein, and (2) tissue protein, or protein that is really a part of the tissue of the body. The former is said to be broken down with comparative ease, the latter being rather stable. From the ratio of phosphoric acid to nitrogen in the urine it is considered that the cleavage of tissue pro- tein begins on the first day of fasting, though it is stored protein especially which is broken down. As the stored protein is exhaust- ed the organism approaches a condition in which it must live entire- ly on its own tissues. The author believes that the increased amount of phosphoric acid observed in the urine after a fast of con- siderable duration, or when the animal is receiving an insufficient amount of food, is due to autolysis of the bones, since there is dur- ing these conditions an increase in the proportion of alkaline earth phosphates to alkali phosphates in the urine. He also believes, in harmony with Forster's conclusions, that when the organism is sup- plied with food poor in phosphoric acid it retains some of the phos- phoric acid from the protein which is broken up. Von Tschirwinski (1889) studied the relative development of the bones and teeth of sheep, and found results not in harmony with the theory of an interdependence between the time of cutting teeth and the development of the bones. Bergell (1898b) states that normal storage of nitrogen and phosphorus is marked by a relation of one to the other like that in muscle. Von Moraczewsky (1901) showed that, in a 6-day balance peri- od, with a case of acromegaly, the phosphorus retention was double the amount of the calcium retention. A 6-day period, however, is insufficient for reliable conclusions as to mineral metabolism. Siven (1901) notes the lack of parallelism between nitrogen and phosphorus metabolism, which fact he relates to the difference of origin of these constituents, since the outgoing phosphorus is de- rived in part each from organic and inorganic phosphorus com- pounds in the body. Dapper (1902) investigated the question as to the method of retention of nitrogen in a man 26 years old. Data from this work are below. In periods II and III the ratios of N:P 2 5 in the amounts retained were 1.69:1 and 1.24 :1, respectively, while the ratio in muscles is 7.6 :1. The considerable amounts of calcium and phosphorus retained must have been deposited largely in the bones. 218 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE DAILY NITROGEN, CALCIUM AND PHOSPHORUS BAL- ANCES WITH A MAN TWENTY-SIX YEARS OLD— Grams Intake Excreta Balances Periods N P2O5 CaO N P2O5 CaO N P2O5 CaO I 6 days.... II 12 days.... Ill 9 days.... 20.247 20.093 24.581 5.111 7.033 7.806 3.547 4.818 4.891 18.07 16.774 22.093 5.617 5.022 5.746 4.237 3.465 4.375 +2.18 +3.32 +2.55 —0.409 +1.961 +2.059 -0.690 +1.354 +0.495 In balance experiments by Tangl (1902a, 1902b) with horses (see Phos. Req. of Horses) we see a case of deficiency of phosphorus in a ration limiting calcium retention, the calcium not being retained in proportion to absorption. On the hay ration the phosphorus was insufficient, while there was considerable calcium retention. In the oats and hay ration, with a reduced calcium intake, there was an increased calcium storage, because of the presence in this ration of phosphorus sufficient to cause retention and allow of the deposi- tion of calcium phosphate in the bones. Gilbert and Posternak (1903) submitted figures showing that the proportion of nitrogen to phosphorus in the urine is so variable that we must consider the phosphorus requirement as in large meas- ure independent of that for nitrogen. Ehrstrom (1903a), (see Nutr. Val. Org. and Inorg. P.) from balance experiments on a man, concluded that phosphorus and ni- trogen metabolism need not run parallel, and also that the organism has not the same tendency to establish a phosphorus equilibrium that it has a nitrogen equilibrium, regarding which conclusions there is no doubt, since they simply signify that the organism can store phosphorus in the bones in large measure irrespective of the status of the nitrogen metabolism. Liithje and Berger (1904) reported results of five balance ex- periments with human beings having for their object the study of nitrogen, phosphorus and calcium metabolism in convalescents from typhus fever. Data from this work are on the following page. In these experiments the object was to bring about maximum nitrogen retention, and then, by a comparison of the constituents of the food and excreta, to determine the form in which the nitrogen had been stored. The authors' computations were based on Katz's analyses of muscle and Zalesky's analyses of bone. PHOSPHOKUS METABOLISM 219 AVERAGE DAILY RETENTION OF NITROGEN, CALCIUM AND PHOS- PHORUS BY MATURE MEN ON HEAVY FEEDING— Grams Condition of subject Diet N Intake P2O5 Intake CaO Intake N Bal- ance P2O5 Bal- ance CaO Bal- ance Weight of subject Length of period in days Typhus convalescent, age 30 years, reduced state Milk, nutrose, sugar 40.246 10.887 5.740 +10.999 +2.832 +1.702 46.9 49.6 10 Typhus convalescent, age 24 years, well nourished Milk, nutrose, sugar, zwei- back 28.337 8.052 5.628 +5.165 +1.291 +0.741 63.3 66.3 12 Typhus convalescent, same as in first period, 16 days later Milk, sugar, nutrose 54.583 12.385 7.760 +11.419 +1.255 +0.454 57.2 58.1 7 Normal Milk, sugar, nutrose 27.337 9.305 5.339 +4.566 +3.381 +1.637 77.5 77.0 7 Normal; same as in first and third periods, after recovery Milk, sugar, nutrose 57.187 12.497 5.910 +6.768 +1.747 +0.144 59.2 61.7 10 From the above data Liithje and Berger compute that in periods 1 and 2 nitrogen, calcium and phosphorus were retained in proportion for the formation of flesh and bone ; that in period 3 there was nitrogen retention without the corresponding quantity of phos- phorus ; and that in periods 4 and 5 there was phosphorus retention in excess of that which could be used with the other food constitu- ents in the formation of flesh and bone. They conclude, therefore, that with a large retention of nitro- gen in a relatively short time, there is usually a quantity of phos- phorus retained which corresponds to the nitrogen as does the phos- phorus of flesh to the nitrogen of flesh ; but there can also be an ex- cess of nitrogen retained, and this is utilized in a different way, per- haps in the form of albumin, as a dead cell-inclusion, corresponding to the cell-deposit of glycogen and fat; and finally, in other cases, more phosphorus may be retained than that corresponding to the relation of nitrogen to phosphorus in flesh. L. F. Meyer (1904a, 1904b) found, in balance experiments with dogs, that nitrogen could be retained from proteins which were so poor in phosphorus as to be incapable of maintaining phosphorus equilibrium. His data show, however, that the nitrogen retention was greater when the phosphorus balance was positive than when it was negative, the nitrogen intake remaining more nearly constant than the phosphorus. Meyer also notes the inclination of the organ- ism to store phosphorus ingested in excess of the maintenance re- quirement, rather than to maintain an equilibrium, as is the case with regard to nitrogen. 220 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Gumpert (1905) (see Sanatogen, feeding exp.) shows, in peri- ods of 3 and 4 days, a retention of P 2 5 of 0.143 and 0.600 gm. co- incident with a loss of CaO of 0.307 and 0.244 gm. Trowbridge and Woodman (1909) found that young, growing steers continued to grow in height, and to build up skeleton even when losing in weight, but the extent to which this involved redis- tribution of body constituents was not shown. A large measure of independence of growth of skeleton and soft parts has been demonstrated by Fleischner (1906) with infants ; by Waters (1908, 1909, 1909-10) with cattle, and by Aron (1910b) with dogs. Kinberg (1911), in a study of nitrogen hunger, considered the proportions of nitrogen, sulphur and phosphorus in the urine. These relations varied irregularly throughout the period, but within com- paratively narrow limits, viz., N:H 2 S0 4 as 5.4 — 5.6:1 and N:P 2 5 as 4.6—6.6:1. Lipschiitz (1911a) and many others express the opinion that in case of lack of a required element in the food, the body takes from less important parts to supply the more important. An important observation of recent date, in this field, is that of Gregersen (1911) who determined in metabolism experiments with rats that even with an abundant intake of phosphorus in assimilable form no phos- phorus is retained from a nitrogen-free diet, thus suggesting a com- plete final dependence of the metabolism of phosphorus upon that of nitrogen. Summary. From a consideration of the above notes we are able to understand that nitrogen and sulphur are somewhat closely interdependent, since they occur associated in the same compounds ; phosphorus is to a considerable extent independent of nitrogen be- cause of its relative abundance in the skeleton, while calcium is still more independent of nitrogen because of the poverty of the nitrog- enous soft parts in calcium. Phosphorus is to some extent inde- pendent of calcium because of its association with nitrogen in the soft parts, but calcium is closely dependent on phosphorus because it is not stored in considerable amounts except as combined with phosphorus. We must regard with considerable skepticism attempts to de- termine with much particularity the sources of excreted constitu- ents by their relative amounts. PHOSPHORUS METABOLISM 221 METABOLISM EXPERIMENTS WITH INORGANIC PHOSPHATES J. Lehmann (1859) found in balance experiments with a calf that alkaline earth phosphates could be absorbed and retained. He concluded that many foods were lacking in bone-forming constitu- ents, and that hay was the best bone-forming food. Von Gohren (1861) conducted, a balance experiment with a lamb on a ration of meadow hay, to which was added, in one period, calcium and magnesium phosphates. Data from this test are be- low. AVERAGE DAILY BALANCE DATA FROM A LAMB ON A RATION OF MEADOW HAY WITH AND WITHOUT ADDED CALCIUM AND MAGNESIUM PHOSPHATES— Grams Period and days Ration Food CaO Food MgO Food P2O5 Balance CaO Balance MgO Balance P2O5 I — 7 days.. 11— 6 days.. Meadow hay Meadow hay, 10 g-m. alkaline earth phosphates 7.081 10.421 2.395 2.428 3.187 7.440 +0.053 +1.183 +0.164 +0.275 +0.227 +1.821 These data show that lambs will digest and retain very much more calcium, magnesium and phosphorus than was present in this meadow hay, if these elements be added to the hay as alkaline earth phosphates. Two lots of lambs, of four each, were fed on these same rations for 45 days. The lot which received the phosphates gained in live weight 7.2 percent more than the one which received no phos- phates. VonGohren noted that the urine of lambs is practically free from phosphorus and that the calcium also is found mostly in the feces. Blondlot (1861) states that the administration of doses of 0.125 gm. sodium hypophosphite produces no phosphorous acid test in the urine, but that doses of 0.50 gm. give the characteristic green flame. Gamgee, Priestley and Larmuth (1876-77) compared the so- dium salts of pyro-, ortho- and meta-phosphoric acids by subcutan- eous, intravenous and oral administration to frogs, rabbits and dogs. The ortho- salt was found to be inert, the pyro- salt markedly poison- ous to the heart, when introduced into the circulation, and the meta- salt also poisonous but to a less marked degree. Paquelin and Joly (1877) administered 2 gm. of sodium pyro- phosphate daily for 5 days to a woman, and found during this peri- od and the following 5 days that apparently all of the pyrophospho- 222 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 ric acid was eliminated in the urine unchanged. The sodium pyro- phosphate had a slight diuretic effect. One qualifying circum- stance, however, requires mention. The diet was not maintained entirely uniform during this experiment. Paquelin and Joly (1878) administered to a woman during 5 days a total of 5 gm. of sodium hypophosphite. They concluded that the hypophosphites also are excreted unchanged in the urine, and that they have a diuretic effect. Vermeulen (1884) studied the physiological action of hypophos- phites, but without important results. He believes that they pass through the body unchanged. He determined hypophosphites in urine by first removing the phosphates by uranium precipitation and then determining total phosphates in a KC10 3 +HC1 digest of the filtrate. J. Neumann (1893b) published results of two calf-feeding ex- periments, in one of which calcium phosphate, and the other cal- cium carbonate, was added to a skim milk diet. The calf used in the first experiment was 5*/2 weeks old, and its weight at the beginning of the experiment was 65.63 kg. The calf used in the second trial was 8 weeks old, and weighed 80.87 kg. at the beginning. AVERAGE DAILY CALCIUM AND PHOSPHORUS BALANCES WITH CALVES ON A DIET OF SKIM MILK AND MINERAL SUPPLEMENTS— Grams Date of periods CaO Intake CaO Outgo CaO Balance P2O5 Intake P2O5 Outgo P2O5 Balance Diet Sept. 10-14.. Sept. 15-16.. Sept. 17-19.. 24.63 27.38 29.19 12.86 14.42 16.77 +11.77 +12.96 +12.42 30.46 32.83 34.33 14.91 16.87 18.39 +15.55 +15.96 +15.94 15000 gm. skim milk. Same plus 7.5 gm. calcium phosphate. Same plus 12 g-m. calcium phosphate. Sept. 29-30.. Oct. 1-2.... Oct. 3-7.... Oct. 8-9.... Oct. 10-14.. 26.252 27.478 30.008 26.407 26.412 14.562 14.854 15.820 15.285 14.776 +11.690 +12.624 +14.188 +11.122 +11.636 34.039 33.863 33.980 34.240 32.247 19.511 19.712 19.327 19.696 19.947 +14.528 +14.151 +14.653 +14.544 +14.300 16000 gm. skim milk. 16000 gm. skim milk. Same plus 7.5 gm. calcium carbonate. 16000 gm. skim milk. 16000 gm. skim milk. Thus it appears that both calcium carbonate and phosphate, when added to skim milk, are digested and, in considerable meas- ure, retained by the young calf ; and further that calcium carbonate increased, not only the calcium storage, but also the phosphorus re- tention. Neither the calcium nor the phosphorus of the phosphate were so well retained as the same elements in the milk, but the cal- cium of the carbonate was retained in about the same proportion as the calcium of the milk. It was found that both of these mineral supplements tended to reduce the gain in live weight. PHOSPHORUS METABOLISM 223 Weiske (1895b) mentions experiments by Graff enberger show- ing that on an oat ration rabbits digested a little more protein, fat, crude fiber and nitrogen-free extract without tricalcic phosphate than when this salt was added to the diet ; and also experiments of his own where calcium carbonate was added to a ration of meadow hay, with rabbits, in which the mineral supplement seems to have reduced to a slight extent (1.35 percent) the digestibility of the pro- tein, but to have increased to the extent of 10.11 percent the digesti- bility of the nitrogen-free extract. Boddaert (1896) found calcium and sodium hypophosphites rapidly excreted, apparently as such, in the urine, whether adminis- tered per os or subcutaneously, with rabbits, dogs and men. Massol and Gamel (1901) added sodium phosphate to a solution of calcium hypophosphite. The solution was then made alkaline, and a precipitate of tricalcium phosphate was formed, leaving so- dium hypophosphite in solution. The same reaction, they state, takes place in the intestine, and the tricalcic phosphate is lost to the organism. The sodium hypophosphite is absorbed, and excreted unchanged by the kidneys, there being no oxidation of phosphorous acid. Thus calcium hypophosphite removes phosphorus from the system in proportion to the amount fed. Panzer (1902), in studying the fate of calcium hypophosphite in the body, reached conclusions by the feeding of the compound, and then by qualitative tests (the green flame with nascent hydro- gen, and the brown silver phosphide precipitate) in the urine, feces, blood and organs. The tests were made in part on a man and in part on dogs. Panzer decided that if the methods were adequate to this in- vestigation calcium hypophosphite fed to a dog is quickly and almost completely absorbed, passes through the organism without being held back anywhere, and is completely eliminated within 24 hours. Martinet (1902a) says that hyperacidity of the urine accom- panied by abnormally small amount of phosphates is frequently an index of hypochlorhydria in the stomach, and that in such cases the administration of phosphoric acid may promote digestion and check fermentation by its eupeptic and antiseptic action. In a later article (1905) Martinet writes that by the use of phos- phoric acid in 48 cases of dyspepsia, 32 cures have been accom- plished, the liver also being benefited ; urea excretion is increased ; indican disappears and glycosuria of hepatic origin is relieved ; also the hepato-pancreatic function is stimulated; intestinal functions are regulated, and diarrhoea suppressed. Treatment with sodium carbonate inhibits the digestive functions, especially of the stomach, while the phosphoric acid treatment, on the contrary, stimulates these functions. 224 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Gouin and Andouard (1902-3) conducted digestion experiments on a heifer, having for their object a study of the effects on metabo- lism of certain inorganic salts. The basal ration was composed of milk, rice meal, clover, peas, and oats. Acid potassium phosphate, in the proportion of 5 grams per 100 kg. live weight, was fed during 8 weeks. Benefit was thought to have resulted. The feeding of 150 grams of powdered bone per day, equivalent to 28.29 gm. P 2 5 , greatly increased the urinary phosphorus, and had a depressing ef- fect on the digestion of protein, carbohydrates and fat. Cautru (1903, 1904a, 1904b) fed pure phosphoric acid in consid- erable quantities to guinea pigs, dogs, ducks, horses and human be- ings, during protracted periods, to determine limits of toleration and therapeutic effects. In large doses, even, the acid is innocuous, and is recommended by Cautru for use in such morbid states as are char- acterized by demineralization and hypoacidity of the urine. As much as 2-4 gm. daily were fed to human beings for more than 5 years ; a dog weighing 8.5 kg. was given gradually increasing doses up to 3 gm. for one month; a duck was given one gram per day; horses were given 30-100 gm. daily; two guinea pigs weighing 350 gm. received 0.5-1.0 gm. daily, etc. Kohler et al. (1904), in experiments with yearling lambs, found that when added to a ration containing comparatively little mineral matter tricalcic phosphate was retained in larger proportion to the intake than were dicalcic phosphate, precipitated bone earths (di- and tri-salts), or calcined bone. After making this observation, he determined with a 6-months-old lamb that the reason the dicalcic phosphate was not so well retained as the tricalcic salt was its lack of calcium. When additional calcium was provided, as calcium lac- tate, the phosphorus of the dicalcic salt was much more efficiently retained than that of the tricalcic phosphate. Of the phosphorus of the precipitated bone earths the retention was 13.1 percent, of the calcined bones 14.2 percent, of the dicalcic phosphate 26 percent and of the tricalcic salt 35.5 percent. With the younger lamb the phosphorus of the tricalcic phosphate was re- tained to the extent of 38.6 percent of the intake, of the dicalcic phosphate, 35 percent when fed alone, and 54.7 percent when fed with calcium lactate. Joulie (1904) administered phosphoric acid in rheumatism of horses. The dose was 10 c.c. phosphoric acid (sp. gr. 1.35) diluted to a liter. He states that such introduction of phosphoric acid diminishes the alkalinity of the blood, and increases the elim- ination of calcium phosphate by the kidneys. PHOSPHORUS METABOLISM 225 Kramer (1907) investigated the effects of ingestion of calcium phosphate on the laxative tendency of beet leaves when fed to cows. It is usually credited with preventing a laxative effect, but in this case the beet leaves were not sufficiently laxative to permit of this observation. Kohler, Honcamp and Eisenkolbe (1907) reported results on further tests of inorganic phosphates with lambs. The authors conclude that the phosphorus of the dicalcic salt is slightly better absorbed and retained than the tricalcic salt, and that the addition of calcium lactate or calcium carbonate is without influence on the degree of retention of the phosphorus. The results of these tests are so variable that it seems to the writers unwise to draw conclu- sions from them. W. Muller and vonWendt (1908) found that precipitated cal- cium phosphate fed to cattle suffering with diarrhoea, from eating partially decomposed beet leaves, served to allay the symptoms, while calcium carbonate accentuated the difficulty. The authors explain the unfavorable effect of the carbonate as due to the neutral- ization of the acid products of bacterial growth, thus favoring fur- ther growth. Oxalic acid was shown not to be the cause of the in- digestion. Aron and Sebauer (1908) concluded from feeding experiments with rabbits and dogs, using calcium-poor rations, to which in some cases bone meal, calcium phosphate or calcium carbonate was added, that the lack of these calcium compounds affected, in noteworthy de- gree, only the bones, these being below normal in dry substance and in the calcium content of the dry matter, the reduction of calcium content of the ash being considered immaterial. The calcium con- tent of the flesh and blood shows no reduction, while the calcium content of the brain is reduced but little. Dibbelt (1909) conducted metabolism experiments with dogs, in which dicalcic phosphate, sodium chloride and magnesium sul- phate were added, in different periods, to a standard basal ration. A part of the data are below. AVERAGE DAILY CALCIUM AND PHOSPHORUS BALANCES WITH DOGS AS AFFECTED BY INORGANIC SALTS— Grams Exp. No. Length of period in days Gain in weight CaO Intake CaO Retention P2O5 Intake P2O5 Retention Diet and treatment 1 2 3 4 15 10 9 11 100 80 0.0500 0.0500 0.0500 0.2178 -0.0478 +0.010 —0.001 -1-0.1388 2.3124 2.3124 2.3124 2.5188 0.6181 1.1882 0.7384 1.0989 Horse meat and pork fat. Horse meat and pork fat; immediately following- Exp. No. 1. Horse meat, pork fat and 12 gm. salt. Horse meat, pork fat and 0.5 g-m. dicalcium phosphate. 5 6 7 23 3.57 9.78 0.0150 0.0160 +0.003 +0.0062 0.6937 0.7400 0.1907 0.3618 Horse meat, fat and salt. Horse meat, fat, salt and magnesium sulphate. 226 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In Exp. No. 1 it appeared that horse meat and pork fat were deficient in calcium, but in Exp. No. 2, with the same diet and same animal, it seemed that the dog had accomplished an adjustment to this diet. The result in No. 3, where salt was fed, was essentially the same, so far as calcium is concerned; at least it did not differ from No. 2 as much as No. 2 differed from No. 1. In Exp. No. 4, however, there was a marked calcium storage coincident with the feeding of dicalcium phosphate. Experiments 5 and 6 were on another dog. There appeared to be an effect by the magnesium sulphate to increase the storage of calcium and phosphorus, but the evidence must be regarded as inconclusive on this point. The preliminary period was short. Some days after the conclusion of Exp. 6 this dog was killed, and the calcium content of its tissues was compared with the same from another dog which had received the same ration except that dical- cium phosphate replaced magnesium sulphate. The soft parts of the dog receiving magnesium sulphate were found much richer, but the bones poorer, in calcium, than those of the dog which received dicalcium phosphate. These differences the author explains by assuming that the magnesium sulphate had reacted with the sol- uble calcium phosphates to form the comparatively insoluble cal- cium sulphate. Dibbelt conducted other experiments in the artificial production of rachitic conditions, and concluded that the principal difference between these conditions and rachitis is in the cause of the calcium deficiency, this being due in the latter case especially to anomalies of absorption and secretion, rather than to calcium shortage in the food as a first cause. Crawford (1910) fed sodium metaphosphate, sodium pyrophos- phate and sodium orthophosphate to rabbits in a study of cotton- seed meal poisoning. The feeding of 2 gm. of the metaphosphate daily for two days caused the death of the subject, as also did the feeding of 0.5 gm. of the pyrophosphate per day for 7 days. The dos- age was enormous. The author's conclusion that cottonseed meal poi- soning was due to pyrophosphates has not withstood criticism. Delaini (1911) submits data showing that human beings and dogs eliminate in the urine practically the whole amount of hypo- phosphite of sodium administered either per os or subcutaneously, but that rabbits otherwise dispose of perhaps 20 percent of the in- take. Patta (1910) administered to a dog, by injection, varying doses of sodium hypophosphite. If administered in large amounts about half of all introduced can be recovered, unoxidized, in the urine; PHOSPHORUS METABOLISM 227 when smaller amounts are administered, the proportion recovered is less, being as low as one-sixth at times. Hence it is maintained that the phosphorus of hypophosphites can be retained by the or- ganism. Starkenstein (1911) theorizes to the effect that the reason that pyrophosphoric acid and metaphosphoric acid are poisonous when injected into the organism, while orthophosphoric acid is not, is that these acids are formed from orthophosphoric acid by condensa- tion of molecules, and loss of water. Experiment showed that if orthophosphoric acid be used in sufficient amount and concentra- tion, it also poisons — 10 c.c. of 14 percent Na,HP0 4 , injected, pro- duces the same effect as 2 c.c. of 5 percent pyrophosphate solution. The poisonous salts are more alkaline. Is the poison due to alka- linity? Blake found that they are more easily dissociated, which would mean greater alkalinity. Dissociation tests of the acids by reaction toward different indicators, by conductivity, and by heat of reaction all show that the pyrophosphoric acid has higher true acidi- ty than the orthophosphoric acid. Two of the H + of the pyrophos- phoric acid split off as easily as one of the orthophosphoric acid; therefore probably the tetrapyrophosphate of sodium dissociates to give as much OH" as does the tertiary salt of the ortho acid. Withers and Ray (1913) submit evidence controverting the idea that cottonseed meal poisoning is due to pyrophosphates. See also Etzinger (1874) on digestibility of bones by dogs; Charrin and Desgrez (1896) on subcutaneous injection of salts, in- cluding sodium phosphate; Vosgien and Geroline (1899) on reten- tion of inorganic phosphates by rabbits; Charrin (1905) on subcu- taneous injection of salts, including sodium phosphate; Cook (1909, Met. Phytin), and Draeger (1911) on the assimilation of calcium phosphates. MISCELLANEOUS EFFECTS OF INORGANIC PHOSPHATES ON GROWTH Graffenberger (1893) found that the feeding of calcium phos- phate to rabbits did not increase the calcium and phosphorus content of their litters. , Knauthe (1898) submits data from experiments with carp, showing that the addition of meat ash to rations of meat meal and corn meal, or meat meal and rice meal, serves to increase both nitro- gen retention and gain in weight. W. P. Wheeler (1903) reported more efficient use of food, and more rapid growth of chicks, from the addition of rock phosphate to a ration of vegetable foods. As a source of calcium and phospho- rus for chicks, however, Wheeler recommends raw or cooked bone. 228 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Klien (1908) found, in feeding* experiments on growing pigs, that when added to a ration of milk, potatoes, barley and rye, cal- cium carbonate had about the same value as calcium phosphate in causing increase of weight. Moore, Roef and Knowles (1908) found that newts would re- spond to the presence of both acid and alkali phosphates in the water in which they were kept, by increased growth, provided the salts were present in certain optimum concentrations. In excess- ive amounts monosodium phosphate was depressant, the disodium salt producing excitation. The same salts were fed to guinea pigs and dogs in quantities sufficient to cause pathological symptoms, and the histological con- dition of the organs and tissues is described. For other materials on the effects of phosphates on general growth see Mai (1869), W. Cohn (1870), J. Lehmann (1873, 1877), Passon (1905), Carlier (1907, 1909) and Forbes and associates (1914). Summary : Alkaline earth phosphates can be absorbed and re- tained by sheep when added to a ration of hay. Calcium phosphate and calcium carbonate can be absorbed and retained by the calf when added to a skim milk diet. Calcium car- bonate, under these circumstances, increases the phosphorus reten- tion, calcium thus appearing to be a limiting factor in the use of the phosphorus of milk. Both the carbonate and the phosphate tend to reduce the gain in live weight. Tricalcic phosphate depressed to a slight extent digestibility of oats by rabbits, and of a mixed ration of milk, grain and green feeds by a heifer. Calcium carbonate seems to depress the digest- ibility of protein and to increase the digestibility of nitrogen-free extract of oats with rabbits. Phosphoric acid has been found to stimulate gastric functions in cases of hypochlorhydric dyspepsia. By subcutaneous injection of phosphates it is learned that the ortho-salts, in moderate amounts, are inert ; the pyro-salts are mark- edly poisonous; while the meta-salts are also poisonous, but to a less marked extent. Lack of consistent results makes difficult the use of data on the comparative values of the various calcium salts fed to live stock. We have very little evidence that the inorganic salts of other than the ortho-phosphoric acid are of value as bearers either of phosphorus or of base. The hypophosphites especially are, through- out therapeutic literature, spoken of as inactive. In contradistinction to the effects of phosphates on the bones the ingestion of inorganic phosphates produces slight effect on body growth in general. PHOSPHORUS METABOLISM 229 METABOLISM OF NUCLEOPROTEINS AND NUCLEIC ACIDS * GENERAL DISCUSSION Anabolic Processes. It is evident from various observations that the animal organism is capable of building up for itself nuclear material from other forms of protein. The first definite evidence of such synthesis in the body was that, from Miescher's (1881, 1896) observations on Rhine salmon, where it was concluded that the nuclein-rich reproductive substance of these fish must form from the material of the muscles, for nearly the entire development of the reproductive organs and their sperm and ova takes place while the fish are taking no food, the fibres of the body muscles los- ing much of their protein at the same time. This view with regard to salmon was strengthened by the investigations of Paton and his as- sociates when they found that the amounts of phosphorus lost by the body muscles account quantitatively for the gain of phosphorus in the ovaries and testes. (Paton, et ah, 1897-8; Paton, 1898). Ac- cording to Milroy (1908), it is not true of herring that the genitalia derive their protein from the protein of body muscle, neither the protein nor the phosphorus figures indicating such a relation in his examinations. These studies were inconclusive because of the lack of complete chemical accounting for the entire fish. Building up nuclein from purin-free protein must, according to the reports of Burian and Schur (1897), take place in the bodies of suckling animals. From xanthin-base nitrogen and nuclein phos- phorus , determinations on the body substance of suckling pups at different ages, and on the mother dog's milk, it appeared that the increase of nuclein in the young animal is too great to be accounted for by the very small amount of nuclein taken in the milk. Schmoll (1904) discusses the chemical origin of leucocytes, con- sidering the question of the synthesis of nucleins by the adult hu- man organism. Leucocyte counts in the blood, and uric acid and purin base determinations in the urine, while patients were on a normal mixed diet, or on diets rich or very poor in purins, are used as evidence of the synthesis of leucocytes (nuclein) from the albu- min of the food without the necessity for food purins. McCollum (1909) also considered nuclein synthesis in rats. The subjects were fed in groups of 3 on (1) normal food, (2) a ration free from organic phosphorus and from purins, (3) a ration such as the last with purins from liver and hydrolyzed beef muscle added, or (4) with casein added to the basal ration. The bodies of some * For general discussions see A. Schittenhelm: Der NueleinstoftVeehsel ; Oppenheimer's Handbuch, Vol. IV, 1st half, 489-539; also Brugsch and Schittehhelm (1910), and Schau- mann (1910). 230 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 were examined as to percent of skeleton and percent of fat-free tis- sue, but the evidence as to the synthetic powers of the organism is mainly the rate of gain in live weight of the animals. He con- cluded that the phosphorus needed by an animal for nuclein form- ation can be drawn from inorganic phosphates, and that the animal has the power to synthesize the purin base necessary for its nuclein formation from some complexes contained in the protein molecule, and does not necessarily use purin bases of exogenous origin for this purpose. Complete chemical accountings for the bodies of the rats would have been required to establish these points. Other investigations involving the subject of nuclein synthesis are those which are considered, in the section on The Nutritive Val- ues of Organic and Inorganic Phosphorus, as evidence of the pro- duction of growth from rations which are free from inorganic phos- phorus. Evidence of synthetic changes in the nuclear material of devel- oping eggs of silk-worms has been submitted by Tichomiroff (1885), and of hens by A. Kossel (1885, 1886). On the other hand, Shack- ell (1911) was unable to find in the early stages of developing sea- urchin eggs any convincing evidence of the synthesis of nuclear ma- terial from the alcohol-soluble substances of the cytoplasm ; and this corresponds with the observations of Masing (1910), who com- pared unfertilized sea-urchin eggs, or those which, being fertilized, had just formed the membrane, with others which had developed to the morula stage, where there are 500-1000 cells. The nuclear mass has at that time multiplied nearly 100-fold, but the nucleic acid con- tent was not found to be any greater than in the unfertilized eggs. Later (1911b) he made similar examination of the successive stages of the embryos of rabbits, and of the livers of embryonal rabbits. Both the whole embryo and the embryo liver showed decreasing percentage content of nuclein as development progressed, though the absolute amount increased. Sections of the livers showed under the microscope a corresponding decrease in the relative size of the nucleus. Masing's work, then, makes it appear that there is a large supply of nuclear material at the start to provide for growth. There is little evidence as to the habit of the body with regard to the details of anabolism during growth and repair. There is abundant evidence that the organs contain within themselves en- zymes which can break down nucleins, but whether they do in fact break them down to the stage of free purins and phosphoric acid be- fore building them into new cellular matter is not known. It may be advantageous and habitual for the cleavage, whether in the di- gestive tract or in the tissues, to be stopped at an earlier stage. The PHOSPHORUS METABOLISM 231 investigations of Taylor (1907) and of Robertson (1907, 1909a, 1911) indicate that syntheses may, under the right circumstances, be brought about by the same enzymes which under other circum- stances cause cleavage. The attempts which have been made at artificial synthesis of nucleoproteins or nucleins are reported and discussed in the chap- ter on the chemistry of these compounds. Katabolic Processes. Three distinct methods of experimental study have been employed in attempts to learn of the extent to which nucleoproteins are broken down in the life processes of the organism, the transformations involved and the places at which they occur. First, isolated enzymes or digestive secretions from the ali- mentary tract have been allowed to act on the substances in ques- tion under controlled conditions and at favorable temperatures, the extent and nature of the change taking place in the digestive tract of the animal being judged by the results of these artificial diges- tions, in vitro. To this may be added the consideration of changes which may be brought about by such bacteria as are found in the intestine. It is to be recognized that observations of this kind lose somewhat in significance from being made under artificial condi- tions ; but, on the other hand, there is an advantage in the limita- tion of variable or unknown factors. A second type of studies made outside the body, and therefore under unnatural conditions, but probably being to an important de- gree indicative of the body processes, has been followed extensively in recent years. We refer to studies with comminuted organs in sus- pension, or with water extracts of organs, as to their autodigestion, especially as related to purins, and their capacities to transform ad- ded nucleic acids or their derivatives. Evidently the organs and tissues generally have within themselves enzymes, or the means for producing them, by which they may break down and transform their own nucleoproteins, or partially digested nucleoproteins or nucleic acids which may be brought to them. The third method of study makes observations of the actual re- sults in living subjects from introduction of the substance, either in the diet or by some form of injection. DIGESTION STUDIES WITH ENZYMES FROM THE ALIMENTARY TRACT Popoff (1894) concluded from digestions of thymus gland with ferment preparations that ' 'Solution of nuclein material occurs only very slightly in the stomach, but to a considerable extent in the in- testine, through the agency of the pancreatic juice. Here the nu- 232 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 clein bodies as such are taken into solution, so that it may be in- ferred that they are also resorbed as such." Very little inorganic phosphorus appeared in the solution. Milroy (1896) found most of the phosphorus from natural or artificial nucleins in organic form after trypsin digestion. A. Neumann's (1898) examination of the a- and /?-nucleic acids and nucleothyminic acid showed that none of these were attacked by pepsin-hydrochloric acid, but that they were dissolved by alka- line intestinal fluids. Umber (1901) made long peptic and tryptic digestions of the nucleoprotein from pancreas. About nine-tenths of the nucleopro- tein passed into solution under the influence of the pepsin. The pepsin first brought about a separation of a protein from a pentose- rich nucleic acid, which was soluble and could be absorbed, but which was not further broken down by pepsin. The digestion of the protein component, after being split off from the nucleic acid, proceeds in the ordinary way with the formation of primary and secondary albumoses, peptone and lower cleavage products. Trypsin had a like effect to that of pepsin, and required less time. Araki (1903a) carried out artificial tests which seemed to indi- cate that the nuclear substance of the red corpuscles of bird's blood is rather quickly dissolved by trypsin; that there is in thymus ex- tract an enzyme which has a like but slower action; also that by both trypsin and the thymus extract nucleic acid is changed from the less soluble (gelatinizing) form which Kossel and Neumann called a-acid to the more readily soluble (non-gelatinizing) /?-acid, and no further digestion occurs except after long-continued action ; and that erepsin splits nuclein and dissolves nucleic acid. Nakayama (1904) compared the action of erepsin from a dog with that of trypsin and of weakly alkaline ex- tracts from the intestines of cattle and rabbits. Commercial trypsins did not digest nucleic acids. The erepsin slowly digested the sodium salts of nucleic acids from the intestine of cattle, from thymus, from the spleen of cattle, and from the spermatozoa of Muraenosox cinereus. In each case phosphoric acid was found in the resulting solution. The extract of intestine acted on the nucleic acid much as did the erepsin. In connection with the study of enzyme action on caseinogen Plimmer and Bayliss (1906) tested the artificial digestion of yeast nuclein with trypsin, and found about four-fifths of its phosphorus in solution as phosphoric acid after 21 days. PHOSPHORUS METABOLISM 233 Mitra (1911) considered the age at which nuclease appears in the stomach of the infant. An infant a year old was fed on milk, on milk with flour and on porridge made from legumes ; and at inter- vals of 0.5, 1, 1.5 and 2 hours after -ingestion portions were removed from the stomach and tested with connective tissue and muscle fibre. No nuclease nor connectivase was found. A similar experiment on a child of fifteen months seemed to show the presence of both. Levene and Medigreceanu (1911c) have made various kinds of nucleic acid digestion tests, among them some such as are now under discussion. Inosin, guanosin, cytidin, guanylic acid, pyrimidin nucleo- tides, yeast nucleic acid and thymus nucleic acid were studied. The glucosides (inosin, guanosin and cytidin) were not changed in the neutralized gastric, pancreatic or intestinal juices. The mononu- cleotides (guanylic acid and the pyrimidin nucleotides) .were not af- fected by gastric or pancreatic juice; but with intestinal juice changes were produced which are interpreted as showing that phos- phoric acid was split off from the guanosin or pyrimidin complex, with no further change of these complexes. The action was much slower in the case of the pyrimidin nucleotides than in that of the guanylic acid. The complex nucleic acids (yeast nucleic acid and thymus nucleic acid) were little changed by gastric or pancreatic juice, but considerably (rapidly in the case of the yeast nucleic acid) by intestinal juice. The changes are interpreted as indicat- ing a cleavage into phosphoric acid and an organic complex, but no reducing substance (carbohydrate) was set free. These experi- ments seem to indicate that nucleic acids are not digested or ab- sorbed by the stomach, but that in the intestine phosphoric acid is split off under the action of intestinal juice. The going into solu- tion which others had observed as apparently brought about by trypsin may have been without this splitting, being simply such a change as Araki noted. According to Schaumann, "Schittenhelm (Arch, f . klin. Med.81, 1904, 423) found that in disease of the pancreas the digestion of nuclein suffers, and he thinks that the chemical experiments cor- respond with the microscopical observations. In considering the latter, he points to the observations of Schmidt, who showed long ago that in failure of pancreatic ferment unchanged cell nuclei of the food are passed with the feces." Abderhalden and Schittenhelm's (1906a) artificial digestions of the sodium salt of a-thymonucleic acid with the juices of the diges- tive tract, and with water extracts of pancreas and intestine, showed no determinable change by the gastric juice, and with the 234 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 pancreatic juice none which went far enough to yield purin bases, though there was some unknown kind of alteration which made the nucleic acid more readily soluble, and increased the amount of di- alyzable matter. The extracts of pancreas and intestine, however, produced an abundance of purin bases and a clear solution. Inter- preted as indications of the place in which cleavage occurs within the body, these experiments point to the understanding that nucleic acids do not undergo deep cleavage by digestive enzymes within the intestine but only in and beyond the intestinal wall. EFFECTS OF BACTERIA IN THE ALIMENTARY TRACT Bacteria in the digestive tract, especially in the large intestine, and perhaps also such as are introduced with the food, may have considerable to do with the disposition of nucleins. These micro- organisms show the power, not only to cleave nucleins in the ways shown above, but also to change amino-purins to oxypurins, even as far as uric acid. Doubtless they produce such effects, as they re- main for considerable time in contact with the food material. It should be remembered that these bacteria further complicate the studies of nuclein digestion because the purins of the feces may come from their bodies, Schittenhelm and Tollens say to the extent of 31.3 percent. Such purins would be the result of constructive as well as destructive activities of the bacteria. Schittenhelm (1903) has shown that nearly all of the nuclein substance of feces disappears as they undergo autoputref action ; and Schittenhelm and Schroeter (1903a, 1903b, 1904) showed that bac- teria may bring about a deep cleavage of yeast nucleic acid. Schaumann tells us that Emmerich and Low (Zeit. f . Hygiene 36, 1901, 9) found in pathogenic bacteria enzymes which were ef- fective to dissolve, not only their own nucleoproteins, but also those of other species of bacteria. Plenge (1903) showed that some bacteria have the power to liquify the sodium salt of a-nucleic acid from thymus and that the action is brought about by means of an enzyme. Vegetable moulds were shown by Iwanoff (1903) to contain or produce an enzyme which completely cleaves nucleic acids. Hahn and Geret (1900) found evidence of an enzyme in the ex- pressed juice of yeast which has a cleaving action on proteins, in- cluding nucleoproteins, which is much like that of trypsin but calls for slightly acid solution. It frees the bases and amino-acids di- rectly, without forming peptones, and but very little intermediary albumose. After one hour's digestion at 37°C. the greater part of the phosphorus was converted to the form of phosphoric acid. The enzyme is designated as yeast endotrypsin, and it is thought that it may play an important part in disassimilation. PHOSPHORUS METABOLISM 235 FERMENTS OF INDIVIDUAL ORGANS The enzymatic actions studied in various organs have included those of nucleases, guanase, adenase, xanthoxidase and uricase. All of these except the nucleases concern only the purin fraction of the compounds. Since the oxypurins, xanthin and hypoxanthin, are thought to be absorbable, and the amino-purins not, and since any processes concerned with the disposal of the nuclein substance may be of importance in the consideration of nucleins as foods, we are in- cluding a number of the many references to studies of the location of purin enzymes, but we discuss only those on nucleases, as having a direct bearing on the freeing of phosphorus from the molecule. The most productive workers in this field have been Jones and Schittenhelm and their co-laborers. References are listed below. The article by Wells (1910) summarizes in detail the enzymes which had, at the time of his writing, been located in the organs of dif- ferent animals. Nuclease was found in all organs tested, but other ferments were rather variously distributed, and it is noticed that species differ in this respect. Pighini's (1910,1911) optical methods used in determining nu- cleic acid cleavage by blood serum are called in question by Amberg and Jones (1911a). They find no cleavage of thymus nucleic acid by blood sera ; yeast nucleic acid, they say, is altered in some way, and perhaps decomposed, but without liberation of either phospho- ric acid or purin bases. A. J. Juschtschenko (1911) (see also Yushchenko, 1912) has studied the relative nuclease content of the blood, muscles and var- ious organs of man, dog, horse, cow, rabbit, hen and pike-perch, by making inorganic phosphorus determinations on uniformly pre- pared solutions containing salt-water extracts of the organs and sodium nucleate solution, after the mixed solution had stood for 40- 42 hours in a thermostat at 37-38°. The data reported show the milligrams of P 2 5 set free from the nucleate by one gram of organ. The general conclusions from the work are thus expressed : "Liver, kidney, spleen, pancreas, and thyroid gland contain sig- nificant quantities of nuclease; brain, suprarenals, lungs, and lym- phatic glands contain smaller quantities; heart, blood, muscle and serum are poor in nuclease. The blood of dogs, rabbits and cattle is richer in nuclease than that of man. The livers of man, horse, cattle, rabbit and pike-perch are richer in nuclease than that of the dog. In most organs of young dogs the nuclease content is less than in the same organs of grown dogs. The organs of man are in general rich in nuclease." Later (A. Juschtschenko, 1911), thyroidectomy was found to reduce the nuclease content of the organs and blood of young dogs and the blood of rabbits. 236 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 E. K. Marshall, Jr. (1913) has determined that the ferments of the thymus can not digest all of the nucleic acid of the gland, but leave a portion completely unaltered. According to Levene and Medigreceanu (1911d), "there exist in the organism several enzymes, which act harmoniously, leading to the disintegration of nucleic acids. The function which hither- to has been ascribed to the 'nucleases' is in reality performed by at least three enzymes, or rather three groups of enzymes, which have to be designated by special names. The part of each enzyme is to render the nucleic acid molecule susceptible to the action of one of the other enzymes, thus producing successive disintegration." Nucleinases are those enzymes which cause the dissolution of the nucleic acid molecule into nucleotides. Nucleinase is present in practically all organs and in the pancreatic juice. It is not present in gastric juice. Nucleotidases split nucleotides into phosphoric acid and a car- bohydrate-base complex. They are present in the plasma of all or- gans, and in the intestinal juice; they are not present in gastric or in pancreatic juice. "Also in regard to nucleotidases the possibility is not excluded that there exists more than one enzyme of the same group, and this for the following reasons [There is] gradation in the stability of the nucleotides towards chemical agencies [and] towards enzymes. Thus, guanylic acid is hydrolyzed readily into phosphoric acid and guanosin by the plasma of the pancreatic gland, whereas there could not be ascertained the occurrence of the anal- ogous cleavage of inosinic acid, of pyrimidin nucleotides, nor of the complex nucleic acids, through the action of the same plasma. On the other hand the cleavage takes place in all nucleotides under the action of the extract of the intestinal mucosa." Nucleosidases split nucleosides (purin ribosides) into their com- ponents. They are present in the plasma of most organs examined but not present in the plasma of the pancreas, nor in gastric, pan- creatic or intestinal juice. "Pyrimidin Complexes. Comparatively little information is obtained regarding the mechanism through which these substances undergo disintegration in the animal organism. The only evidence of the possible existence of enzymes bringing about cleavage of these complexes may be found in the older observation of Levene, that in course of prolonged autolysis of organs, free pyrimidins are formed." PHOSPHORUS METABOLISM 237 A viscosity method has been used by de la Blanchardiere (1913) in investigation of nucleolytic enzyme activity. From such obser- vations, made with organ extracts and pancreatic secretion, it is concluded that nuclease is not identical with any proteolytic en- zyme ; also that the processes of liquefying and of cleaving the nu- cleic acid are distinct, the work either of two different nucleases or of two different groups of the enzyme molecule. References on organ tests: Horbaczewski, 1889, 1891a, 1891b, 1893, Schwiening, 1894; Spitzer, 1899; Kutscher, 1901; Araki, 1903a; Jones, 1904a, 1904b, 1904c; Jones and Partridge, 1904; Schittenhelm, 1904a, 1904b, 1905a, 1905b; Jones and Winternitz, 1905; Jones, 1905; Schenck, 1905; Burian, 1905; Sachs, 1905; Bloch, Bruno, 1906; Abderhalden and Schittenhelm — resume of literature— 1906a; Schittenhelm and Schmidt, 1906, 1907a, 1907b; Jones and Austrian, 1906, 1907a, 1907b; Arinkin, 1907; Mendel, L. B., and Mitchell, 1907-8; Jones, 1908; Steudel, 1908a, 1908b; Kiinzel and Schittenhelm, 1908, 1909; Schit- tenhelm, 1908, 1909a, 1909b; Wells and Corper, 1909; Leonard and Jones, 1909; Rohde and Jones, 1909; Miller and Jones, 1909; Straughn and Jones, 1909; Winternitz and Jones, 1909; Jones, 1910; Wells, 1910; Pighini, 1910, 1911; Amberg and Jones, 1911a, 1911b; Jones, 1911a, 1911b; A. J. Juschtschenko, 1911; A. Juschtschenko, 1911; Medigreceanu, 1911; Levene and Medigreceanu, ' 1911b, 1911d; Mihari, 1911; Corper, 1912; Schittenhelm and Wiener, 1912; Yushchenko, 1912; Levene and LaForge, 1913; de la Blanchardiere, 1913; Mar- shall, 1913. ANIMAL EXPERIMENTS ON THE DIGESTION OF NUCLEOPROTEINS AND NUCLEIC ACIDS Loewi (1900, 1900-01) conducted metabolism experiments, mostly with human subjects, in which observations were made of the effects on excretion of adding various nuclein substances to a mixed diet. In the work reported in the first paper thymus gland was substituted for beef in an otherwise constant mixed diet with a subject suffering from myelogenic leukaemia, and with a normal subject; the effect of adding pancreas to a constant mixed diet was also studied; and a search was made for allantoin in human urine after the ingestion of pancreas, desiccated pancreas or thymus. It was concluded (in part) that : The excretion of uric acid in leukae- mics is not different from that in normal individuals; there is di- uresis. The ratio of uric acid to P 2 5 does not increase with math- ematical precision following thymus feeding. Both the items are increased by the gland. Increased katabolism does not occur, since the nitrogen figures return to normal shortly after discontin- uing thymus feeding. No allantoin could be found in the samples of human urine examined. The work reported in the second paper includes similar tests with salmon nuclein, pancreas nuclein, yeast nuclein, nucleic acid and "Nutrose" (a neutral sodium casein preparation from milk) ; also thymus was fed to a dog having ligated pancreatic ducts, and P 2 5 was estimated in the excreta ; and the uric acid excretion in the urine was compared in three subjects on the same ration. 238 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 02 £ O ■— !03CO^OC3}CMlOi— 1 HH O-HOOOOO-HO fa o Ph +++++++++ fa 3 Ed M 53 CNCNO'«^'-H**t* fa O G -^^HCMOilOCMCOlOlO fa w Pm cotoco^co-^coioco H r: O H t— NMto co fa (XI mooioco — io-jicdcm 55 t^lO-*(MI>r~^<00O mi^mr--mcomcoTO fa t-i P inoeoiot^ocoxt- OHOHOHOOO «• oogocgocMint^cio oooocoiNinoot^'j ANCES,WITH 900-01)— Gram PERIMENT I P* s o cocoo5-siomE^oo"»i ■^ir*-^oiocM'** M+jg+jCM jj fa H »— i ■Ml CM -4- 1 K+ + Z -o A e * -d •d Ed ■§ c 3 -d 5 rt a 5 * m * fa HH ~ >> ■"Ji^HCOi-KMi-ICOi— ICN 53° •e fa o M K£>t>£K£K Cm !>> CO t—i— IO rHCNieO ++ + CD innoo ocmcm +++ m-^ti-n r-r^co" COCOIO lO-WCO ENIOCO 03C33CN TJ1T}od oaco-*^ CO CO CO IOC5CO t^oooo ■d E-d i- :i i- •d^-d Ed i Ed i* SS tic R. *"* s fl> X o P. R p H bo fcj 3 HO" 09 ■3 ■d-a a fH-a H cS 3 <3 3 C3 o - rH |M uO S o •l)t- K . - 04 W*S "o N nco ^ CO CO CO +> e 0) .S u V P. ■d .8 'C f leucocytes in the blood, which may reach as high as 100 percent. This leucocytosis appears soon after nuclein is given, decreases gradually, and may entirely disappear after 18-24 hours. The nuclein leucocytosis appears constant also in different pathological conditions." Schaumann also says that, "Goldscheider and Jacob (Zeit. f. klin. Med., 25, 1894) had somewhat different results in their ex- periments on these phenomena. They used carbolic-glycerin ex- tracts of different organs, which they applied by injection. In these cases no changes in the blood were caused by the extracts from pan- creas, liver, kidneys, or thyroid gland, while those of spleen, bone marrow and thymus gave positive effects, though differing. The last two produced a pronounced influence in the direction of hypoleu- cocytosis, while the spleen extract injection caused a hyperleucocy- tosis, the intensity and duration of which were several times as great as with the giving of spleen pulp per os." From among the many other observations on leucocytosis the following may be of interest. Some have asserted that the in- crease of leucocytes is only an apparent increase due to a transfer of these corpuscles from the central circulation to the peripheral. Delano Ames and A. A. Huntley (1897) disproved this theory by their experiments on dogs. Comparison was also made of the re- spective numbers of the three varieties of leucocytes, which are sup- posed to be three stages in their development — small mononuclear, large mononuclear, and polynuclear cells. It was noticed that the administration of ether produced an increase, the extent of which seemed to depend on the amount of ether used. "2. Before the administration of nuclein the counts showed that the number of leucocytes in the peripheral and in the central circulation was prac- tically the same 3. Following the administration of the nu- clein solution there was immediately, that is by the end of five or PHOSPHORUS METABOLISM 249 ten minutes, a noticeable increase in the number of leucocytes in both the central and peripheral circulation. 4. At this time the per- centage increase was most marked in the young, mononuclear forms while the proportion of polynuclear elements was proportion- ately low. 5. The longer after the injection of nuclein the greater was the actual increase and the number seemed steadily to rise in both the peripheral and central circulation." According to Schittenhelm and Bendix (1905), yeast nucleic acid, and a-thymus nucleic acid injected in rabbits as the sodium salt, both caused at once hypoleucocytosis, followed in 6-8 hours by great hyperleucocytosis, which corresponds with Renner's observa- tions (See p. 258)., According to Schaumann, Walter Hannes ob- served as high as 144 percent increase of leucocytes. Renner's greatest increase was even higher, 452 percent. Meisen (1909, 1911) finds that nucleic acid, subcutaneously in- jected in growing dogs, produces hyperleucocytosis, without injuri- ous accompaniments ; that the relative number of mononuclear neu- trophils is not increased; that with repeated injections there is an up-and-down variation in the number of red blood corpuscles and in the haemoglobin content; and that injections kept up for a long time cause a greater firmness in growing bones. Kaupp (1911) reports certain blood observations on horses af- ter nuclein injections, the conclusions from which may be summed up thus : Nuclein increases the number of leucocytes in the blood, the number of polymorphonuclear leucocytes, and the power of phagocy- tosis of the individual leucocyte. Nuclein decreases the time of co- agulation of the blood. To obtain the best results in cases of infec- tious diseases, nuclein should be given hypodermically. To obtain desirable results in cases of hemorrhage, the nuclein should be given intravenously. M. Tschernoruzki (1912) made a special study of the effects of introduction of the sodium salt of nucleic acid in dogs. The dogs receiving the nucleate either per os, subcutaneously, intraperitoneal- ly or intravenously showed a more or less marked hyperleucocytosis, and a somewhat larger number of red corpuscles than an untreated dog. As judged from the number of leucocytes found, the strong- est hyperleucocytosis resulted in the case of intravenous injection (353.3 percent of the value in the control dog), next in the case of subcutaneous administration (204.8 percent), then intraperitoneal introduction (187.6 percent) and last, in the case of introduction per os (162.5 percent). The animals quickly acquired a certain immun- ity to considerable quantities of the nucleate. 250 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Timoshok (1912) studied the influence of sodium nucleinate (from yeast) on rabbits infected with Staphylococcus pyog. aur. The nucleinate caused a rapid polynuclear hyperleucocytosis, and altera- tion of enzyme functions, the latter varying either directly or in- versely as the changes occurring in Staphylococcus infection without sodium nucleinate. There were noted: (1) Parallel increase in lipoly- tic and catalytic activity of the organs; (2) parallel decrease in the sugar-synthesizing energy of the organs, and the diastatic and cata- lytic energy of the blood ; (3) increase in the lipolytic power of the serum (a decrease occurred in the absence of sodium nucleinate) ; (4) decrease in the diastatic and sugar-synthesizing energy of the organs, and the anti-trypsin content of the serum (an increase oc- curred when sodium nucleinate was absent). MISCELLANEOUS EFFECTS OF NUCLEAR MATERIAL . Milroy and Malcolm (1899) reported a histological study with rabbits and guinea pigs which was concerned with the intracellular wanderings of granulated leucocytes under the influence of nucleic acid, thymic acid, adenin, guanin, cytosin and metaphosphoric acid. Bang (1901b) tried the effects of injection of guanylic acid and of /?-nucleoprotein with dogs. Both of these compounds greatly reduced the rate of coagulation of blood, the nucleoprotein most markedly. Blood pressure fell off noticeably after the injection. Immediately after the injections the dogs were always restless, and breathed irregularly and quickly; then the excitement passed and was followed by partial narcosis for some minutes. In the case x>f guanylic acid injection, the urine became alkaline about half an hour after injection. After 24-48 hours it was again acid. There was no more ammonia present than normal, and the alkaline reaction could not be explained. Albumin was present to the extent of 1-3 parts per thousand, which, it was thought, might be due to the low- ering of blood pressure or to the acid having toxic action on the kidneys. Galeotti (1900) reports tests of the effects of bringing nucleo- proteins extracted from animal tissues or from bacteria, into con- tact with different animal organs or self-moving cells. The report is that nucleoproteins exercise considerable influence on protoplasm in general, the results of the influence varying according to the cells concerned. For some cells the influence was of the nature of ex- citement, and for some it was paralyzing. Mendel, Underhill and White (1903) report experimental studies of the physiological effects of nucleic acid, particularly that from wheat embryo, tritico-nucleic acid. Observations of the fol- lowing kinds are reported : PHOSPHORUS METABOLISM 251 (1) Manometer tracings of blood pressure after intravenous injections, (2) "coagulation time of blood taken from femoral vein after injections, (3) coagulability, composition and rate of flow of lymph from thoracic duct, (4) immunity effects, (5) urine examina- tion after feeding, and intravenous, intraperitoneal, subcutaneous and rectal injection, (6) urine and feces examination and leucocyte count after feeding and rectal injection. The authors' concluding summary is: "The more important observations recorded in this paper indi- cate that the vegetable nucleic acid obtained from the wheat embryo resembles, in its physiological effects, the guanylic acid of the pan- creas. Introduced in sufficient doses into the circulation, it may produce a fall in arterial pressure ; a change in the coagulability of the blood ; an increase in the flow of lymph and a change in its com- position; and perhaps, also, a degree of immunity toward subse- quent injections. "The ingestion of nucleic acid is followed in man by an in- creased output of uric acid, and in the dog by the excretion of al- lantoic These products correspond in either case to only a portion of the purin radicals introduced. In animals allantoin excretion was also observed after the introduction of vegetable nucleic acids into the body per rectum, intravenously, intraperitoneally, and sub- cutaneously. Some features of intermediary purin-metabolism are discussed." Guerrini (1903) made injections of 1 percent aqueous or sodium carbonate solutions of nucleoproteins from the liver and brain of white mice and of dogs into the peritoneal cavity of dogs and white mice, following with histological examination of the organs of the animals after different intervals of time, varying from one-half hour to 72 hours. Both excitations and degenerations were always found to have taken place in the parenchymatous cells of the livers and of the kidneys, the intensity and the time of appearance of the changes being in evident relation to the quantity of nucleopro- tein injected. Both kinds of changes were evident in both the nuclei and the cytoplasm of the affected cells. Enea (1903) carried out injection experiments with rabbits, from which he concluded that nuclein extracted from pathogenic and non-pathogenic bacteria has a toxic action; that in maximum doses it causes immediate death by destruction of leucocytes and co- agulation of the blood, while in lesser doses it produces gradual pro- gressive destruction of leucocytes, but not general coagulation, death occurring after different lengths of time that are proportion- al to the quantities of nuclein injected. It also increases the nor- mal bactericidal power of blood serum, the effect not being specific for the organism from which the nuclein was taken. 252 . OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Schittenhelm and Bendix (1904) found gelatinizing, unab- solved nucleic acid in the urine of rabbits after the subcutaneous injection of the sodium salt of a-thymonucleic acid; and the same acid injected in the ear vein of rabbits led to the appearance t)f red and white corpuscles, nucleic acid cylinders, and the unchanged nu- cleate in the urine ; also purin bases were present ; the kidneys were disturbed. Addition of uric acid to the nucleate injected made lit- tle difference, save that uric acid was included in the nucleic acid cylinders. Desgrez and Zaky (1904a, 1904b, 1904c, 1905) concluded that in guinea pigs and dogs the nuclein of yeast, or its nucleic acid, fa- vored retention, especially of proteins, and a mineralization of the skeleton. (See Growth' and Comp. of An. as Aff. by Comp. of Glyc.) Snowman (1905) mentions an experiment of Miyake, reported in Med. Annual for 1905, in which the injection of 1 c.c. of 0.5 per- cent solution of nucleic acid into the peritoneum of a guinea pig caused the animal to resist a subsequent inoculation with Bacillus coli of 20 times the normal minimum fatal dose. Pouchet and Chevalier (1906) report that injection of nucleic acid (from pancreas and from fish roe) causes an increase of leuco- cytes. An intravenous injection of 0.004 gm. per kilogram body weight in dogs causes acceleration of heart beat. With a larger dose (0.020 gm. per kilo.) an irritating action is produced upon the endo- cardium, and there is a decrease in blood pressure, and a slower rate of heart beat. After a certain time the original blood pressure is regained, and the heart beat becomes normal. The effects are thought to be due to action on the central nervous system. Knapp (1908) states, after his experiments with rats, that con- tinued use of sodium nucleate causes nephritis and fatty degenera- tion of the liver. Achard and Redfield (1911) used a nuclein solution as a wound dressing and consider that it promoted healing. Much literature that we have not seen, on the bactericidal effect of nucleins, is re- viewed. M. Tschernoruzki (1911, 1912, also Czernoruzky, 1912) has re- cently reported an interesting study on the action of nucleic acids on the fermentative processes of the animal organism. Think- ing that the resistance which the organism acquires against infec- tion from the use of sodium salts of nucleic acids is not due to leu- cocytosis alone, because it differs from the effect of aleuronate, which produces equally great leucocytosis, but may be in part due to alterations of the fermentative functions of the organs, he put to test the individual organs of dogs, after long periods of ingestion or injection of nucleic acid. PHOSPHORUS METABOLISM 253 Five young poodles of the same litter were used. One was kept as a control, and the other four received Merck's sodium nucleate, from yeast : No. 1 intravenously, No. 2 intraperitoneal^, No. 3 sub- cutaneously, and No. 4 by mouth. Treatment was begun at about the second month and continued 4*4-5 */£ months, with increasing doses up to 1.5 gm. per kilogram of body weight. Rise of temper- ature and leucocytosis, as results of the treatments, were noted. All of the dogs were killed and the organs removed, ground, dried, ex- tracted with physiological salt solution, and the extract tested for ferments. The organs examined were; liver, lungs, spleen, brain, kidneys, muscles and thymus ; and the following were the tests ap- plied : Fermeats tested for Tests Protease (Tryptase) Amount of extract required to digest completely 0.5 c.c. casein solution Amylase Amount of extract required for digestion of starch to dis- appearance of iodine reaction Diastase (Dextrinase) Amount of sugar produced from starch Oatalase Potassium permanganate method (Results are not reported because conditions of preparation of organ extract would vitiate results) Nuclease Inorganic phosphorus produced from nucleic acid in 48 hrs. at 37.5°C. Lipase " Acid split off from 1 percent monobutyrate solution Jjecithase Acid split from 2 percent lecithin emulsion (All results here were negative.) The author's conclusions are in part as follows : Introduction of nucleic acid into the animal organism has a marked influence on its fermentative activities. As to the signifi- cance of the manner of its introduction — the least effect on ferment- ative functions was observed with subcutaneous and the greatest with intravenous injection. As to the ferment — the greatest changes were found with the amylolytic ferment. With regard to the organs — the greatest variations in the sense of increase of fermentative energy were in the brain, the lungs, the muscles and the thymus. In the brain the value for amylase was 400 times as high as the normal, for diastase 4.4 times, and for protease 10 times. In the lungs the value for amylase was increased 250 times, in the muscles — for amylase 6.4 times, in thymus — for lipase 2.5 times. The brain considerably exceeded the other organs in the number of ferments evident, as well as in the amount of change in their action. Merck's sodium salt of yeast nucleic acid produced no evil effects in the animal organism under continual use of large doses, and with different ways of introduction. Perhaps the therapeutic signifi- cance of nucleic acid preparations in various diseases finds some ex- planation in the facts here reported. Especially in progressive paralysis, the benefit may be due to changes in the fermentative functions of the brain. The strengthening of the neucleolytic function in the blood was greatest in the polynuclear white corpus- cles. Of the organs examined, there seemed to be great increase 254 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 of nuclease content in the thymus, pancreas and liver, less of an in- crease in bone-marrow and spleen, and decrease, or but slight in- crease, in brain, lungs, kidneys and muscles. The dog which re- ceived the nucleate by mouth showed the greatest nuclease varia- tion from normal, considering all the organs together; and the one receiving it subcutaneously, the least. In addition to these effects on ferments, and to a hyperleucocy- tosis and increased purin metabolism, Tschernoruzki (1912) noted in the dogs a rise in body temperature and certain derangements in general condition, whatever the method of introduction. In the case of parenteral injection, doses, which approached the value of 1 gm. per kg. of body weight produced rather marked disturbances, such as loss of appetite, apathy, etc. After a dose of 1.5 gm. per kg. of body weight the intravenously injected dog showed symptoms like those of poisoning, but the dog receiving it by way of mouth showed no disturbance of general condition, with the exception of diarrhoea. At the close of this second paper Tschernoruzki 's conclusions were as follows: There is a strong analogy in the action upon the organism of nucleic acid, and infection. Both result in hyperleuco- cytosis, rise in temperature, disturbance of general condition, and increase of metabolism; and both react upon the fermentative processes, and bring about a certain degree of immunity. Nucleic acid is then apparently an agent which stimulates the natural pro- tective agencies of the organism, especially the leucocyte function. This explains the therapeutic significance of nucleic acid and gives basis for its use in medicine. His final conclusion is that "by intro- duction of nucleic acid into the animal organism the nucleolytic function of the latter is increased." Brossa (1912) made an attempt to determine what he calls the biological value of a-nucleic acid of thymus, for a dog and for hens, by applying to the nitrogen intake and output the following form- ula, which he attributes to Thomas : d_[ C -(a-b)] 100 a-b A nitrogen-free diet was fed for a brief time, followed by a like diet with sodium nucleate added, and to the data obtained the form- ula was applied by letting "a" equal the nitrogen taken in the food, "b" that excreted in the feces, "c" that in the urine on the nitrogen- containing food, and "d" that in the urine on the nitrogen-free diet. In this way it is calculated that the biological value of the nucleic acid for the dog is 60 percent, and for the hens 80 percent. PHOSPHORUS METABOLISM 255 Goubau and VanGoethem (1913) determined by intravenous in- jection experiments with dogs that the effects, on the circulation, of injecting nuclein, and nucleohistone are quite comparable with those of proteins in general. The primary effect is thromboplastic, and is followed by a more lasting antithrombic influence. Nucleins paralyze, slightly, the motor center of the heart immediately after the injection, with an enormous increase in the frequency of the beats, followed by a secondary enfeebling of the beats. See also Bokay (1877), Gumlich (1894), Jacob and Bergell (1898), A. Neumann (1898). SIGNIFICANCE OF NUCLEOPROTEINS AND NUCLEIC ACIDS AS AC- TIVE AGENTS IN SPECIFIC BODY PROCESSES From the mere fact of the presence of nucleoproteins at the heart of every cell, it may be inferred that they are of great import- ance in the life and the activity of cells, as Kossel has repeatedly re- marked. As to just what is their part we cannot say, but we may quote Gustav Mann as to one probability. As a result of his ex- tensive study and observation, chemical and microscopical, he says (Chemistry of Proteids, 1906, p. 454) : "What we call life is simply the manifestation of special chem- ical compounds, and if we see microscopically that every manifest- ation of metabolism is accompanied by enormous changes in the nu- cleoproteids, and that the rapidity with which nucleoproteids or the nuclear basophil chromatin reacts to food substances is directly pro- portional to the ease with which the food is absorbed, we cannot ar- rive at any other conclusion but that the nucleoproteids are the agencies by which amino-acids are built up into the cell plasm." Herlitzka and Borrino (1902a, 1902b, 1903), following Bottazzi, attribute catalytic actions to the nucleohistone of cell nuclei and the nucleoprotein of the cytoplasm. They tested the isolated nucleo- histones and nucleoproteins of liver, thymus and kidney as to their power to decompose carbonates, and to free the C0 2 (the power was possessed by both), to break down haemoglobin (possessed by both kinds of compounds, but only those of liver), to form urea (possessed by neither), and to digest sugars (most action by com- pounds from kidney, especially from its nuclei). Fibrin ferment and blood coagulation action have been ascribed to nucleoproteins and nucleohistones of blood and of muscle. The in- jection of nuclear matter from various sources has been followed by intravascular coagulation. (See Halliburton, 1892; Halliburton and Brodie, 1894 ; Pekelharing, 1895, 1896 ; Lilienf eld, 1895 ; Huis- kamp, 1901a). Other substances, however, introduced in this way 256 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 have also led to such consequences. It is Pekelharing's (1895,1896, 1914) idea that the fibrin-ferment, thrombin, forms from a nucleo- protein of the blood or organ, it being perhaps a calcium compound of the nucleoprotein. On the other hand, several observations which have been quoted above show that injections of nucleic acids affect the blood in such manner that its coagulation is considerably retarded. Doyon and his associates (Doyon, 1912 ; Doyon and Dubrulle, 1912 ; Doyon and Sarvonat, 1912a, 1912b, 1912c, 1913a, 1913b, 1913c, 1913d, 1913e, 1913f ; Doyon, Dubrulle and Sarvonat, 1912, and others) have re- ported investigations of this power in vitro. Anticoagulating bodies were extracted from liver, testicles, intestine, thymus, pan- creas, mesenteric ganglia, beer-yeast, blood corpuscles of birds, and haematogen of egg. Most of these were nucleic acids or nucleo- proteins ; and it is held that a phosphorized nucleus is essential to the anticoagulating power. The degree of effect of nucleic acid was compared (Doyon and Sarvonat, 1913c) with that of ortho-, pyro- and meta-phosphoric acids, with the resulting observation that or- thophosphate does not, under the conditions used, exercise any sen- sible anticoagulating action, while the pyro- and meta-phosphates do. The amounts of sodium metaphosphate and sodium nucleate with the same content of phosphorus showed the same degree of antico- agulating action, and these observations are taken as supporting the view that the phosphorus of nucleic acids is present as metaphos- phoric acid. Glycerophosphoric acid and lecithin were inactive in corresponding amounts. Pepsin had been considered by some to be a nucleoprotein, but Pekelharing (1902) showed that the phosphorus content of the preparation may be reduced by purification processes, and that in all probability phosphorus is> not a constituent of pure pepsin. Giacosa and Dezani (1909) also report the isolation from pig stomach of a digestive enzyme which is still active when free from phosphorus. THERAPEUTIC AND PROPHYLACTIC USE OF NUCLEINS AND NUCLEIC ACIDS Some of the effects of isolated nucleic acids in the diet are bene- ficial and some otherwise. If the body be in normal condition, they are well used and are favorable to nitrogen and phosphorus reten- tion; and in pathological cases the same is usually, but not always, true. When the amount of uric acid in the system is excessive, nucleins should be avoided. In special cases they may be of de- cided advantage, especially if injected, because of their effects upon the circulation and upon the number of blood corpuscles, particularly the leucocytes, and upon the power of phagocytosis. They have PHOSPHOKUS METABOLISM 257 been used with apparent benefit in several diseases, and most strik- ingly as a prophylactic measure before surgical operations and child-birth. See the reviews of Aulde (1900), Martinet (1902b) and Meisen (1911) of the literature of nucleins and nucleic acids from the point of view of therapeutics. Larned (1902) describes a series of iron, copper, mercury and silver nucleids as reconstructives and tonics, and as possessed with the power to increase physiologic resistance, and functional activity of secretory organs; and Burnet (1903) says that the metals of these salts are better used when administered in this form than in other forms. Tomlinson (1897) reports that in infantile scrofula, and other cases of enlarged lymphatic glands, with discharges, the stimulation of leucocytosis prevents suppuration, if it has not begun, and in- creases it, if it has begun, so that a more complete cure is brought about. Laumonier (1905) also reports as being brought nearer to nor- mal, under his observation, certain more or less definitely estab- lished physiological states. Backus (1907) reports apparent benefit in the cure of mange. Joseph Hoppe (1907a) made artificial nucleic acid preparations from yeast cells for use with children with nervous disease. Donath (1909) discusses the benefits from nuclein injection in the treatment of progressive general paralysis. He states that the injection at intervals of 5-7 days of 50-100 c.c. of a 2 percent solu- tion of sodium nucleate, with an equal quantity of sodium chloride added, is effective in the initial stages of this disease. The injec- tions caused a rise of temperature and an increase of leucocytes, with increased oxidation, which brought about a destruction of the poisonous metabolic products formed by the disease. Bachrach and Bartel (1907) conducted experiments in which yeast nucleic acid, added to protein media containing tubercle ba- cilli (from man), increased the speed of reduction of their virulence. On the other hand, the addition of yeast nucleic acid to suspensions of the bacilli in distilled water helps to retain the virulence of the organisms, which in distilled water alone become avirulent in but a few days. Ward (1910) reported favorable results from intravenous in- jection of sodium tritico-nucleate in physiological salt solution in the treatment of tuberculosis. The anaemia and oxygen-starvation of the tuberculous patients are associated with a low specific gravity of the blood, due to a decrease in the number of red corpuscles, and, at the same time, the appearance of increasing numbers of poikilo- 258 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 cytes. It is said that under the influence of the nuclein-saline so- lution the poikilocytes rapidly disappear from the circulation, the haemoglobin being eliminated by the liver as bili- rubin and biliverdin; the deformed cells being replaced by new and healthy erythrocytes more rapidly than is possible under any other form of treatment as yet devised. This author, then, thinks that the administration of nu- clein produces its good effects, not chiefly by inducing leucocytosis, but by effecting an upbuilding of erythrocytes and haemoglobin. Though nucleins and nucleic acids may have bactericidal action in themselves (see Vaughan, Novy and McClintock, 1893; Kossel, H., 1894), their use to prevent infection, in cases of exposure of the tissues to pathogenic bacteria, is probably to be attributed more largely to their leucocytosis-producing power. Rentier (1905) re- ported such use of yeast nucleic acid in 133 surgical cases. Usually 50 c.c. of a 2-percent solution of the acid in sodium chloride solution was injected in the breast within 24 hours before the operation. There was a brief hypoleucocytosis, followed by a pronounced hyper- leucocytosis. Apparently the resistance against Bacterium coli and other pathogenic bacteria was increased. The increase of leucocyte count amounted to from 9 to 452 percent (with the exception of S 1 /^ percent in one case where the initial count was very high) ; in 4 cases there was a lowering of leucocyte count, at least temporary, of 9-14 percent; the average increase in 121 cases was 118 percent. For certain undesirable effects to be guarded against see the original. See also Pollak (1906) ; Hannes (1906) ; Anzilotti (1911) ; Achard and Redfield (1911).; also (through Schaumann) v. Mikulicz (Archiv f. klin. Chirurgie 73, Nr. 2), Miyake (Mitteil, aus den Grenzgebieten der Medizin und Chirurgie 13, Nr. 14, 15) and Dudgeon and Ross ( Amer. Jour, of the Med. Sciences 1906, 17 ; Presse medicale 1906, 569). SUMMARY Very little is known as to the details of the anabolic processes in the body ; but it seems probable, though not proved, that the ani- mal organism is capable of synthetic formation of nucleins and nu- cleoproteins, even without purins being supplied as such. Obser- vations reported as evidence of such apparent synthesis with inad- equate supply of purins are of the following types : The development of the genitalia of fish while no food is taken ; the growth of young mammalia when milk is the only article of diet ; the transformations within eggs and embryos ; and apparently normal growth on experi- mental purin-free (or low-purin) diet. However, the evidence is not complete on any one of these points. PHOSPHORUS METABOLISM 259 As to katabolic processes acting on nucleoproteins, nucleins and nucleic acids, tests in vitro by isolated enzymes or by fluids taken from the digestive tract indicate that, though proteins may be sep- arated from nucleoproteins, and nucleins or nucleic acids may be dissolved by gastric juice, this juice does not cause cleavage; that by activated pancreatic juice (trypsin) they are more readily dis- solved, which solution may be due to transformation of the a-form to the /?-form, or the breaking up of polynucleotides into mononu- cleotides, though there may be slow cleavage of the nucleic acids, but not a complete breaking down such as frees the reducing sub- stance and the purin bases; that intestinal juice cleaves more rap- idly, and frees phosphoric acid, but does not break down the gluco- side complex of carbohydrate and base; that within the intestinal wall cleavage may be carried to the state of free purins ; and that the intestinal bacteria may not only bring about complete cleavage, but may further oxidize and transform the purins. Tests in vitro with blood and organ extracts indicate that cleav- age may take place in the plasma of practically all organs such that the phosphoric acid and the purins are freed from the carbohydrate, and that the purins may then be further altered, though the extent of such alteration differs with different organs, with different species, and probably also with different nucleic acids. Animal experimentation, giving fairly direct evidence as to what actually occurs in the particular animal under the particular conditions of observation, also indicates that, in the stomach, nu- cleoproteins and nucleic acids are not broken down, but, in large part, go into solution as such ; that in the intestine there is some breaking down; that this breaking down cleaves off phosphoric acid from the carbohydrate-base complex, but does not free the purins ; and that nucleic acids and their cleavage products are absorbed chiefly from the lower portions of the small intestine (the lower jejunum and the ileum). We may suppose, then, that nucleins or nucleic acids are in con- siderable part taken up by the walls of the digestive tract without change, and in part also after partial cleavage, and that cleavage may be carried further within the intestinal walls, or at the place of storage. The ingestion or injection of nuclein or nucleic acids is fol- lowed by an increased purin output from the body, which probably originates in part in the purin component of the nucleic acid intro- duced, and in part in the nuclear matter of cells which are broken down as a result of the introduction of this substance. The diges- tion itself may call for cell destruction which produces this nuclear 260 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 matter, or the increased formation of leucocytes may be accompan- ied by an increased destruction of such cells. There is also an in- creased phosphate elimination. Another constant accompaniment of the taking of nuclein-rich food is a marked increase in the number of leucocytes, in the blood, following a brief period of decrease. The effect is more pronounced when the nucleic acid is injected. It takes place soon after the in- troduction and lasts for several hours, affecting all the forms of leu- cocytes. The phagocytic power of the individual leucocytes is also said to be raised. Advantage is taken of this favorable result in the injection of nuclear matter as an aid in combating infection. The blood shows other effects of nucleic acid injection which are more or less temporary, and more or less variable with the con- ditions of operation, and with the amount and kind of acid used. There may be an increase in the red corpuscles, a fall in blood pres- sure, and a change in coagulability. While nuclein injection is said to increase the power to resist bacteria, it seems to be somewhat toxic itself. One author (Tschernoruzki) has noted favorable ef- fects upon the fermentative powers (amylolytic, diastatic, catalytic, nucleolytic and lipolytic) of the organs as a result of the introduc- tion of nucleic acid, either intravenously, intraperitoneally, subcu- taneously, or by way of the mouth. This effect may explain some of the favorable results of the therapeutic use of nucleins and nu- cleic acids. The specific activities of nucleoproteins in body cells are not de- termined; but as they are so universally present as the main con- stituents at the points of most pronounced manifestation of life — the cell nuclei — it cannot be doubted that they are vital, active agents. METABOLISM OF CASEIN PRODUCTION OF CASEIN IN THE MILK GLANDS Nissen (1886) observed nuclear changes in the milk gland dur- ing secretion, and suggested that the casein may form by union of nuclein from the nuclei of the epithelial cells with protein from the cytoplasm of the gland cells. Michaelis (1898) added support to this idea by finding an abundance of the epithelial cells free in the lumen of the alveoli during lactation; and Basch (1898, 1903) isolated a nucleic acid from the gland, from which, by allowing this substance to act on an excess of blood serum, he obtained a body having so many of the properties of cow's casein that he felt sure that it was casein. His conclusion was that within the alveoli this nu- cleic acid from the cell nuclei (freed either by destruction of the cells PHOSPHORUS METABOLISM 261 or by an active .secretion of the cells) combines with transudated serum, thus forming a nucleoalbumin, the casein. His theory seemed to be further supported in that he was unable to obtain purin bases from his nucleic acid, the nucleic acid thus correspond- ing with the paranucleic character of casein; but the nucleic acid which J. A. Mandel and Levene (1905) isolated from the mammary gland did show purin bases. This tends to discredit Basch's theory, and other evidence against it is contributed by Lobisch's (1906) studies of nuclein-protein compounds and by Borrino's (1910, 1911) finding a nuclease in the mammary glands present only during lac- tation. This nuclease would indicate a breaking down of nuclein beyond the point of nucleic acid ; so that if the phosphorus of such a compound is used in the synthesis of casein it is by previous destruc- tion of the acid. There may be significance in the coincidence that a nucleopro- tein which Mandel has obtained from mammary glands yields hydro- lytic cleavage products in approximately the same proportions as those formed from casein. Certain experiments reported in 1908 by Michaelis and Rona (1908) imply a relation of the formation of casein to the phenome- non of milk-secretion, the significance of which has not been ex- plained. Subcutaneous injection of casein into guinea pigs and dogs was followed, in females, and in males as well, by a swelling of the mammae, and, at least in one case, by the production of true milk. Examination of the glands showed that the cells had undergone the changes usual with milk secretion. Bergell (1898b) suggests in explanation of certain figures for the phosphorus of the blood ash of lactating female animals, in com- parison with the normal for the species, that a higher phosphorus content of the blood ash may be associated with the milk producing function, but the data submitted do not warrant a positive assertion to that effect. DIGESTION OF CASEIN DIGESTION IN THE STOMACH Rennet action. Casein, unlike other proteins, is usually intro- duced into the stomach in the uncoagulated state, and accordingly, a special coagulating enzyme is provided in the rennin. Doubtless the coagulation is a favorable preliminary step in digestion. The conditions of this coagulation, and the steps of the process, have been discussed in the consideration of the chemistry of casein. The casein is first modified to paracasein, and then precipitated with calcium salts. The same action may be brought about by enzymes 262 OHIO EXPEEIMENT STATION: TECHNICAL BUL. 5 occurring in blood, pancreas and other organs. That of the pancreatic juice is probably useful with milk which passes from the stomach uncoagulated. Whether or not the rennin itself has any- further action on casein, under favorable circumstances, is uncer- tain (Van Herwerden, 1907, and others). Paracasein is found to be more easily and more thoroughly digested (in vitro) than is the un- altered casein (Hosl, 1910). Gaucher has reported observations on the time taken for milk and casein to pass on from the stomach, and the physical state in which the casein passes, as shown by duodenal fistulas. Gaucher (1909) observed three stages in this process in a dog. First, the milk passes out as such during the first quarter of an hour ; second, dur- ing the second quarter of an hour the lactoserum flows out mixed with large clots ; and third, the liquid which flows later, colored by bile, contains fine particles of coagulated casein held in suspension. Peptonizing does not take place in the stomach. Gaucher (1911) made similar observations on a child. At a later date Gaucher (1912) showed with a dog that the digestion of casein in the con- tracted, solid form corresponds only to the third of these stages. According to Gaucher then — of the 7 grams of casein taken in 250 c.c. of milk, 4 grams pass the pylorus in an unaltered form during the first stage; 10-15 minutes' secretion of gastric juice clots the remainder of the milk, and the stomach contractions detach rather coarse fragments of the clot, 1 gram of the casein passing from the stomach in this stage ; the other 2 grams contract to a harder mass, which, as the muscular action of the stomach becomes more and more vigorous, is gradually reduced to a puree, this portion leaving the stomach after the lapse of about an hour. Peptic Digestion. The action of the enzymes of the digestive tract has been studied largely by artificial digestion in vitro. Study of this kind with artificial gastric juice was undertaken in 1870 by Lubavin (1870, 1877). It was early known that as a result of pep- tic digestion there pass into solution albumoses or proteoses (those of casein are designated caseoses) and peptones, and that there re- mains an insoluble nuclein-like body richer in phosphorus than the casein. This body has been called pseudonuclein or paranuclein, and it has been shown that under favorable circumstances it may be itself digested by the pepsin-hydrochloric acid mixture. On fur- ther digestion it is said to become more rich in phosphorus (von Szontagh, 1892, 1893). References to investigations in this field were given in connection with the consideration of comparisons of different caseins in this respect. PHOSPHORUS METABOLISM 263 According to Chittenden and his associates, (Chittenden and Painter, 1885 ; Jackson, 1900) the caseoses contain no organic phos- phorus, but the paranuclein (which they call dyspepton) always con- tains over 2 percent of phosphorus in organic combination. A small part of the phosphorus goes into solution as inorganic phosphoric acid. Clara Willdenow (1893) conducted peptic digestion experiments on casein (in vitro), and found split off a phosphorus-containing body, which contains its phosphorus mainly, not as calcium phos- phate, but in organic combination, agreeing with Lubavin, but not with Chittenden. This substance, from its properties and phos- phorus content, seemed like nuclein or nucleic acid. Willdenow ob- tained but a single body containing phosphorus and sulphur, not like Lubavin's finding of two such separable by sodium carbonate. Von Moraczewski (1895a), under Drechsel's direction, studied the distribution of the phosphorus between nuclein and the filtrate from the same after the digestion of casein with pepsin. Various changes of the conditions of the digestion were made, especially as to the concentration of the solution and the duration of digestion. The results given below are taken from the author's table : PHOSPHORUS OF THE NUCLEIN RESULTING FROM PEPTIC DIGESTION OF CASEIN Concentra- Weight of nu- Nuclein P tion of case- Time of vVeigtit of Weight of clein from 100 to 100 gm. Phosphorus Exp. in solution digestion pepsin casein gm. casein casein P in nuclein No. Percent Days Grams Grams Grams Grama Percent I 4 8 0.44 14.26 13.60 34.20 2.10 n 4 5 1.38 8.15 14.72 37.50 2.40 III 4 3 1.39 10.19 17.40 44.20 2.19 I 4 2 0.5 3.5 14.84 50.54 0.88 I 6 14 0.5 3.00 3.60 20.66 3.87 II 6 10 0.5 3.00 6.67 29.20 3.13 III 6 7 0.5 3.00 8.31 31.31 2.70 I 0.7 5 1.0 5.27 1.29 6.75 4.10 II 0.7 1 1.0 2.68 4.64 18.20 3.15 III 1.44 7 1.0 5.46 5.96 42.61 6.86 IV 1.44 5 1.0 9.11 8.37 26.17 3.43 I 3.45 2 1.5 33.80 16.48 44.51 2.08 II 3.45 10 1.5 33.80 18.17 52.59 2.27 III 3.51 10 2.0 31.61 17.99 56.40 2.44 IV 3.51 2 1.0 31.61 21.10 63.21 2.82 It was concluded that : "The casein does not have all of its phos- phorus in the form of nuclein, for the amount found ranges from 6 to 60 percent, but is never the total phosphorus." "Nuclein from cow's milk does not, even after long digestion, go completely into so- lution The amount is affected by the time of digestion 264 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 and still more by dilution. In very dilute solution the amount of nuclein is very small and it is very rich in phosphorus. With longer time of digestion under these circumstances the amount of nuclein decreases and its phosphorus content increases. In concentrated solutions nuclein precipitates in large amount and loses little phosphorus even on protracted digestion." "In the di- gestion fluid the phosphorus can be precipitated by magnesia mix- ture directly only after long digestion and in very dilute solution. The degree of dilution plays a greater part than the amount of pep- sin or the time of digestion here also " Von Moraczewski's theory is that the nuclein or nucleic acid from it carries down with it some protein body such as unaltered casein or caseoses. Sebelien (1895) found 44 and 33 percent of the phosphorus of casein in the paranuclein portion. Salkowski reported experiments (Salkowski, 1893a, 1893b ; Sal^ kowski and Hahn, 1894-95) from which he felt sure that in peptic digestion the main part of the phosphorus of casein remains in solu- tion in some kind of organic combination, only a smaller part being in the insoluble paranuclein. It was further found that the amount of phosphorus in the paranuclein varied with the conditions of di- gestion, the more unfavorable the conditions, the greater the quan- tity of paranuclein and the greater also the quantity of phosphorus in the insoluble product. The following table is taken from the ar- ticle by Salkowski and Hahn. RESULTS OF PEPSIN DIGESTION OF CASEIN— Percent Exp. Paranu- clein portion Albumose portion Phosphorus con- tent of Percent of total phosphorus in Paranu- clein Albu- mose Paranu- clein Albu- mose II III IV V VI 6.8 6.76 18.5 15.2 21.05 93.2 93.24 81.5 84.8 78.95 2.41 0.55 (?) 2.27 2.18 2.11 0.74 0.87 (?) 0.59 0.58 0.51 19.0 4.3 (?) 41.2 41.9 52.5 81.0 95.7 (?) 58.5 58.1 47.5 r Conditions most favorable. (.Too little of pepsin solution, f or pepsin less active. Salkowski (1896a) reported the conditions most favorable to complete digestion, and later (1899) stated that by carrying on the digestion under retarding circumstances he was able to identify an intermediary phosphorus-containing albumose. He says: "The pep- sin digestion of casein, then, proceeds in three stages: 1. The transformation of the casein into an albumose, 2. splitting off of paranuclein from this, 3. complete solution of the paranuclein and further digestion of the albumose. While it is not possible sharply to separate the second and third stages from one another, — as during the splitting off of the paranuclein a part at least PHOSPHORUS METABOLISM 265 of it goes into solution, — the distinction between the first and sec- ond stages is easy, as the splitting off of the paranuclein takes a def- inite time, and moreover the process can be interrupted before all the casein has changed to albumose." Zaitchek (1904) and von Szontagh (1905), under Tangl's direc- tion, made comparative digestion experiments on casein from dif- ferent kinds of milk, the results of which are evident in the conclu- sions which we quote from Zaitchek. "1. By pepsin-hydrochloric acid digestion experiments we de- termined that woman's, ass's and mare's milk is completely digested, while the casein of cow's, goat's and buffalo's milk under the same conditions of experiment (temperature 38°C, time 72 hours) is only 8, 14 and 15 percent soluble. "2. Each kind of milk which is not soluble in pepsin-hydro- chloric acid without residue leaves a different amount of pseudonu- clein from the casein obtained from it. The- latter gave without ex- ception 2-3 percent smaller pseudonuclein residue than the milk containing the same amount of casein. The casein precipitated from woman's, ass's and mare's milk is as completely soluble as the milk itself. "3. The woman's, ass's and mare's milk not only contains an absolutely smaller amount of casein than cow's, goat's and buffalo's milk, but a smaller portion of the total nitrogen, also, is in the casein. "4. Under like conditions of experiment the different kinds of pure casein give different amounts of pseudonuclein (0-15 percent) . "5. The addition of thymol, toluol and chloroform hinders the casein-dissolving action. The checking effect increases with the amount added. "6. Both the concentration relations and the duration of ac- tion of the pepsin-hydrochloric acid have considerable influence oh the solubility of casein in pepsin-hydrochloric acid. "7. The drying of casein at 110°C. considerably reduces the solubility in pepsin-hydrochloric acid." Some of Robertson's studies (1906-07, 1907, 1909a, 1910b, 1911, Robertson and Biddle, 1911) on the reversible action of enzymes having been made with casein, this work and his theory regarding the matter may be considered in this connection. One of the stages in peptic digestion of casein is quite marked because of the difficultly soluble nuclein (or, better, "pseudo-" or "paranuclein") produced. It has been thought that digestion of the 266 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 casein from cow's milk could not be carried beyond this point by pep- sin, but it has since been shown that under favorable conditions the paranuclein may be made entirely soluble. Now, Robertson has succeeded, by the use of pepsin, in bringing about a synthesis of paranuclein (or one of the constituents of the mixture usually ob- tained as paranuclein) from the products of complete peptic diges- tion of casein. The obvious explanation is that pepsin acts as a cata- lytic agent, accelerating the opposite phases of a reversible reaction, the breaking down of casein into the simpler products, and the union of the simpler products toward the formation of casein. Robertson's theory modifies this interpretation a little in that he thinks that there are two forms of the enzyme active in the two processes. Just as the cleavage products differ from the protein by the elements of water, and may be looked upon as successively more and more hy- drated, so the enzyme is supposed to have two forms, the hydrated and the anhydrous. The hydrated form is effective in introducing water into the protein (accelerating hydrolysis), and the anhydrous form, in removing water, so that the simpler bodies unite with one another (accelerating synthesis). The theory is that the effect is brought about by the aid of a temporary union of the ferment with the protein. Representing the two forms of the ferment by HFFOH and FF, and the protein and its cleavage products by HXXOH and HXOH, the types of reaction concerned in the process- es under discussion may be shown as follows : HXXOH+HFFOH^±HXXFFOH+H 2 (1) HXXFFOH+H 2 CM2HXOH+FF (2) FF+H 2 O^HFFOH (3) In case of hydrolysis (1) represents the union of protein with the hydrated ferment; (2), the breaking up of this union, leaving the protein split, and the ferment in its anhydrous form; (3), the ferment again taking up water to itself from the medium. In syn- thesis the processes are reversed under the influence of the dehy- drated enzyme. The ratio of the velocity of the reaction in the two directions is affected by the concentration of the protein or its cleavage products and of the enzyme, by the temperature, and prob- ably also by the alkalinity or acidity ; and this ratio determines the final equilibrium. Bayliss (1913) believes that Robertson's (1907) synthesis of paranuclein from casein digestion products by the action of pepsin is not a synthesis at all, but a colloidal precipitation without connec- tion with the enzyme. PHOSPHORUS METABOLISM 267 Kuttner (1909) reports a study of peptic digestion of casein from the standpoint of the acidity of its cleavage products. Diges- tions were made with differences in proportion of gastric juice (from dog) , in acidity, in dilution, in temperature and in duration of digestion, with phosphorus and nitrogen determinations on the dis- solved and undissolved portions, and in one series on the alcohol pre- cipitable portion of the products of digestion. During one series the digestive products were removed at stated intervals, and fresh mixture substituted. In each case the digestion was stopped at the time that a precipitation of the products of digestion appeared. The nature of the data is evident from the table on p. 268. It will be ob- served that a considerable increase of acidity occurs during diges- tion. Of this a certain fraction is spoken of as the non-salt-precip- itable acidity, meaning the fraction of the final acidity which is due to some compound not precipitated by thirty percent sodium chlor- ide solution. The author's conclusions are : "There is every reason to believe that casein is cleaved during peptic digestion into a phosphorus- free and a phosphorus-containing portion. While cleavage of the former continues even under the most unfavorable digestive con- ditions, the latter (the paranuclein) is less easily digestible, though its further digestibility is such that even under the most unfavor- able conditions a fraction is always attacked. By the further diges- tion of paranuclein under favorable conditions there results a peptically indigestible fraction (Kiihne's anticomplex) , while the phosphorus-containing constituent which is separated from it by peptid cleavage is further cleaved into more and more highly phosphorized acid-like compounds (paranucleic acids) . These are in the main responsible for the increase in acidity of the digest- ive products observed during the digestion of casein." According to the observations of Miss Goldthwaite (1910), the presence of carbohydrates retards the peptic digestion of casein, and by an amount proportional to the amount of carbohydrate present. Long (1907a) made protracted digestions (nearly two months) of casein from cow's and from goat's milk observing the "free" and "total" acid in the digestion mixtures as digestion progressed, the electrical conductivity of this solution, the weights of undissolved and dissolved solids, and the conductivity and acidity of this dis- solved solid on being redissolved after evaporation. During diges- tion the electrical conductivity values, and the total and free acidity varied regularly. Both acidity values were lower for the goat casein than for cow's casein. A larger residue of pseudonuclein was finally left from the casein of goat's milk ; the dissolved substances were thought to be similar in nature. 268 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 ft O co o O o ft" ft ft ft CO 3 ft O o H CO ft H Oh ft ■a s. ■§ «4-( LQ o QJ 5 a a n rt^ in g ™N, OINt»000-*CO-« in cio CDCDOOOOOCOCiaO "71 tl "71 TJ Tl t! T! "tJ oooooooo OOOOOOOO CO?OOf-"*CDCOCO *— I001OO3001OCOO3 cocM'iric^ooooTn'co CM-tflLQCOCPC-OOOO rocooomcoocoo) io»-Hoicocococcio I-ICMCNCOCQ-^IIOIO TH— l-*0000(M1030 COCOCOi-HCC'JtOTOO »— ir~mODi>iascNico N CO lOlQCfi CCISCo masCOCNKNOOTHOJ COCDCOOOCDlOOrH odcNiTtior^ot^co C010eoCOCMCNi-1i-< •OTCOi— IOCOOS •eoco^'miQio COCOIOCDOSO^CO cdos'^'^ojo^t-J i-l(MW>SllOt^CC05 COJN IQrH 5288SS oooiooioinin rH-Hf-IOJOJtMCN PHOSPHORUS METABOLISM 269 DIGESTION IN THE INTESTINE In the intestine the casein, whether still unaltered, or changed by the pepsin to proteoses and peptones, can be further split by the trypsin of the pancreatic juice and the erepsin of the intestinal se- cretion (Hammarsten, 1911). The paranuclein from casein is also actively digested by trypsin. Brugsch and Masuda (1911) state that the intestinal juice can not digest casein, though an extract of the colon bacillus does so, and that the feces do not possess a casein-cleaving capacity if the pan- creatic secretion be excluded. The absorption of the cleavage products of casein takes place at least principally as amino acids. The form in which the phos- phorus of casein is absorbed has not been determined. Artificial Digestion Tests. Sir William Roberts (1879, 1881) first noticed the curdling of milk by pancreatic extract ; also that the first principal phase of the tryptic digestion of casein is the forma- tion of a modification of the casein, which he and others since have called metacasein. In both of these respects the action is very sim- ilar to that of rennet, as others have noted (Edkins, 1891 ; Halli- burton and Brodie, 1896). Harris and Gow (1892) found the ac- tion lacking in the pancreas of some animals. Von Szontagh (1894) states that on digestion with pancreatic juice the casein is first entirely dissolved, and then a precipitate forms. In an alkaline solution the precipitate is less, but evidently because of a solvent action of the alkali, and not because of digestion of the nuclein. Sebelien (1895) reports digestion experiments with pan- creatin-soda solution. Below are the author's figures for his fourth and fifth series of experiments, in which, with two different concen- trations of casein solution, increasing amounts of pancreatin solu- tion were used (the total volume of digesting solution being kept at 300 cc. by regulation of the volume and concentration of the soda solution added) . In this case the residue precipitated on acidifying DEGREE OF DIGESTION OF CASEIN AND ITS PHOSPHORUS BY PANCREATIN Time of digestion Casein solution Pancreatin Soda solution ._ Percent Casein undissolved Percent Phosphorus undissolved Percent 18 hours 200 cc, 2.076* 200 cc 200 cc 10 cc, 5* 20 cc 40 cc 0.167 0.167 0.167 1.2 2.3 2.5 2.2 2.3 2.3 200 cc, .556* 200 cc 200 cc 25 cc, 1* 60 cc 75 cc 0.208 0.208 0.208 2.5 3.5 2.5 2.1 M « • 1.6 1.2 270 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 the solution after a certain time of digestion was weighed, and tested for phosphorus. This residue was considered to be undis- solved casein. It was but a small percent of the casein by weight, and was not like the pseudonuclein, which forms a much larger part of the casein. As the figures show, the casein and the casein phos- phorus were practically completely digested. Rotondi (1902) reports that casein, from either human or bovine milk, is much more readily digested than fibrin, by both pep- sin-hydrochbric acid and pancreatin, the digestion by pancreatin being especially easy. E. Fischer and Abderhalden (1903) say that more hydrolysis of casein takes place by the combined working of pepsin-hydrochloric acid and pancreatin than by pancreatin alone. Biffi (1898), using a pancreatic ferment prepared from beef pan- creas, concluded that under favorable conditions casein is completely digested by that enzyme, about 4 percent splitting off as tyrosin, with the formation of primary and secondary albumoses and an anti- peptone which correspond to those of fibrin. All of these products are probably free from phosphorus ; but in the soluble product phos- phorus is always present in two forms, a part being directly precip- itable by magnesia mixture and another part being so precipitable only after fusion with soda and potassium nitrate. The phosphoric acid increases with the duration of digestion and with the amount of ferment, at the expense of the organic phosphorus. The organic can further be changed into inorganic by the action of dilute alkali solution or by boiling with barium carbonate. From the tube digestions made by Rachford (1900) it may be said that pancreatic digestion of the casein of cow's milk is facili- tated by the presence of maltose solution, of lime water, or of 0.4 percent sodium carbonate solution. Hydrochloric acid was found very slightly to retard the action of pancreatic juice alone, but greatly to increase the action in the presence of bile. "Bile assists the pancreatic juice in the digestion of casein, but it renders even greater assistance when the casein is partly saturated with hydro- chloric acid." Plimmer and Bayliss (1906) studied the action of enzymes and of alkali on casein. They summarize their results as below: "1. The whole of the phosphorus of caseinogen, except for a very small residue, is converted into a soluble form by the action of trypsin in 24 hours. "2. The curve of its rate of separation is exactly parallel to the curve of the electrical conductivity (see Bayliss, 1904) during the first 7-8 hours ; its rate of separation after this time is less rapid. PHOSPHORUS METABOLISM 271 "3. The small insoluble residue is partly derived from the trypsin, and partly from the caseinogen, and consists most probably of the products of decomposition of nucleoproteid. "4. The 'soluble P 2 5 ' consists of inorganic phosphoric acid — 35 percent — and organic phosphorus, 65 percent. "5. The phosphorus of caseinogen is very slowly converted in- to a soluble form by the action of pepsin. The quantity thus changed is only 70 percent in 149 days, and consists, except for a negligible quantity of inorganic phosphoric acid, of organic phos- phorus. "6. Papain, in its action on caseinogen, is intermediate in power between pepsin and trypsin. In its rate of splitting off of 'soluble P 2 (V and 'soluble nitrogen' it resembles trypsin the more closely, but it is much slower. "It acts best in a neutral or faintly acid medium ; its action is slower in a slightly alkaline medium and is almost inhibited by 0.5 percent sulphuric acid and 0.5 percent sodium carbonate. "7. Ovovitellin, containing lecithin, is very slowly digested by trypsin in comparison with caseinogen. Only one-half of its phos- phorus is converted into a soluble form in 36 days. A similar quantity is probably contained in the lecithin portion of the mole- cule. "8. One percent caustic soda converts the whole of the phos- phorus of caseinogen into 'soluble P 2 5 ' in 24 hours. It resembles trypsin very closely in its rate of action. "9. The 'soluble P 2 5 ' produced by one percent caustic soda in 24 hours consists entirely of inorganic phosphoric acid. In the same time the quantity of 'soluble nitrogen' scarcely increases. "10. The organic phosphorus produced by the action of tryp- sin on caseinogen is not completely converted into inorganic phos- phoric acid by the action of one percent caustic soda. The total quantity of inorganic phosphoric acid produced by trypsin and sub- sequently by one percent caustic soda is 50 percent of the total phos- phorus of caseinogen." Robertson (1906-7), from his study x>f tryptic digestion of case- in in relation to the ion-proteid theory, concludes (in part) that neu- tral casemates in solution undergo fairly rapid autohydrolysis ; that the velocity of hydrolysis of calcium caseinate by trypsin at mod- erate substrate concentration is directly proportional to the amount of trypsin present — at higher substrate concentrations the rate of increase being a little greater. A variety of salts (including di- sodium phosphate) were found to accelerate in different degrees the 272 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 velocity of the hydrolysis of calcium and sodium caseinates ; mono- sodium phosphate had a retarding influence. The special study of the part played by the alkali in the hydrolysis of proteins by tryp- sin (Robertson and Schmidt, 1908), the alkalinity being followed by means of the gas-chain, brings out facts against the view that the OH" ions in a tryptic digest play the part of an accessory catalyzer, though it is suggested that the real catalyzer in these systems may be a hydrolyzable compound of trypsin with the base present. The investigations of Walters (1912a, 1912b) are a continuation of that of Robertson. ANIMAL EXPERIMENTS WITH CASEIN Sandmeyer (1895), testing the possibility of the absorption of the paranuclein from casein, introduced such paranuclein into a dog by means of a probe, and judged of its absorption by phosphorus de- terminations in the urine. If one may draw any inference from such an observation, something more than ^ of the paranuclein was ab- sorbed. The dog received only water for two x)Y three days before the introduction of the phosphorus compound, and the urine showed 0.33-0.37 grams P 2 5 per day; on the paranuclein days 2.864 and 4.296 grams, respectively, of P 2 5 were introduced, and caused 1.34 and 1.84 grams to appear in the urine ; in each case on the day fol- lowing the experiment the urinary phosphorus returned to its pre- vious value (0.33 and 0.30 grams P 2 5 ). Salkowski (1901) fed the iron compound of his so-called para- nucleic acid to rabbits. Absorption occurred, as judged on the evi- dence of finding the iron content of the livers of the rabbits which received this iron compound appreciably above that of those which had a corresponding diet without this substance, and above that of those which received iron in the form of its combination with atmidalbumose (an albumose derived from fibrin), or as fer- ratin. Another study on the absorption of products of the decomposi- tion of casein is that of E. Voit and Zisterer (1909-10) on dogs. They compared the nitrogen-sparing value of undigested casein with that of the products of pancreatin digestion, and those of acid hy- drolysis of casein. ,The conclusion is that the physiological value of protein bodies is unfavorably influenced by a deep cleavage, and that, in general, protein bodies do not undergo complete cleavage in the digestive tract, but that certain complexes are absorbed un- changed. No observations were made with regard to the phospho- rus of the casein or its decomposition products. These conclusions are almost identical with those of Abder- halden and Rona (1904, 1905) from their observations as to main- taining life in mice and meeting the nitrogen requirement of a dog. PHOSPHORUS METABOLISM 273 With regard to the mice, the report is that the products of pancre- atin digestion of casein serve about as well as pure casein, while those from the combined digestion by pepsin and pancreatin (fur- ther split) are of less value. Mice fed with the latter, however, live longer than starving mice, while those fed with the products of acid hydrolysis do not. The results with the dog were of like import. In later work, however, Abderhalden (1912) showed that dogs are able to maintain nitrogen equilibrium and retention on products of either acid or enzymatic splitting of proteins, or on a mixture of recognized amino-acids. Marcuse (1896, 1897) carried out nitrogen and phosphorus bal- ance experiments with dogs on a mixed diet in which, in some cases, the protein given was in the form of casein, and in some cases in the form of flesh. In either case a salt mixture including phos- phates was given with the other food, and the conditions were gen- erally too complex for definite inferences ; but, on the whole, both nitrogen and phosphorus retention were greater for the casein peri- ods than for the flesh periods. Schreiber and Waldvogel (1897) report 15 cases of various diseases in which "Sanose," a protein preparation containing 80 per- cent casein and 20 percent albumose, was taken with the food or used in its preparation. It was found useful in cases where meat was either a distasteful or an undesirable article of diet. Urine an- alyses are submitted. Knoepfelmacher (1898a, 1898b, 1899, 1900) studied the use of the casein of milk by infants and older children by a comparison of the nitrogen and organic phosphorus of their feces with the casein nitrogen and phosphorus ingested. The methods used in the earli- est work, comparing the use of mother's milk and cow's milk, he says later were faulty. The following table from Knoepf elmacher's latest work shows no important percentage differences in the diges- tion of either the nitrogen or phosphorus of casein by infants and older convalescent children. NITROGEN AND PHOSPHORUS OUTGO IN THE FECES FROM A DIET OF COW'S MILK— INFANTS AND OLDER CHILDREN Age of child Length of ex- peri- ment Days Intake Milk Casein Grams Casein N Grams Casein P Grams Outgo in feces N Grams Total P Grams Organic P Grams 11 yrs . . . 8 yrs . . . 7 yrs... 0i mos 5 mos. . 6 mos. . 8 mos. . 13,250 10,681 8,815 4,770 3,498 4.400 296.19 341.01 277.64 112.21 79.56 60.52 73.91 46.498 53.538 43.5913 17.6169 12.49 9.683 11.825 2.517 2.898 2.3599 0.9537 0.6762 0.514 0.628 3.6088 3.54 1.723 1.238 0.928 0.664 0.6513 8.33 6.68 3.645 1.149 0.914 0.478 0.856 0.1225 0.12 0.1229 0.0526 0.0237 0.0264 0.0343 274 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Prausnitz and his associates (Micko, Miiller, Poda and Praus- nitz, 1900; Poda and Prausnitz, 1900; Micko, 1900; Miiller, Paul, 1900) made similar feces studies with older people. In the intro- ductory article the whole work is summarized, with a discussion of the general conclusion that the feces are made up in the main of residues from the digestive juices, rather than of food residues, the amount of such juices being modified by the kind of food taken. Poda and Prausnitz concluded that casein, in the form of the prep- aration called "Plasmon," was utilized as well by healthy men as the flesh with which it was compared. Micko looked for paranuclein and for nuclein in the feces. Both were found in the feces from both these kinds of diet, and from ordinary mixed diet ; of each the plasmon feces showed less than the flesh feces. Paul Miiller con- cluded that a diet of cow's milk leaves, with neither infants nor adults, a phosphorus-rich casein residue in the feces. Cronheim and Erich Miiller (1900, 1902) compared the useful- ness, to children, of lecithin and casein, by feeding milk-powder with and without added egg-yolk. The periods were very short ; the re- sults were variable, and the observations are not very significant. In these, and in similar feeding experiments on dogs and on guinea pigs, there was thought to be somewhat better storage of nitrogen from lecithin-containing food than from casein. Comparisons of Raw and Sterilized Milk. Jemma (1899) re- ports from his comparison of the action of enzymes on sterilized and unsterilized milk that pepsin-hydrochloric acid digests unsterilized milk more quickly than sterilized, while both pancreatin and rennin digest the sterilized more quickly. The ad- dition of rennin, pepsin and hydrochloric acid together caused dur- ing the first four hours a greater digestion of sterilized milk, but by longer-continued digestion the unsterilized milk showed a greater peptone mass. By the successive action of pepsin-hydrochloric acid for 15 minutes, then rennin, then pancreatin and bile for 4 hours, the sterilized milk was more completely digested. Cronheim and Erich Miiller (1903, 1908) made raw and ster- ilized milk tests with children, — both healthy children and children having rachitis. With the healthy children no differences in favor of either milk could be established, nor were there marked differen- ces in the rachitic children. Other feeding and metabolism experiments involving the use of casein are those of Gumpert (1905), L. Jacob (1906), Lipschiitz (1910a, 1911b) and Osborne and Mendel (1911a, 1911b and others). Summary. No satisfactory theory as to the production of casein in the milk glands can be offered from the evidence at hand, nor even as to whether the phosphorus for the same is obtained di- rectly from gland nucleoprotein or from the blood. PHOSPHORUS METABOLISM 275 Artificial digestion tests made with juices or enzymes from the stomach show that casein is but slowly acted on by such enzymes, and that the early stages of the process tend to leave the main part of the phosphorus in the form of a difficultly soluble paranuclein, which may, however, in time, be nearly or quite entirely dissolved ; and that the phosphorus-free products formed are always more readily further cleaved than are those containing phosphorus, so that the undissolved residue becomes constantly richer in phospho- rus. It is shown also that the phosphorus which goes into solution during these processes is partly inorganic and partly organic in form; and that the results, and the ease and rapidity of the action of pepsin on the paranuclein are variable for the casein ob- tained from different species of mammalia, the completeness and speed of digestion being greater for the casein of woman's, ass's and mare's milk than for that of cow's, goat's and buffalo's milk. The presence of carbohydrate retards peptic digestion. The ftr- ment-protein reaction which brings about cleavage, may, under fav- oring conditions, be reversed. With regard to the enzymes of the intestine, tests in vitro indi- cate that activated trypsin may cause a completion of the digestion of casein as to both its nitrogen and its phosphorus portions ; that the phosphorus thus passing into solution (very nearly all of that of casein) is more largely organic than inorganic ; that the portion of the phosphorus converted into inorganic phosphoric acid increases with the duration of digestion and with the amount of ferment present; that cleavage proceeds further and more easily under the influence of both pepsin and trypsin than of either alone ; and that bile assists trypsin digestion. Experiments on living subjects seem to show an efficient ab- sorption and use of the constituents of casein by both animals and human beings, older and younger, perhaps even better than the use made of flesh. Undigested casein, however, has been found to serve the body better than do the products of complete pancreatin or acid cleavage (dogs and mice) , indicating that within the organism there is some absorption of casein in a state of incomplete digestive cleav- age. Dogs have, however, been maintained in nitrogen equilibrium on the products of complete cleavage. Evidence has been produced that of ingested milk large portions (four-sevenths of the whole) pass the pylorus unaltered, and later the rest of the casein passes in larger or smaller clots, not pepton- ized. 276 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 While pepsin-hydrochloric acid digests unsterilized milk more quickly than sterilized, yet under the influence of the succession of enzymes present in the digestive tract, the sterilized is said to be more completely digested. So far as we learn, experiments with children have not demonstrated any practical advantage as due to* either condition. THE METABOLISM OF THE COMPOUNDS OF GLYCERO- PHOSPHORIC ACID THE FUNCTION OF LECITHIN IN INTERMEDIARY METABOLISM The method of usefulness of lecithin in its more intimate rela- tions with the life of the animal has been a subject of much spec* ulation, but little productive study. A considerable number of theories have been advanced, which we mention in brief. Bergell (1898b) concluded that in animal as in vegetable cells organically combined phosphorus stands in a causal relation to the function of cell cleavage. Lecithin and nuclein phosphorus, as uni- versal cell constituents, may at least be regarded as necessary to cell cleavage. Loew (1899) suggested that the chief function of lecithin is to serve for respiration, it being the form into which fat must be changed to become combustible in the protoplasm. According to this theory, by the transformation of fatty matter into lecithin the higher fatty acids are offered to the protoplasm in a soluble form, and, after being oxidized, other molecules of fatty acids may enter into the place of the former; thus the same molecules of glycero- phosphoric acid serving repeatedly as vehicles for oxidation of mol- ecules of fatty acids. The idea of the service of lecithin as a storage product has often been suggested. Hanai (1897) considers it in this light. He observed that old tea leaves lose their lecithin in the spring, while the amount of it increases gradually in the young leaves. In the bark of Prunus cerasus the lecithin also decreases as the flower buds form and open. The metabolism of lecithin during the incubation of eggs, as shown by the work of Maxwell (1893), Mesernitzy (1907), Carpiaux (1908), and Plimmer and Scott (1909) (see Incubation) also sug- gests a storage function; an accumulation in anticipation of rapid cell multiplication. Springer (1902) says that lecithins are present most abundant- ly where growth is most active, and that they diminish when the seat of growth shifts to some other place. PHOSPHORUS METABOLISM 277 One instance in harmony with this theory is the comparative richness of the marrow of young bones in lecithin. Its abundance in gland cells, eggs, spermatozoa and pollen (Stoklasa, 1896b) also suggests a connection with intense metabolism, but surely no such idea is exemplified by the high lecithin content of nerve tissue. Burow (1900) finds that the lecithin of the milk of cattle, dogs, and human beings varies directly as the total protein of the milk, and also as the weight of the brain of sucklings of the different species, in relation to their body weight; that is, the greater the relative brain weight, as compared with body weight, the higher is the lecithin content of the milk, reckoned in percent of the protein. The figures are as follows: Species Lecithin of milk Percent Protein of milk Percent Lecithin: Protein X:100 Brain: Body weight 0.054 0.170 0.580 3.87 8.05 1.90 1.40 2.11 3.05 1-370 Dogr 1-30 1:7 Glikin (1908a) compared the lecithin content of the bodies of several species of young birds and mammals, and found that it varied directly as the helplessness at birth. He suggests an essen- tial connection. Parrozzani (1909) believes that lecithin represents the final stage by which non-nitrogenous organic substances acquire the pow- er of combining with nitrogen, especially with amino acids, for the synthesis of albuminoid substances. Hammarsten (1905b) suggests a possible connection between the lecithin content of the bile and the amount of fat to be digested, and cites the very high lecithin content of the bile of the polar bear. Daniel-Brunet and Rolland (1911a) suggest the origin of a part of the biliary lecithin by the observation, which we have not seen substantiated, that the lecithin of the bile of the bull is decreased by castration. As results of his studies of the phosphatids W. Koch has made a number of suggestions as to the function of lecithin. In brief they are as follows: In an early article (1902a) he advanced the idea that the emulsion formed by the lecithins may be the substratum in which the reactions of the cell take place. The precipitation of this emulsion by divalent cations is prevented by univalent and tri- valent cations, as far as investigated, and this observation may fur- nish an explanation of the changes brought about by electrolytes in the living cell. 278 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Somewhat later (1903) Koch suggests that the lecithins of the cell may have two values: (1) They, together with proteins in colloidal solution, may furnish the basis for bringing about the nec- essary viscosity, by the facility with which they are influenced by sodium and calcium ions. (2) They share in the metabolism of the cell, and especially by their unsaturated fatty acids and methyl groups bound to nitrogen, in causing reactions as yet not under- stood. Koch (1905b) noted the constancy of the creatinin excretion by men and a dog, and suggested that the lecithins of the food furnish the methyl groups of the creatinin excreted. Two years later Koch (1907a) also made a physico-chemical study (colloid precipitation and measurement of viscosity) of leci- thin and cephalin which may have a bearing on their significance in the red blood cells. Writing on the significance of phosphatids for the living cell, Koch (1909b) says, — "Reactions may be carried out with colloidal solutions of phosphatids which are very much like those observed with physiological material, both qualitatively and quantitatively. Both carbonic acid and ammonia influence lecithin emulsion even in small concentration, as was to be expected from the H + and OH- con- centration, respectively. Therefore the phosphatides play an im- portant part both in the morphological and the chemical differentia- tion of cells, in that they are able to form precipitation membranes, which one may think of as distributed throughout the protoplasm." Koch et al. (1910) * made a series of pharmacological studies on the phosphatids, especially with reference to their participation in the selection and transmission of substances through the mem- branes of the cell. Among their conclusions are the following : "The greater concentration of potassium in the cells of a tissue as compared to the surrounding lymph spaces or serum can be in part accounted for by the specific affinity for this element of some of the phosphatides, especially kephalin." "There is no evidence that anaesthetics or hypnotics produce changes in the state of aggregation of lecithin or kephalin, which are sufficiently consistent to account for such a general phenomenon as narcosis. There is some evidence, however, that chloroform, as distinguished from pure ether, has the power to form a combination with lecithin, a phenomenon which may be brought into relation with its slow elimination and consequent tendency to produce de- layed poisoning." * W. Koch, 1910b; Koch and Pike, 1910; Koch and McLean, 1910; Koch and Williams, 1910; Koch and Mostrom, 1910. ' PHOSPHORUS METABOLISM 279 Considering the possible participation of the phosphatids, through specific chemical affinities, in the translocation of metab- olites, these authors reached conclusions a part of which are as fol- lows: "The changes in state of aggregation of lecithin produced by sodium chloride are the result of the independent action of the so- dium and chlorine ions, whose effects are in opposite directions. Be- low the concentration of a physiological salt solution (0.12 molecu- lar) the action of the chlorine ion, which decreases the state of ag- gregation of the lecithin, predominates. Above the concentration of a physiological salt solution, the action of the sodium ion, which tends to increase the state of aggregation of lecithin, comes more and more into prominence. "It has been suggested that, when the phenomenon of chloride retention occurs, some change has taken place in the state of aggre- gation of the cell lipoids which allows this action of the chlorine ion to predominate to a still greater extent. "Ammonia and bile salts possess the power of altering the physical state of aggregation of lecithin to such an extent as to per- mit of the conclusion that they can be of functional significance in altering the permeability of cell membranes. "The ability of the tissue metabolites to combine with lecithin, as measured by the changes in the physical state of aggregation produced by their presence, is in some cases considerable, in other cases entirely lacking. Thus hypoxanthin, creatin, creatinin, ad- renalin and ammonia salts show evidence of combination. Inosite is doubtful, and urea is negative. "The amino acids show varying powers of combination. The dicarboxy-acids, like acids in general, tend to increase the state of aggregation of lecithin. Studying the function of the brain phosphatids in the action of strychnin they conclude : "The central nervous system, especially the cord, by its high phosphatide content, is enabled to pick the strychnin out of the blood stream on account of the affinity of the lecithin and kephalin for the strychnin as compared to serum albumin. The strychnin probably enters into the combination with lecithin through some re- lation to its unsaturated fatty acid group (oleic acid). "Strychnin interferes in such a way with the normal relation of these unsaturated fatty acid groups to oxygen as to bring about a more rapid transfer to any easily oxidizable substance." 280 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Loewe (1912a, 1912b, 1912c, 1912d) made a series of physico- chemical studies of lipoids in relation to organic solvents and color- ing matters, the object being to determine the nature of lipoid ac- tion, biologically and pharmacologically. The following quotation expresses his conclusion : "The hypothesis of the solvent function of lipoids and the asso- ciated theory of the taking up of substances by the cells and of nar- cosis is not supported by the examples studied. The study of the 'taking up' by lipoids of the substances considered in this theory shows that the process does not follow the Henry-Nernst absorption laws, but is an adsorption. "The taking up by lipoids of basic coloring matters, organic solvents and organic substances dissolved in water is not a linear function of the concentration, but is determined by an adsorption isotherm. Therefore it is not to be expected that the lipoid com- ponents of the cell membrane, so far as they are not very freely movable on the cell contents, will serve to facilitate the taking up into the interior, but rather their action will be hysteresis." Reicher (1911) believes that the physiologic oxidation of fat, in general, is made possible only through combination in lecithin, an idea in harmony with that of Loew (1899). The feeding of leci- thin-free fats, and the perfusion of a surviving liver with blood and triolein increased the lecithin of the blood. Kovaliova (1912) compared lecithin and other phosphorus com- pounds with reference to their effects on the power of oxidation in rabbits. The phosphorus compounds were administered by subcu- taneous injection in oil suspension. Two hours later the rabbits were injected with 2 gm. benzene, and placed in an apparatus where the respiration coefficient and power of oxidation were determined. Five-hour and 24-hour experiments were performed. Lecithin caused an increase in the elimination of phenol, and in- crease in the respiration coefficient. Sodium nucleate in small doses caused an elevation, but in large doses a depression, of respira- tion coefficient and degree of oxidation. Sodium glycerophosphate caused a depression of the respiration coefficient, but a greater in- crease of the degree of oxidation than that of the two preceding sub- stances. Phytin in aqueous suspension caused an increase in the degree of oxidation, but scarcely affected the respiration coefficient. In Kovaliova's judgment lecithin exerted the more uniform influ- ence on the elevation of the respiration coefficient and degree of oxidation, while the sodium nucleate, in large doses, exerted the most uniform action in lowering them. PHOSPHORUS METABOLISM 281 Stuber (1913a, b) determined the effect of lecithin and choles- terol on the phagocytic index of leucocytes. Cholesterol diminishes the index. Lecithin does not affect it, but prevents the action of cholesterol. The oleic and palmitic acid esters of cholesterol also check phagocytosis, but lecithin does not prevent this action. Maslow (1913) studied the effects of lecithin and other phos- phorus compounds on the functions of the intracellular ferments of dogs. Several litters of young dogs were the subjects of these ex- periments. One was used as a control, being killed at the begin- ning of the experiment; and a second control was fed the usual mixed meat diet, and was then killed before the close of the ex- periment. The remaining animals were divided into four groups. One received a low-phosphorus broth containing pro- tein, carbohydrate and fat in sufficient quantity ; another group re- ceived the same broth plus a phosphate; a third, the same plus glycerophosphate; and a fourth, lecithin. The second group re- ceived either whole milk, or a milk preparation in which the casein was replaced by albumin. Under normal conditions of development there was in all organs a normal development of fermentative energy. On the phosphorus- poor ration the animal declined in weight, and died. The phos- phorus content of the organs decreased, the decrease being mostly in the inorganic phosphorus. Of the organic phosphorus only the lipoid phosphorus decreased. The ferment function of the organs was markedly disturbed ; the catalase, lipase, amylase, diastase, and nuclease were depressed in amount or checked in their development. The addition of inorganic and glycerophosphate phosphorus to the diet were without avail. The organs showed phosphorus im- poverishment and the intracellular enzymes were not favorably in- fluenced. Lecithin, however, increased the phosphorus content of the organs, especially their organic phosphorus content, and stimu- lated the development of the ferments. The author concludes that abundance of assimilable phosphorus in the food runs parallel with abundant ferment formation in the or- ganism, and that a power of the animal to synthesize organic phos- phorus compounds is not probable. For therapy the conclusion was drawn that the use of phosphates and glycerophosphates should be limited, and that lecithin is to be preferred, if increase of the phosphorus content of the organism or activating of the ferments is desired. Mayer and Schaeffer (1913) find that the lipoid content of tis- sues tends strongly to remain characteristic of the species and tis- sue, neither fasting nor overfeeding producing characteristic change, this proportion therefore, appearing to be fundamental and 282 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 permanent. The ratio of fatty acids to lipoid phosphorus is re- markably constant in certain types of cells in different animals, as for instance in kidney and red blood cells. A relation is shown to exist between the capacity of tissues to imbibe water, and the ratio of lipoid phosphorus to cholesterin. In all the species examined the various organs compared as to lipoid phosphorus content (on the fresh basis) in the same order; their content of lipoids apparently being related to physiologic activity, in ways yet to be explained. Lecithin and other phosphatids of the blood corpuscles and of the tissue cells also stand in important relations to the action of poisons of various sorts and origin, an important subject which has not been included in this investigation. THE INFLUENCE OF LECITHIN IN DIGESTION There is prevalent an idea that lecithin is of functional value in the alimentary digestion processes, but the experimental evidence on the subject is not all in support of the theory. Thus Kalabou- koff and Terroine (1907a, 1907b, 1907c) tested lecithin in relation to the cleavage of monobutyrin and olive oil ; also as to its influence in activating pancreatic juice to such cleavage, with and without the presence of bile salts. Such activation by bile salts was quite evi- dent, whether with or without lecithin ; but lecithin showed no such activating power in the case of monobutyrin, and only a slight influence, and at relatively high concentration in the case of olive oil. This conclusion is in harmony with those of 0. vonFiirth and Jul. Schulz. In the second paper were reported similar tests of the activat- ing power of lecithin, and of bile salts with gastric and intestinal juices. "1. The lipase of the glycerin extract of gastric mucous is not at all changed by the addition of lecithin ; it is notably retarded by bile salts. 2. The intestinal lipase is not at all modified by the addition of lecithin; it is activated by bile salts." The third paper had to do with the action of ovo-lecithin on amylase, trypsin and rennet. "Addition of lecithin does not modify saccharification of starch, the digestion of casein and coagulated al- bumin, nor the coagulation of milk by pancreatic juice; the addition of bile salts activates plainly the coagulation of milk by kinased pan- creatic juice." In a later paper (Kalaboukoff and Terroine, 1909) cleavage of an emulsion of pure lecithin (prepared from egg yolk) by pancre- atic juice at 40° was tested by the acidity developed. No increase of acidity was detected when boiled pancreatic juice was used, and but slight increase when unboiled juice was used; this was a little further increased by the presence of bile salts; but even after 66 hours the acidity was but slight (not more than equivalent to PHOSPHORUS METABOLISM 283 2.8 c.c. of N/20 NaOH for 10 c.c. of the mixture). These authors think that the results found by Stassano and Billon (1903a, 1903b) should be referred to cleavage of fatty substances obtained in the course of preparation. Regarding some other phases of this matter these authors say : "The work of Slowtzoff and of Stassano and Billon has shown that lecithin is absorbed and appears in lymph. Bayer has found, and so do we, that if a solution of bile salts be added to a milky lecithin emulsion a clear liquid results. Ultramicroscopically, lecithin emul- sions have a colloidal appearance, with an infinite number of grains, and these almost disappear after the addition of a solution of bile salts." As a result of recent investigations, Terroine (1911) repeats his conclusions : (1) Lecithin does not increase the rate of hydroly- sis of monobutyrin by pancreatic juice, and increases only slightly in more concentrated solution the rate of hydrolysis of oils. (2) It increases the lipolytic action of neither mucous membrane of stomach (in glycerol) nor intestinal lipase. (3) It has no action on the rate of hydrolysis of starch, the digestion of milk casein or coagulated albumin, or the coagulation of milk by pancreatic juice. On the other side of this question we have the work of Moore and Parker, Hewlett, Loevenhart and Souder, Kiittner and Usuki as mentioned in brief below. Moore and Parker (1901) state that the presence of lecithin greatly increases the solvent power of bile toward fatty acids and soaps. The bile salts serve to keep lecithin and cholesterin in solu- tion, and hence aid in their emulsification by the liver. The com- bination of bile salts and lecithin accomplishes the solution of the fatty acids and soaps. Numerical results from this study are given below. SOLUBILITY OF FATS AND SOAPS AS AFFECTED BY BILE SALTS AND LECITHIN Distilled water Bile salts, 5 percent Bile salts, 5 percent +lecithin, 1 percent Practically insoluble Less than t .l* soluble 0.5* soluble 0.5 0.1 Less than 0. 1% 0.7* soluble 4. 0.6 0.2 2.2396 ) apparently colloid sol. f 5.0* soluble 0.2 0.1 (Less than 0.1*) (Much less than 0.1%) Not tried 7.6* 1.0 0.2 0.2 Less than 0. 1* Not tried 11.6% 2.4 0.7 1.4 0.9 4 (Less than 0.1*) (Much less than # . 1*) Practically insoluble Absolutely insoluble 3.2 0.2 Less than 0.1* 7.0* About 0.1* 8.2 1.2 1.0 Not more than 0. 15% Free fatty acids mixed., oleic palmitic stearic Sodium salts mixed oleate , palmitate stearate. Calcium salts, oleate .... palmitate stearate Magnesium salts, oleate palmitate stearate Lecithin Cholesterin 284 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Reiss (1904) determined that lecithin, with chloroform, is able to take up the ferments rennet and trypsin, as neither constituent alone can, and as no other fat tried can with chloroform. It is thought that this may have some significance with ref- erence to the role of lecithin in the body. This is likened to the activating power of lecithin toward the haemolysis of cobra poison. Hewlett (1905) studied the action of lecithin in bile on fat di- gestion by pancreatic juice of a dog. Bile increased the action of pure pancreatic juice on triacetin. (Fat was not used, to eliminate the factor of influence through emulsification. Triacetin is a sol- uble glyceride.) Bile served to increase the action of pure pancre- atic juice on this ester, especially in the first hour. Boiling the bile does not destroy this property, and furthermore, it was found not to reside in cholesterin or the pigments, and was found not to depend on variations in reaction, nor on variation in the amount of calcium salts present. Precisely the same effects of accelera- tion were produced, however, by the addition of lecithin to the pan- creatic juice. Hewlett suggests that this influence is of the nature of a "zymoexcitor." Loevenhart and Souder (1906-7) investigated the relations of lecithin to the digestion of the higher fats by quantitative determin- ations of the activity of pancreatic juice on olive oil, ethyl butyrate, diacetin, triacetin, ethyl acetate and ethyl propionate in the pres- ence and absence of bile salts, and bile. Bile salts, lecithin and bile greatly accelerate the action of pan- creatic juice on all the esters studied. The effect of these accel- erators differs with the particular ester, and differences of experi- mental conditions greatly alter the degree of acceleration, and the relative activity of the bile salts and lecithin. These accelerating capacities are considered to depend to a certain extent on their sol- vent action, and also on their action on the enzyme in some other way. Kiittner (1907) determined, in vitro, the effect of lecithin on the peptic digestion of casein, and on pancreatic digestion of monobutyl glycerin. Lecithin in certain proportions accelerates the enzyme action of gastric or pancreatic juice ; in other greater proportions it retards it. Curves were plotted exhibiting this action. The cause of this action was not determined. Long and Gephart (1908b) report that bile salts, as ordinarily obtained, carry down a phosphorus complex, which, by Hammarsten and others, is regarded as lecithin, and also that bile salts are capa- ble of dissolving and holding 80 percent of their weight of egg lecithin. PHOSPHORUS METABOLISM 285 Usuki (1910) studied the influence of lecithin on fat digestion in the stomach, and in the intestine, by the feeding of dogs; and after certain lengths of time the dogs were killed, the different portions of the alimentary tract separated by tying, and the con- tents removed and analyzed. The results seem unmistakable. The author's conclusions are as below. 1. According to these experiments the digestion of fat took place more slowly after feeding of milk than after feeding of milk- lecithin or milk-yolk mixtures which left the stomach l 1 /^ to 2 hours sooner than the milk alone. While of the pure milk, after six hours, only half had left the stomach, the same result was obtained with the mixtures in four hours. 2. After taking milk more soaps are found in the contents of the small intestine than after the taking of milk-lecithin mixture. On the other hand, after taking milk-yolk mixture, more soaps were found in the feces. This contradictory relation between the con- tents of the small intestine and the feces is probably to be ex- plained by absorption in the large intestine. 3. Splitting of lecithin in the stomach takes place more quick- ly than splitting of neutral fat. ' 4. Lecithin acts favorably on the saponification of the neutral fat. This is to be explained by the fact that the digestion of fat is accelerated by the presence of lecithin. 5. In spite of the lower lecithin content of the milk-yolk mix- ture the digestion of fat after taking said mixture was just as good as after the milk-lecithin mixture. The egg-yolk has this advan- tage because of the fineness of the emulsion and the chemical nature of fat contained in it. 6. It has been discovered that the passing of the milk fat from the stomach did not begin until the percentage content of fat- ty acids had about reached the maximum (2 hours after feeding). This percent of fatty acids was maintained almost unchanged dur- ing the further course of the digestion. Only during the last stages of the digestion does the percent of fatty acids rise again. 7. No lecithin was found in the intestine. It must, therefore, have been split either in the stomach or immediately after entrance into the intestine. Lapidus (1910) announced results of preliminary experiments on the effect of lecithin on animal diastases. Lecithin in certain con- centrations affected unfavorably the activity of salivary, pancreatic, serum and intestinal diastases in water and glycerin extracts, but activated serum diastase in ether solution. 286 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Bickel (1911) suggests an indirect influence of the lecithin, leading to the better digestion and use of phosphorus from other parts of the diet. Minami (1912) found that lipoids are superfluous for diastatic action by saliva. Summary. As a result of these investigations we conclude that under certain experimental conditions, at least, lecithin may assist not only in the solution of fats and soaps, but also may accel- erate the pancreatic cleavage of fats, and even the peptic digestion of casein. These actions vary much with the particular nutrient compounds involved, and with the experimental conditions, especi- ally as regards concentration of the lecithin solution. At certain concentrations there may be acceleration of digestion, and at others a retarding action. In the actual digestion of food within the alimentary tract leci- thin seems to accelerate the digestion of fat, and also the passage of food from the stomach. DIGESTION OF LECITHIN AND GLYCEROPHOSPHATES That the digestion of lecithin may involve its cleavage has been observed by a considerable number of workers, among the earlier ones being Bokay (1877), who concluded from artificial digestion experiments and from feeding experiments with dogs, that lecithin is split in digestion by the fat-splitting ferment of the pancreas, or by putrefactive ferments in the intestine; and Hasebroek (1888), who concluded, from fermentation experiments, that the phospho- rus from lecithin must be absorbed as glycerophosphoric acid ; also Nesbitt (1899a, 1899b), who demonstrated the presence in the in- testine of the dog of choline, neurine and an unidentified ptomaine after the ingestion of egg yolk and the occlusion of the small in- testine at its lower end. See also Gaston (1902). Among the later workers to demonstrate the digestive cleavage of lecithin are Pvogozinski (1910), Usuki (1910), and Yoshimoto (1910). The cleavage products, as a rule, are choline, fatty acids and glycerophosphoric acid, but, according to J. and W. Cronheim (1911), the decomposition may go only to distearyl-phosphoric acid. The phosphorus of lecithin, then, is absorbed, in the main, in the same form as though ingested as glycerophosphate. That a portion of the lecithin is absorbed without cleavage, however, has been demon- strated by Slowtzoff, and others, as will be mentioned at greater length below. PHOSPHORUS METABOLISM 287 Marfori (1905) studied the elimination of glycerophosphoric acid ingested by dogs and men. In men it was found, if adminis- tered in suitable amounts, to be absorbed from the alimentary canal. In dogs it is largely, though not completely, absorbed, a part being decomposed in the alimentary tract. At all events it is an easily assimilable compound. Paul Mayer (1905) finds that the lipase of intestinal juice splits lecithin (racemic) asymmetrically; the dextro-lecithin is then de- composed, and the laevo-lecithin is not. From artificial digestion tests of lecithin with lipase (1906b), and from a study of the race- mic and the laevo- forms as to optical activity, structure and cleav- age products, the author concludes as reported above. The break- down of the dextro-lecithin results in fatty acids and dextro-glycero- phosphoric acid. It is suggested that the asymmetric cleavage, and difference in digestibility of the fractions, may have practical significance ; since the various lecithin products used medicinally do differ in optical activity, that is, are more or less strongly racemized, it cannot be thought surprising that the results of lecithin therapy are very unequal, considering the different reactions of the enzymes on dextro- and laevo-lecithin. Kutscher and Lohmann (1903) found that the ferment of the pancreas caused lecithin to break down into glycerophosphoric acid, fatty acids and neurine. Very little lecithin was decomposed by the gastric enzyme. Stassano and Billon (1903a) concluded from microscopic ex- amination that pancreatic juice does not cleave lecithin, and after feeding pure lecithin and egg yolk (1903b), and then examining the chyle with a polarizing microscope, they concluded that leci- thin, taken pure, — not in albuminoid combination — escapes the digestive juices; and enters the circulation by way of the chyle unaltered. Coriat (1904a) found that neither trypsin nor pepsin can split lecithin, but that lipase is able to do so. Pepsin and trypsin seem even to inhibit the normal enzymatic autolysis of brain tissue through which choline is liberated. Coriat finds that this autolytic enzyme acts only in neutral or slightly alkaline media, that the pro- duction of choline is greater in the latter than in the former, that the enzyme is inactive in slightly acid media, and that this enzyme can be destroyed by heat. Slowtzoff (1906a) finds that lecithin given per os acts the same as lecithin given hypodermically, but that more must be given to produce the same result. In eight experiments dogs were given egg yolk or lecithin, and the lymph was collected for analysis after 4-5 hours ; also in four 288 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 experiments the intestinal contents of a dog, after it had ingested lecithin, were analyzed for lecithin and its splitting products ; fur- ther, digestion experiments were conducted with lecithalbumin, and with digestive ferments in vitro. Slowtzoff determined that lecithin in appreciable quantities reaches the blood unchanged, as well as in a saponified condition, by- way of the chyle, but not by the portal vein ; that lecithin may be saponified by the pancreatic juice, or even by steapsin; that the cleavage of lecithin to glycerophosphoric acid and choline occurs only in the duodenum ; that pepsin-hydrochloric acid digestion does not affect lecithin, but that lecithalbumins are digested, the acid- albumin first formed being combined with the lecithin, in which form lecithin may perhaps be absorbed; the albumose solution resulting from pepsin digestion being free from lecithin. This lecith- acid albumin, when introduced per rectum, disappears. Long (1906b) finds ether-soluble phosphorus in human feces equivalent to 1-5.5 gm. of lecithin per day. He quotes Deucher as finding in the feces of a man with closed pancreatic duct as high as 8 grams of lecithin per day. Long and Johnson (1906) suggest that feces lecithins may be but remotely related to simple distearyl lecithin, and also suggest that the feces lecithin may come in part each from food, intestinal epithelium, bile residues and bacteria. Schumoff-Simanowski and Sieber (1906) state that lecithin (from egg yolk, and Kahlbaum's) is split by steapsin from the pan- creas and from the stomach, more energetically by the former. Plant ferments, in particular that from the seeds of castor-oil plant, break it up also, and in the same way, by splitting off fatty acids. The lipase of blood of different animals does not cleave it. Hamill (1906-7) determined by collection and analysis of chyle from a fistula in the thigh that lecithin administered by the mouth produced a rise in the ether-soluble phosphorus of the chyle. Franchini (1907,1908a) sought to determine the fate of that portion of ingested lecithin which is absorbed without cleavage. By analysis of the bodies of rabbits after the feeding of lecithin he found a distinct rise of the lecithin content of both liver and muscle, but not of brain, following the ingestion of lecithin either during feeding or fast. There was also perhaps a slight increase of leci- thin phosphorus in the excreta. No choline was found in the urine, but formic acid from the cleavage and oxidation of choline is said to have been present. Marfori (1908b) in discussing the assimilation of organic phos- phorus compounds says: PHOSPHORUS METABOLISM 289 "Phosphorus in the form of synthetic glycerophosphoric acid, taken into the body by the mouth, is easily absorbed and assimilated. Lecithin administered either per os or subcutaneously is taken up at once by the tissues for their growth." Clementi (1910) finds that pancreatic juice hydrolyzes the fatty acid radicals in lecithin; an action, however, which is not equally noticeable with all pancreatic juices, but which varies, in a more or less pronounced way, according- to the capacity of the juices to saponify the ordinary glycerides. Intestinal juice lipase, as well as pancreatic juice lipase, acts on lecithin. Mathison (1910) found synthetic glycerophosphoric acid not de- composed by pepsin, or trypsin, or by fresh pancreatic juice, either with or without enterokinase. He thinks, therefore, that glycero- phosphoric acid is absorbed as such, though he notes the possibility that the natural and the artificial products may behave differently in this matter. Brugsch and Masuda (1911) report that the intestinal juice contains a lecithin-cleaving ferment, and also that extracts of the colon bacillus and Staphylococcus cleave lecithin. Grosser and Husler (1912) determined that the mucous mem- brane of the intestine, and the cells of the kidney, contain a ferment which completely splits glycerophosphate solutions. The lungs al- so contain such a ferment, but apparently in smaller amount. The liver and spleen show its presence but sparingly, while pancreas, muscle, heart muscle and blood are free from such a ferment. The feces contain this enzyme but the urine does not. Sodium glycero- phosphate and the glycerophosphoric acid from natural lecithin were alike decomposed by this enzyme. Ehrmann and Kruspe (1913) determined that the exclusion of bile from the alimentary tract greatly lowers the absorption of leci- thin or causes its elimination in considerable quantity in the feces ; and the exclusion of the pancreatic secretion produces a similar though less pronounced effect. These authors conclude that the total lecithin is not split by the pancreatic juice, but that a portion is absorbed as such. See also Forbes and associates (1914) under Nutr. Val. Org. and Inorg. P. Summary. From the above investigations we conclude that lecithin is absorbed in part without digestive cleavage, in part after separation into choline, fatty acids and glycerophosphoric acid, probably in part combined as lecith-acid albumin, and probably also in part after the splitting of the glycerophosphoric acid to glycerin and inorganic phosphoric acid. 290 OHIO EXPEKIMENT STATION: TECHNICAL BUL. 5 The digestive cleavage of lecithin is accomplished by the fat- splitting enzymes of the digestive tract, mostly in the duodenum. Lecithin, as such, reaches the circulation by way of the chyle, but not by the portal vein. The feces lecithin probably comes in part each from food, in- testinal epithelium, bile residues and bacteria. Lecithin taken by the mouth, or hypodermically, may be assim- ilated at once by the tissues. Glycerophosphates are absorbed in part as such, and in part after cleavage to glycerin and inorganic phosphates. BALANCE EXPERIMENTS WITH LECITHIN AND OTHER COMPOUNDS OF GLYCEROPHOSPHORIC ACID Of the many observations on the nutritive value of compounds of glycerophosphoric acid comparatively few are of the nature of complete balance experiments. Additional evidence of this sort, especially comparing these with other phosphorus compounds, would be of value. That the phosphorus of glycerophosphates is absorbed and re- tained is universally admitted. An experiment demonstrating this point is that of Sanson (1896), who fed calcium glycerophosphate to rabbits. Considerable interest has attached to the function of lecithin in infant nutrition, especially as added to the milk in the form of egg yolk. Cronheim and Miiller (1900) compared egg yolk and milk powder as elements in the diet of a child eleven and a half months old. The ration containing the egg yolk was rich in leci- thin ; the other was poor in lecithin. The calorific value of the lat- ter was a trifle higher than that of the former. Nitrogen and phosphorus retention, and gain in weight all showed the ration con- taining the egg yolk to be the more efficient. In a later series of experiments Cronheim and Miiller (1902) made further comparisons of protein phosphorus and lecithin phosphorus in metabolism experiments with children, dogs and guinea pigs. The periods with children averaged four days in length, which is in- sufficient for mineral balance experiments. The results on infants are inconclusive. Five young dogs were fed on similar rations to those received by the children. Egg yolk was compared with plasmon — a milk al- bumin preparation. These dogs were fed for 3 months, after which they were killed, and the bones subjected to study. The egg yolk had produced better bone development, and the marrow produced PHOSPHORUS METABOLISM 291 by the ration containing this food was yellow and rich in fat, while that from the -bones of the dogs which had received plasmon was red and immature. Egg yolk and plasmon were also compared, using guinea pigs as subjects. The animals receiving the egg yolk grew the more rapidly and had the fatter livers. Cronheim and Muller conclude that egg yolk, apparently through its lecithin content, especially favors nitrogen retention, and is to be recommended for use in the early feeding of the child. Zuntz (1900) also conducted an experiment in infant metabo- lism in which a child, eleven and a half months old, was fed for two days on a milk diet in which 6 percent of the dry matter was replaced by egg yolk, followed by a period of the same length on milk alone. While receiving the egg yolk the child retained 24.2 percent of the nitrogen of the ration; on the milk diet the nitrogen retention was 9.9 percent of the intake ; of the phosphorus of the egg ration 33.3 percent was retained, and of the diet of milk alone 17.4 percent was retained. Lebbin (1901) conducted a 2-day metabolism experiment on a man 28 years old, and weighing 65 kg., using eggs, of which 22 were consumed, as the only food. The intake contained 286.28 gm. dry matter, and the outgo 14.28 gm. The eggs contained 39.22 gm. leci- thin, and the excreta 3.517 gm. Massacin (1902) conducted a series of balance experiments with a man suffering from pulmonary catarrh, on a normal mixed diet with lecithin added in certain periods. The addition of lecithin seems to have increased nitrogen retention. Gilbert and Posternak (1903) conducted a balance experiment on a man, for the purpose of learning the effect of lecithin ingestion on phosphorus metabolism. The results are as stated below. AVERAGE DAILY NITROGEN AND PHOSPHORUS BALANCES WITH A MAN ON DIETS WITH DIFFERENT LECITHIN CONTENTS— Grams Nitrogen Phosphorus (P2O5) Food Urine Feces Balance Food Urine Feces Balance 17.96 18.04 15.717 16.730 2.173 2.187 +0.07 -0.88 2.430 2.745 1.840 2.139 0.603 0.576 -0.013 +0.031 3-day preliminary period. 3-day period; same diet as above plus 15 grm. lecithin (total). Thus it appears that the ingestion of 15 gm. commercial leci- thin in two days (as the authors state, though the length of the peri- od seems to be three days) caused a change of phosphorus balance 292 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 from -0.013 gm. P 2 5 to +0.031 gm. P 2 5 . At the same time the nitrogen balance changed in the opposite direction, that is, from a positive to a negative balance. Buchmann (1904) conducted balance experiments with two human subjects, administering lecithin as egg yolk, and in the pure form. A part of the data are given below. DAILY CALCIUM, MAGNESIUM AND PHOSPHORUS METABOLISM WITH ADULT HUMAN BEINGS AS AFFECTED BY INGESTION OF LECITHIN— Grams Subject Length P2O5 P2O5, CaO CaO, MgO MgO, and of period intake percent intake percent intake percent Diet period in days Grams retained Grams retained Grams retained Al 10 6.13 8.56 5.72 21.88 0.43 5.34 Without lecithin. A2 7 7.06 21.83 3.30 7.26 0.30 15.08 With egg yolk. A3 7 7.04 6.32 3.28 2.94 0.30 15.07 Egg yolk and lecithin. Bl 5 5.65 neg. 5.55 neg-. 0.42 neg-. Without lecithin. B2 5 7.66 20.37 4.27 31.71 0.33 61.25 With egg yolk. B3 5 6.10 7.21 5.19 9.69 0.48 54.36 Without lecithin. B4 4 4.35 0.69 2.72 7.74 0.44 43.27 With edestin. Most of this evidence tends to sustain the idea that lecithin increases phosphorus retention, though the result in period A3 is negative. Voltz (1905) showed in a series of balance experiments on nitro- gen metabolism with a dog that in a ration of meat, rice, albumin and lard, the replacement of one-third of the albumin nitrogen by the same amount of lecithin nitrogen increased the nitrogen reten- tion from 0.020 to 0.140 gm. per day. Gumpert (1905) conducted balance experiments with sanato- gen, a preparation of casein and sodium glycerophosphate, with results as on the following page. The subject in this investigation was an adult man. The change in period 4 from meat to sanatogen, and in period 5 from sanatogen back to meat showed that it was possible with this preparation to decrease the loss of phosphorus and calcium existing during the meat periods. Experiment 2 reports an attempt to learn the effects of over- feeding with sanatogen. By its use it was found possible to bring about a marked storage of both nitrogen and phosphorus, which fact gains added significance from the coincident loss in calcium. The plan of the experiment is such, however, that we are unable to judge of the participation of the glycerophosphoric acid of the san- atogen in the production of the results noted. The food in periods 2, 3 and 4 of Exp. 2 was essentially the same as in the meat periods of Exp. 1 except for the addition of 60 gm. of sanatogen. PHOSPHORUS METABOLISM 293 As bearing on matters of general metabolism it is of interest that phosphorus may be stored in a grown man, in considerable quantity, for a number of days at least, irrespective of the calcium balance. DAILY NITROGEN, PHOSPHORUS AND CALCIUM BALANCES ON DIETS CONTAINING DIFFERENT PHOSPHORUS COMPOUNDS Grams Exp. N N P2O5 P2O5 CaO CaO No. Periods Diet intake balance intake balance intake balance 1 Normal mixed 4 days + meat 11.6 +0.38 1.80 -0.404 0.265 -0.339 2 Normal mixed 5 days + casein 11.6 +0.75 1.885 -0.018 0.367 -0.088 3 Normal mixed I 3 days 4 + meat Normal mixed 11.6 +0.45 1.80 -0*252 0.265 -0.222 5 days + sanatogren 11.6 +0.54 2.877 -0.012 0.303 -0.182 5 Normal mixed 1 day + meat 11.6 +0.81 1.80 —0.425 0.265 -0.313 . 1 4 days Normal mixed 12.0 +1.34 1.90 -0.28 0.165 -0.265 2 Normal mixed II 3 days 3 + sanatogen Normal mixed 19.87 +3.99 3.91 +0.552 0.216 -0.214 3 days + sanatogren 19.87 +2.04 3.91 +0.143 0.216 -0.307 4 Normal mixed 4 days + sanatogren 19.87 +3.74 3.91 +0.600 0.216 -0.244 Slowtzoff (1906b) showed in three balance experiments with normal men, himself included, that the ingestion of lecithin caused nitrogen and phosphorus retention, and a decrease of urinary sul- phur, all of which suggest protein synthesis. According to Slowtzoff, Umikoff showed that the protein re- serve is stored in the muscles and liver principally as myosin and myostromin (the phosphorus-containing proteins that remain in muscle after myosin extraction, and which in composition and sol- ubility resemble the nucleoalbumins). Slowtzoff found that 24 hours after the taking of food a trans- formation of the myosin into myostromin took place. Slowtzoff says that if his conclusions and those of Umikoff are correct we may consider the transformation of absorbed protein into the organized, as an enriching of absorbed protein with phosphoric acid and xan- thin substances. Then the action of lecithin would be favorable for this organization, and it would be comprehensible that protein reten- tion is accompanied by retention of xanthin and phosphoric acid. Marfori (1908a, 1908b) concluded that the phosphorus of syn- thetic glycerophosphoric acid, if taken by the mouth, is easily ab- sorbed and assimilated; that glycerophosphates introduced subcu- taneously are not retained, but instead are quickly excreted by the kidneys; lecithin, on the other hand, Marfori found to be retained and assimilated whether introduced per os or subcutaneously. 294 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Togami (1908) sought to determine with a growing dog wheth- er or not ingestion of sodium glycerophosphate, by a healthy animal, would lead to increased phosphorus storage. In a preliminary period on meat alone the dog was storing phosphorus. Then during 6 days there was added to the meat diet, each day, 3.2291 gm. P 2 5 in the form of sodium glycerophosphate. This change produced marked digestive disturbance, with vomiting on the third and fourth days. There was a marked increase in the urinary phosphorus, and a decrease in the phosphorus retention, though the phosphorus balance remained positive. In the after period of 6 days, on meat alone, the phosphorus balance was negative, the daily urinary phos- phorus excretion showing a gradual elimination of accumulated phosphorus from the glycerophosphate. There was, therefore, no evidence of permanent retention of phosphorus from the sodium glycerophosphate. Clearly it was not retained. One should bear in mind, however, that the poverty of the basal ration in calcium, and the fact that the glycerophosphate was fed as a sodium salt were conditions unfavorable for phosphorus retention. J. and W. Cronheim (1910) studied phosphorus metabolism in infants, as affected by lecithin, which was used to replace such part of the milk as to leave the nitrogen and phosphorus of the milk plus lecithin as nearly as possible the same as of the diet of milk alone. With one child the lecithin increased the nitrogen retention from 2.69 percent of the intake to 4.44 percent of the same, and the phos- phorus retention from a negative balance to a positive balance of 15.69 percent of the intake. With a second child the nitrogen retention was reduced from 5.61 to 4.01 percent of the intake, and the phosphorus retention increased from 1.69 to 2.34 percent of the intake. Calcium reten- tion was decreased in both cases. These results must be considered inconclusive. Yoshimoto (1910) studied the effects of lecithin ingestion ori protein metabolism in the dog. Lecithin was added to a basal ration of horse-flesh, bacon, salt and water. In the lecithin periods there was evidently a significant retention of both nitrogen and phosphorus, the effects continuing into the after-periods. A. Loeb (1911) found in balance experiments with two human beings that the retention of lecithin phosphorus was not accom- panied by such a decrease of calcium and phosphorus outgo as sug- gested deposition of the phosphorus in the bones, in fact the calcium outgo was increased. The balance periods were 3-5 days in length — insufficient for the purpose. PHOSPHORUS METABOLISM 295 Bickel (1911a) conducted balance experiments with a man 26 years old and weighing about 66 kg., studying the influence of leci- thin on metabolism. The following table we have calculated from the author's data: AVERAGE DAILY NITROGEN AND PHOSPHORUS (P 2 5 ) BALANCES WITH A MATURE MAN AS INFLUENCED BY LECITHIN— Grams Nitrogen Phosphorus (P2O5) Periods Food Urine Feces Balance Food Urine Feces Balance Diet Fore-period 3 days Main period 5 days After period 5 days 24.530 16.387 19.450 19.416 4.200 3.080 3.416 +3.943 +2.000 +1.698 7.880 9.160 7.880 4.200 4.638 3.996 1.633 2.260 2.014 +2.047 +2.262 +1.866 Normal mixed stand aid diet. Same plus "bioci- tin." Standard diet plus dry egg white. Fore-period 3 days Main period 5 days After period 5 days 16.997 17.356 17.444 4.533 4.520 4.404 +3.000 +2.654 +2.682 7.880 8.400 7.880 3.713 3.814 3.642 1.257 1.280 1.474 +2.910 +3.306 +2.764 Standard diet plus dry egg- white. Standard diet, dry eg-g- white and lecithin. Standard diet plus dry egg- white Lecithin fed in the pure form, or as "biocitin," was apparently- absorbed and retained, at least the feeding of these preparations in- creased the phosphorus retention. The amounts of phosphorus ingested were about twice the maintenance requirement, and the phosphorus retention was remarkably large. See also Bickel (1911b). Patta (1912) reports that, in doses of 0.50-0.75 gm. per- day, lecithin manifests a sparing action toward the phosphorus and nitro- gen balance of the body, this effect being most pronounced when there has been a negative balance during the period preceding the injection. Satta and Gastoldi (1913) report that if animals are in nitrogen and phosphorus equilibrium they eliminate the phosphorus of lecithin principally as inorganic phosphate in the urine, and that if not in equilibrium phosphorus from the lecithin is retained. Summary. The phosphorus of lecithin and glycerophosphates may be absorbed from the alimentary tract and utilized in the tis- sues. Lecithin administered subcutaneously may be retained, but there is some evidence that glycerophosphates, under this condition, are quickly eliminated by the kidneys. Lecithin added in the form of egg yolk to the milk diet of an infant appears to favor nitrogen and phosphorus retention, and gain in weight. Egg yolk as compared with plasmon— a milk albumin prepara- tion — appeared, from experiments with dogs and guinea pigs, to favor the growth of the animal, and the development of the bones. 296 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 As with other phosphorus compounds lecithin and the glycero- phosphates are, in their metabolism, largely independent of nitrogen and calcium, at least during the limited periods covered by most bal- ance experiments. We have in these balance data no evidence of the possession by lecithin or glycerophosphates of any unusual nutritive or stimu- lative values, when added to the normal food of healthy animals. That they do possess a higher degree of usefulness in states of im- poverishment, however, seems probable. Naturally, the apparent results of the administration of these compounds depend on the nu- tritive status of the subject, and on the other dietetic treatments used for comparison. In some states of nutritive derangement they possess life-saving capacity ; in some other more favorable cir- cumstances they may be of no unusual value. GROWTH AND COMPOSITION OF ANIMALS AS AFFECTED BY COMPOUNDS OF GLYCEROPHOSPHORIC ACID Protracted experiments with glycerophosphoric acid com- pounds in the growth of animals have been prolific of results of in- terest, and there is considerable evidence of this nature which indi- cates at least a high degree of usefulness of these compounds in the animal economy, if indeed some of them are not essential to the maintenance of life. Heffter (1891) shows that the lecithin of the liver is decreased by starvation and by phosphorus poisoning. Umikoff (1895) experimented with rats and doves on rations containing various -compounds of phosphorus. They throve only on rations containing lecithin. Danielewsky (1895b) reared tadpoles in lecithin solutions, and reported marked increase in growth in excess of that made by the controls in water without lecithin. He ascribes to lecithin a marked stimulating influence on the processes of multiplication of cellular elements. Danielewsky's observations on the effects of lecithin on the growth of tadpoles have not been corroborated. Danielewsky (1896) conducted injection and feeding experi- ments on chickens and young dogs with lecithin. In each case the animal receiving the lecithin made greater gain in weight than the control. The doses, given at intervals of 3-5 days, were about 5-10 mg. for the chicks, and 20-50 mg. for the pups. The dose was doubled or tripled when given by the mouth. Danielewsky states that lecithin has a stimulating influence, doubtless in connection with the increase of erythrocytes and haemoglobin in the blood. Numerical data are given on page 297. PHOSPHORUS METABOLISM 297 EFFECT OF LECITHIN ON THE GROWTH OF CHICKS AND YOUNG DOGS Series Animals First weight Grams Final weight Grams Treatment Time of experiment I 1 2 (ave.) 2 (ave.) 1 1 1 1 1 1 1 1 1 1 1 97 99.5 249 264 495 560 746 679 495 484 459 487 670 720 785 746 1187 880 4080 3630 4085 2965 7450 11170 7420 6820 6570 5930 Lecithin Control Lecithin Control Lecithin Control Lecithin Control Lecithin Lecithin Control Control Lecithin Control Aug-. 26-Dec. 5 I Aug-. 26-Dec. 5 II Sept. 28-Nov. 13 II Sept. 28-Nov. 13 Ill Aug-. 25-Oct. 30 Ill Aug. 25-Oct. 30 IV June 21-Nov. 28 IV June 21-Nov. 28 V -. June 21-Oct. 18 V June 21-Oct. 18 V..., V June 21-Oct. 18 June 21-Oct. IS VI Jan. 25-Apr. 17 Jan. 25-Apr. 17 Danielewsky (1897) submitted photographs showing lecithin- treated animals surpassing the controls by one-half or two-thirds in length. He also claims that the lecithin affects the psychic devel- opment of the young dogs in a remarkable manner. Desgrez and Zaky (1900) injected subcutaneously into guinea pigs lecithin dissolved in sterile olive oil. The dose was 40-60 mg. per day during 8 or 10 days. The lecithin increased the urinary nitro- gen, decreased the urinary phosphorus outgo, and increased the live weight as compared with controls. The apparent result, of course, depends on. the treatment of the control. Wildiers . (1900) reports negative results from the feeding of lecithin to tadpoles, chickens and dogs. The metamorphosis of the tadpoles was delayed ; no increased growth or increase of red blood corpuscles was produced by hypodermic injection of lecithin in an anaemic dog. Carriere (1901) reports benefit from the use of lecithin with six normal children, during a period of six months. Data are sub- mitted on height and weight before and after treatment ; and state- ments are made as to results on blood and urine, but no data are in- cluded on feces or food. According to Carriere's observations the usual effects of lecithin to increase the urea and decrease the urin- ary phosphorus were at first apparent, but passed away by the end of the experiment. The red blood corpuscles are said also to have been increased by lecithin treatment. Desgrez and Zaky (1901a, 1902a, 1902b, 1902c) conducted a series of studies on the influence of lecithin on the animal organism, especially on the live weight, the development of the skeleton and nervous tissue, and on the composition of the urine. The subjects were guinea pigs, rabbits and dogs. Egg-yolk lecithin was admin- istered by the mouth, or subcutaneously. A part of the results are as follows : 298 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1. Three lots of guinea pigs, 3 in each lot, were fed on bread, bran and cabbage; quantities not stated. One lot served as con- trols; a second received lecithin subcutaneously, and a third re- ceived lecithin in pills. In 43 days the controls gained 480 gm., those treated subcutaneously 670 gm. and those receiving lecithin per os 850 gm. The two lots receiving lecithin excreted more urin- ary nitrogen, but less urinary phosphorus than the controls. 2. Two guinea pigs from the same litter were used as the sub- jects of a similar experiment ; one was used as a control, and the other was injected with lecithin. In one month the control gained in weight 120 gm. ; the injected one 240 gm. 3. Of three dogs from the same litter one was used as a con- trol; one was injected subcutaneously, and the third received the same amount of lecithin in the form of pills. In 27 days the con- trol gained 1480 gm., the injected dog 2050 gm. and the one receiv- ing lecithin per os 2100 gm. The dogs receiving lecithin excreted more nitrogen, but less phosphorus than the control. 4. During inanition it was found with guinea pigs that those injected with lecithin in olive oil lived longer than those injected with olive oil alone. 5. After 60 days treatment as under (1) it was found, by killing the guinea pigs, that both the absolute and relative weight of the brains of the treated guinea pigs were greater than the con- trols, while the weight of the femora of the treated animals ex- ceeded the controls only in the case of those which had received leci- thin by the mouth. 6. After 7 months' treatment as under (2) , the control and the treated animals compared as follows : live weight, 750 gm. :830 gm. ; weight of brain, 2.08 gm.:2.24 gm.; weight of left femur, 1.28 gm. :1.60 gm. 7. Five guinea pigs were used as controls while five others received lecithin by the mouth, from Nov. 1 to Dec. 24. All were killed on Jan. 6 and 7. The controls and the treated animals com- pared as follows: live weight, 2510 gm.:2840 gm.; gain in body weight, 780 gm.:1200 gm. ; brain, 16.56 gm.:18.14 gm.; left femur, 6.16 gm. :6.65 gm. ; phosphorus per 100 gm. of brain, 0.358 gm. :0.373 gm. ; lecithin per 100 gm. of brain, 4.03 gm. :4.19 gm. ; mineral mat- ter per 100 gm. of femur, 66.80 gm.:69.30 gm.; P 2 O g per 100 gm. mineral matter, 39.68 gm.:41.52 gm. 8. Of 2 rabbits one was used as a control, and the other re- ceived daily 0.10 gm. lecithin by the mouth. During 40 days the control gained 200 gm., and the other 350 gm. in live weight. The control and the lecithin rabbit compared as follows: live weight, PHOSPHORUS METABOLISM 299 2020 gm.:2200 gm.; weight of brain, 8.18 gm.:9.28 gm.; weight of femur, 8.26 gm. :8.59 gm. ; total phosphorus per 100 gm. of brain, 0.341 gm. :0.367 gm. ; mineral matter per 100 gm. femur, 61.74 gm.: 62.20 gm.; P 2 5 per 100 gm. mineral matter of femur, 38.01 gm. : 39.91 gm. 9. Of two rabbits from the same litter, one was used as a con- trol, and the other received 0.10 gm. lecithin daily for two and a half months. The control and the lecithin rabbit compared as fol- lows: live weight, 2450 gm. :2170 gm. ; gain in weight, 760 gm. :890 gm. ; brain, 8.92 gm. :9.11 gm. ; femur, 10.14 gm. :8.28 gm. ; phospho- rus per 100 gm. brain, 0.356 gm. :0.347 gm. ; mineral matter per 100 gm. femur, 65.10 gm. :66.94 gm. ; P 2 5 per 100 gm. mineral mat- ter of femur, 37.17 gm. :39.71 gm. 10. Of two dogs of the same age, one was used as a control, and the other received 0.10 gm. lecithin per day from Oct. 1 to Dec. 6. The control and the lecithin dog compared as follows: live weight, 2550 gm. :3780 gm. ; gain in weight, 300 gm. :1380 gm. ; weight of brain, 46.42 gm. ;49.90 gm. ; weight of femur, 10.27 gm. :11.00 gm. ; phosphorus per 100 gm. of brain, 0.365 gm. :0.397 gm. ; lecithin per 100 gm. of brain, 3.73 gm. :4.06 gm. ; mineral mat- ter per 100 gm. femur, 61.03 gm. :62.81 gm. ; P 2 5 per 100 gm. min- eral matter in femur, 38.9 gm. : 37.86 gm. 11. Of two dogs of the same litter, one was used as a control, while the other received daily for two months 0.10 gm. lecithin. Three months later the dogs were killed. The control and the lecithin dog compared as follows : live weight, 13300 gm. : 13340 gm. ; weight of brain, 69 gm. :76 gm. ; weight of femur, 59 gm. :68 gm. ; phosphorus per 100 gm. brain, 0.357 gm. :0.340 gm. ; lecithin per 100 gm. brain, 3.82 gm. : 3.97 gm. ; mineral matter per 100 gm. femur, 62.40 gm. :65.14 gm. ; P 2 5 per 100 gm. mineral matter in femur, 39.07 gm. :39.82 gm. 12. In analyzing the mode of action of lecithin, Desgrez and Zaky found with guinea pigs that sodium glycerophosphate in- creased the urinary nitrogen, but did not alter the phosphorus out- go. Choline, however, subcutaneously injected in quantities of 1 c.c. of a 2-percent solution during 8 months, decreased the urinary phosphorus, and increased the live weight. Betain also decreased the urinary phosphorus, but caused loss in live weight. A part of the author's conclusions are as follows: Lecithin of egg exercises on the nutritive exchanges a favorable influence which is manifested by an increase in the urea, the total nitrogen, and the nitrogen coefficient. There is at the same time a constant decrease in the amount of phosphoric acid eliminated through the urine. The appetite and weight of the animals in- crease. 300 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Lecithin administered to guinea pigs in a state of inanition pro- longs life some days. The loss of weight at the time of death is practically equal in both control and treated animals; the propor- tion of material utilized is the same ; the process of nutrition being better in the treated animals, they survive longer, because of a more perfect elaboration of their reserves and of the protein of their tis- sues. The increase in weight of the treated animals does not mean an increase in fatty tissue. It takes place proportionately in the skeleton and the nervous system. In the skeleton it results in an increasing of the mineral material, particularly of phosphoric acid ; in the nervous system, an increase in total phosphorus and in leci- thin. Springer (1902) reports lecithin injection and feeding experi- ments with guinea pigs, rabbits and dogs. The results were inconclusive as to effects on weight of brain per unit of body weight, and also on weight and length of bones. The total phosphorus and the lecithin of the brain, and the mineral matter of the bone were in each case greater where lecithin had been administered than in the controls. A part of the figures are as follows : EFFECTS OF LECITHIN ON THE COMPOSITION OF THE BRAIN AND BONES OF ANIMALS Experiment and subject Method of adminis- tration of lecithin Percent phosphorus (P2O5) in brain Percent lecithin in brain Percent mineral matter in femur Percent P2O5 in mineral matter of femur II Guinea pigs... . Controls (5) 0.05 gm. in food daily 0.358 0.373 4.03 4.19 66.80 69.30 39.68 41.52 Ill Control (1) 0.100 gm. daily in food 0.341 0.367 61.74 66.20 38.01 39.91 IV Control (1) 0.100 gm. daily in food 0.365 0.397 3.73 4.06 61.03 62.81 38.90 37.86 Hatai (1903) conducted feeding and injection experiments with lecithin on white rats. The treated animals in each of 5 cases gained more in weight than the controls, though the amounts of food consumed were not stated. The relative weight of the cen- tral nervous system of the lecithin rats was normal, as was also its moisture content and the relative development of sheath and axis cylinder. Lusena (1903) determined that the lecithin content of the liver, kidneys and myocardium under acute poisoning by arsenic and by phosphorus did not vary much from the normal, and concluded (a) that in experimental fatty degeneration the larger part of the fat in the degenerated organs is infiltrated fat, (b) that it is not dis- PHOSPHORUS METABOLISM 301 proved that a part of the fat is of endocellular origin, from the transformation of protein, and (c) that though the protein may- change to fat, the lecithin is not the transformation substance, for the lecithin is present within normal variations both in cloudy swell- ing and advanced fatty degeneration. See also Billon and Stassano (1903a, 1903b). Dorn (1904) conducted feeding experiments with rabbits show- ing the nutritive value of lecithin and protylin. Protylin is recom- mended for use in scrofula, anaemia and rachitis. Desgrez and Zaky (1904a, 1904b, 1904c, 1905) compared leci- thin, protylin, nuclein (from yeast), and nucleic acid (also from yeast) , as to their influence on the development and composition of animals. The subjects were dogs and guinea pigs, and the program included feeding experiments, with some metabolism data, and par- tial body analyses. All of these organic phosphorus compounds were found to be of value in causing increase in weight greater than that of the controls. The brain increased in weight, as did the bones ; and the bones in- creased in ash under the influence of these compounds. The auth- ors considered lecithin and protylin to be more valuable, and nucleic acid less .valuable than the other compounds in causing gain in live weight. Since there were no feces phosphorus figures we have no basis for a critical judgment of their conclusions. Pignatti (1906) studied the influence of oral or subcutaneous introduction of various organic phosphorus compounds on the fer- ratin in the liver. Casein, sodium glycerophosphate, and lecithin increased the ferratin of the liver. It was further concluded that by injection of the glycerophosphate the phosphorus content of fer- ratin is not increased, but that casein injection does give a ferratin possessing a high phosphorus content. Morgen, Beger and Fingerling (1906) report results of sheep and goat feeding experiments, with lecithin, as affecting milk pro- duction, which seem to show that in doses of 1-2 gm. this compound increases the secretion of milk and milk solids, fat production, how- ever, being increased only when the basal ration was low in fat. Goldfarb (1907) studied the effects of lecithin on growth with tadpoles and kittens. The tadpoles were kept in lecithin solutions of various strength, from one one-hundred-fiftieth to two percent. The kittens received lecithin subcutaneously, or in the food. The tad- poles so treated showed no greater increase in weight than the con- trols. The kittens which received the lecithin gained, on an aver- age, about 7 percent more than the controls. 302 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In a later series of experiments (1910) similar negative results were obtained with tadpoles and sea-urchin eggs, also in lecithin sol- ution ; with an experiment involving 52 kittens, those receiving leci- thin did not, on the whole, exceed in growth those not receiving lecithin. An experiment on a litter of 5 guinea pigs also gave neg- ative results. Franchini (1907, 1908a) investigated the question of method of utilization of lecithin by the animal organism, in experiments on rab- bits. Two lots of seven rabbits each were used. During three days each rabbit of one lot received 4.799 gm. of pure lecithin, but no other food. The other lot received neither lecithin nor food. Both lots received water to drink. Both lots were killed four days after the beginning of the treatment, twenty-four hours after the last feeding of lecithin. The lecithin feeding increased the lecithin content of the liver and muscle, but not of the brain. Franchini accepts SlowtzofFs conclusion that lecithin, in part at least, is absorbed as such, unsplit. Forbes (1909) compared lecithin with other organic and inor- ganic phosphorus compounds in the growth of swine. Results on more individuals will be necessary to settle many of the points of interest, but the observed effects of lecithin on the composition of the muscles is considered characteristic. The moisture content of the fat-free muscle was higher than in any other lot, as also was the total phosphorus, either as related to the whole meat, the protein or the ash. See also Forbes (1909) ; Common Foods in Rel. to P. Met. and Forbes and associates (1914) ; Nutr. Val. Org. and Inorg. P. W. Cronheim (1912) conducted metabolism experiments, with two mature men on rations varying in lecithin content, from which he concluded that for grown people as well as children lecithin is an important constituent of the diet, and that it has the same value for adults as has often been observed for children, in favoring the replacement of lost nitrogenous material. Wesselkin (1913) made a microchemical study, with rabbits as subjects, of the fatty bodies deposited in the organs as a result of the feeding of lecithin and of egg yolk. Both forms of nutriment caused a deposit of lipoid substances other than true fats. Among these substances were phosphatids and apparently lecithin. The phosphatid formed as a result of the yolk feeding was much in ex- cess of the amount stored as a result of the feeding of the same amount of lecithin in the pure form. The lipoids resulting from feeding egg yolk were mainly cholesterin esters. PHOSPHORUS METABOLISM 303 Salkowski (1913b) reports an increase of the phosphorus of the brain through -the administration of cephalin in pill form to fasting rabbits during four-day periods. It is Salkowski's idea that ceph- alin is stored in the brain. The extent of the work was insufficient to establish so important a point. Summary. In all such experiments as the above the results depend on the comparison of the experimental animals with the con- trols, that is, of the animals receiving the nutrients of interest, with others not receiving them. It is, therefore, obvious that the status of the controls has as much to do with apparent results as does the behavior of the animals receiving the nutrients in question. It is our own belief, therefore, that some at least of the differ- ences in the conclusions of the different investigators are due to differences in the conditions attending the experiments, especially in the state of nutrition of the animals, and their susceptibility to the effects of the experimental treatment, as determined by pre- vious conditions of life. It would therefore be desirable that we know much more than has been recorded in regard to some of the conditions which affect results, but which are not commonly recog- nized as of importance. From the investigations above mentioned one must conclude that whatever the function of lecithin and related compounds in the normal life of animals there are conditions not definitely distinct from the normal in which they do possess certain important specific effects. In comparison with more or less carefully chosen controls, ani- mals receiving lecithin, especially if the basal ration be low in phos- phorus, and the animals in a state of depleted reserves, show in- creased appetite and gain in live weight; phosphorus retention is favored ; there has been observed an increase of ash and of phospho- rus in the skeleton, and of phosphorus in the muscles, and of phos- phorus and lecithin in the brain ; fasting animals live longer if they receive lecithin, and their livers and muscles are increased in lecithin content by ingestion of lecithin. See also Lecithin Therapy. METABOLISM OF PHOSPHOCARNIC ACID Siegfried (1894, 1895, 1896) argues that since phosphocarnic acid contains both phosphorus and iron, and readily forms easily sol- uble compounds with lime and magnesia, and since these compounds are soluble in either neutral, weakly acid or weakly alkaline solu- tions it is fitted to serve as a carrier of phosphorus, iron, lime and 304 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 magnesia throughout the body fluids. Its presence in considerable quantity in milk is in harmony with this view. Taken in meat or its extractives it can render available the lime either of other foods or of drinking water. Siegfried performed experiments on dogs in a study of the re- lation of phosphocarnic acid to muscular work. A nerve in one hind leg was cut ; then the corresponding muscle of the other hind leg was stimulated to activity for one hour by an electric current. The dog was then killed, and phosphocarnic acid determined in the fresh and in the tired muscle. The tired muscle always showed less phosphocarnic acid than the rested one, at one time reaching a ratio of 1:3. Siegfried suggests that the phosphocarnic acid of muscle is an energy-producing nutrient rather than a metabolic waste pro- duct. In the latest experiments described the ischiatic and crural nerves were severed, and then stimulated by an induction current, causing tetanus alternately in the flexor and extensor muscles. Af- ter 1 hour and 50 minutes the dog was killed and the flexors and ex- tensors of both thighs were removed and examined. In a second ex- periment the same nerves were severed, and, after the healing of the wound the dog was exercised rapidly for 1 hour and 15 minutes; then killed, and the muscles examined. A third experiment, similar to the. second was conducted, the time of exercise being 1 hour and 30 minutes. Below are the numerical results. PHOSPHOCARNIC ACID IN TIRED AND RESTED MUSCLES — Grams Weight of muscle Absolute weight of phosphocarnic acid (reckoned as carnic) Phosphocarnic acid in 1000 grn, muscle Phosphocarnic acid used for the muscular work Rested Tired Rested Tired Rested Tired Per 1000 gm. muscle Per 100 parts phospho- carnic acid I., ii in 645 845 955 640 1010 1210 1.6193 1.1116 0.5463 0.5996 0.7344 0.4697 2.40 1.31 0.57 0.93 0.73 0.39 1.47 0.58 0.18 61.3 44.3 31.6 The work of Macleod does not sustain Siegfried's theory. See Macleod (1899), Effects of Exercise on P. Met. Martin Miiller (1897) found very much lov/er values for phos- phocarnic acid in the muscle in new-born infants than in adults. Tarozzi's observations (1899a, 1899b) do not indicate any change in the proportion of phosphocarnic acid in striated muscle during fast. See table on next page. Bonanni (1902) found that the phosphocarnic acid of the mus- cles of rabbits in acute veratrum poisoning was reduced from the normal 1.819 percent (mean of 3 determinations) to 1.426 percent (mean of 4 determinations). PHOSPHORUS METABOLISM 305 Cavazzani (1904a) studied the phosphocarnic acid content of the brains Of dogs after different degrees of nervous excitement. Those killed immediately after morphine sleep showed 0.217 — 0.285 percent ferrinucleon, with 6.61 — 7.04 percent nitrogen, and those killed after absynth excitement showed 0.543 — 0.690 percent ferri- nucleon, with 3.24 — 5.74 percent nitrogen. PHOSPHOCARNIC ACID OF MUSCLES FROM NORMAL AND FASTED DOGS (Tarozzi, 1899) Duration of fast Days Loss of weight Percent Mass of muscle Grams Carnic acid Condition of dog In toto Parts per 1000 '6 28 50 io 28 45 400 450 320 435 595 442 0.618 0.6282 0.5728 0.607 0.787 0.804 1.545 1.396 1.790 Fasted 1.580 1.324 1.818 Panella (1906a) found that the quantity of phosphocarnic acid in the brain (dog) gradually fell off during fast, the decrease being in direct proportion to the duration of the fast. The same was true independent of the water content, which increased during the fast when water but no food was taken. METABOLISM OF PHYTIN FEEDING EXPERIMENTS WITH MEN AND ANIMALS As one of the most important organic phosphorus compounds in foods of vegetable origin, and as that one which contains much the highest percentage of phosphorus, especial attention attaches to the metabolism of phytin. A considerable amount of careful work has been done on phytin within the past few years, and its status as a nutrient is fairly well established. Scofone (1904) found that phytin phosphorus is mostly ab- sorbed, and is excreted largely by the kidneys, as inorganic phos- phate. Giacosa (1904) fed phytin to a dog. The phosphorus of both urine and feces was increased, the inorganic phosphorus of the feces being increased more than the organic. In administering phytin to human beings (1905), in 10 gm. doses, no evidence was noted of urinary elimination of either phytin or inosite. The re- lation of phytin to glycogen was studied by introducing phytin into the stomach of starving dogs and rabbits. The animals which re- ceived the phytin lost the most in weight, and their livers con- tained, as a rule, less glycogen. Giacosa (1907) found the mortal dose much less when injected than when taken by the mouth. 806 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Maestro (1905b) presented balance data with rabbits showing that phytin phosphorus is absorbed, and may be retained. Gilbert and Posternak (1905) reported a balance experiment on a man, in an overfed condition, showing that the addition of phytin to the food increased the phosphorus but decreased the nitrogen re- tention. Data on this test are given below. AVERAGE DAILY PHOSPHORUS METABOLISM WITH A MAN IN AN OVERFED CONDITION AS AFFECTED BY PHYTIN Periods of Five Days — Grams Nitrogen P2O5 Remarks Food Urine Feces Balance Food Urine Feces Balance 22.73 22.73 19.01 20.93 1.69 1.51 +2.03 +0.29 3.68 4.28 2.721 2.960 0.924 1.262 +0.039 +0.062 Diet same as above plus 0.6 gm. P2O5 daily as phytin. .. In another experiment these authors compared phytin phospho- rus with other compounds (see Nutr. Val. Org. and Inorg. P.) ; but the amounts fed were not nearly enough the same to warrant con- clusions other than that the phytin phosphorus was absorbed and retained, but, as before, without beneficial effect on the nitrogen balance, which remained negative. Jordan, Hart and Patten (1906) conducted phosphorus metabo- lism experiments on two milch cows, one animal being used for Ex- periment I and the other for Experiments II and III. The phos- phorus contents of the rations were varied by choice of food and by method of preparation, as in the washing of bran to remove soluble phosphorus compounds. The cows were kept in a warmed room and the excreta were caught by attendants. The cows were milked at 7 a. m., noon, five P. M., and midnight. Inorganic phosphorus was determined by the method of Hart and Andrews. Nuclein and nu- cleo-proteid phosphorus were estimated as that portion of the total phosphorus which was insoluble during a 15-minute extraction with 0.2 percent hydrochloric acid. Soluble organic phosphorus is con- sidered to be the total dissolved by 0.2 percent hydrochloric acid minus the inorganic phosphorus as determined by the Hart and An- drews method. Among the authors' conclusions are the following: The amount of outgoing phosphorus rose and fell with the quantity supplied in the food, though within narrower limits. When the phosphorus supply was abundant, there was a storage of this PHOSPHORUS METABOLISM 307 element in the bodies of the animals, but during prolonged periods, in which the phosphorus supply was deficient, there was withdrawn from the body store about 10 gm. daily. Through katabolic changes the phosphorus of the phytin and that of the unused digested nucleo-bodies was reduced to inorganic combinations, and was excreted, chiefly in the feces, though to a small extent in the urine. The inorganic phosphates of the milk were from three to five times greater in quantity than the total amount of such compounds in the food. The rise and fall in the amounts of outgoing phosphorus com- pounds occurred almost wholly with the inorganic salts found in the egesta. The organic phosphorus bodies of the egesta were but lit- tle affected, if at all, by the proportions of phosphorus compounds in the food. Variations in the phosphorus supply appeared not to modify the appropriation of this element by the milk. No relation whatever appears to exist between the nitrogen ex- cretion and the phosphorus excretion. It was shown, without question, that the physiological effect of the two rations, due to the withdrawal from the bran of such com- pounds as were soluble in slightly acidulated water, differed to a marked degree. With the washed-bran ration as compared with the one containing the unwashed bran, the following differences were observed : a. Drier and much firmer feces with the washed-bran ration. b. A greatly reduced flow of urine following a change from the unwashed-bran to the washed-bran ration, the reverse taking place when a reverse change was made. c. An increase in the flow of milk consequent upon the with- drawal from the ration of the phytin and other water-soluble con- stituents of bran. d. A reduction, sometimes large, in the percentage of fat in the milk consequent upon the withdrawal from the ration of phytin and other water-soluble constituents of bran. e. A decreased production of butter fat during the period in which the washed-bran ration was fed, notwithstanding a some- what increased flow of milk. The following tables set forth some of the main points in this work. 308 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 DAILY PHOSPHORUS METABOLISM WITH MILCH COWS EXPERIMENT I Total P fed Bails'- income and outgo Fed Outgo Feces Milk Urine Rations Total Phosphorus Mar. 10-Mar. 16 12.8 22.7 8.40 14.2 0.10 No. 1 lbs. Oat straw 10 lbs., washed branlO , rice 6 lbs., wheat gluten 1.5 lbs. Apr. 12- Apr. 18 78.7 70.6 55.70 11.0 3.90 No. 2 lbs. lib No. 1 Oat straw 10 lbs., whole bran 10 , hominy feed 5 lbs., wheat gluten Apr. 28-May 1 16.0 23.8 13.29 10.1 0.37 Low phytin and nuclein ration. May 9-May 15 m.H 70.4 56.70 8.8 4.88 No. 2 High ;; ;; ;; May 22-May 26 21.4 22.7 9.59 13.0 0.11 No. 1 Low June 10- June 16 15.1 20.5 9.40 11.0 0.12 No. 1 Low Nucleo-phosphorus Mar. 10-Mar. 16 Apr. 12-Apr. 18 Apr. 28-May 1 May 9-May 15 May 22-May 26 June 10- June 16 12.8 7.6 6.6 3.9 2.6 0.00 78.7 24.4 9.5 7.6 1.9 0.00 16.0 9.0 8.7 6.9 1.8 0.00 83.3 28.6 7.5 5.8 1.7 0.00 21.4 14.2 7.7 5.2 2.4 0.00 15.1 7.5 7.8 5.2 2.6 0.00 No. 1 No. 2 No. 1 No. 2 No. 1 No. 1 Low High Low Sigh Low Low Soluble organic phosphorus Mar. 10-Mar. 16 12.8 2.5 2.0 1.80 0.37 0.00 No. 1 Low Apr. 12-Apr. 18 78.7 51.1 4.1 3.90 0.16 0.00 No. 2 High " Apr. 28-May 1 16.0 4.3 2.0 1.40 0.56 0.00 No. 1 Low May 9-May 15 83.3 52.1 6.2 5.30 0.95 0.00 No. 2 High May 22-May 26 21.4 3.7 2.2 1.70 0.52 0.00 No. 1 Low June 10-Junel6 15.1 4.1 1.6 0.98 0.64 0.00 No. 1 Low Inorganic phosphorus Mar. 10-Mar. 16 Apr. 12-Apr. 18 Apr. 28-May 1 May 9-May 15 May 22-May 26 June 10-June 16 12.8 2.63 14.2 2.8 11.3 0.10 78.7 2.60 56.6 44.1 8.6 3.90 16.0 2.60 13.1 4.9 7.9 0.37 83.3 2.60 57.2 45.6 6.7 4.90 21.4 3.50 12.7 2.6 10.0 0.11 15.1 3.40 11.9 3.2 8.6 0.12 No. 1 No. 2 No. 1 No. 2 No. 1 No. 1 Low High Low High Low Low Weight of cow 1100 lbs., 3 months advanced in lactation period, corrected with approval of one of the authors. Dates of last period PHOSPHORUS METABOLISM 309 DAILY PHOSPHORUS METABOLISM WITH MILCH COWS EXPERIMENT II Dates Total P fed Daily income and outgo Outgo Feces Milk Urine Rations Total phosphorus Dec. 27-Jan. 2 37 42.0 24.4 17.5 0.12 Jan. 13-Jan. 19 18 30.2 11.6 18.5 0.09 Jan. 27-Feb. 2 Feb. 10-Feb. 16 37 20 39.8 29.2 22.1 10.9 17.6 18.2 0.07 0.07 No. 1 Oat straw 10 lbs., washed bran 10 lbs., corn germ meal 6 lbs., rice meal 3 lbs. No- 2 Oat straw 10 lbs., washed bran 10 lbs., wheat gluten 2 lbs., rice meal 7 lbs. No. 1 High nucleo-phosphorus. No.2 Low " ' f Nucleo-phosphorus Dec. 27-Jan. 2 37 21.5 11.4 8.0 3.4 0.00 No. 1 High " Jan. 13-Jan. 19 18 9.5 9.7 6.1 3.6 0.00 No. 2 Low Jan. 27-Feb. 2 37 21.2 11.4 7.9 3.5 0.00 No. 1 High " Feb. 10-Feb. 16 20 11.4 10.5 6.8 3.7 0.00 No. 2 Low Soluble organic phosphorus Dec. 27-Jan. 2 Jan. 13-Jan. 19 Jan. 27 Feb. 2 Feb. 10-Feb. 16 37 18 37 20 11.1 4.1 11.2 4.8 3.3 3.2 3.2 2.9 1.4 1.4 1.5 0.7 1.9 1.8 1.7 2.2 0.00 0.00 0.00 0.00 No. 1 High ' ' No. 2 Low ' ' No. 1 High ' ' No. 2 Low Inorganic phosphorus Dec. 27-Jan. 2 Jan. 13-Jan. 19 Jan. 27-Feb. 2 Feb. 10-Feb. 16 37 18 37 20 4.7 4.7 4.7 4.7 27.3 17.3 25.3 15.8 15.0 4.1 12.7 3.4 12.1 13.1 12.5 12.3 0.12 0.09 0.07 0.07 No. 1 High No. 2 Low ' ' No. 1 High " No. 2 Low " " Weight of cow 966 lbs. DAILY PHOSPHORUS METABOLISM WITH MILCH COWS EXPERIMENT III Dates Total P fed Daily income and outgo Fed Outgo Feces Milk Urine Rations Total phosphorus Mar. 12-Mar. 18 Mar. 30-Apr. 5 26.8 43.5 10.9 14.7 15.8 10.17 0.08 No. 1 Oat straw 10 lbs., wheat bran 10 lbs., rice meal 7 lbs., wheat gluten Wi lbs. No. 2 Oat straw 10 lbs., washed bran 10 lbs., rice meal 7 lbs., wheat gluten 2 lbs. Nucleo-phosphorus Mar. 12-Mar. 18 Mar. 30-Apr. 5 77 16 23.2 9.6 13.4 9.4 9.9 6.1 3.5 3.3 0.00 0.00 No. 1 No. 2 Soluble organic phosphorus Mar. 12-Mar. 18 Mar. 30-Apr. 5 50.2 2.6 1.58 1.86 0.53 0.69 1.05 1.17 0.00 0.00 No. 1 No. 2 Inorganic phosphorus Mar. 12-Mar. 18 Mar. 30-Apr. 5 4.0 4.2 53.4 15.5 33.1 4.2 10.2 11.2 10.10 0.08 No. 1 No. 2 Weight of cow 966 lbs. 310 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Mendel and Underhill (1906) ) conducted a series of balance ex- periments with a female dog on a diet of meat, cracker meal and lard. In some periods sodium phytate was administered subcutan- eously, and in others the salt was given by the mouth. The basal ration was composed of hashed meat 72 gm., cracker meal 76 gm. and lard 14 gm. ; the whole containing 3.84 gm. nitrogen, 1.01 gm. P 2 5 and 520 calories. The numerical data are on page 311. A part of the authors' conclusions are as follows : "Comparatively large doses of phospho-organic acid, used as the sodium salt, can be introduced into animals either per os, subcu- taneously, intraperitoneally, or intravenously, without unfavorable effects. The free acid is more toxic. "No marked or immediate characteristic effects of the sodium salt upon general health or nitrogenous metabolism have been ob- served. The compound is readily absorbed and speedily trans- formed within the organism. Its phosphorus reappears in the ex- creta as inorganic phosphates. No constant relation between the metabolism of nitrogen and of phosphorus was observed. In these details our experience with the dog corresponds with the observa- tions of Jordan, Hart and Patten after feeding phytin to cattle. Our results differ in showing that in both the dog and the rabbit the ex- cess of phosphorus was almost entirely eliminated through the kid- neys rather than in the feces. This may have an important bear- ing on the possibility of producing laxative effects with phytin. "In our experimental animals purgative action could not be constantly provoked. Very large doses were frequently effective. No permanent generalizations can be drawn from the observations made on this point." The authors also call attention to the fact that the effects, with their sodium salt, may differ from the natural phytin, because of the character of the bases present. Horner (1907) conducted balance experiments with a dog and a rabbit which apparently showed that phytin may be absorbed and retained, though the results are not considered by Horner as quite conclusive. McCollum and Hart (1908) demonstrated that calf's blood and liver have, but muscle and kidney have not, the property of cleaving phytate with the production of inorganic phosphate. They cite the conclusion of Scofone that the enzymes of the digestive tract do not alter phytin. Hart, McCollum and Humphrey (1909) conducted a balance ex- periment covering about three and one-half months, with one cow, PHOSPHORUS METABOLISM 311 -;-':"J*- o'ooooboooboaooo-jJCjncii ooooobobobccoaoaoojcjt OOtOHCOOCDOJDtDtCQOCO SwOO)MM00050tOtO(C ^ooOTGC--JCo--JGioro--J>-'ao>*>- J6OHO00O00MO0 3 00 CO -JH -*J~4 JOCOCOtO-J~4 oooooooooopoo -coocototoi-T'tototoco^t t^h-i-S^^Jcnooco^Ci^-rf^co ^-4 b> >&■ oj '*>• cn oo cn oo oo to <33 to | +1+ + +++++ OOHO^OpOHMNI-H io ;_>bo!-» ^-a to^-atococo oscocococccoco;s>.co;e.cnccco roo^cotocoa:cocococoo^co tOtOI-'tOl-'IOJ-'COJ-'COCOJ-'h-' eoeociton-ccoih-cDcocncoco OiOCJicocotocoococopo-JOj c n o O'er 3 o o c->- S C 3 p i° rr n> 2 C C o oi oi p. S3.' re'o" o <->■ J+ o'S' 3 B aw p p 01 ►?) £° OO COCO COCO BB coco o*B a-3 O c+ O rf C 1 B i re 2 re o 2. o 3 o B w oi 3.01 g^ 3 o (B (S (1 O o*3 p.p.p.3 H >d 01 01 01 W Sjppfiit o S.SS5H, C o -•-•-• 5 QOOOO 05OT Oq «q 3 bbp E 5 s « S p p W oi oi O EL EL B 3 B OO; 1 5 3.8 S p 3S 1 oo hj *t $ P* O O O W I W w > W o f » i— i 3 H H 02 a o f Q » 3 312 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 in a study of phytin metabolism. The phytin was as present in wheat bran, and as prepared from the same as a crude potassium salt. Whole bran was compared with washed bran, and with washed bran plus potassium phytate. The washing process removes, of course, a great variety of compounds other than the one of especial interest. These authors reached the conclusion that the phytin of wheat bran does not have a specific effect on the production of milk, or on the production of fat in the milk, and that previous results, appar- ently showing such effects, have been due to the individuality of the subjects. Phytin was found to be diuretic because of its potassium content, and also laxative because of the elimination of phosphorus and accompanying bases by way of the feces. Cook (1909) conducted a metabolism and tissue analysis ex- periment with six rabbits, in the comparison of phytin with sodium phosphates, when taken in excess of the maintenance requirement. Two rabbits were fed on corn, oats, and vegetables as controls ; the remaining four received a ration of carrots, wheat gluten, starch, sugar, olive oil and salt mixture, two receiving phytin in addition, and the other two inorganic phosphorus in the shape of sodium dihydrogen phosphate and disodium hydrogen phosphate. The phytin was prepared from wheat bran. The condensed nitrogen and phosphorus balance data are as follows : AVERAGE DAILY NITROGEN AND PHOSPHORUS (P 2 6 ) BALANCES WITH RABBITS COMPARING PHYTIN AND SODIUM PHOSPHATES— Grams Dates Rabbit No. Weight, averag-e Nitrogen Phosphorus P20s^ Intake Balance Intake Balance Form of phosphorus Nov 17-Feb. 15.... 1 2 3 4 1618 1645 1585 1981 1.154 1.353 " 1.520 1.396 +0.23 +0.32 +0.38 +0.33 0.381 0.415 0.501 0.459 +0.14 +0.14 +0.18 +0.18 Phytin Phytin Phosphate Phosphate Feb. 17-Mar. 15.... 1 3 4 1550 1796 1994 1.479 1.747 1.485 +0.26 +0.40 +0.18 0.456 0.541 0.498 +0.166 +0.166 +0.156 Phytin Phosphate Phosphate These data do not show unmistakable differences in the value of these compounds under the conditions of these experiments. It is worthy of note that the calcium and magnesium balances were positive. A part of the inconclusive character of the results must be due to the fact that there was loss of live weight in 5 out of the 7 periods. The tissue analyses revealed more decided differences, though their significance is as yet unexplained. The autopsy revealed ab- PHOSPHORUS METABOLISM 313 normal conditions in the livers, kidneys and lungs of the rabbits which received the phosphorus compounds, the livers all being pale in color, and enlarged. The livers of the rabbits which had received phytin showed marked fatty degeneration, as also did one of those receiving inorganic phosphates, though in a less pronounced way. The kidneys of the inorganic phosphate rab- bits showed parenchymatous degeneration. Below are analytical data from several tissues of these rabbits. CHEMICAL ANALYSES OF BODIES OF RABBITS AS AFFECTED BY PHYTIN AND INORGANIC PHOSPHATE— Dry Basis— Percent Nitro- Ash Cal- Magne- Ether Phosphoric acid Ether- Ether -alco- gen cium sium extract Total alcohol- soluble hol-soluble in terms of total Bones 4.53 55.56 8.86 0.22 11.33 23.96 0.055 0.23 Livers. . . Blood 7.64 14.72 3.78 5.64 0.00 Trace 0.00 0.00 44.95 Trace 1.98 0.75 0.680 0.008 34.34 1.07 Mean of rabbits 1 and 2 fed organic phos- phorus. Brains. . . 6.12 7.00 0.31 0.09 43.23 3.96 2.350 59.34 Nerves. . . 5.52 6.54 0.21 0.11 46.96 3.72 2.390 64.26 Teeth.... 74.65 24.73 1.35 35.31 Bones 4.68 55.92 7.74 0.15 9.39 26.33 0.061 0.23 Livers. . . Blood.... 9.41 14.42 4.73 4.72 0.00 Trace 0.00 0.00 34.48 Trace 2.56 0.69 0.854 0.008 33.36 1.16 Mean of rabbits 3 and 4 fed inorganic phos- phorus. Brains. . . 6.52 7.17 0.27 0.06 43.89 4.07 1.160 28.51 Nerves. . . 3.72 5.83 0.28 0.06 47.34 4.28 1.470 34.35 Teeth.... 76.10 24.65 1.22 34.70 Bones 4.32 57.47 10.17 0.23 5.12 25.93 0.069 0.27 Livers. . . 11.90 5.67 0.00 0.00 14.47 2.85 1.090 38.24 Blood.... 14.42 4.55 0.44 0.19 Trace 0.58 0.037 6.33 Mean of rabbits 5 and Brains. . . 6.85 7.82 0.51 0.08 38.50 4.16 1.750 42.07 6 normally fed. Nerves. . . 4.39 5.97 0.44 0.12 44.34 3.90 2.310 59.23 Teeth.. . . 75.17 28.35 1.15 35.63 Among the more prominent results here set forth are: (1) the low calcium content of the tissues, (2) the increase above normal of the alcohol-ether soluble phosphorus of the brain of the rabbits which received phytin, and (3) the decrease below normal of the al- cohol-ether soluble phosphorus of the brain and nerves of the rab- bits which received inorganic phosphates. Tyshnjenko (1909) found, in balance experiments with 5 labor- atory assistants on a diet of bread, meat, milk and butter, that the addition of sodium glycerophosphate caused a loss of phosphorus from the body, while phytin led to retention. We have seen only Maly's abstract of this article of 117 pages. Starkenstein (1910) states that the organs of infants contain larger amounts of inosite than those of adults, and that it originates in the inosite-phosphoric acid of the food, which adults are able, in larger part than infants, to decompose, the remainder passing off 314 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 unchanged in the urine. Starkenstein, therefore, regards inosite as of no physiological importance in the animal body, but as a waste product of phosphoric acid metabolism; and he considers inosite- phosphoric acid as of importance only as a source of phosphorus, and also of value in certain pathological conditions of the osseous system. We shall stand in a fair way to clear up these problems when we have discovered a satisfactory method for the estimation of inosite-phosphoric acid. Rogozinski (1910) studied phosphorus metabolism in an adult dog in 7 continuous 5-day experiments, without intervals between collection periods. Phytin, lecithin and sodium phosphate were ad- ded, in different periods, to a diet of meat, rice and lard. Rogozinski found no pronounced influence of these compounds on the nitrogen excretion in the urine, when they were added to a ration sufficient to maintain the animal. The phosphorus of the sodium phosphate appeared quantita- tively in the urine. The total lecithin fed to the dog was split, and the phosphorus excreted in the urine as inorganic phosphate; the phosphorus of phytin passed into the urine to the extent of about 30 percent of the total, the remainder being excreted in the feces, where it could be determined as such ; the nitrogen and phosphorus balances were found to a high degree independent of each other. In a 15-day experiment on himself Rogozinski added 2 gm. of phytin to a mixed diet (which was not analyzed) on the 6th to 10th days. The phytin was completely split in the digestive tract; a small part of the phytin phosphorus was retained, and the rest was excreted as inorganic phosphorus in the feces ; the feces contained an abundance of lecithin-like compounds; no inosite was found in the urine after feeding phytin; the bacteria of human feces can split off inorganic phosphoric acid from phytin. Donath (1911) reports phytin as a powerful stimulant to the appetite, and states that, in dogs having Pawlow fistulae, it increas- es the flow of gastric juice. Sodium phytate in large quantities was found toxic to rabbits by A. R. Rose (1911). It appears that 1.7 gm. per kilogram of body weight is fatal. Rose (1912a) has conducted an extensive study on the metabolism of phytin with a milch cow. Organic as well as in- organic phosphorus of the food was eliminated very largely in the form of inorganic phosphorus in the feces, the amount of phospho- rus in the urine being very small. The addition of calcium phytate increased the potassium, both in the urine and feces, and changed the path of elimination of part of the magnesium from the kidney to the intestine. The calcium added as calcium phytate was almost PHOSPHORUS METABOLISM 315 entirely eliminated by the intestine immediately after administra- tion. The calcium of the urine increased with decreasing phos- phorus in the rations, and decreased when calcium phytate was added. The volume of the milk fluctuated inversely, and the total amount of fat in the milk directly, as the amount of phytin phospho- rus in the rations. The increase of milk flow on removal of the phytin was not a mere dilution. Except for the change (increase) in the amount of fat, the composition of the milk was not material- ly altered. These results sustain the earlier work of Jordan, Hart and Pat- ten, but since the cow used by Rose was one of the same that were used in the work of Jordan, Hart and Patten, the factor of individuality was not excluded. G. di Gregorio (1912) concluded that the administration of 1-2 gm. daily of phytin can check a pathological phosphaturia, and un- der normal conditions can diminish loss of phosphorus. Santonocetto (1912) studied nitrogen metabolism as influenced by the administration of phytin. He concluded that under the in- fluence of phytin the breaking-down of the cells is retarded, and the absorption of food nitrogen is promoted, the fecal nitrogen being reduced 30-75 percent, and the urine nitrogen 7.12 percent. Venturi and Masella (1913) studied the influence of phytin in- gestion on the balance of nitrogen, and on the qualitative distribu- tion of the nitrogenous metabolites. The experimental subject was one of the authors. After a preliminary period of 4 days phytin was administered in the amount of 1.5 gm. daily for 5 days; then followed an after-period of 2 days, and a second phytin period of 5 days, during which 2.5 gm. of the compound was fed daily. The daily data show, in the phytin periods as compared with the fore- and after-periods, a marked decrease of urinary nitrogen, especially in the form of urea, creatinin, hippuric acid and xanthin bases, the uric acid and ammonia remaining without great change. The feces nitrogen was decreased, the nitrogen retention being prominently increased. The authors note the agreement of their results with those of Santonocetto. See also Forbes and associates (1914), Nutr. Val. Org. and Inorg. P. CLINICAL EXPERIMENTS WITH PHYTIN Secheret (1904) made a historical and critical study of phytin therapy, with some observations on animals after the introduction of phytin, and also of 61 human patients under treatment with phy- tin. Most of the animals receiving phytin, otherwise than by the mouth, died soon. Hypodermic injections were, therefore, not 316 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 recommended. Dosage was discussed, and phytin recommended in a great number of pathological states, especially as a general tonic, and to increase the blood corpuscles, in constitutional debility, from whatever cause. Phytin is said to possess a very active pharmaco- dynamic action, exciting the internal nutrition Of the tissues and cells, and increasing the nitrogen elimination. Bardet (1905) has given study to the condition of general "demineralization," which may be due to any such illness of long standing as results in a disturbance of the normal balance be- tween assimilation and losses. Of 17 cases examined 4 are re- ported ; one a woman 23 years old, in a low state because of a long siege of typhoid fever; another was a wet-nurse, aged 28, who was in a debilitated state ; a third was a boy 16 years old who had be- come highly neurasthenic through overstudy; and a fourth was a rapidly growing boy of 14 years, who was in a much disturbed state. Adrian's extract of cereals (containing 4 percent P 2 5 , and consid- erable potassium, magnesium, and some manganese, all in organic combination, largely as a phytin) was administered with sodium methyl arsenate. Fifteen to twenty-five grams of the extract were given in solution each day for 30 or 40 days, which treatment brought about almost complete restoration to normal condition. Since this cereal extract contained in assimilable form nutrients of which the body stood in need, it was probably of some value, though the use of arsenic adds a factor to the problem which makes impos- sible a positive interpretation of the results. Novi (1908) submitted data apparently showing that the ad- ministration of phytin, 1-1.5 gm. per day, decreased the urinary phosphorus in antirabes treatment from 69.11 to 46.07 percent of the intake. Feces figures were not submitted. Novi (1909) reported that in consequence of the injection of 1- percent water solution of phytin into the dorsal lymph sack of the frog the muscle power was 2-4 times higher than normal. Calcium chloride produced no such effect, but a mixture of sodium, mag- nesium and calcium chlorides produced the same effect as the phy- tin, as also did magnesium chloride alone. Sodium glycerophos- phate produced no effect, but calcium glycerophosphate doubled the muscular power. Favorable results from the use of phytin, by ingestion, in cases, of gastric ulcer are reported by Wolpe (1911). Other clinical reports from the use of phytin in human medical practice are those of Loewenheim (1904), Gilbert and Lippmann PHOSPHORUS METABOLISM 317 (1904), Fiirst (1904), Gianasso and Ovazza (1905), Winterberg (1905), Dambre (1905), Wechsler (1905), Maestro (1905a), and Weissmann (1908). The results of these studies have been almost uniformly favorable, as shown by improved state of nutrition gen- erally, increased appetite, improved blood conditions, etc. Emphasis is placed on the fact of the very high phosphorus content of this compound, making' it a superior vehicle for the administration of phosphorus in an organic form. Summary. Conservatism in the acceptance of results in the clinical study of phytin is especially to be recommended because of the abundance of phytin and related compounds in some of our com- mon foods, which produce no marked specific symptoms. On the other hand, we are obliged also to admit the possibility that this compound as administered in an uncombined state may have a dif- ferent effect and method of usefulness from the same as normally combined in foodstuffs. There is not the same ground for this latter hypothesis, however, that there is in the case of lecithin, since phytin even though absorbed as such, unsplit, must be broken up be- fore it can be of use for constructive purposes, phytin not being a constituent of the animal body. A measure of uncertainty must exist in regard to results in this field until an understanding of the chemistry of phytin and related compounds has been attained. It seems quite probable that the name phytin has been associated with a variety of compounds. There is no question, however, as to the absorbability of phytin, nor of its usefulness in animal metabolism. Phytin is readily soluble in the hydrochloric acid of the gastric juice and is mostly absorbed without decomposition by the digestive enzymes. It is decomposed, however, before utilizaton by the tis- sues, and to such extent as it is not retained, its phosphorus is elim- inated in the urine and feces. In cattle and in human beings phytin phosphorus is eliminated as inorganic phosphates, following the usual course as determined by the nature of the diet and by the in- organic bases present. In dogs, however, a considerable part of the phytin phosphorus seems to be eliminated in the feces, where it is found present as phytin. The power to split phytin has been found to reside in the blood and liver, but not in the muscle and kidney of the calf. It may also be decomposed by intestinal bacteria. The contribution of phytin to the laxative and diuretic effects of rations is through the inorganic bases contained. There has not yet been established a definite relation between phytin ingestion and nitrogen metabolism. 318 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 We are not yet able to say whether or not phytin has specific effects in animal nutrition, but there is certainly nothing strikingly characteristic about the method of its use. Apparently, at least its chief superiorities as a phosphorus-carrying nutrient lie in its ready solubility and in its high phosphorus content. THE NUTRITIVE VALUES OF ORGANIC AND INORGANIC PHOSPHORUS METABOLISM EXPERIMENTS WITH MEN AND ANIMALS Considering the phosphorus of the animal body as a whole the most obvious distinction among the various groups of its compounds is that certain of these are organically combined, as a part of the liv- ing tissue, being fundamentally involved in all vital activities, while others are simple salts of the mineral bases either deposited in sup- porting structures or dissolved in the body fluids. In both cases the phosphorus itself, so far as known, is present in the same completely oxidized form as phosphoric acid, differing only in its chemical relationships. This discussion is a considera- tion of the evidence as to the nutritive limitations imposed by these differences in relationship of phosphoric acid. We wish to know whether organic and inorganic phosphorus compounds can serve, equally well, all the requirements of the body for phosphorus under all conditions of life. Let us consider first the eggs of birds, for the egg assuredly contains all of the nutrients required by the fully formed animal. Examining the phosphorus of eggs we find a wealth of organic com- pounds but, at the most, mere traces of inorganic phosphates. It is thus obvious that organic phosphorus compounds can serve all of the needs of the body for this element. But our interest is in food- stuffs ; let us consider the first natural food of young mammals, the mother's milk. Are the comparative amounts of organic and inor- ganic phosphorus related to the food requirements of the young ani- mal, or do they simply represent their relative availability for milk production in the maternal organism ? However this may be, if there is not an adaptation of the character of the food to the requirements of the young, then there must be an adaptation of the method of de- velopment of the young to the possibilities of the food, in either case a harmony of objects to be attained and means for their attainment. In this light we would naturally assume that both organic and inorganic phosphorus compounds are of benefit to the animal; for PHOSPHOEUS METABOLISM 319 both classes are represented in milk by several individuals each, and in the whole literature of the subject there is scarcely a dissenting voice raised against this idea. Both organic and inorganic phos- phorus are absorbed and retained. But now we come to the parting of the ways. If organic phos- phorus can serve all of the requirements of the body for phosphorus, and if inorganic phosphorus can be absorbed and retained, are or- ganic and inorganic phosphorus equally useful for all of the purpos- es for which the body needs phosphorus ? Our economic reason for desiring an answer to this question lies in the relative accessibility of organic and inorganic phosphorus for use as food. If rock phos- phate and old bones can furnish us phosphorus in the forms most advantageous for the growth of animals, we are wasting much money on milk, eggs and beef; for there are much cheaper sources than these, of protein, fat and carbohydrates. In this consideration let us bear in mind that our interest as ag- ricultural scientists is not so much in bare physiological minima as in maximum practical optima, for the whole range of success and profit in animal production lies close to the latter. It would seem that so simple a problem ought readily to be solved, but when we approach the subject by direct experiment we find the course beset with hazards, and there never has been unanimity of opinion as to the facts. Among those circumstances which have contributed to this difference in opinion are: (1) An inclination to ascribe to all animals under all condi- tions of life all capacities of synthesis possessed by any animal un- der any condition, natural or experimental, no matter how great the stress of attending circumstance. (2) Drawing conclusions from very short balance experi- ments, without due regard to states and habits of nutrition as de- termined by previous feeding. (3) A failure to distinguish between physiological minima and practical optima. (4) Drawing conclusions from mere gain in weight, without actual estimation of the compounds of interest or measurements of functional efficiency in the experimental animals and in carefully selected controls, the error in so doing being that it implies the maintenance of constancy of composition and function, the variabil- ity of which, as affected by feeding, is usually underestimated. (5) Drawing conclusions from analyses of parts of animals, a complete chemical accounting being desirable. 320 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 (6) Failure fairly to meet the great difficulties of compound- ing rations which do not differ in essential ways other than in the point of interest, that is, in the condition in which the phosphorus is present. (7) It is quite possible that useful enzymes associated with the organic phosphorus compounds have exerted a determining in- fluence on the results. (8) Unsatisfactory mineral salt accompaniments may have affected the usefulness of the phosphorus compounds in such ways as to result in unfair comparisons. (9) The specific physiological action of isolated compounds of phosphorus, as used in nutrition investigations, undoubtedly dif- fers from the effects of the same compounds in their natural rela- tionships in foodstuffs. We shall review, in brief, the evidence on the subject. W. S. Hall (1896), from feeding experiments with mice, came to the conclusion that the salts organically combined with casein have a value greater than salts not so combined. Steinitz (1898) conducted balance experiments on dogs to com- pare organic and inorganic phosphorus compounds as nutrients. Nutrose, a sodium-calcium-casein compound, vitellin, and myosin prepared from horse flesh were each added, in different periods, to a basal ration of bacon, rice starch and mineral salts ; the casein prep- aration and the vitellin both containing organic phosphorus, and the myosin being at least practically free from organic phosphorus. Phosphorus was stored abundantly on the organic phosphorus ra- tions, but the retention was almost nothing on the myosin ration, though in all cases, including the myosin ration, nitrogen was stored in considerable quantity. The salt mixture used with the organic phosphorus compounds contained chlorides and citrates only, while the salts used with the myosin were calcium, magnesium and potas- sium phosphates, sodium chloride and ferric citrate. The experi- mental periods were 5-9 days each, following 8-day fasting periods. Rohmann (1898) found, in balance experiments on a dog, with a diet of lard, rice starch, salt and either a phosphorus-containing protein, or a phosphorus-free protein and a phosphate, that both nitrogen and phosphorus retention were favored by the phosphorus- containing protein much more than by the phosphorus-free protein and phosphate. With nutrose and vitellin the nitrogen retention was 31.0 and 42.2 percent, respectively, of the intake; while with myosin and edestin the retention was but 0.10 and 0.11 percent of the intake. Likewise with the nutrose and vitellin the phosphorus retention was 8.8 and 20.9 mg. per day, while with myosin and edes- tin the retention was 0.1 mg. per day in both cases. PHOSPHORUS METABOLISM 321 Leipziger (1899) studied metabolism in a dog on a ration which was very low in. organic phosphorus. The ration was composed of edestin, fat, starch, salt, beef extract and water. The dog was fasted for 6 and 10 days, respectively, in the fore-periods of two tests, and then fed for 6 days in each of the two main periods. The phosphorus intake in the two experiments was 1.874 and 2.022 gm. per day, of which 0.016 and 0.022 gm. was organic. The dog retained 0.0078 and 0.095 gm. of phosphorus per day in these tests, the nitrogen and calcium balances also being positive. The subject of these experiments was the dog referred to as "Dog II" in Steinitz's experiments. Leipziger considered that his data did not afford evidence as to the condition in which the phosphorus was retained, but thought that synthesis of phosphorus-containing protein from phosphorus,- f ree protein and phosphates was improbable ; also that the phospho- rus retention was less than it would have been on a ration contain- ing more phosphorized protein. The organic phosphorus content of this ration was sufficient to render impossible the solution of the problem of phosphorized protein synthesis except by negative re- sults — which were not obtained. Zadik (1899) conducted nitrogen and phosphorus balance ex- periments with dogs in the comparison of phosphorized proteins with phosphorus-free proteins and inorganic phosphates. The compounds of interest were casein, vitellin and edestin. The basal ration was composed of starch, bacon, sodium citrate or carbonate, and a salt mixture of phosphates, chlorides, magnesium citrate and sugar. The numerical data contain numerous inconsistencies, on account of which we do not transcribe them, but the errors seem not to be of a degree to modify at all the significance of the results. With casein or vitellin there was marked retention of phosphorus; with edestin and disodium phosphate there was always loss. Zadik concluded that the animal organism does not have the power to build from phosphorus-free proteins and phosphates the organic phosphorus compounds necessary for the life of the cells. The organic phosphorus of casein and vitellin was, under his experi- mental conditions, at least, more useful than the inorganic phos- phates ; also, the phosphorus of vitellin was stored in larger propor- tion to the intake than was the phosphorus of casein. Ehrlich (1900) conducted five balance experiments with dogs in a comparison of phosphorized proteins with phosphorus-free pro- tein plus inorganic phosphates. The results tend to show that the phosphorized proteins, casein and vitellin, have a greater useful- 322 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 ness to the animal, in the sense of favoring- phosphorus retention, than does the phosphorus-free edestin plus inorganic phosphates. The periods were 3-7 days in length. Certain unfavorable condi- tions render the results of doubtful value. Kornauth (1900) compared synthetic "nucleins," prepared after the method of Liebermann, by the precipitation of egg albu- min and blood serum albumin with metaphosphoric acid, with natur- al proteins in the form of meat, skin, aleuronate (a vegetable casein) , casein and conglutin (from lupines) . The results on maintenance of nitrogen equilibrium hardly warrant conclusions, but the evidence is quite satisfactory in showing that, for maintenance of phosphorus equilibrium, very much more phosphorus in the so-called synthetic nucleins is necessary than of phosphorus in the natural proteins. Steinitz (1900) reports several attempts to rear young dogs on artificial food mixtures containing their protein as casein, edestin, milk, nucleoprotein from calf liver, and vitellin. Other foods used were rice starch, lard, bacon and inorganic salts. The experiments were mostly of short duration, on account of unfavor- able termination. None were carried through to a satisfactory demonstration of the sufficiency of the diet. Gottstein (1901) conducted a metabolism experiment with a dog in two five-day balance periods in which casein was opposed to edestin. The results were inconclusive. Ehrstrom (1903a) conducted an experiment which bears on the relative value of organic and inorganic phosphorus. The condensed data are as below. DAILY NITROGEN AND PHOSPHORUS BALANCES WITH A GROWN MAN RECEIVING ORGANIC AND INORGANIC PHOSPHORUS COMPOUNDS— Grams Length of balance period Diet N Intake N Balance P Intake P Balance Freely chosen diet of ordinary foods Proton-bread and milk Ordinary white bread, milk and CaHP04 +17.37 +17.86 +12.55 -1.33 +0.58 -1.81 +2.476 +2.090 +2.271 +0.610 +0.642 +0.227 Less phosphorus was stored when a certain portion of the total was administered as dibasic calcium phosphate than when the same amount was taken as proton — a casein preparation ; in these last two periods, then, we have opposed to each other a typical phosphopro- tein and an inorganic phosphate, added in each case to a bread and milk ration. The author does not submit data showing that the phos- phate ration contained bases, especially calcium, sufficient in PHOSPHORUS METABOLISM 323 amount, relative to acid elements, to render conditions as favorable for phosphorus storage as in the proton ration. The periods also are very short for mineral metabolism work. The data seem to show the organic phosphorus the more useful. Hirschler and Terray (1902, 1905) compared bone dust with eggs as sources of phosphorus for growing dogs. A portion of the data are below. METABOLISM EXPERIMENTS ON DOGS WITH ORGANIC AND INORGANIC PHOSPHORUS COMPOUNDS Daily Amounts — Grams Gain or Intake Phosphorus Daily balances Period loss in and weight of Diet days dog- Kg. N P2O5 CaO Urine Feces N P2O5 CaO 1 4.130 4 days +0.010 5.419 1.377 0.686 0.681 0.139 +0.912 +0.557 +0.344 400 c.c. milk; 23.56 2 . gm. dried meat. 4 days -0.015 5.419 1.377 0.686 0.688 0.217 +0.567 +0.472 +0.350 400 c.c. milk; 23.56 3 gm. dried meat. 4 days -0.005 5.419 1.377 0.686 0.677 0.227 +0.444 +0.499 +0.266 400 c.c. milk; 23.56 4 grm. dried meat. 2 days +0.025 5.419 1.765 1.212 0.656 0.403 +0.784 +0.7U +0.575 Same as above + 5 1 grm. bone dust. 2 days -0.010 5.419 1.377 0.686 0.667 0.361 +0.514 +0.350 +0.087 400 c.c. milk; 23.56 gm. dried meat. 1 3.600 4 days +0.013 4.781 1.097 0.524 0.575 0.114 +0.688 +0.408 +0.176 300 c.c. milk; 23.56 2 gm. dried meat. 4 days -0.005 4.778 1.184 0.541 0.638 0.180 +0.798 +0.366 +0.148 300 c.c. milk; 15.06 gm. dried meat; 3 53.5 gm. egg. 4 days -0.013 4.781 1.097 0.524 0.611 0.353 +0.295 +0.132 +0.177 300 c.c. milk; 23.56 4 gm. dried meat. 2 days +0.020 4.781 1.485 1.050 0.548 0.717 +0.115 +0.220 +0.001 300 c.c. milk; 23.56 gm. dried meat; 5 1 gm. bone dust. 2 days 0.000 4.781 1.097 0.524 0.625 0.379 +0.160 +0.094 +0.288 300 c.c. milk; 23.56 gm. dried meat. Age of dogs, 214-3 months. Since the intake was not maintained constant, there was not an entirely satisfactory basis for comparison of phosphorus in the dif- ferent forms. With one dog the bone dust caused increased reten- tion of nitrogen, calcium and phosphorus, with no increase in urin- ary phosphorus, but naturally a decided increase in feces phospho- rus. With the other dog the bone dust caused diarrhoea, and the results are perhaps affected by this disorder. Because of the rapid decline in the rate of phosphorus storage during the experiment with the second dog, it is impossible to say just what was the effect of the egg on phosphorus storage. Gilbert and Posternak (1905) compared organic and inorganic phosphorus compounds in balance experiments with human subjects. A part of the data are in the following table. 824 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 DAILY NITROGEN AND PHOSPHORUS BALANCES FROM A NORMAL HUMAN SUBJECT WITH ORGANIC AND INORGANIC PHOSPHORUS COMPOUNDS ADDED TO A NORMAL RATION Periods of Five Days Each — Grams Nitrogen Phosphorus (P2O5) Ration Pood Urine Feces Balance Food Urine Feces Balance L 300 g-m. meat; 450 g-m. dried 17.96 17.72 2.45 —2.21 2.43 1.972 0.675 -0.217 bread; 30 g-m. butter; 100 gm. sugar; 1600 c.c. tea. 2. Same as above plus mono- and 17.96 16.62 2.33 -0.99 3.19 2.044 1.337 -0.191 di-calcium phosphates, 0.24 and 0.52 gm. P205- Fore-period, constant diet for 5 days. Same as above plus 0.593 gm. P2O5 17.96 15.85 2.40 -0.29 2.43 1.916 0.643 -0.129 17.96 16.96 2.11 —1.11 3.02 2.272 0.853 -0.102 as calcium glycerophosphate. Same as above without added 17.96 16.24 2.42 -0.70 2.43 1.932 0.683 -0.185 phosphorus. Same as above plus 1 gm. per day 17.96 16.94 2.20 -1.18 3.43 2.100 1.209 +0.121 P2O5 as calcium phytate. Age of subject, 3 years. In this experiment the phosphates and the glycerophosphate were not well retained, though they did serve slightly to reduce the phosphorus loss. Calcium phytate, however, brought about de- cidedly improved phosphorus retention and a positive phosphorus balance. The amount of phosphorus taken in this form, however, was much greater than in the other cases, so that we do not have an entirely fair basis for comparison. In consideration of the char- acter of the basal ration, which must have been very low in calcium, it would also be important to know the calcium contents of the phosphatic supplements. Gouin and Andouard (1905-6), in calf -feeding experiments, used potassium phosphate, bone phosphate, sweetbreads, thymus and protylin as sources of phosphorus. The nature of the data did not give a basis for a close estimate of the values of these com- pounds. The authors make the statement, however, that they found the bone phosphates more "digestible" than vegetable phos- phates. In this connection, we would mention the fact that we have no means of determining the digestibility of such com- pounds. Tunnicliffe (1906) conducted balance experiments with two healthy children, aged, respectively, 2 years, and 2 years and 10 months, comparing organic and inorganic phosphorus compounds, and their effects on nitrogen metabolism. A part of the data are as follows: PHOSPHORUS METABOLISM 325 AVERAGE DAILY NITROGEN AND PHOSPHORUS BALANCES WITH HEALTHY CHILDREN RECEIVING ORGANIC AND INORGANIC PHOSPHORUS WITH THE FOOD— Grams Subject Period Duration of periods in days N Food Urine Feces Balance P Food Urine Feces Balance Diet Boy, 2 years old Fore-period 2 5.95 2.21 0.98 +2.75 0.69 0.13 0.18 +0.38 Mixed normal diet. Organic phos- phorus period 6 8.80 3.93 0.78 +4.08 0.96 0.27 0.20 +0.49 Same plus 20 gm. sanatogen. Inorganic phosphorus period 3 5.76 3.39 0.88 +J.48 Same as first plus 1 gm. Ca3 (P04>2. Girl, 2 yrs. 10 mos, old Fore-period 3 6.53 3.22 0.75 +2.56 0.73 0.31 0.15 +0.27 Mixed normal diet. Girl, 2 yrs. 10 mos. old Organic phos- phorus period 6 9.14 4; 70 0.72 +3.72 0.99 0.43 0.12 +0.44 Same plus 20 gm. sanatogen. Girl, 2 yrs. 10 mos. old Inorganic phosphorus period 3 5.80 2.62 0.76 +2.42 0.87 0.28 0.24 +0.35 Same as first plus 1 gm. Ca3 (P04)2. From these data Tunnicliffe concluded (1) that in the healthy- child the addition of an organic phosphorus compound to the diet is followed by an increase in the amount of phosphorus assimilated by and retained in the body; (2) that the addition of an organic phos- phorus compound to the diet of children increases the amount of ni- trogen assimilated; (3) that the addition of Ca,(P0 4 ) 2 to the food did not increase the amount of phosphorus assimilated or retained by the child, nor did this compound exert any favorable influence upon the assimilation of the nitrogen of the food ; and (4) that the phosphorus contained in the sodium glycerophosphate of casein (sanatogen) is practically entirely assimilated by the body. We would suggest that the periods are too short to give results of great value, and that, since the intake of nitrogen and phosphorus was greater in the organic phosphorus period than in the inorganic phosphorus period, these data do not furnish a fair basis for a com- parison of the nutritive values of these compounds. At the same time it seems probable that the phosphorus of sanatogen is more useful than the same amount of phosphorus in Ca 3 (P0 4 ) 2 . 326 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 LeClerc and Cook (1906) conducted a series of nitrogen and phosphorus balance experiments with three rabbits and a dog, com- paring phytin and inorganic phosphates, in twenty-six five-day peri- ods. The phytin was prepared from wheat bran. This compound and a mixture of disodium hydrogen phosphate and dihydrogen sodium phosphate were added to normal and to phosphorus-poor rations. From the considerable weight of the subjects it would appear that, if not mature, they were at least beyond the period of most active growth. During these experiments the live weights were practically constant ; in half of the periods there was loss of weight, and in none of them was there marked gain in weight. These con- ditions, together with the brevity of the periods, were unfavorable to the demonstration of such differences in value as exist between these nutrients. The authors state that the nitrogen retention was generally lowered by the addition of inorganic phosphates, when fed with a normal food ; and that the organic phosphorus compound from wheat bran is more favorable to nitrogen and phosphorus retention than is inorganic phosphorus. See also Cook (1909), p. 312. Egbert Koch (1906) investigated the question of synthesis of phosphorus compounds from edestin and inorganic salts by the hu-! man being, by means of a feeding experiment on a man. The re- sults are shown in the following table. The subject was in a normal state of nutrition throughout the investigation. AVERAGE DAILY NITROGEN, CALCIUM AND PHOSPHORUS METABOLISM OF A MAN ON RATIONS DIFFERING IN ORGANIC PHOSPHORUS CONTENT Periods of Four Days Each — Grams Rations N Intake Urine Feces Balance P2O5 Intake Urine Feces Balance CaO Intake Urine Feces Balance Weight Initial Final Difference Diff . per day Kg. 100 gin. oatmeal; 1500 c.c milk; 6 gm. NaCl; 20 gm. cane sugar; 100 gm. butter: 500 c.c. tea; 300 c.c. wine; 150 gm. egg white. 100 gm. oatmeal; 48.5 gm. edestin; 6 gm. NaCl; 70 gm. cane sugar; 180 gm. butter; 500 c.c. tea; 300 c.c. wine; 150 gm. egg white; 13.36 gm. Na2HP04; 4.12 gm. CaCO.3; 1.5gm.CaHP04. 13.46 10.31 1.10 +2.05 4.43 2.20 1.90 +0.34 2.991 0.137 2.500 +0.354 63.9 63.0 -0.9 -0.22 12.97 10.10 0.97 +1.65 4.44 2.98 1.42 +0.04 2.997 1.420 3.038 -0.183 63.0 63.7 +0.7 +0.18 Calculated from author's data. In the first ration the phosphorus was present to a considerable extent as phosphoprotein, that is, as casein. In the second ration the phosphorus was present principally as inorganic phosphates. PHOSPHORUS METABOLISM 327 The intake of nitrogen, phosphorus and calcium was maintained practically constant, though the nitrogen intake in the second peri- od was a little less than in the first. In the inorganic phosphorus period the nitrogen storage was less, as also was the phosphorus storage, while the calcium balance changed from +0.354 gm. to — 0.183 gm. From these data the author concluded that "the view that the human organism cannot effect a synthesis from phosphorus-free protein and inorganic phos- phorus salts receives further support from these experiments." Hart, McCollum and Fuller (1909) studied the role of inorganic phosphorus in the nutrition of swine by feeding, slaughter and bal- ance experiments. The daily rations in their first experiment were as follows: DAILY RATION AND AVERAGE GAIN IN WEIGHT Lot 1 Pounds Lot 2 Pounds Lot 3 Pounds Lot 4 Pounds Lot 5 Pounds 1.24 0.65 0.26 0.048 1.20 0.63 0.25 0.046 0.077 1.22 0.64 0.26 0.047 0.038 1.26 0.27 0.67 Ground oats 0.67 Sugar-salt mixture (200 gm. sugar; 100 gm. each NaCl, MgCl2, K2 SO4) Wheat middlings Precipitated calcium phosphate (Ca3 (P04)2 + CaHP04> 0.67 Oil meal 0.22 Grams 1.12 Grams 6.57 Grams 3.84 Grams 5.40 Grams 5.45 Pounds 28.33 Pounds 52.6 Pounds 52.6 Pounds 59.0 Pounds 61.5 There were, all told, 16 pigs in this experiment. The experi- ment covered 95 days, after which one animal from each lot was killed, and some parts were analyzed for dry matter, calcium and phosphorus. The calcium and phosphorus data are as follows : COMPOSITION OF PARTS OF PIGS- -Air-Dry Basis — Percents Bone ash Blood Leg muscle Liver Brain P Ca P Ca P Ca P Ca P Ca Basal, lotl Inorganic, lot 2 . . . Whole bran, lot 4 . . Normal, lot 5 18.48 18.26 18 00 18.20 37.16 36.91 37.12 37.23 0.24 0.31 0.33 0.28 0.035 031 0.038 0.026 0.93 0.81 0.77 0.78 0.030 0.029 0.025 0.041 1.43 1.34 1.35 1.27 0.020 0.030 0.066 0.030 1.49 1.57 1.54 1.43 0.08 0.10 0.09 0.09 The skeleton of one animal from each lot, except the third, was dissected out and subjected to study as indicated by the following data: 328 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 DATA ON SKELETON OF ONE ANIMAL FROM EACH LOT OF EXPERIMENT I (EXCEPT LOT 3) Weight of skeleton, gm Weight of animal, lbs Breaking strength of thigh bone, lbs. per sq. mm Diam. thigh bone at centre, mm Sp. gr. of bones Ash (thigh bone) Basal Phosphate Whole bran 1193 2371 1288 84 123 102 0.63 1.80 1.84 18.00 23.90 18.50 0.977 1.157 1.100 31 percent 55 percent 53 percent Standard 1609 138 1.69 22.00 1.192 46 percent Experiment II was similar to the above, except that near the end of the program one animal from each of five lots was put into a metabolism crate,. and subjected to a balance experiment of 5-12 days' length. The whole program covered 123 days. In this experiment the rations used were as indicated below : Lot 1 — Low phosphorus basal ration. " 2 — " " " " -f-P re dprtated calcium phosphate "3—" " " " +boneash " 4— " " " " +floats (rock phosphate) " 5 — Basal ration with whole bran substituted for washed bran. " 6 — Normal foods. The summarized balance data are as follows: AVERAGE DAILY PHOSPHORUS BALANCES Phosphorus retention Basal Precipitated phosphate Bone ash Floats Whole bran Aver, gain Total Total Total Inorganic in weight phosphorus phosphorus phosphorus phosphorus of lot intake in urine 'in feces in feces Pounds Grams Grams Grams Grams 32 1.08. 0.02 0.52 0.168 42 5.02 0.378 2.45 35 4.08 0.281 2.26 43 4.26 0.253 1.65 58 5.65 0.666 2.66 2.47 0.53 2.22 1.54 2.35 2.36 One animal from each lot in this experiment was killed, and be- low are data from the examination of the skeletons. DATA ON THE SKELETON OF ONE ANIMAL FROM EACH LOT OF EXPERIMENT II (EXCEPT LOT 6) Weight of skeleton, gm , Weight of animal at slaughter, lbs Breaking strength of thigh bone, lbs. per sq. mm Diam. thigh bone at centre, mm Sp. gr. bone Ash) percent (thigh bone) 870 77 0.87 16.0 0.984 33 Precipitated phosphate 950 87 1.70 16.0 1.15 46 950 58 1.77 15.5 1.12 53 Floats 1495 82 1.65 20.00 1.19 57 Whole bran 850 87 1.86 17.00 1.14 54 PHOSPHORUS METABOLISM 829 The authors' conclusions are as follows : "1. On the ration extremely low in phosphorus, pigs made as large gains up to 75 or 100 pounds when starting at weights of from 40 to 50 pounds as animals receiving an abundance of this element. After reaching this point loss of weight began, followed by collapse. "2. When such low phosphorus rations as induced the above symptoms were supplemented with calcium phosphates, no un- toward results appeared. Animals fed a low phosphorus ration, supplemented with inorganic phosphates, made as vigorous a devel- opment as others receiving their phosphorus supply wholly in or- . ganic form. "3. Precipitated calcium phosphates, a mixture of di- and tri- calcium phosphates, gave no better results than did floats, a crude tri-calcium phosphate. "4. Phytin as the supply of phosphorus gave no better re- sults than the inorganic phosphates. "5. A young animal of 40 pounds weight receiving inorganic phosphates, together with other salts as supplementary to a ration very low in mineral constituents, grew to be an animal of 280 pounds weight, bore a litter of fairly vigorous pigs, which on the same ra- tion completed the cycle back to 80 pounds, while animals on the same ration less the inorganic phosphates collapsed in three months, with loss of weight accompanied by a loss of the use of their limbs. "6. Determinations of calcium and phosphorus on the prin- cipar organs and tissues of the animals on the low phosphorus ra- tion showed that they maintained the proportion of these elements constant and comparable to that of normally fed pigs. "7. The percentage of ash in the skeleton of pigs on the de- pleted phosphorus ration was reduced to nearly one-half that of pigs receiving a normal ration, or a phosphorus-poor ration supplemented by an inorganic phosphate. "8. The marked reduction in the quantity of ash of the bones of the animal receiving an insufficient supply of calcium phosphates, together with the ability of the animal to build up a skeleton very rich in calcium phosphate when an abundance of the latter is sup- plied in inorganic forms, strongly points to the possession of a syn- thetic power by the animal which enables it to convert inorganic forms of phosphorus into the organic forms demanded by its body. "9. When animals were starving for phosphorus, they drew this element from the skeleton, but removed calcium and phospho- rus in the proportions found in tri-calcium phosphate. 330 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 "10. The daily phosphorus supply for a 50-pound growing" pig* should be at least 3 gm. A supply of 4 or 5 gm. is probably a safer quantity. "11. The data furnish no positive evidence of the synthesis of nucleo-proteids or other phosphorus-bearing complexes from inor- ganic phosphates in the animal body." On these conclusions we would offer the following comments: The basal ration used in these experiments was exceedingly low in calcium. To have furnished a wholly satisfactory basis for the comparison of the phosphorus compounds studied there should have been no question as to calcium deficiency limiting the usefulness of any of these preparations. In this light, the phosphorus of floats had the greatest advantage, since it was associated with an excess of calcium; the phosphorus of precipitated phosphate came next in order of relation of calcium to phosphorus, while the wheat bran phosphorus was associated with an exceedingly small proportion of calcium. The authors' statement that the calcium and phosphorus con- tent of the principal organs of the body was maintained constant is not sustained by their analytical data. True, there is a marked tendency toward the maintenance of constancy of composition, but, at the same time, there are, in the case of each of the five important parts analyzed, variations in the content of calcium and phosphorus, which, reckoned on the basis of the total amount of the constituent present, constitute marked deviations from the normal. The authors' conclusion No. 8, citing the utilization of inor- ganic calcium phosphate in the building up of the skeleton as an evi- dence of "the possession of a synthetic power by the animal which enables it to convert inorganic forms of phosphorus into the organic forms demanded by its body," seems not to be warranted. Con- clusion No. 11, which claims "no positive evidence of synthesis of nucleo-proteids or other phosphorus-bearing complexes from inor- ganic phosphates in the animal body," is more to the point. These experiments seem not well planned to bring out differ- ences in usefulness of organic and inorganic phosphorus. They do make emphatic one point, however; that the amount of organic phosphorus absolutely necessary to the life of the pig is, at the most, not a large part of the total phosphorus requirement. Holsti (1910) attempted to settle the question of organic phos- phorus synthesis by balance experiments on himself; but, as the rations differed in total phosphorus content by as much as the amounts added to some of them as inorganic phosphorus, there was PHOSPHORUS METABOLISM 331 in reality no basis whatever, for judgment as to the matter of syn- thesis of organic phosphorus compounds. This much was demon- strated, however, that if the total phosphorus of the ration is ad- equate, a very small intake of organic phosphorus (about a third of a gram sufficed in one case) will permit of phosphorus retention, even coincident with nitrogen loss. Holsti's experimental periods, 3-6 days in length, were too short really to settle anything. Under the title "Nuclein Synthesis in the Animal Body," McCollum (1909) published results of feeding experiments with rats on a basal ration composed of phosphorus-free foods, to which were added phosphorus-containing substances to be compared. Among the foods used were edestin, zein, corn starch, wheat starch, butter fat, bacon fat, milk sugar, glucose, cane sugar, and cholesterin. Many flavoring extracts were used in an effort to make the food palatable. Milk ash, calcium phosphate, sodium chloride and ferric chloride were also used in the basal ration, and casein, and hydrolyzed beef and liver were added to this ration in some periods. McCollum concludes that, other things being satisfactory, all the phosphorus needed by an animal for skeleton, nuclein, or phos- phatid formation, can be drawn from inorganic phosphates; also that the animal has the power to synthesize the purin bases neces- sary for its nuclein formation from some complexes contained in the protein molecule, and does not necessarily use purin bases of exogen- ous origin for this purpose. McCollum also placed much emphasis on the idea of palatability as of vital importance in nutrition. Rats 1, 2 and 3 were fed a normal ration, and gained in weight one gram or more per day. Rats 4, 5 and 6 were fed on the organ- ic-phosphorus-free ration and lost weight rapidly. Rats 7, 8 and 9 received the same ration as the above, but, in addition, protein-free hydrolyzed beef and liver. Rats 8 and 9 lost 27 and 28 grams, re- spectively, in 106 days. Rat 7 gained 23 grams in 53 days. It was put into the experiment on June 12, weighing 153 grams. By June 23 it weighed 168 grams. On July 28, thirty-five days later, it weighed 170 grams, though between these dates it had weighed as much as 180 grams. On Aug. 4 it was removed from the experi- ment at a weight of 176 grams. Thus far, then, five out of six rats had failed to maintain their weight; the sixth had made a gain in weight. Rats 18, 19 and 20 were younger than those used thus far. They were fed on the same ration as rats 4, 5 and 6, that is, with- out organic phosphorus. They all gained in weight. At the be- ginning of the test they weighed 37, 35 and 46 grams. In 127, 56 332 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 and 56 days, respectively, they gained 39, 6 and 14 grams, respect- ively, in weight. The one rat which more than doubled its weight was analyzed, along with certain others, for dry substance in the body, fat in the dry matter, and weight and ash of skeleton. (See table below.) Two rats, Nos. 15 and 16, were fed on the same ration plus casein, and gained in weight without difficulty. COMPOSITION OF RATS USED IN EXPERIMENTS WITH VARIOUS RATIONS Fat- and Dry tis- Ether Ash of Skeleton water-free Num- Weight Skeleton sues less extract skeleton tissues Ration ber of rat Grams Grams skeleton Grams Grams Grams Percent of live weight Percent of live weight 1 2 10 147 157 34 6.67 6.50 1.33 38.0 45.0 9.5 8.89 10.80 3.25 3.79 3.85 0.68 4.54 4.14 3.91 19.8 21.79 18.39 Inorganic phosphorus: later phosphorus-free 7 102 7.14 25.5 3.42 4.49 7.00 21.64 Inorganic phosphorus 4 135 9.00 34.8 7.00 4.28 6.66 20.59 • < << 5 96 5.78 22.0 4.01 3.02 6.02 18.94 • • < • 6 88 7.50 20.5 3.63 3.18 8.52 19.17 < • • « 8 103 6.03 21.5 2.67 3.52 5.85 18.28 <> i< 9 78 4.58 14.5 0.86 2.77 5.79 17.49 18 76 4.07 17.5 3.40 2.14 5.36 18.55 By comparing the figures from No. 18, the one rat which doubled its weight, with those from Nos. 1 and 2, which were fed on normal foods, one may satisfy himself by computation that the gain in weight of this one rat could not be made up entirely of fat, water, intestinal content and skeleton. The extent to which the gain in weight was dependent on translocation of constituents was not de- termined. There were no determinations of moisture in the tis- sues, nor of nuclein or lecithin phosphorus, and there were no weights of foods taken. The author states in a letter that there was opportunity for the rats to eat their own feces, a fact which we have found enters in most important ways into the determination of results in such work, apparently first through allowing of repeated use of the lecithin of the bile residues, and second through allowing the animal to avail itself of results of the synthetic capacities of intestinal bacteria. The nature of the analytical data hardly warrants the author's assumption that constancy of composition of the tissues was main- tained, and his reference to the work of Hart, McCollum and Fuller with swine, as sustaining the assumption, is not convincing. One rat was so confined that the excreta could be collected, and a ration was given it of phosphorus-free foods. The amounts eaten were not determined. As an average of 8 days' excretion, PHOSPHORUS METABOLISM 333 terminating four days before the death of the rat, the author sub- mits the figure 0.0063 gm. phosphorus as the daily elimination of this rat, which weighed 180 gm. at its maximum, and 102 gm. at its death. With this figure as a basis, the author computes that dur- ing the course of his experiments certain rats must have entirely changed the phosphorus content of the body, the skeleton excepted. He assumes that the rats did not draw phosphorus from the bones from the fact that the skeletons increased in weight, while the ani- mals lost in weight; but there was no evidence presented to show that there was not withdrawal of organic phosphorus from the skel- eton coincident with the deposit of inorganic phosphates. The general failure of the older rats even to maintain live weight on the organic-phosphorus-free diet, taken together with the ability of the younger rats to gain in weight on this ration, implies either that the older animals! did not eat food enough, or else that the ability of the rat to gain in weight on rations which are free from organic phosphorus compounds, if possessed at an early age, becomes less marked with advance in age. Gregersen (1911) conducted an extensive series of balance ex- periments with rats, the object being to study phosphorus metab- olism, especially the possibility of the synthesis of organic phos- phorus compounds from phosphorus-free edestin and inorganic phosphates. The work includes in all 48 experiments involving 170 balances. The method of handling the rats was that of Henriques and Hansen (Zeitschr. physiol. Chem., 1905, XLIII, 418). The data reported include analyses of food, urine and feces, but no analyses of the rats themselves. The author's tables have been transcribed without material change but for the omission of the separate urine and feces data. His last table we have omitted. Gregersen concludes that the organism is able to synthesize organic phosphorus compounds from phosphorus-free organic ma- terials and phosphates ; in feeding on a nitrogen-free ration the or- ganism is not affected by the presence or absence of phosphates; when the organism is held in nitrogenous equilibrium on a phospho- rus-free albumin-containing ration, the excretion of phosphorus falls off quite considerably, under some circumstances becoming as low as 1/50 — 1/60 the amount of the coincident nitrogen excretion; on a phosphorus-free, albumin-containing ration, which contains cal- cium and magnesium salts, the rats excrete only a minimum amount of phosphorus through the urine, less than one-tenth of that quan- tity which is at the same time excreted in the feces ; on a phospho- 334 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 rus-free ration containing albumin but no calcium or magnesium salts more phosphorus is usually excreted through the urine than through the feces, likewise on a phosphorus-free albumin-free diet which contains calcium and magnesium. It is not claimed that phosphorus-free nitrogenous compounds and phosphates are of equal value with phosphorized nitrogenous compounds. From a superficial inspection of the data one is inclined to agree with Gregersen's conclusion as to the synthesis of organic phospho- rus compounds, and the facts may be as concluded by Gregersen, but, considering the mass of conflicting testimony which has been reported on this subject, we must weigh the evidence with care. In certain respects the proof of organic phosphorus synthesis appears incomplete or imperfect. There were a great number of experi- ments ; but the periods were very short. The data would be much more satisfactory if they included determinations of the amounts of organic and inorganic phosphorus in the bodies of the experimental animals and in controls. Our knowledge of the capabilities of the animal to transfer and to transform phosphorus from one tissue or compound to another, under the stress of necessity, is too limited to warrant unreserved statements as to synthesis of individual com- pounds when such deductions rest upon only the storage of the nu- trient and gain in live weight during periods of a very few days. Re- garding certain points one is left in doubt since the percentage com- position of the salt mixtures was not stated. Turning to Table I, p. 335, these rats were taken from a bread diet, and, without an intermediate period on the experimental ration to free them from the effects of the ration of white bread, but after a fast from one afternoon until 9 :00 o'clock next morning, were put into the balance experiments, in which an abundance of protein in the form of edestin, and of phosphorus in the shape of phosphates, were provided. Now bread is deficient in protein and in phospho- rus, and extremely low in calcium, so low that it could hardly have failed, with these growing rats, to have caused a loss of calcium, and therefore probably also of phosphorus, from the bones, if indeed the rats received nothing but bread. The abrupt change to the ex- perimental ration found the rats much in need of the protein, cal- cium and phosphorus which this ration provided, and there was con- sequently, for a short time, a marked storage of these constituents. This apparent prosperity, however, was not sustained. PHOSPHORUS METABOLISM 335 TABLE I. METABOLISM EXPERIMENTS ON GROWING RATS ON A DIET CONTAINING NO ORGANIC PHOSPHORUS— Daily Balances No. of investiga- tion and weight of subject in grams Length of period in days Food per day Grams Food N Milligrams Food P Milligrams N balance Milligrams P balance Milligrams N:P retained 1 26 1 28 5 5 2.76 2.04 71 52 9.7 7.2 23 6 3.5 1.4 6.6 4.3 2 36 2 38 2 38 5 5 5 3.59 2.58 2.58 92 66 66 12.6 9.1 9.1 32 10 4 4.8 2.3 2.1 6.7 4.3 1.9 3 49 3 54 3 55 4 4 5 5.20 4.70 3.71 134 121 95 18.3 16.5 13.1 42 17 6 7.1 2.6 2.2 5.9 6.5 2.7 4 43 4 48 4 49 4 50 4 45 4 4 5 5 5 4.95 3.56 2.85 2.02 2.48 127 91 73 52 64 17.4 12.5 10.0 7.1 8.7 42 20 10 —24 —11 7.3 2.5 1.5 —1.4 0.0 5.8 8.0 6.7 5 44 5 50 5 51 5 5 5 4.12 3.51 2.92 106 90 75 14.5 12.4 10.3 31 12 — 4 6.1 1.8 -0.5 5.1 6.7 6 63 6 71 6 66 6 65 6 6 6 6 4.57 3.21 3.19 2.91 117 83 82 • 75 16.1 11.3 11.2 10.2 35 3 1 3 3.9 1.3 1.2 1.9 9.0 2.3 0.8 1.6 7 60 7 68 7 62 7 62 5 6 6 ' 6 4.96 2.92 3.47 2.85 128 75 89 73 17.4 10.3 12.2 10.0 30 — 7 2 -10 4.0 0.3 0.9 0.5 7.5 2.2 8 58 8 63 8 68 6 7 7 5.38 4.50 3.83 138 116 98 18.9 15.8 13.5 37 26 6 4.3 4.6 3.3 8.6 5.7 1.8 The ration was composed of edestin 15 percent, sugar 30 percent, fat 42 percent, cellulose 5 percent, sodium phosphate 4= percent, and 4 percent of a salt mixture containing sodium, potassium, and calcium chlorides, sodium bicarbonate, magnesium oxide and iron sulphate. In these 8 series of balances there was in each case a progres- sive decline in the food consumed, and in the nitrogen and phospho- rus storage. These 8 experiments were 10-24 days in length. Three out of the 8 showed minus nitrogen balances, one in the period com- posed of the 13th-18th days, another during the 10th-15th days, and the third during the 5th-llth days. Two of these minus nitrogen balances were accompanied by negative phosphorus balances, and in the third the positive balance was but 0.0003 gm. In 6 cases out of the 8 there was a progressive decrease in the proportion of nitro- gen to phosphorus stored, at the same time that the storage of both elements also declined, as above noted. The daily nitrogen storage in the first period of 4-6 days was 23-42 mg. ; during the second period the storage was from — 7 to +0.26 mg., and in the third period from — 4 to +10 mg. The phos- phorus storage in the first period was from 3.5 to 7.3 mg., in the sec- ond, 0.3 to 4.6 mg., and in the third from — 0.5 to -f-3.3 mg. 336 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 These facts are probably due to the falling- off in food consump- tion, which, of course, would leave the rat with a smaller amount of protein, in excess of the maintenance requirement, available for storage, and also a smaller proportion of the total protein. The above-mentioned decrease in food consumption, if accompanied by a considerable storage of phosphorus in the bones might then account for the decreased proportion of nitrogen to phosphorus which was stored. In this connection we would observe that the palatability of a ration is very commonly a reflection of the usefulness of the food to the animal. The general results of this first series of balance experiments suggest great difficulty in the maintenance of normal conditions of growth. This test is the most thoroughgoing of any in the whole investigation, in that the periods were much longer than in subse- quent series ; also the rats were young, growing animals, and there- fore had less reserve material within the body with which to bridge over temporary nutritional deficiencies. In Table II, p. 337, we have' the second series of experiments. The periods were of three to five days duration. Here we have grown rats, fed with and without protein, and without organic phosphorus compounds. The nitrogen-free ration was composed of sugar, lard, cellulose and salts, including sodium phosphate. The ration contain- ing protein was composed like the above, except for the substitution of edestin for 15 percent of sugar. The albumin-free ration was the same as the nitrogen-free ration, except for the addition of 3 percent of Liebig's extract of beef. In Experiments 9-22 there are 18 changes, either from a nitro- gen-free ration, or from one in which the only nitrogen present was in meat extractives, to a ration where the sole albuminous nitrogen present was in edestin, or changes in the reverse direction. Sodium phosphate was present in all rations whether containing protein or without protein. In each of these 18 changes of food the nitrogen and phosphorus balances changed consistently from -f- to — , or from — to +> according to whether or not edestin was present; that is, there was in every case a storage of both nitrogen and phos- phorus in the presence of edestin, and in no case a storage of either element in the absence of edestin, even though the phosphorus was present in greater amount than when edestin was present. The loss of phosphorus during albumin-free periods was prob- ably due to the katabolism of nitrogenous tissues which contained phosphorus. Then with the introduction of edestin into the ration, the phosphorus-containing proteins were protected, and there oc- curred both nitrogen and phosphorus retention. One can only spec- ulate as to the form in which the phosphorus was retained. The PHOSPHORUS METABOLISM 337 average daily loss of P on the nitrogen-free diet during the whole series was 7.0 mg. The daily retention on the edestin ration aver- aged 2.3 mg. There was not conspicuous uniformity in the ratio of nitrogen to phosphorus either in the loss or the retention of these elements. TABLE II. METABOLISM EXPERIMENTS ON MATURE RATS WITH AND WITHOUT PROTEIN AND WITHOUT ORGANIC PHOSPHORUS— Daily Balances 1 No. of investiga- tion, and weight of subject in grams Length of period in days Food per day Grams Food N Milli- grams Food P Milli- grams N balance Milli- grams P balance Milli- grams N:P retained or lost Ration 9 101 9 107 3 3 7.53 7.53 197 26.4 26.6 -63 61 -4.7 2.9 13.4 21.0 N-free Edestin 10 £39 10 224 10 228 3 3 3 10.00 10.00 8.50 261 35.0 35.3 29.8 —137 14 —107 -12.5 0.9 -9.0 11.0 15.6 11.9 N-free Edestin N-free 11 152 11 146 3 3 10.00 7.80 204 35.0 27.5 —107 33 -7.3 2.0 14.7 16.5 N-free Edestin 12 195 12 188 12 196 3 3 3 9.00 9.00 9.00 235 31.5 31.8 31.5 -81 51 -77 -6.1 4.6 -4.4 13.3 11.1 17.5 N-free Edestin N-free 13 125 13 116 3 4 6.50 5.69 149 22.8 20.1 -59 23 -6.6 2.0 8.9 11.5 N-free Edestin 14 138 14 1 131 14 137 14 vl36 3 3 3 3 6.00 6.00 7.00 5.67 157 148 21.0 21.2 24.5 20.0 -57 17 -57 26 -4.9 0.8 -2.1 1.3 11.6 21.4 27.1 20.0 N-free Edestin N-free Edestin 15 160 15 157 3 4 8.00 6.14 160 28.0 21.7 —63- 27 -6.0 2.4 10.5 . 11.3 N-free Edestin 16 58 16 55 3 4 3.50 3.50 91 11.5 11.6 -44 18 -3.6 2.2 12.2 8.2 N-free Edestin 17 58 17 55 3 4 3.55 .3.18 83 11.7 10.5 -45 13 -4.7 1.8 9.6 7.2 N-free Edestin 18 72 18 ' 72 3 4 5.00 2.93 76 16.5 9.7 -37 1 -2.0 0.3 18.5 3.3 N-free Edestin 19 290 19 283 19 288 19 307 2 1 3 1 12.00 12.00 12.00 12.00 40 304 304 304 49.7 39.3 39.3 39.3 -171 f. -16.2 5.1 10.6 8.3 Albumin-free Edestin 20 175 20 169 20 172 20 174 2 1 2 1 8.00 8.00 8.00 8.00 26 202 ' 202 202 33.1 26.2 26.2 26.2 —107 -8.9 0.7 12.0 26.4 Albumin-free Edestin 21 320 21 317 21 322 21 335 2 1 3 1 12.00 12.00 12.00 12.00 40 304 304 304 49.7 39.3 39.3 39.3 —133 -12.0 3.5 11.1 14.4 Albumin-free Edestin 22 247 22 244 22 251 22 262 2 1 2 2 11.00 11.00 11.00 11.00 36 278 278 278 45.5 36.0 36.0 36.0 -108 J- 49 -8.0 4.5 13.5 10.9 Albumin-free Edestin The nitrogen- free ration was composed as follows: Sugar 45 percent, lard 42 percent, cellulose 5 percent, and salts, including sodium phosphate, 8 percent. The edestin ration was composed as above except that 15 percent of sugar was replaced by edestin. The albumin-free ration was composed like the above nitrogen-free ration except for the addition of 3 percent of Liebig's beef extract. 888 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The experiments in Table III (below) compare powdered beef and sodium phosphate with edestin and sodium phosphate. In this series the edestin period in every case follows immediately after a meat period, without intermediate feeding, certain residua from the meat feeding- doubtless holding over into the edestin periods in such way as to mix results, and to favor the utilization of the edestin ra- tions. This advantage was increased by the fact that the prelim- inary meat period was 10-14 days long, the balances covering, on an average, the last 8.3 days of this period, while the following edestin periods were, on an average, only 3.8 days long. In spite of these manifest advantages to the edestin rations, the meat rations made a slightly better showing in the phosphorus balances. TABLE III. METABOLISM EXPERIMENTS WITH GROWN RATS COMPARING POWDERED BEEF WITH EDESTIN— Daily Balances No. of tion, an subject investiga- d weight of in grams Length of period Food per day Food N Milli- FoodP Milli- N balance Milli- P balance Milli- N:P retained Ration in days Grams grams grams grams grams 23 191 2 7 181 22.8 2.5 ] 23 187 2 7 181 22.8 is 1.3 1 Flesh 23 23 190 192 3 3 7 7 181 181 22.8 22.8 [ 30 2.5 12.0 f J 23 23 191 194 1 3 6.93 6.93 181 181 23.1 23.1 | 17 1.0 17.0 1 f Edestin 24 161 2 6.0 155 19.5 0.1 ) 24 158 2 6.0 155 19.5 1 'i [ Flesh 24 158 3 6.0 155 19.5 i 24 24 158 161 1 3 5.94 5.94 156 156 19.8 19.8 t 8 \ Edestin 25 122 2 5.00 129 16.3 -2.0 1 25 120 2 5.00 129 16.3 -7 -0.4 I Flesh 25 121 3 5.00 129 16.3 \ » 0.8 10.0 [ 25 123 3 5.00 129 16.3 J 25 25 122 121 1 3 4.95 4.95 130 130 16.5 16.5 f » 0.8 18.8 t Edestin 25 122 4 5.00 129 16.3 18 0.5 36.0 Flesh 26 195 3 7.00 179 22.7 -8 1.5 l Flesh 26 188 3 7.00 179 22.7 -3 1.9 y 26 186 3 6.93 180 22.8 -7 1.2 Edestin 27 138 4 5.50 140 18.1 -10 1.0 ! Flesh 27 138 4 5.50 140 18.1 -7 1.3 27 136 3 5.45 141 18.0 —5 0.5 Edestin 28 124 4 5.50 140 18.1 -14 3.3 \ Flesh 28 124 3 5.50 140 18.1 —13 3.1 28 123 5 5.45 141 18.0 -11 1.0 Edestin The flesh ration was composed as follows: Powdered beef 20 parts, sugar 25 parts, lard 44.5 parts, cellulose 5 parts, sodium phosphate 2 parts and salt mixture 3.5 parts. The edestin ration was composed as follows: Edestin 14.93 parts, sugar 25 parts, lard 46.8 parts, cellulose 5 parts, sodium phosphate 3.76 parts and salt mixture 3.5 parts. The two rations had like calorific value and like content of nitrogen, phosphorus, fat, cellulose and salts. In Table IV, p. 339, we have a continuance of the tests reported in the preceding table except that the food per gram of live weight of the edestin rats was intentionally increased by the addition of a certain amount of fat to the ration, the object being to determine if PHOSPHORUS METABOLISM 339 with sufficient calorific value the ration free from organic phospho- rus would cause more phosphorus retention than the ration contain- ing organic phosphorus. Gregersen does not mention the coinci- dent reduction in the amount of food eaten per gram of live weight in the meat periods. The food per gram of live weight of the rats on the flesh ration was in this series one gram per 28.4 grams of live weight, while in the preceding series this ration was eaten in the amount of one gram per 25.9 grams of live weight. With these ad- vantages, together with those of influence of previous feeding and shorter duration of the edestin periods, as previously noted, the ra- tion which was free from organic phosphorus appeared to make a better showing in the nitrogen and phosphorus balances than did the flesh ration. The length of the meat period was in each case 12 days, during the last 6 of which balances were taken, and of the suc- ceeding edestin period the average length was 3.7 days. There were, therefore, four differences of experimental conditions between the two rations compared, differences as to calorific value of the rations, as to length of the experimental period, as to advantage from previ- ous feeding, and as to nature of the phosphorus compounds present; which of course makes it impossible to say what factor has produced the differences in results. TABLE IV. METABOLISM EXPERIMENTS ON GROWN RATS COMPARING POWDERED BEEF WITH EDESTIN Daily Balances No. of investigation, Length Food per day Food N Food P N balance p balance and weight of subject of period Ration in grams in days Milli- Milli- Milli- Milli- Grams grams grams grams grams 29 212 6 8 268 29.9 -35 -2.5 Flesh 29 210 1 8+1 267 29.4 y 33 29 220 2 8+2 267 29.4 2.2 Edestin + fat 29 220 1 6.8+1 227 24.9 ! 29 29 220 219 2 2 8 8 268 268 29.9 29.9 +1.0 0.1 Flesh 30 307 6 10 335 37.4 8.0 1.5 Flesh 30 313 1 10+2 334 36.7 1 30 315 2 10+2 ' 334 36.7 y 52 4.5 Edestin + fat 30 329 1 10+3 334 36.7 1 30 336 4 10 335 37.4 10 1.9 Flesh 31 220 6 8 268 29 .'9 1 -1.6 Flesh 31 31 226 232 1 2 8+1 8+1.5 267 267 29.4 29.4 ) » 0.3 Edestin + fat 31 237 3 8 268 29.9 \- —1.3 31 238 2 8 268 29.9 Flesh The flesh ration was composed as follows : Powdered beef 25 parts, sugar 25 parts, lard 40 parts, cellulose 5 parts, sodium phosphate 2 parts and salt mixture 3 parts. The edestin ration was composed as above except that edestin and sodium phosphate replaced the powdered beef, the nitrogen and phosphorus of the two rations thus being held alike, but in addition the edestin ration contained 1-3 gm. of lard in excess of the amount present in the flesh ration. 340 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Table V, below, sets forth results of a comparison of nitrogen- free, phosphorus-free rations with nitrogen-free, phosphorus-con- taining rations, the phosphorus in these latter rations being pres- ent exclusively as phosphates. The loss of nitrogen and phospho- rus, and the proportion of nitrogen to phosphorus in the loss, was not affected by the differences in these rations. TABLE V. METABOLISM EXPERIMENTS WITH MATURE RATS ON NITROGEN-FREE RATIONS WITH AND WITHOUT PHOSPHORUS Daily Balances No. of investiga- tion and weight of subject in grams 32 138 32 135 32 131 32 129 32 124 33 207 33 197 33 189 33 180 33 177 34 225 34 218 34 207 34 196 34 195 35 175 35 164 35 154 35 148 36 217 36 205 36 194 36 189 37 167 37 157 37 143 37 138 38 202 38 191 38 182 171 Length Food of per day period in days Grams 3 4 3 4 3 4 3 4 4 4 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 6 3 5.0 3 4.85 3 5.0 3 4.85 3 6.0 3 5.82 3 6.0 3 5.82 3 4.85 3 5.0 3 4.85 3 5.0 3 5.82 3 6.0 3 5.82 3 6.0 Food N Food P Milli- grams Milli- grams 15.3 15.3 15.3 15.3 15.3 12.7 12.7 15.3 15.3 12.7 12.7 15.3 15.3 N P balance balance Milli- Milli- grams grams -68 -6.3 -49 -5.3 -49 -4.7 -50 -5.0 -48 -4.1 -92 -10.6 -71 -8.2 -75 —7.1 -60 -5.3 -60 -4.9 -95 —10.2 -76 -6.4 -77 -7.4 -66 —6.0 -61 -6.6 -103 -8.9 -68 -5.1 -60 -5.2 -55 -4.3 -114 —10.5 -81 -6.8 -72 -6.7 -65 -6.3 -97 -9.1 -74 -5.5 -69 —5.4 -64 -4.8 -99 -9.4 -70 -6.1 -64 -5.1 -62 —3.8 N:P retained or lost Ration 10.8 9.2 10.4 10.0 11.7 8.7 8.7 10.6 11.3 12.2 9.3 11.9 10.4 11.0 9.2 11.6 13.3 11.5 12.8 10.9 11.9 10.7 10.3 10.7 13.5 12.8 13.3 10.5 11.5 12.5 16.3 N-free, P-free N-free, P-free N-free, P-contain- ing P-containing P-free P-containing P-free P-containing P-free P-containing P-free P-free P-containing P-free P-containing P-free P-containing P-free P-containing Nitrogen-free, phosphorus-free ration of investigations 35-38 composed as follows: Sugar 50 parts, lard 42 parts, cellulose 5 parts and salt mixture 3 parts. Nitrogen-free phosphorus-containing ration of investigations 35-38, the same as above except for the addition of 3 parts sodium phosphate. Gregersen also presents results of a comparison of nitrogen and phosphorus balances on phosphorus-free rations with and without edestin, and, further, results of a study of the effects of calcium and magnesium salts and sodium carbonate added to the phosphorus- free edestin ration, on the elimination of phosphorus. In the former experiment the phosphorus loss was about the same, whether edes- tin. was present or not, there being no phosphorus retention on the PHOSPHORUS METABOLISM 341 nitrogen-free ration. In the latter test the salts above mentioned were also without influence on the phosphorus loss, though the presence of calcium and magnesium in the intestine served to deflect into the feces a considerable portion of the food phosphorus other- wise excreted in the urine, and also of the metabolic phosphorus of the body, as evidenced by the phosphorus content of the feces from a ration free from phosphorus but containing calcium and magne- sium. Considering this set of experiments as a whole, then, the trend of the evidence suggests organic phosphorus synthesis by rats, but, as a demonstration of such synthesis, it must be regarded as not of conclusive character. As above noted Gregersen believes that the rats synthesized organic from inorganic phosphorus compounds, but he is careful not to claim that the inorganic compounds are as efficiently retained as the organic. Heubner (1911) conducted feeding experiments on young dogs comparing phosphates and lecithin as sources of phosphorus for the growing organism. The dogs had been kept on a low-phosphorus diet until their need for phosphorus was acute. Lecithin appeared much superior to phosphates as a source of phosphorus for growing dogs. Shackell (.1911) studied phosphorus metabolism in the early cleavage of the echinoderm egg. He found no evidence of a syn- thesis of nuclear material from alcohol-soluble constituents of the cytoplasm between the 2-4 celled stage and the blastula stage. Fingerling (1912a) demonstrated that ducks, on a ration which is low in organic phosphorus, produce eggs of normal content of leci- thin and nuclein phosphorus. He concludes that the animal organ- ism possesses the ability to cover its requirement of phosphoric acid for the formation of lecithin and nuclein substances just as eas- ily and completely with inorganic phosphates as with organic phos- phorus compounds. This conclusion could be justified, in so positive a form, only by the use of a ration free from organic phosphorus, and by demon- strating with this ration that the original content of the body and of its parts was maintained without loss of organic phosphorus. J. L. Smith and W. Mair (1912) studied the development of lipoids in the brain of the dog. With reference to the origin of the phosphatid, cerebrosid and cholesterin of the brain they conclude, from the very low content of the mother's milk in these compounds, in connection with the considerable daily deposition of the same in the brain of the suckling (0.045 gm. phosphatid, 0.007 gm. cere- brosid and 0.015 gm. cholesterin), that these compounds are syn- thesized from other substances present in the milk. 342 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 McCollum, Halpin and Drescher (1912) studied lecithin syn- thesis in the hen, by feeding a ration which was very low in lecithin, and determining the lecithin content of the yolks of the eggs laid. The hens also were weighed. The hens were kept from Oct. 31 un- til April 15 on a ration containing 30 percent of skim milk powder and 70 percent of polished rice which had been twice extracted in boiling alcohol for 20-30 minutes. From Oct. 31 until Jan. 30 the three hens had gained, respectively, 1290, 2013 and 1588 grams, or 33, 36.4 and 34.1 percent in live weight. They were assumed not to have lost lecithin during this time, or later while laying eggs. Dur- ing the time from Jan. 30 to April 15 fifty-seven eggs were laid. Their yolks contained 27.65 gm. of phosphorized fats per hen. The food consumption from Oct. 31 until March 1 averaged 58.5 grams per hen per day. The amount of food consumed during the last 45 of the 70 days during which the eggs were laid was not stated. The proof of synthesis of lecithin from the various phosphor- ized proteins of the rice and milk would be more complete if the authors had made determinations of lecithin in the bodies of the hens, and in controls, and had submitted quantitative determinations of the lecithin in the foods, since they were not said to be entirely lecithin-free. Masslow (1913a, b, c) studied the biological significance of phosphorus for the growing organism by means of feeding, metab- olism, tissue analysis and enzyme estimation experiments on young dogs. Normal feeding was compared with feeding on a phospho- rus-poor diet, and with feeding on phosphorus-poor food plus phos- phates, glycerophosphates and lecithin. Casein and albumin were also compared. The phosphorus-poor diet led to emaciation and finally death, the phosphorus content of the organs having diminished. This loss of phosphorus was mainly inorganic. Of the organic phosphorus only the lipoid compounds decreased. The brain and heart appeared not to lose phosphorus, the loss being greatest in liver, intestines, muscles, bone marrow and kidneys. The ferment functions of the organs was markedly disturbed, the depression of the action of lipase, amylase and diastase being especially great, there being also a like tendency as to catalase and nuclease. In these respects the liver suffers most, the brain and heart comparatively little. The addition of inorganic phosphates and glycerophosphates to the low-phosphorus diet did not prevent phosphorus impoverish- ment. Lecithin, however, caused an enriching of the organism in phosphorus, especially in organic compounds other than lecithin. The improvement took place especially in the visceral organs. The ferments were stimulated to greater activity. PHOSPHORUS METABOLISM 343 * Exclusive milk diet, maintained beyond the normal period for such food, led -to disturbances of the enzyme activity and phospho- rus compounds of the tissues similar to those produced by the low- phosphorus diet. Fingerling (1913) fed to goats rations of straw, blood, nuclein, starch, molasses and oil, with the addition of phytin, lecithin, casein, nuclein, nucleic acid or disodium phosphate. No essential differ- ence was observed in the utilization of phosphorus in the different forms. Durlach (1913) compared various phosphorus compounds, or- ganic and inorganic, in feeding and balance experiments with young dogs. The basal ration was poor in phosphorus and consisted of cakes made from isolated foodstuffs and inorganic salts. The pups were kept on the mother's milk for 36-38 days ; then to bring them to a state of high phosphorus requirement they were kept on the basal ration for 15-22 days, after which time they were given the distinctive diets. Comparisons were made of monosodium and monopotassium phosphates with Merck's ovo-lecithin and with a mixture of monopotassium phosphate, lecithin, sodium phytate, casein and sodium nucleate. All of the dogs lost in weight. Re- sults were not conclusive, but seemed to show lecithin to possess superior nutritive value inasmuch as two of the three dogs which received lecithin were the only ones which survived the experiments. E. B. Forbes and associates (1914; Ohio Tech. Bui. No. 6) have conducted five series of feeding, metabolism, and carcass analysis experiments, involving 120 growing swine, in the comparison of the nutritive values of several organic and inorganic compounds of phosphorus. The compounds of interest, in the usual "chemically pure" form, were added, in equivalent amounts, to low-phosphorus standard or basal rations composed, in the main, from comparatively simple manufactured products of plant and animal origin. From about 75 tables of results four only are quoted. Series I, conducted in April and May, 1908, consisted of metab- olism experiments with four pigs, comparing phosphates, glycero- phosphates, hypophosphites, and nucleic acid from yeast. The phosphates, glycerophosphates, and hypophosphites were mixtures, in each case, of salts of sodium, potassium, calcium, magnesium and iron. The basal ration consisted of pearl hominy (corn minus the skin and the germ) , blood albumen, wheat gluten, and salt, with the addition of small amounts of senna when necessary. The experi- mental periods were 10 days in length. That phosphorus in each form was absorbed and retained is unquestionably true, even the hypophosphites increasing the urin- ary phosphorus and phosphorus retention. Considering normal re- 344 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 quirements of calcium for growth, these pigs all suffered from a deficient intake of this element. Conditions were not considered favorable for a close comparison of the nutritive values of the phos- phorus compounds involved. The condensed balance data are pre- sented in the following table : BALANCE EXPERIMENTS WITH GROWING SWINE, COMPARING PHOSPHORUS COMPOUNDS Grams per Day — Analyses by H. S. Woods and A. C. Whittier Series I; 10-day Periods Pig and period No. L,ive weight Initial Final Lbs. Average daily ration N Food Urine Feces Bal- ance Food Urine Feces Bal- ance Food Urine Feces Bal- ance Ca Food Urine Feces Bal- ance Mg Food Urine Feces Bal- ance Food Urine Feces Bal- ance Period I Period II Period III Period I Period n Period III 81.00 87.75 88.25 99.25 100.75 109.00 Hominy 1310.13 Blood albumen 40.25 Wheat gluten 40.25 Senna 2.40 Hominy 1314.71 Blood albumen 41.32 Wheat gluten 41.32 Senna 1.00 Phosphates.. (P).... 0.613 Hominy 1245.63 Blood albumen 39.03 Wheat gluten 39.03 Phosphates.. (P).... 0.573 Hominy 1383.30 Blood albumen 41,44 Wheat g-luten 41.44 Senna 2.40 Hominy 1389.38 Blood albumen...... 43.40 Wheat gluten 43.40 Senna 2.00 Nucleic acid.. (P)... 0.619 Hominy 1288.13 Blood albumen 40.39 Wheat gluten 40.39 Nucleic acid . . (P) . . . 0.574 Hominy 1451.70 Blood albumen 42.65 Wheat gluten 42.70 Senna 2.40 Hominy 1435.19 Blood albumen 44.50 Wheat gluten 44.50 Senna 3.00 Hypophosphites..(P) 0.648 Hominy 1501.13 Wheat gluten 43.44 Blood albumen 43.44 Senna 1.90 Hominy 1276.66 Blood albumen 40.14 Wheat gluten 40.14 Senna 2.00 Glycerophosphates.. 0.609 26.421 17.094 1.662 +7.665 26.720 17.078 1.175 +8.467 25.263 16.084 1.150 +8.029 2.255 1.169 0.203 +0.883 2.272 1.248 0.153 +0.871 2.149 1.138 0.164 +0.847 0.671 0.026 0.309 +0.336 1.287 0.435 0.205 +0.647 1.210 0.464 0.235 +0.511 0.277 0.177 0.142 —0.042 0.251 0.001 0.074 +0.176 0.206 0.005 0.070 +0.131 0.287 0.068 0.121 +0.098 0.296 0.045 0.087 +0.164 0.273 0.059 0.102 +0.112 0.719 0.164 0.274 +0.281 1.114 0.185 0.238 +0.691 1.042 0.310 0.262 +0.470 Period I Period II Period I 4 Period n 84.50 92.25 92.75 102.50 104.25 112.25 89.50 96.50 97.25 109.25 90.25 99.75 100.38 107.00 27.621 18.405 1.506 +7.710 29.292 18.825 1.153 +9.314 27.153 17.778 1.149 +8.226 2.365 1.337 0.198 +0.830 2.399 1.370 0.148 +0.881 2.223 1.185 0.195 +0.843 0.706 0.021 0.268 +0.417 1.332 0.341 0.217 +0.774 1.232 0.562 0.225 +0.445 0.286 0.077 0.160 +0.049 0.285 0.004 0.089 +0.192 0.216 0.009 0.073 +0.134 0.302 0.078 0.115 +0.109 0.343 0.071 0.123 +0.149 0.307 0.125 0.170 +0.012 28.773 18.816 2.083 +7.874 29.057 18.237 1.359 +9.461 2.471 1.386 0.227 +0.858 2.478 1.327 0.167 +0.984 0.738 0.022 0.262 +0.454 1.384 0.630 0.175 +0.579 0.303 0.154 0.110 +0.039 0.320 0.006 0.068 +0.246 0.319 0.090 0.125 +0.104 0.326 0.061 0.122 +0.143 29.563 19.245 2.137 +8.181 25.974 18.094 1.765 +6.115 2.545 1.360 0.229 +0.956 2.208 1.349 0.220 +0.639 0.760 0.019 0.344 +0.397 1.265 0.225 0.368 +0.672 0.287 0.162 0.184 -0.059 0.295 0.008 0.140 +0.147 0.323 0.096 0.125 +0.102 0.575 0.119 0.236 -r 0.220 0.754 0.132 0.226 +0.396 0.814 0.298 0.234 +0.282 0.739 0.389 0.212 +0.138 0.788 0.178 0.194 +0.416 1.063 0.173 0.197 +0.693 0.809 0.131 0.493 +0.185 1.138 0.116 0.330 +0.692 PHOSPHORUS METABOLISM 345 Series II, conducted in November and December 1908, consist- ed of a feeding and carcass analysis experiment, covering 56 days, and involving the use of 30 pigs, five individuals in each of six lots. The basal ration was composed of pearl hominy, blood albumen, wheat gluten, and corn bran. Salt was allowed ad libitum. Corn meal and chalk were introduced into the diet at certain times, for corrective purposes. The phosphorus compounds compared were the same as in Series I. The several lots received rations as fol- lows: Lot 1, basal ration plus hypophosphites Lot 2, basal ration plus hypophosphites and nucleic acid Lot 3, basal ration plus glycerophosphates Lot 4, basal ration plus phosphates Lot 5, basal ration alone Lot 6, check lot, killed at beginning of experiment The pigs in lots 1 and 2 suffered from great weakness and lameness; they were also subject to indigestion. The legs were weak, especially the hind ones, which trembled, and bowed out, and the feet were set far under the body. The fore feet were also sore. The muscular control was poor, and difficulty was experienced in step- ping up 6 inches onto the feeding platform. These pigs lay down most of the time, and if disturbed would at once lie down again. They ate well, but moved slowly, carefully and without spirit. Lot 5, which received no phosphorus supplement, behaved much as did lots 1 and 2, but the abnormal tendencies were less pronounced. Lot 3, receiving glycerophosphates, were entirely normal ; they ran and. played in the best of spirits. Lot 4, receiving phosphates, also remained in good condition, though they were less active than the pigs receiving glycerophosphates. Considerable difficulty was experienced in the management of the feeding, the lots receiving hypophosphites and nucleic acid re- quiring frequent reduction in the amount of food given, and in the amount of the phosphorus supplements. These reductions it was not possible to make up by subsequent increase. On these accounts the food consumption could not be maintained uniform in all lots, a fact which resulted in certain advantage to the pigs receiving glycerophosphates. The nutritional disorders in certain lots, as above noted, were successfully combatted by the feeding of limit- ed amounts of corn, which in this relation appeared to possess marked curative value, perhaps due to its organic phosphorus com- pounds — perhaps to vitamines. The basal ration, by the way, was very poor in fats. 346 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Conclusions as to the effects of the phosphatic supplements were drawn from slaughter weights of parts and organs, and an- alyses of the hams, brains, livers, kidneys, femora, and tibiae, the data in all cases representing composite samples from the 5 indi- viduals in an experimental lot. For a discussion of the results see p. 352. Series III, conducted during November and December 1909 and January 1910, was in every way similar to Series II. The feeding covered a period of 70 days, and the experiment involved 35 pigs in 7 lots of 5 each. The basal ration consisted of pearl hominy, wheat gluten, blood albumen, and corn bran. Salt was allowed ad libitum. To this basal ration were added, at various times during the ex- periment, chalk, soda, sugar and corn meal, in efforts to relieve dif- ficulties in the feeding. Without some corn it seemed impossible to keep the pigs in condition for experimentation. The phospho- rus compounds fed were the same mixtures used in Series I and II. The several lots received rations as follows : Lot 1, basal ration plus hypophosphites Lot 2, basal ration plus nucleic acid Lot 3, basal ration.plus glycerophosphates Lot 4, basal ration plus phosphates Lot 5, basal ration plus phytin Lot 6, basal ration alone Lot 7, check lot, killed at beginning of experiment. Difficulties in the feeding were encountered in all lots except the one receiving glycerophosphates, and the one which received no phosphorus supplement. The pigs receiving glycerophosphates were much the most spirited and active of any in the series. Hypo- phosphites and nucleic acid, when fed in amounts furnishing 2 grams of phosphorus daily to each lot of 5 pigs caused nausea, vom- iting, and indigestion. Phytin appeared to cause indigestion, but not pronounced nausea. As a whole the pigs in this series were much less tolerant of the phosphorus compounds fed (other than glycerophosphates) than those in Series II. In this experiment even the pigs receiving orthophosphates exhibited very limited tol- erance for the mineral supplement. Series IV, conducted in November and December 1910 and Jan- uary 1911, was similar in method to Series II and III, the feeding extending over a period of 70 days, and the experiment involving 45 pigs. The basal ration in lots 1-5 consisted of pearl hominy, wheat gluten, blood albumen, corn bran and agar-agar, salt being allowed PHOSPHORUS METABOLISM 347 ad libitum. To this ration were added, during portions of the ex- periment, mangel wurzels and potassium citrate, for corrective pur- poses. Lots 6-8 received a similar ration, except that corn replaced the pearl hominy used in the food for lots 1-5. The hypophosphites, nucleic acid and phosphates used were mostly of the same lots as those used in the earlier series, but the glycerophosphate mixture was soon exhausted and was then replaced by calcium glycerophos- phate alone. The phytin was in part a commercial product, and in part a preparation of the related compound from wheat bran. The several lots received rations as follows : Lot 1, hominy basal ration alone Lot 2, hominy basal ration plus nucleic acid Lot 3, hominy basal ration plus glycerophosphates Lot 4, hominy basal ration plus phosphates Lot 5, hominy basal ration plus phytin Lot 6, corn basal ration plus precipitated bone flour Lot 7, corn basal ration plus glycerophosphates Lot 8, corn basal ration alone Lot 9, check lot ; killed at beginning of experiment. The pigs in this experiment exhibited, in general, the same symptoms as noted with regard to the previous series. The pigs which received nucleic acid and phytin, and those on the hominy basal ration alone, suffered from much weakness and soreness of the feet and legs. There was also considerable trouble with indigestion in these lots, as also in the phosphate lot, and the pigs receiving phytin showed some appearance of nausea. The glycerophosphate pigs, as usual, were entirely normal, and suffered only from the restricted food allowance necessitated by the unsatisfactory behav- ior of other lots. Lots 1, 2, 4 and 5 ate their own feces ; lots 3, 6, 7 and 8 showed no such tendency. In Series II, III and IV it was necessary, because of digestive disturbance or lack of appetite, to reduce the amount of food allowed to one or more of the experimental lots on 51 dates. The pigs receiving glycerophosphates were not the occasion for any one of these food reductions, their immunity to nutritional disturbance be- ing complete, and in marked contrast to the behavior of all other lots. Each of the other rations contributed in somewhat nearly the same proportion to the occasions for reduction of food allowance. Series V, conducted during March, April, May and June 1913, was a carefully controlled set of balance experiments comparing glycerophosphates and phosphates, and was terminated by a slaugh- ter test and a complete chemical accounting for the bodies of the experimental subjects. Six barrows, all of the same litter, were 348 • OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 used in this investigation. Two were killed as controls at the be- ginning of the study, the other four serving as subjects for the metabolism experiments. With the same low-phosphorus basal ra- tion, two pigs received phosphates, and two glycerophosphates, the mineral bases in these supplements being proportioned, one to an- other, as in sow's milk. The basal ration was composed, as in the earlier studies, from pearl hominy, blood albumen, wheat gluten, corn bran, agar-agar and salt. As in the previous work, much difficulty was experienced in the feeding of the pigs, other than those receiving glycerophos- phates, and frequent readjustment of the level of intake of food was necessary. Very fine grinding, and extensive dilution of the food with water, were tried in efforts to solve the problem of feeding the orthophosphates, but without distinct success. The experiment finally terminated itself in the digestive collapse of one of the pigs receiving phosphates. A part of the balance data, reduced to amounts per kilogram of live weight, are presented in the following tables : PHOSPHOEUS METABOLISM S49 I" ^ ^ v hi v oooo oooo OOPM OOh-tO OOOO oooo HOOtO OOHtO CJlOiOSUl G0--JOCJ1 OOOO OOOO oooo oooo COOOOOi O0tOt—O5 hj H .^ H 11 H, OOOO oooo OCMtO OOH-CO OOOO OOOO h-OOW OOHtO W — 2.S "*3 P 3±.P ft P; S ^ p £ So ^ p 8 5 g, p to p ^dw p"8-.o p ^dbj OOOO oooo oooo oooo COtOH-*Oi CCCOh-O^l oooo oooo oooo oooo oooo oooo I— »OHOJ HOMW OOOO OOOO h-OOCO I— ' O CO tO OOtOOitNS M-tCUUO OOOO oooo oocooo ootow o\coii ffioxoi OOOO oooo oooo> OOOO OJI-'tO-J OOF—tO-J OOOO oooo h^OI— *CC h-O^-OO 02H*-05~a 050105-q OOOO OOOO HOOtO HOOM OCCOCDI— ' h£-02COCO P*lCJ*J Bag*" CD P ^d^l i=< J-* O) i-j o & P O 2. o R B»3pi w P *id*l P H l> Kj S| 5 O H H ^2 o ^ CO ►fl HH O n W m ^ •=j 02 -. n hj O a' 03 <5 td f > 2 S. S" p 3 o M 1 * i-3 HH H> l-H ^ 1 21 M e cT on- d p 3 w 2 p Ml ► w CO c- 1 M © H S| •^ w W "^ p 3 5 ° 5 3 ^CO^<0 oototo OOOJW tooco-g if-oi- 'Cji toooco toooco h- *)— »00t— * PMrj pR2.8 « CO S50 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 hj hj hj hj ~, *d m, oooo oooo OOHKJ OOtOH OOOO OOOO MOOtO h-OOtO OOOO OOOO OOOO OOOO t^toocn west— m oooo OOOO OOOO OOOO K^i— it>o»q COt— *tC05 oooo OOOO >— 'Ol— ^rf* I— 'Oil— i to hd h9 hj h OOOO oooo OOHM OOOtO tC4-~-»JCO CCr.GCOJ OOOO OOOO HOCtO I— 'OOtO OOOO OOOO OOOO OOOO OOOO OOOO OOOO OOOO fci^ p. >■ p < 3 n p. p «J «3 0) Tl p. p 15" P *jej*] o al a W P •TjCl'T) .. 3 (D 3 p* P ^dw SSSs p P ^<=l*Tj _. 3 8 3 £*) oooo oooo h- 'O^-'CO h--0*-*^ p ^c|w pS2.o 3 n> 3 2 o to (t P" OOOO OOOO k-OOtO h-OOlO CntOOSlf* tococxo OOOO OOOO ooww o o to OJ CDOtOOO W^OOOtO IOOOCO POOM OQ0O00 Gil— 00*. OOOO OOOO HOOtO I— 'OOtO tocooso oicnto*. w 3§B -8 OOOO OOOO ootceo ootow QlO-^JtO CDOCDQ0 pooto rooooo CDtO-JGO CCIO--ICO P ^CJ 1 ^ CD td P ^cJ 1 ^ £8 2.8 a 2. S ft • ' * td ft i 6 5* P p- 3^ o j£ a N p p «4 © op. p 2 § O H o ^ t* £ o 8 a o H 2 w I— I r O o > PHOSPHORUS METABOLISM 851 Jti ^ V H h3 ^ ^ 1-9 oooo oooo OOMtO OOOi—» ■ -J!— KOQt- » «*4)— '0005 oooo oooo OKQtO OOOI— » COH- 'QIC?! O^GCCRCC 8^ si ^ (K) 3 trq • IT" p < 3 a p." 1 B<« OOOO OOOO p o o ^ o o o ro oooo OOO— ' oooo HOOM COOOh^Oi oooo OOOO oooo oooo COtOOOi tOtOOrf^ oooo oooo oooo oooo ostoiss-j ton-, tooi oooo oooo homo: ^- o *- ro Cioioooo towwoo oooo oooo l—OOCO OOO'-' COtf».~JtO O0OJO5O5 OOOO oooo OOOO OOOO p $3g SjJSft 8. p p *lQw J7* re I") O HH P ^c^w 2 m » oooo oooo oooo oooo oooo oooo oojcooi ^jSKoo OOOO OOOO POO>— i >— 'OOtO OOOO OOOO ootoco oowto 03000 01 OOM-J MOOU h-"OObO t— 'hO^JI— » Oil— 'O^CO C0OC0 00 (OfflOUl oooo oooo ooccto oo 5 W-OOH^ OOOCOOT t2SE5 MOOU oconoto »jaao P ^<3w « p S -o S p ^c^w pRS.g ,re g£ 3 W >-( © 2. H 352 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Discussion of Results. From the results of Series I it is mani- fest that orthophosphates, hypophosphites and yeast nucleic acid may all be absorbed by swine, and may be retained in considerable quantity for at least 10 days. That the retention of each of these compounds may be permanent seems altogether probable. In the case of hypophosphites this would involve a further oxidation of the phosphorus to the ortho form. Under the conditions of Series II, III and IV, there was some evidence to suggest nutritive superiority of glycerophosphates to orthophosphates, nucleic acid, phytin and hypophosphites, es- pecially in relation to the proportion of muscular tissue and fat in the increase, and the breaking strength and ash per cubic centimeter of volume of the bones, but the evidence was not sufficiently of one sort to establish facts with certainty. It seems quite possible that the amount of exercise taken by the pigs, as determined simply by the state of feeling induced by the phosphorus compounds fed, entirely irrespective of fundamental nutritive effects, may have entered prominently into the determina- tion of the relative development and even the composition of the parts. The final series of experiments, No. 5, was intended to establish any such fundamental differences as there may be in the nutritive values of phosphates and glycerophosphates. It included complete and protracted mineral and nitrogen balances, complete chemical ac- counting for the carcasses of the pigs, determinations of digestibil- ity of the food constituents, determinations of the comparative de- velopment and composition of various organs and tissues, and daily determinations of the nitrogen, creatinin and ammonia of the urine. Throughout this exhaustive study there was marked similar- ity in the results from both rations. Aside from the much greater acceptability of the glycerophosphates than the phosphates there were no differences of note in results from the two pairs of pigs. The result of this series was a remarkably uniform and consist- ent set of observations of many sorts indicating that, at least under the artificial conditions of this experiment, there are no essential differences in the fundamental nutritive value of phosphates and glycerophosphates for purposes of growth in swine. Conclusions. Phosphates, hypophosphites, glycerophosphates, nucleic acid (from yeast) and phytin are all absorbed and retained, and apparently utilized, by growing swine, when added in the pure form to rations low in phosphorus but capable of maintaining phos- phorus equilibrium. PHOSPHORUS METABOLISM 353 Marked differences were observed in the tolerance of swine toward the several phosphorus compounds used. These were found to compare as to acceptability to swine, when fed in quantities fur- nishing equal amounts of phosphorus, in the following order, the most acceptable being mentioned first, and the others in order of de- creasing acceptability : glycerophosphates, orthophosphates, phytin, nucleic acid and hypophosphites. That the differences in acceptability of these isolated compounds similarly affect the foodstuffs in which they are naturally combined seems certainly not to be a fact. These differences in acceptability were not found definitely related to more fundamental nutritive effects. That the particular organic compounds used in this investiga- tion (nucleic acid, phytin and glycerophosphates) have nutritive values, to growing swine, superior to the inorganic compounds used (orthophosphates and hypophosphites) was not shown. No funda- mental differences in the nutritive values of the phosphorus com- pounds studied were established. No basis, therefore, was discovered for a differentiation be- tween the nutritive values of organic and inorganic phosphorus com- pounds generally. It should be borne in mind, however, that no representatives of the two classes, phosphoproteins and lecithins, were included in this investigation, and results obtained under con- ditions of such rigid experimental control may not accurately rep- resent the facts under optimum normal conditions of life. These results are not considered to controvert evidence as to specific ther- apeutic effects of the same compounds in relations other than those considered in this study. The amount of phosphorus which an animal will tolerate, when added to the ration in readily soluble form, is definitely limited at an amount much less than that which will be acceptable in its natural relationships in foods. From the great difficulty experienced in the feeding of yeast nucleic acid and of commercial phytin, as well as the related com- pound from wheat bran, it is concluded that the isolation of such compounds from natural products alters at least their therapeutic effects in such manner that it becomes impossible to state, from in- vestigations of this sort, on pure compounds, what may be their nutritive values in their natural relationships in common foods. It seems unlikely that, with growing animals, any ration com- posed from natural foods, and supplying the nitrogen requirement, will fail to furnish enough phosphorus to maintain phosphorus equi- S54 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 librium. That many rations compounded from common foods are lacking in the amount of phosphorus essential to maximum reten- tion and growth, however, is as certainly true. The addition of comparatively small amounts of corn to rations compounded from simple manufactured products of plant and ani- mal origin may enhance the nutritive value of such rations to an ex- tent out of proportion to the amount of corn added, and may affect the results of such comparisons of phosphorus compounds as are here reported in definite and fundamental ways determined by the state of nutrition of the animal, especially with reference to nutri- tive reserves, resulting from the previous feeding and life activity. In spite of a marked tendency toward constancy of composition, appreciable differences were noted in the chemical constitution of the organs and tissues of different experimental lots of swine, these differences apparently being due in part to the phosphorus com- pounds fed, and in part to other factors which were not controlled. Such differences in the composition of tissues as were observed might be accounted for as due to variations in the liquid content of the parts, the salts varying accordingly, or to the composition of supporting structures or unorganized nutritive materials. There is also, in medical iiterature, a vast amount of clinical evi- dence to which we could refer, much of it flimsy, it is true, but other portions of undoubted value as showing specific effects of lecithin which are not possessed by phosphates, especially the direct contribution of lecithin to the lecithin content of the blood serum and tissues, and to the increase of the red blood corpuscles. See summary, p. 534. . ORGANIC PHOSPHORUS SYNTHESIS IN FASTING SALMON This problem of synthesis of organic phosphorus compounds has also been attacked from another angle in the study of the com- position of salmon at different stages of sexual maturity, these fishes, in the journey to their spawning grounds, traveling long dis- tances, apparently without taking food, and at the same time accomplishing the organization of great masses of the sexual ele- ments. Miescher (1881), in studying the life of the Rhine salmon, came to the conclusion that the very great development of nuclear material in the sexual elements during fast took place at the expense of the muscular system. This investigation was not concluded. Paton et al. (1897-8, 1898) made an extensive series of observa- tions on the metabolism of the salmon during its life in fresh water. PHOSPHORUS METABOLISM 355 Lecithin was determined from the ether extract; inorganic phos- phorus by extraction of the ether-extraction residue with 0.2 per- cent hydrochloric acid, while the phosphorus in nuclein and pseudo- nuclein was determined in the residue from the above-mentioned hydrochloric acid extraction. The matter of especial interest in this investigation is the evi- dence on the subject of synthesis of organic phosphorus compounds. By tissue analysis of fishes from the estuary, and later from the upper river waters, after the development of the ovaries and testes during fast, conclusions were drawn as to the transformation of phosphorus compounds within the body. In the female fish the lecithin of the trunk muscles was found to be insufficient to yield the lecithin phosphorus of the ovaries, the total phosphorus lost by the trunk muscles being only just sufficient to yield the total phos- phorus laid on by the ovaries; but in the male fish more lecithin is lost from the muscles than is -required by the testes. The authors concluded that the phosphorus stored in the muscles as simple phosphates is transferred to the ovaries and testes, and there built up into organic combinations, lecithin consti- tuting an important step in the conversion of the phosphates into true nuclein in the testes, and into the intermediate pseudonuclein, ichthulin, and, later, true nuclein in the ovaries. The study of this problem is attended by difficulties which warrant the suspension of our judgment as to the significance of the results at least until there shall have been made complete chem- ical accountings for the entire bodies of the fishes. Milroy (1908) has made similar studies on the herring, but definite conclusions were not reached as to the source of the phos- phorus compounds of the ovaries and testes. ADVANTAGE OF COMPLEXITY OF ORGANIZATION IN FOOD PHOSPHORUS On a priori grounds a certain superiority of the organic as com- pared with the inorganic phosphorus compounds would be consid- ered to be due simply to the greater complexity of organization, since in the case of those compounds (the lecithins and the nucleins) which are absorbed in part as such, or in a state of incomplete digestive cleavage, a certain amount of synthetic activity is prob- ably spared the animal. As to how important a matter this may be we can only speculate. With reference to nitrogen compounds, how- ever, the maintenance of nitrogen equilibrium with the less com- pletely split proteins seems to be appreciably more efficient than 556 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 with the products of complete digestive cleavage, and the same principle may be operative in the case of some of the organic phos- phorus compounds. Abderhalden and Rona (1904) found, in experiments with mice, that pancreatic digestion products of casein are about as valuable for maintenance of live weight as casein itself, that the further split products of peptic-pancreatic digestion of casein are of less value, though of some use, while the products! of sulphuric acid hydrolysis seemed to be without value. These authors (1905) concluded that a dog was able to utilize pancreatic digestion products of casein, these products consisting of amino acids and complex sub- stances not giving the biuret reaction; but the products of acid hydrolysis of casein were not able to maintain nitro- gen equilibrium. In later work, however, Abderhalden (1912) showed, in protracted nitrogen balance experiments, that dogs are able to maintain nitrogen equilibrium and retention on products of either acid or enzymatic splitting of proteins, or on a mixture of recognized amino-acids. Abderhalden expresses the belief that the organism must have prepared for itself all of the building stones which are specific for phosphatids, since these were not fed. To have demonstrated this point, however, would have required carcass analyses, which were not a part of this investigation. D. D. Van Slyke and G. F. White (1911), in protein digestion and retention experiments with dogs, found that with various meats the rate of digestion and absorption, as indicated by the rate of elim- ination, was, in general, the reverse of the amount of nitrogen retained from each at the end of 24 hours. In explanation of these results the authors (Van Slyke and White) suggest that a larger proportion of the more readily digested proteins may be absorbed in the form of the lowest cleavage prod- ucts, which Carrel, Levene, Meyer and Manson, and also Voit and Zisterer and others, have shown to be less capable than the higher cleavage products to maintain nitrogenous equilibrium. In the light of these experiments it is quite conceivable that, other things being equal, some of the more highly organized organic phosphorus compounds should possess, on account of their state of organization, nutritive value greater than that of the simplest inorganic phosphates. A further advantage to the animal in having the phosphorus compounds of the food organically combined is suggested by the experience of one of us (E. B. F.) in the feeding of pure phosphates along with a low-phosphorus basal ration to swine. The ready solu- PHOSPHORUS METABOLISM 357 bility of the uncombined phosphates results in such a concentration of these salts in the digestive tract as causes nausea or catharsis. Much larger amounts of phosphorus may be utilized in a normal manner if they are gradually liberated in the usual way by the digestive cleavage of the organic complexes with which they are combined. FEEDING EXPERIMENTS WITH RATS AND MICE ON RATIONS COM- POSED OF SIMPLE PURIFIED NUTRIENTS An important addition to the evidence on organic phosphorus synthesis has been made through a number of extensive series of feeding experiments with rats and mice, most of them having for their object especially the study of synthesis of nitrogen compounds in general, by experiments on growth and reproduction, on rations of simple purified nutrients. Rohmann conducted experiments with mice which show that even though phosphoproteins and nucleoproteins be present in abun- dance, and mineral salts be furnished as phosphates, nitrates, chlo- rides and lactates of the more important bases in the body, there may still be nutritive deficiencies in the ration such as gradually, through the course of successive generations, make growth impos- sible. Rohmann (1902, 1907) successfully reared two generations of mice on an artificial food mixture containing its phosphorus as phos- phoprotein, nucleoprotein and phosphates, but failed to rear the third generation. In a later paper (1908) Rohmann reported suc- cess in rearing three generations of mice to maturity on artificial foods, but the fourth generation could not be reared. The nutrients used in these experiments were casein, egg albumin, nucleoprotein from liver, potato starch, wheat starch, margarine, malt, chicken protein and a salt mixture compounded as follows : 10 parts calcium phosphate 40 parts acid potassium phosphate 20 parts sodium chloride 15 parts sodium citrate 8 parts magnesium citrate 8 parts calcium lactate Mendel suggested that Rohmann's final failure was due to de- ficiency in the salt mixture. Stepp ascribed the limited usefulness of this ration to deficiency of lipoids. Falta and Noeggerath (1906) attempted to maintain adult white rats on artificial food mixtures and inorganic salts, including 858 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 calcium phosphate. Neither the rations containing phosphorus- free proteins nor those containing organic phosphorus, as of casein, would keep the animals alive indefinitely, though controls fed on milk, milk powder or horse flesh throve without incident. L. Jacob (1906) kept rats as long as 124 days on artificial mix- tures of foodstuffs, such as casein, cane sugar, cellulose, hog fat and salts. Doves fed on a similar mixture did not do well, perhaps be- cause the feed formed a soft mass in the crop. Knapp (1908) records unsuccessful attempts to maintain rats on artificial foods and salts. Stepp (1909) sought to determine the office of the lipoids of bread, on the growth of mice, » by feeding mice on bread extracted with alcohol and ether, or alcohol, ether and chloroform, in compari- son with others which received the residue plus the extract. The results were inconclusive ; none of the mice throve. At a later date Stepp (1911) conducted a series of 19 experi- ments on the growth of mice on various diets with and without the presence of those lipoid bodies which are soluble in alcohol and ether. Maintenance of life and live weight were the criteria on which conclusions were based. Some of the more important re- sults are as follows : Mice die when fed with food which has been completely freed from lipoids. The length of life is increased if the alcohol-ether soluble constituents are added to the extracted food. Fat plays no significant role itself, though there are in butter and in milk-fat very small quantities of important alcohol-ether soluble materials. These substances are present in greater amounts in milk plasma. Lecithin and cholesterin seem not to be the necessary lipoids. That unknown food constituents may possess properties of much importance, and of a value out of proportion to amount, is in- dicated by experiments by Hopkins (1912) in the feeding of rats. When added to rations of purified products such as pure casein, starch, cane sugar, lard and salts — a very small amount of milk had the effect to maintain normal growth, which otherwise soon ceased. The effect was shown not to be due to greater amount, energy value or palatability of the ration containing the small amount of milk. The nature of the important principle was not determined. One of us (E. B. F.) has had similar experience in feeding ex- periments with pigs on a basal ration of pearl hominy, wheat gluten, blood albumen and corn bran, to which were added various pure phosphorus compounds. It has often been the case, with rations other than those containing glycerophosphates, that in order to keep the pigs alive until the end of the experiment it has been PHOSPHORUS METABOLISM 359 necessary to introduce a small amount of corn into the ration. The improvement was always marked. At these times we were unable to get the pigs into good condition by the addition to the ration of corn oil, mangel wurzels, potassium citrate, calcium carbonate or soda. Osborne and Mendel (1911a) in their first year's investigation on feeding with isolated food substances (reported in Carnegie In- stitution Publication 156, Part I) conducted many experiments in the feeding of white rats on rations of pure casein, cane sugar, starch, lard, agar and inorganic salts. Varying results, as deter- mined by the salt mixtures, were obtained. Weight was main- tained and nitrogen stored over considerable periods of time with both young and mature rats, but without significant gain in weight, on rations containing but a single protein. One animal was main- tained for 217 days on a diet in which the sole protein was glutenin. In the second year's work, as reported in Part II of the above- mentioned publication, a large number of pure proteins were com- pared, some containing phosphorus, and others free from this ele- ment. A noteworthy change of method was the addition to the ration of "protein-free" milk, a preparation made from skim milk by precipitation of the casein with hydrochloric acid, nitration, and evaporation of the whey. Active growth was obtained with casein, ovalbumin, lactal- bumin, edestin, glutenin and glycinin, each as the sole protein constituent of the ration. With gliadin, and hordein there was little or no growth, and with zein weight was not maintained. The use of protein-free milk rendered this series of experiments the first thoroughly successful attempt to induce growth in rats with such isolated food substances. The maximum normal size of the animals was not attained, however,. as the authors state in their later work on fat-free foods (Osborne and Mendel, 1912b), but an increase to several times the initial weight was produced on rations at least essentially free from purins and from organic phosphorus com- pounds. See also Osborne and Mendel, 1911b. In the article entitled "Ueber Fiitterungsversuche mit isolierten Nahrungssubstanzen" (1912b) Osborne and Mendel review their previous work and add reports of further similar tests of the ability of rats to gain in weight on rations in which certain legume and other proteins constituted the sole source of protein nutriment. A table is presented in which the many pure proteins tested are arranged in the order of the gain in weight induced by them, in rats, during 30 days. In the order named, beginning with those which cause loss in weight they are as follows : Phaseolin, zein, gelatin and conglutin, the loss in weight of the last-mentioned being 360 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 least. Then in a series of ascending efficiency are mentioned rye gliadin, hordein, pea legumin, wheat gliadin, vignin, legumelin, hemp-seed glutelin, edestin, glycinin, wheat glutenin, cotton-seed globulin, lactalbumin, maize glutelin, excelsin, squash-seed globulin, ovalbumin, casein, and, most efficient of all, ovovitellin. In a paper on "The Role of Gliadin in Nutrition" Osborne and Mendel (1912c) showed that the gliadins of wheat and rye, as well as the closely related hordein of barley, suffice for the maintenance of rats without growth. A female rat after 178 days with gliadin as its sole protein gave birth to four young. At the end of 30 days 3 were removed. The one remaining with the mother, however, failed to thrive on the gliadin food. The authors consider that in this experiment there must have been a synthesis of those protein "building stones" which were lacking in the intake during the period of gestation and lactation. The rat only maintained its weight previous to mating. During pregnancy the gain in weight was very rapid. Subsequent to pregnancy, however, the loss in weight was pronounced, and the rat died 94 days after bearing young, her weight at that time being only a little more than half that at the time she was mated. No post-mortem was held. A number of charts are presented showing the history of ex- perimental animals subsequent to the period of normal growth on the rations containing but a single protein. After such normal increase in weight, as previously mentioned, these animals reached a period of rapid decline in weight. Body analyses, to demonstrate that the normal increase in weight was in every way normal in char- acter, would be of interest in this connection. In "Maintenance Experiments With Isolated Proteins" these authors state that "every animal has sooner or later declined when fed with mixtures of isolated and purified proteins, carbohydrates and fats together with inorganic matter in the form of crystallized salts. In nearly every case the decline has been sudden, with strong evidence that death would soon have ensued had not the food been changed. In each case immediate recovery has followed a change in the diet, thus showing the experimental foods to be inadequate for prolonged nutrition." They state, however, that "with the aid of 'protein-free' milk it is possible to maintain rats for periods equal to practically their entire adult lives on foods contain- ing a single purified protein." They also note the work of Hopkins (1912) who shows that milk, as well as other natural food materials, contains unknown substances which, even in very small quanti- ties, suffice to induce normal and continued growth, for several weeks, at least, in rats maintained on artificial mixtures of food sub- stances which are otherwise inadequate for growth. PHOSPHORUS METABOLISM 361 Twelve rats were maintained more than 400 days, and 5 rats more than 500 days on rations of isolated foodstuffs free from more than the merest possible traces of purins and of phosphoproteins. In 1912 Osborne and Mendel published a series of four papers all bearing on the subject of protein synthesis, and incidentally on the synthesis of compounds of phosphorus. The method of experi- mentation was modified by the use of an artificial mixture of inor- ganic salts and milk sugar in imitation of the protein-free milk prep- aration which had made possible the success of the work of the previous year. This artificial preparation proved to be the most efficient salt mixture thus far compounded. In their work on "Feeding Experiments With Fat-Free Food Mixtures" (1912a) they were successful in causing growth of young white rats on rations of isolated food substances which were entirely free from fat, and at least essentially free from any compounds of lipoid nature, the pro- tein being present in two cases as casein, in another as casein and edestin, and in another as edestin alone. Phosphorus was present in the last-mentioned only in the authors' "artificial protein-free milk." In this last case the curve of growth shows that the rat in- creased in weight to five times the initial weight in 60 days. It is difficult to believe that any sort of redistribution of constituents could make possible so considerable an increase in body weight with- out extensive synthesis of all of the phosphorus-containing com- pounds necessary to growth. The actual proof of such synthesis by body analysis of the experimental subjects will doubtless be forthcoming at an early date. That the growth was normal in composition was not in any way proven. These authors were unable, however, similarly to induce growth in mice on these rations, and they note the agreement of their experience in this matter with that of Stepp. Osborne and Mendel (1913) once more report their failure even to maintain mature animals indefinitely on artificial foods and inor- ganic salts. They write as follows (p. 132), "It is true that in sev- eral instances we have succeeded in keeping grown rats in health and in apparent nutritive equilibrium on purely artificial food mixtures over periods far longer than the experience of our predecessors had led us to expect. But the outcome has never been satisfactory in the sense of extending over what may be considered as the larger portion of the life-span of an adult animal. Successful maintenance has been secured only when the animals have been fed, in part at least, with foods containing our 'protein-free milk/ The superiority of the latter foods, compared with any purely artificial 362 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 food mixture in repairing the depleted body weight of animals that have begun to decline on the artificial salt mixtures tested is beyond question." But even with the use of this "protein-free milk" growth may not be sustained in rats in a normal manner, "a few stopping after sixty days of growth, others continuing to grow for 100 days or more. After normal growth stops, the animals may remain in con- stant weight for a few days, or grow very slowly, and then suddenly decline and die unless a change is made in the diet The con- clusion seems inevitable, therefore, that the 'protein-free milk foods' are deficient in, or completely lack, something which milk contains and which is indispensable for perfect growth." The importance of certain inorganic elements not usually con- sidered in nutrition investigations, namely iodine, manganese, fluor- ine and aluminium, was demonstrated by the addition of very small amounts of salts of these elements to the artificial protein-free milk previously mentioned. Much better results in feeding experiments were obtained with these elements present than in their absence, and a considerable portion of the favorable results obtained with the previous use of artificial protein-free milk must be considered to have been dependent on inorganic impurities present in the chemicals used in its preparation. The authors show that butter contains the principle which is present in milk, lacking in their "protein-free milk," and which is essential to normal growth. Hopkins and Neville (1912) also fed a large number of rats on the diet used by Osborne and Mendel, but the rats ceased to grow before the attainment of full normal size. Ruth Wheeler (1913) maintained white mice in health for 6 months on an artificial diet containing but a single protein (casein) . Stepp (1913a) experimented on mice, with various lipoid-free diets, plus lipoids of diverse sorts and conditions. The lipoids of brain, egg yolk, etc., possessed life-saving power under certain con- ditions, but this capacity is lost through heating. Stepp speculates as to the probability of the essential nutrient being lecithin. Com- mercial lecithin, however, had not the life-saving power, perhaps because of modification in the course of separation from the natural product containing it, perhaps because another phosphatid is required. Stepp (1913b) has studied the importance to animal life of the lipoids, phosphorized and otherwise, as separated from foods by various solvents. Experimenting with mice — ether seems not to remove essential nutrients from foodstuffs; alcohol ex- traction, however, leaves the food incapable of sustaining life. PHOSPHORUS METABOLISM 363 Acetone extract of egg yolk does not contain the essential nutrients removed by alcohol, nor does an alcohol extract of the acetone extraction residue. Thus it appears that acetone removes a part, but not all, of the essential nutrients of egg yolk which are soluble in alcohol. A mixture of lecithin, cholesterol, cephalin, cerebron and phytin can not compensate for the substances extracted from the diet by alcohol-ether. McCollum and Davis (1913a) obtained normal gain in weight of rats during 75-100 days on a ration of casein, dextrin, agar-agar and inorganic salt mixtures similar to the mineral content of eggs or milk; Such mixtures, however, do not support growth indef- initely. In a later paper (1913b) these authors state that beyond 100-120 days little or no increase in weight can be produced on such rations as the above. That animals fed on these rations of puri- fied food substances are in a physiological state which is nearly nor- mal was concluded from the fact that three female rats produced young after being fed only casein, carbohydrates, lard and salt mix- tures for periods of 108, 127 and 142 days, respectively. These rats had made approximately normal growth for about 80 days on this ration. In none of these cases, however, were the mothers able nor- mally to nourish the young. The authors believe that cessation of growth on such rations as the above is due to the animals' "running out" of some essential organic complex, which they determine to be present in the ether-extract of egg and of butter. Lard and olive oil were not similarly efficacious. The feeding of lecithin and choles- terin gave results which the authors characterize as "very doubt- ful." The data were not presented. In applying to human nutrition and to live-stock feeding the results of these experiments with rats and i mice, it is our feeling that, in spite of the fundamental similarity of the nutritive process- es of all vertebrates, we should consider the possibility of important differences in the metabolism of remotely related forms. In the literature on this subject we note a marked tendency on the part of some whose experience with animals has been confined to laboratory work with a very few species, to ascribe to animals generally, without qualification, the physiological capacities of each species. Many important differences are known to exist in the metabolism of vertebrates. As a single instance of this — some mice can live without drinking. While consuming only air-dry foods the metabolic water produced by oxidation within the tissues is adequate for the performance of all necessary functions. Such a fact should convince us of the need of conservatism in the practi- 364 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 cal application of some of our experimental results before they have been shown actually to possess significance in relation to the animal and the conditions of interest. SUMMARY ON NUTRITIVE VALUES OF ORGANIC AND INORGANIC PHOSPHORUS In the light of our present understanding, we must deplore the imperfect and inconclusive character of much of the work on this subject. That the evidence is not more satisfactory is because it has been only with the gradual accumulation of knowledge in this field that investigators have come to realize the difficulties and the requirements of a positive solution of this problem. Considering the evidence as a whole, however, there is much of value, and even the more imperfect work, in the aggregate, produces a composite impression which can hardly fail to affect opinion ; and that this should be true is perfectly proper, unless there be, through- out the work, a neglect of absolutely essential considerations. Can- dor requires that we admit one such possibility. In many experi- ments, at least, there has been no guarantee of the purity of the organic compounds used, and no particular effort to guard against the influence of other useful compounds associated with the organic phosphorus in the natural products and also in the isolated com- pounds as used in these investigations. This consideration we must bear in mind. It does not invalid- ate all of the work on this subject, but should serve to increase the scientific conservatism with which we naturally regard the evidence. Considering that we have sufficiently qualified our conclusions we shall point out the apparent significance of the foregoing investiga- tions. One point, at least, in this connection, we may regard as defin- itely established. Considering the phosphorus requirement as a whole, an amount of organic phosphorus equal to a very small part of the total is sufficient for growth and reproduction, provided inor- ganic phosphorus be present in sufficient amount. This fact is not likely to be questioned, in consideration of the very small propor- tion of the total phosphorus of the animal which is present in organ- ic combination. That organic phosphorus is absolutely essential to any animal has not been demonstrated. The proof that inorganic phosphorus can serve all of the purposes for which any animal needs phosphorus is incomplete. There is much evidence to imply that with some species, at least, some organic phosphorus compounds are more useful than is inorganic phosphorus in the sense of being more readily and economically utilized, and of maintaining a higher state PHOSPHORUS METABOLISM 365 of vitality as revealed by tissue enzyme estimations, the difference probably depending, in part at least, on the fact of the partial absorption and utilization of organic phosphorus compounds as such, without complete digestive cleavage. An important aspect of this problem is afforded by the results of feeding experiments on rats and mice, with simple purified nutrients, and by the results of investigations of the cause of beri- beri (see Beriberi). In the light of these studies the synthesis of organic phosphorus compounds in a normal manner seems to be dependent on the presence of minute quantities of certain unknown compounds which are found in natural foodstuffs, and which act like enzymes, catalyzers or activators. Such essential compounds are found in milk and in eggs and other natural foods of both plant and animal origin. Lack of such nutrient principles in white rice and patent flour has been shown to be the cause of beriberi, and Funk suggests that a deficiency of the food in these unknown compounds, for which he proposes the name "vitamines," produces a predisposi- tion to many other diseases such as polyneuritis in fowls, epidemic dropsy, experimental scurvy in animals, infantile scurvy, ship beri- beri and pellagra. From the facts of the occurrence of these sub- stances in rice polish, and their absence from polished rice ; and also their presence in whole wheat flour, and absence from white flour, we must conclude that in vegetable foods their distribution is localized in certain tissues. It therefore seems not at all unlikely that the many demonstra- tions of superior nutritive value of organic to inorganic phosphorus compounds have been influenced by other beneficial substances occurring in association with them in natural foods, and contained as impurities in these organic phosphorus compounds as isolated and used in nutrition investigations. As to the relative importance of this factor and others we are as yet unprepared to make positive assertions ; but these recent studies at least raise the question as to whether the apparent superiority of organic to inorganic phospho- rus compounds is due to these organic compounds by themselves, or whether their superiority is dependent on minute quantities of cer- tain associated compounds. However this question may be settled, these studies certainly suggest that, if the natural organic phos- phorus compounds are not of superior usefulness, or are not essen- tial to the maintenance of growth in animals, then other nutrients associated with them in the natural foods are essential, and the result, therefore, is to put a new emphasis on the value of the natural organic foodstuffs as compared with inorganic or artificially synthesized nutrients and certain manufactured foods. 366 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 SOME COMMON FOODS IN RELATION TO PHOSPHORUS METABOLISM MISCELLANEOUS EXPERIMENTS WITH MEN AND ANIMALS The relative capacities of common foods to supply the phospho- rus requirements of animals is a subject of much practical import- ance on which we have comparatively little evidence, and, in view of the fact that the comparisons must be made, if they are to be of practical value, not between individual foodstuffs, but between mixed rations, wholly satisfactory evidence in this field is not to be expected. However, very imperfect evidence is not necessarily without value, and is often the only sort obtainable with reference to our most important problems. EXPERIMENTS WITH HUMAN BEINGS Surmont and Dehon (1903) conducted 24-hour balance experi- ments with human subjects in the comparison of white bread and whole wheat bread, eaten with distilled water only. A portion of the results are as follows : NITROGEN AND PHOSPHORUS BALANCES COMPARING WHOLE WHEAT BREAD WITH WHITE BREAD, WITH TWO HUMAN SUBJECTS IN TWENTY-FOUR HOUR EXPERIMENTS— Grams Bread eaten Nitrogen Phosphorus (P2O5) bread Food Urine Feces Balance Food Urine Feces Balance White Whole wheat. Whole wheat. 680 605 950 950 8.357 11.942 11.941 18.230 7.089 11.445 12.130 12.440 1.573 2.485 3.835 7.021 -0.305 -0.988(1) -4.024 —1.231 1.455 6.419 1.990 9.471 1.859 5.265 1.517 6.422 0.930 2.683 1.118 3.657 -1.334 -1.529 -0.645 -0.608 (*) Should be — 1.988 to correspond with other data. The authors state that the two lots of white bread contained 1.229 percent and 1.257 percent nitrogen, and 0.214 and 0.199 per- cent P,0 5 ; the whole wheat bread 1.974 and 1.919 percent nitrogen, and 1.061 and 0.997 percent P 2 5 - A larger proportion of the phosphorus of the white bread was found in the feces than of the whole wheat bread. The apparent digestibility of the nitrogen was about the same in both cases. The periods, however, were so short that the results have only corrobor- ative value. The very considerable phosphorus deficit on the whole wheat bread diet, with an intake of 6.4 gm. P 2 5 in one case and 9.47 gm. in another is remarkable, since these amounts of phosphorus are much more than enough to maintain phosphorus equilibrium under ordinary conditions. The very short periods give results indicating habit of phosphorus metabolism rather than phosphorus requirements. PHOSPHORUS METABOLISM 367 Surmont and Dehon (1904) continued their study of white and whole wheat bread at a later date. A part of their figures are as follows : AVERAGE DAILY NITROGEN AND PHOSPHORUS BALANCES WITH TWO MEN COMPARING WHITE BREAD WITH WHOLE WHEAT BREAD IN THREE-DAY PERIODS— Grams Kind of bread Nitrogen Phosphorus (P2O5) Food Urine Feces Balance Food Urine Feces Balance 12.213 U.718 12.213 11.718 10.377 10.227 12.034 11.976 2.458 2.566 2.414 2.527 -0.622 -1.076 -2.235 -2.785 6.384 8.163 6.384 8.163 4.004 5.081 4.082 5.125 2.311 2.887 2.238 2.870 +0.069 +0.195 +0.066 +0.168 The subjects were first brought to nitrogen equilibrium on a constant mixed diet ; then followed the 3-day experiments on whole wheat bread as a part of the mixed diet. Nitrogen equilibrium was again established in an intermediate period, and then followed the period in which white bread was consumed with a mixed diet. The authors concluded that the phosphorus of whole wheat bread is as well absorbed as that of white bread, and that the coefficient of retention of the phosphorus of whole wheat bread is higher than that of the phosphorus of white bread. The assimi- lation of the nitrogen of the whole wheat bread was found somewhat lower than with the white bread. Newman, Robinson, Hainan and Neville (1912) compared white and whole wheat breads in digestion experiments of 7 days' dura- tion with four men. The phosphorus of the whole wheat bread, being present in much greater quantity than in the white bread, was apparently absorbed as efficiently, and therefore in larger proportion to absorbed nitrogen, thus approaching the ratio of these constitu- ents as absorbed from efficient mixed dietaries. The nitrogen of the white bread was considered to be absorbed 3.5 percent more efficiently than that from the whole wheat bread. Schlossmann and Moro (1904) attempted to compare metabo- lism of a 28-year-old man on a diet of cow's milk with the same on a diet of human milk, but psychic factors rendered results patho- logical on the latter diet, and the figures therefore have no especial value. In a 2-day period on cow's milk, cream, milk sugar and cognac, with a daily intake of 4.487 gm. CaO and 6.113 gm. P 2 5 , there was a storage of 1.60 gm. CaO and 1.33 gm. P 2 5 . The sub- ject weighed 59.1 kg. 368 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Wiley, and associates (1904, 1906, 1907, 1908a,b) studied metabolism in healthy young men as influenced by various food preservatives. Their results on phosphorus metabolism were rather variable, and were not striking. From the administration of 3 gm. boric acid, or equivalent amounts of borax daily, it would appear that this preservative slightly decreases phosphorus retention. Salicylic acid and salicylates in quantities of 0.210 gm. — 2.00 gm. per day appeared to increase to a slight extent the absorption of phosphorus from the intestine, and to lead to its retention in the body. The positive phosphorus balance was somewhat greater in amount during the preservative period than either before or after. Sulphurous acid and sulphites in quantities of 0.171 gm. to 1.020 gm. (S0 2 ) per day increased the feces phosphorus. The authors assume that this increase is due to inhibited absorption from the intestine. Benzoic acid and sodium benzoate in quantities of 1.0 — 2.5 gm. daily of benzoic acid, or equivalent amounts of sodium benzoate, also caused a slight increase in feces phosphorus ; while formaldehyde in quantities of 100-200 mg. daily caused a slight increase in phospho- rus excretion, with a considerable deflection of phosphorus from feces to urine. The experimental periods were of considerable duration and the scope of the work extensive. The feces were not marked at the beginning and end of periods. Slowtzoff (1909a, 1909b) reports a comparison of calcium, mag- nesium and phosphorus metabolism on a diet containing meat with the same after the substitution of fish for meat. The experiment covered two periods of four days each. Under the influence of the fish the phosphorus and magnesium retention was increased, while the calcium retention was decreased. The author considers especially characteristic the increase of the calcium (about 60 percent) and magnesium (about 44 percent) of the urine above the amount present during the meat period. Of this article we have seen abstracts only. The most important demonstration of specific effects of com- mon foods on phosphorus metabolism is certainly the recent discov- ery of the cause of beriberi. In this disease profound disorganiza- tion of the nervous system is due to the lack of certain important activating principles in polished rice. For a discussion of this matter see Beriberi. PHOSPHORUS METABOLISM 369 EXPERIMENTS WITH DOMESTIC ANIMAIiS Verdeil (1849) analyzed the blood ash of different species of animals, and of dogs on various rations. He concluded that a differ- ence of feed shows quickly in the composition of the blood, and that in the blood of animals eating meat, bread or grains alkali phos- phates are abundant, and carbonates almost lacking, while with herbivora the opposite relation holds. Landsteiner (1892) found no significant differences in the blood ash of rabbits fed on milk, and those fed on hay for 2>y% months. According to Hart, McCollum and Fuller (1909), the blood of four pigs that had received different amounts and kinds of phospho- rus compounds in the diet showed a phosphorus range from 0.24 to 0.33 percent P (aver. 0.29 percent) of the air-dry matter. The cal- cium ranged from 0.026 to 0.038 percent Ca, with an average of 0.033 percent. In the work of E. B. Forbes and associates (1914) the minerals (including phosphorus) of the blood were found to vary, in marked and consistent ways in accord with the character of the food. Ash varied between 0.90 and 1.04 percent, phosphorus between 0.38 and 0.64 percent, inorganic phosphorus between 0.009 and 0.019 percent, calcium between 0.0061 and 0.0085 percent, magnesium between 0.0034 and 0.0059 percent, potassium between 0.172 and 0.230 per- cent, sodium between 0.19 and 0.29 percent, sulphur between 0.120 and 0.166 percent and chlorine between 0.24 and 0.27 percent. Alquier (1905-6), from feeding experiments on horses, conclud- ed that beet molasses would increase the solubility, and therefore the assimilability, of the phosphorus of the ration, through the action of the considerable amounts of sodium and potassium present in this food. On the assumption that the greatly increased amounts of sodium and potassium introduced into the ration by the molasses replaced, in a measure, the calcium of calcium phosphate, it seems quite possible that the suggestion is in accord with the facts. E. B. Forbes (1909) studied, in three experiments involving 120 pigs, the specific effects of a number of common foodstuffs, especial- ly corn (maize) , on the bodies of pigs, largely as to relations of fat, water and phosphorus. Results were obtained from the gain in live weight, and in weight of parts and organs, analyses of the tenderloin (psoas magnus) muscles, livers, kidneys, and cross sections of the body at the 6th rib, and also of the foodstuffs used. The first experiment involved 65 pigs in 13 lots of 5 each. The feeding lasted 60 days, and the pigs in the several lots gained in 370 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 weight from one pound to 1.7 pounds per head and day. This experi- ment was conducted in two series of 6 lots, one receiving food ad libitum, and the other receiving restricted amounts of the same foods, one lot being killed as a control at the beginning of the experi- ment. Two lots (one in each series) received corn alone, and of the remaining ten lots, two each received corn supplemented by linseed oil meal, wheat middlings, corn germ . oil meal, soy beans and digester tankage. The muscular increase was generally in the order of the phos- phorus content of the rations, except with the lots which received tankage, the phosphorus of which was present mostly as bone. The gain of fat and muscle were reciprocal; that is, • arranging the lots according to gain in fat arranged them inversely as the gain in muscle. These facts suggest that common foods do not contain as much phosphorus as is essential to maximum growth of muscular tissue and skeleton. Lecithin phosphorus seemed not to be the dominant factor in this consideration. Corn, which is characterized by low protein, calcium and phos- phorus contents, in comparison with better-balanced rations, pro- duced under-sized, over-fat animals, with small viscera, and deficient muscular development, and bones which lacked in size, strength and ash per unit of volume. The muscles were characterized by low protein and low ash content, and high content of f at^ while the water in the tissue as a whole was low, but in the fat-free meat was high. These effects are due especially to lack of protein and of calcium, which seem to limit the usefulness of the phosphorus. The second and third experiments involved 55 animals in 12 lots, commonly of 5 each. They were conducted in the same general way as the first, but the rations were composed of pearl hominy and blood flour to which were added certain phosphorus-containing preparations of interest. These were (1) water extract of wheat bran, containing an abundance of inosite-phosphoric acid, (2) bone flour, (3) lecithin and (4) sodium phosphate. The same amount of phosphorus was fed in disodium phosphate as in lecithin. In these later experiments studies were also made on the growth and compo- sition of the bones (humeri). For numerical data see tables on pages 371 to 374. The water extract of wheat bran appeared to contribute to the muscle-producing capacity of the ration, and bone flour was decided- ly less useful in this way. Marked characteristics of the muscular tissue produced in these experiments were the low phosphorus content (0.195-0.252 percent) of the muscle produced by rations containing the bran extract, and PHOSPHORUS METABOLISM 371 the very high phosphorus content (0.352 percent) of the muscle produced by the ration containing lecithin. In Experiment II the bones of the pigs which had received bone meal exceeded all others in volume, total ash, ash per c.c. of volume, and breaking strength. Clearly the bone meal was of direct value as a source of nutriment. Observation determined the fact that the deposit of bone salts was largely at the inside of the walls of the bones. In some cases this interior thickening of the walls proceeded almost to the obliteration of the marrow space. Certain- ly there is a much less definite upper limit for the mineral content of the bones than for other tissues. This is in harmony with their prominent storage function for mineral nutriment. FOODS AND MINERAL NUTRIENTS CONSUMED (Forbes, 1909) EXPERIMENT H EXPERIMENT HI Lot Rations Grams Cal- cium Grams Mag- nesium Grams Potas- sium Grams Sodium Grams Sul- phur Grams Phos- phorus Grams Chlo- rine Grams Excess normal acid C. C. 1 Com ,. 2114.2 .189 2.326 5.920 .973 2.981 6.237 .846 217.90 2 Hominy. 1991.8 Blood-flour 165.6 1.092 3.500 7.239 1.358 4.236 6.641 1.491 149.11 Bran-extract, (Larger amt.) 2039.4 3 Hominy. 2068.9 Blood-flour 187.3 Lecithin 4.00 .822 .504 2.166 .921 3.728 1.562 1.518 198.50 4 Hominy. 2074.3 .936 1.675 4.140 1.100 3.980 3.429 1.531 Blood-flour 181.4 176.50 Bran-extract, ( smaller amt. ) 779 . 3 5 Hominy. 1821.2 Blood-flour 162.4 5.747 .552 1.920 .915 3.286 3.610 1.337 54.91 6 Hominy 2068.4 .822 .504 2.166 1.152 3.728 1.562 1.518 188.48 ; 1426.9 .134 1.571 3.998 .657 2.013 4.212 .571 147 14 3 Blood-flour 1548.6 171.9 .842 1.272 3.144 .958 3.214 2.655 1.245 138 63 ....... 589.7 4 Blood-flour 1570.4 174.6 3.406 .463 1.684 .579 3.054 2.390 1.247 : ] . gfj 11.0 5 1286.9 143.3 .616 .332 1.378 .672 2.500 0.934 1.021 122.70 372 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In Experiment III the pigs were younger than in Experiment II, and could be persuaded to eat but a very small amount of the bone meal, the pigs on the ration of corn alone receiving more phosphorus than the lot on the basal ration plus bone meal. The bone data from this experiment, therefore, were less significant. The pigs receiving the maximum amount of bran extract that they would tolerate, and receiving in this ration nearly twice as much phosphorus as another lot which was fed less of the extract, had smaller muscles and also smaller bones, the bones containing slightly less ash per cubic centimeter of volume, and the muscles less phosphorus than in the lot which received less of this supple- ment. The bran extract apparently contained some constituent which, when present in excess, tended to restrict the utilization of the phosphorus of the ration. This extract was very rich in mag- nesium. ANALYSES OF TENDERLOIN MUSCLES (Forbes, 1909)— Percent EXPERIMENT II Lot Rations Water Pratein Fat Ash Phos- phorus Propor- tion of phos- phorus to protein Phos- phorus in ash Corn. Hominy; blood-flour; bran-extract, (larger amount) , Hominy; blood-flour; lecithin Hominy; blood flour; bran-extract, (smaller amount) Hominy; blood-flour; bone-meal.. . Hominy; blood-flour; sodium phosphate Check lot 73.51 73.62 74.63 72.74 72.83 72.87 73.57 19.73 18.85 15.78 20.67 20.13 20.74 18.53 5.17 5.17 4.27 4.22 5.12 5.14 4.97 1.09 1.13 1.10 1.21 1.08 1.10 1.10 .264 .195 .352 .252 .233 .264 .247 1.34 1.04 2.23 1.22 1.16 1.27 1.33 24.22 17.35 32.00 20.83 21.57 24.00 22.45 EXPERIMENT III 1 73.39 76.35 73.74 73.20 72.81 17.68 19.42 21.85 18.30 20.39 6.16 3.04 3.90 4.46 4.52 1.07 1.13 1.03 1.12 1.15 .228 .238 .195 .222 .228 1.29 1.23 .81 1.21 1.12 21.31 2 Check lot 21.06 3 4 5 Hominy; blood-flour; bran-extract .... Hominy; blood-flour; bone-flour 18.93 19.82 19.83 This series of experiments demonstrates a susceptibility of the animal body to change of composition as a result of the character of the food which is often underestimated and still more frequently denied. As an instance of this fact the above and the following tables are submitted. PHOSPHOEUS METABOLISM 373 From these tables we note that the phosphorus in muscle varied in amount, expressed as percent of protein, from 1.04 to 2.23 per- cent, and expressed as percent of ash, from 17.35 to 32 percent. In liver the phosphorus varied between 1.57 and 2.10 percent of the protein, and between 24.18 and 28.23 percent of the ash. In the kidneys, the phosphorus varied between 1.32 and 1.99 percent of the protein, and between 18.47 and 27.90 percent of the ash. These variations in gross proximate analysis are certainly sufficient to indicate profound changes in functional efficiency. Enzyme estimations in connection with such studies would probably be of value. ANALYSES OF LIVERS (Forbes, 1909)— Percent EXPERIMENT II Lots Rations Corn „ Hominy; blood-flour; bran-extract, (larger amount) Hominy; blood-flour; lecithin Hominy; blood-flour; bran-extract, (smaller amount) Hominy; blood-flour; bone-meal.. Hominy; blood-flour; sodium phosphate Check lot Water Protein Pat Ash Phos- phorus Propor- tion of phos- phorus to protein 74.24 18.83 2.81 1.27 .320 1.70 73.52 17.19 2.46 1.37 .356 2.07 71.98 18.72 2.90 1.29 .364 1.94 71.96 20.13 3.01 1.32 .364 1.81 71.81 19.45 2.65 1.39 .361 1.85 72.05 20.58 2.85 1.34 .324 1.57 71.48 14.61 1.84 1.17 .291 1.99 Phos- phorus in ash 25.20 **. I 26.00 28.22 27.58 25.97 24.18 24.87 EXPERIMENT III Corn Check lot Hominy; blood-flour; bran-extract. Hominy; blood-flour; bone-flour.. . . Hominy; blood-flour 70.77 16.08 2.32 1.21 .338 2.10 71.21 18.12 3.01 1.33 .338 1.87 71.65 20.44 2.49 1.30 .367 1.80 70.43 19.31 2.03 1.30 .366 1.90 70.64 20.97 2.26 1.34' .346 1.65 27.93 25.41 28.23 28.15 25.82 See also the tables on the following page. Schkarin (1910) studied the influence of the diet of mother dogs on the development of suckling pups. No characteristic effects were observable of the diets of (1) vegetable food, (2) meat and milk, and (3) eggs and milk. The reason for negative results lies in the impossibility of modifying the composition of the milk, to any considerable extent, through the character of the food. Fingerling (1911a) attempted to solve the question of the bet- ter utilization by ruminants of the phosphorus compounds of cereals and mill feeds than of roughage. After various tests, which threw no light on the matter, he fed, in comparison, fresh grass and hay 374 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 ANALYSES OF KIDNEYS (Forbes, 1909)— Percent EXPERIMENT II Lots Rations Corn i Hominy; blood-flour; bran-extract, (larger amount) , Hominy; blood-flour; lecithin. Hominy; blood-flour; bran-extract, (smaller amount) Hominy; blood-flour; bone-meal Hominy; blood-flour; sodium phosphate Check lot Water 78.21 80.68 78.80 79.99 79.27 80.55 78.80 Protein 15.78 14.34 16.38 15.62 15.49 14.56 16.83 Fat 3.07 2.65 2.89 2.57 2.94 2.72 1.99 Ash 1.17 1.02 1.15 1.10 1.11 1.12 1.20 Phos- phorus .232 .243 .258 .250 .205 .234 .262- Propor- tion of phos- phorus to protein 1.47 1.69 1.58 1.60 1.32 1.61 1.57 Phos- phorus in ash 19.83 23.82 22.43 22.73 18.47 20.90 21.83 EXPERIMENT III 1 Con 76.70 80.32 79.78 77.18 77.84 17.07 14.37 15.79 16.01 17.00 4.95 2.11 3.41 3.92 3.91 1.14 1.12 1.11 1.14 1.07 .313 .249 .249 .318 .294 1.83 1.73 1.58 1.99 1.73 27.46 2 Check lot 22.23 3 4 5 Hominy; blood-flour; bran-extract.... Hominy; blood-flour; bone-flour 22.43 27.90 27.48 DATA CONCERNING DEVELOPMENT OF BONES (Forbes, 1909) EXPERIMENT II Vol- Longer Shorter Lot Rations ume of each humer- us each humer- us Ash per c. c. Breaking 1 strength Length trans- verse diam- eter trans- verse diam- - eter C. C. Grams Grams Lbs. Cm. Cm. Cm. 1 108.0 111.8 118.0 117.6 32.92 33.33 38.97 36.39 .3048 .2981 .3303 .3094 509 575 736 676 13.88 13.68 13.70 13.74 2.25 2.23 2.20 2.34 1.63 ? Hominy; blood-flour; bran-extract, 1.67 3 1.66 4 Hominy; blood-flour; bran-extract, 1.68 5 Hominy; blood-flour; bone-meal 121.3 46.22 .3811 791 14.00 2.28 1.71 6 Hominy; blood-flour; sodium phosphate 112.5 35.59 .3164 624 13.90 2.33 1.69 EXPERIMENT III 1 74.6 65.9 100.1 94.4 93.7 23.82 19.93 28.01 28.97 20.56 .•319 .3024 .280 .307 .219 502 440 641 606 426 13.1 11.64 14.46 13.46 13.56 1.83 1.75 1.98 2.08 1.95 1.37 9 1.30 3 4 R Hominy; blood-flour; bone-flour 1.51 1.46 1.41 PHOSPHORUS METABOLISM 375 made from the same. Of the phosphorus of the hay 53.4 percent was used, while of the phosphorus of the fresh grass 91.0 percent was used. Fingerling, therefore, ascribes the difference in the value of the phosphorus in cereals and mill feeds, on the one hand, and roughages, on the other, to the resistance to digestion of the crude fiber of the latter, and its interference with the digestion of the phosphorus compounds. We would suggest that the nature and amount of the bases present with the phosphorus in these two classes of foods should receive consideration in this connection. There is little calcium in the cereals and mill feeds, and much more in roughage. Weiser (1911, 1912) has studied mineral metabolism with swine on cereal diets, with and without additional calcium. The balance data are as follows : AVERAGE DAILY NITROGEN AND MINERAL BALANCES WITH SWINE ON RATIONS OF CEREAL FOODS WITH AND WITHOUT ADDITIONAL CALCIUM— Grams Duration of period in days Rations Average body weight Kg. P Food Urine Feces Balance Ca Food Urine Feces Balance Mg Food Urine Feces Balance N Food Urine Feces Balance 21 55.88 3.2073 1.0241 2.4841 -0.3009 0.2507 0.1428 1.3936 -1.2857 1.5868 0.1951 0.1206 +1.2711 21.39 15.38 3.19 +2.82 9 53.37 2.6731 0.8134 2.2570 —0.3973 0.1996 0.1384 1.1298 —1.0686 1.3201 0.1337 0.1276 +1.0588 17.82 12.59 2.67 +2.66 8 44.12 2.1467 0.6663 1.1986 +0.2818 0.1559 0:0308 0.3206 -0.1955 1.0593 0.1623 0.3878 +0.5092 14.32 9.04 2.13 +3.15 900 firm, barley; 160 gm. starch 52.55 2.5251 0.7950 1.2572 +0.4729 0.5621 0.0489 0.4934 +0.0198 1.1002 0.2718 0.7058 +0.1226 17.899 8.810 3.670 +5.419 10 1050 gm. corn; 5 gm. calcium car- 47.02 2.8167 0.2714 1.6960 +0.8493 2.1950 0.0766 1.2602 +0.8582 1.2528 0.2075 0.9440 +0.1012 18.78 12.08 2.36 + 4.34 7 )00 gm. barley; 3 gm. calcium car- 49.93 2.5251 0.2862 1.2238 +1.0151 2.1961 0.0778 0.6699 +1.4484 1.1009 0.1962 0.5968 +0.3079 17.760 8.760 3.310 +5.690 376 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Author's Summary. "1. By exclusive feeding on corn, growing swine experience a calcium and phosphorus deficit even when there is storage of flesh and fat. At the same time there is storage of magnesium. "2. By the addition of calcium carbonate, not only was the calcium and phosphorus deficit relieved, but there was even a good retention of calcium and phosphorus. At the same time the stor- age of magnesium was decidedly lowered. The amount of calcium carbonate by which one may reckon on a good calcium retention is about 10-11 gm. per 100 kg. body weight. "3. From what has been said it is evident that the bone requirement of young swine is not sufficiently met by a feed of only corn and barley, but this may easily be helped by adding calcium carbonate." Hagemann (1912) obtained results in metabolism experiments with two wethers and a steer showing that calcium phosphate is more effective to increase nitrogen and calcium retention when finely milled with the feed than when simply mixed with the same at the time of feeding, a result in harmony with that of Herxheimer (1897), who found 18 gm. of calcium carbonate baked in bread more effective to increase the solvent power of the urine for uric acid than 30 gm. of the same fed as a powder. These results suggest that the mechanical relations of food constitu- ents may affect their time, and place of digestion, and, in this way, their final disposition. See also Landsteiner (1892) ; effects of hay and milk on blood-ash of rabbits. E. B. Forbes and associates (1914) have shown, in their experi- ments of Series IV, that corn in the diet of swine has definite specific effects on various items of composition, among these, — the total and inorganic phosphorus of the blood, the lecithin content of the liver, and the composition of the ash of the bones. Forbes, Beegle, Fritz and Mensching (1914) conducted an extensive mineral metabolism experiment with swine, phosphorus balances being included. Five barrow pigs, about 6 months old, and all from the same litter, were the subjects of the experiment. Confined in metabolism crates they were taken through eight 10-day collection periods, separated by 7-day intervals on the next ration to follow, the change of food being made abruptly at the end of the collection period. The five animals were given the same kind of food, the results therefore being based on five repeats. The foods used in the several periods were as follows : 1. Corn. 5. Corn; meat meal. 2. Corn ; soy beans. 6. Corn ; skim milk. 3. Corn; linseed oil meal. 7. Corn. 4. Corn ; wheat middlings. 8. Rice polish ; wheat bran PHOSPHORUS METABOLISM 377 They were, therefore, the common practical foods for swine in this country, except for ration No. 8, composed of rice polish and wheat bran, these feeds being selected on account of their very high content of magnesium as compared with calcium. Corn was fed alone in the first and seventh periods to show any such changes as might be due to the long-continued routine or to increasing age. The observations covered the usual proximate analyses of food- stuffs and feces, daily nitrogen, creatinin and ammonia estimations on the urine, also determinations of sodium, potassium, calcium, magnesium, sulphur, phosphorus and chlorine on foods, urines and feces; and further, a slaughter test on the five animals after the termination of the experiment. The condensed mineral balance data are in the table on p. 378. These figures represent averages of results from five individuals. The uniformity of the results with the several individuals was sufficiently marked to warrant averaging. The phosphorus balances in these experiments were all positive, except for one individual on the ration of corn alone. In no case, however, was there any considerable retention of phosphorus on corn alone. Except in one case the phosphorus retention in the sev- eral periods was in the same order as the intake. This exception was the ration containing the wheat middlings. The peculiarity of the phosphorus of this ration was a large proportion of tritico- nucleic acid, and s the phosphorus of this ration was much less efficiently retained than the phosphorus of the rations containing meat meal and milk. With a much smaller intake of phosphorus in meat meal and in milk the retention was much greater. Two circum- stances unfavorable to phosphorus retention in the wheat middlings ration were the presence of much less calcium and much more mag- nesium than in the meat meal and milk rations. The results were increase in both urine and feces phosphorus. The magnesium of the food is shown to be a prominent factor in the partition of the phosphorus between urine and feces, an increased proportion of magnesium to phosphorus in the food increasing the proportion of feces to urine phosphorus. There was no such prominent effect of magnesium to restrict phosphorus reten- tion. During the feeding of skim milk there were lower proportions of potassium, magnesium, sulphur, chlorine and phosphorus in the feces than during any other period ; and this period was also charac- terized by the maximum percentage retention of the calcium, magne- sium, sulphur and phosphorus intake. S78 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 s o fa fa Q fa cc O fa o JO U I-H CT2 «5 rH & 7) O t— i H s - S§ ft a 0/ I ■a. K° - °Z «d fa So s 01 > fa i^'d s- «jgfa r o fau <£ 0. r.f o if. -, S fa S- 3 > i— i - cmcooc-h cococoos o — <~-- mt— - o cx> cm oc i>- nho:o tp cg m o r^COCD'— l •-t(Nt>i > t— (Ot*-CM t-HOTf>H + + ■gcSc \8-cB5 fe P&« ^i3^*^Oi — < cm ro t— lO-^-rtiGO -rtT#iT* co r-- oi co i>- ct> cm *n> co co :o cm c: :d cm :d cc m cm o co m cm io t* cm o t— i co os m o ccjdoco ocooa oa fl CMlOCO lO-— I SSlO COI>-lO r-^'sicooo iocdoco c*-ooo505 IOHMO CDt-i^O o r 8-cai3 Effort « r 8-cSiS •rt " oi ~ fe pk£ »i « oi y rt o c s E o r 8-c8 5 kpfepg. rt o.S 8 « ^fcpk rt ^ I) 01 J! 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OtSCC^ BJa- : c * rt 01 b — *° P-rt 85^15 CO t^ CO CO CO 03 PHOSPHORUS METABOLISM 379 Corn is shown to be more deficient in calcium than in any other nutrient; its magnesium content is also low, and its phosphorus content allows of but slight retention. At the same time the nitro- gen retention is quite considerable. In spite of the slight retention of phosphorus its amount was considered as insufficient, since there was present hardly more than the amount required for maintenance, at a time of life which would naturally be characterized by exten- sive storage of phosphorus. The results, in general, show that the mineral requirements of swine are apt not to be satisfied during cereal feeding. A dry-lot fattening process probably involves, as a rule, considerable draft upon mineral stores previously accumulated during periods of access to green feeds. In a series of experiments by Forbes and associates (1914), with swine, three lots of pigs (5 in a lot), receiving corn in the ration, differed as a group from five other lots which received no corn in the ration, in several respects, as follows : Each of the three lots of pigs receiving corn had more potassium and magnesium, and more total and inorganic phosphorus in the blood, more magnesium and phos- phorus, and less calcium in the bone ash, and more lecithin in the liver than any one of the five lots which had not received corn. For a summary on common foods in relation to phosphorus metabolism see p. 395. See also the following sections on the phosphorus content of milk, and the phosphates of the bones, as affected by foods. THE PHOSPHORUS CONTENT OF MILK AS AFFECTED BY FOODS The idea of modifying the composition, and thus the nutritive value of milk, through the character of the food, is ancient, and has been the inspiration of many investigations. Some misconceptions among the conclusions reached have been due to the relatively considerable variations in the composition of the milk during the course of the period of lactation. Among the earlier observations were those of Bocker (1849) who stated that administration of calcium phosphate to wet nurses whose milk was poor in lime served to enrich the milk in this constit- uent; and Ssubotin (1866), who studied the composition of dog's milk as affected by various rations, and ascribed an increased casein content to fat in the diet; also Decaisne (1871) who made observa- tions on 43 lactating women during the siege of Paris. Hunger prevailed; the flow of milk was often insufficient to nourish the infants, and 12 died of starvation. The milk was low in casein, fat, sugar and salts, but high in albumin. 380 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Weiske (1871b) studied the effects, on the composition of cow's milk, of adding 20 grams daily of acid calcium phosphate to a ration which the cows had been consuming regularly during a protracted period. There were no differences caused by the use of calcium phosphate, nor did Yvon (1879) succeed in increasing the phosphate of cow's milk in this way; but Hess and Schaffer (1891) claim that they did produce increased phosphorus in the milk ash. The aver- age percent of P 2 5 in the ash of the milk of three cows before feed- ing the phosphate was 25.80 ; after about a month this figure was increased to 27.52, and after about two months to 29.54 ; but it was not shown by the use of controls that the advance in the period of lactation did not enter into the production of this result. Sagnier (1891) reports that Charles Gravier, by special feeding (the nature of the feeding is not revealed), was able to furnish for the hospitals milk containing from 2.30 to 2.50 gms. phosphoric acid per liter. Duclaux (1893) concludes that milk which is sold as rich in phos- phate, because of phosphate feeding, is found not to be so on anal- ysis. Hills's experiments (1887, 1894) with ground-bone feeding showed, first, that the added phosphorus did not all appear in the feces ; then that it increased somewhat the phosphoric acid content of the milk of the cows ; in one case from 0.2142 to 0.2263 percent, and in another from 0.1809 to 0.1909 percent. J. Neumann (1893a) reports an experiment in which the addi- tion of a calcium phosphate preparation to a feed already supplying sufficient quantities of the mineral constituents for milk production seemed to cause a slight rise in the lime and phosphoric acid content LIME AND PHOSPHORIC ACID OF MILK AS AFFECTED BY CALCIUM PHOSPHATE ADDED TO THE FEED (J. Neumann, 1893a) Milk In 1000 gm. milk Percent of ash In gms. per cow Date Kg. Ash Percent Lime Phos- phoric acid Lime Phos- phoric acid Lime Phos- phoric acid Aug-. 22 " 23 " 24 27.16 27.40 28.20 0.77 0.77 0.77 1.4950 1.4804 1.4618 1.9271 1.9594 1.9930 19.42 19.23 18.98 25.03 25.45 25.88 13.53 13.51 13.74 17.44 17.88 18.73 Without added phos- phate: about 80 gm. lime, and 110 gm. phosphoric acid per day per cow " 27 " 31 Sept. 3 r. 7 " 13 " 20 " 28 24.85 25.79 25.67 25.00 25.50 24.30 24.12 0.75 0.75 0.78 0.78 0.'77 0.77 1.4355 1.4371 1.5566 1.4995 1.5252 1.5916 1.5509 1.8865 1.9307 1.9864 1.9868 1.9685 2.0262 2.1323 19.14 19.16 19.96 19.22 (19.81) 20.67 20.14 25.15 25.74 25.47 25.47 (25.56) 26.31 27.69 11.88 12.36 13.32 12.49 12.96 12.90 12.47 15.62 16.61 17.00 16.55 16.73 16.41 17.14 40 grm. lime and 33 g-m. phosphoric acid per day per cow added as calcium phosphate PHOSPHORUS METABOLISM 381 of the milk, but only slight, and only after three or four weeks' duration of the phosphate feeding. The first effect of the phos- phate was suddenly to reduce the milk flow. The milk of three cows was mixed for the analyses quoted on p. 380. The first addition of phosphate was made on the evening of August 24. Sanson (1894) reports the following results, which were con- sidered to show increasing amounts of phosphorus in milk, associ- ated with increasing amounts of Na 2 HP0 4 added to the feed, up to the dose of 22 gms., no further increase occurring with greater amounts of the phosphate. The increased phosphorus of the milk was all in the serum, the casein not showing any increase. PHOSPHORUS OF MILK INCREASED BY PHOSPHATE FEEDING Sanson (1894) Day Dose of sodium phosphate Density of milk P2O5 of milk Increase of P2O5 Grams Grams per 1000 Grams per 1000 1 1.032 Mi 1-438 2 10 1.031 1.984 0.546 3 .12 ' 1.033 2.046 0.608 4 14 1.031 1.946 0.508 5 16 1.029 2.046 0.608 6 18 1.032 2.084 0.646 7 20 1.032 2.145 0.707 8 22 1.036 2.170 0.732 9 24 1.034 2.070 0.632 10 26 1.037 2.108 0.670 11 28 1.034 2.083 0.645 12 30 . 1.033 2.170 0.732 In the experiments of Jordan, Hart and Patten (1906), with two cows, the removal of the phytin from the bran fed was with- out significant change on the phosphorus content of the milk. EFFECTS OF THE INGESTION OF PHOSPHORUS COMPOUNDS ON THE COMPOSITION OF MILK— MEAN DAILY OUTPUT IN MILK Jordan, Hart and Patten (1906) Composition of milk, percent Partition of phosphorus in milk Dates Pro- teids Case- in Fat Sugar Solids Total Gms. Nucleo- phos- phorus Gms. Soluble org-anic Gms. Inor- ganic Gms. Ration Mar. 12-Mar. 18 Mar. 30-Apr. 5 Apr. 24-May 1 May 29- June 4 3.07 3.05 3.22 3.27 2.53 2.45 2.66 2.75 3.28 3.09 3.73 3.29 5.56 5.46 5.47 5.50 11.91 11.59 12.41 12.07 14.7 15.8 3.5 3.3 1.05 1.17 10.2 11.2 Oat straw, bran, rice meal, wheat gluten. Oat straw, washed bran, rice meal, larger amount of gluten. Lipschitz (1906) notes that the feeding of bone meal reduces the milk flow, though increasing the total ash content of the milk, 382 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Hart, McCollum and Humphrey's observations (1909) on one cow, extending over three and a half months, are summed up, so far as the ash of the milk is concerned, by saying : "Variations, within wide limits, in the form and quantity of supply of potassium, mag- nesium, or phosphorus did not influence the percentage content of these elements in the milk." It is to be noticed that the amounts yielded fell off markedly during the experiment. Von Wendt's (1909) conclusion from his work was that neither the fodder nor the added salts exerted much influence on the compo- sition of the milk, the phosphorus, casein and calcium being among the items least affected of all. CHANGES OF AMOUNT AND COMPOSITION OF MILK ACCOMPANYING THE ADMINISTRATION OF CERTAIN SALTS (Von Wendt, 1909) Gain or loss of P2O5 Gain or loss of CaO Dates Supplements Sain or loss in milk Cow or | group Grams Percent Grams Percent Grams Oct. 14-16 30 gm. potassium oxalate —2000 —0.002 -2.3 -0.02 -4.8 Oct. 20 60 gm. potassium oxalate +900 +0.9 -0.03 —2.8 Nov. 2-3 60 gm, potassium oxalate —770 +0.006 +0.011 +0.006 +0.3 Nov. 19-21 75 kg. beet leaves —2150 +0.001 —2.1 -2.8 Cow 149 Dec. 16-18 30 gm. Ca HP04 +480 +0.002 +0.7 +0.042 +5.1 Cow 151 May 26-28 75 gm. Ca HPO4 -300 -0.007 -1.1 +0.026 +2.9 Cow 150 May 26-28 75 gm. Ca HPO4 —300 -0.003 -0.7 +0.024 +3. Cow 150 Dec. 16-18 30 gm. Ca HPO4 -600 +0.001 -0.7 +0.013 Cow 149 Dec. 25-27 90 gm. lime +330 +0.35 -0.007 -0.25 Cow 150 Dec. 25-27 90 gm. lime —400 -0.001 -0.23? +0.021 +0.7 Cow 150 June 29- July 4 15 gm. lecithin —1300 +0.004 —0.8 +0.020 +1.1 Gr. I Apr. 22-24 30 gm. Ca HPO4 +100 +0.002 +0.45 +0.007 +1.4 Gr. II Apr. 22-24 30 gm. Ca HPO4 "-200 +0.001 -0.1 +0.006 +0.27 Gr. Ill Dec. 7-9 90 gm. CaHP04 +700 +0.001 +0.66 0.0 +0.63 Gr. II Dec. 25-27 60 gm. Ca HPO4 -220 —0.004 —0.2 -0.007 -1.0 Gr. Ill Dec 28-30 60 gm. Ca HPO4 +400 +0.001 —0.06 +0.006 -1.0 Gr. I Apr. 16-18 30 gm. lime +1400 -0.001 +1 +2 Gr. II Apr. 16-18 30 gm. lime —500 -0.001 -0.57 -0.006 -1.18 Gr. I Dec. 28-30 90 gm. lime +400 +0.004 +0.9 +0.014 +2.3 Gr. II Dec. 16-18 90 gm. lime +40 ' +0.001 +0.2 +0.042 +5 Gr. Ill Dec. 7-9 90 gm. lime -110 +0.003 +0.15 +0.007 +0.5 Gr. II Oct. 19-21 Beet leaves+Ca HPO4 —460 -0.001 -0.6 -0.6 Gr. Ill Oct. 19-21 Beet leaves+Ca HPO4 —440 -0.4 -0.5 Gr. Ill Nov. 29-Dec. 6 Beet leaves+Ca HPO4 +900 +0.009 +2 +0.013 +2.2 Golding and Paine (1910) planned an experiment to test the possible effects on milk of pasturing cows on land manured with phosphates and potash, as compared with pasturing on unmanured land on grass of very poor quality. The ash, phosphoric acid, potash and lime of the milk (averaged) are the same for the two plots of land. The milk yield was considerably higher from the cows on the manured half, and its fat content higher from the other half. According to the data furnished by Monvoisin (1910), both, inflammation of the udder and tuberculosis affect the composition of the milk, lowering the percent of phosphorus in the ash. His data also show a distinct lowering of the casein content of milk in tuber- culosis, both in absolute amount and in relation to total nitrogenous matter. PHOSPHORUS METABOLISM 383 A. R. Rose and J. T. Cusick (1911) report inconclusive studies of the influence of phosphorus of the food on the yield and composi- tion of goat milk. Fingerling (1911b) studied the effects of diets rich and poor in calcium and phosphorus, and of dicalcium phosphate and calcium carbonate, on the yield and composition of the milk of goats; and also the effects of the same on the calcium and phosphorus balances. The balances responded quickly and consistently to variations in the intake of calcium and phosphorus. The effects of these conditions on the composition of the milk were slight; the effects on the amount of milk produced were more prominent. INFLUENCE OF A LIME- AND PHOSPHORIC ACID-POOR DIET ON THE MILK SECRETION OF GOATS (Fingerling, 1911b) Amount of milk Ash Percent of ash No. of Goat Feed days Grams Grams CaO P2O5 A Lime- and phosphoric acid-rich 1958 13.31 18.09 25.89 14 Lime- and phosphoric acid-poor 1886 13.30 18.04 25.80 14 Lime and phosphoric acid 1547 10.71 19.79 29.85 13 Lime and phosphoric acid 1388 8.90 21.44 30.41 13 Same with dicalcium phosphate 1493 10.35 19.97 28.35 14 Same with dicalcium phosphate and CaC03 1687 11.72 18.68 26.41 17 " Like first period 1958 13.31 18.09 25.89 31 Lime- and phosphoric acid-rich 1011 8.59 23.06 29.89 13 Lime- and phosphoric acid-poor 999 8.66 23.23 29.51 14 Lime- and phosphoric acid-poor 839 7.08 24.22 33.43 14 Lime- and phosphoric acid-rich 787 6.55 26.20 35.35 12 Lime- and phosphoric acid-poor 1061 8.73 22.63 30.39 12 Lime- and phosphoric acid-rich 1011 8.59 23.06 29.89 9 From later experiments it was concluded (Fingerling, 1912b) that neither the activity of the mammary glands nor the composi- tion of the milk was altered by feeding the organic phosphorus compounds. EFFECTS ON GOAT'S MILK OF ADDING DIFFERENT PHOSPHORUS COMPOUNDS TO A BASAL RATION VERY POOR IN PHOSPHORUS Fingerling (1912b) — Grams Ration Milk CaO P2O5 Date Total Gain or loss Total Gain or loss Total Gain or loss A B A B A B A B A A A B Basal ration Basal ration Lecithin added Lecithin added Phytin added Phytin added Casein added Casein added Nuclein added Sodium nucleate added Di-sodium phosphate added Di-sodium phosphate added 1587 1396 1515 1563 1397 1593 1525 1606 1491 1430 1583 1824 —72 +167 —190 +197 -62 +210 -96 -157 -4 +428 1.808 3.060 1.838 3.072 1.813 3.136 1.784 3.083 1.828 1.804 1.883 3.179 +0.030 +0.012 +0.005 +0.076 -0.024 +0.023 +0.020 -0.004 +0.075 +0.119 2.594 4.164 2.626 4.247 2.555 4.351 2.531 4.255 2.595 2.561 2.656 4.401 +0.032 +0.083 -0.039 +0.187 -0.063 +0.091 +0.001 -0.033 +0.062 +0.237 384 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Gaube (1895) has also studied this question, and Jensen (1904, 1905a, 1905b, 1905-6) has made extended investigations in this field, especially with rations of varying proportions of hay and beets to which were added phosphates of sodium, calcium and magnesium, etc. Lanzoni (1918) noted slight modifications of composition of milk through the agency of drugs. See also Vivier (1911). In all cases the variations in phosphorus content of cow's milk which are ascribable to the character of the food are within the range of normal variation. At most these differences are slight, or doubtful, and in no case of practical significance from any point of view. As to the effects of starvation — the evidence is insufficient to warrant conclusions. EFFECTS OF DIET ON THE PHOSPHATES OF THE BONES As the principal store of phosphates in the body the skeleton is of interest in connection with the various circumstances which affect phosphorus metabolism. In judging of the results of experi- mental feeding, however, we must bear in mind the fact that the amount of phosphorus in the bones is so very much greater than that in the remainder of the body that we may be unable to detect changes of composition in the skeleton under the influence of circumstances which produce marked effects on phosphorus metabo- lism as revealed by balance experiments. The skeleton is so vari- able in its development ; its mineral constituents are so much affect- ed by the food, that special attention must be given to the breeding and rearing of experimental subjects for comparative studies on the bones. In our own experiments it has been found possible greatly to decrease the central cavity in the shaft of the long bones of pigs by feeding rations which are rich in bone-forming constituents. There seems to be no definite upper limit of phosphate deposit in the bones, as there is in the soft parts. Chossat (1842) found that pigeons fed on wheat alone died in 8-10 months from malnutrition. The salts were gradually with- drawn from the bones, which became weakened, and the breast bone imperforate. The addition to the diet, of calcium carbonate, appeared to prevent these symptoms, and to maintain the birds in health. Apparently the lack of calcium limited the use of phos- phorus. In a later paper Chossat (1843) makes a full report of his experiments with pigeons, chickens, rabbits, frogs, lizards, eels, tortoises, and other animals. The conclusions were in harmony with the above. Where pigeons lost 40.4 percent of the live weight in starving to death, only 3.4 percent of this loss seemed to come from the bony system. PHOSPHOKUS METABOLISM 385 Among other early studies are those of Boussingault (1846a, 1846b), who investigated the effects of foods on the growth of the skeleton of pigs, and C. Falck (1850), who studied the effects of feeding lime-rich and lime-poor foods on the development of domes- tic fowls. Dusart (1869) determined that a pigeon lost 4.9 gm. of calcium phosphate from the skeleton, and 58 gm. of live weight during 83 days' feeding on wheat. The addition of calcium carbonate to this diet reduced to zero the alkali phosphates of the excreta, which had been half of the total, and caused a return to the normal live weight. Dusart (1870) also published clinical reports of 19 hospital cases of fracture or disease of the bones, under treatment with cal- cium lactophosphate, which he recommends. Weiske (1871a), in experiments with milch-goats, found that where the food was especially poor in calcium and phosphorus, or in calcium alone, death might be caused by these deficiencies before noticeable change was produced in the composition of the bones. Probably greater refinement of experimental procedure would have demonstrated some change in the bones. Weiske found (1872a) that calcium, magnesium and strontium phosphates, when fed to rabbits with a ration of hay and turnips, produced no important change in the composition of the ash of the bones. Weiske and Wildt (1873) fed one lamb on a normal ration, and two more on a basal ration of straw, starch, sugar and casein, which was low in calcium and phosphorus. One of these also received calcium carbonate and the other disodium phosphate. The experi- ment lasted 25 days. The basal ration seems to have contained enough calcium and phosphorus so that the experimental procedure produced in 25 days no consistent change in the composition of the bones. Hofmeister (1873) experimented with 8 lambs, 4 in each of two lots, with a low-phosphorus ration of meadow hay and potatoes, to which he added, with one lot, bone phosphates as precipitated from a hydrochloric acid solution by sodium carbonate. The experiment extended from June 7-Nov. 30. The calcium phosphate caused no appreciable increase in rate of gain in weight. Hofmeister considered* that he had proven the digestibility of precipitated phosphates by lambs, by a comparison of food and feces, but attached little importance to the observed increase of phosphorus in the bones of the lambs which had received the phosphates. In the light of subsequent investigation in this 386 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 field it would appear that the increase in phosphorus content of the bones, as caused by the inorganic phosphates, was sufficient definitely to increase the breaking strength of the bones. The following data are taken from Hof meister's tables. CALCIUM AND PHOSPHORUS CONTENT OF BONES OF LAMBS AS AFFECTED BY ADMINISTRATION OF BONE PHOSPHATES Dry Matter Basis — Percent Jaw bone Shoulder blade Humerus Radius Tibia Ration P2O5 CaO P2O5 CaO P2O5 CaO P2O5 CaO P2O5 CaO Low phosphorus 26.20 31.30 19.60 24.50 19.30 22.60 19.70 23.90 19.70 24.30 Low phosphorus plus bone phos- phates 27.80 33.90 20.50 24.90 19.40 22.90 19.90 24.01 20.30 22.60 Hofmeister also studied the digestibility of superphosphate (laboratory prepared), with two 2-year-old wethers, and concluded that, with small doses, all the water-soluble lime and phosphoric acid are digested from superphosphate; with increased doses, one-third of the water-soluble phosphoric acid is digested, while all the water- soluble and half of the insoluble lime is digested. The facts as he noted them, would, of course, be differently interpreted at this time. Weiske (1874) notes that, of animals receiving the same diet under conditions of practice, some will suffer from fragility of the bones, and others will not. In experiments on rabbits receiving barley and various mineral supplements, Weiske found that the EFFECTS OF DIET ON THE PHOSPHATES OF THE BONES Weiske (1874) Percent, Dry, Fat-free Basis Kind of bone No. of ani- mals Mineral matter P2O5 CaO MgrO Feed Fowl, femur Fowl, femur 1 1 45.64 42.79 16.67 15.98 23.14 21.48 0.64 0.43 Usual Same Normal. Data computed by the compilers. Showed bone disease. Lamb, pelvic Lamb, pelvic Lamb, pelvic 1 1 1 60.46 60.36 60.07 23.94 23.79 23.94 31.78 31.60 31.57 Poor in phosphorus Poor in lime Normal These are bones of lambs in experiments reported in 1873. Data of this experiment com- puted by the compilers. Rabbit Rabbit Rabbit 2 2 2 65.64 65.08 64.42 26.79 25.60 26.06 34.10 33.68 33.34 0.75 0.76 0.76 Calcium-free barley; dist. water Same + Mg--phosphate Same + Sr-phosphate Data computed by compilers. Rabbit 1 65.62 26.26 34.23 0.74 Normal, dist. water 5 mos. old; killed when feeding 1 began. 6Vi mos. old; killed at end o experiment. 7H mos- old. Rabbit Rabbit Rabbit 1 2 2 67.61 69.03 67.55 26.87 27.46 26.95 35.15 35.92 35.28 0.80 0.79 0.73 Normal, dist. water Normal, dist. water No food; dist. water PHOSPHORUS METABOLISM 387 percentage content of bones, in ash constituents, varied in marked degree, but that the composition of the bone ash remained almost constant. The feeding of magnesium phosphate with calcium-free food did not cause a perceptible increase of magnesium in the bones, and only traces of strontium were found in the ash of the bones of animals to which strontium phosphate was fed. With calcium lacking in the food, an animal lives about as long as if the starvation is total. If the removal of the mineral matter is as complete as possible, there is, as in total starvation, a gradual loss of bone sub- stance. E. Voit (1880) shows that lack of lime in the diet of dogs reduces not only the lime but also the phosphorus in the bones, as set forth by the following analyses : ANALYSES OF BONES OF YOUNG DOGS AS AFFECTED BY LACK OF LIME IN THE FEED (E. Voit, 1880)— Percent Age of Bone Part of bone Water con- tent of skel- eton Percent of dry substance pup Days Ash Pe203 CaO Mg-O P2O5 Treatment of • subject 10 Humerus Scapula Humerus Scapula Outer Outer Spongy Spong-y [■ 66.2 -j 32.25 37.21 30.45 35.01 0.06 6.06 17.04 19.64 15.92 18.19 0.39 0.46 0.40 0.35 12.93 15.45 12.25 14.42 /- Killed as control 38 Humerus Scapula Humerus Scapula Outer Outer Spongy Spongy [■64.9-j 41.32 40.29 33.71 36.96 0.18 0.19 6."3i 21.40 21.40 16.87 18.50 0.38 6.' 28 0.33 17.40 12! 93 13.62 ] Fed from the agre 1 of ten days for 28 | days, lime being- J included 38 Humerus Scapula Humerus Scapula Outer Outer Spongy Spongy H 30.94 29.06 26.40 27.22 0.16 0.18 0.16 0.34 16.36 15.29 13.34 14.24 0.32 0.38 0.21 0.19 13.17 i6.*77 10.21 Fed the same as [■last, but without lime J. Cohnheim (1882) notes that Weiske and others, and Zalesky conclude that the composition of the ash of the bones is not affected by the composition of the food, but that Chossat, Bibra, Wegner, Voit and others have determined that the bones of animals, birds especially, become poor in earth salts and easily bent if the food is poor in these constituents. Young lions and leopards become rachitic when fed on meat without bones (Roll, 1860) . Weiske, Dehmel, Kennepohl, Schulze and Flechsig (1885) fed hay, acidified with sulphuric acid, to sheep, from Nov, 4, 1879 to Mar. 11, 1880, and studied the effects of the acid on the composition of the skeleton. The slight differences, indicating a solution of the bone salts by the acid, were regarded by the authors as within the legitimate error of work. A portion of the data are as follows. •388 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 EFFECTS OF ACIDIFIED HAY ON THE COMPOSITION OF THE SKELETON OF SHEEP— Fat and Water Free— Percent Lamb No. Feed Organic matter Mineral matter CaO MgrO CO2 P2O5 1 38.16 39.02 39.96 61.83 60.98 60.04 31.55 31.17 30.34 0.66 0.63 0.77 3.07 3.02 24.26 2 23.88 3 Killed at beginning- of experiment 23.59 At a later date Weiske (1886) reported more data on this same experiment, and showed that the calcium content of the 'flesh was decreased by the sulphuric acid feeding, but the phosphorus was not altered ; which implies that the effect of sulphuric acid on the bones in the experiment of Weiske, Dehmel, et al. (1885) was, at least in part, due to its interference with calcium absorption. Weiske (1888) experimented to determine the effects of adding, during 6 months, neutral precipitated calcium phosphate to the nor- mal food of rabbits. The phosphate had no effect on the gain in weight, nor on the composition of the bones, though there was a slight increase in the dry fat-free skeleton, due to the use of the phosphate. Henry (1889, 1890) reported increase in strength and ash of the bones of corn-fed pigs by the administration of bone meal. Wood ashes were found to give similar though less prominent results. Hard-water, in comparison with rain water, was not shown to increase the ash or strength of the bones. No increase of muscle was found (apparently by visual examination) to result from the feeding of bone meal or wood ashes ; nor was there increase in the weight of the blood or internal organs. Beraz (1890) studied the teeth of dogs as affected by the lime content of the ration. Rations producing rachitic conditions in the skeleton generally did not produce similar effects in the teeth. Weiske (1891a) quotes earlier work showing that dilute sul- phuric acid would, if ingested during a considerable time, lower the calcium content of both bones and muscles, and refers to work of Heitzmann, Hofmeister and Siedamgrotzky showing that if lactic acid be given the specific gravity and ash content of the bones is subnormal, and to work of Auerbach showing that KH 2 P0 4 , when ingested by a dog, greatly increased the ammonia of the urine. Weiske reports work of his own in which NaH 2 P0 4 was fed to rabbits on a ration of meadow hay, oats, beets, and potatoes. The analyses of the skeletons showed no differences of note between the rabbits with phosphate and those without. PHOSPHORUS METABOLISM 389 At a later date Weiske (1891b) reported further results of a similar but more critical study with rabbits, the rations being (1) hay, (2) oats, (3) hay and oats, and (4) oats and NaH 2 P0 4 . From the following data it will be noted that the hay was consistently superior, as a bone food, to hay and oats ; that the hay and oats were better than oats alone; and that the rabbits appeared to receive benefit from the sodium phosphate, in spite of its acidity. COMPOSITION OF THE BONES OF RABBITS AS AFFECTED BY THE FOOD (Weiske, 1891b)— Percent, Dry, Fat-free Bone Bones No. of ani- Mineral P2O5 CaO MgO mals matter 1 64.22 24.89 32.08 0.77 2 63.78 24.82 v 31.83 0.75 2 61.10 23.90 29.62 1.01 2 61.59 24.47 30.25 0.88 1 62.13 23.73 30.84 0.68 2 61.55 23.69 30.66 0.64 2 58.74 22.69 28.47 0.92 1 58.47 22.85 28.55 0.67 1 66.51 26.02 33.86 0.73 2 66.65 26.15 33.62 0.78 2 62.16 24.85 30.85 0.94 L 63.38 24.94 31.76 0.68 1 78.72 33.88 37.28 2.63 2 77.93 33.30 36.73 2.52 2 77.61 33.36 36.18 2.75 1 80.01 34.48 38.08 2.67 Feed All All All All All but long bones and teeth All but long bones and teeth All but long bones and teeth All but long bones and teeth Long bones Long bones Long bones Long bones Teeth Teeth Teeth Teeth Hay Hay and oats Oats Oats +Na H2PO4 Hay Hay and oats Oats Oats + Na H2PO4 Hay Hay and oats Oats Oats + Na H2PO4 Hay Hay and oats Oats Oats + Na H2PO4 Weiske (1892) reported results from feeding rabbits on oats, with trisodium phosphate, monosodium phosphate, tricalcium phos- phate, calcium carbonate, and sodium citrate added to the ration, for different lots of animals. The calcium carbonate greatly improved the oat ration. Acid sodium phosphate made the oats less useful than when fed alone, and the effect on the bones was injurious. Benefit was not observed from the other salts. Arnstadt (1893) discusses malnutrition of the bones of cattle and other farm animals as caused by lack of phosphorus in the food. Pregnant, milk-producing, or young, growing animals were found to suffer most severely. He speaks of liberal allowance of common salt being of benefit when fed with calcium phosphate, through assisting in its assimilation. Weiske (1895a) fed oats, having an acid ash, to rabbits, with and without other food, except for calcium sulphate and tricalcic phos- phate, which were fed to certain individuals. There was a loss of mineral matter from the bones, but no great change in composition. The sulphate and phosphate of calcium proved not to be so valuable as previous experiments had shown the carbonate to be, when added to the same ration. . 390 OHIO EXPEEIMENT STATION: TECHNICAL BUL. 5 Weiske (1895c) reports results of feeding experiments on cal- cium-poor rations, which were rich in phosphorus, and to which were added as supplements, calcium, strontium and magnesium carbon- ates, and calcium sulphate. The alkaline earth carbonates were found of value in the correction of the acidity of the ash of an oat ration, but magnesium and strontium do not physiologically replace calcium in the skeleton, although not inconsiderable quantities of these elements were transferred there. The urine of the rabbits receiving the alkaline earths was turbid and became continually more alkaline. The rabbit on oats alone had an acid urine. The addition of calcium carbonate was much more beneficial in its effect on the gain in weight than were other mineral supplements. COMPOSITION OF THE BONES OF RABBITS, AS AFFECTED BY THE FOOD (Weiske, 1895c)— Percent, Dry, Fat-free Bone Bones No. of ani- Mineral P2O5 CaO MgO mals matter 61.34 23.42 31.43 0.73 60.17 23.76 30.72 1.11 55.47 22.21 23.02 0.61 56.98 23.21 28.36 1.39 56.62 22.69 28.85 0.63 67.14 25.75 34.70 0.85 64.58 25.78 32.98 0.76 62.59 25.76 27.95 0.96 63.89 25.48 32.23 1.48 61.73 24.44 31.63 0.74 78.66 33.63 38.44 2.40 79.35 34.71 38.74 2.61 77.58 34.45 36.96 3.23 77.74 33.37 37.72 2.57 Feed All but loner bones and teeth All but long: bones and teeth All but loug- bones and teeth All but long bones and teeth All but long- bones and teeth Long- bones Long - bones Long- bones Long- bones Long- bones Teeth Teeth Teeth Teeth Oats+ CaC03 O ats +Ca SO4 Oats + Sr CO3 Oats + MgC03 Oats Oats + Ca CO3 Oats + Ca SO4 Oats + Sr CO3 Oats + Mg-C03 Oats Oats + Ca COs Oats + Ca SO4 Oats -+• Mg-C03 Oats Springer (1902) reports that by the application of electricity for 5 minutes daily at the seat of the epiphyseal cartilages of the legs, with rabbits, he induced an added growth of the bones as indi- cated by the following figures : Weight of Femur Length of Femur Grams Treated 1 1.105 Meters 0.066 1.185 0.064 Controls 3 0.977 0.060 0.940 0.059 Aron (1905) states, with reference to bone diseases of animals, as caused by abnormal food, that with high potassium and low sodium intake, bone formation is held below normal, even though sufficient lime and phosphorus be taken, potassium chloride in solu- tion with primary phosphates apparently tending to prevent their change to di- and tri-calcic salts. PHOSPHORUS METABOLISM 391 Rasquin (1905-6) fed two young cocks, one with and one with- out powdered bone, for 120 days. Under the influence of these rations the fowls developed as indicated by the following data: A (With Bone) B (Without Bone) Weight of body, live 2.690 kg 2.470 kg. Weight of body, plucked 2.455 kg 2.240 kg. Weight of body, drawn 2.003 kg 1.790 kg. Weight of skeleton 236 gm 190 gm. Weight of ischium 16 gm 13 gm. Weight of bones of wings 41 gm 30 gm. Weight of bones of legs 83 gm 58 gm. Weight of breast bone 12 gm '. 8 gm. Lime in bones 21.98 pet 20.51 pet. Phosphoric acid in bones 18.77 pet 18.14 pet. Length of branches of breast bone 13 cm 9 cm. Burnett (1906, 1908, 1910) examined the bones of hogs which had received natural foods varying in calcium and phosphorus con- tent, and also others which had received with the food bone meal, calcium carbonate, or disodium phosphate. The development of the bones, as determined by breaking strength, specific gravity, thick- ness of wall, ash content, etc., reflected the character of the food in the usual way. The principal deposit of bone salts seems to have been inside the marrow space, the walls in this way becoming greatly thickened. Summary tables from three series of observa- tions are quoted on pages 392 and 393. These tables from the experiments of Burnett show, more plainly than any of the earlier work, the fact that the mineral con- tent of the bones may be modified, in accordance with the composi- tion of the foods, through a very wide range of variation. If this fact appears more prominent in relation to swine than to other ani- mals it is doubtless due to the unusual mineral content of some of the rations used for swine. Through the feeding of precipitated bone flour, which is readily taken by the larger farm animals, it is probably possible to affect the mineral content of their bones also to such extent as to be of practical importance, especially with horses. See also the experiments of Forbes (1909) p. 374. Alway and Hadlock (1909) compared hog bones produced from corn alone with others produced from corn and bone meal. They state that both lots had practically the same composition, thus indi- cating that the nature of the food has no effect upon the relative proportions of the different constituents of the mineral matter of 392 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 bones. At the same time, considering the bones from corn and bone meal as the standard, the feeding of corn alone is shown, by their ash analyses, to have increased the magnesium by 20 percent, to have increased the phosphorus 1.7 percent and to have decreased the calcium by 1.0 percent of the amounts present. A part of their figures are as follows: Weak bones Strong bones (Corn) (Corn; bone meal) Average percent mineral matter, green bones 25.4 37.2 Average wall thickness, green bones 2.8 mm 4.1 mm. Average breaking strength, green bones ....702.0 lbs 1505.0 lbs. CaO 52.47 percent 52.98 percent MgO 1.24 percent 1.05 percent P 2 5 , 41.61 percent 40.93 percent EFFECTS OF FOODS ON THE BREAKING STRENGTH OF THE BONES OF SWINE (Burnett, 1906) FIRST TEST Killed Feb. 10- Number in lot Ration Av. live weight, lbs. . Breaking strength of radius, lbs Breaking strength of tibia, lbs Av. of two bones in each leg, lbs Lot I Soaked corn 192 723 607 714 Lot II Corn 90% Tankage 10% 247 1308 825 1007 Lot III 3 Corn 90% Ground bone 10 % "257 1561 732 1081 Lot IV Corn 75% Shorts 25% 210 641 783 SECOND TEST Killed March 14— 4 171 768 542 * 634 4 286 1254 834 1057 4 242 1044 779 942 4 Av. live weight, lbs. . . Breaking strength of 204 835 Breaking strength of 662 Av. of two bones in 801 AVERAGE BREAKING STRENGTH OF BONES PER 100 POUNDS LIVE WEIGHT OF HOGS AT TIME OF SLAUGHTER (Burnett, 1908)— Pounds Let Ration Femur Tibia Humerus Radius and ulna All bones 1 276 343 462 559 646 252 309 360 409 465 434 555 685 740 898 341 376 529 611 715 325 ?, 396 3 509 4 580 5 681 PHOSPHORUS METABOLISM 393 rf (« r g cciomojco C M S-O OOCOWCOIO Perce: mini matt green CO CO CO "-* ''Jl nh H «3 ol e* o H M SGcu H irii— i co CM ••31 CO ■* "0 & I-H T-H I— 1 1— 1 rH w « H , o <5 . CO o CD r-l 00 Average circumfer ence of bones in mm. CO CO CC CM CO cDt-cot-c-" o CO H co «*^ , -+* 03 CO W 2 H g Average length ol bones in mm. C5t-CNr-CO rH Fh o S O m w H CD CO CD CO CD O OS 3h u Average breaking trength of bones per 100 lbs. ive weight of hog fc Ph 10C001I-* « O cocnt-cnr- ■WCOTfl'*10 o H ^ !x s* 01 -* Pn © w CO Is A vera breaki streng of bon> . in lbs CCOW«H p 02 p CO CO CM lO CO r— t-oocncci 5? 5 ** 2 £ w c ■^xcpmco.— i coa5t-c»t— ^1 1 i-H r-t t— < r-t rH fe O CQ H U H fe Ph H R o3 i a o a c J3 3 s ■4J 01 ££ ri « J -a "St scfic ai rt rt c I S — — - rt is o s ii :' : ^SEE S3 rt rt c , (sssas _. rt rt c ! ,T3 ** a N : ^"3 rt" i esses o o o o < i c c >c >c >c > OOHOOrt OCM1T305-* CO CO CO CO T»f cm cor- CD CO CMoicMCO'^5 COt— CDOCO ■** CO ~H C<1 CO Hi- 1 CM CO CO CO CD CD 00 CD i— IOCOCOO •hi cm co coco OCMCMCO'"* CD CD CD COt- COCO en cm in cd co cor— co ioin<— ICDCM r-t-ot--H CN CM CO CM-* ■^ICOt— i-HlO r— CD COt— 05 COCOTJICDr— CD'-nmcoco co^m^co « a £ .gSfl :a oi+j^i •'d'O'O'ci a a a a pj cd ra rt ddddd O O O O o ooooo •s a p. a 394 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Hart, McCollum and Humphrey (1909), in their study of phytin metabolism with a milch cow, found that when calcium or phosphorus was deficient in the food, the skeletal tissues appeared to be ready sources of supply. The average quantities of calcium oxide and phosphorus pentoxide metabolized and excreted daily during periods of deficient supply were, respectively, 50 and 60 gm. Schaumann (1910) says, "The idea that the phosphorus elimin- ated in the urine in pronounced phosphorus starvation comes mainly from bony tissue, as Cathcart thinks, is supported by the findings of Hoist with guinea pigs, of Lipschutz and of Heubner with dogs, and by my own with rabbits." (Lipschutz, 1910a; Heubner, 1909; Schaumann, 1908.) Lipschutz (1910a) studied phosphorus metabolism in dogs with rations varying in phosphorus content. A ration very low in phos- phorus produced a moderate increase in weight, but at the end of seven weeks there were protracted muscular cramps and other symptoms of general physiological derangement. An examination of the bones showed a condition which he describes in detail as resembling Barlow's disease. A ration containing the same amount of calcium, but more phosphorus, caused normal development — a case of phosphorus limiting the utilization of calcium. Shortage of calcium limiting the use of phosphorus is much more common in the practical feeding of animals. R. Berg and C. Rose (1910) state that children and soldiers from regions of hard-water have fewer unsound teeth than others from soft-water regions. The facts may be as stated, but are diffi- cult of satisfactory proof, especially as due to the water. Dibbelt (1911) reported on experiments on the production and cure of experimental osteomalacia. For several months a dog was kept on a low-calcium diet to which were added disodium phosphate and sodium chloride. At the end of this time the left fourth rib was removed for analysis. Then after 12 days, in which dicalcium phosphate replaced the sodium phosphate, the right fourth rib was removed. For the next 3 months the dog was kept on a mixed diet with liberal allowance of bones. At the end of this period the left fifth rib was removed. During the experiment the dog gave birth to four young. The analysis of the ribs showed that the with- drawal of calcium, as a result of the diet and the bearing of young, caused an atrophy of the bone tissue, and that the administration of the calcium salt, and omission of the salts causing the withdrawal of calcium, with normal feeding, had the effect to replace this loss. PHOSPHORUS METABOLISM '395 For earlier views on calcification of bone see Aeby (1873b). For other material on the effects of diet on the composition of the bones seeMalyand Donath (1873), Weiske (1873, 1891b), Laurer (1910), and Cagnetto (1911) ; also Hart, McCollum and Fuller (1909), p. 328 of this work, and E. B. Forbes and associates (1914). Summary on Common Foods in Relation to Phosphorus Metabolism Our knowledge of the specific effects of common foods in rela- tion to phosphorus metabolism is slight and fragmentary. The evidence, however, is of such nature as to promise important results from further study. The retention of phosphorus from common foods is, in general, in the same order as the intake, but deficiency of nutrients (as calcium) necessary to the maximum utilization of phosphorus may limit retention, as also may excessive amounts of magnesium in the ration. The deposit of calcium phosphate in the bones is also hindered by inorganic acids, acid salts, calcium-precipitating ions other than phosphorus, and a high potassium and low sodium content of the ration. The human being stores more phosphorus from whole wheat bread than from white bread. In comparing diets containing meat and fish, the latter appears more favorable to phosphorus and magnesium retention but less favorable to calcium retention than the former. White rice does not contain as much phosphorus as the adult human being requires. Red or unpolished rice contains more than enough. Beet molasses has been thought to increase the solubility and therefore the assimilability of the phosphorus of the ration of the horse. Young carnivora suffer from malnutrition of the bones if fed on meat alone, to the exclusion of bone. The low calcium content of cereals and other seeds has been shown, in experiments with growing swine, to limit the retention of phosphorus. The addition of calcium to rations composed of cereal foods serves to increase phosphorus retention. In experiments with growing swine, comparing many common food during the feeding of skim milk there were minimum propor- tions of potassium, magnesium, sulphur, chlorine and phosphorus in the feces, and maximum retention of calcium, magnesium, sulphur and phosphorus. 896 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Experiments with growing swine show that many common foods are lacking in the amount of calcium and phosphorus neces- sary to maximum production of tissues containing phosphorus. Foods have been shown to affect the phosphorus content of the muscle, the liver and the kidneys of swine, as related to the ash, the nitrogen and the fresh substance of these parts. The total and inorganic phosphorus of the blood, the lecithin content of the liver, and the composition of the ash of the bones of pigs have also been shown to be susceptible of variation through the influence of corn in the diet. The bones may be greatly weakened by protracted feeding on foods poor in either calcium or phosphorus, and may be greatly strengthened by the use of foods rich in these elements, the bones appearing to possess an extensive storage function for calcium, magnesium and phosphorus. Rations which, because of deficiency of mineral salts, cause rachitic changes in the skeleton, have not been shown to produce such effects on the teeth, though it has been stated, with some evidence as a basis, that the salts of the drinking water, in the course of time, may affect the quality of the teeth. In the effects of diet on the composition of the bones, the ratio of the mineral constituents, one to another, varies but little ; there is little replacement possible, for instance, of calcium phosphate by strontium phosphate, though there is some change of proportion of calcium, magnesium and phosphorus in response to the character of the food. The total ash content of the bone may be varied almost at will, by the administration of calcium phosphate or by the use of foods containing the same elements. Lack of food also affects the phosphorus content of the bones, since these tissues, along with the soft parts, undergo katabolism in starvation. Calcium phosphate in the diet does not greatly influence gain in body weight, but is readily deposited in the bones, especially within the marrow spaces, having the effect to increase the density and strength of these supporting structures. The variations in phosphorus content of milk, ascribable to effects of food, are within the range of normal variation. PHOSPHOKUS METABOLISM 397 PHOSPHORUS REQUIREMENTS OF ANIMALS GENERAL DISCUSSION In the practical nutrition of human beings, as well as live stock, our interest is especially in conditions of maximum production or efficiency. We never feed for mere maintenance. Maintenance expense is all loss. Our interest is in production, and the amounts and kinds of nutriment necessary to sustain maximum production can with certainty be determined only by production. Optimum conditions, however, are indefinite and variable. We know almost nothing as to the maximum amounts of phosphorus useful to animals in their various states of life and activity, and are quite unable to submit accurate figures as guides to practice. Maintenance requirements are more accurately known, are less variable with conditions, and hence may be more definitely expressed with safety; but as guides in practical nutrition they must be regarded as irreducible minima, and hence as danger signals, and distinctly not as expressions of the amounts which animals should receive. For real guides to practice we must, for the present, at least, depend in large measure on very general recommendations, and on that judgment which is the result of experience. Our opinions as to phosphorus requirements of animals are based principally on balances of intake and outgo, and data on elimination during fast. In the use of such observations we must bear in mind, first, that a given amount either of gain or loss of phosphorus in the body ordinarily represents a larger amount of food phosphorus. If an animal gains one gram per day of phos- phorus on an intake of 3 grams it would not do to assume that 2 grams represents the maintenance requirement. The one gram of phosphorus retained by the animal represents more than one gram of food phosphorus, because there are certain losses inevitably incident to its utilization, and there would, therefore, be a certain retention of phosphorus, with the above mentioned animal, on a 2.Q gram intake; second, a part of the phosphorus katabolized by the tissues and excreted into the intestine is normally reabsorbed and utilized, as is shown by the fact that the phosphorus loss from the body is less on a phosphorus-free diet than during fast, the reason for which is not definitely known, but which is perhaps connected with the greater absorptive activity in the intestine during the digestion of food than during fast, a fact which would make the fasting loss greater than the minimum maintenance requirement, were it not that this factor is offset by the compensating considera- tion that a part of the food phosphorus normally escapes digestion. 398 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 As a matter of fact these two opposing factors must commonly somewhat nearly balance each other in human nutrition, for the minimum maintenance requirement and the fasting loss happen to be so nearly the same that the one may be used as a measure of the other without great error. This is not necessarily so in animal nutrition generally, however, for were the maintenance require- ment determined with foods containing phosphorus in indigestible relations to other nutrients, the loss of indigestible food-phosphorus might exceed the reabsorption of once-digested and excreted phos- phorus from the intestine, so that the fasting loss would not equal the maintenance requirement. The phosphorus maintenance requirement of a horse, for instance, would be less on fresh grass than on hay made from the same grass, because of the greater apparent digestibility of the phosphorus of the former than of the latter. Since it is impossible to feed an animal so as to maintain it in exact phosphorus equilibrium we are obliged to form our judgments as to requirements from data on experiments in which the subject either gained or lost phosphorus. From such data we are unable to state the phosphorus requirement in an exact way, even for the con- ditions of the experiment, since, as above stated, either gain or loss of phosphorus represents more than the same amount of phospho- rus in the food, under ordinary conditions of life. A gram of phos- phorus retention, however, represents the same amount of food phosphorus as a gram of loss of phosphorus from the body, and hence by subtracting the retention of phosphorus from the amount in the food, or adding the amount of the loss of phosphorus to the amount in the food, we are able to say that the requirement is less than the former, and greater than the latter. The smaller the retention or loss the more closely will the apparent requirement, calculated as above, approximate the actual requirement under the conditions of the determination. In order accurately to determine the phosphorus requirements of animals the ration should be so compounded that deficiencies of nutrients other than phosphorus, but essential to the formation of the phosphorus compounds of the body, shall not limit the useful- ness of the phosphorus of the food. These conditions are not always observed in studies of this sort, and to this is due a large measure of the variability of the results. It is true that a considerable measure of independence of other nutrients is to be seen in the retention of phosphorus, but at the same time nitrogen and calcium, especially, are so largely used with phosphorus in the body that coincident negative balances of both nitrogen and calcium would in a short time come to exercise a retarding influence on the retention of phosphorus. PHOSPHOKUS METABOLISM 399 Other factors entering into the determination of results of balance experiments with animals are the food habit, as determined by previous feeding, the nature of the particular foods used, and also the kind, age and weight of the subject. From among the many balance experiments which we might use in this discussion of phosphorus requirements we present a few typical reports of each of the various sorts, without an effort to use them all. On account of the diversity of conditions attending these experiments the computation of mathematical averages would constitute an act of scientific violence of which we desire not to be convicted. The reader will probably be surprised to note the extremely fragmentary character of the data even on minimum phosphorus requirements of animals, while on optimum requirements we have almost no evidence at all. Most writers, thus far, have been obliged to submit computed estimates only, and as yet there is but slight basis for more definite statements. PHOSPHORUS EXCRETION DURING FAST In studying phosphorus excretion during fasting Ajello and Solaro (1893) found, with Giovanni Succi as a subject, that the phosphorus excretion in the urine varied as the loss in body weight, and not as the volume of the urine. C. Lehmann, et al. (1893) showed that the average daily excre- tion of P 2 5 in 10 days fasting with Cetti (urine and feces both included) was 2.675 gm. ; with Breithaupt the average daily P 2 5 excretion during 6 days was 2.28 gm. E. and 0. Freund (1901) investigated the proportions of acid, alkaline and earthy phosphates in the urine during fast. The follow- ing figures show the results : FOI IMS OF PHOSPHORUS IN URINE DURING FAST— Grams P*O e Day of Earthy Acid Alkaline Day Earthy Acid Alkaline Day Earthy Acid Alkaline phos- phos- phos- of phos- phos- phos- Of phos- phos- phos- j phates phates phates fast phates phates phates fast phates phates phates 1 0.19 1.60 1.38 8 0.47 1.08 1.23 15 0.46 0.37 0.95 2 0.26 1.41 1.34 9 0.81 0.85 1.55 16 0.36 0.25 0.63 3 0.48 1.40 1.12 10 0.48 0.54 1.14 17 0.39 0.38 0.96 4 0.21 1.48 1.06 11 0.30 0.45 0.96 18 0.32 0.27 0.69 5 0.27 1.46 1.05 12 0.51 0.46 0.89 19 0.33 0.39 0.74 6 0.45 13 0.35 0.36 0.68 20 0.17 0.26 0.41 7 0.39 0.91 1.22 14 0.41 0.29 0.70 21 0.14 0.23 0.41 Ave. 7 days 0.32 1.21 1.19 0.48 0.58 1.02 0.31 0.31 0.67 From these data it is apparent that, considering the results from a week together, there was during this fast a progressive decrease in both acid and alkaline phosphates, while the earthy 400 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 phosphates were the same in amount in the last week as in the first. Averaging these weekly figures shows that for the whole fast the daily excretion of earthy phosphates was 0.37 gm., of acid phos- phates 0.70 gm., and of alkaline phosphates 0.96 gm., stated as P 2 5 . Gilbert and Posternak (1903) state that the loss of phosphorus in the urine of fasting subjects is as follows. DAILY ELIMINATION OF P 2 5 IN URINE— Grams per Kilo Body Weight Man 0.035 Dog 0.065 Cat 0.110 Cathcart and Fawsitt (1907) have given us the following figures on fasting metabolism. DAILY URINARY EXCRETION OF INORGANIC ELEMENTS DURING FAST S S S •Acidity Day of P2O5 CI (total) (inorganic) (neutral) Exp. C.C N/10 Diet Grams Grams Grams Grams Grams NaOH 4 3.60 7.3 1.39 1.17 0.14 579 Egg and milk 5 3.73 5.4 1.41 1.13 0.18 588 Egg and milk 6 4.14 6.7 1.33 1.07 0.15 582 Egg- and milk 1 2.26 3.2 0.62 0.44 0.13 378 Past 2 2.93 2.0 0.93 0.75 0.12 640 Fast 3 2.98 1.5 0.80 0.65 0.12 687 Fast 4 2.91 1.3 0.86 0.69 0.12 604 Fast 6 2.37 1.0 0.71 0.55 0.12 454 Fast 7 1.84 0.84 0.64 0.50 0.11 358 Fast 8 1.89 0.59 0.62 0.49 0.09 344 Fast 10 1.60 0.39 0.56 0.43 0.10 280 Fast 11 1.54 0.30 0.57 0.42 0.12 256 Fast 12 1.55 0.18 0.58 0.44 0.11 212 Fast 14 1.25 0.24 0.54 0.39 0.11 228 Fast 1 0.45 0.53 0.48 0.31 0.12 108 Starch and cream 2 0.20 0.66 0.28 0.14 0.10 88 Starch and cream 3 0.34 0.97 0.29 0.15 0.10 104 Starch and cream 4 0.89 1.10 0.93 0.73 0.16 156 Egg and milk 5 2.10 4.60 0.93 0.74 0.16 263 Egg and milk Thus the urinary phosphorus excretion during fast varies between 2.98 and 1.25 gm. P 2 5 , the average being 2.10 gm. daily. The decreased excretion of P 2 5 after the end of the fast is notable, and is coincident with a similar fluctuation in the excretion of sulphur. It doubtless signifies synthesis.- Benedict (1907), in connection with his own work on fasting metabolism, reviews the results of earlier investigations. The follow- ing summary is taken from Benedict's discussion. The black-faced type signify the quotient N:P 2 5 . PHOSPHORUS METABOLISM 401 DAILY PHOSPHORUS (P 2 5 ) EXCRETION AND RATIO BETWEEN NITROGEN AND PHOSPHORUS EXCRETION IN URINE OF FASTING SUBJECTS— Grams Succi Breit- haupt Subject I Flora Tosca Day of At At At Cetti J Sohn Keller fast Florence Naples Vienna 4.73 4.89 4.84 7.40 6.21 4.53 5.24 4.41 1.90 2.76 2.69 1.682 3.381 1.58 2.670 1.86 7.87 4.90 5.70 5.22 6.42 5.20 5.37 3.97 5.65 3.55 i 1.930 1.78 2.98 2.597 1.56 1.091 2.303 2.22 1.550 1.90 5.91 4.64 4.07 4.30 5.25 6.51 5.45 3.81 4.58 3.24 2 2.051 1.82 2.75 2.925 1.89 1.491 2.268 2.65 1.830 2.44 7.30 4.64 4.19 3.99 5.25 6.03 6.17 4.41 4.04 4.54 3 2.090 1.95 2.52 3.289 2.53 1.571 2.270 2.65 2.654 2.53 6.64 5.83 4.25 4.17 5.42 5.54 3.20 4 2.120 1.46 2.54 2.974 2.36 5.052 2.934 5 5.90 3.74 4.46 3.73 5.00 4.50 2.394 2.64 2.51 2.871 2.19 1.749 5.18 3.49 4.85 3.79 4.32 4.43 7.23 6 2.150 2.47 2.27 2.667 2.29 2.434 1.069 7 5.53 3.28 4.13 4.09 4.60 8.57 1.865 2.32 2.13 2.663 3.150 0.713 8 5.85 4.18 4.22 5.17 4.86 4.64 1.601 1.48 2.31 1.722 4.442 1.658 9 6.29 4.63 4.19 5.24 4.32 1.360 1.49 2.40 2.065 1.702 10 5.96 4.37 4.24 10.00 4.65 1.246 1.23 1.68 0.948 1.461 Av. ratio Nr*- oco Ht~ OiH t^co CM CM GOO COO ino COCO t^o COi— 1 3 "E HO HO HO OO o_o CMO 13 P oo OO OO oo oo oc> o a) r^-tra T»o id - in in HOI OH TTCO CO CM CJ5CM CM CM d CJ r~o r-o coo in co mo mo h fa oo oo oo oo ©o o'o **$ O CO 3 S 4) moo om (MO 00 CO t^co CM CM o .2 OSHJI IOO> t^t- OS CO I— lf- CMOS ,d com Tti-^fi OCO OCO Iflt* o. "E T*0 OO TXO coco t-o coo Cfl o P HO MO HO HO HO HO /a PM V coco osr- ooo Hin cm m t^co com io co coS CJilO coco coco COO! COOS ■»HCM CM CO cSh eon ooo COH CO-— 1 O"— 1 a coo COO NO coo coo CMO H d _o GOr- >— IIO r-t~- CM-* r-oo cjiin oo -M a) r-i>- coh COO osin CO-* O— 1 i oo HO oo no -*o c^o HO HO HO HO oo « . fca . hi . til . ta . hi . bt Cfl M %Z CO M Cfl M •2 u »rt ra aj rt u a a a p. p. p. oo CM CO ^ OJ m tj i £M CO CM T* H CM CM CM CM o CM 3 to o Cfl Cfl o o O a o o cu a a a a a < o r- CO rS co in U9 Cfl Cfl w at CO tx l>> u I I «* CO 00 r- CO 1-1 1-1 H PHOSPHORUS METABOLISM 413 AVERAGE DAILY NITROGEN, CALCIUM AND PHOSPHORUS BALANCES WITH TWO GROWING BOYS— Grams Boy No. Body weight Initial Final Kg-. N Food Urine Perspiration Feces Balance CaO Food Urine Feces Balance P2O5 Food Urine Feces Balance Exercise taken I Age 14 years, 3 mo. 36.360 36.335 36.335 36.990 12.357 10.040 0.673 1.177 +0.467 14.600 11.094 0.217* 1.800 +1.650 0.823 0.251 0.291 +0.281 0.911 0.250 0.507 +0.154 2.203 1.220 0.436 +0.547 2.669 1.439 0.719 +0.511 Walking- period Normal activity II Age 13 years, 10 mo. 43.005 41.740 41.740 42.640 12.469 12. 162 0.386 0.811 -0.879 15.507 12.908 0.133* 1.216 +1.250 0.829 0.121 0.202 +0.506 0.953 0.215 0.336 +0.402 2.224 1.681 0.376 +0.167 2.826 1.788 0.560 +0.478 WaUrinjr period i Normal activity "Estimated. Summary. From all of these data we conclude that the usual phosphorus requirement of an adult human being of average weight is about 1.5-1.75 gm. P or 3.4-4.0 gm. P 2 5 ; that under special condi- tions of diet and previous feeding this amount may" be reduced to about 0.9 gm. P or 2.00 gm. P 2 5 . Regarding the optimum phosphorus content of the food for immature human beings beyond the age of infancy we have only the above work of Herbst. 414 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 PHOSPHORUS REQUIREMENTS OF INFANTS Very few balance experiments with normal infants are to be found in the literature of the subject from which it is possible to compute their phosphorus requirements. Those which have come to our attention are here recorded in brief. Michel (1896) conducted balance experiments with new-born infants on a milk diet. A part of the data are as follows : AVERAGE DAILY RETENTION OF NITROGEN, CALCIUM AND PHOSPHORUS WITH NEW-BORN INFANTS ON A MILK DIET Age in days Weight, initial and final Grams Milk ■received per day Grams Milk per kg. body weight N Gain per kg. body weight P2O5 Gain per kg. body weight CaO Gain per kg. body weight 5-8 11-14 5-9 7-13 3730 3810 4400 4520 2680 2836 3500 3675 480 835 485 666 127.3 187.2 176.0 185.0 0.312 0.358 0.402 0.302 0.068 0.063 0.062 0.059 0.055 0.045 0.047 0.054 3643 617 168.9 0.3435 0.063 0.050 Michel and Perret (1899) report that a two-and-a-half -months- old infant, weighing 4725 gm. at the beginning, gained 19 gm. per day for 3 days on a daily intake of 1.675 gm. nitrogen, 0.268 gm. P 2 5 and 0.377 gm. CaO, which equals 0.35 gm. nitrogen, 0.056 gm. P 2 5 and 0.079 gm. CaO per kg. body weight. The storage of nitro- gen was 0.785 gm., and of P 2 5 0.121 gm. per day. Blauberg (1900a, 1900b) compared artificial with natural feed- ing of infants. Balance data for calcium, magnesium, and phospho- rus are as follows : DAILY CALCIUM, MAGNESIUM AND PHOSPHORUS BALANCES WITH ARTIFICIALLY FED INFANTS— Grams Subject Diet Income CaO Balance CaO Income MgO Balance MgO Income P2O5 Balance P2O5 3 Normal (?) 6% mo. old. . . Diluted, sweetened and sterilized cow's milk.. "Kufeke mehl" and Sterilized, undiluted .752 .096 1.784 .272 0.154 -0.052 0.924 0.018 .102 .095 .129 .044 0.032 -0.027 0.017 0.018 .725 .465 1.766 0.203 0.149 -0.054 0.507 4 Normal 0.093 PHOSPHOKUS METABOLISM 415 From these data the author concludes that we must consider feeding with cow's milk, even when diluted, to be "supernutrition" as regards the above mineral nutrients, that is, considering the healthy breast-fed infant as the standard. In Keller's experiments (1900b) with infants on milk diet (see table on p. 452) it was found that on human milk satisfactory- gain in weight and retention of nitrogen and phosphorus were induced in infants 2-5 months old (Exp. 2, 6 and 8), weighing 3300 to 4350 gm., on an intake of 1.512-1.875 gm. N and 0.268-0.325 gm. P 2 5 daily, which is equivalent to 361-485 mg. N and 67-81 mg. P 2 5 , or 29-35 mg. P, per kilogram of body weight. In these experiments satisfactory gain in weight was not pro- duced with diluted cow's milk. A maximum gain in weight was pro- duced with whole cow's milk, however, containing much more than the above-mentioned amounts of nitrogen and phosphorus, with a 10-months-old child. From Netter's data (1900) we compute that 6 healthy infants, with an average age of 8.8 months, made the following average daily gains per kilo of body weight on intake per kilo as stated: Intake of milk 144 c.c, of CaO 0.261 gm., and of P 2 5 0.335 gm.; gain of nitrogen 0.204 gm., CaO 0.059 gm., and of P 2 O g 0.061 gm. Rothschild and Netter (1901) studied the effect of variation in the amount of milk taken by infants on the thoroughness of its utilization. Children from 4-10 months old were given milk in amounts varying from 125.5-190.2 gm. per kg. of body weight. The greatest gain of nitrogen and phosphorus per day, per kg. of body weight, occurred with a child 8 months old, weighing 7270 gm., and receiving 177.2 gm. of milk per kg. of body weight daily. On this intake the child gained 0.199 gm. nitrogen, 0.095 gm. CaO, and 0.09 gm. P 2 5 per day, per kg. of body weight. The composition of the milk was not given. E. Miiller (1902) reported experiments with infants 4 and 6 months old comparing the utilization of the constituents of raw and sterilized milk by means of 2 complete balance experiments, each of 7 days' duration, excreta being collected for the last 4 days of each period. The nitrogen and fat of the sterilized milk were better used than of the raw milk. As to calcium and phosphorus the results are inconclusive. A portion of the figures follow. 416 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE DAILY BALANCES OF NITROGEN, CALCIUM AND PHOSPHORUS PER KILOGRAM OF BODY WEIGHT WITH INFANTS ON RAW AND STERILE MILK— Grams Length of period in days Treatment of milk N Food Urine Feces Balance P2O5 Food Urine Feces Balance CaO Food Urine Feces Balance 4 4 4 4 Raw Sterile Raw Sterile 0.592 0.403 0.117 +0.072 0.593 0.357 0.137 +0.099 0.539 0.424 0.068 f 0.047 0.540 0.419 0.067 +0.054 0.272 0.141 0.116 +0.015 0.273 0.092 0.149 +0.032 0.256 0.156 0.047 +0.053 0.256 0.151 0.060 +0.045 0.193 0.002 0.166 +0.025 0.193 0.002 0.181 +0.010 0.195 0.003 0.133 +0.059 0.195 0.003 0.133 +0.059 Bruck (1908) has given us two balance experiments with artificially-fed infants. The calcium and phosphorus figures we have calculated from the author's data. AVERAGE DAILY CALCIUM AND PHOSPHORUS BALANCES WITH ARTIFICIALLY FED INFANTS— Grams Exp. No. Age Initial body weight CaO intake CaO retention Percent CaO retention P2O5 intake P2O5 retention Percent P2O5 retention Duration of period 1 2 3 mo. 8^ mo. 3400 5600 0.602 0.743 0.298 0.299 45.3 40.3 0.720 1.454 0.168 0.300 23.5 26.3 4 days 3 days Thus with intakes of 0.212 and 0.260 gm. P 2 O s per kg. of body weight there was retention of 23.5 and 26.3 percents of these amounts. During these tests these two infants gained in weight 40 grams and 80 grams respectively; the diet was milk, gruel and malt. L. F. Meyer (1908) made a study of mineral metabolism in infants as affected by limited intake of food, by increase in the casein of the food, and by increase of the fat of the food. Two infants were the subjects of these experiments. "A" was 3 months old, and "B" was 10 months old at the beginning of the study. Both were in a normal state of general metabolism, though "A" had a slight umbilical hernia, and "B" had exzema, and during the interim between the low diet and the increased food periods suffered from bronchitis. A part of the numerical data are as follows: PHOSPHORUS METABOLISM 417 AVERAGE DAILY NITROGEN, CALCIUM AND PHOSPHORUS BALANCES WITH NORMAL INFANTS ON MILK DIETS OF VARYING COMPOSITION— Grams Subject and age Length of period in days Diet N Intake Urine Feces Balance CaO Intake Urine Feces Balance P2O5 Intake Urine Feces Balance A 3 months old at beginning 6 3 4 3 Low diet: 200 gm. milk, 800 gm. oat gruel, 20 gm. sugar=44 cal. per kg. High protein: 2211 gm. of 1:5 milk, 49 gm. casein High protein and fat: 2927 gm. of 1:5 milk, 80 gm. salt-free butter, 66 gm. casein High fat diet: 2201 gm. of 1:5 milk, 60 gm. salt-free butter, 66 gm. casein 1.056 0.88496 0.1740 -0.00296 3.5515 1.799 0.2556 +1.4969 3.6296 1.9737 0.202 +1.4539 3.5157 1.7505 0.2171 +1.5481 0.3485 0.0242 0.2657 +0.0586 0.3336 0.0051 0.2911 +0.0374 0.2195 0.0034 0.0226 +0.1935 0.5686 0.4003 0.2772 -0.1089 0.7851 0.4327 0.1668 +0.1856 0.5823 0.3634 0.1042 +0.1147 J B 10 months old at beginning' 3 8 3 4 3 Control: 1500 gm. of 2:3 milk, 1:3 oat gruel, £ gm. sugar=100 cal. per kg. Low diet: 300 gm. milk, 500 gm. oat gruel, 700 c.c. water, 60 gm. sugar=50 cal. per kg. High protein: 4500 gm. of 1:5 milk, 63 gm. casein High protein and fat: 6000 gm. of 1:5 milk, 100 gm. salt- free butter, 84 gm. casein High fat diet: 4500 gm. of 1:5 milk, 75 gm. salt-free butter, 63 gm. casein 5.124 3.3192 0.4439 +1.3609 2.5690 3.3603 0.2232 -0.0145 4.7732 2.947 0.177 +1.6492 4.7732 3.3439 0.214 +1.2153 4.7732 3.4137 0.1815 +1.1780 1.3944 Traces 1.2674 +0.1270 0.4809 0.0030 0.4280 +0.0499 0.5693 0.6273 -0.0580 0.4270 0.5468 -0.1198 0.5693 0.6862 -0.1169 2.0887 0.9649 0.6049 +0.5200 0.6695 0.4451 0.2306 -0.0062 1.1047 0.5101 0.5027 +0.0919 0.8285 0.5220 0.4162 -0.1097 1.1180 0.6483 0.4452 +0.0245 Among the author's conclusions are the following : While losing in weight, on a deficient diet, a child loses mineral matter and nitrogen; after readjustment to the same diet, so that there is but slight loss in weight, the loss in these constituents may- be reduced to zero. Birk's experiments (1909) (see Rachitis), with two normal infants, showed that with 1.55 and 1.51 grams of phosphorus in the food per day there resulted a retention of 21 and 18 percents of these amounts. Tobler and Noll (1910) published mineral balances with a healthy infant two and a half months old. The following table sets forth the more important results. 418 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE DAILY MINERAL METABOLISM WITH A HEALTHY BREAST-FED INFANT TWO AND ONE HALF MONTHS OLD— Grams Intake Urine Feces Balance Percent retained Retention per day an kilo. KO 0.3432 0.2214 0.2390 0.0284 0.2102 0.1124 0.0078 0.0372 0.0184 0.0571 0.0582 0.0085 0.1485 0.0343 +0.1726 +0.2051 +0.0533 +0.1188 50.27 92.64 22.30 56.51 0.0431 Na»0 0.0513 CaO 0.0133 MgO P2O5 0.0297 The milk contained in 100 gm. : ash 0.1596, K 2 0.048, Na 2 0.0309, CaO 0.0334 and P 2 5 0.0294 gm.; length of experiment 6 days; initial weight 4000 gm. ; final weight 4170 grams. From these data we conclude that there was marked retention of phospho- rus on an intake of 0.051 gm. P 2 5 , or 0.022 gm. P per kg. of body weight. Schabad (1910d) reported the following figures for phosphorus excretion by children, which amounts, in relation to food require- ments, should, of course, be considered as below the minimum. AVERAGE DAILY PHOSPHORUS EXCRETION OF HEALTHY CHILDREN ON VARIOUS DIETS Total output of P2O5 per kilo, per day Grams Percent P2O5 in urine per kilo, per day Grams Partition of P2O5 output Urine Percent Feces Percent Naturally nourished children 0.0134 0.0303 0.0229 17 40 65 0.0074 0.0175 0.0185 55.6 57.3 80.6 44.4 42.7 19.4 Artificially fed children j 0.214 1 0.119 57.5 65.2 42.5 34.8 Older children on bread and milk 0.159 0.102 64.4 35.6 Summary. New-born infants (Michel) weighing on an average 3643 grams, and receiving of milk 617 gm. per day, or 168.9 gm. per kilogram of body weight, retain about 0.063 gm. P 2 D , or 0.0275 gm. P, per day ; the infant receiving cow's milk may retain considerably more calcium and phosphorus than the breast-fed baby (Blauberg) ; on human milk infants 2-5 months old made satisfactory retention of phosphorus on an intake of 29-35 mg. per kg. of body weight per day (Keller) ; infants with an average age of 8-9 months retained PHOSPHORUS METABOLISM 419 0.061 gm. P 2 5 , or 0.027 gm. P, on an intake of 0.335 gm. P 2 5 , or 0.146 gm. P (Netter) ; with infants 4-6 months old on artificial feed- ing there was a retention of 0.015-0.053 gm. P 2 5 , or 0.0065-0.023 gm. P, on an intake of 0.256-0.273 gm. P 2 5 , or 0.112-0.119 gm. P, per day (Miiller) ; with infants 3 and 8^ months of age, and intakes of 0.212 and 0.260 gm. P 2 5 , or 0.093-0.114 gm. P, per kg. of body weight, there was retention of 23.5 and 26.3 percents of these amounts (Bruck) ; the breast-fed infant may retain 56.5 percent of an intake of 0.051 gm. P 2 5 , or 0.022 gm. P, per kg. of body weight per day (Tobler and Noll). See also Lehmus (1878). PHOSPHORUS REQUIREMENTS OF CATTLE Weiske (1873) studied the metabolism of calcium phosphate with two 5-6-months-old calves. One calf retained about a half of the 12 grams of calcium phosphate added to the ration per day, while the other did not retain any of the added calcium phosphate, the difference in results apparently being due to the greater consumption of the basal ration by the latter calf, the food furnish- ing the entire calcium and phosphorus requirement. Weiske consid- ered that 16.85 gm. CaO and 21.88 gm. P 2 5 probably represents the full daily requirement of the calves for these elements. F. Soxhlet (1878), experimenting with 3 young calves on a milk diet, determined the average daily storage per kilogram of live weight of the several mineral constituents as follows : DAILY MINERAL RETENTION PER KILOGRAM OF LIVE WEIGHT OF THE CALF RECEIVING MILK ALONE Ash 0.657 gm 53 percent of the food P 2 5 0.274 gm 72.5 percent of the food CI 0.051 gm 3.8 percent of the food CaO 0.286 gm 97.0 percent of the food MgO 0.008 gm 30.5 percent of the food K 2 0.065 gm 20.7 percent of the food Na 2 0.027 gm 29.1 percent of the food Fe a 3 0.0008 gm 38.1 percent of the food J. Neumann (1893b, 1894) found that a calf weighing 65.63 kg., when fed on milk alone, stored both calcium and phosphorus rapidly. On a daily allowance of 15000 gm. skim milk, and an intake of 24.63 gm. CaO, the calf retained 11.77 gm. CaO. The intake of P 2 5 was 30.46 gm., and the retention 15.55 gm. We are not able to present figures of any great value as indi- cating the phosphorus requirements of milch cows. On account of the nature of the case especial value would attach to observations covering the whole period of pregnancy and lactation. Anger (1898) made a study of mineral metabolism in milch cows. The cows were fed the following basal ration : 50 kg. fodder beets, 6 kg. straw, 12 kg. hay per 1000 kg. live weight, and in 420 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 addition a supplementary allowance of grain, mill feed, or commer- cial concentrate, the amounts of the supplements being so adjusted as to furnish the same weight of protein in each case. Anger concludes from this work that the customary assumption that ordinary foods contain as much mineral nutriment as is needed is not justified. The intake and outgo of calcium and phosphorus are in the following table. CALCIUM AND PHOSPHORUS BALANCES WITH MILCH COWS Rations Varied from Period to Period — Grams CaO P2O5 Week of lacta- Milk pro- duced daily tion Kg. Intake Outgo Balance Intake Outgo Balance i 20 9.53 39.05 45.38 -6.33 66.44 47.46 +18.98 22 12.05 41.64 52.88 -11.24 78.72 79.11 -0.39 / 26 10.54 55.78 51.31 +4.47 185.25 143.45 +41.80 8.07 41.86 51.57 -9.71 52.24 92.34 -40.10 34 6.19 49.81 50.20 -0.39 59.84 52.88 +6.96 38 4.71 62.51 49.80 +12.71 97.27 98.88 -1.61 41 3.11 99.07 95.33 +3.74 99.20 95.25 +3.95 44 2.82 52.57 49.34 +3.23 76.49 68.25 +8.24 2 41 8.12 39.65 52.92 -13.27 • 48.35 42.25 +6.10 2 43 7.81 62.32 45.66 +16.66 95.91 84.09 +11.82 , 2 47 5.92 95.27 91.28 +3.99 92.19 85.57 +6.62 2 51 3.91 40.40 36.10 +4.30 +6.43 65.90 73.90 -8.00 2 55 1.32 33.01 26.58 54.94 65.42 -10.48 2 5 15.48 37.16 67.63 -30.47 61.11 85.32 -24.21 3 17 11.32 24.75 30.90 -6.15 63.70 52.81 +10.89 3 20 10.00 40.76 54.17 +13.41 155.32 144.13 +11.19 3 23 9.16 33.36 48.86 -15.50 47.14 78.03 -30.86 4 49 8.17 53.98 52.33 +1.65 90.41 88.66 +1.75 4 56 7.84 55.13 54.91 +0.22 90.13 87.66 +2.47 5 19 • 13.85 54.90 71.89 —16. 99 78.90 93.59 -14.69 5 23 11.43 57.46 60.95 -3.49 57.86 68.91 —11.05 5 26 12.62 58.19 52.14 +6.05 80.70 77.23 +3.47 Note: There was in each period a gain in live weight. From the work of Jordan, Hart and Patten (1906) we take the following data which have a bearing on the phosphorus requirement of milch cows. AVERAGE DAILY PHOSPHORUS REQUIREMENT OF MILCH COWS Weight of cow Lbs. Daily milk produced Kilos Phosphorus fed daily Grams Phosphorus stored daily Grams Rations 1100 966 12.813 12.027 16.715 16.768 14.606 78.7 83.3 37.0 37.0 77.0 8.1 12.9 17.6 17.7 11.6 Oat straw 10 lbs., wheat bran 10 lbs., hominy feed 5 lbs., wheat gluten 1 lb. Oat straw 10 lbs., washed bran 10 lbs., corn germ meal 6 lbs., rice meal 3 lbs. Oat straw 10 lbs., wheat bran 10 lbs., rice meal 7 lbs., wheat gluten 2 lbs. In their study of the metabolism of the ash constituents of wheat bran Hart, McCollum and Humphrey have recorded balance data on high- and low-phosphorus rations with milch cows. These PHOSPHORUS METABOLISM 421 rations furnished decidedly more or decidedly less phosphorus than the cow requires, and so do not make possible a close estimate of their necessities. DAILY PHOSPHORUS BALANCES WITH COWS ON HIGH AND LOW PHOSPHORUS RATIONS— Grams Dec 1-4... Dec 19-22. Dec 27-28. Jan. 6-9 .. Jan. 18-19. Jan. 28-31. Feb. 5 Feb. 10-13. Ration High phosphorus Low High " Low Hig-h " Low High \\ Low Intake Milk Total P2O5 P2O5 outgo P2O5 190.5 30.4 181.5 46.7 27.6 67.6 190.5 27.6 175.2 47.7 23.4 60.3 190.5 23.5 155 41.5 24.1 55.4 190.5 24.7 65.6 46.7 26.4 58.0 Balance P2O5 +9.0 -20.9 +15.3 —12.6 +35.5 -13.9 +124.9 -11.3 Gouin and Andouard (1907) state that if the ration of the calf does not contain legumes and milk it is desirable to add 100 gm. of powdered bone to the daily ration of a calf of 150 kilograms live weight. Khuen (1908) determined in a 7-day period that with a cow weighing 429.1 kg. and producing, on the average, 7014.3 gm. milk per day, an intake of 61.88 gm. CaO, 44.04 gm. MgO and 84.76 gm. P 2 5 resulted in a loss of 18.24 gm. CaO, a gain of 0.21 gm. MgO and a loss of 1.76 gm. P 2 5 . The milk contained 16.31 gm. CaO, 1.97 gm. MgO and 54.60 gm. P 2 5 . From these data we compute that in addition to the amount of phosphorus in the milk a cow must receive more than 70 mg. P 2 5 per kg. live weight. Henneberg (Beitrage zur Begriindung usw., 2 heft, 1864 S. 53.) determined the maintenance requirement of the steer, for calcium and phosphorus, per 100 kg. live weight, as 100 gm. lime and 50 gm. phosphoric acid. Kellner (1907), in computing the requirements of the milch cow, adds to these figures three times the lime and phosphoric acid content of the 20 kg. of milk produced per 1000 kg. of live weight by milch cows, and so obtains as a total 200 gm. of lime and 140 gm. of phosphoric acid as the requirements of milch cows per 1000 kg. live weight. A. R. Rose (1912a) concludes that the phosphorus maintenance requirement of a milch cow is the amount of phosphorus eliminated in the milk plus 26 milligrams (P) per kilogram of body weight. Kellner (1907) computes the calcium and phosphorus retention of the growing calf as 21 gm. CaO and 19 gm. P,0 5 per day during the first year, and states that the food should contain 40-60 gm. of each. 422 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 PHOSPHORUS REQUIREMENTS OF HORSES There is in existence but very little evidence of value on the phosphorus requirements of horses, especially on the optimum amounts under the various conditions of growth. At the same time it is surely true that there is no other animal in which the develop- ment and character of the skeletal tissues contributes in so promin- ent a way to the economic value. As is true with other herbivora, little is to be learned from the quantitative estimation of urinary phosphorus, except in connection with the other factors of a complete balance determination. A few data on the amounts of phosphorus in the urine of horses, however, are perhaps worthy of record. Salkowski (1885 and 1904) found the urinary phosphorus of a horse to be about 0.0107 gm. P 2 5 per 100 c.c, and 0.2199 gm. in 24 hours. F. Smith (1889) found in horse urine sometimes only a trace of phosphorus; at others from 0.13 to 9.45 gm. P 2 5 for 24 hours, an average amount for rest being 1.3 gm., and for work 1.897 gm. P 2 5 . Liebermann (1891c) found the phosphorus content of the urine of 8 stallions to vary between 0.004 and 0.021 percent P 2 5 , and the 24-hour outgo of P 2 5 in the urine of six stallions to vary from 0.118 to 0.481 gm. Tangl (1901) reported balance experiments on mature horses with calcium-deficient rations, an abstract of the report of which we have seen in Maly's Jahresbericht. The original work contains nitrogen, calcium, magnesium and phosphorus balances. The abstract referred to contains little significant material. Tangl states the opinion that the mature horse is able to supply its calcium needs from the same low-calcium fodder, which, with cattle, may produce malnutrition of the bones. Tangl (1902a, 1902b) studied mineral metabolism in growing horses, with rations of low calcium content. Two horses were used in these experiments. In the first experiment they received hay alone ; in the second oats and hay. (See table on p. 423) . The length of the first experiment was 8 days ; of the second 6 days. The percentage composition of the foods is as follows : Experiment I Experiment II Hay Hay Oats N P Ca Mg 1.366 0.146 0.440 0.137 1.844 0.165 0.410 0.209 2.272 0.406 0.143 0.104 PHOSPHORUS METABOLISM 423 AVERAGE DAILY NITROGEN, CALCIUM, MAGNESIUM AND PHOSPHORUS BALANCES WITH HORSES ON LOW CALCIUM RATIONS— Grams Weights N Ca Mg P Initial Final Ave. daily change Kg. Food Food Food , Food Subject No. Urine Feces Urine Feces Urine Feces Urine Feces Rations Balance Balance Balance Balance 109.28 36.38 11.23 11.740 408.0 70.39 14.38 3.43 0.061 8 kg. bay, 19.6 kg. water 1 404.0 50.70 21.31 7.80 11.830 -0.50 -11.81 +0.69 +0.09 -0.151 109.28 36.72 11.33 11.74 445 68.11 14.11 3.46 0.13 8 kg. hay, 26.5 kg. water 2 440 52.91 21.84 7.85 11.85 -0.63 -11.74 +0.77 +0.02 -0.24 183.08 27.23 14.86 24.50 403.8 102.16 8.10 3.44 0.79 5 kg. hay, 4 kg. oats, 1 406.5 48.30 17.23 30.09 23.07 17.3 kg. water +0.45 +32.62 +1.90 +0.33 +0.64 183.08 27.44 14.92 24.50 432.5 90.65 7.69 3.59 0.47 5 kg. hay, 4 kg. oats, 2 436.8 53.45 18.66 10.70 23.50 22.0 kg. water +0.72 +38.98 +1.09 +0.63 +.053 The drinking water contained 0.0049 percent Ca and 0.0013 percent Mg. From these data we compute that 0.27 gm. N in hay, per kg. body weight, is below the maintenance requirement, while 0.45 gm., in hay and oats, results in marked storage ; the phosphorus content of the hay ration limited calcium storage, since with increased phos- phorus but decreased calcium intake, in the second experiment, there was increased calcium storage ; with an intake of 0.063 gm. Ca per kg. live weight there was Ca storage ; with an intake of 0.029 gm. P per kg. there was loss of P, while with an intake of 0.056 gm. P per kg. there was storage of this element. Alquier (1905-6) determined that a working horse of 400-450 kilograms (880-990 lbs.) weight requires the equivalent of about 35 grams daily of P 2 O g for maintenance of phosphorus equilibrium. By the use of a suitable amount of feeding molasses, however, it was found that 25.5 grams of P 2 5 sufficed for the maintenance of equi- librium, the increased usefulness of the phosphorus being considered to be due to increased solubility, and therefore assimilability, being caused by the action of the sodium and potassium salts occurring abundantly in the molasses. 424 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 PHOSPHORUS REQUIREMENTS OF SWINE From the composition of a year's growth of a pig Kellner (1907) computes, allowing three times as much in the food as the animal will store in the body, that the daily requirement of a pig is 12 gm. each of lime and phosphoric acid. Hart, McCollum and Fuller (1909) expressed the opinion that the daily phosphorus supply for a 50-lb. growing pig should be at least 3 gm., and that a supply of 4 to 5 gm. is probably a safer quan- tity. From their experiments with low-phosphorus rations contain- ing 1.12 gm. of phosphorus per kilogram of feed, and from the experiments of Forbes and associates (1914) with similar rations containing 0.92-0.98 gm. of phosphorus per kilogram of feed it appears to be impossible to keep pigs alive indefinitely on food so poor in phosphorus. Doubt attaches to this conclusion, however, because of the fact that these rations were also very low in calcium. In the course of his investigation of nuclein metabolism in swine Schittenhelm (1910) conducted balance experiments which indicated phosphorus requirements. A part of his figures are as follows : AVERAGE DAILY NITROGEN AND PHOSPHORUS BALANCES WITH A YOUNG PIG— Grams Period Duration in days N Food Urine Feces Balance P2O5 Food Urine Feces Balance Ration 1 2 3 5 3 3 8.70 5.84 0.265 +2.595 9.49 6.57 0.682 +2.238 8.70 6.30 0.193 +2.207 3.80 1.20 0.39 +2.21 4.40 1.63 0.695 +2.075 3.80 1.23 0.191 +2.379 1500 c. c. milk 1500 c. c. milk. 20 gm. yeast nucleinic acid 1500 c. c. milk 1 2 3 5 5 5 14.07 5.20 0.55 +8.32 16.12 6.38 0.21 +9.52 14.07 6.40 0.295 +7.375 5.30 1.07 0.45 +3.78 7.65 3.32 0.20 +4.13 5.30 6.'3i 1500 c. c. milk, 300 gm. wheat flour 1500 c. c. milk, 300 gm. wheat flour, 14 gm. yeast nucleinic acid 1500 c. c. milk, 300 gm. wheat flour PHOSPHORUS METABOLISM 425 In the first experiment the pig weighed about 15 kg. and is said not to have gained in weight ; it was 8 weeks old ; in the second experiment the same pig was used, at an age of 4 months. The initial weight was 25.4 kg., and the final weight 31.6 kg. In both experiments there was a large storage of nitrogen and phosphorus. The energy value of the ration in the first experiment was deficient. In the second experiment the increase in weight was 413 gm. daily, with an intake of about 0.5 gm. nitrogen, and 0.2 gm. P 2 5 per kg. of body weight. In balance experiments on young swine Weiser (1912) showed that calcium is the limiting factor in the storage of phosphorus on a ration of corn alone. By the addition of calcium carbonate to the corn ration the phosphorus balance changed from negative to posi- tive. It seems certain, therefore, that cereal foods generally, at least corn and barley, contain enough phosphorus to make storage possible if the calcium is increased to an adequate amount. If, how- ever, the protein were increased to such an amount as would provide for maximum growth, by the addition of a more highly nitrogenous food, it may be that the phosphorus of the ration would then limit production, this depending in large measure on the phosphorus con- tent of the proteid supplement. On rations of corn alone Weiser got negative phosphorus balances on intakes of 0.057, 0.050, and 0.049 gm. phosphorus per kg. live weight, and marked storage on an intake of 0.048, 0.060, and 0.051 gm. phosphorus per kg. live weight on barley and starch, or on corn and calcium carbonate, or barley and calcium carbonate. From the experiments of Forbes, Beegle, Fritz and Mensching (table p. 378) it would appear that actual phosphorus requirements of growing swine are satisfied by corn and the supplementary foods ordinarily fed with corn, but the optimum allowance of phosphorus has not been determined. In these experiments phosphorus was retained on an intake of 48 mg. per kg. of live weight. PHOSPHORUS REQUIREMENTS OF SHEEP No very satisfactory data on this subject have come to hand. Weiske (1880) found that a ration of meadow hay and peas contained enough of each of the mineral nutrients to provide for liberal storage at all stages of maturity. Jordan (1885-6) found that timothy hay and cottonseed meal would sustain large retention of phosphorus, while timothy hay and corn led to moderate retention of phosphorus by a mature wether. On the following page are data from this test. 426 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE DAILY PHOSPHORUS AND NITROGEN BALANCES WITH A MATURE WETHER ON NORMAL FOODS— Grams Nitrogen Phosphorus Rations Food Urine Feces Bal- ance Food Urine Feces Bal- ance 20.72 16.78 5.36 -1.42 8.08 0.00 7.48 +0.60 Timothy hay 600 gm.; cottonseed meal 200 gm. 9.02 4.59 4.12 +0.31 2.72 0.00 2.48 +0.24 Timothy hay 600 g-m.; corn meal 200 gm. The first period was of five days duration and the second of four days. No phosphorus was found in the urine. The following figures from Weiske's work with sheep (copied from Wolff) are of some value as indicating mineral requirement. DAILY RETENTION OF MINERAL MATTER BY SHEEP— Grams Age of sheep Months Live weight Lhs. Potash Soda Lime Magnesia Phosphoric acid 5-6 7-9 10-12 13-15 51 66 77 85 2.04 2.89 3.05 2.65 0.84 1.05 0.81 0.72 1.56 2.00 1.81 2.07 0.12 0.32 0.38 0.35 1.09 1.65 2.50 3.14 As to how many times these amounts of the mineral nutrients the food should contain, however, we are unable to say with assur- ance. Wolff states that the food of young animals reared artificially should contain 2 to 3 times as much lime and phosphoric acid as that actually required as nutriment by the animals. Kellner (1907) computes, on the basis of Weiske's experiments with lambs 4-5 months old, that the daily retention per 50 kg. of live weight is 2.5-3.8 gm. of lime and 2.0-4.1 gm. of phosphoric acid. Then, allowing 2-3 times these quantities in the food, he recom- mends 22 gm. of lime and 25 gm. of phosphoric acid per 100 kg. live weight per day. PHOSPHORUS REQUIREMENTS OF DOGS In investigations by Forster (1873a), with dogs on low-ash experimental rations, one dog weighing about 24 kg. was in phospho- rus equilibrium on about 0.04 gm. P 2 5 , or 0.017 gm. P, per kg. body weight, daily. This may be considered as a minimum figure for artificial conditions. Munk (1894) showed that a mature dog weighing normally 17.16 kg. lost, per day, during fasting, 4.68 gm. nitrogen and 0.98 gm. P 2 O s in the urine, and 0.158 gm. nitrogen and 0.187 gm. P 2 5 in the PHOSPHORUS METABOLISM 427 feces, a total therefore of 4.838 gm. nitrogen and 1.167 gm. P 2 5 per day, and 0.0282 gm. nitrogen and 0.068 gm. P 2 5 , or 0.030 gm. P, per kg. per day. In metabolism studies by Zadik (1899), with dogs, there was, during the 5th period, very nearly a condition of phosphorus equi- librium. A dog weighing 10706 gm. stored 0.009 gm. phosphorus per day in a 6-day period on a total intake of 0.634 gm. phosphorus per day, or 0.059 gm. per kg. live weight. The phosphorus was present mostly in casein. The nitrogen balance was negative, the average daily loss being 0.37 gm. on an intake of 7.25 gm. Kornauth (1900), in comparing the utilization of different pro- teins with dogs, obtained balance figures which show that, when the principal source of the protein is meat, an intake of about 55-60 mg. P, per kilogram of body weight per day, is close to the maintenance requirement. On a diet of meat, lard, crackers and bone ash, Hawk and Gies (1904) maintained a dog weighing 16.96 kg. for 12 days on an intake of 2.456 gm. of phosphorus per day, with a daily phosphorus balance of +0.045 gm. Of this 2.456 gm. phosphorus, 1.779 gm. was pres- ent as bone ash. With another dog, on the same ration, these authors fed the animal 1.95 gm. phosphorus daily (apparently not 0.195 gm. as stated in the original), of which 1.423 gm. was present as bone ash. The dog weighed 11.85 kg., and the daily phosphorus balance was —0.030 gm. The apparent phosphorus requirement with these dogs was about 0.140-0.170 gram per kg. live weight, the amount being due in part to the comparatively insoluble form in which most of the phosphorus was fed. Heubner (1909) determined that the phosphorus retention of the normal growing dog was 0.14 gm. per kilogram of body weight per day. The lowest amounts of calcium and phosphorus on which stor- age took place in Biernacki's experiments (1909) were 0.044 gram CaO and 0.248 gram P 2 5 , or 0.108 gm. P, per kg. live weight. The amount of phosphorus stated, however, is considerably above the maintenance requirement. In an investigation on the use of organic and inorganic phos- phates in foods, Lipschutz (1911b) determined that, in a 7-day bal- ance experiment, a dog weighing 2450 gm. at the beginning, and 428 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 2700 gm. at the end, with a ration of rice, egg albumin, sugar, palmi- tin, and a salt mixture, including phosphates, and with a daily phosphorus intake of 0.651 gm., or 0.252 gm. per kg. live weight, the retention was 0.084 gm., or 0.033 gm. per kg. live weight per day. Kochmann and Petzsch (1911), in their study of calcium metabolism in the dog, made observations on phosphorus metabo- lism from which it is possible to estimate the phosphorus require- ment. The dog in question was mature, and weighed 10300-11040 gm. in the periods in which there was a retention of P 2 5 . In 4 periods of 4-5 days each the P 2 5 balances, on an intake of 3.671 gm., were +0.308, +0.121, —0.260 and +0.622. With an intake of 2.800 gm. P 2 5 the phosphorus balance was negative. The phos- phorus requirement, therefore, appeared to be about 0.114-0.142 gm. per kg. body weight. From these data it appears that the fasting loss is about 0.030 gm. per kilogram (Munk) ; the maintenance requirement on an arti- ficial low-ash diet about 0.017 gm. per kilogram (Forster) ; the main- tenance requirement on a more nearly normal diet, with the phos- phorus present mostly in casein, was 0.059 gm. per kilogram (Zadik) ; with a diet containing meat and bone ash the requirement seemed to be about 0.140-0.170 gm. per kilogram (Hawk and Gies) ; retention took place on intakes of 0.108 gm. (Biernacki), and 0.252 gm. (Lipschiitz), while Heubner showed that the normal retention is about 0.140 gm. per kilogram in a growing dog. The data are insufficient for the establishment of general conclusions. PHOSPHORUS REQUIREMENTS OF RATS From the experiments of Gregersen (1911) we deduce the observations that on a nitrogen-free, phosphorus-free diet the daily elimination of phosphorus per 1000 gm. of body weight is 36.& milligrams, the eight figures averaged varying from 28 to 54.5 milli- grams. From an experiment by McCollum (1909), we compute from a determination with a single individual on a phosphorus-free diet, that the outgo was 35.0 milligrams per 1000 gm. of body weight. The experiments of neither author afford satisfactory evidence for judgment as to the maintenance or growth requirement on normal foods. PHOSPHORUS METABOLISM 429 PHOSPHORUS METABOLISM AS AFFECTED BY VARIOUS CONDITIONS EFFECTS OF ALTITUDE ON PHOSPHORUS METABOLISM Von Wendt (1910) studied metabolism in man at three differ- ent altitudes in the Alps. Complete nitrogen and mineral balances were made. A portion of the data are as follows : DAILY NITROGEN AND MINERAL BALANCES WITH A MAN AT DIFFERENT ALTITUDES IN THE ALPS— Grams Day Altitude Meters N Food Urine Feces Balance S Food Urine Feces Balance Fe Food J Urine 1 Feces Balance Ca Food Urine Feces Balance P Food Urine Feces Balance 1 2 3 4 3000 3000 3000 3000 22.58 13.64 1.42 +7.52 22.58 16.02 1.42 +5.14 22.58 18.23 1.42 +2.93 22.58 15.70 1.42 +5.46 1.706 1.062 0.343 +0.301 1.706 1.343 0.343 +0.020 1.106 0.963 0.343 +0.400 1.706 1.123 0.343 +0.240 0.156 0.101 +0.055 0.156 0.101 +0.055 0.156 0.101 +0.055 0.156 0.101 +0.055 0.160 0.177 0.200 -0.217 0.160 0.197 0.200 -0.237 0.160 0.140 0.200 -0.180 0.160 0.157 0.200 -0.197 0.861 0.661 0.197 +0.003 0.861 0.774 0.197 -0.110 0.861 0.766 0.197 -O.102 0.861 0.828 0.197 -0.164 1 2 3 4 4560 4560 4560 4560 22.58 14.58 1.22 +6.78 22.58 14.38 1.22 +6.98 22.58 15.00 1.22 +6.36 3.30 9.97 1.22 -7.89 1.706 0.892 0.282 +0.532 1.706 0.928 0.282 +0.496 1.706 1.086 0.282 +0.338 0.435 0.672 0.282 -0.519 0.156 0.140 +0.016 0.156 0.140 +0.016 0.156 0.140 +0.016 0.003 0.140 -0.137 0.160 0.193 0.239 -0.272 0.160 0.137 0.239 -0.216 0.160 0.143 0.239 —0.222 0.083 0.124 0.239 -0.280 0.861 0.585 0.184 +0.092 0.861 0.658 0.184 +0.019 0.861 0.555 0.184 +0.012 0.284 0.508 0.184 -0.408 1 2 3 3000 3000 3000 22.58 14.25 1.06 +7.27 22.58 15.88 1.06 +5.64 3.30 11.90 1.06 -8.86 1.706 0.950 0.331 +0.425 1.706 1.198 0.331 +0.177 0.435 1.112 0.331 -1.008 0.156 0.096 +0.060 0.156 0.096 +0.060 0.003 0.096 -0.093 0.160 0.191 0.216 -0.247 0.160 0.176 0.216 -0.232 0.083 0.145 0.216 -0.278 0.861 1.057 0.241 -0.437 0.861 1.140 0.241 -0.520 0.284 0.629 0.241 -0.586 430 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The first and third series of balances were at an altitude of 3000 meters, and the second at 4560 meters. The nitrogen balance was positive at all times, except on the two days of low nitrogen intake. Reckoned as muscle substance, more than 100 gm. per day was formed. The parallel sulphur balances showed that the nitrogen storage was protein synthesis. The phosphorus balance was posi- tive only during the second period, at the higher altitude. Von Wendt believes, from the relative amounts of nitrogen, sulphur, phosphorus and iron stored, that during the first and third periods, at the lower altitude, there was much haemoglobin synthesized, while synthesis of muscle substance predominated during the second period at the higher altitude. THE INFLUENCE OF AMOUNT OF FOOD ON PHOSPHORUS METABOLISM The plane of nutrition on which an animal is living, as deter- mined by the nutrients available, naturally involves phosphorus as well as the other essential constituents. Thus Pettenkofer and Voit (1866) showed that on a medium diet the phosphorus in the urine was one-third more than during fast, the subject being at rest in both cases, and Forster (1873a), experimenting with foods poor in mineral matter, showed that the less the quantity of the salt-poor foods ingested the greater was the loss of phosphorus, due to tissue katabolism, from the body. An observation of similar portent was made by Weiske (1894), who found that in the less well-developed rabbits of the same age and kind the bones and also, to a less extent, the teeth, contained subnormal percentages of mineral matter. Gevaerts (1901), in a study of feeding white rats on phospho- rus-free foods, submits data which we have condensed as in the table on p. 431. From these data we see that in the white rat the phosphorus excretion in the urine from a phosphorus-free diet is very much less in amount than that present in the urine during starvation ; and also that on a ration of sucrose and edestin, or on sucrose and ovalbumin, there is much less phosphorus in the urine than on a ration of sucrose alone, the edestin and ovalbumin naturally being more efficient than the sucrose to spare the phosphorus-containing pro- teins of the body. Sherman (1902) showed, in experiments with human subjects, that nitrogen, sulphur and phosphorus balances all reflected consist- ently, by change of sign, the change from restricted to liberal diet, or the reverse. (Exp. 7-10 p. 432.) The diet was crackers, butter and milk. It is elsewhere shown that nitrogen, sulphur and phos- phorus balances need not, of necessity, show interdependence in their variations. PHOSPHORUS METABOLISM 431 AVERAGE DAILY EXCRETION OF PHOSPHORUS IN THE URINE OF WHITE RATS ON PHOSPHORUS-FREE DIETS— Grams Experiment, period and days Ration Initial live weight Gain or loss in weight Loss in phosphorus 1 A— 3 210 198 -8.3 -7.7 0.0093 IB— 3.. 0.0141 2 A— 2 182 159 147 145 -11.5 -4.0 -2.0 +7.0 0.0060 2 B— 3 0.00085 2c 1.. 0.0050 2D— 1 0.0033 3 A— 2. 181 158 155 146 -11.5 —1.0 -3.0 +8.0 0.0060 3B— 3 0.0010 3 c— 3 0.0040 3D— 1 0.0038 4 A— 2 202 183 168 160 -9.5 -5.0 -2.7 +8.0 0.0060 4B— 3 0.0010 4 C— 3 0.0031 4 D— 1.. . . 0.0017 5 A— 6.. 255 180 153 152 148 147 128 128 —1.7 —27.0 -1.0 —4.0 —1.0 -6.3 0.0 +1.0 0.0041 5 b— 1 0.0124 5C— 1 0.0019 5D— 1 0.0008 5E— 1.. 0.0013 5F— 3... 5G— 2 BH-2 0.0018 6 A— 1 240 213 195 178 173 -27.0 -6.0 -5.7 -2.5 +4.0 0.0124 6B— 3.. 0.0005 6D— 2. 6E— 2 0.0015 7 A— 1 280 252 -28.0 -0.7 0.0124 7B— 3 0.0029 8A-L 290 268 —22.0 -2.5 0.0072 8B— 2 0.0036 432 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE DAILY NITROGEN, SULPHUR AND PHOSPHORUS BALANCES WITH A MAN ON A DIET OF CRACKERS, BUTTER AND MILK— Grams N S P Food Experi- Dura- Fuel tion in days value Cals. Pood Urine Food Urine Food Urine No. Remarks Feces Feces Feces Crack- Milk Butter Balance Balance Balance ers 15.82 1.12 2.29 14.31 0.95 1.41 0.74 0.06 0.78 1 4 2908 +0.77 15.82 14.67 0.67 +0.11 1.12 0.97 0.06 +0.10 2.29 1.48 0.73 300 2040 40 Fore-period 2 4 2901 +0.48 15.82 14.16 0.70 +0.09 1.12 0.93 0.06 +0.08 2.29 1.42 0.78 300 2040 40 Loss of sleep 3 4 2913 +0.96 +0.13 +0.09 300 2040 40 After-period 12.05 0.94 1.40 10.43 0.72 1.03 0.74 0.09 0.44 4 4 2082 +0.88 18.52 16.50 0.87 +0.13 1.44 1.12 0.08 -0.07 3.07 1.74 1.10 405 1000 60 Low protein 5 4 2607 +1.15 +0.24 +0.23 120 3060 High protein 9.76 0.69 1.60 10.94 0.75 1.12 0.27 0.02 0.37 Intermediate 6 4 1555 -1.45 -0.08 +0.11 120 1530 period 10.11 0.70 1.58 ' 11.51 0.83 1.00 0.45 0.04 0.60 ■ 7 j 5 1660 -1.85 20.22 15.52 1.04 -0.17 1.40 1.06 0.09 -0.02 3.16 1.49 1.42 150 1500 Food restricted Twice as much 8 1 5 3336 +3.66 10.11 12.87 0.43 +0.25 0.70 0.88 0.04 +0.25 1.58 1.32 0.50 300 3000 as above 9 5 1656 -3.19 -0.22 -0.24 150 1500 Food restricted f 20.70 16.58 1.05 1.36 1.21 0.10 3.26 1.49 1.37 Twice as much 10 3 3329 +3.07 +0.05 +0.40 300 3000 as above Marked loss of sleep for 3 successive nights resulted in a small increase in the excretion of nitrogen, sulphur and phosphorus, the proportions not being markedly abnormal. The increased elimina- tion due to loss of sleep did not appear until the third day, while changes resulting from alteration of the diet were always percept- ible on the first day. The elimination of phosphorus by the intes- tine was large and variable. PHOSPHORUS METABOLISM 433 Kaufmann and Mohr (1903) studied metabolism during over- feeding 1 , following an extended period of underfeeding, with two adult human beings. Both subjects were in bodily health during this experiment on forced feeding. The average daily nitrogen, phosphorus and calcium storage in grams was as indicated below : Days N P2O5 CaO Body Subject Intake Retention Intake Retention Intake Retention weight Kg-. 1 2 7 10 18.38 16.45 4.90 5.67 6.20 5.36 1.35 1.37 6.07 4.46 2.34 2.10 62.0 57.7 The amount of retention is much influenced by the state of impoverishment existing, and the nutriment available. Hawk (1903) compared phosphorus balance on moderate and excessive protein intake. With the excessive ingestion of protein there was a negative phosphorus balance, while with less protein in the fore- and after-periods there were positive balances. The inges- tion of 4.96 gm. per day of phosphorus (stated as P 2 5 ) provided for storage of this element in men of 56.2-60 kg. body weight. In this experiment the sudden marked increase in phosphorus intake coincident with excessive protein ingestion, so greatly increased the urinary phosphorus excretion as to change a positive to a negative balance. DAILY PHOSPHORUS BALANCES ON ADULT MEN WITH VARYING AMOUNTS OF PROTEIN IN THE FOOD— Grams Subject Period Leng-th of period in days Pood P2O5 Urine P2O5 Feces P2O5 Balance P2O5 Rations H H H I II III 4 1 4 4.96 5.82 4.96 2.58 3.28 2.57 2.07 3.01 1.92 +0.32 -0.47 +0.47 300 gm. crackers, 60 gm. butter, 1650 gm. milk. 225 gm. beef, 250 gm. crackers, 60 gm. butter, 1375 gm.milk 300 gm. crackers, 60 gm. butter, 1650 gm. milk. R R R I II III 4 1 4 4,96 5.82 4.96 3.15 3.62 2.78 1.52 3.45 1.89 +0.29 —1.25 +0.29 Same as Period I above. Same as Period II above. Same as Period III above. Initial weight of H — 56.2 kg., of R — 60 kg. 434 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 C. Tigerstedt (1904) reported results on nitrogen and phospho- rus balance experiments with a young man 20 years old on rations varying in phosphorus content. Daily balances, as stated by the author, we have computed to average daily balances per period. A part of the results are as follows : AVERAGE DAILY PHOSPHORUS BALANCES WITH A MAN TWENTY YEARS OLD ON DIETS OF DIFFERENT PHOSPHORUS CONTENT Grams Nitrogen Phosphorus Days Balance Food Urine Feces Balance Daily rations 1 2 3-14 15-17 18-22 23-25 -13.06 -7.36 +1.23 -2.43 +2.91 -0.14 0.027 traces 2.545 2.515 3.056 1.757 0.649 0.730 1.333 1.506 1.612 0.956 ( mean 0.134 1.102 1.291 1.253 0.935 -0.756 —0.864 +0.200 -0.282 +0.191 -0.134 100 gm. meal, 18 grin, sugar, 207 gm. sago gruel, 3 gm. salt. 70 gm. sugar, 270 gm. jelly, 120 gm. potato, 80 gm. apple. About 70 gm. butter, 750 c.c milk, 150 gm. bread, 430 gm. potatoes, 150 gm. roast meat, 150 gm. groats. Same as above plus additional milk and a casein preparation, and on days 19-22, 160 gm. zweiback. First two days, 275 gm. groats, 180 gm. rye bread, 100 gm. butter; third day, 200 gm. groats, 140 gm. rye bread, 80 gm. butter. Weight of subject, 62 kg. The first two days' rations were very low in both nitrogen and phosphorus. The results of these two days show that the phospho- rus arising from the mucous coat of the intestine and from the intes- tinal juices, which appears in the feces, is about 0.134 gm. daily, but this amount may vary with the amount and kind of food. This phos- phorus is less in amount when food is given which is poor in both nitrogen and phosphorus than in starvation, since such foods serve to protect body proteins, etc., from katabolism. The negative balance on days 15-17 was due principally to imperfect absorption from the intestine. The last ration was a vegetable dietary, except for the butter. The negative nitrogen balance and the low calcium content of the cereal diet were both unfavorable to phosphorus retention. From the work of Liithje and Berger (1904) (see p. 218) we would infer that calcium and phosphorus storage might take place as rapidly in a normal man, who had never been ill, as in a typhus convalescent; but the nitrogen retention seems to be much more marked in the convalescent, in a greatly reduced state, than it is in either the well-nourished convalescent or the normal cases. The maximum phosphorus retention with a convalescent was 2.832 gm. P 2 5 , and in a normal case 3.381 gm. P 2 5 daily, in each case for 10 days. PHOSPHORUS METABOLISM 435 Sweet and Levene (1907) made observations on nuclein metabo- lism in a dog with an Eck fistula which show the same ability of the organism to adapt itself to different planes of nuclein metabolism that has frequently been noted with regard to total nitrogen and phosphorus. Vozarik (1909) studied the effect of variation in the amount of protein in the diet on the acidity, ammonia, phosphorus and total nitrogen in the urine of children. Increase of protein increased the outgo of all these constituents, both per day and per c.c. of urine. Biernacki (1909) sought to determine with dogs the effects of "supernutrition" on mineral metabolism. The foods added to the standard diets were butter, sugar and eggs. In each case the animal, though previously storing phosphorus, accomplished an increased storage through the ingestion of excessive amounts of these foodstuffs. Of calcium also there was in each case an increased storage, or decreased loss, when the supplementary foods were given. The conduct of mineral metabolism experiments on a really satisfactory basis requires that there be intermediate periods of sufficient length to allow the effects of previous feeding to disappear. Numerical data from this study are below. DAILY NITROGEN, CALCIUM AND PHOSPHORUS BALANCES WTH DOGS— Grams Exper- iment No. Periods Change in weight of dog Nitrogen balance CaO intake CaO balance P2O5 intake P2O5 balance Diet I Fore-period 8 days Fat period 8 days After-period 8 days 5700 -250 5450 +170 5620 -210 -0.0264 +0.5232 +0.2886 0.0915 0.2385 0.0915 -0.0019 +0.1673 +0.0261 1.4127 1.7007 1.4127 +0.3527 +0.8059 +0.4547 Meat; rice Meat; rice; butter Meat; rice II Fore-period 6 days Sugar period 6 days After-period 6 days 5290 +110 5400 +380 5780 -160 +0.8316 +1.5553 +0.4855 0.0927 0.0927 0.0927 -0.0328 -0.0159 -0.0301 1.4776 1.4776 1.4776 +0.5297 +0.6736 +0.4845 Meat; rice Meat; rice; sugar Meat; rice in Fore-period 6 days Fat period 6 days After-period 6 days Protein period 6 days 5620 -120 5500 +290 5790 -50 5740 +130 -0.1618 +0.6057 +0.5142 +1.6857 0.1289 0.2737 0.1289 0.1475 -0.0090 +0.1147 -0.0092 -0.0818 1.6643 1.9523 1.6643 1.5778 +0.5540 +0.5769 +0.2515 +0.6692 Milk; rice Milk; rice; butter Milk; rice Milk; rice; eggs' 436 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The increased nitrogen storage resulting from the addition of butter and sugar indicates that these foods were able to replace a portion of the protein previously used for energy production, thus allowing its participation, along with calcium and phosphorus, in synthesis of tissue. Heubner (1909) conducted an experiment with young dogs to study the effects of phosphorus starvation. He used 6 pups taken at weaning time, when 38 days old, and fed them various rations for 7 weeks, after which a part were killed, and their tissues examined. Two were fed on a low-phosphorus ration composed of egg-white, rice, sugar, palmin, ferric saccharate, and sodium, potassium, calcium and magnesium chlorides. Three others received a similar ration containing casein from cow's milk, and with a part of the alkali chlorides replaced by diphosphates, so that the food contained one percent more phospho- rus than the above. The sixth dog had bread, milk, meat, potato, rice and fat. The phosphorus-starved dogs were undersized, their bones became curved, and the abnormality of their diet was in many ways outwardly evident. An examination of the bones seemed to show decided differences in appearance from those of cases where calcium starvation was known to prevail. The feces of these dogs did not contain phosphorus in amounts corresponding with the food ; in fact, the phosphorus content of the dry feces was somewhat nearly the same throughout, and apparent- ly not related to the food. The phosphorus content of the urine, however, varied greatly, and in accord with the amount in the food. Data from this work are below. DATA SHOWING PHOSPHORUS STORAGE IN YOUNG DOGS Weight Phosphorus Phosphorus storage No. of dog Age of dog in weeks in urine per day, per kg. Nutrition Kg. Percent Grams 1 5 1.13 0.38 Mother's milk 6 5 1.00 0.51 Mother's milk 3 7 1.60 0.029 o.'ii Cow's milk 3 10.5 2.80 0.039 (0.026) Mixed diet 3 17 4.80 0.052 0.037 Mixed diet 1 7 1.32 0.39 0.13 P in excess 1 10.5 1.80 0.17 0.19 P in excess 6 11 2.20 0.17 0.23 P in excess 2 7 1.23 0.0024 0.008 P hunger 2 10.5 1.70 0.001 0.015 P hunger 5 11 1.90 0.0007 0.016 P hunger Kochmann (1911) studied calcium metabolism, as affected by other food constituents, with three grown dogs fed on dog biscuit and water. Below are his figures on phosphorus balance for Dog III. PHOSPHORUS METABOLISM 437 Nine out of twelve of the periods were 5 days in length. That 5 days is too short a period for mineral metabolism studies is mani- fest as we compare periods 2, 3 and 4, the intake being the same, and periods 8-12, the intake in these also being the same. In the urinary figures there is some evidence of delayed outgo as the intake changed; and in the two series above mentioned the change in balance is due principally to variation in feces phosphorus, the urinary phosphorus remaining much more nearly constant. The change in feces phosphorus, especially its progressive decrease in periods 9, 10, 11 and 12, was not interpreted by the author. It is of interest to note that with each of the three increases in intake there was a degree of improvement in retention which the animal was unable to sustain, since in the next period after the one in which the intake was increased there was in each case a decreased retention. AVERAGE DAILY PHOSPHORUS (P 2 5 ) METABOLISM OF A DOG RECEIVING VARIOUS AMOUNTS OF FOOD— Grams Length Weight Intake of Intake Urine Feces Balance Period of period in days of dog dog biscuit P2O5 P2O5 P2O5 P2O5 1 4 6750 100 2.191 1.159 1.326 -0.293 2 5 6600 150 3.286 1.200 1.937 +0.149 3 3 6450 150 3.286 1.243 2.104 -0.061 4 5 6280 150 3.286 1.212 2.262 -0.188 5 5 6260 175 3.833 1.219 2.038 - 1-0.576 6 5 6320 175 3.834 1.305 2.502 0.027 7 6 6355 200 4.381 1.250 2.251 -0.880 1 8 5 6420 200 3.994 1.300 2.467 -0.227 9 5 6460 200 3.994 1.078 2.908 -0.012 10 5 6300 200 3.994 1.056 2.815 -0.123 1 n 5 6350 200 3.994 1.016 2.190 - -0.788 12 5 6450 200 3.994 1.085 2.020 - -0.889 Summary. Balance experiments show us that increased intake of phosphorus usually causes increased elimination in the urine with carnivora, in the feces with herbivora, and according to the nature of the diet with omnivora, the increased outgo being accom- panied by increased retention or production at such times as so determined by the general state of nutrition. Marked increase in intake, incidental to excessive protein ingestion, is sometimes accom- panied by decreased retention; but this is not the usual course of metabolism. During deficient intake of phosphorus, or of food in general, the consumption of phosphorus-free food may decrease the loss of phosphorus, through the protection afforded the body tissues from katabolism. 438 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Even in a normal state of nutrition, grown animals are able to increase their bodily reserves of phosphorus to a considerable extent; and after sickness the retention may be very extensive, though apparently not of a different order from that characteristic of normal growth. As with other nutrients, the retention of phosphorus is not directly as the intake, but, other conditions being favorable, as the amount of intake in excess of the maintenance requirement. After a certain point is reached, however, the rate of increased retention coincident with increased intake, begins to fall off, and, with indefin- itely continued increase of intake, a point is reached where further increase does not lead to further storage, but only to elimination. Animals have a capacity to adapt their phosphorus exchanges to different planes of metabolism, as with other nutrients, nitrogen in particular. PHOSPHORUS METABOLISM DURING FAST In fasting there is a very considerable loss of phosphorus from the body, due to the katabolism of the phosphorus-containing pro- teins which are consumed for energy production, a much greater loss, naturally, than where foods are used which are poor in phos- phorus. In this latter case phosphorus-containing tissues are protected, and a portion of the katabolized phosphorus may be retained and utilized along with nutrients supplied by the food. Considerable attention has also been given the possibility that the bones lose phosphorus during starvation. I. Munk (1887) announced conclusions reached in the course of fasting experiments with Cetti as follows : In fasting, the phospho- rus of the urine is united partly with potassium, and partly with calcium and magnesium. The ratio of phosphorus to nitrogen in the urine is about 1 :4.5 during fast, while in the soft parts of the body the ratio is about 1 :7. The increase of phosphorus outgo is both absolute and relative to nitrogen. The calcium excretion is 3 or 4 times that amount which would correspond to the break-down of flesh. Magnesium excretion also is much greater than as if derived from the flesh, but during fast the calcium exceeds the magnesium. From these facts he concluded that the bones contribute to the phos- phorus loss during starvation. F. A. Falck (1875), in studying hunger metabolism in a dog, determined that a dog weighing 21210 grams lost in body weight 10380 grams, and in phosphorus 31.7 gm. P 2 5 , equivalent to 5706 gm. fat-free fresh dog-flesh, while starving to death in 61*4 days. PHOSPHORUS METABOLISM 439 Luciani (1889) made a general physiological study of fasting, with Succi as a subject. In 1889 he reported results from a 30-day fast from which it would appear, in harmony with the conclusions of Munk, that the nitrogen and the phosphorus of the urine have in part an independent origin early in the fasting period, as well as late, but most markedly so after a considerable duration of the fast. A portion of his figures are as follows : DAILY PHOSPHORUS ELIMINATION IN THE URINE DURING FAST Day of ; fast P Grams N/P Day of fast P Grams N/P Day of fast P Grams N/P 1 0.842 16.3 11' 0.620 12.7 21 0.325 11.9 2 0.898 12.2 12 0.441 16.2 22 0.313 10.2 3 0.920 15.0 13 0.137 25.6 23 0.459 10.4 4 0.925 13.8 14 0.434 12.2 24 0.344 16.1 5 1.031 12.4 15 0.449 11.4 25 0.257 27.4 6 0.932 13.0 16 0.468 11.7 26 0.341 17.7 7 0.814 11.5 17 0.531 11.6 27 0.375 14.3 8 0.691 12.1 18 0.439 12.3 ' 28 0.349 16.0 9 0.593 13.1 19 0.415 12.3 29 0.345 11.8 10 0.544 12.4 20 0.382 11.5 30 0.444 14.9 C. Lehmann, Miiller, Munk, Senator and Zuntz (1893) reported on fasting experiments with Cetti, the professional faster, and Breit- haupt, a cobbler. Both drank water at will, Cetti took no exercise ; Breithaupt assisted in laboratory work. DAILY NITROGEN AND PHOSPHORUS EXCRETION IN THE URINE BY CETTI AND BREITHAUPT DURING FAST— Grams Cetti Breithaupt Day of fast N P2O5 Day of fast N P2O5 1 13.545 2.597 1 10.01 1.56 2 12.586 2.925 2 9.92 1.89 3 13.121 3.289 3 13.29 2.53 4 12.393 2.974 4 12.78 2.36 5 10.695 2.871 5 10.95 2.19 6 10.100 2.667 6 9.88 2.29 7 10.885 2.663 8 8.903 1.722 9 10.833 2.065 10 9.467 0.948 The average daily fecal nitrogen and phosphorus (P 2 O g ) with Cetti were 0.316 gm. and 0.205 gm., respectively, and with Breit- haupt, 0.113 gm. and 0.140 gm., respectively. The phosphorus of the urine was found to be largely combined with potassium, more than twice as much as was present in com- bination with calcium and magnesium. Calcium and magnesium were present in the urine only as phosphates. By computation the authors show that it would have been impossible for the flesh to have given up all of the phosphorus excreted. In both cases a part of the phosphorus is shown to have come from the bones. 440 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Gusmitta (1893) studied the composition of the bones of sev- eral rabbits and a dog as affected by starvation. The method was amputation of parts, and comparison with others remaining after recovery of the animal from the mutilation, and after subsequent starvation to death. The bones were shown to lose during starvation, in weight, volume and specific gravity, and to increase in porosity ; there was a slight increase in water, and a uniform decrease in all organic and inorganic constituents. The results seem indisputable in showing that starvation causes katabolism of bones as well as soft parts. The following figures set forth results with the dog experi- mented upon. CONSTITUENTS OF BONES OF A DOG AS AFFECTED BY STARVATION Dry weight of bones Total inorganic matter Iron phosphate Calcium carbonate Magnesium phosphate Calcium phosphate Grams Percent Grams Percent Grams Percent Grams Percent Grams Percent Rig-ht cubitus and radius, dog - in nor- mal state, 11.917 g-m. Left cubitus and radius, after star- vation, 10.382 gm. 7.261 6.556 60.93 63.16 0.1090* 0.0134 0.16 0.13 0.6888 0.5391 5.78 5.20 0.1120 0.0809 0.94 0.78 6.4208 5.9011 53.88 56.84 * Apparently erroneous; 0.0169 would harmonize with percentage figure. I. Munk (1894) reported a fasting experiment with a dog which shows more clearly still than his previous experiment on Cetti that in inanition a part of the nitrogen, calcium, magnesium and phosphorus loss is from the bones. Some of his figures are as fol- lows: EXCRETION OF NITROGEN AND PHOSPHORUS BY A MATURE FASTING DOG— Grams Body weight Day Kg. N P2O5 P205:N Prelim. 17.16 16.82 1.99 1:8.4 1 16.94 5.59 0.820 1:6.8 2 16.48 5.31 0.901 1:5.9 3 15.85 5.32 0.996 1:5.3 4 15.58 5.28 1.106 1:4.8 5 15.35 5.18 1.268 1:4.1 6 15.16 4.29 0.776 1:5.5 7 14.95 4.66 1.020 1:4.5 8 14.77 3.79 1.210 1:3.1 9 14.49 3.59 0.883 1:4.1 10 14.27 3.74 0.851 1:4.4 Total loss 2.89 46.75 9.83 1:4.8 Average 0.29 4.68 0.98 Feces Total Average 1.576 0.158 1.874 0.187 1:0.84 PHOSPHORUS METABOLISM 441 During the preliminary period of several days the diet was liberal and normal; ratio of P 2 5 :N in urine as 1:8.4. During fast this ratio was 1 :4.8. The increased phosphorus in the urine, the author computes, would be equal to that in 32 pi. fresh bone ; the extra calcium would imply the destruction of 31.5 gm. fresh bone, and an excess of magnesium was also to be accounted for. Heymans's (1896) work on metabolism during starvation of the rabbit shows that there is a premortal rise in phosphorus excretion. If the fast is discontinued by the giving of food the phosphorus excretion in the urine is very much less than either the normal or the fasting excretion, until an equilibrium is reestablished. Weiske (1897), experimenting with rabbits, studied the effects of starvation on the composition of the bones, teeth and other tissues. Four rabbits from the same litter were used, No. 1 as a control, being killed at the beginning of the experiment, and the three others after fasting for 7-11 days. Below are some of the data from the analysis of the carcasses. COMPOSITION OF BODIES OF RABBITS AS AFFECTED BY STARVATION No. 1 No. 2 No. 3 No. 4 Loss of Different Parts — Percent Flesh Skeleton. . . Pelt Stomach. . . Intestines , Liver Lungs Kidneys. . . Heart Spleen 39.7 45.0 4.1 Gain 23.3 20.3 20.8 34.8 24.7 50.4 56.5 54.4 68.1 62.5 26.3 29.5 29.7 23.4 73.9 69.6 43.4 3.3 20.0 27.7 48.2 62.5 53.3 26.9 Gain 71.7 Weight of Parts in Grams Flesh, water and fat-free Skeleton, water and fat-free. Fat in flesh Fat in skeleton Fat in flesh and skeleton 210.33 147.00 133.39 88.79 95.65 101.26 41.32 4.75 4.98 11.58 0.52 0.53 52.90 5.27 5.51 Analysis of Bones Other Than Leg Bones — Percent of Water- and Pat-free Organic substance , Mineral substance. . CaO MgO COS P205 Remainder 38.06 39.73 37.74 61.94 60.27 62.26 32.35 31.76 32.52 0.71 0.69 0.62 2.50 2.70 2.88 24.17 24.02 24.67 2.21 1.10 1.57 39.35 60.65 31.77 0.64 2.48 24.22 1.54 Analysis of Teeth — Percent of Water- and Pat-free Organic substance. Mineral substance. . CaO MgO..„ CO2 P2O5 Remainder 23.42 21.80 21.52 76.58 78.20 78.48 37.88 38.75 38.92 - 2.49 2.44 2.49 0.77 0.99 1.10 33.91 34.21 34.46 1.53 1.81 1.52 22.09 77.91 38.83 2.37 1.45 34.24 1.02 442 OHIO EXPERIMENT STATION: TECHNICAL BTJL. 5 It will be noted that the leg bones were not included in the above analyses. In an effort to harmonize these results, which do not show a loss of phosphorus from the bones, with those which have been discussed, we can only suggest that the bones constitute, rela- tively, so large a store of the mineral constituents of the body that a considerable change in amounts of urinary constituents would repre- sent but a slight change in the skeleton as a whole, so slight a change, in fact, that its detection by skeleton analysis would be very likely to be missed through the inevitable errors involved in the comparison of a starved animal with another individual serving as a control ; further, the fasting time was short ; the skeleton yields but slowly to influences which tend to modify its composition. Hoover and Sollmann (1897) studied phosphorus metabolism during fasting in hypnotic sleep covering a period of 8 days. The analysis of the urine is as follows : ANALYSIS OF URINE DURING HYPNOTIC FAST— Grams Date Quantity C.C. Urea Uric acid P2O5 NaCl Total N Water taken July 15 1350 36.21 0.824 3.381 12.330 20.978 .J " 16 570 22.62 0.617 2.303 6.837 12.369 .. 1? 470 22.99 0.450 2.268 3.863 12.370 750 " 18 530 25.24 0.538 2.270 3.964 14.013 750 "19 & 20 1020 58.18 0.866 5.052 5.954 27.988 1150 " 21 410 20.67 0.375 2.434 2.496 10.791 875 " 22 660 28.26 0.572 3.150 2.419 14.504 1250 " 23 820 34.13 0.908 4.442 3.296 21.582 750 The loss in weight during the last 7 days was 5896 gm. ; loss of nitrogen, 113.617 gm. ; nitrogen estimated as albumin 710 gm., — estimated as muscle 3341 gm. There was some evidence of an increased proportion of phos- phorus to nitrogen during the progress of the fast. The average loss of phosphorus during the last 7 days was 3.131 gm. P 2 5 . Nemser (1899) studied the effects of starvation on the total and nuclein phosphorus content of the muscles, liver, kidney and intes- tine of white mice. Eighty mice were used. The data seem to indicate that the percent of loss in weight of parts was greater than the gain in percent of total and nuclein phosphorus, which implies that the total and nuclein phosphorus contributed to the loss during fast. Sedlmair (1899) studied the losses sustained by the various organs of the cat during starvation, by comparing three cats of the same litter, one well-fed, with two which were starved. One cat, starved for 36 days, and losing in weight 1764 gm. from an initial PHOSPHORUS METABOLISM 443 weight of 3368 gm., apparently lost in this time only 0.564 gm. CaO, which was computed to represent 3.4 gm. of fresh bone substance, or less than 1 percent of the total weight of the skeleton. Below are figures which distribute the total loss to the various parts sus- taining decrease in weight. LOSSES IN DRY SUBSTANCE OF DIFFERENT TISSUES OF CATS DURING STARVATION— Percents of Total Loss Cat B Cat C Muscular system Skin Mesentery Bones ■ Liver Intestines Kidneys Pancreas Heart Lungs Spleen Brain Spinal marrow Central nervous system Urinary bladder, aorta, trachea and eyes Blood 62.23 57.06 11.33 16.50 8.51 7.56 5.67 8.17 5.03 4.41 3.00 3.18 0.80 0.73 0.22 0.41 0.39 0.31 0.24 0.30 0.25 0.25 +0.17 +0.10 0.05 0.07 +0.12 +0.03 0.16 0.06 2.29 1.11 100.00 100.02 The bones were in most cases made richer in water by starva- tion, while the dry substance decreased, both absolutely and in per- cent, these effects being most noticeable in the long bones. The principal loss of weight from the bones was fat, though all other constituents of the bones shared in the loss. Schulz and Mainzer (1901) studied nitrogen and phosphorus metabolism in 3 starving rabbits and 1 starving dog. They observed in each case the usual premortal rise in the urinary excretion of both nitrogen and phosphorus, but no significant change in ratio of nitrogen to phosphorus in the urine just prior to death. Death occurred before the disappearance of fat from the body. Rubow (1905) found the lecithin content of 9 normal dogs' hearts to vary between 7.05 and 8.80 percent ; while of fat the same hearts contained 3.60 to 3.99 percent, in the dry substance. Two dogs which were starved to death, had in the hearts 7.3 and 7.67 percent of lecithin, and the same hearts contained 3.67 — 3.71 percent of fat, on the dry basis. In normal dog muscle the lecithin varied between 4.44 and 5.27 percent, while the fat content of the same samples was 14.16 and 6.30 percent, respectively, on the dry basis. In the muscle of the starved dogs was found 3.08 and 3.74 percent of lecithin, and 2.58 and 2.74 percent of fat, on the dry basis. From these figures it would appear that in starvation the dog uses both fat and lecithin from the muscles, but the withdrawal of either fat or lecithin from the heart is doubtful. 444 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Van Hoogenhuyze and Verploegh (1905), in studying creatinin excretion, publish urine analyses of a human subject, covering 15 days of fast, preceded and followed by one day with food. The figures are as follows: URINE ANALYSIS OF A FASTING MAN— Grams Date Total N Creatinin P2O5 X/P2O5 Remarks June 10 13.99 1.087 2.670 5.2 Food 11 8.76 0.904 1.550 5.8 Beginning- of starvation period " 12 8.38 0.577 1.830 4.6 ' 13 10.73 0.581 2.654 4.0 ' 14 9.40 0.634 2.934 3.2 In the morning-, 50 c.c. sulphatic water drunk ' 15 7.87 0.603 1.749 4.5 In the morning-, 50 c.c. sulphatic water drunk ' 16 7.73 0.590 1.069 7.2 In the morning-,100 c.c. sulphatic water drunk ' 17 6.11 0.469 0.713 8.5 From 11 to 1 o'clock, muscular work ' 18 7.70 0.689 1.658 4.6 19 7.35 0.715 1.702 4.3 20 6.80 0.602 1.461 4.6 ' 21 6.14 0.453 1.097 5.6 ' 22 6.97 0.566 1.312 5.3 ' 23 5.62 . 0.548 1.114 5.0 ' 24 4.08 0.426 0.869 4.7 ' 25 4.38 0.715 0.539 8.0 Food taken at 10 o'clock at night '" 26 7.23 1.028 0.145 49.9 Food At the beginning of this fast the nitrogen, phosphorus and creatinin decreased from the quantities excreted during the day before, and, as tissue protein was attacked, the ratio of N to P 2 5 in the urine became less. The muscular work of the 7th day of fasting had the effect temporarily to lessen the excretion of nitro- gen, creatinin and phosphorus, especially phosphorus, though the excretion of all these constituents rose on the next day, the creatinin and phosphorus elimination remaining high for three days. At the end of the fast the total nitrogen and creatinin excretions rose markedly, but the phosphorus excretion sank to a lower figure than at any time during the fast, or on the day before the fast, which implies a lessened katabolism of phosphorized proteins, and the replenishment of depleted phosphorus reserves. Cathcart and Fawsitt (1907) reported on a 14-day fasting experiment, with a 31-year-old man, with a 3-day preliminary and 5-day after-period, in which nearly one-fifth of the phosphorus seemed to have some source other than the soft parts. During this fast the elimination of purin bases and phosphorus decreased on the first day, the organism apparently sparing its nucleoproteins. The minimum purin excretion was on the third day of the fast, after which it increased, reaching the normal on the tenth day. The phosphorus outgo, however, steadily decreased, as also did total nitrogen, and both were less in amount on the last day of the fast than on any other. Thus purins increased as phosphates and total nitrogen decreased. That nucleins participated in the tissue katabolism is, of course, clear. The fate of the nonpurin nitrogen and of the phosphorus from the nucleins, however, is not so certain, PHOSPHORUS METABOLISM 445 but since the outgo of total nitrogen and phosphorus was under- going a progressive decrease it would be fair to assume that these constituents were retained, or else, if eliminated, that they served to protect equivalent amounts of the same in a higher state of organization or combination. Wellman (1908) studied mineral metabolism in fasting rabbits. In experiments of 12-15 days duration it was determined that full- grown rabbits, in starving to death, lost 6.5 to 7.7 percent of the fat-free, dry substance in the bones, and about 14 percent of the fresh weight. Of this loss of fresh weight nearly half was fat. The absolute loss of calcium from the bones was determined as 1.8 gm., of phosphorus 0.6 gm., and of dry substance 8.7 gm. Falta and Whitney (1908) also found in the fasting metabolism of the dog such an increase in the proportions of Ca and P to N in the urine as indicated a probable participation of the bones in the increased katabolism. Grund (1910) concluded that the ratio P:N in the organs of animals tends strongly to remain constant under different conditions of nourishment as well as in fasting, though some variation in this proportion was noted in the liver of the hen and the dog. Accord- ing to recent observations (1912a, 1912b, 1913) the relative amount of protein phosphorus in muscle, in relation to total phosphorus and to the protein nitrogen, is not altered by the wasting away due to hunger, but is materially increased by degeneration due to the sever- ing of nerves or to severing from the bone. Atrophy, from division of the nerve, and from severing from the bone, alike, cause a dissolv- ing of phosphorus-free proteins, with a sparing of phosphoproteins. Wolf and (Esterberg (1911) made a study of nitrogen, sulphur and phosphorus metabolism in the dog as affected by fasting or underfeeding. The feeding of a comparatively small amount of pro- tein was found to reduce the phosphorus excretion to a very low amount. The feeding of starch and fat seemed to have little or no effect on phosphorus excretion. During fasting the ratio of phosphorus to nitrogen in the urine was so high that it seemed prob- able that some of the phosphorus came from the bones. Wilson and Hawk (1914) found that during fast the urinary acidity and phosphates varied, in general, together, increasing amounts of acid in the blood or lymph (due to increased fat katabolism in the presence of decreased carbohydrate oxidation) leading to a change of di- to monophosphates, and the excretion of the excess of the latter. Thus increased phosphate excretion and urinary acidity followed an increased formation of acids in the body. Increased neutralization of acids with ammonia led to decreased urinary acidity and phosphates. 446 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Among the other changes produced by starvation, in the phos- phorus compounds of animals, may be mentioned the reduction in the lecithin phosphorus of the liver as noted by Heffter (1890, 1891) and the decrease in the phosphocarnic acid in the brain as observed by Panella (1906a). Summary. Fasting causes an increased outgo of phosphorus. From the evidence that non-purin nitrogen, as well as nuclein com- pounds and skeleton, all contribute to this outgo we conclude that its production is due to generalized katabolism. The proportionate losses sustained by the various tissues are difficult of determination. The loss from the skeleton, however, is appreciable, though but a very small part of the total phosphorus present. The lecithin phos- phorus of the liver and muscles, and the phosphocarnic acid of the brain have been shown to contribute to the phosphorus loss in star- vation. There is evidence that the first deficiencies of phosphorus are met principally by non-purin compounds, that there is a progressive decrease of phosphorus outgo from this source, and also of total phosphorus, until the premortal rise, but increasing contributions from nuclear and skeletal compounds. There is evidence of a further utilization by the animal of the phosphorus of katabolized nuclein compounds after the elimination of the purin nitrogen. The feeding of protein during starvation has a much greater effect to reduce phosphorus loss than the feeding of starch and fat. PHOSPHORUS METABOLISM DURING INCUBATION As typical of metabolism generally, especial interest attaches to the simplified physiology of the egg. C. Voit (1877a) sought to determine if there is a loss of lime from the egg-shell, and utiliza- tion of the same in the growth of the skeleton of the incubating chick. He concluded that there was no such loss of lime from the shell, but his method of demonstration leaves one in doubt as to the fact. During incubation A. Kossel (1885) found that the phospho- protein of the hen's egg changes to nucleoprotein, because, while the yolk nuclein of the fresh egg does not yield purin bases, the nuclein of the hatched chick does yield guanin and hypoxanthin. Maxwell (1893) studied the phosphorus exchanges in the incu- bating hen's egg but the analytical data indicate that his methods were unsatisfactory. PHOSPHORUS METABOLISM 447 Mesernitzy (1907) states that the lecithin of the fresh hen's egg, dried, is about 15.35 percent, estimated from the phosphorus of the ether extract. During incubation he found this amount reduced by about a half. Carpiaux (1908) shows that with the decrease of lecithin in the incubating hen's egg there proceeds an accompanying decrease in the lime of the shell, and an increase of inorganic phosphates. The following data substantiate these conclusions. ANALYSES OF HENS' EGGS DURING INCUBATION— Grams Exp. No. Agre in Weight CaO P2O5, P2O5, P2O5, Lecithin days inorganic organic total 1 0.040 0.075 0.150 0.225 1.050 2 6-7 62.63 0.128 0.146 0.274 1.022 3 6-7 55.43 0.0377 . 4 7-8 64.70 0.0360 0.127 0.135 0.262 0.945 5 13-14 54.20 0.0478 6 14-15 54.62 0.0616 0.137 0.108 0.245 0.760 7 18 55.90 0.1357 0.182 0.050 0.232 0.350 8 19 53.10 0.1487 9 21 0.2022 Plimmer and Scott (1909) compared the phosphorus distribu- tion in the body of the chick at the time of hatching with that of the fresh egg, and of the egg at different times during incubation. They draw the following conclusions with regard to the sources of the different phosphorus compounds of the young bird's body : "There is not much change in the ether-soluble phosphorus until about the 16th or 17th day of incubation, when a very rapid disappearance of the ether-soluble phosphorus bodies commences. At this time it corresponds with a great increase in the amount of inorganic phosphate in the chicken; therefore it is impossible to avoid the conclusion that these glycerophosphoric acid compounds have been converted into inorganic phosphate for the calcification of the bones .... Some of the inorganic phosphate must have come from the vitellin, since all of it disappeared. Probably some of it, however, was converted into nucleic acid, as the latter increased .... There is a gradual absorption of the protein phosphorus bodies of the yolk by the developing chicken before there is a change in the lecithin bodies." General Conclusion: "That the glycerophosphoric acid gives rise only to inorganic phosphate in the developing chicken and is not transformed into any combination with protein. There is no evi- dence of a synthetic process occurring in the developing egg as regards the phosphorus compounds unless the probable transforma- tion of the phosphoprotein into nucleoprotein be so considered." Data from this study are in the following table. 448 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 FH £ bo w bo M W W cw © In hH n 0) H «* 85 C/2 O ^S o u o w H «! ffl bo bo W S i— i U i— i • F* CO ft: CO PO S-H « rp Oh o W Q n CO 3 cu i— i S-i CO 03 iTi O W O. W Oh to n Oh fc O S3 £ H o «M H P co M s HH CU (3 w H °bo . . .10 .cocm . . . • -00 -lOCO • • • ■ -iH T-lr-( • • o ■ • -O -OO ;0 ho ho H(DO . . ..-1 . . t^COCO • • • i— < • • CMCNCM • • "rH ■ • 1-° o 2 .9-- <0.S _d . . . . • ."*31 < 010 • • • • ■ CM "CMCM • • o o . . .10 . ho u cu . . .00 .Tf(^H . . • • -co "o ^ • • 3 o . . .O .OtJI .CO • . -co -inr^ 'Go » • 'iO -IOCD *CD ho ho (MCM"— 1 -CM .(MO) • coooo> 'O •r^o) • rH -i-H -OJlO • t d S.2 After 7 days " 13 " " 14 " " 16 '" " 17 ; " 19 ; " 20 PHOSPHORUS METABOLISM 449 Hanes (1912a, 1912b) finds, by microchemical studies, that phosphorized fats are abundant in the liver of the incubating chick during the first two weeks ; during the third week these compounds diminish, the phosphorus apparently serving for calcification. It is suggested that the pathological calcification of arteriosclerosis results from a splitting in situ of phosphorized fats, with subsequent formation of calcium salts. See also Tornani (1909). Robert and Wasteneys (1913) concluded that serious errors vitiated the results of Massing and of Shackell in their studies on the phosphorus changes in developing sea-urchin eggs. Frdm an investigation of their own they submit the following data : FORMS OF PHOSPHORUS IN DEVELOPING SEA-URCHIN EGGS Percent of Total P Stage of development Soluble in alcohol Soluble in water Insoluble Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2 39.5 36.5 35.2 46.5 38.8 35.1 36.7 47.6 30.8 29.6 40.6 37.8 23.8 15.9 34.0 23.9 20.6 Plutei 27.1 The conclusions are as follows: "1. During the development of the eggs of Strongylocentrotus purpuratus to blastulae the proportion of P which is present in the form of phospholipines (lecithin, etc.) undergoes appreciable dimin- ution. The further development of the blastulae into plutei is accompanied by a further diminution in the proportion of phospho- lipines in the larvae. "2. The development of the eggs of S. p. to blastulae is accom- panied by a considerable increase in the proportion of P which is soluble in boiling water and insoluble in alcohol, and by a decrease in the proportion of P which is insoluble either in alcohol or in boil- ing water. The further development of the larvae to plutei is accompanied, on the contrary, by a decrease in the proportion of water-soluble phosphorus and an increase in the proportion of insol- uble P." PHOSPHORUS METABOLISM IN INFANCY RELATIVE VALUE OF DIFFERENT KINDS OF MILK Human milk is low in the ash constituents generally, and also low in phosphorus, as compared with all other milks. It is also low in casein as compared with most of them, including cow's milk. It 450 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 has, however, a relatively high content of both phosphatid and nucleon, and of Wroblewski's opalisin, and a relatively large part of its phosphorus is in an organic form. Each of these items has been brought forward as an explanation of the undoubted fact that child- ren thrive better, and form bones and tissues more rapidly, when they are fed on mother's milk than when artificially fed. In composition, the milk of equidae is most nearly like human milk. Schlossmann (1897) considered ass's milk with a view to its value as a substitute for human milk, but decided that it would be unsatisfactory on account of its high proportion of protein to fat. Aside from differences in amounts, the caseins are found to be different in kind, the evidences being reported in our discussion of the chemistry of casein on pages 43 and 44. Soxhlet (1893) discusses differences between human and cow's milk, with consideration of suitable modifications of cow's milk to prepare it for the human infant. He says that cow's milk gives coarser clots with rennet ferment, and contains about twice as much casein and 6 times as much calcium, and has 3 times as high acidity, as human milk. Addition of water, or heating the cow's milk, reduces the size of colts ; but in each case other effects produced are unfavorable. Decoctions of grains, etc., affect the clots as do corre- sponding amounts of water, but add starch ; if malt extract be added the resultant mixture may be of higher nutritive value. Since cow's milk contains 4 times as much phosphoric acid and 6 times as much calcium as human milk, more calcium phosphate is supplied than is needed for bone-formation, and the excess is excreted, mostly as calcium soaps in the feces. Both kinds of milk contain the essen- tial soluble calcium, principally in the form of calcium citrate. Julius Lehmann (as reported by Hempel, 1894) and Hammar- sten (1895) both proposed modification of cow's milk for the use of human infants, on the basis of the consideration that the most significant difference between human and cow's milk is in the ratio of albumin to casein, and of casein to fat. Stoklasa (1897) gives the range of lecithin in cow's milk as 0.90-1.13 gm. per liter (representing an average of 0.091 gm. P 2 5 ), and that in human milk as 1.70-1.86 gm. per liter (representing an average of 0.153 gm. P 2 5 ). In cow's milk the total P 2 5 is 1.81 gm. per liter, and therefore the lecithin phosphorus forms 5 percent of the total ; while in human milk there is but 0.44 gm. P 2 5 per liter, and the lecithin, therefore, forms a much larger proportion, 35 per- cent, of the total. PHOSPHOKUS METABOLISM 451 Blauberg (1897c), studying the nutrition of infants on woman's milk and on cow's milk diet, found about the same differences in the mineral constituents of the feces as in ash of the two kinds of milk, especially a larger calcium and phosphorus, and lower iron content in the feces from cow's milk. Blauberg (1897b) reports an analysis of Mellin's Food. For comparison with these figures we have computed to the dry basis the proximate food constituents of human milk as reported by Ellenberger, Seeliger and Klimmer (1902), and the mineral con- stituents as reported by Soldner (1902) . From these data it would appear that the use of Mellin's Food with milk would tend to substitute carbohydrates for fats, and to decrease the calcium. The phosphorus content, on the dry basis, is not less than in human milk, and as Mellin's Food is used not with human but with cow's milk, which contains much more calcium and phosphorus than does human milk, it seems likely that the use of this proprietary food would not have the effect to starve the infant for any of the mineral nutrients. COMPARISON OF THE DRY MATTER OF MELLIN'S FOOD AND HUMAN MILK Water, percent Percetnts of dry substance Protein Fat Carbo- hy- drate Mineral matter K2O Na20 CaO MgO P2O5 CI Mellin's food Human milk 6.31 86.4 8.2 11.8 2.22 35.3 85.15 48.5 3.93 1.84 1.15 0.65 0.41 0.26 0.025 0.28 0.047 0.039 .0.38 0.23 0.081 0.43 For other analyses of infant foods see Blauberg (1897a), Klautsch (1896), and vonSzontagh (1902). Keller (1898) determined that when feeding infants on cow's milk more phosphorus is found in the urine than under normal feed- ing, and also a very much larger proportion of phosphorus to nitro- gen. Keller (1900b) studied nitrogen and phosphorus metabolism with infants, both normal and suffering. from gastrointestinal dis- orders, on normal and on artificial milk feeding. By the addition of Na 2 H P0 4 to human milk the phosphorus retention was increased in either well or sick infants. Phosphorus retention was greater on human milk than on cow's milk. Balance data from Keller's studies are in the following table. '452 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 O S w. -3 o M *(Niecocoao-H'«i.ogoccoinoQ'*iOi-[ OCOOOCO.-ICOaOCOOSOOCOOO''* ooooooooooooo bo o +■> u u a fa *> .-1 CO Ifi 00 OS OS CO r~ t» CM 1(5 T* 00 WaOOSmr^-HCO-HOi-H-WCOCT! i— ICO miN>OCDlOH^l«Cl5H o .9 P comcoo-^cooooooicooccDoo rHlOOt^OjMCOX3!»OCMm t-iC-cor-cDcoior-iooococooj <-IOi-IOOOOOCOOOrtO CO 0) o S fa Tflomas^iflcOTWCooico.— ion CMO}t~Or-OCOTt<:j}OCO''*CO .— tCOC0CMr-HCOCM •u ■- ^ a © s ■*< Jllll W i-H COCOOOO HMOOHO 2 (4 ft-S^ at*?'- ^ OSlOSOCMiJtOS co^xcocmcmcni CO iHl-il-H — -li-Hi-l TH *>0 a * « a XI o 5-^ u •E^'pm OS 00 T t— CM OS CO OS cm coco -win co CO a £8^ V> £ iocotjicocmco CO o r O +J IS rHlHlHlHt-HlH 3 .y £•§ ap CONlOO'*'* ■»JI w OOOOOO CO o SSooQ o'oo'ocso o °.S£ cor^^r^cMT* CO . r- u H i-HOi-HOOO o — oooooo o 3 a >, P4 u •Hn osoo^oor^co •^■n. CD OS CO CO $ a i OOOOuO a Q Pra OOOOOO o ta H o o u CM u +S 1 ooosiracoojg co O In i-H CM CM CM CM i-t CM s» oooooo o o a * CM cocposoos-n CM CM CM CO CM CM ITS H S CM oooooo o H «4 OSCMOOSt^cM intooiT-icoco CO CO ■**•"»< CO CO CO co OOOOOO o H s U U fc L| U U, < cm t-»-^ corpus l ~ l j 1 12 iHCMCO^lOCO a rt J «-l o a A a^'^ 3 3 rt Organic water e tract mi nucleon nucleon H/F ITS CMOS CD •^co^;o HHrtri 1-1 CM ■p a (4 t o .S.S .H rt O-P P- HI 1— ItHiH-H 1-t a o w o s 0-<5HOSIM COCOCO- rt a M Pm fa r- os co «5 g n 4J O CS CM CM CM OOOO CM fc CM CO .HO co co co 00 ^f( tf1 ^tl V CO 1»l o oooo Eh ■ ^ . s ftilK < COCO B u 1 * ^i 2 1 PHOSPHORUS METABOLISM 469 I. Kaup (1902) concluded that there was no increased protein metabolism because of exertion. There were no food phosphorus figures. The author states that moderate exercise even resulted in phosphorus retention. Maillard (1908a, 1908b, 1909) studied urinary phosphorus elimination by ten soldiers during 6 days, on an ordinary mixed diet, the object being to determine the effects of muscular exercise and the use of wine. No effects of the use of wine could be noted, on the nitrogen and phosphorus elimination; but the exercise, which was arranged to increase in severity from day to day, in two three- day experiments, caused an undoubted increase in phosphorus elimination in the urine. The total acidity of the urine increased, as well as the undetermined nitrogen, and also to a slight extent the uric acid; while the urea decreased a little, and the ammonia nitrogen, purin bases, nitrogen precipitable by silico-tungstic acid, and total nitrogen were unchanged. The following figures show the effects of exercise on phosphorus elimination in the urine. Exercise 1st d ay- Light 2nd day- Medium 3rd day Severe 4th day Light 5th day Medium 6th day Severe P2O5 P:N 1.76 1:48.4 1.72 43.3 2.54 33.1 1.91 44.4 2.25 35.4 2.52 33.4 Scaffidi (1910-11) studied purin metabolism as affected by the fatigue of mountain climbing. A portion of his phosphorus figures follow : DAILY PHOSPHORUS BALANCES AS AFFECTED BY FATIGUE OF CLIMBING HIGH MOUNTAINS— Grams Intake Urine Feces Urine and feces Balance Balance Day P2O5 P2O5 P2O5 P2O5 P2O5 N 1 3.634 2.29 0.58 2.87 +0.764 +2.45 2 ' ' 2.42 0.37 2.79 +0.844 +3.149 3 Fatigue 2.712 0.96 3.67 -0.036 -2.001 4 2.938 0.48 3.41 +0.224 +0.544 +2.039 5 2.75 0.34 3.09 +1.089 6 2.54 1 2.70 1.900 0.48 2.38 +0.32 +2.055 2 * * 1.593 0.34 1.93 +0.77 +4.525 +2.190 3 Fatigue 2.363 0.919 0.672 1.839 +0.40 4 2.70 1.995 1.37 2.93 -0.23 +3.155 5 1.870 0.725 2.31 +0.39 +3.915 1 3.237 1.89 0.78 2.67 +0.567 H 1-2.019 2 ' ' 1.84 0.24 2.08 +1.157 - -5.069 3 2.62 0.25 2.87 +0.367 +0.159 - -3.239 4 Fatigue 2.328 0.75 3.078 -4.439 5 2.88. 1.25 4.23 -0.993 -3.489 1 6 2.14 0.64 2.78 +0.457 !-3.219 470 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Scaffidi concluded that during muscular activity there is no increase of uric acid elimination, though there is a slight increase of purin base elimination ; also as follows : Immediately after ending the muscular work, there is a more or less pronounced, but always quickly developing and very evident increase in the amount of uric acid eliminated. During the same time the purin base elimination falls. The increase of uric acid elimination is more pronounced if the work, and especially the rest following, is not taken at too high an altitude. The total purin nitrogen always rises on the working day ; often this increase continues also for two days following the work. This increase is due almost entirely to the increased formation of uric acid, which, as may be concluded from the phosphorus content, comes also from purin bases which result from a tearing down of nucleoproteids. The elimination of phosphorus rises considerably above the normal mean on the working days and on the following days. The total elimination of nitrogen is not much changed from that of the resting days, if one leaves out of account the cases when the work undertaken was too great, because of lack of previous training, or because it was carried to excessive duration or intensity. The ammonia content of the urine, and still more the acidity, rise in consequence of labor. See also Hammond (1863) and Lepine and Jacquin (1879). Summary. Ordinary activity does not cause appreciable increase in phosphorus outgo, nor does more vigorous exercise of a man in training. Severe exercise, however, carried to the point of tissue destruction, and this is by no means a rare condition in the lives of many people, may cause marked increase in the outgo of phosphorus, uric acid and other tissue katabolites. The principal part of the increased phosphorus outgo occurs after the termination of the exercise, and the supernormal outgo may persist during several days. The phosphorus outgo is not increased by sweating or massage. The increased phosphorus outgo after severe exertion is with- out doubt due to tissue destruction, the muscles probably contrib- uting at least the principal part. PHOSPHORUS METABOLISM DURING PREGNANCY Hugounenq (1899a) studied the retention of minerals by the human fetus. The retention of minerals by the fetus is slight at first, but very active at the end. At birth the infant contains about 100 gm. of salts. During the last three months of gestation the fetus acquires twice as much mineral matter as previously. PHOSPHORUS METABOLISM 471 Ver Eecke (1900) studied metabolism as affected by pregnancy in rabbits. There was in each of 16 cases a very marked decrease in phosphorus elimination during- the last week of pregnancy, to a figure usually below that for the period of sexual inactivity, or for the first week of pregnancy. Between the first and last weeks of pregnancy, that is, during the gestation period, the phosphorus excretion was, in twelve cases out of 16, higher than during either the first or the last week. The author connects the decrease in phosphorus outgo with increase in activity of the mammary glands. Jagerroos (1902), studying nitrogen metabolism in pregnancy, with dogs, reaches the conclusion that in this condition there is a marked tendency toward parallelism in nitrogen and phosphorus metabolism, as is natural, considering the fact that the pregnant female is in a sense a growing animal, and also that there is usually, in the first half of the pregnancy, a period of increased protein decomposition. Michel (1899) shows the increasing demand of the developing human fetus for phosphorus. The figures are to be found on p. 110. Schkarin (1910) fed female dogs on various diets, vegetable and animal, and determined the total phosphorus of the pups at birth, and after suckling for varying lengths of time. The effects of the maternal diet on the composition of the unborn young were not pronounced. Hoffstrom (1910) studied metabolism of a woman during preg- nancy, the subject receiving an ordinary mixed diet. Among the author's conclusions are the following: 1. The phosphorus excreted through the urine was less during the second half of the period than the first, and during this time was comparatively constant in amount. 2. The organism exhibits a greater tendency to hold back nitrogen than phosphorus. 3. The average phosphorus storage was 0.331 gm. daily, and the cumulative storage of phosphorus reached 56 gm. at the end of the experiment. 4. Of this amount 18 gm. were deflected to the fetus, and consequently 38 gm. were retained by the mother organism itself. This was calculated according to figures of Michel for phosphorus content of the fetus at different stages of development. Numerical data from this work are contained in the following table : 472 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 DAILY PHOSPHORUS METABOLISM DURING PREGNANCY Grama Week of Total Cumula- Ratio Ratio Ratio preg- Intake Urine Feces outg-o Balance tive N:P N:P N:P nancy P P P P P retention P Intake Urine Feces 17 2.060 0.955 1.033 1.988 +0.072 0.504 5.8 10.8 0.9 18 2.300 0.886 0.823 1.709 +0.591 4.641 6.5 12.7 1.1 19 2.862 1.178 0.679 1.857 +1.005 11.676 6.1 11.6 1.2 20 2.471 1.099 1.195 2.294, +0.177 12.915 6.4 11.7 1.0 21 2.304 0.991 1.000 1.991 +0.313 15.106 6.5 12.4 1.1 22 2.223 1.006 0.735 1.741 +0.482 18.480 6.5 11.1 1.6 23 2.231 1.067 0.781 1.848 +0.383 21.161 7.1 10.7 1.6 24 1.482 0.774 0.390 1.164 +0.318 23.387 7.0 12.7 1.4 25 1.927 0.851 0.854 1.705 +0.222" 24.941 7.1 13.4 1.0 26 1.835 0.653 0.784 1.437 +0.398 27.727 7.0 15.4 1.1 27 1.569 0.763 0.512 1.275 +0.294 29.785 7.0 11.8 1.2 28 1.458 0.854 0.536 1.390 +0.068 30.261 7.3 11.7 1.1 29 1.998 0.907 0.652 1.559 +0.439 33.334 6.9 11.4 1.3 30 1.754 0.935 0.694 1.629 +0.125 34.209 7.3 10.4 1.3 31 2.136 0.966 0.779 1.745 +0. 391 36.946 6.5 9.8 1.2 32 1.892 0.895 0.620 1.515 +0.377 39.585 7.1 10.8 1.2 33 1.834 0.903 0.677 1.580 +0.254 41.363 6.2 10.5 1.3 34 1.909 0.845 0.574 1.419 +0.490 44.793 6.3 9.8 1.5 35 1.994 0.898 0.523 1.421 +0.573 48.804 6.0 9.2 1.5 36 1.670 0.919 0.623 1.542 +0.128 49.700 6.7 9.7 1.6 37 1.957 0.962 0.607 1.569 +0.388 52.416 6.1 9.2 1.5 38 1.972 0.936 0.635 1.571 +0.401 55.223 6.4 9.6 1.9 39 1.872 0.947 0.710 1.657 +0.215 56.728 7.2 11.5 1.7 40 1.149 0.832 0.467 1.299 -0.150 55.828 6.2 9.3 1.3 Mean 1.952 0.918 0.703 1.621 +0.331 6.6 11.1 1.3 See also Mosler (1853). The extent of the general anabolic excess during pregnancy is indicated by the work of E. Landsberg (1912), who found in human pregnancy a retention of nitrogen four times as great, and of sulphur and phosphorus twice as great, as that required for the fetus. E. and J. Hermann (1912) reported a study of the lipoid content of the blood of normal and pregnant women, and of new-born infants. The phosphatid content is found about the same for the three bloods, though the proportion of the total fat varies, because both cholesterin esters and neutral fat are high in the blood of the pregnant woman, and low in that of the infant. SLEEP Laehr (1890) studied metabolism as affected by sleep, with himself as the subject. He concluded that the calcium, magnesium and phosphorus outgo were not influenced by sleep. Breisacher (1891) determined phosphorus and nitrogen in the urine in three 8-hour periods per day, during ten consecutive days, in a study of the effects of sleep on metabolism. No figures were given on the composition of the food. Averages of the daily deter- minations are as follows : RELATION OF P 2 0; TO N IN URINE 12-8 A. M P.03:N=1:5.29 8 A. M.-4 P. M P 2 5 :N=1:7.46 4 P. M.-12 P 2 5 :N=1:6.93 PHOSPHORUS METABOLISM 473 The subject slept during the period from 12 P. M. to 8 A. M. The author does not come to a definite conclusion as to the reason for the low proportion of nitrogen to phosphorus in the urine formed during sleep. A more rapid elimination of food nitrogen than of food phosphorus would produce such variation in the rate of excre- tion as the author noted. Sherman (1902) investigated metabolism as affected by loss of sleep (see table, p. 432), on three successive nights. Increased elimination did not occur until the third day, while changes resulting from alteration of the diet were always perceptible on the first day. There was slight increase in the outgo of nitrogen, sulphur and phosphorus, the proportions not being markedly abnormal. PHOSPHORUS METABOLISM AS AFFECTED BY THIRST AND WATER DRINKING Landauer (1894) states that deprivation of water increases both nitrogen and phosphorus outgo, but that after reaching a cer- tain limit the phosphorus elimination returns to the normal. Straub (1899) investigated metabolism during thirst in dogs. The phosphorus elimination in the urine during periods of 3-4 days without water intake was undoubtedly increased, though only to a slight extent, about 5-10 percent. In connection with his thorough investigation of the effects of water-drinking on metabolism Hawk (1905) has studied phosphorus metabolism, with results as indicated below. DAILY PHOSPHORUS BALANCES WITH NORMAL MATURE MEN ON A STANDARD DIET WITH VARYING AMOUNTS OF WATER— Grams Subject Length of Initial Gain and period body Food Urine Feces or Diet period in days weight Kilos P2O5 P2O5 P2O5 loss P2O5 1-1 2 61.51 5.226 2.340 2.39 +0.496 Milk 1800 g-m., butter 75 firm. 330 g-m., water 500 c. c crackers 1-2 2 5.226 2.650 2.36 +0.216 Same as above plus 4500 c c. water 1-3 4 5.226 2.310 2.33 +0.588 Same as No. 1 2-1 1 4.960 2.509 Milk 1650 g-m., butter 60 g-m., 300 gm., water 900 c. c. crackers 2-2 2.754 Standard, same as above 2-3 " 2.406 Standard 2-4 " 2.568 Standard + 3100 c C. water 2-5 " 2.653 Standard + 3100 c c water 2-6 ** 1.840 Standard 3-1 " 2.120 " 3-2 " 2.275 '* 3-3 " 2.330 " 3-4 " 2.479 Standard + 3100 c c. water 3-5 " 2.700 ■ C • < <• •( >• 3-6 ** 2.167 Standard 4-1 2 62.8 •* 2.925 1.89 +0.145 *' 4-2 2 " 3.227 2.20 -0.462 Standard + 3100 c. c. water 5-1 2 61.4 " 2.766 1.92 +0.279 Standard 5-2 ' 2 " 2.943 2.02 -0.003 Standard + 3100 c. c. water 5-3 2 2.823 1.93 +0.207 Standard 474 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 From this work the author concludes that copious water drink- ing increases the excretion of phosphorus in the urine, this increase being due to increased cellular activity, and the accompanying katab- olism of phosphorus-containing bodies. In every instance the excretion of P 2 5 was increased above the normal on each day of the water period, the maximum excretion occurring on the second day of the increased ingestion. From the evidence at hand, then, one must conclude that either great thirst or unusual consumption of water will increase the phos- phorus excretion, though further work is required for the positive determination of the source of this phosphorus and, therefore, the significance of this increased outgo. See also Breed (1851a). EFFECTS OF TIME OF DAY ON PHOSPHORUS METABOLISM This is one of those subjects which seems readily explainable on the basis of a few results, but puzzling after the accumulation of an abundance of evidence. A number of extended studies of the question have been made, but there is no such unanimity of findings, or prospect of useful conclusions, as inclines one to attempt to harmonize and summarize the results. From the papers of W. Kaup (1856b), Haxthausen (1860), Pettenkofer and Voit (1866), Edlefsen (1878, 1881), Speck (1882), Breisacher (1891), Roeske (1897), Vogt (1906) and Sarvonat and Gentz (1911) we conclude that this is a matter which is much influenced by personal idiosyncrasy, environment, and various condi- tions of the general method of life, such for instance as the time of the chief meal of the day (W. Zuelzer, 1876). We shall mention in detail only two of the later papers. Sherman and Hawk (1900) studied the urinary elimination by human beings of nitrogen, sulphur and phosphorus as affected by the time of day. As measured by 3-hour periods, the rates of elimination of nitrogen and sulphur run closely parallel, and normal- ly show a tendency to rise during the morning, reaching a maximum after the midday meal, with a slight fall in the following period, and another rise after the evening meal. During the night the excre- tion usually reaches the minimum. The excretion of phosphorus describes a curve altogether differ- ent from that of nitrogen and sulphates, rising steadily from the middle of the morning until the time of retiring, then falling during the hours of sleep, and continuing to fall for three hours after rising, reaching a minimum after breakfast. PHOSPHORUS METABOLISM 475 After the ingestion of a considerable amount (63.7 gm.) of extra protein .in lean beef with the breakfast, the nitrogen began to rise in the first three hours, the sulphur and phosphorus a little later. The nitrogen excretion regained the normal in 36-39 hours, the sulphur a little earlier, and the phosphorus about 12-15 hours after the ingestion of the beef. Hawk and Chamberlain (1904) also studied this question with results as below. VARIATION IN THE COURSE OF PHOSPHATE EXCRETION IN THE URINE AS AFFECTED BY TIME OF DAY AND INCREASED INGESTION OF PROTEIN Averages of Results from Two Normal Men — Grams July July July July July July " July July July Period 16 17 18 19 20 21 22 23 24 1 6:30 a. m.— 9:30 a. m. 0.076 0.086 0.137 0.127 0.191 0.165 0.100 •0.194 0.149 2 9:30 a. m— 12:30 p. m. 0.126 0.191 0.254 0.257 0.402 0.317 0.233 0.325 0.276 3 12:30 p. m— 3:30 p. m. 0.302 0.321 0.411 0.422 0.509 0.432 0.383 0.427 0.409 4 3:30 p. m. — 6:30 p. m. 0.229 0.243 0.308 0.330 0.409 0.337 0.383 0.351 0.349 5 6:30 p. m— 9:30 p. m. 0.264 0.329 0.399 0.339 0.411 0.390 0.366 0.431 0.335 6 9:30 p. m.— 6:30 a. m. 0.747 0.882 0.876 0.881 1.098 1.007 0.909 0.850 0.880 7 Total for day 1.745 2.050 2.387 2.358 3.022 2.702 2.375 2.580 2.402 Average initial weight of subjects, 56.14 kg.; diet, crackers 100 gm., butter 20 gm., whole milk 550 gm. per meal; 3 meals per day, except on July 20, when the morning meal was changed to crackers 50 gm., butter 20 gm., milk 450 gm., and lean beef 100 gm. In the excretion of phosphorus in the urine two distinct rises were seen each day. The maximum rate was reached after the midday meal. The rate of excretion reached the minimum during the first period in the morning. The maximum rate of phosphate excretion, due to the ingestion of meat in the morning meal, fell in a period between those in which the maxima of nitrogen and sulphate occurred, that is, after the nitrogen maximum and before the sulphate maximum. With one subject the normal rate of nitrogen and phosphate excretion was regained on the second day following the increased proteid ingestion ; the normal rate of sulphate excretion was regained one day later, i. e., three days after the inges- tion. With the other subjects the normal rate of excretion was not regained in any case until the fourth day following the increase in proteid food. 476 OHIO EXPERIMENT STATION: TECHNICAL BUL. 6 PART V PHOSPHORUS METABOLISM IN DISEASE ACID INTOXICATION The phenomenon of acidosis is a general condition rather than a specific disease, and is a symptom in many disorders of nutrition. The immediate cause is a disturbance of the phosphate-carbonate balance in the blood which constitutes its central agency for the maintenance of that approximate neutrality which is necessary to the function of respiration. Having to do especially with compounds other than those of phosphorus, we shall but briefly consider this important matter, and for a fuller discussion the reader is referred to vonNoorden's Metab- olism and Practical Medicine. The general subject of acid intoxication was opened up by the experiments on acid poisoning with animals by F. Walter (1877). Acidosis as relating to human nutrition, however, has to do partic- ularly with acid products of disordered metabolism, rather than with acid poisons introduced from without, and this field of learning was developed after Stadelman (1883) discovered /?-oxybutyric acid, and demonstrated the fact that diabetic coma is at least largely due to this compound. In general, excess of acid in the system may be brought about in two ways, first by actual excess of acid products, and second by such a deficiency of bases as leaves the organism subject to disturbance by a relative excess of the normal acid products of katabolism, the one being in effect quite as truly an acid excess as the other. The acids of the body which may enter into the disturbance of its approximate neutrality are of diverse origin. They may be (1) inorganic acids from outside the body, (2) unoxidized organic products of acid reaction, (3) acid salts, and acid ions of dissociated neutral salts, and (4) sulphuric and phosphoric acids resulting from protein katabolism ; while relative excess of acids may come about (1) through abnormally low intake of alkalis and alkaline earths, (2) through excessive outgo of these same elements, and (3) through deficiency of intake in mineral matter generally, for even a neutral salt may serve for acid neutralization through its anion entering into relations which mask its potential acidity, as for instance with proteins, thus freeing its cation. PHOSPHORUS METABOLISM 477 The acids actually involved in the production of acidosis as we know it in practice are principally /3-oxybutyric and related com- pounds, in actual acid excess, and the sulphuric and phosphoric acids of normal katabolism, in relative excess of acids. The reasons are somewhat obscure for the failure of the body, in acidosis, to oxidize /?-oxybutyric acid in the normal manner, and for its consequent appearance along with the derived diacetic acid and acetone in the urine. They appear, however, to be either deficiency of carbohydrates, as in starvation and fever, or defective powers of oxidation of carbohydrates, as in diabetes ; the coincident oxidation of carbohydrates apparently being necessary to the normal oxidation of /3-oxybutyric as well as other fatty acids. Actual acidity of the blood in the living animal, however, is impossible. These acids do not circulate free, but are neutralized for the protection of the organism. Normally the alkalis used for the neutralization of the acid products of katabolism are furnished principally (1) by the oxidation of organic salts of the fixed alkalis and alkaline earths in the food (especially among herbivora) , (2) by the separation of acid urine from alkaline blood, (3) by metabolic ammonia deflected from urea formation (especially among omnivora and carnivora) , (4) also to a less extent by carbonates and ammonia in the food, and (5) by ammonia split off from proteins in the alimentary tract by bacteria. The automatically variable agency for the compensation of varying amounts of acid in the system is the above-mentioned deflec- tion, for acid neutralization, of a part of the ammonia normally contributed by the tissues to the blood as carbamate, and synthe- sized to urea in the liver, the acids thus forming ammonium salts, and appearing as such in the urine. The amount of ammonia thus rendered available for acid neutralization is quite considerable (omnivora and carnivora) , but at the same time it is not unlimited, and the protection afforded is not perfect, even as far as it goes, since the ammonium salts themselves become somewhat toxic through ionization (Wilbur, 1904) . Our usual measure of acid excess in the system, then, however produced, and of whatever nature, is the ammonia of the urine (that is, total ammonia — free NH 3 and NH 3 in salts), though an actual estimation of the disturbing compounds in the blood, as well as the estimation of the inorganic elements of the excreta, afford us supple- mentary information. 478 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In brief, the effects of acidosis are dyspnoea, and attendant nervous symptoms, terminating in stupor and collapse, the causes being the expulsion of carbon dioxide from the blood by stronger acids, its accumulation in the tissues, and the resultant interference with their oxygenation. Herbivora and carnivora differ greatly, as Walter showed, as to their resourcefulness in adjusting themselves to excess of acids. In herbivora the food normally furnishes a superabundance of alkali, and therefore these animals have neither need nor capacity for the extensive use of ammonia in acid-neutralization which is character- istic of flesh-eating animals. Acid poisoning, therefore, causes a much greater increase in the excretion of fixed bases by herbivora than by omnivora and carnivora. So far as known the prevalence of acidosis as differentiated from calcium starvation in domestic animals has not yet been demonstrated. In human beings acidosis is well known in a great variety of diseases and conditions, especially in diabetes, gastrointestinal dis- orders in children, fever, starvation, pregnancy, burns and anaes- thesia. Taylor (1904) made a study of human metabolism on an ash- free diet. Metabolism was disturbed apparently by sulphuric and phosphoric acids of metabolic origin. VonNoorden (1907) states that the blood, muscles and glands give up no appreciable amounts of alkali in diabetic acidosis, but that there is, at the same time, a loss from the body, apparently from the bones, of calcium, magnesium and phosphorus. By subse- quent work of many other investigators these facts are well estab- lished. In gastrointestinal disorders of children there is an acidosis which was explained by the work of Keller, Steinitz and Freund. Keller (1897) found an increased elimination of ammonia in the urine always accompanying an increase in the fat of the food. Stein- itz (1903) showed that this increased ammonia excretion was due to .an acidosis caused by a withdrawal of alkali from the body in soaps formed from fats in the intestine, and excreted in the feces. W. Freund's (1905) observations make it apparent that the increased soaps in the feces are calcium soaps, a part of the calcium normally excreted as calcium phosphate uniting with the fatty acids to form soaps, leaving an equivalent amount of the phosphorus to unite with alkalis and ammonia, the urinary excretion of which is thereby increased. PHOSPHORUS METABOLISM 479 VonJaksch (1885) first showed that the acidosis of fever is of the acetone variety. The acetone bodies, /?-oxybutyric acid, diacetic acid and acetone, are much less abundant in fever than in diabetes, and also less abundant than in complete inanition. In general, considering the causes and degrees of acidosis in fevers, the quantitative excretion of the acetone bodies is controlled much more directly by inanition, fat destruction, the seat of the infection, and the individuality of the patient than by the temper- ature, or by the severity of the infection. As bearing on the effects of experimentally produced dyspnoea on phosphorus elimination, Saccone (1907) found that restriction of respiration in a dog, by the application of a Sayre corset, caused an increase in urinary phosphorus which disappeared after two days. We may consider, then, that the connection of acid intoxication with phosphorus metabolism is through the participation of phos- phates in the maintenance of neutrality ; through the contribution of phosphoric acid to the total acids of the body ; through the interfer- ence of abnormal amounts of acid with synthesis and retention of phosphorus compounds, especially in the bones; and otherwise in still more general ways. A few references to articles which we have consulted on acid intoxication are the following: Allard (1907) ; Ewing (1908) ; Folin (1907) ; Nichols (1908) ; Rossi, F., : Inner Respiration of Tissues and Its Relation to Death from Hunger and to Acidosis (Bui. Sci. Med., 81, 149-54) ; Szili (1906) ; Talbot (1907) and Fitz, Alsberg and Henderson (1907). ACROMEGALY That this disease is due to an excess of function of the hypoph- ysis, or pituitary body, is the prevailing opinion, and one of the more satisfactory investigations in support of this hypothesis is the work of Lewis (Johns Hopkins Hosp. Bui. 16 (1905), 157.), who found, by histological demonstration, hyperplasia of the chromophile cells of the anterior lobe, and other conditions indicative of glandular activity. Schiff (1897a, 1897b) used hypophysis and thyroid preparations in metabolism studies with acromegalics. The nitrogen and phos- phorus of the diets were estimated by calculation, but in the urine and feces were determined by analysis. He found that both the hypophysis and thyroid preparations react strongly on the sluggish 480 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 metabolism of acromegalics, producing diuresis, and increased nitro- gen and phosphorus excretion, with protracted after-effects, and loss of weight. Tauszk and Vas (1899) noted retention of phosphorus and loss of calcium in acromegaly, and negative results from the use of pituitary and thyroid tablets. VonMoraczewski (1901) conducted metabolism studies in acro- megaly, with analyses of food, urine and feces, and with blood exam- ination. Hypophysis and thyroid tablets produced no change in the phosphorus retention, which was considerable throughout this experiment. In a second experiment treatment with yohimbine, elemental phosphorus, silver nitrate and oxygen all lessened the retention, or caused a loss of phosphorus. Oswald (1902), experimenting with a dog, found no definite peculiarities of metabolism following ingestion of powdered pituitary body in quantities of 1-2 gm. Edsall and Miller (1903) conducted a well-controlled metabo- lism study of acromegaly. The percentage of retention of phos- phorus was 20.88, nitrogen 12.86, and calcium 9.32 in a 7-day period. The phosphorus retention was over a gram a day. The relation of stored calcium and phosphorus shows that the latter could not have been used to any considerable extent in the growth of bone. The subject was an imbecile. Malcolm (1904) studied metabolism in the dog as affected by the ingestion of pituitary gland substance. The glandular and "nervous" portions of the gland were fed separately in different periods, and in others the whole gland. The calcium and phosphorus balances led the author to the conclusion that the nervous portion is probably the more active and has the effect to cause the katabolism of bone tissue. Franchini (1904) published results of an experiment in the feeding of hypophysis tablets to a case of acromegaly. This treat- ment produced nothing decisive in results on phosphorus metabo- lism. In a later study (1908b) Franchini published blood analyses from three cases of acromegaly. The data, however, are insufficient to warrant the drawing of conclusions. Still later (1910) Franchini published metabolism and blood data on rabbits and guinea pigs under injection treatment with hypophysis extracts prepared from cattle and horses. These extracts caused a marked loss of calcium and magnesium and a less marked loss of phosphorus. In the circu- lating blood was found an increase of calcium and magnesium. PHOSPHORUS METABOLISM 481 Thompson and Johnston (1905) made metabolism studies on dogs under pituitary treatment by ingestion per os. The feces were not analyzed for phosphorus. Pituitary substance appears to stim- ulate metabolism in the dog, as shown by increased outgo of nitrogen (urine and feces), of urea, and to a less degree of urinary phos- phates, effects which do not pass away immediately when the feed- ing of pituitary body is discontinued. Diesing (1909) expresses the opinion that the hypophysis gland controls phosphorus metabolism, and that its function is physiologic- ally opposite to that of the thyroid in such sense that when the hypophysis stores too much phosphorus, too little connective tissue is formed, leading to myxoedema, or in younger individuals to cretinism, having the same effect as atrophy of the thyroid. When the hypophysis is below normal it causes an excessive growth of bone and connective tissue. The disease acromegaly is the result. Medigreceanu and Kristeller (1911) injected sterile extract of anterior lobes of hypophysis into an acromegaly patient on two days during a 20-day period of observation. Previous to the first injec- tion the patient was in phosphorus equilibrium. The injection caused a marked loss of phosphorus. Between the first and second injections the organism showed a decided tendency to reestablish equilibrium, but on the second injection a marked loss of phospho- rus was again produced. See also Delille, (1909). Mochi (1909) studied elimination of nitrogen, calcium, magne- sium and phosphorus in the urine of 8 starving rabbits, of which 6 were treated by injection with extract of fresh lamb hypophysis, one with extract of nerve substance, and one, the control, left without treatment. The author found that, as compared with the control, the animals treated with extract of hypophysis died sooner than the others, but lost less in weight; further, the relation between the nitrogen and phosphorus of the urine showed such an excess of the latter as to indicate destruction of bone tissue. The relation of nitrogen to P 2 5 in the urine was as follows : Control, 6.5:1 ; treated with hypophysis, 4.6, 4.3, 4.5, 5.9, 5.3 and 4.5 : 1, and treated with extract of nerve substance 8.1:1. (Bibliography of 26 refer- ences.) Mochi (1910) reported further experiments with extract of pituitary body injected into fasting guinea pigs. In complete balance experiments he learned that the increased loss of phosphorus under the pituitary treatment is without doubt due to tearing down of bone tissue. A part of the numerical results are as follows : 482 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE DAILY LOSSES OF NITROGEN, PHOSPHORUS AND CALCIUM BY GUINEA PIGS TREATED WITH EXTRACT OF HYPOPHYSIS Grams No, of Treatment Duration of experiment in days N P Ca Partition of loss of phosphorus animal From muscle From hone I n in IV Fasting 1 only Fasting and injection fC cc «< 18 17 16 15 1.6436 0.3718 0.4376 1,0127 0.0934 0.1516 0.1069 0.1050 0.0943 0.2776 0.1571 0.1154 0.0183 0.0215 0.0498 0.1334 0.0854 0.0552 Rubinraut (1912) studied metabolism in acromegaly, and deter- mined that hypophysis ingestion increased calcium and phosphorus elimination, and caused marked loss of these elements, while thyroid therapeutics decreased the rate of elimination of calcium and phos- phorus in proportion to the amount of thyroid preparation ingested,, the balances becoming positive. Potassium iodide resembled hypoph- ysis in its effects, increasing to a considerable extent both calcium and phosphorus outgo. Aschner (1912) has made a recent and extensive study of the function of the hypophysis. By extirpation experiments with dogs he obtained results varying in intensity inversely as the age of the animal. In very young dogs the removal of the hypophysis had the effect to retard general development in marked and characteris- tic ways, affecting the size, hair, claws, teeth, bones, sexual develop- ment, etc. These effects were produced by removal of the whole gland, or the frontal lobe, not by removal of the posterior lobe. Aschner notes the contradictory evidence on results of hypoph- ysis administration, and cites a case of his own, a hypoplastic dwarf in whom there was supposed to be under-functioning of the hypophysis, who under hypophysis treatment increased in height 7 cm. in 5 months. Aschner cites the work of Benda, M. Sternberg, Erdheim, B. Fischer, v. Frankl-Hochwart, Marburg and others in support of his belief that acromegaly is caused by over-functioning or disordered functioning of the hypophysis, a part of the symptoms being due to its internal secretion, and a part to its local action on surrounding parts of the brain. The true dwarf condition appears to be due, at least largely, to hypo-functioning of the hypophysis, while giantism is rather definitely associated with an excessive functioning of this gland. The connection, therefore, of this matter with phosphorus metabolism is general in character, but with an especial bearing on the growth of the bones. PHOSPHORUS METABOLISM 483 ALCOHOLISM Romeyn (1887) showed that the ingestion of 35-50 c.c. of alcohol by fasting men temporarily decreased urinary phosphorus elimination, an effect which was followed in a few hours, first by an increased elimination, and then soon by a decrease toward the normal for the fasting subject. The increased nitrogen elimination was much less marked. Saccone (1907) found that alcoholic intoxication reduced the urinary phosphorus in dogs, due perhaps to retarded metabolism. Salant and Hinkel (1910), experimenting with 4 dogs, found that subacute alcohol intoxication in well-fed dogs causes a moder- ate diminution of total nitrogen and total sulphur, and a much greater diminution of total and inorganic sulphates and of phos- phates in the urine. There is also a tendency to retention of chlor- ides. Schittenhelm (1909a) notes that both Pollak and Bloch recog- nize the fact that in alcoholism, as in gout, there is characteristic delay and diminution of uric acid elimination, and that Landau in extensive studies has come to the conclusion that the elimination of endogenous purins is increased, but that exogenous uric acid is decreased by the loss of ability of the kidneys to let uric acid pass, a phenomenon as yet unexplained. Schittenhelm conducted experiments with dogs in an effort to clear up this point. Since dogs excrete their purins mostly as allan- toin, while men excrete the same as uric acid, it was thought that if there were disturbance in ferment action, which results in slowing up excretion, it would probably show as a change in the end products; and if on the other hand there is a specific injury to the kidney filtration of uric acid, it would hardly hold to the same extent for the more easily soluble allantoin. Chronic alcoholism was established by feeding meat scrap treated with ethyl and amyl alcohol; and daily urinary nitrogen studies were made both during feeding and fast. As in alcohol experi- ments with men there was delayed purin elimination, as shown, in this case, by the allantoin. The dog had no appearance of nephri- tis, and microscopic examination of the kidneys showed no evidence of disease, from which it would appear that there was delayed purin metabolism. Baskoff (1909b) found that alcohol poisoning reduced the leci- thin content of the liver of the dog. Choumova-Sieber (1910; same as Sieber, 1909a) studied the effects of alcoholism on the organism by the administration of daily doses of alcohol to three dogs during several months. One or two 484 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 months after the last dose they were killed, and the organs analyzed for phosphatids. Compared with three control animals they showed a decrease in the phosphatid content of all the organs examined except the kidneys: brain 11.41 percent, mucosa of stomach, 4.15 percent, intestinal mucous membrane 3.22 percent, spleen 3.18 per- cent, liver 2.74 percent, heart 2.52 percent, lungs 1.34 percent, intes- tinal wall 1.10 percent, and others. The percentage of decrease was reckoned, not on the phosphatid content of the organs, but on the total dry matter of these parts, and hence the losses noted must be considered as astonishingly large. Examination of the individ- ual records in both groups shows that in almost all cases each of the controls exceeds all of the experimental subjects in the phosphatid content of the organs. This article contains a bibliography of 56 references. PHOSPHORUS METABOLISM IN ARTHRITIS AND CHRONIC RHEUMATISM Stokvis (1876), studying phosphorus excretion in arthritis, finds that the urinary phosphorus combined with calcium and mag- nesium is considerably reduced during the whole of the attack, and that even after the ingestion of lime or magnesia the earthy phos- phates remain much reduced in the urine, while in a normal subject increased intake of calcium carbonate or magnesia increases the earthy phosphates of the urine. Godefroy (1903) finds in the urine, in chronic rheumatism, sub- normal amounts both of phosphates and total phosphorus, but on the other hand much more than the normal amount of incompletely oxidized phosphorus. Joulie (1904) administered phosphoric acid to rheumatic horses in doses of 10 c.c. of the acid (sp. gr. 1.35) diluted to one liter. The excretion of calcium phosphate by the kidneys is said to have been increased. See also vonNoorden and Belgard (Berliner klin. Wochenschr., 1894). BERIBERI Beriberi is a nutritional disorder especially prevalent among the rice-eating peoples of the world, but is not unknown even in America, where it has been found in certain fishing settlements of Labrador in which the people, during several months of the year, live largely on white bread and tea. The disease is characterized by profound nervous disorganization, and appears to be essentially the same as the multiple polyneuritis of fowls. Volumes have been written to show that beriberi is caused by deficiency of phosphorus in the food ; also great works to show that PHOSPHORUS METABOLISM 485 it is due to poisons of fungous origin. Both of these positions have been generally abandoned, and the problem seems to be at least near to solution, if indeed it is not already solved, on quite another basis. It is, therefore, considered unnecessary to review the earlier litera- ture of the subject. Prevailing opinion as to the dietetic basis for the disease was set forth by a resolution of the Far Eastern Association of Tropical Medicine after a conference at Manila, in March 1910. The resolu- tion was as follows : "Resolved, that in the opinion of this Association sufficient evidence has now been produced in support of the view that beriberi is associated with the continuous consumption of white (polished) rice as the staple article of diet, and the Association accordingly desires to bring this matter to the notice of the various govern- ments concerned." H. Schaumann (1910) has given us an exhaustive treatise on beriberi in relation to the phosphorus of the food, and for a review of the literature up to 1910 the reader is referred to this mono- graph. His position at the time of this publication was that beriberi is a metabolism disease which is due to an insufficient supply of organic phosphorus to the tissues, this being caused, in general, by too small a content of organic phosphorus in the food, and in other cases, apparently much less frequently, by insufficient absorption of organic phosphorus compounds when they are supplied in sufficient quantities in the food. Schaumann considers that scurvy, Barlow's disease, rickets and osteomalacia may have a similar etiology. As indicating some of the details of opinion at that time, and subsequently, we submit the following notes. Fraser and Stanton (1910) conclude that beriberi is caused by white rice, and that the tendency of rice to cause beriberi varies inversely as the total phosphorus. De Haan (1910) coincides with these conclusions, and finds that neither lack of salts nor nucleins in the rice is the cause of beriberi. Highet (1910) agrees in his findings with the above. Shibayama (1910) considered monotony of diet as a predispos- ing factor. He believed the disease to be due to a specific organ- ism. Kilbourne (1910) reported that beriberi varied in prevalence in companies of native Philippine scouts inversely as the phosphorus and potassium of the diet. 486 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Aron (1910a) reported that diets such as cause beriberi contain insufficient phosphorus to maintain health, and that diets similar to those thought to cause beriberi cause polyneuritis in chickens. Experiments with chickens showed that the addition of phytin was of some benefit in this condition. Aron and Hocson (1910) reported the following balance data: AVERAGE DAILY PHOSPHORUS AND NITROGEN BALANCES Periods Four Days Each — Grams Period and subject P2O5 N Food Intake Urine Feces Out- put Bal- ance Bal- ance a 1-normal Polished rice, bread, sugar, dried fish, coffee 1.674 1.138 0.526 1.664 +0.01 +0.38 Rice diet b 1-normal Same 4- 75 gm. rice bran, (less bread and rice; more sugar than above) 5.460 1.378 3.460 4.838 +0.62 +0.57 Phosphorus added as rice bran c 2-normal Polished rice, bread, sugar, coffee, bacon 1.498 1.735 0.733 2.468 -0.97 -4.67 Lower in N and P than above d 2-normal Same + 12 gm. egg albumen, (less sugar, morecoffee than above) 1,878 1.568 0.610 2.178 -0.30 -1.98 Protein added to above e 3-beriberi patient Same as above + 6 gm. phytin, (more coffee than above) 4.913 1.563 1.845 3.408 +1.50 -2.08 Phosphorus, as phytin, , added to above f 3-beriberi patient Rice, bread, sugar, coffee, bacon 1.103 1.553 0.730 2.283 —1.18 -2.29 Diet such as sup- posed to cause beriberi g 3-beriberi patient Same as above but with bacon containing less N and P2O5 1.045 1.240 0.843 2.083 -1.03 -1.94 Diet such as sup- posed to cause beriberi h 3-beriberi patient Same as above + 6 gm. phytin 3.700 1.313 2.885 4.198 -0.50 -1.81 Phosphorus, as phytin, added to above l 3-beriberi patient Same as above but with less bread and coffee 3.638 1.093 3.090 4.183 -0.54 -2.77 Phosphorus, as phytin, added to above i 3-beriberi patient Same as above _ but with more and differ- ent bacon 1.013 0.703 0.655 1.358 —0.35 -1.22 No phosphorus added k 3-beriberi patient Same as above + 12 gm. egg albumen, but with less sugar 1.263 0.840 0.790 1.630 -0.37 —2.09 Protein added 1 3-beriberi patient Rice, bread, sugar, fish 1.908 1.133 0.913 2.045 -0.13 -0.58 Typical Filipino diet; about same as first ration above From this work some of their conclusions are as follows : "A diet consisting of bread and rice (both poor in phosphorus), some fat (bacon) and sugar, furnishing 40 calories, 0.15 gm. N and 0.025 gm. P 2 O r , per kilo body weight does not cover the demands of the body for N and P 2 5 , and therefore leads to N and P 2 5 loss from the body. Addition of protein reduces the N loss of the body and the loss of P 2 5 . PHOSPHORUS METABOLISM 487 "The addition of phosphorus in the form of phytin prevents a loss of that constituent from the body, and if sufficient of this element is added a storage of phosphorus after a period of phospho- rus starvation takes place. The loss of nitrogen from the body is reduced by the addition of phytin, as compared with a corre- sponding period in which phytin is not given. "A diet consisting of fish, bread, rice, sugar, etc., furnishing 37 calories, 0.2 gm. N and 0.032 gm. P 2 5 per kilo of body weight, is sufficient to keep a man in N and P 2 5 equilibrium. "The addition of rice bran has a tendency to produce a slight storage of P 2 5 ; the rice polish in this respect corresponding to phytin. The phosphorus, both of rice and of phytin, is excreted almost entirely in the feces. "It is highly probable that living for an extended period on a one-sided almost exclusively vegetable diet, which is characterized by its poverty in phosphorus and in protein, may result in beriberi. "The process of polishing rice removes a fine skin and the outer layers (bran) ; this rice bran is rich in phosphorus, especially in its organic soluble form (phytin) ; the content of phosphorus of the rice is considerably reduced by the removal of the bran. "Polished rice, poor in phosphorus, may cause beriberi in man if it is the main constituent of the food, but it is harmless if sufficient other nourishment, rich in phosphorus and protein, is taken. The same polished rice causes a polyneuritis in chickens. White bread, a food of similar chemical composition as regards phosphorus and protein, cannot sustain monkeys in normal health if it forms the entire diet. "The addition of phytin (the organic phosphorus compound of rice bran) considerably reduces the deleterious effects of white rice on chickens." In connection with their study of rice as the cause of beriberi Aron and Hocson (1911a, 1911b) report the following balance data (see next page) from experiments with healthy men. The subjects subsisted on diets composed largely of rice of different conditions as to milling. The husked rice contained 0.7- 0.8 percent P 2 5 , undermilled rice 0.45-0.60 percent P 2 5 , and over- milled rice 0.15-0.35 percent P 2 5 . In addition to rice the subjects received bread, fish, sugar, bacon, coffee, etc. The results show that an intake of less than 1.65 gm. P 2 5 per 50 kg. of body weight (0.033 gm. P 2 5 per kg.) is insufficient to cover the demands of the body for phosphorus. The only cases, Nos. 3, 6, and 11, in which 488 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 the balance was positive were those which received unpolished rice, rice bran or phytin. Experiments Nos. 3 and 4 show that the body loses phosphorus on a diet of white rice, and that when this is replaced by red rice, the amount of phosphorus exceeds the demand of the body. When the phosphorus intake was high the excess was excreted almost entirely in the feces. AVERAGE DAILY PHOSPHORUS METABOLISM WITH HUMAN SUBJECTS ON DIETS COMPOSED PRINCIPALLY OF DIFFERENT SORTS OF RICE— Grams No. and length of Subject Body weight Kilos Food P2O5 Outgro P2O5 Balance P2O5 P2O5 metabolism per 50 kg-, body weight experiment Urine Feces Intake Balance 3 4 days Prisoner C 49.0 3.22 1.03 2.03 +0.16 3.30 +0.16 4 4 days .. 48.6 1.42 1.04 0.83 —0.45 1.45 -0.45 5 4 days B 64.0 1.50 1.74 0.73 -0.97 1.15 -0.80 6 4 days " 64.0 4.91 1.56 1.85 +1.50 3.85 +1.20 7 4 days .. 64.0 1.88 1.57 0.61 -0.30 1.45 -0.30 9 6 days D 43.5 1.28 0.80 0.49 -0.01 1.65 -0.01 10 4 days A 52.5 1.57 1.14 0.52 +0.01* 1.60 +0.01* 11 4 days .. 52.5 5.46 1.38 3.46 +0.62 5.20 +0.60 12 3 days E 54.0 1.77 1.23 0.76 -0.22 1.65 -0.20 13 3 days .. 54.0 1.77 1.11 0.73 -0.10 1.65 -0.10 14 3 days G 45.9 ' 1.45 0.95 0.58 -0.08 1.60 -0.10 * To harmonize with other data, should he — 0.09. Jebbink (1910) concluded, from dietetic studies, that deficiency of nuclein phosphorus in the ration is responsible for the production of beriberi. Janin (1910) got sufficient benefit from the administration of organic phosphorus compounds to lead him to believe that deficiency of these in the diet causes beriberi. Kajiura and Rosenheim (1910), as also Eijkmann, found that barley, fed with rice, relieves symptoms of polyneuritis in poultry, and that this improvement is not due to the alcohol-soluble proteid. PHOSPHORUS METABOLISM 489 Fujitani (1910) found that the very thin skin of half-hulled rice has the power to protect from polyneuritis, but although its phosphorus was present mostly as phytin, that phytin itself does not produce the same benefit. Chamberlain, Bloombergh and Kilbourne (1910) found that potassium and phosphorus salts did not cure polyneuritis, and that the idea of deficiency of phosphorus as the cause of beriberi must be abandoned. These authors found polyneuritis curable by a dialyzable substance which could be extracted from rice polish with either cold water or cold alcohol. Teruuchi (1910) found that phosphorus excretion is not very different in beriberi from the same under normal conditions; further, that the protective principle in unhusked rice and in oats, while not injured by 100° heat, is destroyed by heating to 130°-135°. Teruuchi confirmed the conclusions of Eijkmann, Fraser and Stanton that the neuritis-protecting principle in rice hulls is soluble in warm alcohol, and that it is probably not a phosphorus compound. Simpson and Edie (1911) report that an exclusive diet of white- flour bread, with pigeons, produced degenerative changes in the peripheral nerves, and death; while a diet of whole-wheat bread resulted in health and gain in weight. These authors cured this disease by the feeding of yeast. Chamberlain and Vedder (1911) report the multiple polyneu- ritis of poultry and beriberi as essentially the same. They found that extracts of rice polish, and also of white beans, would cure polyneuritis. Chamberlain (1911) also notes the disappearance of beriberi from the native Philippine scouts with a change of diet. Breaudat and Denier (1911) also cured beriberi with rice bran. Fraser and Stanton (1911a, 1911b) note the fact of the low phosphorus content of rice which causes beriberi, and present a gen- eral review of the evidence as to the cause of beriberi. They state that cooking of the unpolished rice under pressure at 120° for two hours destroys that principle which prevents polyneuritis. Attempts to isolate this active principle are described. The protective sub- stance was found to be soluble in 0.3 percent HC1, and in alcohol. Phytin was shown not to be the protective compound. Cooper and Funk (1911) report that exclusive diets of starch, inulin, cane sugar, and dextrine will cause polyneuritis in poultry; that phytin, edestin, casein, or egg-yolk will not cure the same, but that yeast both prevents and cures polyneuritis. Yeast press juice also cures, as does yeast juice after 24 hours hydrolysis with 490 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 20 percent sulphuric acid. Another curative preparation was made from rice polish by solution in alcohol, precipitation by phosphotung- stic acid, and decomposition with baryta. This compound was free from phosphorus, carbohydrates and proteins. Schaumann (1911a) concludes that the protective effect of various preparations such as phytin, yeast-lecithin, pepsin-hydro- chloric acid extract of phaseolus, rice-bran-phosphatid, and purified hydrochloric acid extract of rice bran and yeast, is due to the com- bined presence of organic phosphorus and an "activator," which acts on these compounds in such manner as to render them com- pletely available to the organism. Chamberlain and Vedder (1912) concluded that beriberi was not due to phosphorus shortage nor to acid intoxication. Funk (1912a) found that polyneuritis of birds causes a decreased nitrogen and phosphorus content of the brain, and is caused by the lack of a basic alcohol-soluble substance which is necessary to the functioning of the nervous tissues ; that the medul- lary sheath, both within and without the central nervous system, is more or less degenerated, that polyneuritis can be cured by prepara- tions from milk, yeast, and lime juice; that the cure is very rapid, the functioning of the axis cylinder being restored before the regen- eration of the medullary sheath is completed. Edie, Evans, Moore, Simpson and Webster (1912) isolated from yeast an anti-neuritic basic compound which they call "torulin," and with which they plan to conduct experiments relating to beriberi. The approximate percentage composition was C 40.5, H 8.07, N 13.32, O 38.11. Three possible structural formulae are suggested as follows: C 7 H 17 N 2 5 C 7 H 16 N0 2 (HN0 3 ) N(CH 3 ) 3 C 4 H 7 2 (HN0 3 ) Suzuki, Shamimura and Odake (1912) have isolated a basic compound from rice bran which they believe to be the active prin- ciple in the prevention of beriberi. It is an organic compound, soluble in alcohol, insoluble in ether, is precipitated by phosphotung- stic acid from solutions acidified by sulphuric acid, and from water solution by tannin. It was isolated in crystalline form as the picrate. This compound they call "orizanine." Of the pure com- pound obtained from the picrate, 5-10 mg. given per os or subcutane- ously suffice to cure a pigeon suffering from rice polyneuritis. Simi- lar results were obtained with chickens, mice and dogs. They regard orizanine as an essential nutrient. Dogs fed on meat and PHOSPHORUS METABOLISM 491 polished rice die in a few weeks with symptoms of starvation. They remain in health if 0.3 gm. of orizanine is added to the daily diet. Judged from their therapeutic action, grains and vegetables in gen- eral contain orizanine. Milk, eggs, fish and meat, and their alco- holic extracts were without effect on pigeons suffering from the polished-rice disease. With dogs, however, the alcoholic extract of meat is practically as effective as orizanine in preventing or curing the disease. Details of the chemistry of the compound are given. Schaumann (1912a) now practically accepts the position and conclusions of Funk and his associates, and expresses his belief, based on experimental observations, that the anti-neuritic extract with which he is working contains a phosphatese, or several such, and suggests that this "activator" serves alike in plant and animal physiology in playing a part in the synthesis of organic phosphorus compounds. Wieland (1912) fed mice on polished rice, and studied the organic and inorganic phosphorus of their bodies with reference to Schaumann's earlier hypothesis connecting beriberi with insuffi- ciency of certain organic phosphorus compounds in the diet. His results did not sustain the theory. MacLean (1912b) found a substance in animal tissues which possesses an anti-neuritic capacity. It is soluble in water and in alcohol. It is not a lipoid, but is precipitated by excess of acetone from both its water and alcohol solutions. It contains phosphorus apparently only as an impurity. This substance is likely to be pres- ent as an impurity in lecithin as ordinarily prepared, and MacLean suggests that to this fact must be due the variable results from the use of lecithin in beriberi. Eijkmann (1911) found that the protective principle in rice hulls is effective after peritoneal injection. Fraser and Stanton (1914) state that a satisfactory measure of the degree of polishing of rice, in relation to the cause of beriberi, is its total phosphorus content. A rice which contains less than 0.4 percent P 2 O g cannot safely be permitted to form the staple article of a diet for man. See also Moszkowski (1911), Vedder and Clark (1912), Vedder (1912), Strong and Crowell (1912), Gouzien (1912), Fargier (1912), Hulshoff-Pol (1912), Heiser (1912), Grijns (1912), Yamigawa, Koyana, Midorikawa and Mogi (1912), Onodera, Nakamura and Tateno (1912) and Gregg (1913). 492 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 THE BLOOD IN RELATION TO PATHOLOGICAL PHOSPHORUS METABOLISM A review of the literature of the diseases of the blood, and of the composition of the blood in disease reveals a considerable measure of uncertainty and disagreement in the fragmentary evidence, and comparatively little which is characteristic in the associated phosphorus metabolism, a condition due in part, at least, to the natural variability in the composition of the blood in harmony with the number and diversity of its general service func- tions. The bearing of the blood diseases on nuclein metabolism as indicated by uric acid excretion has been discussed somewhat recently by H. Strauss in von Noorden's Metabolism and Practical Medicine, to which we refer the reader. We shall, however, review a portion of the literature of other aspects of the subject. PHOSPHORUS METABOLISM IN CHLOROSIS AND ANAEMIA Vannini (1904), in five balance experiments, in a study of chlorosis, found absorption of fats, carbohydrates and proteins normal, and a nitrogen storage in four cases out of five. The obser- vations on the mineral nutrients seem to us neither significant nor characteristic. With calcium, magnesium and phosphorus the positive balances were about as numerous as the negative balances, but the latter slightly exceeded the former in magnitude. VonMoraczewski (1897c) also conducted balance experiments with 3 cases of chlorosis, and 2 of anaemia from carcinoma. In anaemia there was retention of nitrogen, calcium and phosphorus in both of the cases. In chlorosis there was, in each case, nitrogen retention, but the results with calcium and phosphorus varied with the haemoglobin content of the blood. With 25 percent of the normal haemoglobin there was loss of both calcium and phosphorus; with 40 percent haemoglobin there was retention of calcium and loss of phosphorus ; with 60 percent haemoglobin there was retention of both calcium and phosphorus. The addition of sodium chloride and CaHP0 4 to the food caused nitrogen economy. VonMoraczewski concluded that anaemia itself causes no protein decomposition. Stejskal and Erben (1900b) report results of a balance experi- ment on a fatal case of pernicious anaemia. Absorption from the alimentary tract was poor. The salts in the urine were related, one to another, quantitatively, as in the food; and as to calcium, magnesium and phosphorus the patient was practically in equilibri- um. There was a slight storage of nitrogen. The authors stated PHOSPHORUS METABOLISM 493 the opinion that pernicious anaemia is caused by inanition or auto- intoxication following stomach-intestinal atrophy, or to a disturb- ance of function of the intestinal mucous coat. Von Moraczewski (1900b) published results of a study of four cases of pernicious anaemia. He states that the results show, first of all, the impaired power of assimilation of the organism. There is but little loss of nitrogen on nitrogen-poor food, and little storage of nitrogen on adding nitrogen to the food. Oxidation appeared deficient, as in chlorosis, leukaemia and nephritis. The increased calcium outgo indicates katabolism of bone substance. The addi- tion of lime caused a general improvement of symptoms, with decreased nitrogen outgo, and improved oxidation of sulphur com- pounds. The main characteristics of pernicious anaemia he states as follows : (1) Increased lime excretion; (2) the small values of the out- go, especially of nitrogen and phosphorus ; (3) apathy of the organ- ism; (4) lack of accommodation of the outgo to the intake; and (5) usually increased (relatively) chlorine excretion. PHOSPHORUS METABOLISM IN LEUKAEMIA Von Moraczewski (1898b) published balance experiments with one case each of leukaemia and pseudoleukaemia. The following are among his conclusions : Leukaemia is a nitrogen and phosphorus disease ; pseudoleukae- mia is a nitrogen disorder. Almost all constituents of the food were found to be retained abnormally in leukaemia, because of a lack of katabolism, more in respect to phosphorus and nitrogen than to chlorine and calcium. In the case of pseudoleukaemia the nitro- gen and calcium elimination was impaired to a greater extent than that of the chlorine and phosphorus. With the case of leukaemia the inhalation of oxygen caused an increased calcium and phospho- rus outgo. The addition of sodium chlorate and calcium phosphate caused an increase in the nitrogen and phosphorus outgo, and an increase in the red blood corpuscles, with a decrease of leucocytes. The treatment with thyroid tablets was beneficial ; elimination was increased and the balances became normal. The retention of phos- phorus is noted by the author as being contrary to the theory that leucocytosis should cause phosphorus excretion. Milroy and Malcolm (1898) observed that in splenomedullary leucocythaemia the pathological condition seemed to consist in a hindrance to the leucocytic breaking-down, as evidenced by an abso- lute diminution of phosphorus, and a marked relative decrease of 494 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 the same in proportion to the total nitrogen in the urine. The alloxuric nitrogen, as a whole, was, relatively to the total nitrogen, increased in leucocythaemia, both the bases and the uric acid appar- ently participating in this increase. In a later publication Milroy and Malcolm (1899) presented conclusions from a further study of leucocythaemia. In a case of lymphatic leucocythaemia they found a marked diminution in the absolute amount of phosphorus excreted, and also a relative decrease compared to the amount of nitrogen excreted. The alloxuric excre- tion (uric acid and bases) was hardly affected. In a case of medullary leucocythaemia where the number of leucocytes was rapidly falling, the phosphorus excretion was found to have undergone no diminution, while the alloxuric excretion underwent marked diminution , which the authors suggest might be explained by Ehrlich's theory of leucocythaemia being a form of active leucocytosis, due to the action of some body circulating in the blood, and acting as a positive chemotactic agent on the myelocytes and fully formed polymorphonuclear leucocytes in the marrow. White and Hopkins (1899) published urine analyses for two leukaemic patients, in comparison with those from two normal sub- jects. The feces were not analyzed. They observe, in harmony with von Moraczewski (1898b), the lack of relation between leucocytosis and the excretion of products such as phosphorus and alloxuric bodies, which result from katabolism of nucleins. Stejskal and Erben (1900a) conducted complete balance experi- ments with cases of lymphatic and myelogenic leukaemia. In one very anaemic case of chronic lymphatic leukaemia, on a normal mixed diet, there was in a 5-day experiment a marked nitrogen retention, 5.2 gm. per day, indicating, according to the authors, diminished powers of oxidation. Coincident with this nitrogen storage was a retention of 0.17 gm. P 2 5 and 0.8 gm. chlorine, and a marked loss, 0.35 gm., of calcium. Of magnesium, the output exactly equalled the intake. The authors attribute the loss of calcium to decomposition of bone. In a case of leukaemia Uenalis et myelogenes, with the blood in much better condition than in the above case, and with a greater intake of nitrogen, there was a slight loss of this element as well as of phosphorus, while there were slight gains of calcium and mag- nesium. According to Balthazard (1901b) an increased lecithin content of the liver is general in diseases involving the destruction of leucocytes. PHOSPHORUS METABOLISM 495 Y. Henderson and Edwards (1903) studied one case of lym- phatic leukaemia during a period of six and a half months. In spite of an enormous leucocytosis (175000-380000 per cu. mm., of which 96 percent were lymphocytes), and in spite of the alternation of periods of great increase and marked diminution in the number of circulating corpuscles, the excretion of phosphorus and of uric acid was at no time excessive. The leucocytosis was considered as due, not to a general increase in nuclein metabolism, but to a failure in the normal destructive processes. During the progress of this study, the phosphorus elimination, which was subnormal at the beginning, decreased to a much smaller figure. The nitrogen, also subnormal at the beginning, also decreased, but not to so great an extent as the phosphorus ; so that there was an increase in the pro- portion of nitrogen to phosphorus in the urine. At the same time the uric acid, which was somewhat above normal at the beginning, increased its proportion to total nitrogen to a considerable extent, and its proportion to phosphorus very greatly, while the absolute uric acid excretion increased slightly. The decreased nitrogen and phosphorus outgo were sjiown to be due, not to decreased nuclein katabolism, for uric acid excretion increased during the study, but to retention. Henderson and Edwards harmonize the conflicting observations of Milroy and Malcolm (1898, 1899), who noted decreased phospho- rus excretion, and of von Moraczewski (1898b), who noted a marked phosphorus retention, of Magnus-Levy (1898), who recorded a great increase in phosphorus excretion during 40 hours, with slight leucocytosis, and of White and Hopkins, who concluded that there is no necessary proportionality between the number of circu- lating leucocytes and the excretion of those products (P 2 O g and alloxuric bodies) which result from the breakdown of nucleins, by showing that these apparent differences are probably due to these experimenters having made their observations at different stages in the progress of the disease, as made clear by the changes in amount and proportion of urinary constituents during their own much more protracted observations. Symmers (1904-5a) found the excretion of organic phosphorus in the urine pronounced in lymphatic leukaemia, there being a marked change in the ratios of organic phosphorus, both to total phosphorus and to nitrogen, while the ratios of nitrogen to inorgan- ic and total phosphorus showed little if any departure from the normal; and Erben (1908) found in leukaemia that the plasma was normal, but the erythrocytes were low in iron and potassium, and high in sodium, chlorine, total phosphorus and lecithin. 496 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 PHOSPHORUS METABOLISM AS AFFECTED BY BLOOD DISSOLUTION Kiihnau (1896-7) experimented on a considerable number of dogs by subcutaneous and intraperitoneal injections of various poisons and foreign substances as follows : pyrogallic acid, blood of a dog poisoned by pyrogallic acid, blood of a normal dog, red blood corpuscles from normal dog blood suspended in physiological salt solution, magnesium sulphate plasma of a pyrogallic-acid-poisoned dog, serum of a pyrogallic-acid-poisoned dog, normal dog's blood ser- um, normal human blood serum, typhoid serum, leukaemia serum, NaCl solution of haemoglobin, sodium indoxylsulphate, cinnabar and pure carbon. Among the author's conclusions are the following: In blood dissolution there occur considerable leucocytosis, and the fol- lowing changes in metabolism; (a) an increased excretion of uric acid and purin bases, (b) a rise at the beginning, in the excretion of phosphoric acid, with considerable diminution following it, (c) an increased excretion of chlorine, and (d) chlorine and phosphoric acid excretion stand in an inverse relation to each other, in that increased excretion of the one corresponds to retention of the other. PHOSPHORUS OF THE BLOOD AS AFFECTED BY BLOOD DISEASES Anaemia. Von Moraczewski (1896) observed that in lead colic and anaemia the blood relations are identical, and the urine also shows the same similarity. In these cases there was an increase of chlorine and decrease of phosphorus in the blood, and in the urine a decrease of chlorine and increase of phosphorus, especially the alkaline earth phosphates. Masing (1911a) made a comparison of the blood of rabbits and geese, in a normal state, with the same after the production of an anaemic condition by bleeding, and by injection of phenylhydrazine. In rabbit serum both the total and lipoid phosphorus were increased by the production of an anaemic condition, thus differing from chlorosis, as observed by Erben (1902) ; and in the erythrocytes the proportions of total, lipoid and nuclein phosphorus to total nitrogen were increased by the production of anaemia. In the goose blood similar results were obtained. The increase in the lipoid and nuclein phosphorus of the corpuscles is regarded as evidence of the youth of these cells. Chlorosis. Erben (1902) reported analyses of the blood of three chlorotics. He found the lecithin content of the serum low, as also the phosphorus of the ash of the serum, pointing to a decom- position of erythrocytes. There was an increase above normal in the calcium and magnesium of the serum. In the erythrocytes PHOSPHORUS METABOLISM 497 the fat and lecithin, as well as the ash constituents, were above normal; the cholesterin subnormal; while the iron is decreased in proportion to haemoglobin. Considering the blood as a whole, the protein, lecithin, cholesterin, phosphorus, potassium and iron were subnormal, while the fat, calcium and magnesium were above nor- mal. Leukaemia. E. Freund and Obermayer (1891) report an anal- ysis of leukaemic blood, and compare it with analyses of normal blood by Jarisch, Schmidt, and Becquerel and Rodier. A part of the figures are as follows: COMPARISON OF COMPOSITION OF ASH OF LEUKAEMIC AND NORMAL BLOOD Leukaemic Normal (Freund and Obermayer) (Jarisch) P 2 5 16.92 8.82 so 3 12.31 7.11 CI 17.82 30.74 K 2 15.65 26.55 Na 2 38.52 24.11 CaO 0.47 0.90 MgO 0.07 0.53 Fe 3 3 2.24 8.16 104.00 106.92 equivalent to CI 4.00 6.92 100.00 100.00 Erben (1908) also reports a case of leukaemia in which the lecithin and total phosphorus of the erythrocytes were in excess of the normal. PHOSPHORUS OP THE BLOOD AS AFFECTED BY VARIOUS DISEASES Erben reported analyses of the blood and also of the serum and erythrocytes in a number of pathological conditions. Data from his articles of 1902, 1903, 1905, 1907 and 1908 are included in the table on the following page. Von Moraczewski (1896) published extensive series of blood and urine analyses of patients with pneumonia, nephritis, syphilis and other diseases. In one case of pneumonia there was in the blood a subnormal chlorine content, followed by an increase after the crisis, and a high phosphorus content due to leucocytosis. See table on page 499. Peritz (1908) studied the relation of lecithin to lues, tabes and paralysis. In the first contribution, figures were reported for the ether-soluble phosphorus in the feces of cases of tabes and tabo- paralysis as affected by injections of lecithin. The results are of doubtful significance. 498 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 HUMAN BLOOD AS AFFECTED BY VARIOUS DISEASES Diagnosis Parts per 1000 of substance Percent of ash Portion of blood Leci- thin Grams P2O5 Grams CaO Grams Mg-O Grams P2O5 CaO Mg-O 2.193 1.769 1.715 1.668 3.545 1.455 2.421 1.226 1.452 0.964 5.800 1.536 1.601 5.232 2.251 2.387 1.060 1.822 0.805 2.391 2.97 2.15 0.957 3.163 2.000 1.780 1.817 0.292 0.206 0.228 0.476 0.477 0.341 0.120 0.247 0.764 0.093 0.144 0.100 0.130 0.219 0.095 0.277 0.097 0.460 0.57 0.53 0.873 0.127 0.111 0.135 0.257 0.238 0.219 0.180 0.175 0.117 0.145 0.165 0.355 0.247 0.357 0.359 0.232 0.156 0.177 0.185 0.067 0.01 0.21 0.085 0.202 0.164 0.077 0.049 0.055 0.033 0.043 0.025 0.012 0.024 0.060 0.054 0.065 0.008 0.005 0.006 0.009 0.056 0.077 0.113 0.03 0.02 0.063 0.042 0.036 2.46 2.33 8.82 5.83 6.11 4.12 7.31 3.17 8.64 1.14 1.63 1.26 1.52 2.68 1.18 3.26 1.18 3.08 2.43 0.90 2.68 2.30 2.11 1.58 1.86 1.87 4.09 2.79 4.48 4.21 2.83 1.94 2.02 2.25 0,59 ii ii 0.56 • I it 0.53 :: :: Nephritis, subchronic parenchymatous Nephritis, chronic parenchymatous. .... Same with secondary shrunken kidneys Nephritis, chronic parenchymatous, * shrunken kidneys, uremia, hypertrophy 0.40 0.55 0.30 0.16 31 •i ii •1 ii 0.68 O.fift 0,74 •• •• Nephritis, subchronic parenchymatous Nephritis, chronic parenchymatous Same with secondary shrunken kidneys Chronic uremia, secondary shrunken 0.10 0.06 0.07 0.11 0.66 NephritiSi chronic parenchymatous*. . . . 0.08 ■ • < < * Also shrunken kidneys, uremia, and hypertrophy of the heart. In another article (1908-9a) Peritz states that he has found that the feces of patients having tabes or tabo-paralysis were rich in lecithin, while in the normal man the feces contain only a trace or 0.2 to 0.7 gm. per day. There was also an increase in the lecithin of the serum. The lecithin content of the serum in tabes and par- alysis is given as 4.7 to 6.15 parts per 1000. In a third article, of about the same date as the above, Peritz (1908-9b) submits lecithin estimations on the serum of a large number of cases of lues, tabes, paralysis, and other nervous affec- tions. The figures for the same disease vary remarkably. We dis- cover nothing characteristic in them. Peritz (1910) reported further work of a similar nature. Both ingestion and injection of lecithin in syphilis, tabes, neurasthenia, and the normal state are shown to increase the lecithin in the serum. Lecithin determinations were also made in the fat of the bone marrow of cases of dementia paralytica. These figures varied from to 4.21 percent of lecithin in the fat. PHOSPHORUS METABOLISM 499 «2 * « <=> s ^ S w Q O jz; w ^ (si H «*! sz; i— i « o j m u "? bo a; -^ .9 a 6 c4 8^ ft S3 .2 2.2 M o W -sin 2.2 •2.2 ffl u COCO "CO ■»«coa5r-.ioiocoT*(a»oom • «-> •tf* • ■« •COCX) ••— I • oo -o •oo ■© i— iTjtcqsoeMosooor^t^eNisocn • .cocdocm • r-c---»CMCiocD CMOOoioiocooocMr^oocMco . -cMCMr-oo . -r^r-coioor- COlOlOOT*r-1t^TJ1CMCOCMCMr ooooooooooooo • oooo • co oo in -rr io m •oo oooo ooooooooooooo •oooooo oo oo • OSr- .00 io c^ cm co it- ooco cm coior-oo cm . .-ticM-tno — tt--COCMt— IO QO CO CM 00 C-- IO 03 TJ1 O t-- CD CO O ° .COlOCOCft • • CO 00 ""* 03 •* CM r- < ^H CO i— I CO CM i— I CD Tfl 0O CO CO •* • •lOTftlOCO - - 02 03 O0 t> 00 IO HrtrtOOOHOOOOOO oo'ooooooooooo • oooooo •oooooo •oco .-SI .TJIO *CO •IT-CO -IO O© CUNNMOL^OOOOOOOOOOffJ tmco oooooot^ocoro -1CDI--C0-3110CO~H - .. © n iccB'Jooootiioooo ooo moooooo U-rH CM CM -* IO —4 CO -31 O IO -* Cn IO U-H-* OOO^HOTCOCoS rt°. ti- 2SSSSS2SSS22 SJ!2 «22£ - = = &_,0 ^,« ©©©©©©©©©©O© j_,00 i^,©©©©©© HoH ooo'ooo'oooooofHoo^oooo'oo NO -CO ©-31 'Ttl CMCO 'CM .NOjtMBOcgruNooainooomooomooiNMTiit-. .— -li-H-31^t003'^1lOCOCO©-31t^©-31iOt^.'*+ooooooooooo^oooooo •oooo'o'oo'ooo'ooo'o'o'ooo'o'oooooo h o> COCOiO-*-*1CSCMCCroiNCOCOC^TtlC73C-J03C>a!IO -31CC0^HCMCOr^O5CO-*1COTf1'— last-COt—OO^lCOCOOi— HCD"^0010t— 1 CMC-JNCN10ai>JCMCMCM0qCMCOCM>-WCMCMC>)CNCMCJOQC^CMC^CMC^ ooooooo'ooooo'o'ooooooooooooocJ g: : : : feg: fe§: : i : s z fes uS ™ y -^ a C u (i> oT *» ft.9 -r -!» « b .2 .2 ft* S'E'E fi ■axs-a-d Pi ty Hi 53- TTT Fever day 0.0016 ^ a Fever-free day .0.0398 Case IV Small variations for fever- and fever-free-periods r v Fever day 0.0067 Case v Fever-free day 0.0234 Case VI Fever da y 0.0042 Fever-free day 0.0699 The relation of P 2 5 to nitrogen in the urine was also character- istic, as follows : P 2 5 :N P 2 5 :N Fever After Fever Case 1 1.60:100 6.4:100 Case 2 0.46:100 8.3:100 Case 3 0.78:100 14.9:100 Case 4 0.58:100 5.7:100 Case 5 2.45:100 5.5:100 Case 6 0.36:100 8.8:100 The total nitrogen and ammonia increased during fever, but the proportion of ammonia nitrogen to total nitrogen increased five- fold. The sodium, potassium and chlorine of the urine greatly increased in fever, the phosphates varying in the other direction, the decrease of the phosphates beginning before the onset of the fever. Paton, Dunlop and Macadam (1899) studied phosphorus excre- tion in the urine of female dogs as affected by diphtheria infection (controlled by antitoxin), and by fast. As an average of results from four experiments the authors found a normal daily urinary PHOSPHORUS METABOLISM 517 excretion of 1.63 gin. P 2 5 , during the fast 1.35 gm., and during fever 1.24 gm. P 2 5 ; the percent of P 2 5 relative to total nitrogen, 21.9 normal, 25.7 in fast, and 17.9 in fever. These figures they consider to show that in fever nuclein compounds are less rapidly- decomposed than others containing a lower proportion of nitrogen to phosphorus, or else that katabolic phosphorus is retained. Von MoraczewsM (1899) studied urinary excretion in various febrile conditions. Considering the course of an attack of fever as divisible into six periods, von MoraczewsM observed the interrela- tionship of urinary constituents, and plotted curves to ishow the initial rise in chlorine and fall in phosphorus, followed by a more protracted fall in chlorine and rise in phosphorus elimination, and then an increase of chlorine and decrease of phosphorus to the normal. Variation in these two constituents was always in the opposite direction. At a later date von MoraczewsM (1902) published observations on a case of acute pneumonia in a man from whom the spleen had been removed, because of tumor, seven months previously. With rise in temperature the white blood corpuscles fell from 70000 to 8000, to rise again when the temperature began to fall, and finally to return to the normal level. With rise of number of white blood corpuscles was associated a parallel decrease in urinary phosphorus from 0.833 to 0.041 gm. P 2 5 daily, and associated with this, a cor- responding decrease of potassium elimination. Calcium, however, varied in the reverse direction. This behavior of the phosphorus excretion is attributed to the increased requirement of phosphoric acid by the increased formation of white corpuscles; while the increased elimination of calcium is perhaps to be explained by an increase in the katabolic processes in the bone marrow associated with the growing number of white corpuscles in the blood. Von MoraczewsM (1900a) conducted 18 nitrogen and mineral balances in simple inflammation of the lungs. The nitrogen loss in fever was found to be dependent on the intake ; that is, the great- er the intake, the smaller the loss. The chlorine retention was much higher in fever than when the temperature was normal. The calcium outgo was also decreased in fever while the phosphorus balances were usually negative and seemed to follow the nitrogen. Sommerfeld and Caro (1902) studied metabolism in three child- ren who were convalescent after scarlet fever, with a diet of milk alone. A part of the results are as follows : 518 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 AVERAGE DAILY NITROGEN AND PHOSPHORUS BALANCES WITH THREE CHILDREN CONVALESCENT FROM SCARLET FEVER, ON A DIET OF PURE MILK— Grams Case No. and age Milk taken C.C. Nitrogen Phosphorus Food Urine Feces Balance Food Urine Feces Balance I 514 years II 7 years III 6 years 3000 3700 2562.5 16.395 19.978 13.688 13.350 15.423 12.530 0.965 1.058 0.535 +2.080 +3.497 +0.623 3.140 3.875 2.688 0.663 0.955 1.205 2.035 2.205 1.540 +0.442 +0.715 +0.057 Computed from authors' data. The periods were 4 days each in extent. Cases I and III devel- oped nephritis hemorrhagica in connection with scarlet fever, but recovered, No. I quickly, No. Ill slowly. The above experiments were conducted when the patients had been free from fever for several days, but remained in bed. During the balance experiment Case III showed in the urine, leucocytes, red blood corpuscles and kidney epithelium. The phosphorus retention with these children was in Cases I and II quite considerable ; in Case III, however, there was a loss of phosphorus with an intake which would normally cause retention even in an adult. Achard, Laubry and Thomas (1902) studied the question as to whether the retention of chlorine, which occurs in the organism in so marked a degree in the course of certain acute diseases, is accom- panied by a retention of phosphates. Daily determinations of chlorine and phosphorus in the urine were made in the different stages of a number of diseases. Then injections of sodium glycero- phosphate were made, and the urine examined again. The diseases studied were typhoid fever (7 cases), pleurisy (1 case), pneumonia (5 cases), rheumatism (1 case), pulmonary tuberculosis (1 case), and asystole (3 cases). The authors found no parallelism between the chlorine and phosphates. Increased phosphorus excretion was a frequent occurrence in acute cases of infectious diseases. In typhoid fever it was often greatly increased, and was observed before the chlorine crisis, while there was no general improvement, and while the fever continued. Increased phosphorus excretion occurred even in the period of the acme of certain diseases, as was observed in typhoid fever and infectious icterus, and so can not be looked upon as a forerunner of recovery. This increased phospho- rus excretion was often independent of the diet. The injected glycerophosphate was usually quickly eliminated in the urine. PHOSPHORUS METABOLISM 519 According to Gouraud (1902), the phosphorus elimination is similar in pneumonia and typhoid fever, in absolute value as well as in relation between phosphates and alkalis. The urinary phosphates are diminished in amount during the time when the temperature is at its highest point, and also as long as that temperature lasts. At the beginning of convalescence there is a "phosphate crisis" which varies in intensity according to the disease and its duration. During the fever the percent of phosphate earths in the total phosphates falls from 45 to 30, and sometimes even as low as 10. The phenom- enon is reversed at the moment of crisis, which affects especially the earth phosphates, and the percent of phosphate earths in the total phosphates increases to 50 or 60, and sometimes to 70 or 80. Gouraud (1903) believes that in fever the disintegration of phosphorus-containing compounds is markedly increased, but that the katabolic processes are not carried to the final liberation of inorganic phosphoric acid, the result being such an accumulation of organically combined phosphorus as induces degenerative histologi- cal alterations. Urinary phosphates are decreased, the proportion of P to N signifying the seriousness of the malady. In convales- cence enzymatic activities become normal and the accumulated organic phosphorus is katabolized and eliminated, the result being a phosphaturic crisis. Death is sometimes preceded by such a rapid disintegration of tissues rich in phosphorus as results in an ante- mortem phosphaturic crisis the proportion of P to N in the urine becoming above normal. Garratt (1904) has made an extensive study of metabolism in fever. He is of the opinion that apparent retention of katabolized phosphorus in fever is due to the escape of the phosphorus-rich pro- teins from the oxidation which overtakes phosphorus-poor proteins, and that increased excretion of phosphorus in fever should be regarded rather as a result of the action of the toxin on tissues rich in phosphorus than of the influence upon these tissues of the pyrexia itself, therefore that it has but little direct connection with the febrile state. This article contains 51 references. According to Molyakov (1912), sodium nucleate, given in the first stages of scarlet fever, causes a great increase in the number of polynuclear leucocytes. See also W. Zuelzer (1876) and S. Weber (1901). Summary. From these notes it is apparent that in fevers the amount of the phosphorus outgo is the resultant of the effects of a considerable number of agencies differing in the nature as well as the degree of their influence, as determined by the cause and dura- tion of the fever, and the state of nutrition of the patient. 520 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Among those conditions making for increased outgo of phospho- rus are (1) increased tissue destruction, (2) acidosis and (3) chlor- ine retention; among those tending to restrict the outgo are (1) decreased intake, and (2) retention for growth of white blood cor- puscles and pathogenic organisms. In the post-febrile stages there are unusual tendencies making for phosphorus retention, to com- pensate for previous losses, but this retention is often masked by an increased outgo due to leukolysis, excretion of metabolic wastes previously delayed by unfavorable conditions, and excretion of products derived from the pathogenic organism. There seem, therefore, to be no invariable rules as to interrelationships of urin- ary constituents in fevers generally. GLYCEROPHOSPHATE THERAPY On account of the facts that lecithin phosphorus is absorbed largely in the condition of glycerophosphoric acid, and that lecithin is exceedingly expensive, considerable interest has attached to the therapeutic use of salts of glycerophosphoric acid. Pasqualis (1894) found that the calcium glycerophosphate increased the urinary phosphorus a day sooner than did calcium phosphate, but after examination of the blood, concluded that the calcium phosphate was only slightly slower in getting into the circu- lation. The glycerophosphoric acid was not found unchanged in the urine, even when large amounts were given per os, or even by injection. By a new method he finds abundant glycerophosphoric acid in the blood. Pasqualis states that in every organ the glycero- phosphoric acid is converted into glycerin and phosphoric acid. Robin (1895) published urine analyses from several cases of a number of diseases under treatment with sodium glycerophosphate by subcutaneous injection. The author discusses at length, and in detail, various improved conditions of metabolism said to be due to this treatment. In view of the fact that no feces analyses were made we are unable to judge of the validity of the claims. De Stella (1897) conducted injection and feeding experiments with sodium glycerophosphate on two rabbits and a dog. Urine analysis shows increased excretion of sodium chloride, urea and phosphorus. Bardet (1900) distinguishes between the therapeutic effects of acid and neutral sodium and calcium glycerophosphates. The acid glycerophosphates can not be used hypodermically. Urinary excretion of phosphates was not appreciably increased by the inges- tion of acid glycerophosphates, even when given in large doses. The PHOSPHORUS METABOLISM 521 acid glycerophosphates increase the titratable acid of the urine. In large doses, 15-25 gm. per day, they have a purgative and chologogue action. West (1902) reports "immediate and pronounced" results from the administration of sodium and calcium glycerophosphates in 8 cases of neurasthenia. Street (1902) reports that glycerophosphates are most useful in all cases of nervous impairment due to overwork, or excess of any kind, in premature advance of age, and in senility, attended by gen- eral debility ; the benefits from their protracted use are said to be striking, and their value considerable in chronic neuralgia, in sciat- ica (by hypodermic injections along the nerve), and in convalescence from la grippe, and acute infectious diseases. Street notes that Magnin of Paris asserts that in diabetes he has seen the sugar remarkably diminished by glycerophosphates. To get the best results it is said to be necessary to administer the glycerophosphates from three to six months, sometimes longer, though occasional inter- ruptions of several weeks in the course of very prolonged adminis- tration are beneficial. Fabiani (1903) reports favorable results from glycerophos- phate therapy by oral or subcutaneous introducton in 8 cases. J. Smolenski (1904) investigated the therapeutic effect of glycerophosphates with 19 infants, one to eleven-and-a-half months of age, and 15 children of one to five years. Both sodium and calcium salts were used. No case showed unfavorable results ; many showed improved appetite, and gain in weight. Novi (1904), in balance experiments on himself, studied phos- phorus metabolism as affected by antirabes treatment, with and without accompanying sodium glycerophosphate, taken per os or hypodermically. The numerical results are as follows : AVERAGE DAILY PHOSPHORUS BALANCES WITH A MAN AS AFFECTED BY ANTIRABES, WITH AND WITHOUT SODIUM GLYCEROPHOSPHATE— Grams Period Length in days Intake P2O5 Urine P2O5 Feces P2O5 Bal- ance P2O5 Percent of total P2O5 ingested Urine Feces- Total 1 4 2.4485 1.3345 0.8023 +0.3117 54 32 86 Normal 2 3 2.2827 1.3452 0.9702 -0.0327 59 42 101 Cure, nonvirulent material 3 5 2.1346 1.4573 0.7119 -0.0346 68 33 101 Cure, virulent material 4 5 2.0041 1.4150 0.8190 -0.2300 70 40 110 Cure, virulent, and 0.200 gm. sodi- um glycerophosphate, per os. 5 1 2.3387 1.4858 0.5889 +0.2640 63 25 88 Cure, virulent material 6 3 2.3161 1.4177 0.6581 +0.2403 61 28 89 Cure, virulent, and 0. 20 or 0.30 gm. sodium glycerophosphate injected 7 1 2.2335 1.3319 0.5788 +0.3228 59 25 84 Cure, virulent material 622 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 The author attributes to the antirabes treatment, both virulent and nonvirulent, a tendency to increase the urinary phosphorus, this being due, Novi thinks, to the same constituent of the injected material, which, while not itself toxic, produces a leucocytosis and a coexistent or following leucolysis. The figures show that the administration of sodium glycero- phosphate, either hypodermically or per os, increased the feces phos- phorus, and increased the loss, or decreased the retention, of this element. Ingestion per os did not reduce the urinary phosphorus, reckoned as percent of the intake, but hypodermic injection may perhaps have done so to a slight extent. Frey (1906) has obtained favorable results from therapeutic use of a glycerophosphate preparation containing iron and bromine. It would seem, therefore, that there is at least similarity in the action of glycerophosphates and lecithin, though the probability is, of course, that the glycerophosphates are less effective through the lack of those functions or superiorities of lecithin which depend on its partial absorption in an unchanged condition. For an extended review on glycerophosphate therapy see Merck (1911). PHOSPHORUS METABOLISM IN GOUT Gout is due to uric acid retention, and results from deranged nuclein metabolism. Observations on phosphorus elimination with nuclein-free and nuclein-containing diets, therefore, have been used in judging of the progress of this disease. Purin base elimination 3nay be even more significant as indicating incomplete formation of 'uric acid or urea. Hans Vogt (1901) conducted nitrogen and phosphorus balances with a man suffering from gout, for the purpose of studying nuclein metabolism in this condition. Balance data are below. AVERAGE DAILY NITROGEN AND PHOSPHORUS BALANCES WITH GOUT PATIENT AND CONTROL AS AFFECTED BY INGESTION OF PANCREAS— Grams Gout patient Control Body weig-ht of gout patient Kg. Body ■weight of control Kg-. Periods N Intake Balance P Intake Balance N Intake Balance P Intake Balance Diet Fore-period 6 days Main period 5 days After-period 4 days 14.58 +1.16 19.31 +3.79 14.58 -0.33 2.621 -0.513 4.861 +1.016 2.621 -0.535 14.58 -1.76 19.31 +2.01 14.58 —2.38 2.621 -0.938 4.861 +0.865 2.621 -1.108 68.2-68.4 68.4-68.95 68.9-68.5 64.3-64.9 64.9-64.9 64.9-64.8 White bread, egg's, sirloin steak, butter, cream, beer Same plus calf's pan- creas Same as first period PHOSPHORUS METABOLISM 523 The subject suffered from acute gout, caused by beer drinking, and also from kidney trouble. For comparison a second subject was included, a servant ordinarily healthy, but suffering from nervous dyspepsia during this experiment. The gout patient was 35 years old and formerly healthy. On Apr. 26, 1900, typical symptoms of gout appeared. After treatment with sodium salicylate he showed improvement. The experiment lasted from May 8-May 23, 1900. Conclusions. 1. There was a nitrogen loss in the preliminary and after periods, due to insufficient intake, but a retention by both subjects during the gland-feeding period. 2. The nuclein is entirely split in the organism. The phos- phorus is excreted normally but the nitrogen is retained. This nitrogen retention is not due to a disturbance of the secretory activ- ity of the kidneys, since the phosphorus outgo is normal. 3. Much uric acid was retained by the gout patient in compari- son with the control. This is the result of a disturbance of metabo- lism, which may be due either to a decrease of oxidation or splitting, or to synthetic processes. The outgo of phosphorus as compared with the outgo of nitrogen, and therefore of uric acid, in the case of the gout patient is very small, compared to the control. This points to a tardiness of nuclein metabolism of the gout patient. Whether the large retention of nitrogen is due also to this is not to be conclud- ed from these results. Waldvogel (1902) reports a metabolism study at the time of a gout attack, in which the urine was examined as to its amount, specific gravity, total nitrogen, uric acid, total phosphorus and disodium phosphate. In conclusion he says, in part : "The gouty attack may be thus characterized: The nitrogen retention between attacks shows itself to be a slowing up of the elimination of the decomposition products of nucleins, the values for both uric acid and phosphoric acid being small, without any recognizable kidney insufficiency "But while the phosphate shows gradual increase for a long time after reduced nuclein metabolism, the elimination of uric acid rises sharply from the first day of the attack, and it is also increased in the blood bringing about an elevation of 8 without kidney insuffi- ciency making its appearance. The uric acid apparently rises from that being produced in the joints, the solution of it in the alkaline blood taking place so rapidly that the kidney secretion cannot immediately make up for the subsequent appearance of uric acid in the blood. 524 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 "Also before the attack the relative amount of P 2 5 and uric acid in the urine is different; on the second day before the attack the amount of uric acid is already low, that of phosphoric acid not. Not till the day before the attack does the value for P 2 5 fall, while that of uric acid does not appear further reduced." The data are, in part, as follows : URIC ACID AND PHOSPHORUS ELIMINATION IN THE URINE OF A GOUT PATIENT FOLLOWING ACUTE ATTACK Waldvogel (1902)— Grams Day Uric acid Total P2O5 Na2HP04 1 0.652 0.58 0.18 Day of attack 2 0.769 0.71 0.14 3 0.770 0.61 0.19 4 0.824 0.74 0.24 5 0.742 0.81 0.34 6 0.679 0.88 0.34 7 0.758 1.48 0.70 8 0.723 1.43 0.60 9 0.620 1.25 0.67 10 0.768 1.57 0.64 11 0.615 1.37 0.41 12 0.540 1.21 0.51 24 0.644 Two days before a second attack 25 0.479 1.20 6.42 Kaufmann and Mohr (1902) found that the values of endogen- ous alloxur nitrogen and the ratio of uric acid-nitrogen to base-nitro- gen both fell within normal limits in gouty subjects. They give the following nitrogen and phosphorus balances for 5 gouty persons on nuclein-free diet. NITROGEN AND PHOSPHORUS BALANCES WITH GOUT PATIENTS ON A NUCLEIN-FREE DIET— Grams Days Calories intake Nitrogen Phosphorus (P2O5) Case No. Intake Balance Intake Balance Case 1 2 3 4 5 4 3 4 3 8 3200 ? 2900 3200 2350 12.5-13.9 21.25 15.65 13.6-19.3 15.1-16.7 +1.55 +4.72 -2.56 +0.24 -1.55 8.'45 6.25 6.35-7.35 5.78-6.33 +i.'i6 -0.77 +0.71 +0.13 Chronic gout Chronic gout, "very fat" Acute attack Acute attack Less acute attack Judging from the values and the signs of the nitrogen and phos- phorus balances, it is supposed that in Case 2 there was a building up, and in Case 3 a breaking down of phosphorized nitrogenous tissue, while in Cases 4 and 5 it appears that the material used was non-phosphorized protein. PHOSPHOKUS METABOLISM 525 These authors also made observations on the absorption and retention of the nitrogen and phosphorus of thymus when added to the purin-free diet, and in one case of chronic gout they added to the diet alternately "dark" (filet) and "light" (veal or haddock) meat ; the dark meat gave no greater increase of alloxur bodies than did the light meat. Ciuffini (1910) compared the calcium, magnesium and phospho- rus balances on a normal subject, a subject with chronic gout, and a subject having gout with paroxysms. Balances were noted during 4-day periods on mixed diet and on the same without meat (consid- ered to be purin-free). Urinary phosphorus elimination appeared to be markedly low in chronic gout. In paroxysms there was phospho- rus retention before, during and after the highest point of the attack. Rotky (1910) studied nuclein metabolism in various diseases of human subjects, including gout, and he reports purin nitrogen observations on urine and feces, and P 2 5 on the urine. Uric acid excretion resulting from a milk diet was considered to be of endogen- ous origin. Exogenous excretion was studied by means of the addition of guanin, hypoxanthin or thymus to the diet. Serious disturbance of ferment activity was noted in gout, the food purins not being converted to uric acid, and in acute nephritis there was delay of excretion, and but a small part of the purin nitrogen was excreted as uric acid, while urinary purin bases showed a small increase. But slight variation was noticed in the values of phos- phoric acid. With regard to the etiology of gout as an anomaly of purin metabolism the reader is referred to the discussion of Brugsch and Schittenhelm (1910). The discussion includes normal nuclein metabolism, as well as its disturbances, and was preceded by exhaustive investigations on certain phases of the subject (Schitten- helm, 1907 ; Brugsch and Schittenhelm, 1907a, 1907b, 1907c, 1907d, 1907e, 1907f, 1908-9, 1910; Brugsch, 1909). The reports of others are also extensively quoted. According to these authors uric acid, originating in nucleins, becomes so abundant in the blood as to exceed its solubility limits in that fluid, and hence urate is deposited in the tissues, which deposit causes the arthritis, pain and fever of gout. The deposits are found to be mainly monosodium urate. Brugsch and Schitten- helm distinguish a metabolic gout and a kidney gout, according as the uricaemia is. due to disturbances in the purin metabolism or in the excretion. Disturbance of the kidneys may follow ae a second- ary result in metabolism gout. 526 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In metabolism gout they find uric acid constantly present in the blood, even on purin-f ree diet (endogenous uricaemia) , and in four- fifths of all the cases which they observed the uric acid excretion, during intervals between attacks, was subnormal or at a low normal value, and the base-N excretion was within normal limits. The ratio of uric acid-N to purin base-N excretion tends toward the low- er limit of normal variation. At the time of attack, and immediate- ly afterward, the uric acid elimination was high. The accumulation of exogenous uric acid (exogenous uricaemia) is not considered a retention in the usual sense, but a temporary increase due to a depression and a retardation of urea-formation, from the purin bodies fed, and of the decomposition and elimination of uric acid. According to Brugsch and Schittenhelm this depression and retard- ation involve the whole fermentative system of uric acid formation and decomposition (especially nuclease and purin deamidase, togeth- er with the uricolytic ferment, and to a far less degree the xanthin oxidase). See Miller and Jones (1909) for evidence throwing some doubt on this point.) This weakening of the ferment activity raises the uricaemia to hyperuricaemia, and the symptoms of gout appear. Of phosphorus metabolism during gout we may say then that in acute attacks the balances may be positive or negative. The phosphorus excretion falls before the attack, is low during the attack, and rises slowly thereafter during a considerable period. In chronic gout the urinary phosphorus excretion is low. INFLUENCE OF HEMORRHAGE ON PHOSPHORUS METABOLISM Hawk and Gies (1904) in their work on the influence of exter- nal hemorrhage on dogs included determinations of phosphorus balance. The experiments here reported were on two dogs, normal at the beginning of the work, and subjected to anaesthesia, opera- tion and hemorrhage. The blood was let from the saphena. The diet was meat, crackers and bone ash. As a result of the many alterations in the usual course of metabolism blood letting seems slightly to favor phosphorus retention. The excess of ingested over excreted phosphorus was least in the anaesthesia period (Exp. 1, period 3) and greatest in the anaesthesia-operation period (Exp. 1, period 4) . Although the results were not very striking, operation and hemorrhage, in most instances, seemed to cause slight retention of phosphorus; etherization favored increased elimination. The anaesthetic used was principally ether, with a very little chloroform at the beginning. PHOSPHORUS METABOLISM 527 INFLUENCE OF EXTERNAL HEMORRHAGE ON PHOSPHORUS METABOLISM IN THE DOG— Daily Balances— Grams Exp, and period 1-1 1-2 1-3 1-4 1-5 1-6 1-7 2-1 2-2 2-3 Initial weight of animal Kilos 16.96 16.29 15.81 15.44 15.18 13.76 13.35 11.85 11.40 10.47 Treatment Normal Anaesthesia.operation and 1st hemorrhage, 2.93 percent, body weight Anaesthesia alone Anaesthesia and operation, (no hemorrhage) 2nd hemorrhage, 3.22 percent, body weight 3rd hemorrhage, 3.51 percent, body weight 4th hemorrhage, 3.26 percent, body weight Normal Anaesthesia and operation.... Anaesthesia, operation and hemorrhage, 3. 11 percent, body weight Meat 0.557 0.557 0.558 0.558 0.543 0.527 0.527 0.440 Food phosphorus Crack- ers 0.094 0.070 Lard 0.026 0.013 Bone ash 1.779 1.423 Total 2.456 2.456 2.457 2.457 2.442 2.426 2.426 1.950 Phos- phorus balance Days in period +0.045 +0.123 +0.015 +0.163 +0.022 +0.065 -0.180 -0.030 -0.025 -0.017 HOOKWORM Vannini (1900) conducted balance experiments with human beings under treatment for anaemia from hookworm (Anchylostoma). Nothing characteristic was observed in the phosphorus metabolism. ICTERUS The connection of icterus (jaundice) with phosphorus metabo- lism is comparatively slight and indirect. Neither the overloading of the blood with bile, nor the deflection of bile from the intestine, nor yet the injury which sometimes occurs to the liver cells bears directly on phosphorus metabolism in important ways. Through under-nutrition there is sometimes an increased katabolism of body protein, and hence an increased production and secretion of endogenous uric acid, but only to such extent as is due to the increased tissue destruction. The increased gastric secretion of hydrochloric acid, which is usually present, probably has a demonstrable bearing on phosphorus metabolism, as also the charac- teristic calcium soap stools. Phosphorus metabolism in icterus has been studied by Simnit- ski and Rodoslawow (1902) and by Schilling (1901a). Simnitski and Rodoslawow found urinary phosphorus nearly normal, though often increased, with relative amounts of constituents normal. 528 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Schilling notes that Szydlowski has stated in a dissertation, "Beitrage zur Mikroskopie der Faces," that in icterus the feces contain no crystals of tricalcic phosphate. Schilling, however, finds that although in this disease the feces do not usually contain many phosphate crystals, when the food has a high "phosphorus content they are readily seen. He finds no relation between the fatty-acid content of the feces and the amount of phosphate crystals present. LECITHIN THERAPY The use of lecithin as a drug has been, in large part, due to its prominence as a constituent of the brain, and to the established increase of formation of its cleavage products in degenerating nerve tissue. The conception of administering nutriment to the brain, direct, is attractive, and nothing is more natural than that this idea should have been accepted by the ignorant, overworked by the humbug, and stoutly denied by the honest radical. The extreme claims of many advocates of the use of lecithin in therapeutics have tended so strongly to discredit any such element of truth as they may have expressed that it is with reluctance that we attempt to point out the probable facts. Approaching the task with the assumption of the sincerity of the average man, we have eliminated some of the obviously unrelia- ble claims, have toned down many of the remainder, and have sought to call attention especially to those points on which there is general agreement. With this explanation it seems unlikely that the following reports should require any further discount than is implied in the suggestion that there is more inclination to record and to publish the favorable than the unfavorable results, and we can only speculate as to the number of unfavorable results which have been suppressed. Baud (1858a, 1858b) reported beneficial effects, in cachexia, from the administration of phosphorized fatty matter extracted from the medulla oblongata of animals. B. Danielewsky (1895a, 1895b) states that subcutaneous injec- tion of lecithin in dogs increased the number of red corpuscles up to 800,000 to 1,000,000 above normal, and also increased the haemo- globin. This improvement occurred in a few days after the injec- tion, and lasted for some time. Serono (1897a, 1897b, 1902) conducted subcutaneous injection experiments with specially purified lecithin on a considerable num- ber of pathological human subjects. There resulted gain in appe- tite, and improvement in general condition, a rapid increase of red PHOSPHOKUS METABOLISM 529 blood corpuscles, and usually increased nitrogen elimination in the urine. Lecithin was found to possess a tonic effect comparable to arsenic, but acting more quickly. Haemoglobin increased very slowly, and only after improvement of general conditions. Serono considers it better to administer lecithin hypodermically than by ingestion, because of digestive decomposition in the alimen- tary tract, and would use egg lecithin rather than brain lecithin, because of admixture of the latter with toxic substances, in doses of 20-30 centigrams or more per day. Magnani (1898) found injections of lecithin helpful during treatment of the eyes, by improving the condition of the cornea, when it had suffered during lowered nutrition of the whole organism. Muggia (1898) reports nutritive and curative value of lecithin and egg yolk injected in cases of anaemia and infantile athrepsia. Saint-Aubin (1900) conducted hypodermic injection experi- ments on five patients, with a solution obtained by filtration of a mixture of vaseline oil and egg yolk, after heating to coagulate nucleins and albuminoids. The treatment led to increase of weight and of urea elimination, due, it is said, to stimulation of cellular multiplication, and in consequence, of activity of the elements. Phosphorus and uric acid outgo were also increased. Morichau-Beauchant (1901) has made an extended study of lecithin therapy, and reviews experimental work beginning with that of Danielewsky in 1895. From his conclusions we condense the following : Lecithin administered either subcutaneously or through the stomach stimulates the appetite, and leads to rapid gain in weight. It stimulates growth, and increases the number of red blood corpus^- cles and the percent of haemoglobin. It increases urea, total nitrogen elimination, and the coefficient of utilization of nitrogen, and diminishes uric acid. In tuberculosis lecithin is of service in the earlier stages, not as a specific, but as an auxiliary. Similarly it may be of value in diabetes through improving general conditions. It also gives good results, as a rule, in anaemias, cachexias and convalescence. In neurasthenia it is especially valuable. Lecithin is active in doses of 0.2-0.3 gm. per os and 0.05-0.10 gm. subcutaneously each day, or every other day. Lecithin is not toxic in any dose. Subcutaneous administration is indicated in gastrointestinal disorders. 530 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Suzor (1901) treated two patients by subcutaneous injeetion of raw egg yolk in water solution, and obtained stimulation and im- provement of general condition. Iljin (1901) conducted feeding experiments with a laboratory helper, on mixed diets of normal foods, to which brain and egg yolk were added as carriers of organic phosphorus. From this work we have seen only Maly's brief abstract. The organic phosphorus compounds were considered to have promoted nitrogen retention. Zaky (1901a, 1901b) studied the effects of lecithin ingestion, in doses of 0.3 gm. per day, on urinary elimination. In experiments with three subjects there appeared to be slight increase of total nitrogen and urea, with decrease of uric acid and phosphorus. The changes were so small as to be of doubtful significance unless estab- lished by most critical experiment. Answering Robin's criticism to the effect that egg yolk increases uric acid excretion Zaky says that it is the nuclein and not the lecithin of the egg which increases the uric acid elimination. Aries (1902), on the strength of clinical experience, considers lecithin a valuable remedy in all cases where "denutrition" is to be combatted, especially in old age. He speaks of it as a bioplastic and morphogenic agent. Maillon (1902) made a clinical and physiological study of leci- thin by administration of 0.3-0.8 gm. doses in 34 cases of tubercu- losis, chlorosis and nervous diseases, basing conclusions principally on an examination of the urine. He found the amounts of phos- phoric and glycerophosphoric acids in the urine unchanged by the lecithin treatment. The ingestion of lecithin, however, was of benefit; in some cases it improved the appetite; in chlorosis it caused a constant increase in body weight, but in tuberculosis the increase in weight was irregular. The effect on the urine, if there was any at all, was indirect. Stassano and Billon (1902a, 1902b, 1902c, 1902d) conducted a series of experiments on the effects of lecithin injection on the blood elements in animals. Lecithin in physiological salt solution injected into the auricular vein of rabbits produced hyperleucocy- tosis lasting for several days, and an increase in the ratio of mono- to polynuclear forms. Intraperitoneal injection, with guinea pigs, caused an increase of both forms of leucocytes. In the lecithin exudate the ingestion of the polynuclear forms by the very large mononuclear leucocytes proceeded with a rapidity suggesting that the mononuclear forms, especially, had taken up the lecithin injected, and nourished them- selves therewith. PHOSPHORUS METABOLISM 531 Further intravenous injections, with rabbits, led to a progress- ive increase of red blood corpuscles after each injection. Tests showed an increased vitality of these cells, as implied by increase of resistance to saline solutions of various strengths. A different reaction of the red corpuscles to stains, implying increased acidity of the nuclear chromatin, was noted. By experiments with frogs it was determined that, after having grown large and granular at the expense of the lecithin, the mono- nuclear leucocytes left the blood vessels, and spread into the system. Endothelial cells in their turn retained lecithin, becoming crammed with granulations, and their nuclei showing marked activity. Courtial (1903) showed that 1-2 gm. of lecithin taken by healthy people produced no notable differences in the composition of the urine ; no increase of either phosphorus or uric acid. Fratta (1904) conducted hypodermic injection experiments on a human being weighing about 70 kg. with marrow, sodium hypo- phosphite, glycerophosphates, lecithin and cura antirabica. Glycero- phosphates and lecithin, injected hypodermically in doses of 0.20 gm. daily, increased urinary nitrogen elimination, but decreased the urinary phosphorus excretion. The cura antirabica increased both nitrogen and phosphorus elimination. Heim (1904) describes "bioson" as a protein-iron-lecithin combination and recommends it as a blood building and nerve strengthening preparation. The iron content is 0.24 percent, and the lecithin 1.27 percent. It is, therefore, not nearly so rich in lecithin as many common foods. Migliaccio (1904) used lecithin injections in cases of anaemia, atrophy and rachitis, — all infants. The results were rapid and sometimes notable gain in weight, notable improvement in the blood, and in gastrointestinal functions. The urinary phosphorus showed increase in all cases. Silberstein (1904) discusses and recommends phosphorus treatment with "hemaprotagon" tablets, a preparation containing iron from the blood, and unaltered lecithin. Cases of melancholia, tuberculosis and lues are discussed, and the preparation recommend- ed for such cases. W. Koch (1905a) stated that for the clinical administration of lecithin to the adult, in the pure form, there is at present no good scientific basis. Koch based this view on the fact that, in the ordinary diet, one consumes vastly more lecithin than is recom- mended by the promoters of the clinical application of lecithin. He 632 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 suggests, however, that with the infant the case may be slightly different in that the lecithin content of cow's milk diluted with water may be reduced to a figure very much below that in human milk. Snowman (1905) gives clinical reports of benefit from the use of sanatogen, especially improved blood conditions in chlorosis, chorea, and tuberculosis. For suggestions regarding the therapeutic use of lecithin activ- ated by Rontgen rays, radium rays, and by other means see Werner (1905). Von Oefele (1907) found "bioson" to be utilized in a normal way during health, and in sickness to be utilized more perfectly than other articles of diet. Errani (1908) treated 10 pathological cases by lecithin injec- tion. He concluded that lecithin promotes and accelerates the nor- mal bioplastic and morphogenetic processes, improves the metabo- lism and the constitution of the blood, and, in consequence, the general condition of the organism. Kleinertz (1908) reported several instances of improvement in clinical condition after the use of "biocitin," a lecithin preparation of Altmann, containing 10.7 percent of lecithin. Sleeswyk (1908) found that the bactericidal action of certain lecithin solutions on typhus bacilli, as reported by Bassenge, was due to their acid reaction, caused in all probability by impurities in the commercial products used. W. Koch (1909a), in discussing phosphorus compounds as brain foods, calls attention to the facts that, in comparison with other parts of the body, the nervous system does not have a very large amount of total phosphorus, though it has of lecithin; that the amount is not materially less in cases of dementia praecox, but is reduced in general paralysis ; that the commercial phosphorus prep- arations used as drugs (hypophosphites, glycerophosphates, phytin, lecithin) are much less effective for supplying the requirements for growth of the brain than are phosphorus-rich foods, some of them not being taken up by the system, the amount usually recommended of any kind giving very insignificant addition to the amount of phos- phorus taken with the daily food, and there being no conclusive evi- dence that they have any effect on the growth of the brain. J. and W. Cronheim (1910) made clinical observations on the use of lecithin with 18 cases, all of them undernourished, and most of them tuberculous. The authors conclude that under such con- ditions lecithin induces remarkable increase of appetite, and conse- quent gain in weight. PHOSPHORUS METABOLISM 633 Renshaw and Atkins (1910) studied the bactericidal properties of lecithin and of choline salts. Such a property of lecithin was shown to be at the most very slight, while there was no indication of bactericidal effect of the choline salts. Buslik and Goldhaber (1911) conducted metabolism experi- ments with a lecithin-albumin, "glidine." This preparation appeared superior to scraped meat as to digestibility, absorbability and action on intestinal putrefaction ; it permitted forced protein storage, and had a favorable effect on the haemoglobin content of the blood of anaemics ; in the last respect, however, "f erroglidine" was still more efficient. Borschim (1911) applied lecithin to the back of a rabbit to determine its effect on iodine absorption. The result was negative. Lecithin has no such effect. Milkowicz (1911) found that lecithin and cholesterol in vitro cause a great increase of phagocytosis of Staphylococcus and tubercle bacilli. An iodine-lecithin-protein preparation is described by Isaac (1911) and C. Neuberg (1911) under the name "iodocitin." Bain (1912) obtained from the oral administration of lecithin to rabbits marked increase of both red and white corpuscles and of haemoglobin, and the same results from subcutaneous injection. The increase of white corpuscles was chiefly in the mononuclear lymphocytes. Similar experiments were conducted with phytin. Introduced by mouth, the improvement in blood conditions was slight ; introduc- tion by injection produced marked increase in red corpuscles, but less increase of white corpuscles and of haemoglobin than was obtained by the injection of lecithin. Bain regards lecithin especially as a metabolic stimulus, its effects on the nervous system being secondary to improvement in general state of nutrition. In our own experience we have found that egg yolk, presum- ably through its lecithin content, often improves the digestion of milk by infants, but also that infants differ widely in their tolera- tion of egg yolk. Among other articles on lecithin therapy are those of Coulombe (1901), who presents a historical review up to 1901, with summary and 56 references; Hartenberg (1901), Huchard (1901), Lancer- eaux and Paulesco (1901), Labbe (1903), Fiirst (1903), Grimm (1903), H. C. Wood Jr. (1903), Goliner (1905), G. Landsberg (1906) and Berkley (1908), the above consisting of clinical reports 534 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 of the use of lecithin in a great variety of pathological cases char- acterized, in general, by low state of nutrition, as after wasting diseases, the results in almost all cases reported being marked improvement of general condition. It would appear from these clinical reports that, in states of impoverishment, lecithin is a valuable aid in the restoration of the organism to normal condition. Claims of stimulation of growth under normal conditions are not so well sustained, and are difficult of adjudication by reason of the fact that the normal state is vari- able and not definitely definable. The benefit derived from lecithin treatment is usually spoken of as improvement of the general state of nutrition, especially as evidenced by improved appetite and digestion (which indeed might be accountable for most of the rest), increased weight, red blood corpuscles, haemoglobin, and white blood corpuscles, especially of the mononuclear form. It is said not to be specific in disease, but to be of value in many derangements, — anaemia, cachexias, conval- escence, neurasthenia, etc., through the elevation of the general plane of nutrition. As to the relative efficiency of lecithin administered as a drug and taken in natural foods we have not information sufficient to warrant a positive statement. If lecithin, taken in the pure form, actually has the capacities which it is said to possess, this may be due to the more extensive absorption in an unsplit condition which seems probable when the compound is administered uncombined with nutrients requiring digestive cleavage. See also notes on lecithin therapy in the discussion of tubercu- losis. For an extended review on lecithin therapy see Merck (1912). MALNUTRITION OF THE BONES OF THE DOMESTIC ANIMALS The facts of the existence of malnutrition of the bones as a disease of the domestic animals, the dependence of this disorder primarily on deficiencies in the mineral elements of the food, as determined by its nature and by soil and climate, and the ready response of this ailment to improved treatment, either by change of food, use of fertilizers on the pastures, or direct feeding of bone- producing salts, have been demonstrated many times over in a long series of agricultural investigations in which there is such general agreement that individual statement of the results is unnec- essary. The following notes suggest some of the kinds of observa- tions to be found in the literature. PHOSPHORUS METABOLISM 585 According to Moussu and Dollar (1905) osseous cachexia was known in Norway as early as 1660. Koloff (1866) recognized pregnancy, lactation and digestive disorders as predisposing conditions, and (1869) doubled the calcium and phosphorus content of meadow grass by the use of fertilizers. E. Voit (1880), experimenting with dogs and pigeons on lime- poor and other diets, produced rachitic conditions by withholding lime.^ Animals of the larger breeds suffered more acutely from lime deficiency than those of the smaller, slower-growing breeds. The bones were decreased in phosphorus, magnesium and iron as well as calcium, by the calcium starvation. Nessler (1873) demonstrated the connection of this disorder with the nature of the underlying rocks from which the soil was derived. Stilling and von Mering (1889) produced malnutrition of the bones in a dog by use of a lime-poor diet. Maier (1894) reports this disorder in pigs fed on low-calcium foods. Von Seelhorst, Georgs and Fahrenholtz (1900) showed that the moisture of the soil affected the phosphorus of the forage. Phosphatic slag gave best results as a fertilizer, through increasing the phosphorus of the plants. Lane (1906) discusses this bone disease in army horses, mules and donkeys in South Africa. Dibbelt (1911) found that pups in utero developed normally while the mother lived on such a low-calcium diet as caused puerperal osteomalacia. Schenke (1903) quotes Pott and Dammann in their recom- mendations of the amounts of precipitated calcium phosphate to feed to farm animals. Pott advises its constant use with young animals ; swine for 6 months, colts and calves for the first year. ' Dammann advises feeding to a pregnant and milk-giving sheep 12-20 gm., a sow the same, a mare and a cow 30-50 gm. Wherever there is fear of disturbance of normal bone formation, foals and calves may be given 8-15 gm. daily, and pigs and lambs 3-6 gm. For young fowls Pott advises a teaspoonful of bone meal for each 12-14, for hens 2-3 gm. each. Under normal conditions such additions are not necessary, but with food poor in calcium or phosphorus they may be needed. Schenke summarizes a large number of investigations on the use of precipitated calcium phosphate for animals. Dobrowolsku (1911) has found that phosphorized cod-liver oil is fairly efficacious in relieving the experimental malnutrition of the bones which results from the establishment of fistulae in the alimentary tract. 536 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 See also the following articles having to do with malnutrition of the bones in domestic animals: Bopp (1838), Haubner (1854, 1867), Nessler (1866-71), H. Bauer (1868), Karmrodt (1867), Stoh- mann (1869), Tripier (1874), Haubner (1875), C. Voit (1877b), J. Cohnheim (1882), Stutzer (1888), Kellner, Kohler and Barnstein (1894, 1895), Lafevre (1894), Winkler (1891), Bongartz (1894a), Uhlich (1895), Robert (1895), Bonnetat (1903), Moussu and Dollar (1905), Alquier (1905-6), Grandeau (1905), Lewite (1905-8), Klimmer and Schmidt (1906), Ingle (1907, 1908, 1909), Scheunert, Schattke and Loetsch (1911), and Neubauer and Hillkowitz (1912). We have not considered infectious osteoporosis and "big-head." MENTAL AND OTHER NERVOUS DISORDERS In considering the influence of mental diseases on the phospho- rus metabolism of the body as a whole, as measured by the excreta, we should bear in mind the facts that the central nervous system is not highly vascular, and not the seat of intense metabolic change, that it contains but a very small part of the total phosphorus of the body, and that violent functional disorders are accompanied by but slight observable change in its chemical composition. There is, therefore, no reason to anticipate marked alterations in the total phosphorus outgo directly related to changes in mental condition. We have at hand, however, certain evidence which bears on this matter, and we submit the same, in brief, with the suggestions that such effects of mental disorder as have been observed on the phosphorus outgo are at least largely due to the indirect influence of' the disease on general metabolism, especially on muscular activity, and on secretion and absorption in the alimentary tract, with the result of altered rates and paths of elimination ; that in some cases the mental disorder may be the effect rather than the cause of a metabolic disturbance; and, further, that in the absence of feces analyses the urine data are of doubtful value in this relation. NERVE DEGENERATION AND PHOSPHORUS METABOLISM In the search for recognizable products of nerve tissue katabo- lism, which might serve as measures of this process, considerable attention has been bestowed upon choline, particularly in the cerebrospinal fluid, since Noll, Mott, Halliburton, Donath and others have demonstrated a slow autolytic liberation of lecithin and choline in degenerating nerve tissue. It has been suggested that choline, which is slightly toxic, and the highly poisonous neurine, which differs from choline by one molecule of water, may contribute to the production of the intoxication of fatigue, as well as acute mental and nervous disorders. PHOSPHORUS METABOLISM 537 Coriat (1904a) found that choline was produced by autolysis of nerve tissue, and also by putrefactive decomposition of lecithin or tissues rich in lecithin, but not by cleavage by pepsin or trypsin. Nesbitt (1899b) and Hoesslin (1906) have found choline and neurine as products of intestinal putrefaction, and Kutscher and Lohmann (1906) have demonstrated neurine in the urine of human beings. According to Halliburton (1905) the existence of choline in nor- mal cerebrospinal fluid, and in salt water extracts of fresh nervous tissue, implies chemical activity in living nerve substance, the more active part, the gray matter, yieldng the greater amount of choline. Halliburton considers that the cerebrospinal fluid functions as the lymph of the brain, and that the increased choline content of the cerebrospinal fluid in dementia paralytica is directly due to decom- position of brain substance. In harmony with this idea is the find- ing by Donath (1904) of a coincident increase of phosphoric acid in cerebrospinal fluid in cases of degenerative lesion, the phosphoric acid also being considered as a product of lecithin decomposition. PHOSPHORIC ACID (P 2 5 ) CONTENT OF CEREBROSPINAL FLUID IN DISEASE (Donath, 1904) Grams Diagnoses Maximum Minimum Mean Anaemia Neurasthenia Epilepsy Hysteria Pulmonary tuberculosis with melancholia Water on the brain Sclerosis multiplex Tumor on the brain Tabes dorsalis Progressive paralysis 0.0070 0.0278 0.0209 0.0093 0.0026 0.0116 0.0051 0.0286 0.0426 0.0508 0.0064 0.0028 0.0036 0.0065 0.0026 0.0082 0.0045 0.0068 0.0046 0.0052 0.0067 0.0153 0.0086 0.0076 0.0026 0.0100 0.0048 0.0177 0.0203 0.0219 These figures show the highest phosphoric acid content of the cerebrospinal fluid in those diseases which are accompanied by rapid degeneration of nerve tissue. For comparison with the above we have Mestrezat's average figure (1911) for the percent of P 2 5 in 20 normal cerebrospinal fluids of 0.003. Mott and Halliburton (1901) found choline in the cerebrospinal fluid and also sometimes in the blood in cases of severe nervous lesion, especially in paresis, and Donath (1903b) considers choline a factor in the causation of epileptic seizures, though he did not find choline in the cerebrospinal fluid in functional disorders not associ- ated with tissue destruction. See also Masoin (1904). 538 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Mott and Halliburton (1901) studied Wallerian degeneration, both chemically and microscopically. In a number of cats they severed both ischiadici. The animals were killed after different intervals of time had elapsed after this operation. The normal blood of the cat contains but the merest trace of choline, but in three or four days after the operation it became quite evident, at which time degeneration of the nerve was recognizable by Marchi's reaction. These symptoms were most pronounced in the nerve which had been severed for 8 days. Coincident with the increase of choline in the blood was an increase in the moisture content of the nerve and a decrease in the phosphorus content of the solid substance of the nerve. Regeneration was fairly complete after 100-106 days, the first sign of the return of phosphorus being the beginning of the response to the myelin reaction on the 60th day. In the normal nerve the phosphorus content is a little over 1 per- cent; in the regenerated nerve a little less than 1 percent. The moisture content returns to normal when regeneration begins. These authors state that excessive degeneration of nerve tissue, either in general paralysis of the insane, or in other diseases, is accompanied by an accumulation of choline in the blood, and of loss of phosphorus in the nerve accompanying the change of lecithin to non-phosphorized fat. Noll (1899) also conducted similar experiments with dogs and horses. Mott and Barratt (1899) studied the chemistry of nerve degen- eration by analysis of the spinal cord after hemiplegia. (See table, p. 539.) Their conclusions were as follows: "On the degenerated side of the cord in simple hemiplegia it was found that (1) a breaking up of the phosphorized fat occurs; (2) the amount of lecithin present is diminished; (3) the amount of fat present is in excess ; (4) the amount of extractives soluble in ether is increased; (5) the proteid residue diminishes in amount pari passu with the increase in extractives; (6) the phosphorus in the residue diminishes at a still greater rate than the residue itself; (7) the percentage of phosphorus in the half -cord as a whole is dim- inished ; (8) the ether extract has an appearance of butter instead of being crystalline." Coriat (1904b) has studied the cerebrospinal fluid in different nervous diseases, 29 cases being investigated. Choline was found in 25 and quantitatively determined in 21 ; was absent in alcoholic depressive hallucinosis, a senile paranoiac condition, melancholia and senile dementia. It was invariably present in general paralysis, PHOSPHOEUS METABOLISM 539 but no parallel could be observed between the amount of choline, the weight of the brain, and duration of the disease. The largest amounts of choline seemed to be associated with the highest per- centages of protein, both being considered as measures of the extent of destruction of the central nervous system. Cholesterin was always absent. See also Apelt and Schumm (1908) and Coriat (1903), (34 cases discussed, 21 titles and references). LECITHIN AND PHOSPHORUS DETERMINATIONS ON THE TWO HALVES OF SPINAL CORD AFTER LEFT HEMIPLEGIA Mott and Barratt (1899)— Percent Case Lecithin P in ether extract P in residue P in half-cord Left side Right side Left side Right side Left side Right side Left side Right side I II 18.9 20.7 22.2 23.5 1.72 1.84 2.14 1.93 0.92 1.03 0.97 0.98 1.19 1.38 1.38 1.42 Ziveri (1909) also found choline in the cerebrospinal fluid in paralysis. Symmers (1904-5a, 1904-5b) reports that in nervous diseases of the degenerative type the absolute and relative amounts of organic phosphorus of the urine are increased, sometimes to an enormous extent, to such amounts as he considers could not be derived directly from the destruction of nervous tissues, but rather from increased production of phosphorized endogenous metabolic compounds, or as a result of lessened oxidation, with organic phos- phorus compounds as end products. W. Koch and W. H. Goodson (1906) published a prelim- inary study of nerve tissue degeneration. A normal human brain was compared with the brain of a paralytic, and a healthy spinal cord of a dog with a degenerated cord. (See numerical data p. 130.) They found a smaller content of solids in the degenerated brain than in the normal. Nucleoproteins were increased, according to the authors, by leucocytes, proliferating blood vessel elements and neu- roglia cells. The cord of the dog, which was allowed to degenerate for 19 days after cutting, had a composition like that in the degenera- tion of general paralysis. The proportions of constituents remained unchanged, but the absolute amounts were reduced. The authors state in conclusion, "Nervous tissue more than any other tissue, both in pathological and in experimental degeneration, tends to keep its relative composition constant, which observation is in harmony with the results obtained in starvation." 540 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 W. Koch and Mann (1909) published analyses of brains of nor- mal human beings and also of brains of four cases each of dementia praecox and general paralysis. (See p. 132.) The variations in the diseased brains were within the range of variation in the normal brains. The present state of knowledge of the chemistry of the brain and of methods for its study do not begin to answer the require- ments of such an investigation. Pighini (1911) tested for esterase the blood serum in various nervous diseases such as epilepsy, idiocy from contusion of the brain, dementia praecox, alcoholism, etc., by the speed of reaction against neutral sodium monobutyrate, as shown by developing acidity; and compared these observations with others on normal serum. In most of these cases the action was slower than normal, although quite evident. Similar tests with a lecithin emulsion in sodium chloride solution showed the presence in all these cases of a lipase which would split lecithin, but only in the presence of a small amount of MnS0 4 . It reached its maximum activity within the first hour. The optical method (Pighini, 1910) was used for testing the serum for nuclease. In cases of alcoholism and epilepsy there seems to be less activity of the nuclease of the serum than is normal, dur- ing the interval between attacks ; while in acute maniac depressive insanity, and in the period following epileptic attacks, this activity is increased. Pighini and Nizzi (1912) report that neither in normal condi- tions nor in dementia praecox, epilepsy or progressive paralysis is there a specific esterase or lecithase in the cerebrospinal fluid. PHOSPHOEUS METABOLISM IN EPILEPSY Lepine and Jacquin (1879) made a study of the proportions of phosphorus and nitrogen in the urine of 10 hospital patients and a number of dogs. In the case of certain epileptics, the relation of phosphorus to nitrogen is much lower than normal during the inter- vals between attacks. In the case of the same patients the relative amount rises remarkably immediately after the attack. Sometimes, without there being an attack, but only a threat of an attack, the relative amount rises, and this rise may be due to the alkaline earth phosphate. Lailler(1884) reached similar conclusions, to the effect that at the time of the crisis or immediately thereafter, the urine con- tains an increased amount of phosphorus and a small amount of PHOSPHORUS METABOLISM 541 urea, but when the crises succeed each other rapidly both phospho- rus and urea are increased, while in the intervals between crises the urine is normal. Mairet (1884c) investigated metabolism in various mental dis- orders. In epilepsy he found during attacks an increased urinary excretion of nitrogen and phosphorus. Loewe (1910) also found an increased excretion of phosphorus in the urine of the day of the attack, both organic and inorganic phosphorus sharing in the increase, the latter, however, more than the former. W. Koch (1904) published a brain analysis of an epileptic who had died in an attack. The analyses are submitted as provisional, and especially as illustrative of the methods of analysis. COMPOSITION OF HUMAN BRAIN (EPILEPTIC) Koch (1904) Percent Corpus Callosum Cortex (Prefrontal) Water 67.97 84.15 Simple proteids 3.20 (by difference) 5.00 (by difference) Nucleoproteids 3.70 3.00 Neurokeratin 2.70 (Chittenden) 0. 40 (Chittenden) Extractives 1.51 1.58 Inorganic salts 0.82 0.87 Lecithins 5.19 3.14 Kephalin and myelin 3.49 0.74 Amido lecithins trace trace Phrenosin and kerasin 4.57 1.55 Cerebrin acids trace none Cholesterin „ 4.86 0.70 Sulphur compound 1. 40 1. 45 99.41 102.58 Loewe (1911) determined total and organic phosphorus in the urine of 33 subjects for 1-12 days after attack of various psychoses and neuroses. There was noted an increased organic phosphorus elimination after epileptic and some other attacks, and a probable increase in certain phases of paralysis and delirium tremens, though no increase in a number of other diseases in the psychiatric realm, as for instance in kakatonia. A. Bornstein (1911) found also a slight increase in the serum lecithin in epileptics. This increase of the lecithin content of the blood serum is regarded as indicative of increased katabolism of brain lipoids. PHOSPHORUS METABOLISM IN PARALYSIS Ewald (1883) determined phosphorus in the urine in 8 cases of paralysis agitans, chorea and senile tremor in periods of 2-33 days, also in four controls in periods of 12-23 days. The urinary phos- phorus excretion was not in any way characteristic. Gurther (1883), in somewhat extended studies, reached the same conclusion with cases of hystereoepilepsy and paralysis agitans. 542 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Robert and Parisot (1906) studied 4 cases of paralysis agitans with trembling and one without. In the former cases they thought that the urinary phosphorus excretion was subnormal, but the basis for their conclusion is slight or of doubtful value. Barratt (1899), studying the water and phosphorus in the central nervous system in general paralysis of the insane, and other conditions, found that there was a decrease in the phosphorus of the hemispheres of the brain associated with evidence of nerve-degen- eration and of chromatolysis ; and with increase in percent of water, but not proportional to any one of these conditions, or to the age of the patient, or the wasting of the hemispheres. A similar condi- tion was found in the spinal cord. Halliburton (1905) says that in general paralysis the cerebro- spinal fluid is richer than normal in solids, especially in proteins, the most abundant of which being nucleoprotein ; and much richer also in choline, both nucleoprotein and choline being considered as products of degeneration of nerve substance. See also Mott and Halliburton (1901) and Halliburton (1901b). Glikin (1909a) estimated the lecithin of the marrow of the long bones of diseased persons. He found in five cases of dementia paralytica and in one of tabes (ages 33-43) no lecithin or only a trace, while in five others (ages 30-43) he found 1.195-4.21 percent. Where lecithin was absent phosphorus and iron were also absent. Glikin gives the following figures for the lecithin content of the fat of bone marrow of normal men. Age 34 3.30 percent lecithin Age 56 2.02 percent lecithin Age 61 2.21 percent lecithin Age 70 2.33 percent lecithin Age 70 2.76 percent lecithin Age 88 1.83 percent lecithin A. Bornstein (1909) found an increased lecithin content of the blood serum in paralysis. Lecithin was determined by the Glikin chloroform method. He gives as the normal content of lecithin in blood serum 2.0-2.4 percent; in progressive paralysis he found 2.7- 3.5 percent, and in taboparalysis 2.9 percent. In a later publication (1911) Bornstein reports 11 cases of paralysis in which the lecithin content of the blood serum was deter- mined. Two were considered as within the normal range of varia- tion, these being 1.86 and 2.38 percent, while the remaining 9 cases ranged from 3.07-4.60 percent lecithin in the serum. PHOSPHORUS METABOLISM 543 PHOSPHORUS METABOLISM IN INSANITY Mairet (1884c), in studying phosphorus metabolism in mania, divided the observations into four periods covering different por- tions of the attack of the disease, as follows : agitation, depression, remission and convalescence. The results of this investigation are indicated by the accompanying table. URINARY EXCRETION IN MANIA Periods Nitrogen Phosphorus Total Alkaline earth Alkali Increased Diminished Increased Increased Diminished Increased Diminished " " In melancholia there was an increase in the alkaline earth phos- phates, and a decrease of the alkali phosphates of the urine. Lailler (1884) reached similar conclusions; that in acute delir- ium or mania there is a marked excess of urea and of phosphorus in the urine, in mania with excitement a slight excess of phospho- rus only, while in simple mania, or in melancholia without agitation, the urine is normal. In acute melancholia, or with excitement, there is a notable increase in the urea and a slight increase in the phosphorus of the urine. Modica and Audenino (1901) found in 10 cases of insanity, caused by immoral practices, a reduction in the alkali phosphates of the urine, and in certain cases also the total phosphorus. In 5 healthy guinea pigs and 2 healthy dogs, whose frontal lobes had been removed, the alkaline earth phosphates of the urine decreased, and finally disappeared, while the total phosphorus and the nitrogen also diminished. Folin and Shaffer (1902) studied the metabolic accompani- ments of a 48-hour periodicity of general nervous disturbance diagnosed as maniac depressive insanity. On the nervous days there was an increased amount of phosphorus, and an increased pro- portion of nitrogen and sulphur to phosphorus in the urine. The authors concluded, from the constancy of the percentage of alkali phosphates on four consecutive days, that the increase of phospho- rus elimination on the nervous days was not due to katabolism of bone tissue. As explaining the above phenomenon the authors suggest that "there exists in this patient on every second day a condition some- what analogous to diabetes, in virtue of which the system, or some part of it, is unable to assimilate (organize) a part of the phosphate 544 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 absorbed from the digestive tract. The non-assimilated phosphate is eliminated on the same day, and the total amount of phosphate eliminated on the 'nervous' days is therefore greater than the amount eliminated by a normal person absorbing the same amount of phosphoric acid from the digestive tract. On the alternating days, on the contrary, a correspondingly less amount of phosphate than the normal is eliminated, because on those days the system repairs the loss sustained on the preceding days." The authors suggest further that it is the nervous tissues which are subject to the abnormal periodicity of ability to assimilate phosphates which produces the above described phenomenon. In view of the small amount (about 12 gm. P 2 5 ) of the total phosphorus of the central nervous system, however, it is unlikely that the very considerable variations (about 0.66 gm. P 2 5 ) in the daily urinary phosphorus excretion could depend directly on the state of nutrition of the nervous system. This investigation included no feces phosphorus estimations. At a later date Folin, Shaffer and Hill (1904), after a careful study of the urines of insane patients and numerous controls, reached the following conclusions: "From a constructive, positive point of view it must be admitted that they teach very little that is tangible concerning mental dis- eases, except for the strong suggestion which they contain that in general paralysis we have a disease which may be associated, at one stage or another, with some demonstrable metabolism disorder. "From the destructive, negative or critical point of view, it is believed that the data given prove the untrustworthiness of all those metabolism experiments, old and new, which report a 'char- acteristic' increase or diminution of any of the urinary constituents included in this research as associated with any particular one of the ordinary mental disorders. "It is not claimed that such abnormal metabolism may not exist, but simply that the experiments recorded in the literature are insufficient to demonstrate the fact." Nizzi (1912) reports that the elimination of nitrogen and phos- phorus is increased in the initial stage of maniac-depressive insan- ity and decreased in the chronic stage. E. Mendel (1872) reported urine analyses from 110 patients suffering from various types of insanity and other nervous diseases, and of healthy persons ; also the same from a dog and rabbit with the brain pierced by a -needle. In the light of our present under- standing, however, the results do not seem significant. PHOSPHORUS METABOLISM 545 ADMINISTRATION OP PHOSPHORUS COMPOUNDS AND OTHER DRUGS IN MENTAL AND NERVOUS DISEASES Pfeiffer and Scholz (1899) made a general metabolism and res- piration study of senility and paralysis agitans as affected by thyreoidin.* Figures on phosphorus balance are as follows : AVERAGE DAILY PHOSPHORUS BALANCES IN SENILITY AND IN PARALYSIS AGITANS AS AFFECTED BY THYREOIDIN— Grams Exp. No. and Condition and Treatment Intake Urine Feces Balance duration age of subject P2O5 P2O5 P2O5 P2O5 in days X la- 6 Par. agitans, 57 Without thyreoidin 3.3155 2.3893 3.1798 -2.2374 lb-5 57 With 3.3593 2.8518 2.8703 —2.3628 2a-4 57 Without " 3.9479 2.2340 3.4550 —1.7411 2b-3 57 With 4.3216 2.4538 3.4550 —1.5872 3a-4 58 Without 2.7716 2.0570 2.4057 —1.6911 3b-3 58 With 2.4491 2.2669 2.5385 —2.3563 5a— 6 Healthy 76 Without " 2.0906 1.2095 3.7702 —2.8891 5b-5 76 With 2.2473 1.8921 2.3958 -2.0406 6a-4 81 Without 2.0906 1.3567 2.5346 -1.8007 6b-5 81 With 2.2655 1.7317 2.4873 -1.9535 These results show both classes of subjects to be indifferent to thyreoidin, and that with paralysis agitans there is a marked increase of phosphorus outgo which, on the diet used, appeared in the urine. The authors regard this high phosphorus outgo as due more especially to senility than to paralysis agitans. Gilbert (1901) reports general improvement in neurasthenics from the use of lecithin, either in pills, or in subcutaneous injec- tions. Kaufmann (1902) studied excessive protein ingestion in sub- acute neurasthenia, with a man 22 years old, on various simple diets, to which were added, in certain periods, the whites of 30-39 eggs per day. This excessive protein ingestion caused a reduction in the excretion of phosphorus by both urine and feces. Martinet (1903) writes that phosphate therapy is beneficial both in accidental and in long-standing psychasthenia, but in psychatoxies with excitement phosphate therapy aggravates the pathological state. He bases his conclusions on clinical observa- tions and examination of the urine after administration of officinal phosphoric acid, with sodium acid phosphate added to decrease the acid taste. Donath (1903a) treated progressive paralysis, as well as toxic and infectious psychoses, 9 cases in all, by injection with a salt solu- tion composed as follows : K 2 S0 4 0.25 gm., KC1 1.0 gm., NaCl 6.75 gm., K 2 C0 3 0.40 gm., Na 2 HP0 4 +12H 2 3.10 gm., and distilled water * Thyreoidin is the same as thyroidin, an iodine-free, crystallizable compound from the thyroid; an amorphous iodine- and phosphorus-containing compound from this gland is known variously as thyrein, thyreiodin, thyroiodin, thyroiodinin and iodothyrin. 546 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1000 gm. This solution was nearly isotonic with the blood. The treatment acted as a stimulus to the body functions generally, as indicated by improved heart action and appetite, increased diuresis, and particularly as a tonic to the nervous system as shown by improved handwriting, freshening of defective memory, increased accuracy of speech and singing, and ability to solve problems, etc. Gordon (1906) reported clinical observations on 56 cases of nervous disease under treatment with phosphorus-containing drugs, with and without other drugs. Of the 56 cases 28 were multiple syphilitic affections of the nervous system, 5 were cases of tabes, 14 were neurasthenics, 2 melancholia, 3 cases of obcessions, and 4 of cerebral softening. Phosphorus-containing compounds had a bene- ficial effect on the asthenia, and apparently also improved the special symptoms of the disease itself. Glycerophosphates gave the best results. J. Hoppe (1907b) has studied the effects of adding thyroid tab- lets to the food in treatment of three kinds of idiocy, namely myxoedema, cretinism, and "mongoloid" idiocy. In myxoedema there was a marked increase in the apparent absorption of calcium and phosphorus, and a noticeable growth of the bones. In cretinism there was but slight improvement in the absorption of calcium and phosphorus ; and in mongoloid idiocy none at all. As a result of a vegetable diet and rectal feeding with 5 gm. Na 2 HP0 4 daily, the patient with myxoedema showed improved calcium absorption and a simultaneous increase in the phosphorus outgo in the urine. See alsoTuczek (1884). Peritz (1908, 1908-9a) found an apparent increase of lecithin in the feces in lues, tabes and paralysis ; and also that lecithin injection would reduce the increased lecithin elimination in these diseases. A large number of estimations of the lecithin in the blood serum in the above-mentioned, and in other diseases, were presented (1908-9b). The results were characterized by much variability. Lack of normal controls leaves uncertain the significance of these observations by Peritz. In a later paper Peritz (1910) shows that the lecithin of the blood serum responds readily, by marked increase, when lecithin is administered per os. Taghamuro (1908) administered lecithin by injection in dorsal tabes. There resulted improvement of digestion, appetite and general condition, as well as increase of red blood corpuscles and haemoglobin. Nerking (1909) made an extensive study of the effects of leci- thin on narcosis. With considerable numbers of rats, dogs and rabbits, with the usual use of controls, he treated them with anaes- PHOSPHORUS METABOLISM 547 thetics as follows : ether, chloroform, morphine, scopolamin, ureth- an, urethan-chlorhydrate, novokain-suprarenin, tropakain and stovain, usually as subcutaneous, intraperitoneal or intralumbar injections, and followed these treatments with injections of 1-10 per- cent solutions of purified lecithin. In all cases the lecithin injection is said to have produced a favorable effect on narcosis (1) in short- ening the period, (2) in the alleviation of unfavorable after-effects and (3) in causing a rapid return to normal condition. Donath (1909) experimented with 2-percent sodium nucleate, containing an equal quantity of sodium chloride, by subcutaneous injection in 21 cases of general paralysis, in amounts of 50-100 c.c. at intervals of 5-7 days. The injections caused a rise of tempera- ture, in some cases to 40.5° (average 38.5°), and an increase of leucocytes to 61000 (mean 23000). Out of 21 cases 10 were suf- ficiently improved to return to ordinary duties (the oldest case was of two years' standing) ; in 5 cases there was improvement, while 6 remained unimproved. Klieneberger (1911) and Lowenstein (1911) report negative or unfavorable results from the use of sodium nucleinate, by injec- tion, in progressive paralysis. For the successful use of a lecithin preparation in migraine see Schottin (1911). Leubuscher (1913) gave elemental phosphorus in 10000 parts of oil in 9 severe old cases of epilepsy. Seizures were reduced by 30 or 40 percent in 3 cases, "about 65 percent in two others ; in 3 others the improvement was less striking, while with one patient there was no improvement. The greatest benefit was obtained in the more severe cases. Phosphorus was administered for 15 months with 6 cases. NEPHRITIS Fleischer (1881) found in chronic interstitial nephritis a decreased excretion of phosphorus, and a parallelism between nitro- gen and phosphorus elimination by the kidneys. Sodium phosphate ingestion led to quantitative excretion of the phosphorus within 24- 48 hours in a normal subject, but in nephritis produced little or no increased phosphorus excretion — a difference not noted with respect to potassium bromide and sodium salicylate. In a case of lighter acute nephritis there was a marked lowering of urea and phospho- rus in the urine. . VanAckeren (1890-91) and Kornblum (1892) found neither a parallelism of urea and phosphorus elimination nor a consistent low- ering of phosphorus elimination. 548 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Von Moraczewski (1896) showed that in parenchymatous neph- ritis the urine contains less chlorine and more phosphorus, especially calcium and magnesium phosphate, than normal. In interstitial nephritis, however, there was no increase of urinary phosphorus; in fact a decrease of calcium and magnesium phosphates. Uric acid was found increased in parenchymatous nephritis and normal in the interstitial variety. Mohr (1903) published food and urine analyses from four nephritic patients. The data show plainly that the phosphorus outgo is normally affected by variations in the intake. Von Koziczkowsky (1903-04) found in nephritis that the outgo of phosphorus varied normally with the intake, and that the phos- phorus outgo was indirectly proportional to the sodium chloride retention. Kovesi and Roth-Schulz (1904) found in nephritis that the proportion of the total phosphorus of the food, which appeared in the feces, varied widely without ascertainable relation to food or pathological conditions. In their cases they found almost constant phosphorus retention, reaching very high figures in cases of rapidly developing oedema ; but they noted no relation between phosphorus elimination and the form or symptoms of the disease, or the nitro- gen or sodium chloride of the urine, or the total of dissolved mole- cules in the same. These authors consider it desirable to restrict the absorption of phosphates in nephritis, and they note von Noor- den's suggestion that calcium carbonate be given with the usual milk diet in the accomplishment of this purpose. Von Noorden (1907, II, p. 467) mentions a number of investi- gations, in addition to those which we have seen, in which the capacity of the kidneys to eliminate phosphorus was not lowered, and also several investigations showing that the observation of von Koziczkowsky of the reciprocal relation of phosphates to sodium chloride was not the rule; hence, we are unable to say that there is any invariable character of the urinary phosphorus elimination in nephritis, though a retention of phosphorus, apparently an expression of a lowered capacity of excretion, is very common. The significance of this variation is not known. Erben (1903, 1905) found low values for phosphorus in the blood in subchronic nephritis, in chronic parenchymatous nephritis, and also in cases of secondary shrunken kidneys. J. Miiller and H. Reinbach (1913) studied digestive lipaemia in a case of subacute nephritis. Lecithin was present in the serum to the extent of 0.688 percent. PHOSPHORUS METABOLISM 549 NUCLEIN THERAPY This subject is discussed in connection with normal nuclein metabolism ; see p. 256-258. OEDEMA L. E. Meyer (1905) studied the effect of variations in the water, sodium chloride and phosphorus content of the food of a nursing infant suffering from idiopathic oedema. The addition of 200 c.c. of distilled water per day to the food was without marked influence on mineral metabolism. Where sodium chloride was fed in vary- ing quantities the storage varied directly with the intake; and where sodium phosphate was added, the sodium chloride intake remaining constant, there was marked increase in the storage of both phosphorus and sodium chloride, and a decrease to more nearly the earlier retention figures when the phosphate was with- drawn. Meyer was of the opinion that the above-mentioned stor- age was pathological in character, in that the outgo was not nor- mally responsive to the intake ; that the cause was a functional dis- turbance of the epithelial cells of the uriniferous tubules, resulting in an overloading of the blood with salts, and therefore in oedema; and further, that the treatment for idiopathic oedema should be the decrease of the salt content of the food to the lowest possible amount. EFFECTS OF MISCELLANEOUS ORGANIC PHOSPHORUS-CONTAINING COMPOUNDS AND PREPARATIONS ON PHOSPHORUS METABOLISM TETBA-ETHYL-PHOSPHONITTM IODIDE Lindemann (1898) studied the pharmacology of tetra-ethyl- phosphonium iodide by injection experiments with frogs, rabbits, guinea pigs and dogs. The action of this substance is character- ized by loss of coordination, and general paralysis, which soon extends to the respiratory center and then to the heart, which is invariably found engorged and in diastole. The lungs are strong- ly hyperaemic and oedematous, while in chronic intoxication there are characteristic changes in the liver and kidneys, consisting in hydropic, albuminotic degeneration of the protoplasm. A full statement of details is given. This substance is excreted in the urine together with a compound giving an insoluble precipitate with barium, and a volatile base, which, with sulphuric acid, forms a non- volatile salt. The only marked pathological change in the urine was a decided haemoglobinuria, appearing in the last stages of chronic intoxication. It is therefore shown that neither the lower oxidative stages of phosphorus, nor phosphine, nor the organic phosphorus compounds have the action of elemental phosphorus. 650 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 FEESAN Kornauth and Czadek (1900) experimented with fersan, a beef -blood preparation containing much iron and phosphorus in a mixture of organic combinations. With this preparation two experiments were conducted, one on geese and another on a man. Two geese were fed 240 gm. corn each per day. In addition one received pellets of fersan. In two 7-day periods the one receiv- ing corn alone gained 70 and 230 gm. in live weight, while the one receiving the fersan with its corn gained 195 and 235 gm. during the same periods. In four balance experiments of 6-15 days duration with a man, two periods with fersan and two without, there was no evidence that this preparation could improve a mixed diet containing an abundance of meat, though the authors state that it was almost completely absorbed and assimilated. EOBOEAT Laves (1900) studied the nutritive value of prepared proteins, roborat (prepared from grain; contains lecithin), tropon (from flesh and blood), aleuronate (from wheat), and plasmon (from milk). Experiments of short duration were conducted on the author and on a dog, with artificial digestion tests. Urine analyses furnished the basis for conclusions. The results are not significant. OAEBOHYDEATE-PHOSPHOEIC ACID ESTEES Euler (1912b) investigated the disposition by animals of the carbohydrate-phosphoric acid ester synthesized by yeast. His con- clusions are in part as follows: This ester is split by an intestinal enzyme and by Bacterium coli; also by an enzyme contained in the kidney of the horse. The cal- cium salt of this ester is broken down and the inorganic phosphate excreted as such. The calcium salt of glycerophosphoric acid ester is likewise broken down by Bacterium coli. The calcium salt of the phosphoric acid ester when fed to dogs goes over largely into the urine as inorganic phosphate. From a study of the literature Euler concludes that organic phosphorus in the food is absorbed both split and unsplit. He sug- gests that "it is especially to be emphasized that the physiological effects of phosphates are to be sought in their remarkably strong influence on the vital enzyme reactions." Euler and Funke (1912) report an experiment with the carbo- hydrate-phosphoric acid ester formed by phosphatese, the compound being fed to a rabbit. At least three-fourths of the ester under- went cleavage. For discussions of nuclein, lecithin and phytin therapy see pages 256, 528 and 315 respectively. PHOSPHORUS METABOLISM 551 PROTYLIN Protylin is a phosphorus-containing preparation obtained by the dialysis of egg-albumin after protracted treatment with ortho- phosphoric acid. It is a white, tasteless powder, insoluble in water, and contains 2.7 percent of phosphorus. Gnezda (1903) reported clinical observations on 11 cases of cachexia with carcinoma, hysteria, anaemia, nervous dyspepsia, etc., under treatment with protylin, which is said to have proven itself a valuable tonic, as evidenced by increase of appetite, body weight and haemoglobin, and improved psychic conditions. Siegmann (1904) published similar observations on 32 cases, as also did Marian von Bilgorajski (1904) on 10 cases, the latter recommending especially the iron and bromine compounds. See also Dorn (1904). Laumonier (1905) submits other clinical data. Pouchet and Chevalier (1905) conducted injection experiments with a protylin solution, 38 gm. protylin and 2 gm. soda per liter of water, with dogs. After injection for 2 hrs. 35 minutes into a chloralized dog a sharp decrease in blood pressure occurred, and an accelerated and subsequently retarded cardiac action. Afterward the blood pressure slowly became normal, if the injections were continued more feebly, the cardiac action being accelerated simul- taneously. In the case of a dog with section of the pneumogastric nerves there took place a more marked acceleration of cardiac action, the blood pressure decreased less ; on the other hand a lessening of the cardiac energy followed, which caused the heart to fail to become completely filled. These authors consider that the action of this phosphorus preparation on the circulation accounts to a great extent for its influence on the general nutrition. Timpano (1906) reports increase of appetite and of strength, reviving of psychic activity, and improvement of general state of nutrition from the feeding of protylin to 18 cases, and Gallenga (1906) made similar observations on 22 cases. R. O. Neumann (1906) showed that protylin, when used in varying amounts to replace equivalent amounts of cheese protein in a mixed ration, does not increase either nitrogen or phosphorus retention. When added to a full diet, however, its nutritive value was apparent, and both nitrogen and phosphorus storage were increased. Fjodoroff (1907) determined that the ingestion of lecithin, nucleic acid and phytin often causes a slight increase of free hydro- chloric acid in the stomach, sufficient in cases of chronic 552 OHIO EXPEKIMENT STATION: TECHNICAL BUL. 5 hyperacidity to cause heart-burn, and to constitute a contraindica- tion of their use. In such cases protylin, even in 5-gram doses, is well tolerated. See also Maestro (1905a), Wechsler (1905) and Laguesse (1905). OSTEOMALACIA Osteomalacia in human beings is a disease of entirely unknown cause, one of the prominent symptoms of which is an absorption of the salts of bone, and their replacement by tissues not having the normal content of bone salts. It is not due primarily to lack of phosphorus or calcium compounds in the food, nor to rapid growth, or pregnancy, or lactation, or senility, though naturally these may all be contributory or accentuating conditions ; and it is not curable by the use of foods which are rich in bone-forming constituents. The literature of this disease is very voluminous, and of it we have seen but a small part. There is little variety in the experi- mental findings, and our materials are perhaps typical of the whole body of facts as published. LACTIC ACID IN OSTEOMALACIA An early idea regarding human osteomalacia was that the bone salts were removed by an excess of acids in the system, and lactic acid seems occasionally, though not as a rule, to have been found in the blood, urine and bones. C. Schmidt (1847) reported a case of bone disease involving all of the bones of one leg, resulting in the absorption of nearly the whole of the mineral substance, the periosteum remaining intact. Analysis showed the soft inner portion of the bone to contain much lactic acid; but Virchow (1852) discusses a fatal case of puerperal osteo- malacia in which the bones had become filled with a soft jelly-like substance of alkaline reaction. O. Weber (1867) found lactic acid in osteomalacic bones, and Steiner (1869) made an anatomical study of senile osteomalacic bones, and concluded that the disease was due to removal of the lime salts by lactic acid. Note: Osteomalacia of the domestic animals, which is also known as malnutrition of the bones, halisteresis of the bones, fragility of the bones, and osseous cachexia, differs from human osteomalacia. This malady is without doubt due principally to deficiency of the food in bone-forming constituents, though perhaps also in part to a lack of correct proportion between the mineral elements, bone-forming or otherwise, contained in the food. This condition is readily curable, simply by the removal of the cause, though in the course of time affected animals may become more or less unresponsive to improved conditions of diet. The principal predisposing conditions are pregnancy, lactation, growth, starvation, and unhygienic surround- ings. The use of the term osteomalacia for this simple malnutrition of the bones is confusing and should be abandoned. PHOSPHORUS METABOLISM 553 Mors and Muck (1869) found much lactic acid in the urine of one case of osteomalacia, a little in the urine of another, and none in the urine of a third case. They consider excessive production of acid to be the cause of osteomalacia. In one case, which was cured by the use of a nourishing diet, cod-liver oil, and a salt mixture of calcium carbonate, calcium phosphate and ferric hydrate, the lactic acid disappeared from the urine as the bones began to harden. See also von Jaksch ; Ueber die Alkalescenz des Blutes; Zeitschr. f. klin. Med., 13, 355. Heitzmann (1873) administered lactic acid subcutaneously, and with the food, to 7 cats, 5 dogs, 2 rabbits and 1 squirrel, the food being low in lime ; and the experiments were continued for sev- eral months, in one case at least, 13 months, followed by examination of the bones. After two weeks' administration, by either method, the car- nivora showed rachitic symptoms, swelling of the epiphyses of the long bones and of the union of the costal cartilage with the ribs. The swellings mentioned increased visibly by the 4th and 5th weeks ; the long bones became crooked, and the microscopic examination of the epiphyses agreed perfectly with that of rachitic children. After 4 to 5 months' feeding of the acid the long bones became as flexible as fish bones. The microscopic structure of the bones after 4 to 11 months' feeding of lactic acid was analogous to that of people who had died from osteomalacia. With the three herbivora the case was different. The walls of the long bones of the rabbits became very thin, but remained brittle; while with the squirrel, which was fed for 13 months, the walls of the bones became very thin and also flexible. Unfortunately in these experiments there were present two pos- sible demineralizing agencies, and it is impossible to say whether the results were due to calcium starvation alone, or in part to the effects of the acid. Vogt (1875) injected into the tibiae of living rabbits, five weeks old, 2 drops of pure lactic acid. The rabbits were killed 5 months afterward. The periosteum was rather easily separable from the rough surface of the bones, and there appeared to have been hyperplasia of the bone tissue, the bones having increased to nearly 3 times the size of the uninfected tibiae. Parallel punctures without acid produced no such result. All things considered, however, these facts probably no longer have significance. Rudolph Wegner (1876) reports that 1:3 or 1:5 lactic acid solution injected under the periosteum causes solution of the bony substance, 1:10 acid showing no such effect. 554 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Heiss (1876) investigated the question as to effect of lactic acid ingestion on the composition of the bones of a dog. The feed- ing of lactic acid covered 308 days. The body of the dog was then analyzed. The acid was administered at first in quantities of 1-2 gm. daily, but during the greater part of the test in quantities of 7-9 gm. daily. The author discovered no solution of the mineral constituents of the bone by the lactic acid. Langendorff and Mommsen (1877) reported a study of a fatal case of osteomalacia in a man 35 years old. Lactic acid was not found in the bones. Baginsky (1881) fed 3 young dogs, all from the same litter, on a ration which was nearly free from lime. To this diet was added, with one dog, 2 gm. lactic acid per day, with another 2 gm. calcium phosphate, and the third received the basal ration alone.. The calcium starvation caused by the basal ration led to a reduction of the ash of the bones, and with the dog receiving lactic acid this effect was said to be still further emphasized. Hofmann (1897) cites cases of osteomalacia in which no lactic acid was present in the urine. Klotz (Jahrb. f. Kinderheilk., 70, 1-61) found that large doses of lactic acid disturb fat and mineral metabolism. The disturb- ance of the fat metabolism may have been due to the neutralization of the alkali carbonates by the acid, with the consequent formation of insoluble calcium and magnesium soaps. The phosphoric acid, no longer combined with the calcium as insoluble phosphate, could combine with the alkalis to form soluble compounds, thus increasing the absorption of phosphorus. Siedamgrotsky and Hofmeister (1879) published results of experiments with sheep and goats which received in addition to a ration of normal foods and pasture grass either lactic, sulphuric or hydrochloric acid. The results are in the nature of analyses of the bones, balance experiments and analyses of the milk. In our opinion the results are inconclusive. Moritz Levy (1894) finds that lactic acid removes carbonate more rapidly from normal bone than it does phosphate, and that therefore there is no solution of bone salts by an acid in osteomal- acia. This view of the matter prevails at this time. Bonnamour and Escallon (1913) studied, with a rabbit, the effects on the bones, of intravenous injection of lactose. During 3 months one rabbit received 378 gm. of lactose in this manner in doses of 10 gm. each. Analysis of the bones revealed the follow- ing: PHOSPHORUS METABOLISM 555 COMPOSITION OF BONES AS AFFECTED BY LACTOSE INJECTION Pecent of Dry Bones Control Treated CaO 31.66 24.8 MgO 0.60 3.04 p 2 5 16.63 25.5 These data imply a decalcification and an increase of magne- sium and phosphorus. COMPOSITION OP THE BONES IN OSTEOMALACIA A considerable number of analyses of osteomalacic bones have been published. One of the earlier studies is that of Mors and Muck (1869). Their figures show considerable variation in the proportions of the different mineral constituents, with a marked loss of mineral matter generally, and of calcium, a less marked loss of phosphorus, and an increase in magnesium. See also von Gohren (1865), Huppert (1867) and Regnard (1879). The method by which these changes are produced, as stated by Pommer (1885), is through a continuous building of new lime-free bone tissue, a cessation of the deposition of lime, and a local removal of lime from parts containing it. See also von Recklinghausen (1891). Moritz Levy (1894) submits extensive figures on the composi- tion of osteomalacic bones, and numerous citations of the work of others. He concludes that the relation 6 PO 4 :10 Ca, of normal bones, remains the same during osteomalacia, and that the removal of phosphate takes place in the same quantitative relation as that of the carbonate. Levy's own figures, however, show changes in the proportion of phosphorus to calcium of various degrees up to one-twelfth of the total. Galimard and Konig (1905) publish analyses of bones from a case of infantile osteomalacia which show a marked change from the normal relation of calcium, magnesium and phosphorus. The following figures are from this article. PERCENTAGE COMPOSITION OF NORMAL AND OSTEOMALACIC BONES— Fat- and Water-free Basis Normal Femur Osteomalacic Femur Ossein 33.35 55.03 Phosphate of lime 55.84 38.07 Carbonate of lime 6.33 6.38 Phosphate of magnesia 0.79 0.49 McCrudden (1906a) found in the osteomalacia of horses, in harmony with the conclusions of Roloff (Virchow's Archiv, 37, 433), Huppert (Archiv der Heilkunde, 1866, 1867, 8, 345) and 556 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Chabrie (Les phenomene chimique de Fossification, Paris, 1895, p< 65), that there was a decrease of calcium and phosphorus, and an increase in magnesium and sulphur. The following data are from this article. PERCENTAGE COMPOSITION OF RIBS OF HORSE, NORMAL AND OSTEOMALACIC Osteomalacic Normal CaO I 20.09 0.50 16.55 0.35 II 18.35 0.46 16.00 0.38 I 33.48 0.11 23.66 0.11 II 33.12 MgO 0.10 P2O5 23.22 s 0.09 Cappezzuoli (1909b) publishes analyses of long bones and flat bones from a case of osteomalacia. He found a general demineral- ization of the bone, but a greater proportionate loss of calcium than magnesium. In the long bones he found Ca:Mg: :100:2.66 and in the flat bones Ca:Mg: :100:3.85, while in the normal the ratio of Ca to Mg is as 100:1.14. McCrudden (1910b) published a chemical analysis of bone from a case of human osteomalacia. The results are as follows : CaO.. MgO.. P205.. s These figures show a great decrease of calcium, a less marked decrease of phosphorus, and four-fold increase of both magnesium and sulphur. While the cause of the deposition of the salts in bone is not definitely known we must admit (1) that there resides in the osteogenous tissue some property which acts like an affinity for the bone salts, (2) that this affinity tends to maintain a constant, proportion between the salts deposited, but (3) that variations in the composition and reaction of the blood, as affected by disease, modify the character of this affinity, as indicated by the composi- tion of the bones, in definite and consistent ways, even if not to unlimited degrees. METABOLISM IN OSTEOMALACIA Mohr, in von Noorden's "Metabolism and Practical Medicine," tabulates calcium, magnesium and phosphorus balances by von Lim- beck (1894), S. Neumann (1894a, 1894b) von Korczynski (1902), PHOSPHORUS METABOLISM 557 Sauerbruch (1902), Goldthwait et al. (1905), and Hotz (1906). Improved physical condition of the patients was usually accom- panied by increased retention of the bone salts. Phosphorus reten- tion was commonly disproportionately large, as compared with calcium, and both may be retained in moderately severe cases. Von Korczynski (1902) reports balance data with two cases of osteomalacia snowing that if the disease is not too far advanced, and the course of the disease not rapid, and no marked cachexy is apparent, the organism retains phosphoric acid. Earlier metabolism studies showed considerable losses of cal- cium, magnesium and phosphorus, not only in the urine but also in the feces, in bronchial mucous in cases associated with bronchial catarrh, and also in the milk. See Pagenstecher (1862), Gusserow (1862), Schmuziger (1875) and von Limbeck (1894). Raspopoff (1884) finds the proportion of the urinary phospho- rus united to alkaline earths greater, and that united to the fixed alkalis less, in osteomalacia than the normal. See also Raspopoff (1885). Metabolism studies by S. Neumann (1894a, 1894b) show that a predominating loss of phosphorus may change to as considerable a retention of the same as the case improves, while on the other hand the work of His (1902), on a case of infantile osteomalacia, shows that improvement may be due to improved retention of calcium, the preexisting positive phosphorus balance remaining without striking change. The existence of typical osteomalacia in children was attested by investigations of Siegert (1898), who cites a previous demon- stration by von Recklinghausen, and also by the above-mentioned work of His. Very few studies of the blood in osteomalacia have come to our attention. Fehling (1891) reports subnormal alkalinity in the blood in severe cases; Pende (1905) found improved blood condi- tions as determined by corpuscle count, etc., after phosphorus therapy. Kobler (1888) also publishes blood ash analyses contrasted with the normal, with results as follows : COMPOSITION OF ASH OF BLOOD Osteomalacia Normal (Jarisch, mean of four) 7.2 16.04 19.925 34.160 9.35 0.52 12.85 8.49 Sulphuric anhydride 6.85 29.59 25.565 Soda 23 169 0.872 0.512 7.86 558 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 A somewhat recent idea as to the cause of osteomalacia is that of Hoennicke (1904, 1905) (10 pp. bibliog.), who considers it due to disease of the thyroid gland. Zunz (1913) found in osteomalacia no characteristic variation in protein metabolism. The phosphorus metabolism was quite variable, but a high fecal outgo was rather constant, this elimina- tion being usually reduced by ovariotomy, with increased tendency to retention. TBEATMENT OF OSTEOMALACIA In the treatment of osteomalacia His (1902) notes favorable results from the use of phosphorus, first by Trousseau in 1868, later by Wegner in 1872, cites Sternberg's report (1893) of eight cured cases from the clinics of Busch, Strumpell, Matterstock and Nothnagel, and also his own description of four other such cases, also favorable results by von Limbeck (1894), Kosminski (1896), Bernstein (1898), Heubner (1898), Siegert (1898) and Littauer (1899). He also cites unfavorable reports of the use of phospho- rus in osteomalacia by Gelpke (1891), and Fehling and Wetzel (1899). Other favorable reports from the use of phosphorus in osteo- malacia have been published by Warschauer (1890), Sternberg (1893), H. Fischer (1894), Pende (1905) and Hotz (1906). Treatment with oophorin and thyraden by Senator (1897) led to improvement in the patient, but treatment with oophorin by Bernstein (1898) failed, after which phosphorus therapy caused a cure. Ovariotomy has given good results in a number of cases but there is no established connection between the ovaries and osteo- malacia. Beneficial results seem to be due especially to the preven- tion of loss of calcium and phosphorus from the body through the various incidents of the sexual life. Fehling (1891) reports cases of ovariotomy, and later (1895) 10 cures from 12 such operations. Curatulo and Tarulli (1895) also report favorable results from ovariotomy. S. Neumann (1896) reported metabolism data with cases of ovariotomy in osteomalacia. This treatment is regarded as radical, and not generally to be recommended, though benefit resulted in case the disease was not too severe. Results were unfavorable in advanced cases. Goldthwait, Painter, Osgood and McCrudden (1905) made bal- ance experiments on a 16-year-old osteomalacic, both before and after ovariotomy. Before the operation there was a marked loss PHOSPHORUS METABOLISM 559 of calcium, a slight loss of phosphorus, and a slight storage of mag- nesium. Some months after the operation the patient was found to be storing calcium, but magnesium and phosphorus determina- tions were not made. These authors consider that their balances indicate a partial replacement of calcium by magnesium previous to the operation. McCrudden (1906b) reports a later metabolism study on the same case of osteomalacia that was studied a year and a half before by Goldthwait et al, and found much improved by ovariotomy. At the time of this later study the patient was once more in a critical condition, and was found to be losing calcium and magnesium, but retaining phosphorus. As to the connection of the ovaries with osteomalacia it was McCrudden's idea that ovariotomy is without influence on the ultimate cause of the disease. In accord with this idea we have the results of Luthje (1903), who showed by the removal of testes and ovaries from dogs that neither organs bear any noticeable relation to phosphorus metabo- lism generally. Curshmann (1911) describes cases of osteomalacia in men and women of middle age and old age. Cure was effected by the use of phosphorized cod-liver oil. In this connection we have also considered the paper of Ogata (1911-12). PHOSPHORUS METABOLISM AND THE PARATHYROIDS The extirpation of the parathyroids reveals an essential relation of these glands to metabolism, the nature of which is as yet but imperfectly understood. The following notes show that they are intimately connected with phosphorus metabolism. Morel (1910) notes that parathyroidectomized animals fail to heal fractures as rapidly as normal animals, the conversion of carti- lage to bone being slower. Removal of the thyroid was without appreciable effect on the bones. Also (1909) administration of thyroid substance was without effect on the bones of adult animals, while the feeding of parathyroid extract to young rabbits favored the growth of the bones independently of the calcium of the ration. Erdheim (1911) found that in rats, after parathyroidectomy, the new bone produced is very poor in calcium, and that the skeleton generally takes on an osteomalacic or rachitic character. T. Bauer (1911) supports Erdheim's findings of parathyroid hyperplasia in human osteomalacia, which hyperplasia is ascribed, to an increased functional demand for calcium. 560 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Erdheim also finds that after parathyroidectomy the dentine calcifies late, or not at all; that the enamel is deficient, and that fractures of the bones are frequent, but that if parathyroids be successfully transplanted into these parathyroidectomized animals the dentine calcifies; if they are then removed the dentine forms without calcium. Toyofuku (1911) made a histological study showing that deficient deposition of calcium is responsible for the structural change observed. These above observations regarding calcium involve, of course, phosphorus, without which calcium could not be used in the normal growth of bone. Greenwald (1911, 1913a) found that parathyroidectomy caused a very marked phosphorus retention, which was followed, but not until the appearance of tetany, by an increased excretion of phosphorus. In another paper (1913b) Greenwald reported that after parathyroidectomy the phosphorus of the blood is increased, the greater part of this increase being in the fraction which is insoluble in the usual lipoid solvents, but is soluble in a mixture of dilute hydrochloric or acetic and picric acids. Some of Greenwald's data are as follows : PHOSPHORUS IN BLOOD FROM A THYRO-PARATHYROIDECTOMIZED DOG AND A NORMAL DOG— Milligrams of Phosphorus per Kilo of Blood Phosphorus Parathy- roidectomized 214 Normal 214B Total 436 162 8 233 33 370 140 Extracted, with alcohol and ether 7 192 31 PHOSPHORUS IN SERUM FROM PARATHYROIDECTOMIZED AND NORMAL DOGS— Milligrams of Phosphorus per Kilo of Serum Phosphorus Parathyroidectomized Normal Dog- 215 Dog- 217 Dog- 219* Dog 216 Dog 218 Dog 220 Total In acetone extract. In acid extract 212 149 62.3 222 128 76.7 291 194 87.7 3.8 157 131 26.9 244 186 44.4 2.4 167 110 45.5 * Complete thyroidectomy in this case. PHOSPHORUS METABOLISM 561 Paladino (1913) considers that after the removal of the thyroid and parathyroid glands urinary phosphorus elimination increases greatly, but Greenwald (1913c) states that the removal of these organs reduces phosphorus elimination, and that there is no increase until the appearance of tetany, the increase then being due to the muscular activity. PHOSPHATURIA Phosphaturia is not a disease; it is a symptom or condition which may result from many causes, normal as well as pathological. It is said to exist when, from whatever cause, there is a sediment of alkaline earth phosphates, which may be mixed with carbonates, in the fresh urine. The immediate cause of the sediment is a relative increase of alkalis or alkaline earths, or a relative decrease of acid elements. In either case the increase or decrease may be. relative without being absolute; hence the existence of the phos- phate sediment is no indication of the amount of phosphorus in the urine. It may occur with increased or decreased phosphorus so long as there is sufficient relative increase of alkali or alkaline earth. The method of formation of the sediment is a change, under the influence of alkalis or alkaline earths, of a portion of the soluble diacid-phosphates normally present into the insoluble monacid or normal phosphates. This condition may result from any such change of diet as increases to a sufficient extent the carbonates or oxidizable organic compounds containing alkalis or alkaline earths ; or through, any such influence as decreases the acids normally excreted in the urine, such circumstances for instance as the ingestion of much protein, or continued washing out of the stomach, both of which result in the separation from the blood of unusual amounts of hydrochloric acid, and liberate within the blood corresponding amounts of alkali. Another form of phosphaturia, common in children, is due to a deflection of calcium from feces to urine without necessary change in the amount of either calcium or phosphorus, the cause not being established beyond possibility of doubt, but commonly thought to be such pathological condition of the large intestine as results in decreased ability to excrete calcium. Aside from matters of diet and digestive disorder it seems likely that nervous influences affect metabolism in such ways as to cause phosphaturia, either combined or not with the juvenile form above described; and phosphaturia is common in many diseases; for instance, in some cases of diabetes mellitus, in tuberculosis, and in such bone diseases as ostitis and osteomvelitis. 562 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 It is impossible for us to attempt a review of the enormous literature of this subject, but we shall record observations on a few of the more important articles which have come to our attention. One of the more important of the earlier works on phosphaturia is that of Teissier (1875, 1877). He found that it was a condition not particularly connected with alterations in skeleton or nerves. A part of his conclusions are as follows : "Permanent phosphaturia always indicates serious trouble in the general nutrition. The trouble may be profound enough to show all the apparent symptoms of sugar diabetes. But phos- phaturia of the diabetic form is not an essential disease, but js rather a morbid complication which may be sympathetic of various affections. It may be a simple form of nervousness which accom- panies pulmonary phthisis, or may be a precursory sign of it. It may be symptomatic of sugar diabetes, latent or transformed. Diabetic phosphaturia may, like sugar diabetes, exert an evil influ- ence on the progress of traumatic lesions." "Phosphates are abundant in the urine of consumptives at the beginning of the disease ; they diminish somewhat as the period of tuberculous cachexy arrives. They diminish in true chlorosis. They increase in diseases of the brain, and of the marrow. They increase in chronic rheumatism. They decrease generally in the course of fever and ague. They do not increase in spite of most abundant feeding in the course of convalescence; rather they are diminished." Ralfe (1887) classified cases of phosphaturia coming to his attention (1) as associated with disturbances of the nervous system, (2) as associated with pulmonary disease, (3) as alternating or coexisting with saccharine diabetes and (4), as without special con- nection. Sendtner (1888) showed in a single case that excess of lime was the cause of phosphaturia. Peyer (1889) also considered excess alkalinity of the blood as the cause of phosphaturia. Klemperer (1899) considered phosphaturia as due to hyper- acidity and motor insufficiency of the stomach, the former taking abnormal amounts of acid from the blood, and the latter by retain- ing it over-long in the stomach, delaying its return in compensating amount by way of intestinal absorption. Klemperer considered both of these symptoms as due to nervous affection. PHOSPHORUS METABOLISM 563 Leo (1902) reported a case of phosphaturia in which there was abundant phosphate and carbonate precipitate in the urine, the same apparently not being due either to motor insufficiency or hyperacidity of the stomach, since the stomach secretion was alkal- ine at night and early morning. Soetbeer (1902) made a very satisfactory comparison of a child exhibiting phosphaturia with one in normal condition. The phospho- rus in the urine was about the same, slightly higher with the healthy child, but the lime more than two-and-a-half times as much in the urine of the patient as in that of the control. The phosphorus of the feces was the same in both cases, as also was the lime insoluble in water but soluble in hydrochloric acid, while the water-soluble lime was 0.037 gm. in the patient and 0.310 gm. in the control; thus the essential difference in these cases was that the control had excreted water-soluble lime into the intestine, while the patient had excreted the same into the urine, the difference apparently being due to intestinal catarrh in the pathological case. Soetbeer and Krieger (1902) reported a case of phosphaturia where the ratio P 2 5 :CaO was sometimes 1.5 or 2 to 1 instead of 12 to 1, the normal relation, while the urinary elimination of lime rose to almost 0.7 gm. daily, the normal being about 0.2 gm. See also Panek (1900) and De Lange (1903). Gouraud (1903) investigated the ratio of P to N- in the urine of patients suffering from pneumonia, typhoid fever, rheumatism, cerebrospinal meningitis, tubercular meningitis, diabetes, neuras- thenia and false and true phosphaturia. No account was taken of food or feces phosphorus. Gouraud distinguishes between false phosphaturia, ordinarily due to alkalinity of the urine, and true phosphaturia, due especially to (1) dyspepsia, especial- ly hyperchlorhydria, which produces only a minimum increase, (2) tuberculosis, with phosphaturia as a complication, (3) diabetes, in its more serious forms, and (4) nervous states, partic- ularly neurasthenia. Von During (1905) studied 60 cases of phosphaturia. He concluded that cases of phosphaturia, attributed to neurasthenia, were actually caused by urethritis and prostatitis, the neurasthenia being secondary, and sometimes late in appearing. Von During explains it by the idea that the whole urogenital tract is inflamed by "inflammation of any part, the parenchyma of the kidneys thus becoming impaired. Albuminuria was found often to accompany phosphaturia. 664 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Moll (1905) describes a case of hysteria combined with intes- tinal catarrh in a child 51/2 years old. A symptom of the case was an abnormal amount of calcium phosphate in the urine. While in this condition the child lost 2.5 kg. in weight in 2.5 months. After a change of diet, and an avoidance of lime-rich foods there was an improvement in general health, and a gain in weight of 2 kg. in V/% months, the urine being normal. With a return to the original diet came a return of the old symptoms, and an increase of calcium and phosphorus, especially the former, in the urine. The diet was once more changed to fat, meat, sugar and fruits, thus avoiding lime, with the result that the urine became normal, and the nervous and digestive disturbances disappeared. Moll (1909) published an extensive report of further studies of this same subject, having reference to breast-fed infants. In this study he made a comparison of the composition of the urine of healthy infants and those suffering from defective digestion, the following are a part of the conclusions from parts I and II of the report : The urine of healthy breast-fed infants contains no phosphorus, or only traces. The dyspeptic infant excretes more or less phosphorus through the urine. This amount may be decreased by a short fasting period (24 hours), a reduction of the number of feedings, or artificial evacuation of the intestine. If the trouble is relieved the phospho- rus content of the urine decreases; if it continues the phosphorus increases. In inanition, in the ordinary sense of the word, if not carried too far, no phosphorus, or only a trace is excreted in the urine of the healthy child. If a breast-fed child gains in weight and shows no sign of ill- ness, and has no phosphorus in the urine, then it is healthy. If the breast-fed infant gains in weight, and with insufficient food intake shows no phosphorus, or only traces, in the urine, it is starved. If the child does not gain or lose in weight, and, with appar- ently insufficient nourishment, excretes large quantities of phospho- rus in the urine, it has a digestive disorder. Thus the content of the urine in phosphorus is a gauge of the condition of the infant. Loss in weight and a high percentage of phosphorus in the urine run in a parallel way ; also gain in weight and a low percent- age of phosphorus in the urine. PHOSPHORUS METABOLISM 565 The same milk reacts differently on different children. It is possible then to determine the effect on the child of a change of milk by testing the urine (24 hours) for phosphates. If benefit has been received the urine will show a considerable decrease in phos- phorus. In part III of this article Moll discusses acute gastroenteritis in infants, and submits experimental data showing the effect of treatment on the phosphorus of the urine. In the height of the disease there is a high percentage of phos- phorus in the urine, which is quickly decreased by stopping the food intake, and substituting a water diet. These low values persist during convalescence. When breast-feeding is resumed the values of urine phosphorus increase again. The tolerance of the organ- ism toward new food is measured by the urine analysis. The organ- ic phosphorus present in severe stages of the disease disappears with the decrease of total phosphorus in the urine. In cases which have a fatal termination this fasting therapy does not produce the above results, that is, improvement, in general, accompanied by decreased urine phosphate. Organic phosphorus in the urine of the breast-fed child is to be regarded as a pathological symptom. Von Moraczewski (1905) gave attention to the balance of acid and basic elements in pathological urines. His table as published is in need of recalculation. A part of the conclusions are as follows : The chronic phosphaturia which does not show the clinical symptoms of neurasthenic phosphaturia, or of the phosphaturia of children, is characterized by an abnormal relation of phosphorus and calcium elimination in the urine, in that, coincident with a decrease of lime salts there is an increase, though small, of phos- phorus. By treatment with alkalis the elimination of the metalloid ions (chlorine, sulphur and phosphorus) is relatively more favored than that of the metal ions, by which the relative elimination returns to the normal. One and the same individual may have this form of phosphaturia and gout ; that is, (a) may show phosphaturia alter- nating with oxaluria or gout; (b) may suffer generally with phos- phaturia and then with gout, or the reverse. Normal urine contains more acid ions than the urine of phosphaturia or gout. Tobler (1905) published data on two cases of phosphaturia. As usual the lime in the urine was high, while the phosphorus remained within normal limits, but the lack of food analyses limits the use of the data. 566 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Doctor (1906) says that phosphorus estimations show that it is incorrect to designate every case of alkalinuria as phosphaturia. Langstein (1906) observes the presence of phosphaturia in cases of disease where there was not the usual excess alkali or excess removal of acid as a cause. He mentions sexual phos- phaturia, due to a complication of diseases of the urogenital system ; neurasthenic, causing reflex effects on kidney secretions ; that form which von Moraczewski describes as associated with uric acid diathesis, and finally the juvenile form. Langstein describes in detail the nervous symptoms accompanying the juvenile form. He considers overnourishment on foods such as milk and eggs, which are rich in lime to be the cause, and found that dietetic changes on this basis caused marked improvement. In his cases there was no intestinal catarrh demonstrable. Langstein emphasizes the diver- sity of causes of phosphaturia. Novi (1908) reports a reduction of the urinary phosphorus, in phosphaturia following antirabes treatment, from 69.11 percent of an intake of 2.457 gm. to 46.07 percent of an intake of 2.451 gm., plus 0.603 gm. P 2 5 in the shape of phytin, the difference being ascribed to this phosphatic supplement. Klemperer (1908), in an extended study of various phases of phosphaturia, found that doses of 0.3 gm. oxalic acid plus 3.0 gm. NaHCOg greatly decreased the absorptive power of the kidneys for calcium, as also did very small quantities of HgCl 2 (1 drop 3 times daily of a 5-percent solution, in all 1.0 mg. in 24 hours). PHYTIN THERAPY This subject is discussed in connection with normal phytin metabolism; see p. 315. RACHITIS Rachitis has been known for a very long time; at least it was described by Glissonius in 1650. It is a disease of infants which involves phosphorus metabolism, especially through its effects upon the bones, but also apparently in other ways as well. In the literature of the subject much prominence is given to the element calcium, but we may bear in mind the fact that one could as consistently put the same emphasis on phosphorus, for these two elements are used together in the tissues most involved in rachitis. In rickets calcification of the bones is irregular; areas of partial ossification lying between areas of greatly enlarged cartilage cells. The bone is deeply imperforate by projections into it of both mar- row and periosteum, and the marrow spaces are enlarged and irreg- ular. Absorption of formed bone is not prominent as in osteomal- acia, but, rather, a failure of the osteoid tissues to calcify. PHOSPHORUS METABOLISM 567 Associated with rickets there are often gastrointestinal disor- ders, and anaemic changes in the blood. The blood alkalinity, how- ever, was found normal by Stoltzner. The largest part of the literature of rachitis has to do with its etiology. This aspect of the problem, however, remains unsolved. Among the possible causes, lack of calcium in the food, deficient absorption of calcium, and acid intoxication, none fits the facts in a thoroughly satisfactory manner. From an enormous literature, of which we do not attempt to review the whole, the following are some representative fragments. Among the earlier papers which we have seen are those of Gobley (1844, 1846), who used ray's liver oil with success in rickets, Virchow (1853), who histologically distinguished rickets from osteomalacia, G. Wegner (1872), who studied the effects of the element phosphorus repeatedly administered in small doses, and suggested the probable benefit from such treatment in rachitis, Heitzmann (1873), who thought he produced rickets and osteomala- cia with lime-poor food and lactic acid, and Roloff (1875a, 1875b, 1879), who also produced and cured artificial rickets, and considered it actual rickets, which he thought due to lack of phosphorus in the food. Seeman (1879) considered rickets as caused by deficient absorp- tion of lime, due to digestive disorders originating in a subnormal hydrochloric acid content of the gastric juice. Zander (1881) also had the idea of a deficiency of hydrochloric acid in the gastric juice, and considered this due to an excess of potassium and phosphorus in the mother's milk causing an undue elimination of sodium and chlorine. These observations were based on the study of the composition of the milk of the mothers of normal and of rachitic children. E. Voit (1880) recognized the futility of feeding calcium salts in actual rickets, and recommended attention to the gastrointestinal disorders often accompanying rickets, considering these the cause, and their relief the cure. Kassowitz (1881, 1884a, 1884b, 1886) experimented with phos- phorus therapy in rickets. He considered that it reduced the morbidly increased vascularization of the osteoid tissue from which result the characteristic bone conditions. Kassowitz experimented with many thousands of cases (1890) and recommended "lipanin," olive oil and phosphorus. Other statements of his ideas and results are found in his publications of 1901, 1910, 1912. He expresses the idea that rachitis is not due to lack of lime in the food. Phosphorus, 568 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 he finds, causes hardening of the bone through the laying on of lime salts, and he speaks of rickets as involving an abnormal increase of tissue building, and an abnormal fullness of blood in the bone- making tissues. J. Cohnheim (1882) published an extended discussion of the disease, as also did Pommer (1885), who considered it as originating outside the skeleton. Baginsky (1882) studied demineralization by lactic acid in con- nection with rickets. Anna Schabanowa (1889) reported successful use of phospho- rus in rickets. Korsakov (1892) could not cause rickety conditions by feeding lactic acid. Phosphorus therapy caused varying results, some successful, others not. Strontium salts used in rickets caused a certain amount of ossification. Cremer (1891), however, found that strontium phosphate would not prevent the appearance of rickety symptoms in a dog on a low calcium diet, though it was deposited to some extent in the bones. Brubacher (1890) shows that in congenital rickets the body is characteristically poor in ash. On the dry, fat-free basis calcium and phosphorus are both markedly deficient, the former decidedly more so than the latter. The analysis of the ash shows calcium quite deficient, and phosphorus only slightly so. Magnesium seems not to vary in an important way. Brubacher agreed with Kassowitz in the belief that organs other than the bones were not subnormal in calcium. Herter (1898) experimented with fat-free rations on swine, having in mind the alleged low fat content of the milk of women whose children have grown rachitic on the mother's milk, and also the occurrence of rickets in children fed largely on condensed milk. He concluded that fat starvation causes a very imperfect absorption of phosphorus from the intestine; but since complete balance data were lacking we must suspend judgment on this matter. There was nothing resembling or suggesting rickets, however, in the results from the fat-free rations. Jviidel (1893b) satisfactorily demonstrated that lime was absorbed from the carbonate and the acetate, and was also excreted, by rickety children, in a normal manner. Chabrie (1894, 1895) developed an interesting theory of the causation of rickets by the interference of lactic acid in the normal formation of collagen from chondrin, a process necessary to normal ossification, and also the interference of lactic acid with the normal precipitation of calcium phosphate and calcium carbonate in bone by PHOSPHORUS METABOLISM 569 the fixation by lecithin of the carbonic acid holding them in solution, lecithin thus being necessary to normal utilization of inorganic phosphates. For details see the original. He also explained the partial replacement of calcium of the bones by magnesium, in osteo- malacia, by reference to a different relation to lecithin. Flieger (1897) reported many unsuccessful attempts to cure rickets with phosphorus in cod-liver oil. The reason for his fail- ure is not apparent. Zweifel (1900) advanced the theory that deficient hydrochloric acid of the gastric juice, as caused by lack of common salt in the diet, interfered with the solution of calcium salts, and so caused rickets; and Delcourt (1899) stated that he had produced in bones lesions similar to those of rickets by means of potassium phosphate. Leichnam (1903) found phosphorus, calcium, magnesium and chlorides all above normal, and urea below normal, in the urine in rickets, indicating a profound state of denutrition. W. Stoeltzner (1903) reports negative results from phosphorus therapy in much complicated cases. He recommended phosphorus, however, (1904) to be administered in cod-liver oil, and suggested as a cause of rickets the functional insufficiency of some organ analogous to the thyroid gland. Burger (1904) reports benefit from the use of protylin; Fiirst (1904), from the use of phytin; Lepski (1905) reported negative results from phosphorus in cases complicated by severe general illness; Pfaundler (1904) observed no difference in calcium ion absorption by finely ground bone and cartilage of normal and rick- ety children, which, so far as it goes, argues against the idea of an inability of rachitic bones to absorb calcium salts in the normal manner. Birk (1909) reports that phosphorized cod-liver oil increased mineral retention in rachitis, and that calcium retention varied inversely as fecal soap formation. (See table below.) CALCIUM AND PHOSPHORUS METABOLISM- RACHITIC CHILDREN -HEALTHY AND Diet Condition Child CaO intake Grams CaO retention Grams Percent CaO reten- tion P intake Grams P retention Grams Percent P reten- tion Length of period Without phosphorized cod-liver oil Healthy Rachitic Sch. K T F 2.175 2.600 1.417 1.629 +0.942 +0.515 +0.021 -0.135 43 20 2 4.654 4.517 1.862 2.238 +0.995 +0.834 -0.159 -0.110 21 18 3 days ■ • With phosphorized cod-liver oil Healthy Rachitic Sch. K T P 2.726 2.390 1.247 1.694 +1.200 +0.573 +0.120 +0.111 44 24 10 7 5.716 4.158 1.504 2.386 +1.951 +0.708 -0.131 +0.201 32 17 '8 1 1 •• 570 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 In these data we see no positive evidence of an increased reten- tion of phosphorus, as a result of the. administration of phosphor- ized cod-liver oil, though there seems to have been an improved retention of lime. Aron (1908) found the milk of the mothers of rachitic children to be subnormal in lime, as also did Ramacci (1910). W. Cronheim and E. Miiller (1908) found, in balance experi- ments on normal and rickety children, that nitrogen assimilation and absorption was normal in the latter, and their experiments showed nothing markedly abnormal about the mineral balances, all of which were positive. Dibbelt (1908, 1909) considered, in view of the complete- ness of utilization of the calcium normally present in woman's milk, that under abnormal conditions, it is likely that the calcium content is often deficient. He also concluded that feeding with cow's milk must often lead to calcium-loss through lower retention, especially through the gastrointestinal disorders prevalent at wean- ing time. Dibbelt considers that the low content of infants' food in calcium phosphate is a predisposing factor in the development of rickets. He puts much emphasis on gastrointestinal disorders as a causative factor. The following balances from Dibbelt's work are of interest, because, in the light of Schabad's conclusions, they may be consid- ered to represent a case, not of actual, but of pseudorachitis, since there were positive nitrogen and phosphorus balances coincident with a marked loss of calcium, which last became strongly positive simply through a change of food. DAILY NITROGEN, CALCIUM AND PHOSPHORUS BALANCES WITH NORMAL AND RACHITIC INFANTS— Grams Subject No. 1. Healthy; age 25 days No. 2. Rachitic; age 7 months No. 2. Rachitic; same subject as above but at a later date Intake Outgo Retention Retention per kg-, body weight. . Intake Outgo Retention Retention per kg. body weight. . Intake Outgo Retention Retention per kg. body weight. . 2.0320 1.5419 +0.4901 +0.1223 4.1310 2.7885 +1.3455 +0.2790 5.0400 3.1E46 +1.8854 +0.3840 CaO 0.6098 0.9022 -0.2924 -0.0731 1.1070 1.4460 -0.3390 -0.0704 1.5030 0.7495 +0.7555 +0.1547 P2O5 0.7913 0.8840 -0.0933 -0.0248 1.4870 1.4227 +0.0623 +0.0123 1.9058 1.8256 +0.0802 +0.0160 Diet Cow's milk; af- ter change from mother's milk Specially pre- p ared butter- milk Undiluted whole cow's milk PHOSPHORUS METABOLISM 571 Dibbelt reported success in increasing the calcium content of woman's milk from 0.575 to 1.852 parts per 1000 by adding calcium salts to the food, and that he decreased the calcium content of dog's milk from 4.56 to 1.94 parts per 1000 by use of a ration low in calcium, these results being in marked contrast to the prevailing trend of evidence on this matter. Bahrdt and Edelstein (1910) submit data showing the calcium content of normal woman's milk to be 0.0426 percent CaO, and that of the milk used by rickety infants (sustained by many collected figures) as 0.0315 percent CaO. Efforts on the part of the authors to increase the calcium content of the milk of a woman met with apparent success in the feeding of calcium carbonate, but with calcium lactate the results varied widely, from much below the normal to a little above. Lauxen (1909) studied elemental phosphorus administration to young normal dogs, having in mind the bearing of the results on phosphorus therapy in rachitis. Phosphorus was shown to affect the character of the leucocytes in a manner opposite to that of rachitis. In the bones there was a pronounced hardening, with a decrease of osteoclasts. New bone formation was of a type characteristic of phosphorus action, and decreased the marrow spaces. Histological observation led to the conclusion that phosphorus has a paralyzing effect on the bone cells, decreasing both apposition and resorption, but leaving a residual balance in favor of apposition. The spleen was much enlarged, possibly on account of taking up blood-forma- tion to compensate for restriction of this function in the bone marrow. Dibbelt (1909), in experiments on dogs with experimental rickets, concluded that human rickets is due to anomalies of absorp- tion and secretion. A late expression from Dibbelt (1910a) is a theory to the effect that the cause of rickets is an abnormal amount of calcium-precipitating compounds, such as phosphorus, from decomposition of undigested casein, and carbonates from carbohy- drate fermentation, in the intestine. Flamini (1907) submits calcium balance experiments with both normal and rickety children under phosphorus therapy. With the former there was a change from a calcium retention of 37.4, 36.S and 26.6 percent of the intake to 37.8, 46.1 and 36.5 percent after 12-15 days of phosphorus treatment, while with rickety children the increase was from 59.7, 56.8 and 55.7 percent to 68.2, 72.6 and 61.1 percent after 12-20 days of phosphorus therapy. 572 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Gassmann (1910) finds that lime, phosphoric acid and carbonic acid all decrease in amount in rachitic bones, together with a loss of water. The relations Ca:P0 4 :C0 3 are about the same for normal and rachitic bones. Magnesium is considerably increased in rachitic bones. ANALYSIS OF NORMAL AND RACHITIC BONES— (Gassmann, 1910) Aschenheim and Kaumheimer (1911) find calcium and phos- phorus diminished in the muscles of rachitic children, by compari- son of their figures for pathological cases with Tobler's figures for the normal. In 6 cases the phosphorus in the muscle varied from 0.3849 to 0.6485 percent, the normal being given as 0.325 percent. In these same six cases the calcium varied between 0.0255 and 0.0570 percent, while the normal is given as 0.0650. Schabad has conducted a most excellent series of studies of calcium and phosphorus metabolism in rachitis which contributes much to our understanding of the subject. A large part of Schabad's study has centered in the use of elemental phosphorus in the treat- ment of this disease. A comparison of its action on rachitic and normal children (1907) .shows that in rickets phosphorus assists in the absorption and retention of calcium, and also changes the com- position of the bones toward the normal, but that in health it produces no such effects. See also Schabad (1908) and (1909a). Phosphorus administered with cod-liver oil has given best results in increasing retention of both calcium and phosphorus, both sub- stances taking part in the improvement (1909b), while with the addition of calcium acetate a still larger retention of both calcium and phosphorus is brought about. Calcium citrate and calcium phosphate added to phosphorized cod-liver oil, not only are not retained, but they decrease the phos- phorus retention. The acetate, however, as above noted, had the PHOSPHORUS METABOLISM 573 opposite effect, and also brought about an increased nitrogen reten- tion; though at the same time both the nitrogen and fat of the feces were increased (1910a, 1910b). Lipanin, olive oil and sesame oil cannot replace the cod-liver oil in the treatment of rickets (Schabad and Sorochowitsch, 1911a), and the repeated heating of cod-liver oil to 100° for an hour does not destroy its beneficial action in rickets (Schabad and Sorocho- witsch, 1912). Schabad submits many data to show that only the maximum normal calcium content of human milk can cover the real needs of the infant for calcium, and that there are doubtless many occasions when it is insufficient (1909d). That a deficiency of the milk in calcium should be the cause of rickets he considers not to be exclud- ed by his studies. Rickets is histologically, but not otherwise, distinguishable from the experimental or natural malnutrition of the bones of domestic animals, which Schabad calls pseudorachitis. In order to identify a condition in human beings parallel with the pseudorachitis of the domestic animals Schabad assumes that both actual and pseudorachitis exist in human beings without our being able to distinguish them. There is conflict in the evidence as to whether or not the calcium of tissues other than bones is subnormal in rachitis. Per- haps the difference in observations on this point is due to the con- fusion of actual and pseudorachitis. Schabad finds the calcium content of the milk of the mothers of rickety children slightly lower than normal, and the content of organic nutrients somewhat above normal, so that the calcium con- tent as related to the calorific value is considerably below the nor- mal. He finds, however, that rachitis can occur with a maximum cal- cium content of the milk, so that it is impossible to consider a .deficiency of the milk in calcium as the only cause (1909e, 1911a) though is has not been so definitely related to any other condition. It has been found impossible to increase the calcium content of woman's milk by feeding. The effect of rachitis on phosphorus excretion is shown by the following table from Schabad (1910d) (page 574). For all groups the phosphorus excretion in rickets is distinct- ly higher than in health, and amounts in the urine are smaller. In developing rickets calcium and phosphorus excretion increase together, with excess of the latter, and the increased phosphorus excretion usually exceeds the bone-equivalent of the accompanying calcium, so that participation of other phosphorus-rich tissues is 574 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 apparent. The increase of phosphorus excretion is in the feces, the urine phosphorus decreasing toward the normal, and the normal excess of urine phosphorus over feces phosphorus is changed to an excess of feces phosphorus over urine phosphorus. In convalescence, the total phosphorus excretion is subnormal, and the normal excess of urine phosphorus over feces phosphorus returns. AVERAGE DAILY PHOSPHORUS EXCRETION BY RICKETY AND HEALTHY CHILDREN OF THE SAME AGE, AND ON THE SAME DIET Diet Condition P2O5 excretion per kilo per day Relative P2O5 excretion, in percent of amount sriven Partition of P2O5 excre- tion Excess in feces in re- lation to Ca3(P04)2 Total Grams In urine Grams Total In urine Urine Feces CaO P2O1, Breast fed Healthy, 4-5 mos. ProgT. rickets, 5-13 mos. 0.023 0.034 0.018 0.021 65.3 122.2 52.8 72.2 80.6 60.8 19.4 39.2 64.8 52.8 .... Infants on cow's milk Healthy, 3-6 mos. Progr. rickets, 5-8M mos. 0.214 0.186 0.119 0.077 79.7 94.6 45.1 39.3 65.2* 39.3** 34.8 60.7 22.8 14.1 Older children on mixed diet Healthy, 4-5 yrs. Progr. rickets, 4 yrs. 0.159 0.091 0.102 0.040 80.3 102.1 51.5 44.9 64.4 44.1 35.6 55.9 22. 39'.8 Developed rickets, lyr. 5 mos.to 2 yr. 7mos. 0.178 0.069 71.8 27.5 38.2 61.8 (16.5) 23.3 Convalescent, 2 0.103 0.077 65.5 48.7 74.4 25.6 25.6 (64) * We figure this to be 55.6, and the feces figure 44.4. * * We figure this to he 41.4, and the feces figure 58.6. The calcium balance (1910c) varies during the course of a case of rickets from negative or subnormally positive in the progressive stage, to decided retention, which begins some time before the clini- cal manifestations of improvement, and later, in convalescence, to a retention of two or three times the normal amount, which, on com- plete recovery, falls again to the normal. Increased excretion of calcium in the progressive stage of the , disease is through the feces; calcium excretion at this stage is- sub- normal in the urine, and in the convalescent stage above normal in the urine. These facts, Schabad states, rule out the acid theory of the cause of rickets. Schabad finds that increased calcium excretion in the feces increases the phosphorus retained in the intestine, and conversely that increased phosphorus excretion in the feces increases the cal- cium retained in the intestine; therefore, since in rickets the feces phosphorus exceeds the bone-equivalent of the feces calcium, the increased excretion of phosphorus, and not of calcium, is the primary factor. PHOSPHORUS METABOLISM 575 Schabad (1909c) finds that in advanced stages of rickets the relation of calcium to phosphorus in the bones changes, the calcium decreasing, and the phosphorus increasing, whereby the normal relation of calcium to phosphorus of 100:75-85 is changed to 100: 70-75, the decrease in total ash affecting the calcium more than the phosphorus. Cattaneo (1909) also notes departure from the normal composi- tion of the bones in rachitis, especially in the direction of a relative increase in magnesium. Rickets complicated with tetany, Schabad (1910-11) finds not to differ as to calcium and phosphorus metabolism from uncompli- cated rickets. Schabad states (1909e) that in pseudorachitis there is no such excess of phosphorus over calcium in the feces as in true rickets, but rather, an excess of calcium over phosphorus, a fact which sug- gests a different degree of participation of organs other than the bones in the production of the mineral losses in these diseases. As shown by the table below, Ogata (1911-12) finds the calcium phosphate as well as other mineral salts of the bones much lowered by rickets. Different proportionate reduction in the amounts of the several salts result in an altered relation of one to another in the bone. There was marked increase of collagen and fat. ANALYSES OF NORMAL AND RACHITIC BONES Ogata (1911-12)— Percent Bones of 2-mos.-old healthy child Bones of patient having- rachitis* Tibia Ulna Femur Tibia Humerus Ribs Vertebrae 65.32 34.68 57.54 1.00 6.02 0.73 33.86 0.82 64.07 35.93 56.35 1.00 6.07 1.66 34.92 1.01 20.60 39.40 14.76 0.80 3.00 1.02 72.20 7.20 33.64 66.36 26.94 0.81 4.88 1.08 60.14 6.22 18.88 81.12 15.60 '2i66 2.62 81.22 37.19 62.91 32.29 67.71 Magnesium phosphate Fat * The age of the rachitic patient was not stated. Schabad (1911b, 1911c) reported unfavorable results from the use of the phosphorized cod-liver oil treatment in two cases of so- called late rickets. Schabad and Miss Sorochowitsch (1911b) studied calcium, phosphorus, nitrogen and fat metabolism in rachitic infants as affected by various preparations of cod-liver oil and inorganic salts. The phosphorized cod-liver oil alone was found sometimes to be with- out beneficial effect. The most liberal calcium retention resulted from the use of calcium acetate with cod-liver oil emulsion. Phos- 576 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 phorus was retained in considerable quantity .from cod-liver oil emulsion with hypophosphites of sodium and calcium. C. Meyer (1913) found in metabolism experiments with rickety infants that phosphorized cod-liver oil caused a marked reduction in the feces phosphorus, and improved retention of this element. Gessner (1913) considers that rickets is the result of a dis- turbed fat metabolism, among the causes of which are deficiency of fat in modified milk, a retarded state of metabolism generally, and the prevalence of acid-reacting metabolites in the blood and lymph. He believes that the efficiency of phosphorized cod-liver oil in the treatment of rickets is due to its ready emulsification and absorp- tion, the presence of phosphorus facilitating its oxidation. Gessner likens this relation of fat and phosphorus to that naturally existing in the phosphatids of the body. Schloss (1913a, 1913b) reports results from metabolism studies on a breast-fed infant under treatment for rachitis. The calcium balance was very unfavorably influenced by phosphorized cod-liver oil alone, but with calcium acetate added the balance became and continued to be strongly positive; the. phosphorus balance was affected much as was that of calcium, though in much lower degree ; a moderate retention of magnesium in the fore-period became strongly negative under the influence of phosphorized cod-liver oil and of calcium acetate; phosphorized cod-liver oil diverted alkalis from urine to feces, and the addition of calcium acetate led to large loss of alkalis. In a later study Schloss (1914a) confirmed previous observa- tions on the ineffectiveness of phosphorized cod-liver oil alone, and the effectiveness of the oil and calcium salts together on the calcium balance. It was found, further, that the calcium salt alone is ineffective, and that two calcium-casein preparations are of value in combination with phosphorized cod-liver oil. Regarding the phosphorus metabolism, the introduction of cal- cium acetate increased the feces phosphorus; the addition of cod- liver oil slightly improved the balance, while the oil alone caused only a different distribution of the phosphorus elimination. An improvement of the phosphorus balance comparable with that of calcium was first seen when the calcium caseonphosphate and plas- mon were used. In a third paper Schloss (1914b) reported once more that phos- phorized cod-liver oil has an unfavorable influence on nitrogen, cal- cium and phosphorus metabolism, and also that CaHP0 4 with phos- phorized cod-liver oil very greatly improved the calcium and especi- ally the phosphorus balance. Plasmon and CaHP0 4 were said to have identical effects on calcium and phosphorus metabolism. PHOSPHORUS METABOLISM 577 Kochmann (1912), in balance experiments on dog's, studied the effect of elementary phosphorus on calcium metabolism. The phosphorus was administered in quantities of 1 to 2 mg. daily. By using* larger doses than is possible in human medicine he obtained positive results such as have not been obtained from the use of phosphorus without cod-liver oil with human beings. The effective quantities equalled or exceeded the toxic dose without exercising an influence beyond the time of administration. During the use of the phosphorus there was a regular and con- tinuous reduction in the preexisting positive phosphorus balance until it finally became a minus figure. The calcium balance, which was negative during the preliminary feeding, was considerably im- proved during the phosphorus therapy, but did not become positive until calcium chloride and phosphorus were given together, which produced marked retention of both calcium and phosphorus. Mag- nesium metabolism ran parallel to nitrogen metabolism and was not influenced by phosphorus therapy. Ribbert (1913) reports a more or less complete destruction of the cartilage cells in rachitis. From the finding of these cell- changes in light cases and at the onset of the disease, he concludes that this condition is* a primary one, apparently due to some toxic substance which hinders calcium deposition in cartilage and bones. Ribbert considers the origin of this causative toxic substance to lie in metabolic disturbances resulting from wrong feeding. Kassowitz (1913) states his own experience and also cites results of others showing that cod-liver oil is not essential to the utilization of phosphorus in rickets, that phosphorus is of value in combination with other oils, and of some value alone. Kassowitz cites cases in which prolonged treatment with cod-liver oil alone was not beneficial. He does not believe that the calcium balance points the way either to the cause or treatment for rachitis. See also Dibbelt (1913), Diesing (1913), and Bamberg and Huldschinsky (1913). The etiology of rachitis remains an unsolved problem. THE THYROID GLAND IN RELATION TO PHOSPHORUS METABOLISM The thyroid gland exercises important control over metabo- lism, especially of nitrogenous compounds. Ingestion of thyroid gland substance increases tissue katabolism, while deficiency in the thyroid causes cessation of physical, mental and sexual development, and also retards ossification of epiphyseal cartilages, leading to the characteristic deformity of cretins. Oxygen consumption and carbon 578 OHIO EXPERIMENT STATION: TECHNICAL BUL, 5 dioxide secretion are diminished, and metabolism generally is great- ly depressed. There is considerable evidence that the disturbance of functions is due to toxic products of protein metabolism, which, through the agency of the thyroid secretion, are normally rendered innocuous. The thyroid stands in close relation to the reproductive func- tions, as is shown by its hypertrophy at the time of sexual maturity, during the menses, and during gestation and lactation. Myxoedema and cretinism are associated with subnormal thyroid secretion; while there is some evidence that exophthalmic goiter depends on excessive thyroid secretion. Ord and White (Brit. Med. Jour. 1893, 2, 216), studying thyroid treatment in myxoedema, note a slight increase in phos- phorus excretion, though Houghhardy and Langstein (Jahrb. fur Kinderh. 61, 634, 1905) observed a retention of calcium and phos- phorus, apparently due to the growth of bone in a growing child. Roos (1895) found in experiments on healthy animals that the administration of thyroid gland substance in large doses caused a marked increase in nitrogen elimination (much more than the nitrogen so introduced) lasting for several days, and also increased sodium chloride and phosphorus elimination. The increased elim- ination of chlorine lasted only 2 or 3 days, and then sank abruptly to a markedly subnormal amount, while the increased phosphorus elimination, like that of nitrogen, persisted. The administration of thyroid substance to a dog, deprived of the thyroid gland, increased the nitrogen and chlorine elimination to a greater degree than when given to a normal animal, but the phos- phorus elimination remained considerably below normal. Roos concludes that the thyroid gland has a decided influence on phosphorus metabolism, the nature of which is as yet obscure, though he notes evidence sustaining the idea that phosphorus assim- ilation requires the assistance of a secretory product of the thyroid. Bayon (1903) reported confirmation of the findings of others in such experiments, that broken bones heal less quickly and com- pletely in animals from which the thyroids have been removed, and that administering thyroid-gland preparations to such animals favors the healing, though not entirely making up for the loss of the glands; also that such preparations favor healing of fractures in normal animals as well. Scholz (1905) has made thoroughgoing studies of metabolism in three cretins, covering periods of 95, 95 and 117 days respectively, and including complete mineral balances. The work covers the PHOSPHORUS METABOLISM 579 normal state of metabolism, and the modifications produced by the ingestion of thyroid tablets, and of 5 gm. trisodium phosphate per day. Scholz states that metabolism of cretins is to be regarded as sluggish. The urine is decreased, and uric acid, creatinin and sodium chloride are excreted in subnormal quantities; but urea, xanthin bases, ammonia and sulphur compounds appear to have normal values. The alkaline earths show increased excretion. The phos- phorus retention is considerable, on a small intake, and does not increase proportionately with increased intake. There was nothing characteristic in the effects of thyroid treatment on phosphorus metabolism. Aeschbacher (1905-6) studied the thyroid glands of 61 persons. He found the phosphorus content determined mainly by the richness of the gland in nuclei, but in part by the phosphorus content of the colloid. As noted by Kocher, there was generally a reciprocal rela- tion between the quantities of phosphorus and iodine in the gland, due principally to the fact that glands rich in the iodine-containing colloid are relatively poor in cells. The thyroids of women were found larger and also richer in iodine, than the thyroids of men, while the phosphorus value reaches the higher figure in the thyroids of men. Diseases which cause circulatory disturbances cause a marked decrease in the iodine content of the gland, but for phospho- rus this relation does not hold. Saccone (1907) removed the thyroids and parathyroids from a dog, after which the urinary phosphorus rose to three times the previous amount. The feeding of a phosphatic diet containing liver, brain and sodium glycerophosphate did not bring about normal con- ditions, but instead, increased especially the organic phosphorus of the urine ; the feeding of the normal diet, however, with the addition of thyroidin reduced the excretion of phosphorus to the original level. Underhill and Saiki (1908) in the few urinary phosphorus estimations made in their metabolism study note a low phosphorus elimination "possibly indicating when considered with the low purin and allantoin excretion, a low rate of nuclear disintegration. A large output of purin-nitrogen and a low output of phosphorus were observed after the continued administration of large doses of thyroid tissue." Soli (1909) finds that rabbits, if deprived of thyroids at a suf- ficiently early age, fail to develop the skeleton normally, while guinea pigs and chickens did not show this result so readily. 680 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Bircher (1910) removed the thyroid glands from 6 rats of the same litter. To three of these he fed thyroidin tablets. The others were used as controls. The former showed much the more rapid calcification of the bones. A. Juschtschenko (1911) observed that thyroidectomy reduced the nuclease content of young dogs. Schafer (1912) found that the addition of small amounts of thyroid tissue to the diet of white rats greatly increased the food consumption, especially in quite young individuals, with acceleration of growth, and retention of nitrogen. Phosphorus excretion is increased if the ingestion of thyroid is increased beyond a certain point. A. I. Ushenko (1913) found that after thyroidectomy the ratio of phosphorus to nitrogen in the urine first increased and then decreased, synthetic processes suffering acute disturbance in tissues containing nitrogen and phosphorus. A. S. Juschtschenko (1913) found in experiments on dogs that thyroidectomy causes a general disturbance of phosphorus com- pounds in the organs and tissues. There was general increase of the inorganic phosphorus of brain, heart, pancreas and liver, and general decrease of the total and organic phosphorus of the same. In the kidneys there was increase of the total phosphorus, generally both organic and inorganic; and in the serum organic and total phosphorus increased, while inorganic decreased. Hyperthyroidism was also found to cause disturbance in the phosphorus distribution in the tissues, these disturbances being in some respects just the opposite to those observed in athyroidism. The organic and total phosphorus are decreased in brain, muscle and heart, but are increased in liver, kidney, pancreas and serum. Inorganic phosphorus was less in all the tissues than in the normal animal, a fact almost completely opposite to the results in thyroid- ectomy. Confusion has resulted, in the study of the function of the thy- roid, through the removal along with the thyroid of the parathy- roids, or parts of the same, or through their injury. Further study will more sharply differentiate their functions. Karl Droge (1913) has recently reported experiments with young dogs in which observations were made on the effects during the suckling period of removal of thyroids, spleen or testicles. Six pups of a litter of eight were used. Dog I was killed about 12 hours after birth ; dog II was kept as a control ; the thyroid dogs I and II, the spleen-dog, and the testicle-dog were operated upon on the day PHOSPHORUS METABOLISM 581 the weight had about doubled, the 10th day ; ail were suckled by the mother. On the 23rd day the thyroid-dog I was killed to allow better nourishment of the others ; the remaining dogs were killed as soon as there was any tendency shown to take other food than the mother's milk, the 28th day. The bodies were analyzed as a whole for moisture, fat and ash, and for nitrogen, calcium, magnesium and phosphorus in the fat-free dry substance and in the ether extract. We quote but two of the 44 tables of results given. Conclusions should await results with more individuals. LIME, MAGNESIUM AND PHOSPHORUS CONTENT IN RELATION TO NITROGEN (Droge, 1913) Day of operation Day killed Nitrogen content Gm. To 1 gm. nitrogen Dog CaO Gm. MgO Gm. P2O5 Gm. Dog I iotii 1st 28th 23rd 28th ii 6.87 . 30.63 21.85 17.06 19.48 35.34 0.31 0.49 0.56 0.44 0.74 0.52 0.003 0.008 0.008 0.008 0.004 0.005 0.43 Dog- II ... 0.60 0.46 0.62 0.67 0.54 PHOSPHORUS CONTENT IN FAT-FREE DRY SUBSTANCE AND IN ETHER-SOLUBLE SUBSTANCE (Droge, 1913) P2O5 in fat-free dry substance P2O5 in ether-soluble substance Dog Weight of fat-free dry substance Grams P2O5 Percent P2O5 Grams Weight of ! ether-soluble substance Grams P2O5 Percent P2O5 Grams Total P2O5 Grams Dog I 61.1372 277.70 193.72 146.27 182.51 324.70 4.85 6.51 5.09 7.16 7.01 5.81 2.965 18.078 9.860 10.473 12.794 18.865 10.6278 280.507 208.570 255.656 178.810 312.977 ? 0.09 0.14 0.045 0.11 0.07 ? 0.252 0.292 0.115 0.197 0.219 2.965 Dog II 18.330 Thyroid-dog I 10.152 10.588 12.991 19.084 MORBUS BASEDOWII— EXOPHTHALMIC GOITER Scholz (1895) conducted a complete balance experiment with a woman suffering from exophthalmic goiter, and with a healthy person on the same treatment for comparison, two periods of 3-5 days with each subject, one without treatment, and the other with dried thyroid gland substance. Without treatment the goiter patient stored 7.437 gm. nitrogen and 1.06 gm. P,0 5 per day; with thyroid treatment the nitrogen storage was 7.09 gm. per day, while the P 2 5 balance fell from +1.06 to— 2.09 gm. 582 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 Under thyroid treatment the nitrogen storage with the healthy subject fell from +3.76 to +2.64 gm., while the P 2 5 balance fell from —2.996 to —3.831 gm. There was not any great increase of phosphorus elimination in the urine with either subject, but in the feces the phosphorus elim- ination of the goiter patient was increased 10-fold, and that of the healthy subject about 25 percent. Since there was no such increase of nitrogen outgo as of phosphorus it was evident that the latter was due to phosphate rather than nuclein katabolism. Von Noorden mentions the similar result of thyroid treatment on nitrogen metabolism by Hirschlaff (Zeit. klin. Med. 36, 200, 1899), and, on the other hand, the greatly increased excretion of nitrogen as observed by Matthes (Verhandl. der Cong. f. inn. Med. (1897), 232) and by David (Zeit. Heilkunde 17, 439). That the behavior of Scholz's patient under thyroid treatment was typical seems not yet to have been demonstrated, though Roos (1895) and others obtained similar results, though less marked, in experiments with animals. Berkley (1908) is said to have administered an alcoholic solu- tion of lecithin in cases of exophthalmic goiter with strikingly favor- able results. Berkley used this solution for alternate weeks with glycerophosphates, quinine and gentian, but with unfavorable results from the glycerophosphates. With lecithin there was gain in weight and relief from nervous symptoms, while with glycero- phosphates there was loss in weight, and increase of nervous phenomena. Lecithin treatment was stated to be out of place with a disturbed digestion, and not of value without the assistance of a milk diet. Tschikste (1911) considers, as a result of a metabolism study, that the iodin-free nucleoprotein which Oswald isolated from the thyroid has an influence in exophthalmic goiter opposite to that of thyreoidin (iodothyrin) . In this article Tschikste reviews many articles on this subject that we have not consulted. TOXINS, ANTITOXINS AND PHOSPHORUS METABOLISM Dalmastri (1901) studied phosphorus metabolism as affected by rabes antitoxin. Metabolism generally was considerably increased during the cure, as shown by an increased loss of nitro- gen and especially of phosphorus, and by loss of weight. The period following showed a prompt return to normal, so far as nitro- gen and body weight go, but a slow return as to phosphorus. PHOSPHORUS METABOLISM 583 Dmitriewski (1900) determined the effects on metabolism in the dog of poisonous substances extracted from Bacillus pyocyaneus and Bacillus coli communis, and also of diphtheria toxin. In carefully controlled subcutaneous injection experiments, it was found that all three of these poisons led to increased nitrogen and corresponding phosphorus elimination. Novi (1904) published balance data on phosphorus metabolism during antirabes treatment, both with the virulent and non-virulent material, and also with and without sodium glycerophosphate, the latter being administered in one period per os, and in another by in- jection. Beyond a slight increase of urinary phosphorus, under the influence of the antirabes treatment, the figures did not show any notable departures from the normal, though the author states that the cure by both the toxic and the non-toxic treatment caused a leucocytosis and a following or coexistent leucolysis. See also Decroly (1898). Lederer and Stolte (1911) made mineral analyses of human and of dog hearts under normal conditions and under the influence of diphtheria or scarlet fever toxins. The authors did not find that these diseases alter the composition of the heart. TUBERCULOSIS The effects of tuberculosis on phosphorus metabolism are of several kinds; (1) characteristic changes, both quantitative and qualitative, in the phosphorus-containing lipoids of the tissues, (2) pathologic retention and deposit of phosphates in the calcification of degenerative tissues, (3) deranged organic functions of the parts affected, which of course may be of most diverse character, (4) others due to the fever induced by this disease, (5) still others due to anaemia, and lastly, (6) those of general starvation. The relation of tuberculosis to phosphorus metabolism, therefore, is largely gen- eral and incidental, and little attention has been given to the matter, except in relation to diet. In times past much prominence has been given the idea of flooding the system with lecithin, as in the very liberal ingestion of eggs, and benefit derived from such treat- ment has often been ascribed in large part to the phosphorus com- pounds present. Lecithin and nuclein phosphorus have also had a place in the treatment of this disease by parenteral injection. At present, however, an appreciation of the greater importance of the environment and general management has detracted emphasis from this particular dietetic consideration. 584 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 ' PHOSPHORUS METABOLISM IN TUBERCULOSIS Passing over the earlier work referring to this matter, we note Mitulesco's (1902) observation that in times of hemorrhage from the lungs in tuberculosis there is a decrease of urinary nitrogen and phosphorus. In a later article (1903a), he reports complete nitro- gen and phosphorus balances on four cases of tuberculosis. Three were given tuberculin treatment; the fourth hygienic and dietetic treatment only. The last mentioned patient improved slowly, and the positive nitrogen and phosphorus balances were increased. The three patients on tuberculin treatment were further advanced in the disease, all showing negative nitrogen and phosphorus balances. Under this treatment these balances all became positive, the improvement being greater than with the other case. In another paper Mitulesco (1903b) reports nitrogen and phosphorus balances on eight cases of tuberculosis. In general the loss of nitrogen and phosphorus in the urine was excessive, and the balances negative. There was a high content of organic phosphorus in the urine. The nitrogen and phosphorus in the urine were less at times of hemorrhage. The loss of appetite, impairment of digest- ive functions, increased cell destruction, the resultant anaemia, and the excessive losses of material by the body combine to lower the resistance of the organism to the infection. Zickgraf (1910) compares phosphorus and chlorine elimination in tuberculosis and chlorosis. He finds the average phosphorus excretion to be, as Teissier stated, lower in chlorosis than in tuber- culosis ; but the difference is too small, and the individual variations too great, to make possible diagnostic use of the distinction. Mulier (1911) concludes that the phosphorus excretion in tuber- culosis is not characteristic, and has no differential diagnostic value. DEMINERALIZATION IN TUBERCULOSIS Ott (1903) reports complete mineral and nitrogen balances with three cases of tuberculosis. He admits the presence of "demin- eralization" in certain cases, but finds that it is not a regular symp- tom. Steinitz and Weigert (1904, 1905) analyzed the body of a tuber- culous child, and compared the analysis with others by Sommerfeld on a child dead from gastrointestinal disorder, another such by Steinitz, and six analyses of new-born infants by Camerer and Soldner. They find in their study no grounds for belief in the theory of demineralization. PHOSPHORUS METABOLISM 585 Amat (1906), also studying demineralization, considered the possible diminution of the phosphorus content of the body through the use of white flour as a predisposing factor to tuberculous infec- tion. In, this 'study mice were fed on breads made from white roller process flour, and flour ground between stones. The latter was eaten in larger quantity and produced a considerable gain in weight ; the former (the roller process flour) was not eaten in quite so great quantities, and did not maintain the body weight. No evi- dence was submitted on demineralization. A. Mayer (1907) studied metabolism in tuberculosis, and from complete nitrogen and mineral balances with five patients in 3-day periods draws conclusions as to demineralization. In these cases he finds a retention of calcium and phosphorus and a marked reten- tion of chlorine. There was a slight tendency to retain potassium and a greater one to retain sodium. There was no apparent rela- tion between the phosphorus and nitrogen balances. Mayer's posi- tive mineral balances lead him to the belief that demineralization is not characteristic of tuberculosis. Sarvonat and Rebattu (1910) made ash, calcium and phospho- rus estimations on normal and tuberculous guinea pigs. They con- clude that the total ash of the body of the tuberculous guinea pig decreases about one-tenth, this loss being due to the animal's grow- ing thin in flesh. The absolute amount of phosphorus was less in the body of the tuberculous animals, and the proportion of phosphorus to total ash was much less, while the authors state that the phos- phorus in the ash of the soft parts was sometimes the higher in the tuberculous animals. The absolute and relative calcium content of the bones of the tuberculous animals is decreased. The propor- tionate loss of calcium from the bones is much greater than from the soft parts. In these cases, then, there was demineralization — as, in fact, there is in starvation from whatever cause. PHOSPHORUS COMPOUND THEBAPY IN TUBERCULOSIS Gilbert (1901) states that consumptives and neurasthenics thrive remarkably under the influence of pills containing 0.1 to 0.5 gm. lecithin. Lecithin was also administered subcutaneously in injections of 0.05 to 0.15 gm., repeated every second day. The appetite and weight increased, and no evil effects were observed, even on prolonged administration. One subject, already in a hos- pital for four months, gained 3.5 kg. in one month. Other details of improvement were noted. 586 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 According to Morichau-Beauchant (1901) the efficacy of leci- thin therapy in tuberculosis is inversely as the stage of advancement of the disease. Gilbert and Fournier (1901) report that egg lecithin in doses of 0.10 to 0.50 gm. per day by the mouth, or 0.05 to 0.15 gm. every other day by injection, led to general improvement, and gain in appetite, and in weight, in cases of pulmonary tuberculosis. Claude and Zaky (1901a) found by feeding and injection experiments with lecithin, on tuberculous men and guinea pigs, increased urinary nitrogen and decreased urinary phosphorus, associated with gain in weight and general improvement. The leci- thin did not affect the development of the disease. The improve- ment was marked in early stages of the disease, but perceptible even in those which were hopelessly advanced. See also (1901b) and (1901c). Mouneyrat (1902a) experimented with disodium-methyl-arsen- ate and nucleic acid for tuberculosis. He reports results with 120 patients. The daily dose was 30 c.c. of a solution, taken in two portions, the whole containing 0.05 gm. methyl arsenate of sodium and 0.20 gm. nucleic acid from herring milt. Both red and white blood corpuscles increased rapidly, and general improvement was marked after varying periods of treatment up to a maximum of a month, the Koch bacillus disappearing from the spittle in many cases. This treatment is said to have succeeded where lecithin treatment failed. Colombet (1902) administered this same arsenic-phosphorus compound with 33 cases of chronic tuberculosis. Two cases died; 3 did not receive benefit ; the remainder were said to be improved in a notable manner, especially those having the disease in the first or second degree. Many symptoms of improvement were noted, among them diminution of phosphaturia. Ward (1910) attempted to relieve the progressively increasing anaemia of tuberculosis by intravenous injection of nuclein-saline solution. This treatment caused a rapid disappearance of the poikilo- cytes, and their replacement by new and healthy erythrocytes. The data presented show that the nuclein treatment markedly increased, nearly to the normal, the haemoglobin, the number of red cells, and the specific gravity of the blood. Of the 15 cases reported, 2 died, 9 recovered and became free from tubercle bacilli, 4 were improved but not yet cured, while one refused the treatment after the firit period. The solution used was composed as follows : PHOSPHORUS METABOLISM 587 Approximately 6 grains of sodium triticonucleate were dissolved in each ounce of physiological salt solution, and standardized to 1 mg. organic phosphorus to each cubic centimeter. This solution was used in the quantity of 1 oz. per 20 lbs. body weight of patient. Otolski and Biernacki (1912) made total phosphorus and phos- phatid phosphorus determinations on the organs of rabbits vaccin- ated with dead tubercle bacilli, and compared these with similar determinations on the organs of a control rabbit. The data reported show unmistakably lower content in both these items in the kidneys, heart and lungs of the treated animals than in the control; in the livers it was lower in some cases but higher in most. The results showed a loss of lecithins and an increase of jecorins in the liver, and at the same time a marked increase in the phosphorus content of the lecithin. Griniew (1913) has made an investigation of the quantitative differences in the lipoids and the lipoid phosphorus of several organs of guinea pigs, brought about by chronic tuberculosis. The organs were extracted consecutively with acetone, benzol, petroleum ether, alcohol and ethyl ether, and the amounts of the extracts and of their phosphorus content were recorded. Full data are given and compared in tables. We quote three condensed tables (p. 588) and a part of the author's conclusions. Conclusions: "During tuberculous infection the chemical constitution of the cells of nearly all organs and tissues changes in its lipoidal part. This change is of qualitative as well as quantita- tive character. It shows itself in the diminution of the quantity of phosphorus in the lipoids and by the replacement of different forms of lipoids by each other "During tuberculosis: (a) In nearly all the organs the sum of all the lipoids and the phosphorus diminishes as compared with the normal. (b) The quantity of cholesterin is increased in certain organs, diminished in others, (c) In all the organs there is less of the extracts containing lecithin, (d) In all the organs there is more of the extracts containing cephalin and myelinous materials, (e) In all the organs the diminution of the quantity of lipoidal material of the filtrate from the acetone and of the phosphorus contained in it is pronounced, (f ) The quantity of materials extracted by benzol is comparatively increased in most of the organs, (g) It appears that a part of the lecithin passes into cephalin or analogous compounds, (h) The organs which suffer most in their lipoid content and in phosphorus contained there are : the lungs, the spleen, the medulla, the liver. The lungs suffer most." 588 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 LIPOIDS OF THE ORGANS OF NORMAL GUINEA PIGS Griniew (1913) — Percent, Dry Basis Mixture of Lecithin Cephalin Phosphatids Lipoids of the lipoids of benzol extract +Cd the alcohol extract solu- of the acetone and Phos- Phos-l compounds ble in water alcohol ex- tissues phorus phatids Ex- tract Ex- tract tracts P P Ex- tract P Ex- tract P Ex- tract P Liver 0.3 15.9 1.2 0.022 0.5 0.011 2.7 0.03 2.04 0.03 9.5 0.18 Kidneys 0.2 14.1 1.0 0.023 0.34 0.008 3.7 0.015 4.1 0.06 5.0 0.09 Brain 0.6 31.8 0.5 0.007 2.5 0.05 2.05 0.4 5.4 0.07 2.8 0.06 Heart 0.15 12.2 0.73 0.018 0.31 0.006 1.65 0.003 3.7 0.05 5.7 0.08 Muscles 0.16 7.3 0.9 0.016 0.35 0.008 1.8 0.008 2.02 0.02 2.2 0.03 Lung-s 0.2 9.7 1.56 0.03 1.1 0.038 1.83 0.006 2.3 0.05 2.8 0.07 Spleen 0.25 14.4 0.3 0.04 0.6 0.055 2.08 0.0002 7.5 0.12 3.8 0.07 Medulla 0.04 1.9 0.15 0.003 0.17 0.004 0.43 0.43 0.004 0.7 0.01 ( x ) Apparently the heading "Lipoids" given here in the original is a mistake. LIPOIDS OF THE ORGANS OF TUBERCULOUS GUINEA PIGS Griniew (1913) — Percent, Dry Basis Orgrans and tissues Phos- phorus Phos- phatids Lecithin Cephalin, myelin, and others Phosphatids of benzol ex- tract and of the Cd com- pounds Mixture of the lipoids of the filtrate from the acetone The portion of the alco- hol extract soluble in water Ex- tract P Ex- tract P Ex- tract P Ex- tract P Ex- tract P Liver Kidneys Brain Heart Muscles Lungs Spleen Medulla 0.13 0.09 0.56 0.15 0.08 0.14 0.14 0.02 9.5 13.7 28.6 11.7 9.4 10.0 11.5 2.2 0.58 0.97 0.42 0.65 0.59 0.37 0.16 0.08 0.011 0.03 0.012 0.02 0.011 0.011 0.003 0.0009 0.8 0.73 4.7 1.02 0.30 0.4 0.6 0.12 0.022 0.0093 0.072 0.02 0.0084 0.007 0.006 0.0022 1.9 7.1 16.9 3.1 3.5 2.1 2.4 0.83 0.01 0.02 0.4 0.007 0.01 0.015 6.005 3.4 0.3 2.6 2.9 0.8 3.1 0.8 0.04 0.05 0.004 0.04 0.04 0.01 0.03 0.006 0.0005 2.6 4.6 3.8 3.9 4.1 3.2 7.5 1.1 0.04 0.02 0.05 0.06 0.04 0.07 0.1 0.01 COMPARISON OF LIPOIDS AND PHOSPHORUS IN NORMAL AND TUBERCULOUS ORGANS— (Griniew, 1913) Percent, Dry Basis Organs and tissues Liver . . . Kidneys Brain... Heart.. . Muscles. Lung-s.. . Spleen... Medulla. Phosphatids Nor- mal 15.9 14.1 31.8 12.2 7.3 9.7 14.4 1.9 Pathol. 9.5 13.7 28.6 11.7 9.4 10.0 11.5 2.2 Differ- ence -6.4 -0.4 -3.2 -0.5 +2.1 4-0.3 -2.9 +0.3 Nor- mal 26.6 32.1 47.9 19.5 16.8 21.1 29.8 8.7 Lipoids Pathol. 21.5 34.8 50.4 19.1 13.6 19.7 22.4 4.9 Differ- ence -5.1 +2.7 +2.5 -0.4 -3.2 -1.4 -7.4 -3.8 Nor- mal 0.30 0.20 0.60 0.15 0.16 0.20 0.25 0.04 Phosphorus Pathol. 0.13 0.09 0.56 0.15 0.08 0.14 0.14 0.02 Differ- ence -0.17 -0.11 -0.04 -0.08 -0.06 -0.11 -0.02 PHOSPHORUS METABOLISM 689 BIBLIOGRAPHY 1897 Abderhalden, Emil: Zur quantitativen Analyse des Blutes, Zeit. physiol. Chem., 23, 521-531. 1898 Abderhalden, Emil: Zur quantitativen vergleichenden Analyse des Blutes, Ibid., 25, 65-115. 1899a Abderhalden, Emil: Die Beziehungen der Wachsthumsgeschwindigkeit des Sauglings zur Zusammensetzung der Milch beim Kaninchen, bei der Katze und beim Hunde, Ibid., 26, 487-497. 1899b Abderhalden, Emil: Die Beziehungen der Zusammensetzung der Asche des Sauglings zu derjenigen der Asche der Milch, Ibid., 26, 498-500. 1899c Abderhalden, Emil: Die Beziehungen der Zusammensetzung der Asche des Sauglings zu derjenigen der Asche der Milch beim Meerschwein- chen, Ibid., 27, 356-367. 1899d Abderhalden, Emil: Die Beziehungen der Wachsthumsgeschwindigkeit des Sauglings zur Zusammensetzung der Milch beim Hunde, beim Schwein, beim Schaf, bei der Ziege und beim Meerschweinchen, Ibid., 27, 408-462; 594. 1905 Abderhalden, Emil: Abbau und Aufbau der Eiweisskorper im tierischen Organismus, Ibid., 44, 17-52. 1912 Abderhalden, Emil: Futterungsversuche mit vollstandig abgebauten Nahrungsstoffe, Ibid., 77, 22-58. 1907 Abderhalden, Emil, and Casimir Funk: Beitrag zur Kenntniss der beim Kochen von Casein mit 25%iger Schwefelsaure und mit starker Salzsaure entstehenden Spaltungsprodukte, Ibid., 53, 19-30. 1906 Abderhalden, Emil, and Andrew Hunter: Hydrolyse des im Eigelb des Huhnereies enthaltenen Proteins ("Vitellin"), Ibid., 48, 505-512. 1910 Abderhalden, Emil, and Leo Langstein: Vergleichende Untersuchung liber die Zusammensetzung des Caseins aus Frauen- und Kuhmilch, Ibid., 66, 8-12. 1909 Abderhalden, Emil, E. 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Zeit. 31, 46-64. 1871 Bauer, Jos.: Der Stoffumsatz bei der Phosphorvergiftung, Zeit. Biol. 7, 61-85. 1878 Bauer, Jos.: Ueber die Eiweisszersetzung bei Phosphorvergiftung, Ibid. 14, 527-541. 1911 Bauer, T.: Ueber das Verhalten der Epithelkorperchen bei der Osteo- malacic, Frankfurter Zeit. Path. 7, 231-237. 1913 Baumann, A.: Ueber den stickstoffhaltigen Bestandteil des Kephalins, Biochem. Zeit. 54, 30-39. 1885 Baumstark, F.: Ueber eine neue Methode, das Gehirn chemisch zu erforschen, und deren bisherige Ergebnisse, Zeit. physiol. Chem. 9, 145-210. 1904 Bayliss, W. M. : The Kinetics of Tryptic Action, Archives des sci. biol. 11, Suppl., 261-296. St. Petersburg. 1913 Bayliss, W. M.: Researches on the Nature of Enzyme Action. III. The Synthetic Action of Enzymes, Jour, of Physiol. 46, 236-266. 1903 Bayon, P. G.: Erneute Versuche iiber den Einfiuss des Schilddrusen- verlustes und der Schilddrusenfiitterung auf die Heilung von Knochen- bruchen, Verhandl. Physik.-Med. Gesellsch. zu Wiirzburg, N. F. 35, 249-311. 1893 Bechamp, A.: Sur la caseine et le phosphore organique de la caseine, Compt. rend. Acad, des sci. 117, 1085-1088; also Bui. Soc. chim. de Paris 61 (ser. 3, Vol. 11) (1894), 152-176. 1901 Bechhold, Heinrich: Ueber Phosphorsaureester von Eieralbumin, Zeit. physiol. Chem. 54, 122-127. 1844 Becquerel, Louis Alfred, and A. Rodier: Recherches sur la composition du sang dans l'etat de sante et dans l'etat de maladie, Paris. 127 pp. Trans, by Eisemann 1845, 162 pp. 1907 Benedict, Francis Gano: The Influence of Inanition on Metabolism, Car- negie Inst. Pub. 77. 1870 Beneke, F. W.: Zur Wiirdigung des Phosphors. Kalkes in physiolog. und therapeut. Beziehung, Marburg. 48 pp. See Jahresb. d. in- u. aus- landisch. ges. Med. 151 (1871), 137-139. 1906 Bensley, R. R.: An Examination of the Methods for the Microchemical Detection of Phosphorus Compounds Other than Phosphates in the Tissues of Animals and Plants, Biological Bui. 10, 49-65. 1910 Benson, C. C, M. B. Tamblyn and Students: A Day's Metabolism, Jour. Home Economics 2, 658. 1890 Beraz, Heinrich: Ueber die Bedeutung des Kalkes fur die Zahne, Zeit. Biol. 27, 386-397. 1910a Berg, Ragnar: Ueber das Ausscheidung von per os eingeftihrten Phos- phaten, besonders der Calciumphosphate, Biochem. Zeit. 30, 107-142. 1910b Berg, Ragnar: Ueber Phosphorsaurestoffwechsel, Chem. Zeitung 34, 1049. 1911a Berg, Ragnar: Eine Fehlerquelle bei Versuchen iiber Phosphorsaure- stoffwechsel, Zeit. physikal. u. diatet. Ther. 15, 98-100. PHOSPHORUS METABOLISM 597 1911b Berg, Ragnar: Schlusswort, Ibid. 15, 240-242. 1910 Berg, R., and C. Rose: Ueber den Mechanismus der Beeinflussung der korperlichen Entwicklung durch die Wasserharte, Biochem. Zeit. 27, 204-222. 1906-7 Berg, William N., and William J. Gies: Studies of the Effects of Ions on Catalysis, with Particular Reference to Peptolysis and Tryptolysis, Jour. Biol. Chem. 2, 489-546. 1898a Bergell, Peter: Ueber den Phosphorsaurestoffwechsel unter normalen und pathologischen Verhaltnissen, Fortschritte der Med. 16, 1-18. 1898b Bergell, Peter: Die Bedeutung der Phosphorsaure im menschlichen und thierischen Organismus, Inaug. Diss., Berlin. 1900 Bergell, Peter: Darstellung des Lecithins, Ber. deut. chem. Gesell. 33, II, 2584-2586. 1901 Bergell, Peter: Ueber das Spaltung des Lecithins durch den bei voll- standigem Darmverschluss abgesonderten Darmsaft, Centralbl. allgem. Path. u. path. Anat. 12, 633, 634. 1905 Bergell, Peter, and A. Braunstein: Ueber das Lecithin und Bromlecithin, Ther. der Gegenwart 46 (N. S., 7), 156-160. 1908 Bergell, Peter, and Leo Langstein: Beitrage zur Chemie der Frauen- milch. I. Mitt. Unterschiede zwischen dem Kasein der Frauen- und Kuhmilch, Jahrb. Kinderheilk. 68, 568-576. 1901 Bergmann, W.: Ueber die Ausscheidung der Phosphorsaure bei Fleisch- und Pflanzenfressern, Arch. exp. Path. u. Pharm. 47, 77-81. 1908 Berkley, H. J. : Action of Lecithin in Exophthalmic Goiter, Johns Hopkins Hosp. Bui., Sept., 1908; through Jour. Amer. Med, Assoc. 51, 1464. 1903a Bernard, Leon, Bigart and H. Labbe: Sur la secretion de lecithine dans les capsules surrenales, Compt. rend. Soc. de biol. 55, 120-122. 1903b Bernard, Leon, Bigart and H. Labbe: Importance de la lecithine dans les fonctions de la capsule surrenale et sa secretion par cet organe, La presse med. 11, 119, 120. 1912 Bernardini, Luigi: (Chemical Composition of the Rice Embryo), Atti. accad. Lincei 21, I, 283-289; through Chem. Abs. 6 (1912), 1171. 1908 Bernazky, Stanislaw Stanislawovitch : (Das Lezithin des Knochenmarks normaler und immunisierter tiere und die Verteilung des Phosphats im Organismus), St. Petersburg. 60 pp.; through Biochem. Centralb. 7 (1908), 827. 1898 Bernstein: Die Oophorinbehandlung bei Osteomalacic, Munch, med. Wochenschr. 45, 1, 427, 428. 1878 Bertram, Julius: Ueber die Ausscheidung der Phosphorsaure bei den Pflanzenfressern, Zeit. Biol. 14, 335-382. 1899 Bertz, F. : Ueber die chemische Zusammensetzung der Zahne, Inaug. Diss., Wiirzburg. 36 pp.; through Jahresb. u. d. Fortschr. d. Thierchem. 30 (1900), 457, 458. 1816 Berzelius, Jacob: Untersuchungen uber die Zusammensetzung der Phos- phorsaure, der phosphorigen Saure und ihrer Salze, Gilbert's Annalen der Physik 53 (new ser. 23), 393-446. 1902 Bethe, Albrecht: Ueber einige Educte des Pferdegehirns, Arch. exp. Path. u. Pharm. 48, 73-86. 1858 von Bezold, A. : Das chemische Skelett der Wirbelthiere, Zeit. wissensch. Zool. 9, 240-269; through Jahresb. ii. d. Fortschr. d. Chem. u. verwand- ter Theile andere Wissensch. 1858, 553, 554. 598 OHIO EXPEEIMENT STATION: TECHNICAL BUL. 5 1844 von Bibra, Ernst: Chemische Untersuchungen liber die Knochen und Zahne des Mensehen und der Wirbelthiere, mit Rucksichtnahme auf ihre physiologischen und pathologischen Verhaltnisse, Schweinfurt. 430 pp. Through Schrodt, 1876. 1853 von Bibra, Ernst: Ueber das Gehirn, Annalen der Chem. u. Pharm. 57, 201-224. 1854a von Bibra, Ernst: Vergleichende "Untersuchungen iiber das Gehirn des Mensehen und der Wirbelthiere, Mannheim. 134 pp. 1854b Von Bibra, Ernst: Ueber das Ruckenmark und die Nerven/ Annalen der Chem. u. Pharm. 91, 1-33. 1911a Bickel, A.: Zur Kenntnis des Lezithinstoffwechsels, Internat. Beitrage z. Path. u. Ther. der Ernahrungsstorungen 3, 171-179. (1911b) Bickel, A.: Ueber Mastkuren, Med. Klinik (Berlin) 12, 441,442; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 473. 1852 Bidder, F., and C. Schmidt: Die Verdauungssafte und der Stoffwechsel. Eine physiologisch-chemische Untersuchung, Mitau u. Leipzig. 413 pp. 1874 Biedert, Philipp: Neue Untersuchungen und klinische Beobachtungen iiber Mensehen- und Kuhmilch als Kindernahrungsmittel, Arch. path. Anat. u. Physiol. 60, 352-379. 1880 Biedert, Philipp: Die Kinderernahrung im Sauglingsalter, Stuttgart. 372 pp. 153 refs. Later editions; 1893, 1897. 1884 Biedert, Philipp: Untersuchungen iiber die chemischen Unterschiede der Mensehen- und Kuhmilch, Stuttgart. 69 pp. 1887 Biedert, Philipp: Ueber die Eiweisskorper der Mensehen- und Kuhmilch, Deut. med. Wochenschr. 13, 105, 106; see also Schroter, 1887. 1897 Biedert, Philipp: Die Kinderernahrung im Sauglingsalter und die Pflege von Mutter und Kind, Stuttgart. 1912 Bienenfeld, Bianca: Beitrag zur Kenntnis des Lipoidgehaltes der Pla- centa, Biochem. Zeit. 43, 245-255. 1909 Biernacki, E.: (Ueberernahrung und Mineralstoffwechsel), Tygodnik lekarski 4, 197-206 (Polish); through Zentralbl. ges. Physiol, u. Path. d. Stoffwechsels 10 (new ser. 4) (1909), 449-455; 481-496; and 11 (new ser. 5) (1910), 240. 1910 Biernacki, E.: Ueber die Beziehungen zwischen dem Umsatz mineralischer und organischer Nahrungsstoffe, Wien. klin. Wochenschr. 23, 850-854. 1898 Biffi, Ugo: Zur Kenntniss der Spaltungsprodukte des Caseins bei der Pankreasverdauung, Arch. path. Anat. u. Physiol. 152, 130-157. 1904 von Bilgorajski, Marian: Klinische Erfahrungen mit Protylin und dessen Eisen- und Bromkombinationen, Wien. klin. Rundschau 18, 188-190; 206-208. 1903a Billon, F., and H. Stassano: Sur la maniere d'etudier l'action des com- poses phosphores organique naturels et synthetiques, Comp. rend. Soc. de biol. 55, 276. 1903b Billon, F., and H. Stassano: Action de quel que composes phosphores sur la nutrition, Ibid. 55, 277-279. 1899 Bing, H. J.: Untersuchungen iiber die reducirenden Substanzen im Blute, Skand. Arch. Physiol. 9, 336-411. 1901 Bing, H. J.: Ueber Lecithinverbindungen, Ibid. 11, 166-175. 1910 Bircher, E.: Zur Wirkung der Thyreoidintabletten auf das normale Knochenwachsthum, Arch. klin. Chir. 91, 554-566. PHOSPHORUS METABOLISM 599 1909 Birk, Walter: Untersuchungen iiber den Einfluss des Phosphorlebertrans auf den Mineralstoffwechsel gesunder und rachitischer Sauglinge, Monatsschr. Kinderheilk. 7, 450-475. 19H Birk, Walter: Zur Physiologie des neugeborenen Kindes. IV. Mitteilung. Der Stoffwechsel des Kindes wahrend der ersten Lebenstage bei kiinstlicher Ernahrung, Ibid. 10, 1-11. 1867 Bischoff, Ernst: Ueber die Ausscheidung der Phosphorsaure durch den ThierkBrper, Zeit. Biol. 3, 309-323. 1894 von Bitto, Bela: Ueber die Bestimmung des Lecithingehaltes der Pflanzensamen, Zeit. physiol. Chem. 19, 488-498. 1913 de la Blanchardiere, P.: Ueber die Wirkung der Nuclease, Ibid. 87, 291- 309. 1897a Blauberg, Magnus: Ueber die chemische Zusammensetzung einiger "Nahrsalz," nebst kurzen Bemerkungen iiber die Bedeutung der Mineralstoffe fiir den Organismus, Arch. Hygiene 30, 95-124. 1897b Blauberg, Magnus: Weitere Untersuchungen iiber Kindernahrungsmittel, nebst kurzen Bemerkungen iiber die mikroscopische und bacteriolog- ische Prufung derselben, Ibid. 30, 125-155. 1897c Blauberg, Magnus: Ueber die Mineralbestandtheile der Sauglingsfaces bei natiirlicher und kiinstlicher Ernahrung wahrend der ersten Lebens- woche, Ibid. 31, 115-141; also Inaug. Diss., Berlin, 1897, abstract Jahresb. ii. d. Fortschr. d. Thierchem. 27 (1897), 382, 383. 1900a Blauberg, Magnus: Experimented Beitrage zur Frage iiber den Mineral- stoffwechsel bei kunstlich ernahrten Saugling, Zeit. Biol. 40 (N. S. 22), 1-35. 1900b Blauberg, Magnus: Ueber den Mineralstoffwechsel beim naturlich ernahrten Saugling, Ibid. 40 (N. S. 22), 36-53. 1906 Bloch, Bruno: Die Umwanglung der Purinkorper im Saugetierorgan- ismus, Biochem. Centralb. 5, 521, 561, 817, 873. 1900 Bloch, Ernst: Ueber das Plasmon (Caseon) als Eiweissersatz, nebst Beitragen zur Lehre vom Eiweissstoffwechsel, Zeit. diatet. u. physikal. Ther. 3, 482-504. 1861 Blondlot: Sur la recherche toxicologique du phosphore par la coloration de la flame, Compt. rend. Acad, des sci. 52, 1197-1200. 1912- Bliihdorn: Eine Demonstration des Einflusses der Reaktion auf den 1913 Umsatz von Kalk und Phosphorsaure im Dickdarm der Sauglings, Monatsschr. Kinderheilk., Originalen, 11, 68-79. 1896 Blumenthal, Ferd.: Ueber die Produkte der bakterischen Zersetzung der Milch, Arch. path. Anat. u. Physiol. 146, 65-85. 1879 Blyth, A. Winter: The Composition of Cow's Milk in Health and Disease, Jour, of the Chem. Soc. 35, 530-539. 1896 Boddaert, A.: Contribution a l'etude de Taction des hypophosphites sur la nutrition, Archives de pharmacodynamic 2, 195-207. 1849 Bocker, Friedrich Wilhelm: Beitrage zur Heilkunde, insbesondere zur Krankheits-, Genussmittel- und Arzneiwirkungs-Lehre, nach eigenen Untersuchungen, vol. I, 139-187. 1911 Bogdanow, B. A.: (Zur Frage iiber die Ausscheidung des organischen Phosphors (im Ham) bei einigen Infektionskrankheiten), Russki Wratsch 1911, Nr. 17, 18, 19, 20; through Jahresb. U. d. Fortschr. d. Thierchem. 41 (1911), 485. 600 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1877 Bokay, A.: Ueber die Verdaulichkeit des Nucleins und Lecithins, Zeit. physiol. Chem. 1, 157-164. 1910a Bolle, Alwin: Ueber den Lecithingehalt des Knochenmarks von Mensch und Haustieren, Inaug. Diss., Erlangen. 25 pp. 1910b Bolle, Alwin: Ueber den Lecithingehalt des Knochenmarks von Mensch und Haustieren, Biochem. Zeit. 24, 179-190. 1902 Bonanni, A.: (Die Phosphorfleischsaure der Muskeln bei Veratrinver- giftung), Accad. med. di Roma 29, through Jahresb. u. d. Fortschr. d. Thierchem. 32 (1902), 537, 538. 1906 Bonanni, A.: Ein weiterer Beitrag zur Kenntnis der menschlichen Galle. Die Gegenwart gebundener Glykuronsaure in der Galle; Ausscheidung der Borneol-Glykuronsaure durch dieselbe, Bullett. d. reale Accad. med. di Roma. Anno. 32; through Jahresb. ii. d. Fortschr. d. Thierchem. 36 (1906), 468-470. 1894a Bongartz: Ueber seuchenartiges Auftreten der Knochenbriichigkeit im Fruhjahr und Sommer dieses Jahres, Berlin, tierarzte Wochenschr. 435-437. 1894b Bongartz: Ueber seuchenartiges Auftreten der Knochenbriichigkeit im Fruhjahr und Sommer dieses Jahres, Fiihling's Landwirtsch. Zeitung 43, 666-668; see Grandeau, 1905. 1913 Bonnamour, Albert Badolle, and Escallon: Decalcification et lesions osseuses chez le lapin sous l'infiuence du lactose en injections intra- veineuses, Compt. rend. Soc. de biol. 74, 1106, 1107. 1903 Bonnetat, L.: Action des engrais phosphates sur la composition chem- ique des fourrages, Jour, d'agri. prat. 5, 343-345. 1906 Boos, William F.: Ueber Darstellung und Zusammensetzung der Myko- nukleinsaure aus Hefe, Arch. exp. Path. u. Pharm. 55, 16-20. 1909 Boos, William F.: On the Reducing Compound of Yeast Nucleic Acid, Jour. Biol. Chem. 5, 469-475. 1838 Bopp: Stimmen liber die als Knochenbriichigkeit erkannte Krankheit des Rindviehes in der Provinz Rheinheffen, Magazin. f. d. ges. Thierheilk. 4, 322-350. 1902a Bordas, F., and de Raczkowski: Variation de l'acide phosphorique suivant l'age du lait, Compt. rend. Acad, des sci. 135, 302, 303; also Annales de chim. anal. 7 (1902), 370, 371. 1902b Bordas, F., and de Raczkowski : De l'infiuence de l'ecremage sur la repar- tition des principaux elements constitutifs du lait, Compt. rend. Acad. des sci. 135, 354, 355: also Annales de chim. anal. 7 (1902), 372, 373. 1903 Bordas, F., and de Raczkowski: Diminution du taux des lecithines dans les laits chauffes, Compt. rend. Acad, des sci. 136, 56, 57; also Annales de chim. anal. 8 (1903), 168, 169. 1909 Bornstein, Arthur: Die chemische Zusammensetzung des Blutes bei progressiver Paralyse, Monatsschr. Psychiat. u. Neurol. 25, 160-168. 1911 Bornstein, Arthur : Ueber die Lecithinamie der Geisteskranken, Zeit. ges. Neurol, u. Psychiat., Originalien 6, 605-608. 1905 Bornstein, Karl: Ueber den Schwefel- und Phosphorstoffwechsel bei abundanter Eiweisskost, Arch. ges. Physiol. 106, 66-79. 1906 Borri, Andrea : Untersuchung iiber die phosphorhaltigen Bestandteile der Darmschleimhaut, Arch. Verdauungskrankheiten 12, 301-307; also Studio sui composti fosforati della mucosa intestinale, La Clinica med. ital., Milano 45, 458-464. PHOSPHORUS METABOLISM 601 1910 Borrino, Angiola: Sulla nucleasi della ghiandola mammaria, Archivio di fisiologia, 8, 73-80. 1911 Borrino, Angiola: Sull' origine della caseina del latte, Rivista di clinica pediatrica 9, 211-220; through Zentralbl. Biochem. u. Biophys. 13 (1912), 744, 745. 44 refs. 1911 Borschim, S.: Ueber den Einfluss des Lecithins auf die Resorption der Haut, Biochem. Zeit. 35, 471-477. 1907 Bosworth, Alfred W.: Chemical Studies of Camembert Cheese, N. Y. Agr. Exp. Sta., Tech. Bui. No. 5. Pp. 23-39. 1913 Bosworth, Alfred W. : The Action of Rennin on Casein, Jour. Biol. Chem. 15, 231-236; also N. Y. Agr. Exp. Sta. (Geneva), Tech. Bui. No. 31, 7 pp. 1913 Bosworth, Alfred W., and Lucius L. Van Slyke: Preparation and Com- position of Basic Calcium Caseinate and Paracaseinate, Jour. Biol. Chem. 14, 207-210. 1903 Bottazzi, F.: Proprieta di nucleoproteidi estratti dalla placenta muliebre (Properties of Nucleoprotein Extracted from Human Placenta.), Bollett. d. r. Accad. med. di Genova 18, 247-250. 1846a Boussingault, M.: Sur le developpement de la substance minerale dans le systeme osseux du pore, Annales de chim. et de phys., 3rd ser.,16, 486-493. 1846b Boussingault, M.: Untersuchungen Tiber die Entwickelung der mineral- ischen Substanzen in dem Knochensystems des Schweins, Annalen der Chem. u. Pharm. 59, 322-330. 1830 Braconnot, H.: Memoire sur le caseum et sur le lait; nouvelles ressources qu'ils peuvent offrir a la societe, Annales de chim. et de phys., 2nd ser., 43, 337-351. 1907 Braddon, W. L.: The Cause and Prevention of Beri-Beri, London and New York. XIII + 544 pp. Through Exp. Sta. Record 24 (1911), 66, 67. 1913 Brahm, C: Nukleinsauren und Spaltprodukte; Oppenheimer's Handbuch der Biochemie, Erganzungsband, 80-104. 1902 Brand, J.: Beitrag zur Kenntniss der menschlichen Galle, Arch. ges. Physiol. 90, 491-522. 89 refs. 1903-4 Braunstein, A. : Beobachtungen uber die Ausscheidung der Chloride, der Phosphorsaure, des Stickstoffs und Ammoniaks bei Carcinom, Zeit. f. Krebsforschung 1, 199-224. 1911 Breaudat, L., and Denier: The Use of Rice Bran in the Prevention and Cure of Beriberi, Annales de lTnst. Pasteur 25, 167-189. 1851a Breed, D.: Ueber den Gehalt des normalen Urins an Phosphorsaure, Annalen der Chem. u. Pharm. 78 (n. s. 2), 150-157. 1851b Breed, D.: Analyse der Asche des menschlichen Gehirns, Ibid. 80, 124. 1891 Breisacher, Leo: Zur Physiologie des Schlafes, Dubois-Reymond's Arch. Physiol. 321-334. (1911) Brennemann, Joseph: (Ein Beitrag zur Kenntniss der Ursache und Herkunft der harten Brockel im Sauglingsstuhl), Amer. Jour. Dis- eases of Children 1, 341-359; through Jahresb. u. d. Fortschr. d. Thier- chem. 41 (1911), 298. 1912 Brossa, G. A.: Ueber die biologische Wertigkeit der a-Nucleinsaure, Arch. Anat. u. Physiol,, physiol. Abt., 191-196. 602 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1892a Brown-Sequard: Effets physiologique d'un liquide extrait des glandes sexuelles et surtout des testicules, Compt. rend. Acad, des sci. 114, 1237. 1892b Brown-Sequard: Effets produits sur de nombreux morbides par des injections sous-cutanees d'un extrait liquide retire des testicules, Ibid. 114, 1318, 116, 856. 1890 Brubacher, Heinrich: Ueber den Gehalt an anorganischen Stoffen, besonders an Kalk, in den Knochen und Organen normaler und rachit- ischer Kinder, Zeit. Biol. 27, 517-549. 1908 Bruck, A. W.: Ueber den Mineralstoffwechsel beim kiinstlich genahrten Saugling, Monatsschr. Kinderheilk. 6, 570-579. 1908 Brugsch, Theodor: Zur Physiologie und Pathologie des Nukleinstoff- wechsels, Med. Klinik, Berlin, 4, II, 1841-1843. 1909 Brugsch, Theodor: Zur Stoffwechselpathologie der Gicht. VIII. Mitteil- ung. Die Harnsaure im Blute, bei der Gicht, ihre Bindungsweise— Gicht und Leukamie — die Absorption der Harnsaure durch den Knorpel und ihre Verhinderung, Zeit. exper. Path. u. Ther. 6, 278-299. (1911) Brugsch, Th., and N. Masuda: Ueber das Verhalten des Diinndarmsaftes und -extraktes, ferner des Extraktes einiger Bazillen (Coli, Strep tok- ken) gegeniiber Kasein, Lecithin, Amylum. Ein Beitrag zur funk- tionell-diagnostischen Priifung der Faces auf Fermente des Pankreas, Ibid. 8, 617-623; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 295. 1907a Brugsch, Theodor, and Alfred Schittenhelm : Zur Stoffwechselpathologie der Gicht. I. Mitteilung. Der Harnsauregehalt des Blutes bei purinfreier Kost, Ibid. 4, 438-445. 1907b Brugsch, Theodor, and Alfred Schittenhelm: Zur Stoffwechselpathologie der Gicht. II. Mitteilung. Beziehung zwischen Blut und Harnsaure, Ibid. 4, 446-450. 1907c Brugsch, Theodor, and Alfred Schittenhelm: Zur Stoffwechselpathologie der Gicht. III. Mitteilung. Der endogene und exogene Harnsaure- und Purinbasenwerth bei der chronischen Gicht, Ibid. 4, 480-531. 1907d Brugsch, Theodor, and Alfred Schittenhelm: Zur Stoffwechselpathologie der Gicht. IV. Mitteilung. Ueber den Befund von Harnsaure in Organen. Ibid. 4, 532-537. 1907e Brugsch, Theodor, and Alfred Schittenhelm: Zur Stoffwechselpathologie der Gicht. V. Mitteilung. Ueber den Abbau von Glykokoll und Alanin beim gesunden und gichtkranken Menschen, Ibid. 4, 538-550. 1907f Brugsch, Theodor, and Alfred Schittenhelm: Zur Stoffwechselpathologie der Gicht. VI. Mitteilung. Pathogenese der Gicht, Ibid. 4, 551, 557. 1908-9 Brugsch, Theodor, and Alfred Schittenhelm: Zur Stoffwechselpathologie der Gicht. VII. Mitteilung. Das Verhalten verfutterter Purin- basen bei der Gicht, Ibid. 5, 215-226. 1910 Brugsch, Theodor, and Alfred Schittenhelm: Der Nukleinstoffwechsel und seine Storungen (Gicht, Uratsteindiathese u. a.). Jena, 1910. 166 pp. 1905 Buchner, Eduard, and Wilhelm Antoni: Existiert ein Coenzym fiir die Zymase ? Zeit. physiol. Chem. 46, 136-154. 1908 Buchner, E., and F. Klatte: Ueber das Ko-Enzym des Hefepresssaftes Biochem. Zeit. 8, 520-557. PHOSPHORUS METABOLISM 603 1904 Bttchmann, L.: Beitrage zum Phosphor- Stoffwechsel, Zeit. diatet. u. physikal. Ther. 8, 67-74; 148-160. 1894 Biilow, K.: Ueber Glycerinphosphorsaure, Arch. ges. Physiol. 57, 89-92. 1904 Biirger, Max: Ueber Protylin und seinen Wert als Nahr- und Heilmittel, insbesondere bei rachitischen Zustanden im Kindesalter, Therapeut. Monatssh. 18, 302-306. 1913a Burger, M., and Beumer: Zur Lipoidchemie des Blutes, Berlin, klin. Wochenschr. 50, 112-114. 1913b Biirger, M., and Beumer: Ueber die Phosphatide der Erythrocytenstrom- ata bei Hammel und Menschen, Biochem. Zeit. 56, 446-456. 1873 Bunge, G.: Ueber die Bedeutung des Kochsalzes und das Verhalten der Kalisalze im menschlichen Organismus, Zeit. Biol. 9, 104-143. 1874 Bunge, G. : Der Kali-, Natron- und Chlorgehalt der Milch, verglichen mit dem anderer Nahrungsmittel und des Gesammtorganismus der Sauge- thiere, Ibid. 10, 295-335. 1876 Bunge, G.: Zur quantitativen Analyse des Blutes, Ibid. 12, 191-216. 1885a Bunge, G.: Ueber die Assimilation des Eisens, Zeit. physiol. Chem. 9, 49-59. 1885b Bunge, G. : Analyse der anorganischen Bestandtheile des Muskels, Ibid. 9, 60-62. 1886 Bunge, G.: Eine Bemerkung zur Theorie der Drusenfunction, Arch. Anat. u. Physiol., physiol. Abt., 539, 540. 1889 Bunge, G.: Ueber die Aufnahme des Eisens in den Organismus des Sauglings, Zeit. physiol. Chem. 13, 399-406. 1901 Bunge, G. : Der wachsende Zuckerkonsum und seine Gef ahren, Zeit. Biol. 41, 155-166. 1904a Burian, Richard: Zur Kenntniss der Bindung der Purinbasen in Nuclein- sauremolekul, Ber. deut. chem. Gesell. 37, I, 708-712. 1904b Burian, Richard: Zur Frage der Bindung der Purinbasen im Nuclein- sauremolekul, Zeit. physiol. Chem. 42, 297, 298. 1905 Burian, Richard: Ueber die oxydative und die vermeintliche synthet- ische Bildung von Harnsaure in Rinderleberauszug, Ibid. 43, 497-531. 1907a Burian, Richard: Weitere Beitrage zur Kenntnis der Diazoaminoverbind- ungen der Purinbasen, Ibid. 51, 425-437. 1907b Burian, Richard: Pyrimidinderivate aus Purinbasen, Ibid. 51, 438-456. 1897 Burian, Richard, and Heinrich Schur: Ueber die Nucleinbildung im Saugethierorganismus. I. Mitteilung, Ibid. 23, 55-73. 1900 Burian, Richard, and Heinrich Schur: Ueber die Stellung der Purin- korper im menschlichen Stoffwechsel. Drei Untersuchungen, Arch, ges. Physiol. 80, 241-343. 1901 Burian, Richard, and Heinrich Schur: Ueber die Stellung der Purin- korper im menschlichen Stoffwechsel, Ibid. 87, 239-354. 1903 Burian, Richard, and Heinrich Schur: Das quantitative Verhalten der menschlichen Harnpurinausscheidung, Ibid. 94, 273-336. 1903 Burnet, James: On the Therapeutic Value of the Nuclein Compounds, Therapeutic Gazette (Detroit) 19, 312, 313. 1906 Burnett, E. A.: Fattening Pigs on Corn and Tankage, Nebr. Agr. Exp. Sta. Bui. 94, 12pp. 1908 Burnett, E. A.: The Effect of Food on Breaking Strength of Bones, Ibid. 107, 11-39. 604 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1910 Burnett, E. A.: The Effect of Food on the Strength, Size and Composi- tion of the Bones of Hogs, Neb. Agr. Exp. Sta. Rept. for 1910, pp. 178-208. 1900 Burow, Robert: Der Lecithingehalt der Milch und seine Abhangigkeit vom relativen Hirngewichte des Sauglings, Zeit. physiol. Chem. 30, 495-507. 1905 Burow, Robert: Beitrage zur Entscheidung der Frage, ob die Casei'ne verschiedener Tierarten identisch sind, Diss., Basel. 28 pp. 1910 Burow, Robert: Ueber das Vorkommen eisenhaltiger Lipoide in der Milz. Vorlaufige Mitteilung, Biochem. Zeit. 25, 165-170. 1907 Burr, Anton: Eigenschaften und Zusammensetzung der Schweinemilch, Milch-Zeitung 36, 565, 566. 1911 Buslik and Goldhaber: Stoffwechselversuche mit Lecithineiweiss Dr. Klopfer (Glidine), Zeit. f. physikal. u. diatet. Therap. 15, 93-97; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 548. 1911 Buxton, B. H., and A. H. Rake: (Wirkung der Verdiinnung auf die Aus- flockung von Kolloiden), Jour. Med. Research 22, 483-500; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 8, 9. 1868 Byasson, H.: Essai sur la relation qui existe a l'etat physiologique entre l'activite cerebrale et la composition des urines, Paris. {1911) Cagnetto, G.: Die Veranderungen der normalen chemischen Bestandteile des Knochengewebes nach Behandlung mit Strontiumsalzen, Patho- logica 3, 137-139; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 361. 1896 Camerer, W. Sr.: Harnsaure, Xanthinbasen, und Phosphorsaure im menschlichen Urin, Zeit. Biol. 33 (N. F. 15), 139-155. 1904 Camerer, W. Sr.: Analysen vom menschlichen Urin, Ibid. 45 (N. F. 27), 1-22. 1896 Camerer, W., and Soldner: Analysen der Frauenmilch, Kuhmilch und Stutenmilch, Ibid. 33 (N. F. 15), 535-568. 1900 Camerer, W. Jr.: Die chemische Zusammensetzung des Neugeborenen. Second article. Ibid. 40 (N. F. 22), 529-534. 1902a Camerer, W. Jr.: Die chemische Zusammensetzung des neugeborenen Menschen. Third article. Ibid. 43 (N. F. 25), 1-12. 1902b Camerer, W., Jr.: Beobachtungen und Versuche liber die Ammoniakaus- scheidung im menschlichen Urin mit Beriicksichtigung noch weiterer stickstoffhaltiger Urinbestandteile und Bestimmung der Aciditat nach Lieblein, Ibid. 43 (N. F. 25), 13-45. 1900 Camerer, W. Jr., and Soldner: Die chemische Zusammensetzung des Neugeborenen. First article. Ibid. 39 (N. F. 21), 173-192. 1903 Camerer, W. Jr., and Soldner: Die Aschenbestandteile des neugeborenen Menschen und der Frauenmilch, Ibid. 44 (N. F. 26), 61-77. 1902 Camerer, W., Soldner and Herzog: Die chemische Zusammensetzung des neugeborenen Menschen, Ibid. 43 (N. F. 25), 1-12. 1909a Capezzuoli, Cesare: Ueber die eisenhaltigen Korper der Milz, Zeit. physiol. Chem. 60, 10-14. 1909b Capezzuoli, Cesare: Mineralstoffzusammensetzung der Knochen bei Osteomalacic, Biochem. Zeit. 16, 355, 356. 1907 Carlier, Albert: Experiences sur l'influence du phosphate alimentaire (Poudre d'os) sur l'elevage des gorets, Annales de Gembloux 17, 373- 377. PHOSPHORUS METABOLISM 605 1909 Carlier, Albert : Experiences sur Pemploi de la poudre d'os et de la craie moulue dans Palimentation des gorets, Ibid. 19, 166, 167. 1892 Carnot, Adolphe: Recherche du fluor dans les os modernes et les os fossiles, Compt. rend. Acad, des sci. 114, 1189-1192. 1908 Carpiaux, E. : Contribution a l'etude de l'assimilation du phosphore et de la chaux pendent la vie embryonaire du poussin, Bui. Acad, royal Belgique, CI. d. sci., 1908, 283-295. 1904 Carre, P.: Sur les ethers phosphoriques de la glycerine, Compt. rend. Acad, des sci. 138, 47-49. . 1901 Carriere, G.: Influence de la lecithine sur les echanges nutritifs, Compt. rend. Acad, des sci. 133, 314-316. 1911 Casanova, Carlo: (Ueber Eierlecithin, iiber eine charakteristische Farb- reaktion und iiber Verfalschung desselben), Bol. Chim. Farm. 50, 509-513; through Zeit. Unters. d. Nahrungs- u. Genussmittel 23 (1912), 695. 1907 Cathcart, E. P., and C. E. Fawsitt: Metabolism during Starvation. Part II. Inorganic. Jour, of Physiol. 36, 27-32. 1909 Cattaneo, Cesare: Ulteriori ricerche sul rapporto fra calce e magnesio nella ossa dei rachitici, La pediatria 17, 497, 498; through Ch. Abs. 4 (1910), 2668. 1903 Cautru, Fernand: Sur l'innocuite absolue de l'acide phosphorique, Bui. gener. de therapeut. 145, 223-227. 1904a Cautru, Fernand: Innocuite et indications de l'acide phosphorique, Les nouveaux remedes 20, 535-538. 1904b Cautru, Fernand: L'acide phosphorique; son innocuite; ses principal es indications en therapeutique, La presse med., Paris, 12, 588, 589. 1904a Cavazzani, Emilio : II nucleone nei centri nervosi, Gazzetta degli ospedali e delle cliniche 25, 200, 201. 1904b Cavazzani, Emilio: Das Nucleon im Ostrea edulis (L.), Centralbl. Physiol. 18, 666-668. 1896 Ceconi, A.: Sul valore della determinazione della sostanze organiche fosf orate nelle orine normali e pathologische, 7th Congr. f . innere Med., Rom., 1896; through Jahresb. ii. d. Fortschr. d. Tierchem. 27 (1897), 362. 1898 Ceconi, A.: Eliminazione del fosforo organico durante l'alta febbre e la grave dispnea, II Morgagni, 1898, No. 3; through Centralbl. innere Med. 19, 883. 1894 Chabrie, C: Recherches sur les transformations chimiques de la sub- stance fundamentale de cartilage, Annales de chim. et de phys. 333 (ser. 7, Vol. 3), 524-563; also (briefer reports) Compt. rend. Acad, des sci. 118 (1894), 1057-1060; Bui. Soc. chim. 66 (1894), 420, 421; 1895 Chabrie, C: Considerations sur les phenomenes chimiques de l'ossifica- tion, Compt. rend. Acad, des sci. 120, 1226-1228. 1911 Chamberlain, Weston P.: The Eradication of Beriberi from the Philippine (Native) Scouts by Means of a Simple Change in their Dietary, Phil- ippine Jour, of Sci., B., 6, 133-146. 1910 Chamberlain, W. P., H. D. Bloombergh and E. D. Kilbourne: A Study of the Influence of Rice Diet and of Inanition on the production of Multi- ple Neuritis of Fowls and the Bearing thereof on the Etiology of Beriberi, Ibid. 6, 177-208. 606 OHIO EXPEEIMENT STATION: TECHNICAL BUL. 5 1910 Chamberlain, W. P., and E. B. Vedder: A Contribution to the Etiology of Beriberi. Ibid 6, 251-258. 1911 Chamberlain, W. P., and E. B. Vedder: A Second Contribution to the Etiology of Beriberi, Ibid. 6, 395-404. 1912 Chamberlain, Weston P., Edward B. Vedder, and Robert R. Williams: A Third Contribution to the Etiology of Beriberi, Ibid. 7, 39-52. 1905 Charrin, A.: Action des matieres minerales sur les echanges et la resistance de l'organisme, Compt. rend. Soc. de biol. 59, 112-114. 1896 Charrin, A., and Desgrez: Action des solutions mineralisees sur l'organ- isme, Ibid. 48, 805, 806. 1895 Chatin, A., and A. Muntz: Existence du phosphore en proportion notable dans les huitres, Comp. rend. Acad, des sci. 120, 1095-1097. 1908 Chavan, Paul: Influence de la fumure phosphatee et potassique sur la composition chimique du fourrage de prairie naturelle, Annuaire agric. de la Suisse 9 (22 of Ger. edition), 193-206. 1886 Chevalier, Josephine: Chemische Untersuchung der Nervensubstanz, Zeit. physiol. Chem. 10, 97-105. 1897 Chittenden, R. H.: The Protagon of the Brain, Proc. Amer. Physiol* Soc, Science 5 (N. S.), 909. 1885 Chittenden, R. H., and H. M. Painter: Casein and its Primary Cleavage Products, also Caseoses, Casein Dyspeptone and Casein Peptone, Studies from Lab. of Physiol. Chem., Yale Univ., II, 156-199; III, 66-105. 1842 Chossat, Charles: Note sur le systeme osseux, Compt. rend. Acad, des sci. 14, 451-454. 1843 Chossat, Charles: Recherches experimentales sur l'inanition, Mem. pre- sented a l'Acad. des sci. de lTnst. de France 8 (ser. 2), 438-640. 1910 Choumowa-Sieber, N. O.: Influence exercee par l'alcool sur la teneur des organes des animaux en phosphatides, Archives des sci. biol., l'Inst. imper. de med. exper. a, St. Petersbourg 15, 373-392. 56 refs. 1910 Ciaccio, C: Contributo alia distribuzione ed alia fisio-patologia cellulare dei lipoidi, Arch. f. Zellf. 5, 235-363; through Zentralbl. Biochem. u. Biophys. 11 (N. F. 2) (1910-11), 322, 323. 1910 Ciuffini, Publio: II ricambio del calcio, del magnesio, e dell' acido fosfor- ico nell' uomo sano e nel gottoso, II Policlinico, sezione med. 17, 156- 180. 1901a Claude, H., and A. Zaky: Le lecithine dans la tuberculose (Note prelim- inaire), Compt. rend. Soc. de biol. 53, 821-823; also Compt. rend. Acad, des sci. 133, 486-488. 1901b Claude, H., and A. Zaky: La lecithine dans la tuberculose, La Presse med. 9, II, 173-178. 1901c Claude, H., and A. Zaky: La lecithine dans la tuberculose. Gazette des hospitaux civils et militaires 74, 1084. 1910 Clementi, A.: Intorno all'azione della lipasi pancreatica ed enterica sulla lecitina, Archivio di fisiologia 8, 399-408; through Zentralbl. Biochem. u. Biophys. 11 (1911), 435. 1901 Cocchi, A.: Sopra il nucleoproteide della placenta umana, Lo Speri- mentale 55, 503-508. 1908 Cohen, L. J., and William J. Gies: A Study of "Protagon" Prepared by the Wilson and Cramer Method, Proc. Soc. Exp. Biol, and Med. 5, 97-100. PHOSPHORUS METABOLISM 607 1889 Cohn, Conrad: Ueber den Einfluss der Caries auf die chemische Zusam- mensetzung des Zahnbeines, Inaug. Diss., Berlin. 25 pp. 1907 Cohn, Michal: Kalk, Phosphor und Stickstoff im Kindergehirn, Deut. med. Wochenschr. 33, 1987-1991. 1913 Cohn, R.: Ueber den "Riickgang" der Lecithinphosphorsaure, Chem. Zeitung 37, 581-583. 1870 Cohn, Wilhelm: Ueber die Verwendung des basisch phosphorsauren Kalkes bei der Aufzucht von Vieh, Annalen der landwirtsch. Woch- enbl., Konig. Preussisch. Statten, pp. 431-433. 1882 Cohnheim, Julius: Vorlesungen iiber allgemeine Pathologie; ein Handbuch fiir Aerzte und Studierende. 2nd edition. Berlin. 2 Vols. 1908 Cohnheim, Otto: Die Physiologie der Verdauung und Ernahrung. 23 Vorlesungen fiir Studierende und Aerzte. Berlin and Wien. 479 pp. 1902 Colombet, Andre: De la medication arsenio-phosphoree organique dans le traitement de la tuberculose pulmonaire chronique, These de Paris. 1903 Concetti, Luigi: La cura del fosforo nel rachitismo, Rivista di clinica pediat. 1, 24-37. 1909 Cook, F. C: Metabolism of Organic and Inorganic Phosphorus, U. S. Dept. Agr., Bur. Chem., Bui. No. 123. 1911 Cooper, E. A., and Casimir Funk : Experiments on the Causation of Beri- beri, Lancet 1911, II, 1266, 1267. 1903 Coriat, Isador H.: A Contribution to the Chemistry of Nerve Degenera- tion in General Paralysis and Other Mental Disorders, Amer Jour. Insan. 59, 393-416. 1904a Coriat, Isador H.: The Production of Cholin from Lecithin and Brain- Tissue, Amer. Jour. Physiol. 12, 353-362. 1904b Coriat, Isador H.: Chemical Findings in Cerebrospinal Fluid, Amer. Jour. Insan. 60, 733-761. 1905 Coriat, Isador H. : A Review of Some Recent Literature on the Chemistry of the Central Nervous System, Jour, of Comp. Neurol. 15, 148-159. 1912 Corper, Harry J.: Chemistry of the Dog's Spleen, Jour. Biol. Chem. 11, 27-35. 1912 Costantino, A.: Beitrage zur Muskelchemie. II. Ueber den Gehalt der glatten und quergestreiften Saugetiermuskeln an organischem und anorganischem Phosphor, Biochem. Zeit. 43, 165-180. 1834 Couerbe, J. P.: Du cerveau, considere sous le point de vue chimique et physiologique, Annales de chim. et de phys. 152 (ser. 2, 56), 160-193. 1901 Coulombe, Edmond: La lecithine de l'oeuf, son emploi therapeutique, These de Paris. 56 refs. 70 pp. 1891 Courant, Georg: Ueber die Reaktion der Kuh- und Frauenmilch und ihre Beziehungen zur Reaktion des Caseins und der Phosphate, Arch. ges. Physiol. 50, 109-165. 1903 Courtial, C: Etude de la lecithine et des jaunes d'oeufs industriels, These Montpellier; through Jahresb. u. d. Fortschr. d. Thierchem. 33 (1903), 870. 1903 Cousin, H.: Sur les acides gras de la lecithine de l'oeuf, Compt. rend. Soc. de biol. 55, 913-915; also Compt. rend. Acad, des sci. 137, 68-70. 1906 Cousin, H.: Sur les acides gras de la cephaline, Compt. rend. Soc. de biol. 61, 23-25; also Jour, de pharm. et de chim. 24 (1906), 101-108. 608 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1904 Cramer, W. : On Protagon, Cholin, and Neurin, Jour, of Physiol. 31, 30- 37. 1910 Crawford, Albert C: A Poisonous Principle in Certain Cottonseed Meals, Jour, of Pharm. and Exp. Ther. 1, 519-548. 1891 Cremer, Max: Demonstration eines mit kalkarmer Nahrung unter Zugabe von Strontiumphosphate ernahrten jungen Hundes, Sitzungs- ber d. Gesell. f. Morph. u. Phys. in Mtinchen 1891, 124-126. 1910 Cronheim, J. and W.: Weitere Untersuchungen liber die Bedeutung des Lecithins fur den Stoffwechsel des Sauglings, Zeit. physikal. u. diatet. Ther. 14, 257-267. 1911a Cronheim, J. and W.: Erwiderung, Ibid. 15, 101. 1911b Cronheim, J. and W.: Schlusswort, Ibid. 15, 359. 1912 Cronheim, W. : Die Bedeutung des Lecithins im Stoffwechsel des Erwach- senen, Ibid. 16, 262-272. 1900 Cronheim, W., and Erich MUller: Zur Kenntnis der Bedeutung des organ- isch gebundenen Phosphors fur den Stoffwechsel des Kindes, Jahrb. Kinderheilk. 52, 360-364. 1902 Cronheim, W., and Erich MUller: Versuche iiber den Stoff- und Kraft- wechsel des Sauglings mit besonderer Beriicksichtigung des organisch gebundenen Phosphors, Zeit. diatet. u. physikal. Ther.6, 25-44; 92-114. 1903 Cronheim, W., and Erich MUller: Untersuchungen uber den Einnuss der Sterilisation der Milch auf den Stoffwechsel des Sauglings unter besonderer Berucksichtigung der Knochenbildung, Jahrb. Kinderheilk. 57, 45-63. 1908 Cronheim, W., and Erich MUller: Stoffwechselversuche an gesunden und rachitischen Kindern mit besonderer BerUcksichtigung des Mineral- stoffwechsels, Biochem. Zeit. 9, 76-126. 1895 Curatulo, G. E., and L. Tarulli: Einnuss der Abtragung der Eierstoke auf den Stoffwechsel, Centralbl. Physiol. 9, 149-152; through Jahresb. u. d. Fortschr. d. Thierchem. 25 (1895), 498, 499. 1911 Curschmann, H.: Ueber Osteomalacia senilis und tarda, Med. Kiinik 7, 1565-1571. 1912 Czernoruzky, M. W.: (Ueber die Wirkung der Nukleinsaure auf die fer- mentativen Prozesse im Tierorganismus), Wratschebnaja Gazeta, 1912, No. 14 and 15; through Zeit. Immunitatsforschung, II. Teil, Referate 5 (1912), 328. (See also Tschernoruzki, 1911.) 1866 Dahnhardt, C: Zur Chemie der Lymphe, Arch. path. Anat. u. Physiol. 37, 55-67. 1870 Dahnhardt, C: Zur Caseinbildung in der MilchdrUse, Arch. ges. Physiol. 3, 586-598. 1913a Dakin, H. D., and H. W. Dudley: The Racemization of Proteins and their Derivatives Resulting from Tautomeric Change. II. The Racemiza- tion of Casein, Jour. Biol. Chem. 15, 263-269. 1913b Dakin, H. D., and H. W. Dudley: The Action of Enzymes on Racemized Proteins and their Fate in the Animal Body, Ibid. 15, 271-276. 1901 Dalmastri : II zicambio dell' azoto e del f osf oro durante la cura antirabica Bollett. delle sci. med. di Bologna, Apr. 1901, No. 4; through Jahresb. u. d. Fortschr. d. Thierchem. 31 (1901), 766, 767. 1905 Dambre, L. A.: Contribution a Petude de la medication phosphoree. La phytine, Thesis, Toulouse. 64 pp. Through Biochem. Centralbl. 5 (1906-7), 133. PHOSPHORUS METABOLISM 609 1911a Daniel-Brunet, A., and C. Rolland: De l'influence du sexe et de la castra- tion sur la quantite des lipoi'des de la bile chez les bovides, Compt. rend. Acad, des sci. 153, 214, 215. 1911b Daniel-Brunet, A., and C. Rolland: Contribution a l'etude chimique et physiologique de la glande hepatique des bovides, Ibid. 153, 900-902. 1895a Danilewsky, B.: Ueber die Blutbildende Eigenschaft der Milz und des Knochenmarks, Arch. ges. Physiol. 61, 264-274. 1895b Danilewsky, B.: De l'innuence de la lecithine sur la croissance et multi- plication des organismes, Compt. rend. Acad, des sci. 121, 1167-1170; also Westnik mediciny (1896) 1, 1 and 269-285 (Russian). 1896 Danilewsky, B.: De l'influence de la lecithine sur la croissance des animaux a sang chaud, Compt. rend. Acad, des sci. 123, 195-198. 1897 Danilewsky, B.: Influence des lecithines sur le croissance, Compt. rend. Soc. de biol. 49, 475, 476. 1902 Dapper, Max: Ueber Fleischmast beim Menschen, Inaug. Diss., Marburg. 1871 Decaisne, E. : Des modifications que subit le lait de f emme par suite d'une alimentation insuffisante. Observations recueillies pendant le siege de Paris, Gaz. med. de Paris, p. 317. 1898 Decroly, O.: Etude de Taction des toxines et antitoxines sur la nutrition generale, Archives internat. de pharmacodynamic 4, 385-489. 1911 Delaini, G.: Sul comportamento degli ipofosfiti nell- organismo animale, Archivio de fisiologia 9, 329-340. 1899 Delcourt, Albert : Le rachitisme, Ann. Soc. des sci. med. et naturelles de Bruxelles 8, 1-104. 1909 Delille, Arthur: L'hypophyse et la medication hypophysaire. (Etude experimentale et clinique.) These de Paris. 366 pp., 14 pages refs. 1905 Dennstedt, M., and Th. Rumpf : Weitere Untersuchungen uber die chem- ische Zusammensetzung des Blutes und verschiedener menschlicher Organe in der Norm und in Krankheiten, Zeit. klin. Med. 58, 84-162. 1906 Desgrez, A., and Bl. Guende: Influence de l'acide phosphorique, des phos- phates mono et disodiques sur les echanges nutritifs, Compt. rend. Acad, des sci. 142, 1440-1442. 1907 Desgrez, A., and J. Posen: Sur la determination de la molecule elaboree moyenne et ses variations, dans l'organism animal, sous 1'influence des composes mineraux du phosphore, Compt. rend. Soc. de biol. 63, 455- 457. 1900 Desgrez, A., and A. Zaky: De l'influence des lecithines sur les echanges nutritifs, Ibid. 52, 794, 795. 1901a Desgrez, A., and A. Zaky: Influence des lecithines de l'oeuf sur les echanges nutritifs, Ibid. 53, 647-649; also Compt. rend. Acad, des sci. 132, 1512-1514. 1901b Desgrez, A., and A. Zaky: Influence de le lecithine sur l'elimination de l'acide urique, Compt. rend. Soc. de biol. 53, 830-832. 1902a Desgrez, A., and A. Zaky: De l'influence des lecithines sur le developp- ment du squelette et du tissu nerveux, Ibid. 54, 501-504; also Compt. rend. Acad, des sci. 134, 1166-1168. 1902b Desgrez, A., and A. Zaky: Analyse du mode d'action des lecithines sur l'organisme animal, Compt. rend. Soc. de biol. 54, 730, 731; also Compt. rend. Acad, des sci. 134, 1522, 1523. 610 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1902c Desgrez, A., and A. Zaky: Etude de l'influence des lecithines sur l'organ- isme animal, Jour, de physiol. et de path, gener. 4, 662-671. 1904a Desgrez, A., and A. Zaky: De Tinfluence comparee des composes organ- iques phosphores sur la nutrition, sur le developpment et la composi- tion des tissus animaux, Revue Soc. sci. hygiene alimentaire (1904) 825-831. 1904b Desgrez, A., and A. Zaky: De l'influence comparee des composes des organiques phosphores sur la nutrition, Compt. rend. Soc. de biol. 57, 392-395; 440-443. 1904c Desgrez, A., and A. Zaky: Influence comparee de quelques composes organiques du phosphore sur la nutrition et la developpement des animaux, Compt. rend. Acad, des sci. 139, 819-821. 1905 Desgrez, A., and A. Zaky: Etude de l'influence de quelques composes organiques phosphores sur l'organisme animal, Jour, de physiol. et de path, gener. 7, 213-220. 1898 Deucher, P.: Stoffwechseluntersuchungen bei Verschluss des Ductus pancreaticus, Correspondenzblatt fur Schweizer Aerzte 28, 321-329; 361-366. 1909 Dhere, Ch., and H. Maurice: Influence de l'age sur la quantite et la repartition chimique du phosphore contenu dans les nerfs, Compt. rend. Acad, des sci. 148, 1124, 1125. 1910 Dhere, Ch., and H. Maurice: Influence de l'age sur la quantite et la repartition chimique du phosphore contenu dans la rate, Compt. rend. Soc. de biol. 69, 311, 312. 1867a Diaconow, C: Ueber die phosphorhaltigen Korper der Hiihner und Storeier, Hoppe-Seyler's med. -chem. Untersuchungen (1867), 221-227. 1867b Diaconow, C: Ueber das Lecithin, Ibid., 405-411. 1868a Diaconow, C: Das Lecithin im Gehirn, Centralbl. f. med. Wissensch. 6, 97-99. 1868b Diaconow, C: Ueber die chemische Constitution des Lecithins, Ibid. 6, 2, 3; 434, 435. 1908 Dibbelt, W.: Die Pathogenese der Rhachitis. I. Arbeiten a. d. Gebiete d. path. Anat. u. Bakteriol. aus dem path. anat. Inst. Tubingen 6, 670- 709. 50 refs. 1909 Dibbelt, W.: Die Pathogenese der Rhachitis. II. Ibid. 7, 144-214. 31 refs. 1910a Dibbelt, W.: Weitere Beitrage zur Pathogenese der Rhachitis, Verhandl. deut. path. Gesell. 14, 294-299. 1910b Dibbelt, W. : Die Bedeutung der Kalksalze f iir die Schwangerschaf ts- und Stillperiode und der Einflusz einer negativen Kalkbilanz auf den miitterlichen und kindlichen Organismus, Ziegler's Beitrage 48, 147- 169. 1911 Dibbelt, W.: Die experimentelle Osteomalacic und ihre Heilung. Zugleich ein kritischer Beitrag zur Histogenese der Skeletterkrankungen, Arbeiten aus dem path. Inst. Tubingen 7, 559-588. 1912 Dibbelt, W.: Neue experimentelle Untersuchungen iiber die Pathogenese der Rachitis, Deut. med. Wochenschr. 38, I, 316-318. 1913 Dibbelt, W.: Die Aetiologie der Rachitis und der Kalkstoffwechsel, Ibid. 39, 551, 552. 1909 Diesing, Ernst: Beitrag zur Kenntniss der Funktion der Stoffwechsel- driisen, Zentralbl. ges. Physiol, u. Path. d. Stoffwechs. n. ser. 4, 209- 216. PHOSPHORUS METABOLISM 611 1913 Diesing: Beitrag zur Aetiologie der Rachitis, Deut. med. Wochenschr. 39, 552. 1909 Dietrich, M.: Ueber phosphorhaltige Caseinpeptone, Biochem. Zeit. 22, 120-130. 1902 Dietrich, Th.: Getrocknete Biertreber, Landwirtsch. Versuch. Stat. 56, 207-256; 257-262. 1904 Diffloth, Paul: Du role de quelques agents physiques et chimiques dans l'insolubilisation des phosphates du lait, Bui. des sci. Pharmacol. 10, 273-279. 1900 Dmitriewski, K.: Stoffwechsel bei wiederholter Injection von bacteriellen Giften, Russ. Arch. f. Path., klin. Med. u. Bacteriol. 9, 375-391; through Jahresb. u. d. Fortschr. d. Thierchem. 30 (1900), 778, 779. 1911 Dobrowolsku, W. M.: (Die Behandlung der Hunde mit erweichten Knochen), Verhandl. d. Gesellsch. russ. Aerzte 1911, 195; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 360. 1906 Doctor, Earl: Ueber die chemische Reaction des Harnes mit Riicksicht auf die Phosphaturie, "Urologia" a Budapesti Orvosi Ujsag melleklete, Budapest, 4, 88, 89; through Pester med.- chir. Presse 43 (1907). 1885 Dogiel, A.: Einiges uber die Eiweisskorper der Frauen- und der Kuhmilch, Zeit. physiol. Chem. 9, 591-615. 1913 Dohrn, Max: Beitrag zum Nucleinstoffwechsel, Ibid. 86, 130-136. 1876 Domenico, Pecile: Guanin im Schweineharn, Annalen der Chem. u. Pharm. 183, 141-144. (1911) Donath, Hedwig: Ueber die therapeutische Anwendung des Phytinum liquidum in der Kinderheilkunde, Wien. klin. Wochenschr. 24, 1192-1197; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 842. 1903a Donath, Julius: Die Behandlung der progressiven Paralyse, sowie toxischer und infectioser Psychosen mit Salzinfusionen, Allgem. Zeit. Psychiat. u. psychischgerichtl. Med. 60, 583-605. 1903b Donath, Julius: Das Vorkommen und die Bedeutung des Cholins in der Cerebrospinalfliissigkeit bei Epilepsie und organischen Erkrankungen des nervensystems, nebst weiteren Beitragen zur Chemie derselben, Zeit. physiol. Chem. 39, 526-544. 1904 Donath, Julius: Die Phosphorsauregehalt der Cerebrospinalfliissigkeit bei verschiedenen Nervenkrankheiten, Ibid. 42, 141-148. 1909 Donath, Julius: Die Behandlung der progressiven allgemeinen Paralyse mittels Nuklein-Injektionen, Wien. klin. Wochenschr. 22, 1289-1292. 1890 Doremus, Ch. A: Ueber Elefantenmilch, Molkereizeitung p. 67; through Jahresb. u. d. Forschr. d. Thierchem. 20 (1890), 147. 1904 Dorn, Franz: Beitrag zur Phosphorbehandlung mit besonderer Beriick- sichtigung der Wirkung des Protylin-Roche bei Rachitis und Skrofulose, Deut. Aerzte-Zeitung, 265-270. 1910 Dornic and Daire: Contribution a l'etude de la lecithine du lait, Annales des falsifications 3, 533-538. 1912 Doyon, M.: Extraction de l'antithrombine des testicules et de l'intestin, Compt. rend. Soc. de biol. 72, 925, 926. 1912 Doyon, M., and P. Dubrulle: Formation d'une substance anticoagulante phosphoree sous l'influence de l'autodigestion de l'intestin, Ibid. 73, 546. 612 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1912 Doyon, M., P. Dubrulle and F. Sarvonat: Digestion pepsique de la nucleo- proteide extraite de l'intestin. Comparaison du pouvoie anticoagu- lant de la substance initiale et du residu, Ibid. 73, 720. 1912a Doyon, M., and F. Sarvonat: Proprietes anticoagulantes de l'acide nucleinique extrait de l'intestin, Ibid. 73, 546, 547. 1912b Doyon, M., and F. Sarvonat: Proprietes anticoagulantes des acides nucleiniques d'origine animale et vegetale, Ibid. 73, 619. 1912c Doyon, M., and F. Sarvonat: Proprietes anticoagulantes des acides thymo-nucleinique et thymique, Ibid. 73, 644. 1913a Doyon, M., and F. Sarvonat: Proprietes anticoagulantes de l'acide nucleinique extrait des globules du sang des oiseaux, Ibid. 74, 312. 1913b Doyon, M., and F. Sarvonat: Action anticoagulante de l'hematogene, Ibid. 74, 368, 369. 1913c Doyon, M., and F. Sarvonat: Action comparee des divers phosphates sur la coagulation du sang, Ibid. 74, 460. 1913d Doyon, M., and F. Sarvonat : Action comparee du nucleinate de soude sur la coagulation du sang et sur la coagulation du lait, Ibid. 74, 765, 766. 1913e Doyon, M., and F. Sarvonat: Pouvoir glycolytique du sang preleve pen- dent l'intoxication provoquee par les peptones, Ibid., 74, 779. 1913f Doyon, M., and F. Sarvonat: Nucleinate de soude et pouvoir coagulant du serum, Ibid. 74, 872-874. 1911 Draeger, Ewald: Ueber die Assimilation und Verwertung verschieden- er Kalkphosphatpraparate im Tierkorper, Diss. Rostock 1911, 51 pp. Through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 893. 1886 Drechsel, E.: Ueber einen neuen, schwefel- und phosphorhaltigen Bestandtheil der Leber, Jour, prakt. Chem. 33, 425-432. 1913 Drbge, Karl: Ueber Veranderungen in der chemischen Konstitution des Tierkorpers nach Exstirpation der Milz, der Hoden und des Schild- drusenapparates, Arch. ges. Physiol. 152, 437-477. 1914 Du Bois, Gaston: The Chemistry and Properties of Glycerophosphates (Glycerinophosphates) Jour, of Ind. and Engin. Chem. 6, 122-128. 1884a Duclaux, Emile: Sur les matieres albuminoides du lait, Compt. rend. Acad, des sci. 98, 373-375. 1884b Duclaux, Emile: Sur la constitution du lait, Ibid, 98, 438-441. 1884c Duclaux, Emile: Action de la presure sur le lait, Ibid. 98, 526, 528. 1893 Duclaux, Emile: Sur les phosphates du lait, Annales de llnst. Pasteur 7, 2-17. 1894 Duclaux, Emile: Le lait, Paris. 371 pp. 1905 von During, E.: Ueber Phosphaturie, Med. Klinik, Berlin, 1, 513-516. 1903-4 Dunham, Edward K.: The Lecithin Content of Fatty Extracts from the Kidney, Proc. Soc. Exp. Biol, and Med., New York, 1, 39-41. 1904-5 Dunham, Edward K. : Further Observations upon the Phosphorized Fats in Extracts of the Kidney, Ibid. 2, 63, 64. 1905-6 Dunham, Edward K.: Analogies between the Phosphorized Fats Obtained from the Brain and Kidney, Ibid. 3, 67-70. 1908 Dunham, Edward K. : The Isolation of Carnaubic Acid from Beef Kidney, Jour. Biol. Chem. 4, 297-299. 1910 Dunham, Edward K., and C. A. Jacobson: Ueber Carnaubon: Ein glycer- infreies Phosphatid, lecithinahnlich konstituiert mit Galaktos als Kern, Zeit. physiol. Chem. 64, 302-315. PHOSPHORUS METABOLISM 613 1897-8 Dunlap, J. C, D. Noel Paton and R. Stockman : On the Influence of Mus- cular Exercise, Sweating and Massage, on the Metabolism, Jour, of Physiol. 22, 68-91. 1913 Durlach, Ernst.: Untersuchungen liber die Bedeutung des Phosphors in der Nahrung wachsender Hunde, Arch. exp. Path. u. Pharm. 71, 210- 250. 1869 Dusart, L.: Recherches experimentales sur les proprietes physiologiques et therapeutiques du phosphate de chaux, Archives gener. de med., 6th ser., 14, 670-695. 1870 Dusart, L.: Recherches experimentales sur les proprietes physiologiques et therapeutiques du phosphate de chaux, Ibid. 15, 66-84. 1913 Eckles, C. H., and R. H. Shaw: The Influence of the Stage of Lactation on the Composition and Properties of Milk, U. S. Dept. Agr., Bur. Animal Ind., Bui. 155, 88 pp. 1912 Edie, E. S., W. H. Evans, B. Moore, G. C. E. Simpson, and A. Webster: The Anti-Neuritic Bases of Vegetable Origin in Relationship to Beri- beri, with a method of Isolation of Torulin, the Anti-Neuritic Base of Yeast, Biochemical Jour. 6, 234-242. 1911 Edie, E. S., and G. C. E. Simpson: The Preparation of Various Foodstuffs (Especially Wheat and Rice) : Its Effects on their Content of Organic Phosphorus Compounds and its Relation to Disease, British Med. Jour. 1, 1421, 1422. 1891 Edkins, J. Sydney: The Changes Produced in Casein by the Action of Pancreatic and Rennet Extracts, Jour, of Physiol. 12, 193-219. 1878 Edlefsen, G.: Ueber das Verhaltniss der Phosphorsaure zum Stickstoff im Urin, Centralbl. med. Wissensch. 16, 513-523. 1880 Edlefsen, G.: Ueber die Ableitung der specifischen Gallenbestandteile und des Glycogens neben Harnstoff aus der Formel des Hamoglobins und iiber den relativen Wert der aus den Blutkorperchen abzuleitend- en Phosphorsaure im Urin, Ibid. 18, 657-661; 675-677; 691-696. 1881 Edlefsen, G.: Verhaltnis der Phosphorsaure zum Stickstoff im Urin, Deut. Arch. klin. Med. 29, 409-480. 1882 Edlefsen, G.: Ueber die Stoffwechselvorgange beim Fieber, Mitt, des Vereins schleswig-holst. Aerzte 1882, No. 3; abs. Centralbl. med. Wis- sensch. 20 (1882), 783. 1901 Edlefsen, G.: Ueber die Hauptunterschiede zwischen der Kuhmilch und Frauenmilch und den Werth und die Bedeutung der Ersatzmittel fur Muttermilch, Munch, med. Wochensch. 48, 7-11. 1896 Edmunds, Arthur: Notes on Rennet and on the Coagulation of Milk, Journal of Physiology 19, 466-476. 1903 Edsall and Miller: A Contribution to the Chemical Pathology of Acro- megaly, Med. Bui., Univ. Penn. 16, 143-150. 1860 Edwards, A, M.: Etudes chimiques et physiologiques sur les os, Annales sci. naturelles, [4] Zool. 14, 113-192. 1900 Ehrlich, Ernst: Stoffwechselversuche mit P-haltigen und P-freien Eiweisskorpern, Inaug. Diss., Breslau. 28 pp. 1913 Ehrmann, R., and E. Kruspe: Die Verdauung des Lecithins bei Erkrank- ungen des Magendarmkanals, Berlin klin. Wochenschr. 50, 1111. 1903a Ehrstrom, Robert : Zur Kenntniss des Phosphorumsatzes bei dem erwach- senen Menschen, Skand. Arch. Physiol. 14, 82-111. 614 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1903b Ehrstrom, Robert: Ueber den Nahrwerth des Caseinklystiere nebst Bemerkimgen iiber den Phosphorstoffwechsel, Zeit. klin. Med. 49, 377- 392. 1903 Eichelberg, Simon: Ueber den Einfiuss der Driisengifte Atropin und Pilokarpin auf den Stoffwechsel, insbesondere auf die Ausscheidung von Stickstoff, Phosphorsaure und Harnsaure, Inaug. Diss., Marburg. 48 pp. 1911 Eijkmann, C: Polyneuritis Gallinarum und Beriberi, Arch. Schiffs- u. Tropenhygiene 15, 698-712. 1899 Ellenberger: Die Eigenschaften der Eselinmilch, Arch. Anat. u. Physiol. 33-52. 1902 Ellenberger: Die Zusammensetzung und die Eigenschaften der Esel- inmilch, Ibid. Suppl., 313-322. 1902 Ellenberger, Seeliger and Klimmer: Die Eigenschaften und Zusammen- setzung der Eselinmilch, Arch, wissensch. u. prakt. Thierheilk. 28, 3 and 4, 247-298; through Jahresb. u. d. Fortschr. d. Thierchem. 32 (1902), 989, 990. 1895 Emery, F. E., and B. W. Kilgore: Fertilizing Constituents of Various Rations of Cotton-seed Hulls and Meal Recovered in Manure of Animals, North Carolina Agr. Exp. Sta. Bui. 118, 245-253. 1909 Emmett, A. D.: Chemistry of Animal Feces. Second paper. The Determination of Fatty Matter in Animal Feces by Ether and Carbon Tetrachloride, Jour. Amer. Chem. Soc. 31, 693-695; 871. 1906 Emmett, A. D., and H. S. Grindley: The Chemistry of Flesh. III. A Study of the Phosphorus Content of Flesh, Ibid. 28, 25-63. 1909a Emmett, A. D., and H. S. Grindley: The Chemistry of Flesh. (Seventh paper.) A Preliminary Study of the Effect of Cold Storage upon Beef and Poultry. (First communication.) Jour. Ind. and Engin. Chem. 1, 413-436. 1909b Emmett, A. D., and H. S. Grindley: Chemistry of Flesh. (Eighth paper.) A Preliminary Study of the Effect of Cold Storage upon Beef and Poultry, (Second communication.) Ibid. 1, 580-597. 1903 Enea, Domenico: Azione della nucleina da batterii patogeni e non patogeni sul potere battericida del siero di sangue normale, La Riforma medica, Napoli, 19, 1292-1296. 1910 Engel, and A. Frehn: Der Caseingehalt der Frauenmilch, Berlin, klin. Wochenschr. 47, I, 436-438. 1871 Engelmann, Geo. J.: Schwefelsaure- und Phosphorsaure- Ausscheidung bei korperliche Arbeit, Arch. Anat. u. Physiol. (1871) 14-30. 1913 Eppler, Julius: Untersuchungen iiber Phosphatide, insbesondere iiber die im Eigelb vorhandenen, Zeit. physiol. Chem. 87, 233-254. 1902 Erben, Franz: Ueber die chemische Zusammensetzung des chlorotischen Blutes, Zeit. klin. Med. 47, 302-320. 1903 Erben, Franz: Studien iiber Nephritis. I. Ibid. 50, 441-463. 1905 Erben, Franz: Studien iiber Nephritis. II. Ibid. 57, 39-69. 1907 Erben, Franz: Ueber den Lezithingehalt der Erythrocyten bei Diabetes mellitus, Zentralbl. innere Med. 28, 1090-1093. 1908 Erben, Franz : Die chemische Zusammensetzung des leukamischen Blutes, Zeit. klin. Med. 66, 278-301. PHOSPHORUS METABOLISM 615 1911 Erdheim, J.: Ueber den Kalkgehalt des wachsenden Knochens und des Callus nach der Epithelkorperchenexstirpation, Frankfurter Zeit. Path. 7, 175-230. 1906 Erlandsen, A. W. E.: Undersogelser over Hjertets Phosphatides (Die Phosphatide des Herzens.) Diss., Copenhagen. 157 pp. Through Jahresb. ii. d. Fortschr. d. Thierchem. 36 (1906), 495-498. 1907 Erlandsen, A. W. E.: Untersuchungen uber die lecithinartigen Substan- zen des Myocardiums und der quergestreiften Muskeln, Zeit. physiol. Chem. 51, 71-155. 1908 Errani, Galdino: Contributo clinico all' uso della lecitina, La'Rassegna di terapia 3, 833-846. 75 refs. 1874 Etzinger, Johann: Ueber die Verdaulichkeit der leimgebenden Gewebe, Zeit. Biol. 10, 84-110. 1907 Euler, Hans: Gleichgewicht und Endzustand bei Enzymreaktionen, Zeit. physiol. Chem. 52, 146-158. 1912a Euler, Hans: Ueber die Wirkungsweise der Phosphatese. III. Mitteil- ung, Biochem. Zeit. 41, 215-223. 1912b Euler, Hans: Verhalten der Kohlenhydratphosphorsaureester im Tier- korper, Zeit. physiol. Chem. 79, 375-397. 1913 Euler, Hans: Om Kolhydratfosforsyra och fosfaters biokemiska roll (Biochemical Role of Phosphates and Carbohydrate-phosphoric Acid Esters), Svensk. kern. Tidskrift 25, 168-175; through Chem. Abs. 8 (1914), 715. 1912 Euler, Hans, and Helmer Backstrom: Zur Kenntnis der Hefegarung, II. Mitteilung, Zeit. physiol. Chem. 77, 394-401. (1911) Euler, H., and A. Fodor: Ueber ein Zwischenprodukt der alkoholischen Garung, Biochem. Zeit. 36, 401-410; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 782. 1912 Euler, Hans, and Yngoe Funke: Ueber die Spaltung der Kohlenhydrat- phosphorsaureester, Zeit. physiol. Chem. 77, 488-496. 1912a Euler, Hans, and David Johansson: Ueber den Einflusz des Toluols auf die Zymasen und auf die Phosphatese, Ibid. 80, 175-181. 1912b Euler, Hans, and David Johansson: Versuche uber die enzymatische Phosphatbindung, Ibid. 80, 205-211. 1913 Euler,. Hans, and David Johansson: Ueber die Reaktionsphasen der alko- holischen Garung, Arkiv Kemi, Min. Geol., 4, 1-17; through Chem. Abs. 7 (1913), 3511. 1911a Euler, Hans, and Sixten Kullberg: Untersuchungen uber die chemische- Zusammensetzung und Bildung der Enzyme. III. Mitteilung. Zeit. physiol. Chem. 71, 14-30. 1911b Euler, Hans, and Sixten Kullberg: Ueber die Wirkungsweise der Phos- phatese. I. Mitteilung. Ibid. 74, 15-28. 1911 Euler, Hans, and Gunnar Lundeqvist: Zur Kenntnis der Hefegarung, Ibid. 72, 97-112. 1911 Euler, Hans, and Hjalmar Ohlsen: Ueber den Einfluss der Temperatur auf die Wirkung der Phosphatese, Biochem. Zeit. 37, 313-320. 1912 Euler, Hans, and Hjalmar Ohlsen: Ueber die Wirkungsweise der Phos- phatese. II. Mitt. Zeit. physiol. Chem. 76, 468-477. 1911 Euler, Hans, and Beth af Ugglas: Untersuchungen uber die chemische Zusammensetzung und Bildung der Enzyme. II. Mitteilung. Ibid. 70, 279-290. 616 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1883 Ewald, C. A.: Ueber Phosphorsaureausscheidung bei Paralysis agitans und verwandten Formen der Zitterlahmung, Berlin, klin. Wochenschr. 484-486; 502-504. 1908 Ewing, James: Acidosis and Associated Conditions, Archives of Inter- nal Medicine 2, 330-354. 1903 Fabiani, Pietro: II potere curativo dei glicerofosfati, del Cav. Cesare Sebastiani, Nuova scuola med. napolitana 19, fasc. 12, 1-8. 1850 Falck, C: Die Knochencaries u. ihr Verhaltniss zum phosphors. Kalk, Schmidt's Jahrbiicher der in- und auslandischen gesammten Med. 67, 261-263. 1872 Falck, C. Ph. : Ueber die Ausscheidung von in das Blut gebrachtem phos- phorsaurem Natron durch die Nieren, Arch. path. Anat. u. Physiol. 54, 173-184. 1875 Falck, F. A.: Ueber den Stoffwechsel des hungernden Hundes, Sitzungs- ber. der Gesell. zur Beforderung der ges. Naturwissensch. zu Marburg, 1875, No. 7, 83-90. 1908 Falk, Fritz: Ueber die chemische Zusammensetzung der peripheren Nerven, Biochem. Zeit. 13, 153-172. 1906 Falta, W., and C. T. Noeggerath: Futterungsversuche mit kiinstlicher Nahrung, Beitrage z. chem. Physiol, u. Path. 7, 313-322. 1908 Falta, W., and James Lyman Whitney: Zur Kenntnis des Eiweiss- und Mineralstoffwechsels pankreasdiabetischer Hunde, Ibid. 11, 224-228. 1912 Fargier: Beriberi et riz fraichement decortique, Ann. hyg. med. colon. 15, 491-497. 1907 Fauvel, Pierre: Quelques experiences sur la valeur alimentaire de differents pains, Revue internat. des falsifications 20, 19, 20. 1881 Feder, Ludwig: Der zeitliche Ablauf der Zersetzung im Thierkorper, Zeit. Biol. 17, 531-576. 1891 Fehling, H.: Ueber Wesen und Behandlung der puerperalen Osteo- malakie, Arch. Gynakol. 39, 171-196. 1895 Fehling, H.: Weitere Beitrage zur Lehre von der Osteomalakie, Ibid. 48, 472^498. 1912 Feinschmidt, J. : Die Saureflockung von Lecithinen und Lecithin-Eiweiss- gemischen, Biochem. Zeit. 38, 244-251; through Zeit. Unters. d. Nahrungs- u. Genussmittel 24 (1912), 629. 1913 Fenger, Frederic: On the Iodine and Phosphorus Contents, Size and Physiological Activity of the Fetal Thyroid Gland, Jour. Biol. Chem. 14, 397-405. 1914 Fenger, Frederic: The Influence of Pregnancy and Castration on the Iodine and Phosphorus Metabolism of the Thyroid Gland, Ibid. 17, 23-28. (1911) Fernbach and Schoen: (Einige Bemerkungen iiber den Reaktions- mechanismus der proteolytischen Diastasen), Compt. rend. Acad, des sci. 153, 133-136; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 732. 1907 Ferrata, A., and G. Moruzzi: Ueber das Verhalten von Phosphorverbind- ungen in der Darmschleimhaut im Hungerzustand sowie nach Verabreichung von Nahrungsstoffen, Arch. Verdauungskrankh. 13, 223-232. PHOSPHORUS METABOLISM 617 1910 Fetzer, L. W.: The Lecithin Content of Milk under Pathologic Conditions, Meeting Amer. Assoc, for the Adv. of Sci., 1910, report in Science, n. s. 33 (1911), 339. 1912 Feulgen, R.: Das Verhalten der echten Nucleinsaure zu Farbstoffen. 1. Mitteilung, Zeit. physiol. Chem. 80, 73-78. 1913a Feulgen, R.: Das Verhalten der echten Nucleinsaure zu Farbstoffen. II Mitteilung, Ibid. 84, 309-328. 1913b Feulgen, R.: Ueber eine Nucleinsaure aus der Pankreasdriise. I. Mit- teilung, Ibid. 88, 370-376. 1911a Fingerling, Gustav: Die Verwertung der in den Futtermitteln enthalten- en Phosphorverbindungen durch Wiederkauer, Biochem. Zeit. 37, 266- 271. 1911b Fingerling, Gustav: Beitrage zur Verwertung von Kalk- und Phosphor- saureverbindungen durch den tierischen Organismus. I. Einfluss kalk- und phosphorsaurearmer Nahrung auf die Milchsekretion, Land- wirtsch. Versuch. Stat. 75, 1-52. 1912a Fingerling, Gustav: Die Bildung von organischen Phosphorverbindungen aus anorganischen Phosphaten, Biochem. Zeit. 38, 448-467. 1912b Fingerling, Gustav: Einflusz organischer und anorganischer Phosphor- verbindungen auf die Milchsekretion, Ibid. 39, 239-268. (1913) Fingerling, Gustav: (The Utilization of Calcium and Phosphorus Com- pounds by the Animal Organism. II. The Use of the More Important Phosphorus Compounds by Ruminants), Landwirtsch. Versuch. Stat. 79-80, 847-870; through Chem. Abs. 7 (1913), 3355. 1903 Finzi, Roberto: Modificazioni della composizione della saliva per alter- azioni della crasi del sangue dovute ad iniezioni endovenosa di fosfato neutro di sodio, Bollett. delle sci. med. di Bologna 74, 23-32. 1903 Fischer, Emil, and Emil Abderhalden: Ueber die Verdauung des Caseins durch Pepsinsalzsaure und Pankreasfermente, Zeit. physiol. Chem. 40, 215-219. 1894 Fischer, Heinrich: Casuistischer Beitrag zur Behandlung der Osteomal- acie, Prager med. Wochenschr. 19, 425-427. 1907 Fitz, R., C. L. Alsberg and L. J. Henderson: Concerning the Excretion of Phosphoric Acid during Experimental Acidosis in Rabbits, Amer. Jour. Physiol. 18, 113-122. 1907 Fjodoroff, Vladimir Stepanovitch : Ueber den Einfluss organischer Phos- phorpraparate (Lecithin, Nucleinsaure, Phytin und Protylin) auf die Magenverdauung, Diss., St. Petersburg, 103 pp. Through Russische med. Rundschau 6 (1908), 729-731. 1907 Flamini, M.: L'azione del fosforo sul ricambio del calcio in bambini normali ed in bambini rachitici, Archivio di farmacol. 6, 653-663. 1881 Fleischer, R.: Klinische und pathologisch-chemische Beitrage zur Lehre von den Nierenkrankheiten, Deut. Arch. klin. Med. 29, 129-192. 1906 Fleischner, E. C: The Relation of Weight to the Measurements of Child- ren During the First Year, Archives of Pediat. 23, 739-757. 1876 Flesch, M.: Zur Physiologie der Knochen-Resorption, Centralbl. med. Wissensch. 14, 524, 525. 1909 Fletcher, William: Rice and Beriberi, Jour, of Tropical Med. and Hyg. 12, 127-135. 1897 Flieger, Erdmann: Ueber die Behandlung der Rhachitis mit Phosphor, Inaug. Diss., Breslau. 39 pp. 618 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1871 Flint, Austin, Jr.: On the Physiological Effects of Severe and Protracted Muscular Exercise; with Especial Reference to the Influence of Exer- cise upon the Excretion of Nitrogen, N. Y. Med. Jour. 13, 609-697. 1900 Forberg: Welchen Einfluss iiben Nahrungsmittel und Trinkwasser auf Entwicklung und Bestand der Zahne aus ? Odontolog. Blatter 1900, Nr. 24; through Centralbl. Stoffwechsel u. Verdauungskrankh. II (1901), 107. 1873 Fokker, A. P. : Ueber das Vorkommen von gelosten Erden und Phosphor- saure im alkalischen Blute, Arch. ges. Physiol. 7, 274-284. 1905 Folin, Otto: Laws Governing the Chemical Composition of Urine, Amer. Jour. Physiol. 13, 66-115. 1907 Folin, Otto: The Acid Intoxication Theory, Jour. Amer. Med. Assoc. 49, 128-131. 1912 Folin, Otto, and W. Denis : Tyrosine in Proteins as Determined by a New Colorimetric Method, Jour. Biol. Chem. 12, 245-251. 1902 Folin, Otto, and Philip Shaffer: On Phosphate Metabolism, Amer. Jour. Physiol. 7, 135-151. 1904 Folin, Otto, Philip A. Shaffer, and L. A. Hill: Some Metabolism Studies with Special Reference to Mental Disorders, Amer. Jour. Insanity 60, 699-732; 61, 299-364. 1909 Forbes, E. B.: Specific Effects of Rations on the Development of Swine, Ohio Agr. Exp. Sta. Bui. 213, 239-305; Mo. Agr. Exp. Sta. Bui. 81, 3-69. 1914 Forbes, E. B., and associates: The Metabolism of Organic and Inorganic Phosphorus Compounds, Ohio Agr. Exp. Sta. Research Bui. 6. 1914 Forbes, E. B., F. M. Beegle, C. M. Fritz and J. E. Mensching: A Chemical Study of the Nutrition of Swine, Ohio Agr. Exp. Sta. Bui. 271, 225-261. 1913 Forbes, E. B., F. M. Beegle, and J. E. Mensching: Mineral and Organic Analyses of Foods, Ohio Agr. Exp. Sta. Bui. 255, 211-231. 1914 Forbes, E. B., and C. M. Fritz: The Effects of the Ensilage Process on the Solubility of Floats, Jour. Ind. and Engin. Chem. 6, 222, 223. 1910 Forbes, E. B., A. Lehmann, R. C. Collison, and A. C. Whittier: Methods for the Quantitative Estimation of Inorganic Phosphorus in Vegetable and Animal Substances, Ohio Agr. Exp. Sta. Bui. 215, 459-489. 1910 Forbes, E. B., A. C. Whittier, and R. C. Collison: The Mineral Nutrients of Blue-Grass, Ohio Agr. Exp. Sta. Bui 222, 39-52. 1873a Forster, J.: Versuche liber die Bedeutung der Aschenbestandteile in der Nahrung, Zeit. Biol. 9, 297-380. 1873b Forster, J.: Beitrage zur Ernahrungsfrage, Ibid. 9, 381-410. 1876 Forster, J.: Ueber die Verarmung des Korpers, speciell der Knochen an Kalk bei ungeniigender Kalkzufuhr, Ibid. 12, 464-474. 1884 Forster, J.: Beitrage zur Kenntnis der Kalkresorption im Thierkorper, Arch. Hyg. 2, 385-411. 1793 Fourcroy, A. F.: Examen chimique du cerveau de plusieurs animaux, Annales de chim. 16, 282-322. 1908 Frankel, Sigmund: Ueber Lipoide, Verhandl. des Kongr. innere Med. 25, 564-569. 1909a Frankel, Sigmund: Gehirn-Chemie, Ergebnisse d. Physiol. 8, 212-253. 75 refs. PHOSPHORUS METABOLISM 619 1909b Frankel, Sigmund: Ueber Lipoide. VI. Mitteilung. Ueber eines neues Verfahren der fraktionierten Extraktion der Gehirnlipoide, Biochem. Zeit. 19, 254-265. 1908 Frankel, Sigmund, and Carlo Bolaffio: Ueber Lipoide. I. Ueber das Neottin, ein Triaminomonophosphatid, Ibid. 9, 44-53. 1909 Frankel, Sigmund, and Ludwig Dimitz: Ueber Lipoide. VIII. Mitt. Ueber die Spaltungsprodukte des Kephalins, Ibid. 21, 337-347. 1910 Frankel, Sigmund, (and Kurt Linnert) : Ueber Lipoide. IX. Mitt. Ueber das Sahidin aus Menschenhirn, Ibid. 24, 268-276. 1909 Frankel, Sigmund, Kurt Linnert and G. A. Pari: Ueber Lipoide. V. Mitt. Ueber die Phosphatide des Rinderpankreas, Ibid. 18, 37-39. 1909 Frankel, Sigmund, and Ernst Neubauer: Ueber Lipoide, VII. Mitt. Ueber Kephalin, Ibid. 21, 321-336. 1909a Frankel, Sigmund, and Alexander Nogueira: Ueber Lipoide. II. Mitt. Ueber die ungesattigten Phosphatide der Niere, Ibid. 16, 366-377. 1909b Frankel, Sigmund, and Alexander Nogueira: Ueber Lipoide. III. Mitt. Ueber die Wechselwirkung der ungesattigten Nierenphosphatide mit Farbstoffen, Ibid. 16, 378-382. 1909 Frankel, Sigmund, and G. A. Pari: Ueber Lipoide. IV. Mitt. Ueber die Phosphatide des Rinderpankreas, Ibid. 17, 68-77. 1904 Franchini, Giuseppe: Sul ricambio materiale in uno caso di acromegalia, Bollett. delle sci. med. di Bologna 75 (4 of ser. 8), 8; through Biochem. Centralbl. 3 (1905), 522. 1907 Franchini, Giuseppe: Ueber den Ansatz von Lecithin und sein Verhalten im Organismus, Biochem. Zeit. 6, 210-225. 1908a Franchini, Giuseppe: Ricerche sulla lecitina, colina et acido formico, Archivio di farmacologia 7, 371-389. 1908b Franchini, Giuseppe: Beitrag zum chemischen und histologischen Studium des Blutes bei Akromegalie, Berlin, klin. Wochenschr. 45, II, 1636-1639. 1910 Franchini, Giuseppe: Die Funktion der Hypophyse und die Wirkungen der Injektion ihres Extractes bei Tieren, Ibid. 47, I, 613-617; 670-673. 719-723. 1910 Francis, C. K., and P. F. Trowbridge: Phosphorus in Beef Animals. Parts I and II, Jour. Biol. Chem. 7, 481-501; 8, 81-93. 1913 Francois, M., and E. Boismenu: Le glycerophosphate de calcium commer- cial, Jour, de pharm. et de chim. [ser. 7] 7, 448-457, 492-497. 1909 Frank, Franz, and Alfred Schittenhelm: Ueber die Umsetzung verfiit- terte Nucleinsaure beim normalen Menschen, Zeit. physiol. Chem. 63, 269-282. 1910 Frank, L. : Ueber den Einfluss kalkarmen Futters auf den kalkgehalt der Kuhmilch, Chem. Zeitung 34, 978, 979. 1894 Frankfurt, Saloman: Ueber die Zusammensetzung der Samen und der etiolierten Keimpflanzen von Cannabis sativa und Helianthus annuus, Landwirtsch. Versuch. Stat. 43, 143-179. 1912 Fraps, G. S., and J. B. Rather: Composition and Digestibility of the Ether Etract of Hays and Fodders, Texas Agr. Exp. Sta., Tech. Bui. No. 150. 29 pp. 1910 Fraser, Henry, and A. T. Stanton: The Etiology of Beriberi. Philippine Jour, of Sci., B, 5, 55-64. 620 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1911a Fraser, Henry, and A. T. Stanton: The Etiology of Beriberi, Studies Inst. Med. Research, Federated Malay States, No. 12; through Jour. of Tropical Med. and Hyg. 14 (1911), 248, 249. 1911b Fraser, Henry, and A. T. Stanton: The Etiology of Beriberi, Jour, of Tropical Med. and Hyg. 14, 333-341; 349-358; 365-374. 1914 Fraser, Henry, and A. T. Stanton: Unpolished Rice and the Prevention of Beriberi, The Lancet 186, 96-98. 1904 Fratta, Aldo: Rapporto fra l'azione della cura antirabica e quella del glicerofosfati e della lecitine, Bollett. delle sci. med. di Bologna 75, 593-610. 1841 Fremy, Edmond : Recherches sur le cerveau, Annales de chim. et de phys. 173 (2 of ser. 3); 463-488; also Jour, de Pharm. 27, 453. 1842 Fremy, Edmond: Ueber das Gehirn, Jour. f. prakt. Chem. 25, 29-50. 1855 Fremy, Edmond : Recherches chimiques sur les os, Annales de chim. et de phys. 214 (43 of ser. 3), 47-109. 1842 Frerichs: Ueber die chemische Zusammensetzung der menschlichen Knochen, Annalen der -Chem. u. Pharm. 43, 251-255. 1891 Freund, E., and F. Obermayer: Ueber die chemische Zusammensetzung leukamischen Blutes, Zeit. physiol. Chem. 15, 310-318. 1901 Freund, E. and O.: Beitrage zum Stoffwechsel im Hungerzustand, Wien. klin. Rundschau 15, 69-91. 1905 Freund, W.: Zur Wirkung der Fettdarreichung auf den Sauglingsstoff- wechsel, Jahrb. Kinderheilk, 61, 36-50. 1906 Frey, Ernst: Therapeutischer Wert der Glycerophosphate, Budapest! Orvosi Ujsag 4, 145; through Pester med.-chir. Presse 42 (1906), 862, 1911 Frey, Ernst: Jodid, Nitrat, Sulfat, Phosphat werden durch Sekretion in den Harnkanalchen ausgeschieden, Arch. ges. Physiol. 139, 512-531. 1911 Friedenthal, Hans: Ueber die massgebende Rolle der Salze der Frauen- milch bei der Ernahrung im Sauglings- und ersten Kindesalter, Munch. med. Wochenschr. 58, II, 2385-2387. 1911 Fromherz, K.: Zur Kenntniss der Wirkungsweise der Phenylcinchonin- saure auf den Purinstoffwechsel des Hundes, Biochem. Zeit. 35, 494- 502. 1912 Frouin, Albert, and Pierre Gerard: Sur la composition minerale du sue pancreatique de chien et de vache, Compt. rend. Soc. de Biol. 72, 98-100. 1903 Fiirst, L. : Ueber Ernahrungs- und Funktionsinsuffizienz des Gehirns, Therapeut. Monatsh. 16, No. 8; through Jour. Amer. Med. Assoc. 41, Pt. II, 1378. 1904 Fiirst : Einige Erf ahrungen iiber des Phytin als Antirhachiticum und als Mittel gegen Psychasthenie, Centralbl. Kinderheilk. 9, 409-413. 1907 von Fiirth, Otto, and Ernst Jerusalem: Ueber die chemische Stellung der Pankreasnucleinsaure (Guanylsaure), Beitrage z. chem. Physiol, u. Path. 10, 174-187. 1908 von Fiirth, Otto, and Ernst Jerusalem: Ueber die chemische Stellung der Pankreasnucleinsaure (Guanylsaure), II. Mitteilung, Ibid. 11, 146- 150. 1910 Fujitani, I.: Contribution to the Etiology of the Disease Resulting from Feeding Birds on Rice, Archives internat. de pharmacodyn. et de ther# 20, 288-309. PHOSPHORUS METABOLISM 621 1902a Fuld, Ernst: Ueber die Verbindungen von Eiweisskorpern mit Metaphos- phorsaure, Beitrage z. chem. Physiol, u. Path. 2, 155-168. 1902b Fuld, Ernst : Ueber Milchgerinnung durch Lab, Ergebnisse d. Physiologie 1, Abt. 1, 468-504. 114 refs. 1907 Funatsu, T.: On Different Forms of Phosphoric Acid in Press Cakes, Bui. Coll. Agr. Tokyo 7, 457-459. 1911 Funk, Casimir: The Chemical Nature of the Substance which Cures Polyneuritis in Birds Induced by a Diet of Polished Rice, Jour, of Physiol. 43, 395-400. 1912a Funk, Casimir: The Effect of a Diet of Polished Rice on the Nitrogen and Phosphorus of the Brain, Ibid. 44, 50-53. 1912b Funk, Casimir: Preparation from Yeast and Certain Foodstuffs of the Substance the Deficiency of Which in Diet Occasions Polyneuritis in Birds, Ibid. 45, 75-81. 1912c Funk, Casimir: The Substance from Yeast and Certain Foodstuffs which Prevents Polyneuritis ( Beriberi), British Med." Jour., 1912, II, 787, 788. 1913 Funk, Casimir: Studies on Beriberi. Further Facts Concerning the Chemistry of the Vitamine-fraction from Yeast, Ibid. 1913, I, 814. 1894 Gabriel, S.: Chemische Untersuchungen fiber die Mineralstoffe der Knochen und Zahne, Zeit. physiol. Chem. 18, 257-302. 1900 Galeotti, Gino: Azione dei nucleoproteidi sulle cellule e sui tessuti. Ricerche sperimentali, Lo Sperimentale 54, 435-463. 1905 Galimard, J., and P. Konig: Recherches sur l'analyse des os dans un cas d'osteomalacie infantile, Jour, de pharm. et de chim. 21 (ser. 6), 352- 357. 1906 Gallenga, Pietro: Contributo alio studio della medicazione fosforato con i preparati organici di fosforo, II Policlinico 13, 115-129; 167-174. 1879a Gamgee, Arthur, and Ernst Blankenhorn: Ueber Protagon, Arch. path. Anat. u. Physiol. 77, 389-397. 1879b Gamgee, Arthur, and Ernst Blankenhorn. Ueber Protagon, Ber. deut. chem. Gesell. 12, 1229-1234. 1879c Gamgee, Arthur, and Ernst Blankenhorn: Ueber Protagon, Zeit. physiol. Chem. 3, 260-283. 1880 Gamgee, Arthur, and Ernst Blankenhorn: Ueber Protagon, Zeit. f. physiol. Chem. 3, 260-283. 1903a Gamgee, Arthur, and Walter Jones: Ueber die Nucleoproteide des Pankreas, der Thymus und der Nebenniere, mit besonderer Berfick- sichtigung ihrer optischen Aktivitat, Beitrage z. chem. Physiol, u. Path. 4, 10-22. 1903b Gamgee, Arthur, and Walter Jones: On the Nucleoproteids of the Pan- creas, Thymus, and Suprarenal Gland, with Especial Reference to their Optical Activity, Amer. Jour. Physiol. 8, 447-455. 1903c Gamgee, Arthur, and Walter Jones: Die optische Activitat der Nucleo- prote'ide des Pankreas, des Thymus und der Nebennieren, Ber. deut. chem. Gesell. 36, 914. 1876- Gamgee, Arthur, John Priestley and Leopold Larmuth: On the Differ- 1877 ence in the Poisonous Activity of Phosphorus in Ortho,- Meta- and Pyro-phosphoric Acids, Jour, of Anat. and Physiol. 11, 255-272. 1898 Garratt, G. C: On the Sequence of Certain Changes in the Urine Produced by Exercise and by Turkish Baths, Jour, of Physiol. 23, 150-162. 622 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1904 Garrat, G. C: Observations on Metabolism in the Febrile State in Man, Medico-Chirurg. Transact. 87 (69 of ser. 2), 163-326. 1908 Gassmann, Theodore: Chemische Untersuchungen der Zahne, Zeit. physiol. Chem. 55, 455-465. 1910 Gassmann, Theodore: Chemische Untersuchungen von gesunden und rachitischen Knochen, Ibid. 70, 161-170. 1913 Gassmann, Theodore: Die Darstellung eines dem Apatit-Typus entsprechenden Komplexsalzes und seine Beziehungen zum Knochenbau, Ibid. 83, 403-408. 1902 Gaston, Zephirin, Octave, Laurent-Boulenger : Das Lecithin, durch seine Anwendung im Organismus erzeugte Reaktionen. These de Lille, 1902. 136 pp. Through Jahresb. u. d. Fortschr. d. Thierchem. 32 (1902), 659. 1895 Gaube, J.: La mineralisation du lait, Compt. rend. Soc. de biol. 47, 489, 490. 1909 Gaucher, Louis: Sur la digestion gastrique de la" caseine, Compt. rend. Acad, des sci. 148, 53-56; also Compt. rend. Soc. de biol. 66, 25-27; 536- 538. 1911 Gaucher, Louis: Sur la digestion de la caseine, Compt. rend. Acad, des sci. 153, 891, 892. 1912 Gaucher, Louis : Recherches sur la digestion du lait. Digestion gastrique du caseine, Compt. rend. Soc. de biol. 72, 354, 355. 1877- Geoghegan, Edward G.: Ueber die anorganischen Gehirnsalze, nebst 1878 einer Bestimmung des Nucleins im Gehirn, Zeit. physiol. Chem. 1, 330-338. 1911 Gerard, Ern., and M. Verhaeghe: Contribution a. l'etude chimique des lipoi'des des organes animaux, Jour, de pharm. et de chim. 3 (ser. 7), 385-390. 1875 Gerber, N.: Recherches sur le lait, Bui. Soc. chim. 28 (23 of ser. 2), 342- 344. 1899 Gerhardt, Dietrich, and Wilhelm Schlesinger: Ueber die Kalk- und Magnesiaausscheidung bei Diabetes mellitus und ihre Beziehung zur Ausscheidung abnormer Sauren (Acidose), Arch. exp. Path. u. Pharm. 42, 83-108. 1913 Gessner, W.: Ueber die Wirkung des Phosphors im Phosphorlebertran bei Rachitis als Inflammator, Berlin, klin. Wochenschr. 50, 688-690. 1901 Gevaerts, Jacques: Diete sous phosphore, La Cellule 18, 7-33. 1895 Giacosa, G.: Analyse des cendres d'un nouveau-ne, Archives ital. de biol. 22, 252-258. 1904 Giacosa, Piero: Sulla fitina (sale calcico-magnesiaco dell' acido anidrossimetilendifosforico) e suo comportarsi nell* organismo, Giornale d. reale Accad. di med. di Torino 67, 414-416. 1905 Giacosa, Piero: Sul comportamento nell' organismo della fitina, Ibid. 68, 369-374. 1905- Giacosa, Piero: SulP azione della fitina in relazione alia funzione glico- 1906 genica, Atti d. reale Accad. sci. di Torino 41, 651-655. 1907 Giacosa, Piero: SulP azione farmacologica della fitina, Giornale d. reale Accad. di med. di Torino 70, 290-295. 1909 Giacosa, P., and S. Dezani : Studi sulla secrezione stomacale, Atti d. reale Accad. d. sci. di Torino 44, 521-534. PHOSPHORUS METABOLISM 623 1905 Gianasso, A. B., and V. Ovazza: Valore terapeutico della fitina in pedia- tria, Giornale di ginecol. e di pediat. 5, 227-231. 1899 Giertz, K.H.: Zur Kenntniss der Pseudonucleine, Zeit. physiol. Chem. 28, 115-122. 1907 Gies, William J.: Further Observations on Protagon, Jour. Biol. Chem. 3, 339-358. 1913 Giffhorn, Heinrich: Beitrage zur Kenntnis des Stoffwechsels, besonders der Mineralien, im Sauglingsalter. III. Der Einfluss von Fettzulagen auf den Stoffwechsel verdauungsgesunder Kinder bei molkenarmer und molkenreicher Ernahrung, Jahrb. f. Kinderheilk. 78, 531-564. 1901 Gilbert, A.: Lecithin in Therapeutics, Therapeut. Monatsh., Nov. 1901; through Jour. Amer. Med. Assoc. 37, Pt. II, (1901), 1711. 1901 Gilbert, A., and L. Fournier: La lecithine en therapeutique, Compt. rend. Soc. de biol. 53, 145-148. 1904 Gilbert, A., and A. Lippmann: Du principe phospho-organique des graines vegetales. L'acide anhydro-oxymethylene-diphosphorique, La Presse med. 545-547; 577-580. 1903 Gilbert, A., and S. Posternak: La medication phosphoree envisagee au point de vue des echanges nutritifs de l'organisme (etude critique et experimental ), L'Oeuvre med. -chir., No. 36, 48 pp. 117 refs. 1905 Gilbert, A., and S. Posternak: Ueber die Phosphortherapie vom Stand- punkte des Stoffwechsels aus betrachtet, Aertzliche Rundschau (Munchen) 15, 398-402; 411-415. 1888 Gilson, Eugen: Beitrage zur Kenntniss des Lecithins, Zeit. physiol. Chem. 12, 585-602. 1884 Girard, Aime: Memoire sur la composition chimique et la valeur alimen- taire des diverses parties du grain de froment, Annales de chim. et de phys. 3 (ser. 6), 289-355. 1903 Girard, Aime, and L. Lindet: Le froment et sa mouture. Traite de meunerie, 355 pp. 1907 Glikin, W.: Ueber den Lecithingehalt des Knochenmarks bei Tieren und beim Menschen, Biochem. Zeit. 4, 235-243. 1908a Glikin, W.: Zur biologischen Bedeutung des Lecithins. I. Mitteilung, Ibid. 7, 286-298. 1908b Glikin, W.: Ueber den Eisengehalt der Fette, Lipoide und Wachsarten, Ber. deut. chem. Gesell. 41, 910-915. 1909a Glikin, W.: Zur biologischen Bedeutung des Lecithins. II. Mitt. Ueber den Lecithingehalt bei Degenerationen im Zentralnervensystem, Biochem. Zeit. 19, 270-273. 1909b Glikin, W.: Zur biologischen Bedeutung des Lecithins. III. Mitt. Ueber den Lecithin- und Eisengehalt in der Kuh- und Frauenmilch, Ibid. 21, 348-354. 1909c Glikin, W.: Zur biologischen Bedeutung des Lecithins. IV. Mitt. Ueber den Gehalt des Blutes bei Polycythaemia rubra megalosplenica an Phosphor und Eisen in Lipoidform, Ibid. 22, 461-463. 1903 Gnezda, Julius: Ueber "Protylin," Deut. Aerzte-Zeitung, 193-196. 1844 Gobley, Nicholas Theodore: De la presence du phosphore dans l'huile de foie de raie, Jour, de pharm. et de chim. 6 (ser. 3), 25, 26. 1846 Gobley, Nicholas Theodore: Recherches chimiques sur le jaune d'oeuf, Ibid. 9, 81-91; 161. 624 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1847 Gobley, Nicholas Theodore: Recherches chimiques sur le jaune d'oeuf, deuxieme memoire, presente a l'Academie royale des sciences. Examen comparatif du jaune d'oeuf et de la matiere cerebrale Ibid. 11 (ser. 3), 409-417; 12 (ser. 3), 1-13. 1850a Gobley, Nicholas Theodore: Recherches chimiques sur les oeufs de carpe, Ibid. 17 (ser. 3), 401-417; 18 (ser. 3), 107-119. 1850b Gobley, Nicholas Theodore: Recherches chimiques sur la laitance de carpe, Ibid. 19 (ser. 3), 406-421. 1851 Gobley, Nicholas Theodore: Recherches chimiques sur les matieres grasses du sang veineux de l'homme, Ibid. 21 (ser. 3), 241-254. 1856 Gobley, Nicholas Theodore: Recherches sur la nature chimique et les proprietes des matieres grasses contenus dans la bile, Ibid. 30 (ser. 3), 241-255. 1877 Gobley, Nicholas Theodore: Chemische Untersuchungen fiber das Gehirn, Arch. Pharmacie 10 (ser. 3), 445-452. 1903 Godefroy, Maurice: Recherches sur 1 'elimination du phosphore urinaire dans les cas graves de rhumatisme chronique, These de Paris. 52 pp. 1861 von Gohren, Theodor: Ueber eine Beigabe von Knochenerde zum Futter der Thiere, nebst einem physiologisch-chemischen Versuch liber deren Verdaulichkeit, Landwirtsch. Versuch. Stat. 3, 161-178. 1865 von Gohren, Theodor: Knochen von einem knochenbruchigen Rinde, Centralbl. ges. Landescultur in Bohmen, 1865, p. 344; through Jahresb. u. d. Fortschr. d. Agricultur-Chemie, for 1866, 9 (1868), 345, 346. 1904 von Gohren, Theodor: Schweinemilch, Milch-Zeitung 33, 777. 1907 Goldfarb, A. J.: A Study of the Influence of Lecithin on Growth, Proc. Soc. Exp. Biol, and Med. 4, 159, 160. 1910 Goldfarb, A. J. : Does Lecithin Influence Growth ? Arch. Entwicklungs- Mechanik der Organismen, 29, 255-274. 1910 G61ding r John, and S. G. Paine: Note upon the Composition of Milk Yielded by Cows Fed on Pasture Manured with Phosphates and Potash, Analyst 35, 246, 247. 1905 Goldthwait, J. E., C. F. Painter, R. B. Osgood, and F. H. McCrudden: A Study of the Metabolism in Osteomalacia, Amer. Jour. Physiol. 14, 389- 402. 1910 Goldthwaite, N. E.: Effects of the Presence of Carbohydrates upon the Artificial Digestion of Casein, Jour. Biol. Chem. 7, 69-81. 1905 Goliner: Beitrag zur Wirkung des Lecithins, Reichs-Medicinal-Anzeiger, Leipzig, 30, 7. 1912 Goodman, E. H.: Nuclease in Carcinoma, Jour, of Exp. Med. 15, 477-484. 1906 Gordon, Alfred: The Role of Organic Phosphorus in the Treatment of Asthenic Conditions in Various Nervous Diseases, Medicine, Detroit, 12, 896-899. 1898 Gossmann, Heinrich: Ueber die anorganischen Bestandtheile der Bauch- speicheldriise (Pankreas) und der Niere, Inaug. Diss., Erlangen. 23 pp. 1901 Gottstein, Ernst: Ueber das Verhalten von Kalcium und Magnesium in einigen Stoffwechselversuchen mit phosphorhaltigen und phosphorfreien Eiweisskorpern, Inaug. Diss., Breslau. 34 pp. 1911 Goubau, Fernand: Sur la repartition de la nucleohistone dans les tissus des mammiferes, Bui. Acad, royale de med. de Belgique 25, 921, 946. PHOSPHORUS METABOLISM 625 1913 Goubau, Fernand, and Maurice Van Goethem: Etudes sur l'anaphylaxie par les nucleines. I. Memoire: Action de l'injection intraveineuse de nucleine et de nucleohistone sur la circulation chez le chien, Archives internat. de physiol. 13, 289-304. 1902-3 Gouin, Andre, and Pierre Andouard: Nouvelles recherches sur la nutri- tion des jeunes bovides, Bui. Sta. agron. Loire-Inferieure, 66-100. 1903 Gouin, Andre, and Pierre Andouard: De la reaction de l'urine des bovides, Compt. rend. Soc. de biol. 55, 1600-1602. 1905-6 Gouin, Andre, and Pierre Andouard: Recherches sur l'alimentation des jeunes bovides, Bui. Sta. agron. Loire-Inferieure, 52-82. 1907 Gouin, Andre, and Pierre Andouard: Recherches sur l'alimentation des jeunes bovides, Soc. de 1'aliment. rationelle du betail, Compt. rend. 11th congr. 11-28; 127-130. 1908 Gouin, Andre, and Pierre Andouard: Modes d'elimination des phosphates dans l'espece bovine, Compt. rend. Soc. de biol. 64, 133-135. 1902 Gouraud, F. Xavier: Courbe d'elimination des phosphates dans la pneu- monie et la fievre typhoide, Ibid. 54, 373, 374. 1903 Gouraud, F. Xavier: Des echanges phosphores dans l'organisme normal et pathologique: des phosphaturies, These de Paris. 135 pp. 1894 Gourlay, Frederick: The Proteids of the Thyroid and the Spleen, Jour. of Physiol. 16, 23-33. 1912 Gouzien, P.: Le beriberi au Tonkin, Ann. hyg. med. colon. 15, 445-491. 1891 Graffenberger, Louis: Leber die Zusammensetzung der Kaninchen- knochen im hohen Alter, Landwirtsch. Versuch. Stat. 39, 115-126. 1893 Graffenberger, Louis: 1st bei der Fiitterung trachtiger Tiere die Beigabe von neutralem phosphorsaurem Calcium zu normal beschaffenem Futter fiir die Nachkommen derselben von Nutzen? Ibid. 41, 57-64. 1905 Grandeau, L.: Les maladies des os et ralimentation phosphatee. Rachit- isme. Osteomalacia, ou cachexie osseuse. Jour, d'agriculture pratique 120 (9 of ser. 7), 726, 727. 1902 Grandis, V., and Oscar Copello: Studi sulla composizione chimica delle ceneri della cartilagine in relazione col processo di ossificazione, Archivio per le scienze mediche 26, 175; through Jahresb. u. d. Fortschr. d. Thierchem. 32 (1902), 523, 524. 1908 Granstrom, E. : Zur Frage iiber den Einfluss der Sauren auf den Calcium- stoffwechsel des Pflanzenfressers, Zeit. physiol. Chem. 58, 195-214. 1911 Greenwald, Isidor: The Effect of Parathyroidectomy upon Metabolism, Amer. Jour. Physiol. 28, 103-132. 1913a Greenwald, Isidor : Further Metabolism Experiments upon Parathyroidec- tomized Dogs, Jour. Biol. Chem. 14, 363-367. 1913b Greenwald, Isidor: On the Phosphorus Content of the Blood of Normal and Parathyroidectomized Dogs, Ibid. 14, 369-379. 1913c Greenwald, Isidor : Bemerkungen zu der Mitteilung von Raffaele Paladino, "Untersuchungen iiber einige Veranderungen des Stoffwechsels bei Tieren nach Exstirpation der Schilddriise und der Parathyroiden," Biochem. Zeit. 54, 159, 160. 1911 Gregersen, J. P.: Untersuchungen iiber den Phosphorstoffwechsel, Zeit. physiol. Chem. 71, 49-99. 1913 Gregg, D.: Infantil Beriberi in the Philippines, Boston Med. Surg. J. 168, 676-679; title through Chem. Abs. 7, (1913), 2779. 626 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1912 di Gregorio, Giovanni: La fitina nel ricambio organico fosforato fisiologico, Bollett. delle sci. med. di Bologna 83, 233-247; through Zentralbl. Biochem. u. Biophys. 14 (1912-13), 476. (1912) Grijns, G.: Kritische opmerkingen over "Jets over de atiologie van Polyneuritis gallinarum, in verband met verzuurde rijst." (Etiology of Polyneuritis gallinarum in Connection with Sour Rice) Geneesk. Tijdschr. Nederiandsch-Indie 52, 50; through Zentralbl. Biochem. u. Biophys. 14 (1912), 399. 1903 Grimm: Lecithin und seine Bedeutung fur die Kinderernahrung als organische Phosphorverbindung, Vereinsblatt der pfalzischen Aerzte 19, 194-197. 1905 Grindley, H. S., and A. D. Emmett: The Chemistry of Flesh. (Second paper.) Improved Methods for the Analysis of Animal Substances, Jour. Amer. Chem. Soc. 27, 658-678. 1913 Griniew, D. P.: Les lipoi'des et leur teneur en phosphore (dans differents organes et tissus) pendant l'infection tuberculeuse chronique, Archives des sci. biol. 17, 363-396. 1913 Grosser, Paul: Ueber den Einflusz des Kochens auf das physikalisch- chemische Verhalten von Frauenmilch, Kuhmilch, und Buttermilch, Biochem. Zeit. 48, 427-431; through Chem. Abs. 7 (1913), 1929. 1912 Grosser, Paul, and Joseph Husler: Ueber das Vorkommen einer Glycero- / phosphatase in tierischen Organen, Ibid. 39, 1-5. 1910 Grund, Georg: Organanalytische Untersuchungen liber den Stickstoff und Phosphorstoffwechsel und ihre gegenseitigen Beziehungen, Zeit. Biol. 54, 173-229. 1912a Grund, Georg : Ueber die chemische Veranderung des Muskels unter dem Einfluss der Entartung, Centralbl. innere Med. 33, 173-177; through Chem. Abs. 6 (1912), 1923. 1912b Grund, Georg: Zur chemischen Pathologie des Muskels, Arch. exp. Path. u. Pharm. 67, 393-407; through Chem. Abs. 6 (1912), 2780. 1913 Grund, Georg: Zur chemischen Pathologie des Muskels, II. Der Einfluss der Inaktivitatsatrophie auf die Stickstoff- und Phosphorverteilung im Muskel, Ibid. 71, 129-138; through Chem. Abs. 7 (1913). 1892 Grundzach, Ig.: (Ueber die Asche des normal en Koths), Gazeta lekarska 1892, p. 48; through Jahresb. ii. d. Fortschr. d. Thierchem., 22 (1892), 311, 312. 1903 Guerrini, Guido: Dell azione dei nucleoproteidi sulle cellule del parenchima epatico, La Riforma medica, Palermo-Napoli 19, 707-709. 1894 Gumlich: Ueber die Aufnahme der Nucleine in den tierischen Organis- mus, Zeit. physiol. Chem. 18, 508-512. 1905 Gumpert, E. : Beitrag zur Kenntniss des Stickstoff-, Phosphor-, Kalk-, und Magnesia-Umsatzes beim Menschen, Med. Klinik 1, 1037-1041. 1883 Gurther, Gustav: Ueber Veranderungen im Stoffwechsel unter dem Einfluss der Hypnose und bei der Paralysis agitans, Arch. Psychiatrie 14, 17-39. 1893 Gusmitta, Mario: Sur les alterations des os produites par l'inanition, Archives ital. de biol. 19, 220-232. 1862 Gusserow: Beitrag zur Lehre von der Osteomalacic, Monatsschr. Geburtskunde 20, 19-25. 1896-7 Gutinkov, S. : Zur Lehre von der chemischen Zusammensetzung des menschlichen Gehirns, Allgem. Zeit. Psychiat. 53, 270-329. PHOSPHORUS METABOLISM 627 1910 de Haan, J.: On the Etiology of Beriberi, Philippine Jour, of Sci., B, 5, 65-71. 1907 Hamatainen, Juho, and Waino Helme: Ein Beitrag zur Kenntnis des Eiweissstoffwechsels, Skand. Arch. Physiol. 19, 182-200. 1909 Haensel, E.: Ueber den Eisen- und Phosphorgehalt unserer Vegetabilien, Biochem. Zeit. 16, 9-19. 1912 Hagemann, O.: Beitrag zur Wirkung der Mineralsubstanzen im Tier- korper, Arch. ges. Physiol. 146, 455-483. 1900 Hahn, M„, and L. Geret: Ueber das Hefe-Endotrypsin, Zeit. Biol. 40, 117- 172. 1895 Haiser, F. : Zur Kenntniss der Inosinsaure, Monatshef te Chem. 16, 190-206. 1909 Haiser, F., and F. Wenzel: Ueber Karnin und Inosinsaure, Ibid. 30, 147- 164; 377-386. 1912 Hall, L. D., and A. D. Emmett: Relative Economy, Composition and Nutritive Value of the Various Cuts of Beef, Univ. 111. Agr. Exp. Sta. Bui. No. 158. 233 pp. 1896 Hall, Winf. S.: Einige Bemerkungen iiber die Herstellung eines kunstlichen Futters, Arch. Anat. u. Physiol., 142-153. 1892 Halliburton, W. D.: The Proteids of Kidney and Liver Cells, Jour, of Physiol. 13, 806-846. 1893 Halliburton, W. D.: The Chemical Physiology of the Animal Cell. British Med. Jour. 1, 501-506, 572-577, 627-632. 1894 Halliburton, W. D.: The Proteids of Nervous Tissue, Jour. Physiol. 15, 90-107. 1895 Halliburton, W. D.: Nucleo-proteids. Supplementary paper, Ibid. 18, 306-318. 1901a Halliburton, W. D.: The Physiological Effects of Extracts of Nervous Tissues, Ibid. 26, 229-243. 1901b Halliburton, W. D.: The Chemical Side of Nervous Activity. I. The General Composition of Nervous Structures. II. Metabolism in Nervous Tissues. III. The Chemical Pathology of Hyperpyrexia, and the Chemical Pathology of General Paralysis of the Insane. IV. The Chemistry of Nerve Degeneration. The Croonian Lectures on the Chemical Side of Nervous Activity. 1901, London. Through Lancet, 1901, 1, 1659, 1660; 1741, 1742. 1905 Halliburton, W. D.: Die Biochemie der peripheren Nerven, Ergebnisse d. Physiol. 4, 23-83. 1894 Halliburton, W. D., and T. Gregor Brodie: Nucleo-albumins and Intra- vascular Coagulation, Jour, of Physiol. 17, 135-173. 1896 Halliburton, W. D., and T. Gregor Brodie: Action of Pancreatic Juice on Milk, Ibid. 20, 97-106. 1889 Halliburton, W. D., and W. M. Friend: The Stromata of the Red Cor- puscles, Ibid. 10, 532-549. 1908 Halpern, Mieczyslaw: Beitrag zum Hungerstoffwechsel, Biochem. Zeit. 14, 134-142. 1906-7 Hamill, J. Molyneux: Observations on Human Chyle, Jour, of Physiol. 35, 151-162. 1877 Hammarsten, Olof : (Zur Kenntnis des Caseins und der Wirkung des Labfermentes,) Nova Acta Regiae Soc. Sci. Upsaliensis in Memoriam Quattuor Saec. ab Univ. Upsaliensi Peractorum. 1877. Through Jahresb. u. d. Fortschr. d. Thierchem. 7 (1877), 158. 628 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1883 Hammarsten, Olof : Zur Frage, ob das Casein ein einheitlicher Stoff sei, Zeit. physiol. Chem. 7, 227-273. 1885a Hammarsten, Olof: Ueber den Gehalt des Caseins an Schwefel und iiber die Bestimmung des Schwefels in Proteinsubstanzen, Ibid. 9, 273-309. 1885b Hammarsten, Olof: Studien iiber Mucin und mucinahnliche Substanzen, Arch. ges. Physiol. 36, 373-456. (1893) Hammarsten, Olof: Till kaunedomen om Nukleoproteiderun, (Zur Kenntniss der Nucleoprote'ide), Upsala Lakareforenings Forhandl. Bd. 28; through Jahresb. ii. d. Fortschr. d. Thierchem. 23 (1893), 35-37. 1894 Hammarsten, Olof: Zur Kenntniss der Nucleoproteide, Zeit. physiol. Chem. 19, 19-37. (1895) Hammarsten, Olof: Nagra ord om olikheterna mellan qvinnomjolk och komjolk, Upsala Lakareforenings Forhandlinger, Bd. 30; through Jahresb. ii. d. Fortschr. d. Thierchem. 25 (1895), 206, 207. 1896 Hammarsten, Olof: Ueber das Verhalten des Paracaseins zu dem Lab- enzyme, Zeit. physiol. Chem. 22, 103-126. 1901 Hammarsten, Olof: Untersuchungen iiber die Gallen einiger Polarthiere. I. Ueber die Galle des Eisbaren. Erster Abschnitt, Ibid. 32, 435-466. 1902 Hammarsten, Olof: Untersuchungen iiber die Gallen einiger Polarthiere. I. Ueber die Galle des Eisbaren. II. Abschnitt, Ibid. 36, 525-555. 1904 Hammarsten, Olof: Untersuchungen iiber die Gallen einiger Polartiere. II. Ueber die Galle des Moschusochsen, Ibid. 43, 109-126. 1905a Hammarsten, Olof: Zur Chemie des Fischeies, Skand. Arch. Physiol. 17, 113-132. 1905b Hammarsten, Olof: Zur Chemie der Galle, Ergebnisse d. Physiol. 4, I. Abt., 1-22. 1908 Hammarsten, Olof: Zur Frage nach der Identitat der Pepsin- und Chymosinwirkung, Zeit. physiol. Chem. 56, 18-80. 1911 Hammarsten, Olof: A Text Book of Physiological Chemistry, from the Author's 7th German Edition. 1881 Hammerbacher, Friedr.: Quantitative Verhaltnisse der organischen und unorganischen Bestandtheile des menschlichen gemischten Speichels, Zeit. physiol. Chem. 5, 302-308. 1863 Hammond, William A. : On the Excretion of Phosphoric Acid through the Kidneys, Physiological Memoirs, Philadelphia, 1863, 29-41. 1897 Hanai, T.: Physiological Observations on Lecithin, Tokyo Imper. Univ., Coll. Agr. Bui., 2, 503-506. (1911) Handovsky, Hans, and Rich. Wagner: Ueber einige physikalisch-chem- ische Eigenschaften von Lecithinemulsionen und Lecithineiweiss- mischungen, Biochem. Zeit. 31, 32-46; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 42. 1912a Hanes, Frederick M.: Ueber das Vorkommen und die Bedeutung von anisotropen Lipoiden in der Leber des Huhnerembyros, Centralbl. allgem. Path. 23, 533-537; through Chem. Abs. 6 (1912), 2627, 2628. 1912b Hanes, Frederick M.: Lipoid Metabolism in the Developing Chick and its Relation to Calcification, Jour. Exp. Med. 16, 512-526; through Chem. Abs. 7 (1913), 1909. 1906 Hannes, Walther: Resistenzerhohung des Peritoneums gegen Infektion mittels Nukleinsaure, eine prophylaktische Massnahme, um die Morbiditat und Mortalitat nach der abdominalen Radikaloperation des gebarmutterkrebses herabzusetzen, Zentralbl. Gynakol. 30, 681-692. PHOSPHORUS METABOLISM 629 1910 Harden, Arthur, and Roland V. Norris: The Fermentation of Galactose by Yeast and Yeast- juice (Preliminary Communication), Proc. Roy. Soc. of London 82 B, 645-649. 1906 Harden, Arthur, and William John Young: The Alcoholic Ferment of Yeast- juice, I. and II. Ibid. 77 B, 405-420; 78 B, 369-375. 1908a Harden, Arthur, and William John Young: The Fermentation of Mannose and Laevulose by Yeast- juice, Proc. of the Chem. Soc. 24, 115, 116. 1908b Harden, Arthur and William John Young: The Alcoholic Ferment of Yeast-juice. III. Proc. Roy. Soc. of London 80 B, 299-311. 1909 Harden, Arthur, and William John Young: The Alcoholic Ferment of Yeast- juice. Part IV. The Fermentation of Glucose, Mannose and Fructose by Yeast- juice, Ibid. 81 B, 336-347. 1911b Harden, Arthur, and William John Young: Ueber die Zusammensetzung der durch Hefepresssaft gebildeten Hexosephosphorsaure. I. and II. Biochem. Zeit. 32, 173-176; 177-188. 1912 Harden, Arthur, and William John Young: Der Mechanismus der alkohol- ischen Garung, Ibid. 40, 458-478; through Zeit. Unters. d. Nahrungs- u. Genussmittel 25 (1913), 456. 1910 Harden, J.: (Neuere Untersuchungen liber alkoholische Garung), Jour, of the Inst, of Brewing 1910, 623; through Zeit. Unters. d. Nahrungs- u. Genussmittel 23 (1912), 164, 165. 1892 Hardin, M. B.: I. On the Available Phosphoric Acid and the Water- Soluble Potash in Cotton Seed Meal. II. On the Methods of Prepar- ing Solutions of Cotton Seed Meal for Precipitation of the Phosphoric Acid. III. On the Occurrence of Meta-Phosphoric Acid and Pyro- Phosphoric Acid in Cotton Seed Meal, So. Carolina Agr. Exp. Sta. Bui. No. 8, New Series. 16 pp. 1892 Harris, V. D., and Wm. J. Gow: Ferment Actions of the Pancreas in Different Animals, Jour, of Physiol. 13, 469-492. 1907 Hart, E. B. : Variations in the Amount of Casein in Cow's Milk, Twenty- fourth Ann. Rept. Wis. Agr. Exp. Sta. 111-116. 1909 Hart, E. B. : A Volumetric Method for the Estimation of Casein in Cow's Milk, Jour. Biol. Chem. 6, 445-451. 1903 Hart, E. B., and W. H. Andrews: The Status of Phosphorus in Certain Food Materials and AnimaL By-products, with Special Reference to the Presence of Inorganic Forms, N. Y. Agr. Exp. Sta. Bui. 238, 181-196; also Amer. Chem. Jour. 30, 470-485. 1909 Hart, E. B., E. V. McCollum, and J. G. Fuller: The Role of Inorganic Phosphorus in the Nutrition of Animals, Amer. Jour. Physiol. 23, 246-277. 1909 Hart, E. B., E. V. McCollum, and G. C. Humphrey: The Role of the Ash Constituents of Wheat Bran in the Metabolism of Herbivora, Ibid. 24, 86-103; also Univ. Wis. Agr. Exp. Sta., Research Bui. No. 5. 1910 Hart, E. B., and W. E. Tottingham: The Nature of the Acid-Soluble Phos- phorus Compounds of Some Important Feeding Materials, Univ. Wis. Agr. Exp. Sta., Research Bui. No. 9, 95-106; also Jour. Biol. Chem. 6, 431-444. 1901 Hartenberg, P.: La lecithine dans la therapeutique des affections du systeme nerveux, 11th Congr. des med. alienistes et neurologistes de France, p. 392. 630 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1913 Hartwell, Burt L.: The Percentage of Total Phosphorus in Flat Turnips as Influenced by the Amount Available in Soils, Agr. Exp. Sta. of R. I. State Coll., Bui. 154, 121-148. 1911 Hartwell, Burt L., and Frederick S. Hammett: The Effect of Phosphorus Manuring on the Amount of Inorganic Phosphorus in Flat Turnip Roots, Jour. Ind. and Engin. Chem. 3, 831, 832. 1910 Hartwell, Burt L., and Wilhelm B. Quantz: The Phosphorus of the Flat Turnip, Proc. Amer. Soc. Biol. Chemists, 1909, in Jour. Biol. Chem. 7, xxxviii. 1888 Hasebroek, Karl: Ueber das Schicksal des Lecithins im Korper, und eine Beziehung desselben zum Sumpfgas im Darmcanal, Zeit. physiol. Chem. 12, 148-162. 1903 Hatai, Shinkishi: The Effect of Lecithin on the Growth of the White Rat, Amer. Jour. Physiol. 10, 57-66. 1854 Haubner: Wissenschaftliche und praktische Mittheilungen. Ueber einige dyskasische Knochenkrankheiten, Magazin f. d. ges. Thierheilk, 20, 197-242. 1867 Haubner: Sitz. Ber. d. Ges. f. Natur- u. Heilk. zu Dresden, 1867; through Jahrbticher der in- u. auslandisch. ges. Med. 151 (1871), 138. 1875 Haubner: Rachitis und Tuberkulose beim Rinde nach Huttenrauchfutter, Jahresb. d. Ges. f. Nat. u. Heilk. 76, 115. 1903 Hawk, P. B.: On the Time Relations of Proteid Metabolism, Amer. Jour. Physiol. 10, 115-145. 1905 Hawk, P. B.: On the Influence of Copious Water Drinking, Univ. Penn. Med. Bui. March, 1905, 53 pp. 1904 Hawk, P. B., and Joseph S. Chamberlain: A Study of the Variations in the Course of the Nitrogen, Sulphate and Phosphate Excretion, as Observed in Short Periods Following a Small Increase in the Proteid Ingested, Amer. Jour. Physiol. 10, 269-289. 1904 Hawk, P. B., and William J. Gies: The Influence of External Hemorrhage on Chemical Changes in the Organism, with Particular Reference to Proteid Catabolism, Ibid. 11, 171-236. 1860 Haxthausen, Hugo Augustus Ab.: Acidum phosphoricum urinae et excre- mentorum, Inaug. Diss., Halle. 1886 Heckel, Ed., and Schlagdenhauffen: Sur la presence de la lecithine dans les vegetaux, Compt. rend. Acad, des sci. 103, 388-390. 1890 Heffter, Arthur: Lecithin in der Leber, Inaug. Diss., Leipzig; through Chem. Centralbl. 1 (1891), 459. 1891 Heffter, Arthur: Das Lecithin in der Leber und sein Verhalten bei der Phosphorvergiftung, Arch. exp. Path. u. Pharm. 28, 97-112. 1903 Heffter, Arthur: Die Ausscheidung korperfremder Substanzen im Harn. I. Teil: Anorganische Verbindungen, Ergebnisse d. Physiol. 2, I. Abt. 95-129. 1904 Heim, Max.: Klinische Erfahrungen mit "Bioson," einer Eiweiss-Eisen- Lecithin-Verbindung, Berlin, klin. Wochenschr. 592-597. 1912 Heiser, V. G.: Beri-beri: with a Suggestion for Governmental Aid in its Eradication, Med. Record 81, 516, 517. 1876 Heiss, Ernst: Kann man durch Einfiihrung von Milchsaure in den Darm eines Thieres den Knochen anorganischen Bestandteile entziehen? Zeit. Biol. 12, 151-169. PHOSPHORUS METABOLISM 631 1873 Heitzmann, C: Ueber kiinstliche Hervorrufung von Rhachitis und Osteo- malacie, Wien. med. Presse, 1873, 1035-1037; Wien. med. Anzeiger 113 (1873); and Wien. med. Jahrbiicher 1874. 1894 Hempel, Walther: Die Milchuntersuchungen Professor Dr. Julius Leh- mann's Arch. ges. Physiol. 56, 558-578. 1906 Henderson, Lawrence J.: Equilibrium in Solutions of Phosphates, Amer. Jour. Physiol. 15, 257-271. 1908a Henderson, Lawrence J.: A Note on the Union of the Proteins of Serum with Alkali, Ibid. 21, 169-172. 1908b Henderson, Lawrence J.: Concerning the Relationship between the Strength of Acids and their Capacity to Preserve Neutrality, Ibid. 21, 173-179. 1908c Henderson, Lawrence J.: The Theory of Neutrality Regulation in the Animal Organism, Ibid. 21, 427-448. 1909 Henderson, Lawrence J.: On the Neutrality Equilibrium in Blood and Protoplasm, Jour. Biol. Chem. 7, 29-35. 1911 Henderson, Lawrence J. : A Critical Study of the Process of Acid Excre- tion, Ibid. 9, 403-424. 1913 Henderson, Lawrence J.: The Regulation of Neutrality in the Animal Body, Science, N. S. 37, 389-395. 1907 Henderson, L. J., and O. F. Black: Concerning the Neutrality of Proto- plasm, Amer. Jour. Physiol. 18, 250-255. 1908 Henderson, L. J., and O. F. Black: A Study of the Equilibrium between Carbonic Acid, Sodium Bicarbonate, Mono-sodium Phosphate, and Di- sodium Phosphate at Body Temperature, Ibid. 21, 420-426. 1903 Henderson, Yandell, and Gaston H. Edwards: Nuclein Metabolism in Lymphatic Leukaemia, Ibid. 9, 417-424. 1897 Henriques, Valdemar: Ueber die reducirenden Stoffe des Blutes, Zeit. physiol. Chem. 23, 244-257. 1903 Henriques, V., and C. Hansen: Ueber den Uebergang des Nahrungsfettes in das Huhnerei und iiber die Fettsaure des Lecithins, Skand. Arch. Physiol. 14, 390-397. 1889 Henry, W. A.: Experiments in Pig Feeding, Sixth Ann. Rept. Wis. Agr. Exp. Sta., pp. 6-41. 1890 Henry, W. A. : Feeding Bone Meal and Hard Wood Ashes to Hogs Living on Corn, Univ. Wis. Agr. Exp. Sta. Bui. No. 25, 3-10. 1719 Hensing, Joh. Th.: Examen chemicum cerebri, ex eodemque phosphorus singularis omnia inflammans, Diss., Giessae; through Thudichum, 1901. 1912 Herbst, O.: Beitrage zur Physiologie des Stoffwechsels im Knabenalter mit besonderer Berucksichtigung einiger Mineralstoffe, Jahrb. Kinder- heilk. 76, Erganzungsh., 40-130. 1913 Herbst, O.: Calcium und Phosphor beim Wachstum am Ende der Kind- heit, Zeit. Kinderheilk. 7, 161-192. 1900 Herlant, Leon: Untersuchungen iiber die Nucleinsaure aus unreifer Lachsmilch, aus Kalbsthymus und aus Hefe, Arch. exp. Path. u. Pharm. 44, 148-159. 1902a Herlitzka, Amedeo, and Angeola Borrino: Ricerche sull' azione chimico- fisiologica dei nucleohistoni e dei nucleoproteidi, Giornale d. reale Accad. di med. di Torino 65 (8 of ser. 4), 351-357. 1902b Herlitzka, Amedeo, and Angeola Borrino: Ricerche sull' azione biochem- ica di alcuni nucleohistoni e nucleoproteidi, Lo Sperimentale 56, 656- 673. 632 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1903 Herlitzka, Amedeo, and Angeola Borrino: Recherches sur Taction bio- chemique de quelques nucleo-histones et nucleo-proteides, Archives ital. de biol. 39, 1-17. 1866 Hermann, Ludimar: Ueber die Wirkungsweise einer Gruppe von Giften. Anhang Ueber das Vorkommen von Protagon im Blute, Arch. Anat. u. Physiol. 27-40; also Hoppe-Seyler's Med. Chem. Unters. 1, 140. 1912 Hermann, Edmund, and Julius Hermann: Ueber den Lipoidgehalt des Blutes normaler und schwangerer Frauen sowie neugeborener Kinder, Biochem. Zeit. 43, 47-55. 1898 Herter, C. A.: An Experimental Study of Fat Starvation with Especial Reference to the Production of Serous Atrophy of Fat, Jour, of Exp. Med. 3, 293-314. 1897 Herxheimer: Untersuchungen iiber die therapeutische Verwendung des Kalkbrodes, Berlin, klin. .Wochenschr. 34, 423-425. 1897 Herzog, Alois: Flachs und Leinen, Mitt. d. Verband. d. osterr. Flachs- u. Leinen-inter. 2, Nr. 19; through Biedermann's Centralbl. f. Agricul- turchem. 26 (1897), 570. 1891 Hess, E., and Schaffer: Ueber den Einfluss des verfiitterten precipitierten phosphorsauren Kalkes auf die chemische Zusammensetzung der Milchasche, Landwirtsch. Jahrb. d. Schweiz 5, 76-78. 1896 Hess, N., and E. Schmoll: Ueber die Beziehungen der Eiweiss und Para- nucleinsubstanzen der Nahrung zur Alluxurkorperausscheidung im Harn, Arch. exp. Path. u. Pharm. 37, 243-252; also Verhandl. d. 14th Congr. f. innere Med. 1896, 330-332. 1909 Heubner, W.: Versuche iiber den Phosphorumsatz des wachsenden Organismus, Verhandl. d. 26. Versamml. d. Gesell. Kinderheilk., 81. Versamml. d. Gesell. deut. Naturforsch. u. Aerzte, Salzburg 1909, 149- 161. 1910 Heubner, W.: Ueber die Phosphorausscheidung eines Neugeborenen, Arch. exp. Path. u. Pharm. 62, 253-257. 1911 Heubner, W.: Versuche iiber den Nahrungsphosphor, Munch, med. Wochenschr. 58, II, 2543, 2544. 1908 Heubner, W., and M. Reeb: Ueber Menge und Verteilung des Phosphors in einigen Nahrungsmitteln, Arch. exp. Path. u. Pharm., Sup., 265- 272. 1905 Hewlett, Albion Walter: The Effect of the Bile upon the Ester-splitting Action of Pancreatic Juice. A preliminary communication. Johns Hopkins Hosp. Bui. 16, 20, 21. 1904a Heymann, Felix: Zur Einwirkung der Castration auf den Phosphor- gehalt des weiblichen Organismus, Arch. Gynakol. 73, 366-405. 1904b Heymann, Felix: Zur Einwirkung der Kastration auf den Phosphor- gehalt des weiblichen Organismus, Zeit. physiol. Chem. 41, 246-258. 1896 Heymans, J. F.: Recherches experimental es sur l'inanition chez le lapin, Arch, de pharmacodynamic 2, 315-353. 1906 Hiestand, O.: Historische Entwicklung unserer Kenntnisse iiber die Phosphatide. Beitrage zur Kenntnis der pflanzlichen Phosphatide, Diss., Zurich. 202 pp.; through Jahresb. ii. d. Fortschr. d. Thierchem. 37 (1907), 1141, 1142. 1910 Highet, H. Campbell: Beriberi in Siam, Philippine Jour, of Sci., B, 5, 73-79. PHOSPHORUS METABOLISM 633 1885 Hiller, E.: Vergleichende Untersuchungen am Skelett eines Vogels, Landwirtsch. Versuch. Stat. 31, 319-335. 1887 Hills, J. L.: Test of the Availability of Bone Meal when Fed to Milch Cows, Vt. Agr. Exp. Sta., Ann. Rept. 1, 81-84. 1894 Hills, J. L.: The Effect of Feeding Bone Meal on the Character of the Milk, Ibid. 7 (for 1893), 106, 107. 1902 Hirschler, August, and Paul Terray: Ueber die Bedeutung der anorgan- ischen Salze im Stoffwechsel des menschlichen und tierischen Organis- mus, Math. u. naturwiss. Ber. aus Ungarn 20 (Pub. 1905), 145-238; also Math, es termeszettudom. Ertesito, 1902, 20, 477-561, through Jahresb. ii. d. Fortschr. d. Thierchem. 32 (1902), 696, 697. 1905 Hirschler, August, and Paul Terray: Ueber die Bedeutung der anorgan- ischen Salze im Stoffwechsel des Organismus, Zeit. klin. Med. 57, 137- 184. 1902 His: Zur Phosphortherapie bei Osteomalacic, Deut. Arch. klin. Med. 73, 546-554. 1898-9 Hitzig, Theod. : Ueber das Verhalten des Harns bei Febris intermittens, Ibid. 62, 358-389. 1900 HIad'ik, J.: Zur Kenntniss der Alkalescenzbestimmung in kleinen Blutmengen, Zeit. klin. Med. 39, 194-201. 1904 Hoennicke, Ernst: Zur Theorie Osteomalacic Zugleich zur Lehre von den Krankheiten der Schilddriise. Vorlaufige Mittheilung, Berlin, klin. Wochenschr. 41, 1154-1156. 1905 Hoennicke, Ernst: Ueber das Wesen der Osteomalacic Ein Beitrag zur Lehre von den Krankheiten der Schilddriise. Nebst Bemerkungen iiber den seelische Zustand bei der Knochenerweichung, Halle. 78 pp. 1910 Hosl, J. : Ueber Unterschiede in der tryptischen und peptischen Spaltung des Caseins, Paracaseins und des Paracaseinkalkes aus Kuh- und Ziegenmilch, Inaug. Diss., Bern. 31 pp. 1906 von Hoesslin, Heinrich: Ueber den Abbau des Cholins im Tierkorper, Beitrage z. chem. Physiol, u. Path. 8, 27. 1909 von Hoesslin, Heinrich: Experimentelle Untersuchungen zur Physiologie und Pathologie des Kochsalzwechsels. Mit Beitragen iiber die Wirkung des Kochsalzes auf dem Umsatz und die Ausscheidung von Stickstoff und Phosphorsaure, Zeit. Biol. 53 (N. F. 35), 25-92. 1910 Ho ff strom, K. A.: Eine Stoffwechseluntersuchung wahrend der Schwang- erschaft, Skand. Arch. Physiol. 23, 326-420. 1897 Hofmann, K. B.: Augenblicker Milchsauregehalt des Harnes bei Osteo- malakie, Centralbl. innere Med. 18, 329-333. 1873 Hofmeister, V.: Ein Futterungsversuch mit Lammern, Landwirtsch. Versuch. Stat. 16, 126-183. 1910 Holsti, Oesten: Zur Kenntniss des Phosphorumsatzes beim Menschen, Skand. Arch. Physiol. 23, 143-153. 1890 D'Hont, Fred.: Contribution a l'etude du lait, Courtrai. 6 pp. 1911 Hoobler, B. Raymond: The Role of Mineral Salts in the Metabolism of Infants, Amer. Jour, of Diseases of Children 2, 107-140. 1912 Hoobler, B. Raymond: Mineral Salts and Their Relation to the Dietary of Infants and Young Children, Archives of Pediat. 29, 208-214. 1911 Hooper, D.: Phosphorus in Indian Foodstuffs, J. Proc. Asiatic Soc. of Bengal 7, 313-322; through Chem. Abs. 6 (1912), 1638, 1639. 634 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1897 Hoover, G. F., and Torald Sollmann: A Study of Metabolism During Fasting in Hypnotic Sleep, Jour. Exp. Med. 2, 405-411. 1912 Hopkins, F. G.: Feeding Experiments Illustrating the Importance of Accessory Factors in Normal Dietaries, Jour, of Physiol. 44, 425-460. 1912 Hopkins, F. G.: and Allen Neville: A Note Concerning the Influence of Diets upon Growth, Biochem. Jour. 7, 97-99. 1862 Hoppe, Ernst Felix Immanuel (Hoppe-Seyler) : Untersuchungen iiber die Constitution des Zahnschmelzes, Arch. path. Anat. u. Physiol. 24, 13-32. 1866a Hoppe, Ernst Felix Immanuel (Hoppe-Seyler): Beitrage zur Kenntniss der Constitution des Blutes, Med. chem. Untersuch., 133-150. 1866b Hoppe, Ernst Felix Immanuel (Hoppe-Seyler): Ueber einige Bestand- theile der Maiskorner, Ibid., 162, 163. 1878-9 Hoppe, Ernst Felix Immanuel (Hoppe-Seyler) : Ueber Lecithin und Nuclein in der Bierhefe, Zeit. physiol. Chem. 2, 427-429. 1907a Hoppe, Joseph: Die Verwendung des Nukleogens bei nervenkranken Kindern, Therapie der Gegenwart 48 (N. F. 9), 501-503. 1907b Hoppe, Joseph: Ein Beitrag zur Kenntnis des Mineral- Stoffwechsels der Idioten, Zeit. Erforsch. u. Behandl. d. jugendlichen Schwachsinns 1, 29-35. 1889 Horbaczewski, J.: Untersuchung iiber die Entstehung der Harnsaure im Saugethierorganismus, Sitzungsber. d. Math, -naturw. Classe d. Kaiserlich. Akad. d. Wissensch. (Vienna), 98, Abt. 3, 301-318. 1891a Horbaczewski, J.: Beitrage zur Kenntniss der Bildung der Harnsaure und der Xanthinbasen, sowie der Entstehung der Leukocytosen im Saugethierorganismus, Ibid. 100, Abt. 3, 78-132. 1891b Horbaczewski, J.: Beitrage zur Kenntniss der Bildung der Harnsaure und der Xanthinbasen, sowie der Entstehung der Leukocytose im Saugethierorganismus, Monatshefte Chem. 12, 221. 1893 Horbaczewski, J.: Bemerkungen zum Vortrage des Hrn. Alb. Kossel.: "Ueber Nucleisaure." Arch. Anat. u. Physiol., physiol. Abt., 109-115. 1907 Horner, Oskar: Zum Verhalten des Phytins im Organismus, Biochem. Zeit. 2, 428-434. 1906 Hotz, Gerhard: Phosphorsaure- und Kalkstoffwechsel bei Osteomalacie unter dem Einfluss der Phosphortherapie, Diss., Basel. 32 pp.; also Zeit. exp. Path. u. Ther. 3, 605-632. 1894 Houdet, V.: Contribution a Tetude du colostrum de la vache, Annales de lTnst. Pasteur 8, 506-513. 1901 Huchard, H. : L'emploi therapeutique de la lecithine, Jour, des praticiens, 439-441. 1899a Hugounenq, L.: Recherches sur la statique des elements mineraux et particulierement du fer chez le foetus humain, Jour, de physiol. et de path, gener. 1, 703-711; also Compt. rend. Soc. de biol. 51, 337, 338; and Compt. rend. Acad, des sci. 128, 1054-1.056. 1899b Hugounenq, L.: La composition minerale de l'enfant nouveau-ne et la loi de Bunge, Compt. rend. Acad, des sci. 128, 1419-1421; also Compt. rend. Soc. de biol. 51, 523-525. 1900 Hugounenq, L.: La composition minerale de l'organisme de l'enfant nouveau-ne, Jour, de physiol. et de path, gener. 2, 1-5. 1905a Hugounenq, L., and A. Morel: Recherches sur l'hematogene, Compt. rend. Acad, des sci. 140, 1065-1067. PHOSPHORUS METABOLISM 635 1905b Hugounenq, L., and A. Morel: Recherches sur la formation de l'hemo- globin chez l'embryon, Ibid. 141, 848, 849. 1901a Huiskamp, W.: Ueber die Eiweisskorper der Thymusdriise, Zeit. physiol. Chem. 32, 145-196. 1901b Huiskamp, W.: Ueber die Elektrolyse der Salze des Nucleohistons und Histons, Ibid. 34, 32-54. 1903 Huiskamp, W.: Beitrage sur Kenntniss des Thymusnucleohistons, Ibid. 39, 55-72. (1912) Hulshoff-Pol, D. J.: Jets over de atiologie van Polyneuritis gallinarum in verband met verzuurde-rijstvoeding. (Etiology of Polyneuritis gall- inarum in Hens in Connection with the Feeding of Sour Rice), Geneesk. Tijdschr. Nederlandsch-Indie 52, 11; through Zentralbl. Biochem. u. Biophys. 14 (1912), 399. 1867 Huppert, H.: Analyse eines osteomalacischen Knochens, Archiv der Heil- kunde 8, 345-351. 1901-2 Hutchison, Robert, and J. J. R. MacLeod: A Contribution to our Know- ledge of the Chemistry of Red Bone Marrow, Jour, of Anat. and Physiol. 36, 292-295. (1911) Ibrahim, J.: Kaseinklumpen im Kinderstuhl im Zusammenhang mit Rohmilchernahrung, Monatsschr. f. Kinderheilk. 10, 55-64; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 298. 1901 Iljin, M. D.: (Der Einfluss der organischen Phosphorverbindungen auf die Stickstoffablagerung im menschlichen Korper), Russkij Wratsch 5, 574; through Jahresb. ii. d. Fortschr. d. Thierchem. 31 (1901), 733. 1906 Iljin, M. D.: (Gegenseitige Beziehungen der Lecithine, des Phytins und der Nucleinsauren auf der Grundlage ihrer Konstitution.) Russkij Wratsch 1906, 5, 390-392; through Biochem. Centralbl. 5 (1906-7), 534- 538. 1897 Imbert, M., and G. Belugon: Chaleur de neutralisation de l'acide glycerophosphorique, Compt. rend. Acad, des sci. 125, 1040-1042. 1907 Ingle, Herbert: Osteoporosis in Animals, Jour, of Compar. Path, and Ther. 20, 35-48. 1908 Ingle, Herbert: The Mineral Constituents of Foods, Jour, of Agr. Sci. 3, I, 22-31. 1909 Ingle, Herbert: The Importance of the Mineral Constituents of Foods, Jour, of the Roy. Inst, of Public Health 17, 736-747." 1904 Inouye, Katsuji: Ueber das Vorkommen einer Lavulinsaure bildenden Atomgruppe in Nucleinsauren, Zeit. physiol. Chem. 42, 117-120. 1906 Inouye, Katsuji: Ueber die Nucleinsaure aus den Spermatozoen des Hamo, Ibid. 48, 181-184. 1905 Inouye, Katsuji, and Y. Kotake: Ueber die Darmnucleinsaure, Ibid. 46, 201-205. (1911) Isaac: Das Jodocitin, Med. Klinik (Berlin) 7, . 1541-1545; through Jahresb. ii. d. Fortschr. d. Thierchem. 41.(1911), 890. 1903 Iwanoff, Leonid: Ueber die fermentative Zersetzung der Thymonuclein- saure durch Schimmelpilze, Ibid. 39, 31-43. 1907 Iwanoff, Leonid: Ueber die Synthese der Phosphoorganischen Verbind- ungen in abgetoteten Hefezellen, Ibid. 50, 281-288. 1900 Jackson, Holmes C: On the Phosphorus Content of the Paranuclein from Casein, Amer. Jour. Physiol. 4, 170-177. 636 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1906 Jacob, Ludwig: Futterungsversuche mit einer aus den einfachen Nahrungsstoffen zusammengesetzten Nahrung an Tauben und Ratten, Zeit. Biol. 48 (N. F. 30), 19-62. 1898 Jacob, Paul, and Peter Bergell: Ueber den Einfluss nucleinhaltiger Nahrung auf Blut und Stoffwechsel unter besonderer Beriicksichtigung des Phosphorsaurestoffwechsels, Zeit. klin. Med. 35, 171-211. 1902 Jaeckel, Hermann: Ueber die Zusammensetzung des menschlichen Fettes. Ein Beitrag zur Analyse der Fette, Zeit. physiol. Chem. 36, 53-84. 1902 Jagerroos, B. H.: Studien iiber den Eiweiss-, Phosphor- und Salzumsatz wahrend der Graviditat, Arch. Gynakol. 67, 517-590. 1897 de Jager, L.: Over de werking van labferment, Nederl. Tijdschrift voor Geneeskunde (2r.) 33, 253-267; through Jahresb. ii. d. Fortschr. d. Thierchem. 27 (1897), 276-279. 1910 de Jager, L.: Ueber den Gehalt des Harnes an Ammoniak, Phosphor- saure, sauren Korpern und Aminosauren, Zentralbl. ges. Physiol, u. Path, des Stoffwechsels 11 (n. s. 5), 241-255. 1876 von Jaksch, Rudolf: Ueber das Vorkommen von Nuclein im Menschen- gehirn, Arch. ges. Physiol. 13, 469-473. 1885 von Jaksch, Rudolf: Ueber Acetonurie und Diaceturie. 156 pp. 1910 Janin: Sur une theorie phosphatique du beriberi, Soc. de med. et d'hyg. tropicale, Caducee 1910, No. 2, p. 25; through Arch. Schiffs- u. Tropen- hygiene 15 (1911), 36. 1877 Jarisch, Adolf: Untersuchungen iiber die Bestandtheile der Asche des Blutes, Wien. med. Jahrbucher, 39-64. 1910 Jebbink, G. J.: Over het nucleingehalte van menschelijk voedsei en voor- al van Indische versnaperingen, Diss. Amsterdam. 124 pp. Through Jahresb. u. d. Fortschr. d. Thierchem. 40, 640. 1912 Jegorow, M. A.: Zur Kenntniss der Eigenschaften des Phytins, Biochem. Zeit. 42, 432-439. 1899 Jemma, R.: Sulla digestione artificiale del latte, Clinica med. italiana 1899, No. 6; through Centralbl. innere Med. 21 (1900), 671, 672. 1888 Jenkins, E. H.: Observations on the Chemical Composition of Certain Grasses, Conn. Agr. Exp. Sta. Rept. for 1888, Pt. II, 100-102. 1904 Jensen, Orla: Ueber den Einfluss der Mineralbestandteile des Futters auf Milch, Molkerei-Zeitung 14, 505-508; 517-519. 1905a Jensen, Orla: De l'influence des elements mineraux du fourrage sur le lait, Revue gener. du lait 4, 273-285; 297-306. 1905b Jensen, Orla: Lasst die Milch sich in ihrer Zusammensetzung durch das Futter beeinflusst? Landwirtsch. Jahrb. d. Schweiz, 1905, Separate abdruck; through Biedermann's Centralbl. f. Agricultur-Chem. 35 (1906), 285-287. 1905-6 Jensen, Orla: La composition du lait peut-elle etre influencee par l'aff ourragement ? Revue gener. du lait 5, 103-110; 121-128; 152-161; 178-185; 198-205. 1897-8 Jerome, William J. Smith : The Formation of Uric Acid in Man, and the Influence of Diet on its Daily Output, Jour, of Physiol. 22, 146-158. 1899 'Jerome, William J. Smith: Further Proofs of the Origin of Uric Acid from Nuclein-compounds and Derivatives, Ibid. 25, 98-103. 1887a Jolly L.: Sur les phosphates et leurs fonctions dans les etres vivants, Paris. Archives gener. de med. 1, 627. PHOSPHORUS METABOLISM 637 1887b Jolly, L. : Les phosphates. Leurs f onctions chez les etres vivants, Paris. 578 pp. 1898 Jolly, L.: Recherches sur le phosphore organique, Compt. rend. Acad, des sci. 126, 531-533. 1904a Jones, Walter. : Ueber das Enzym der Thymusdruse, Zeit. physiol. Chem. 41, 101-108. 1904b Jones, Walter: On the Enzyme of the Suprarenal Gland, Proc. Amer. Physiol. Soc, in Amer. Jour. Physiol. 10, XXV. 1904c Jones, Walter: Ueber die Selbstverdauung von Nucleoproteiden, Zeit. physiol. Chem. 42, 35-54. 1905 Jones, Walter: Ueber das Vorkommen der Guanase in der Rindermilz und ihr Fehlen in der Milz des Schweines, Ibid. 45, 84-91. 1908. Jones, Walter: On the Identity of the Nucleic Acids of the Thymus, Spleen and Pancreas, Jour. Biol. Chem. 5, 1-26. 1910 Jones, Walter: Ueber die Beziehung der aus wasserigen Organextrakten gewonnenen Nucleinfermente zu den physiologischen Vorgangen im lebenden Organismus, Zeit. physiol. Chem. 65, 383-388. 1911a Jones, Walter: Concerning Nucleases, Jour. Biol. Chem. 9, 129-137. 1911b Jones, Walter: On the Physiological Agents Which are Concerned in the Nuclein Fermentation, with Special Reference to Four Independent Desamidases, Ibid. 9, 169-180. 1912 Jones, Walter: On the Formation of Guanylic Acid from Yeast Nucleic Acid, Ibid. 12, 31-35. 1906 Jones, Walter, and C. R. Austrian: Ueber die Verteilung der Fermente des Nucleinstoffwechsels, Zeit. physiol. Chem. 48, 110-129. 1907a Jones, Walter, and C. R. Austrian: On Thymus Nucleic Acid, Jour. Biol. Chem. 3, 1-10. 1907b Jones, Walter, and C. R. Austrian: On the Nuclein Ferments of Embryos, Ibid. 3, 227-232. 1904 Jones, Walter, and C. L. Partridge: Ueber die Guanase, Zeit. physiol. Chem. 42, 343-348. 1914 Jones, Walter, and A. E. Richards: The Partial Enzymatic Hydrolysis of Yeast Nucleic Acid, Jour Biol. Chem. 17, 71-80. 1908 Jones, Walter, and L. G. Rowntree: On the Guanylic Acid of the Spleen, Ibid. 4, 289-295. 1902 Jones, Walter, and G. H. Whipple: The Nucleoproteid of the Suprarenal Gland, Amer. Jour. Physiol. 7, 423-434. 1905 Jones, Walter, and M. C. Winternitz: Ueber die Adenase, Zeit. physiol. Chem. 44, 1-10. 1885-6 Jordan, W. H.: The Manure Residue of Corn Meal and of Cotton-Seed Meal, Ann. Rept. Maine Agr. Exp. Sta. for 1885-6, 42-46. 1906 Jordan, W. H., E. B. Hart, and A. J. Patten: A Study of the Metabolism and Physiological Effects of Certain Phosphorus Compounds with Milch Cows, Amer. Jour. Physiol. 16, 268-313; also N. Y. Agr. Exp. Sta., Tech. Bui. 1, 59 pp. 1904 Joulie, H.: L'acide phosphorique dans Palimentation des herbivores, Rev. agr. Reunion 10, 84-86. 1912 Juchler, Theodor: Die mineralischen bestandteile der Bauchspeicheldriise, Inaug. Diss., Zurich. 24 pp. Through Zeit. Kinderheilk. Referate 4 (1913) 166, 167. 638 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1868 Jiidell, Gustav: Zur Blutanalyse, Hoppe-Seyler's Med. Chem. Unters. 386-393. 1913 Jundell, I. : Untersuchungen iiber den Stoffwechsel bei der Dyspepsie und der alimentaren Intoxikation, Zeit. Kinderheilk. Originalien 8, 235- 290. 1911 Juschtschenko, A.: Die Schilddriise und die fermentativen Prozesse, Zeit. physiol. Chem. 75, 141-168. 1911 Juschtschenko, A. J.: "Ueber den Nucleasegehalt verschiedener Organe des Menschen und der Tiere, Biochem. Zeit. 31, 377-384. 1913 Juschtschenko, A. S.: Zur Physiologie der Schilddriise: Gehalt an Phos- phor, Stickstoff und Lipoiden bei Thyroidektomierten Tieren, Ibid. 48, 64-85. 1906 Kaas, Karl: Ueber den Phosphorgehalt von Huhnereiweiss, Monatshefte Chem. 27, 403-409. 1910 Kajiura, S., and O. Rosenheim: A Contribution to the Etiology of Beri- beri, Jour, of Hyg. 10, 49-55. 1907a Kalaboukoff, and Emile F. Terroine: Sur l'activation des ferments par la lecithine. I. Action de la lecithine sur la lipase pancreatique, Compt. rend Soc. de biol. 63, 372-374. 1907b Kalaboukoff, and Emile F. Terroine: Sur l'activation des ferments par la lecithine. II. Action de la lecithine sur les lipases gastrique et intestinale, Ibid. 63, 617-619. 1907c Kalaboukoff, and Emile F. Terroine: Sur l'action de la lecithine sur les ferments. III. Action de l'ovolecithine sur l'amylase, la trypsine et le lab, Ibid. 63, 664-666. 1909 Kalaboukoff, and Emile F. Terroine: Action du sue pancreatique et des sels biliares sur l'ovolecithine, Ibid 66, 176-178. 1897 Kalinin, A. A.: Untersuchungen iiber die Ausscheidung von Kohlensaure, Stickstoff und Phosphor und den Sauerstoffverbrauch in der Latenz- periode des Fiebers bei Kaninchen und Hunden nach subcutaner Infec- tion mit Bouillonculturen von Pyocyaneus- und Diphtheriebacillen, Centralbl. allgem. Path. u. path. Anat. 8, 518-524. 1913 Kaminer, Gisa, and Ernst Mayerhofer: Ueber den klinischen Wert der Bestimmung des anorganischen Phosphors im Harne unnaturlich ernahrter Sauglinge, Zeit. Kinderheilk. 8, 24-49. 1867 Karmrodt, C: Untersuchungen von Wiesenheu, Zeit. des landwirtsch. Vereins f. Rheinpreussen 35, 376-378; through Jahresb. f. Agricultur- chem. 11 & 12 (1868-9), 488 and 545. 1881 Kassowitz, Max: Die normale Ossification und die Erkrankungen des, Knochensystems bei Rachitis und hereditarer Syphilis. I. Theil: Normale Ossification, Wien. med. Jahrbucher, 1881. 1884a Kassowitz, Max: Die normale Ossification und die Erkrankungen des Knochensystems bei Rachitis und hereditarer Syphilis. II. Theil: Rachitis. 1. Abtheilung. 2. Abtheilung. Die Pathogenese der Rachitis, Ibid. 1884. 1884b Kassowitz, Max: Die Phosphorbehandlung der Rhachitis, Zeit. klin. Med. 7, 36-74; 93-139. 1886 Kassowitz, Max: Die Symptome der Rachitis auf anatomischer Grund- lage bearbeitet. I. Abtheilung. Verbildungen und Funktionsstorung- en der Extremitaten, Leipzig. 130 pp. 1890 Kassowitz, Max: Zur Theorie und Behandlung der Rachitis, Beitrage z. Kinderheilk. 1890, book 1, 106-149. PHOSPHORUS METABOLISM 639 1901 Kassowitz, Max: Zur Theorie der Rachitis, Wien. med. Wochenschr. 51, 1753-1758'; 1807-1813; 1857-1860. 1910 Kassowitz, Max: 15. Vorlesung: Knochenwachstum und Rachitis (15 pp.); 16. Vorlesung: Die Ursachen der Rachitis (14 pp.); 17. Vor- lesung: Vorbeugung und Behandlung der Rachitis (18 pp.), Praktische Kinderheilkunde, in 36 Vorlesungen, Berlin. 1912 Kassowitz, Max: Ueber Rachitis. II. Osteochondritis rachitica, Jahrb. Kinderheilk. 75 (25 of ser. 3), 194-212; 334-349; 489-505; 581-600. 1913 Kassowitz, Max: Weitere Beitrage zur Rachitisfrage. III. Phosphor und Lebertran, Deut. med. Wochenschr. 39 II, 1716-1720. 1909 Kastle, Joseph H., and Norman Roberts: The Chemistry of Milk, in "Milk and Its Relations to the Public Health," Bui. No. 56, Hygienic Lab. of the Pub. Health and Marine-Hosp. Service of U. S., 313-425. 373 refs. 1908 Katayama, T.: Ueber die Zusammensetzung der Kuhmilch verschieden- er Rassen mit besonderer Berucksichtigung ihres Kalk- und Phos- phorsauregehaltes, Landwirtsch. Versuch. Stat. 69, 342-358. 1896 Katz, Julius: Die mineralischen Bestandtheile des Muskelfleisches, Archiv ges. Physiol. 63, 1-85. 1902 Kauf mann,- Martin : Ein Beitrag zur Frage der Fleischmast, Centralbl. Stoffwechsel- und Verdauungs-krankheit, 3, 239-241. 1902 Kauf mann, M., and L. Mohr: Beitrage zur Alloxurkorperfrage und zur Pathologie der Gicht, Deut. Arch. klin. Med. 74, 141-162; 348-369; 586- 614. 1903 Kauf mann, M., and L. Mohr: Ueber Eiweissmast, Berlin, klin. Wochen- schr. 8, 161-163. 1902 Kaup, Igo : Ein Beitrag zu der Lehre vom Einflusse der Muskelarbeit auf den Stoffwechsel, Zeit. Biol. 43, 221-255. 1856a Kaup, Wilhelm: Beitrage zur Physiologie des Harnes. 2. Ueber die Auf saugung von Harnbestandtheilen in der Blase, Arch, physiol. Heilk. 125-164. 1856b Kaup, Wilhelm: Beitrage zur Physiologie des Harnes, Ibid. 556-566. 1911 Kaupp, B. F.: The Effect of Nuclein on the Blood, Amer. Veterinary Review, 40, 306-315. 1897 Keller, Arthur: Zur Kenntniss der Gastroenteritis im Sauglingsalter, Ammoniak-Ausscheidung, Jahrb. Kinderheilk. 44, 25-52. 1898 Keller, Arthur: Phosphorstoffwechsel im Sauglingsalter, Zeit. klin. Med. 36, 49-76. 1900a Keller, Arthur: Organische Phosphorverbindungen in Sauglingsharn, ihr Ursprung und ihre Bedeutung fur den Stoffwechsel. Zeit. physiol. Chem. 29, 146-184. 1900b Keller, Arthur: Phosphor und Stickstoff im Sauglingsorganismus, Arch. Kinderheilk. 29, 1-95. 1880 Kellner, Oskar: Versuche iiber die Entbitterung und Verdaulichkeit der Lupinenkorner, Landwirtsch. Jahrbticher 9, 977-998. 1907 Kellner, Oskar: Die Ernahrung der landwirtschaftlichen Nutztiere, Berlin. Fourth edition. 621 pp. 1894 Kellner, O., A. Kohler, and F. Barnstein: Untersuchungen verschiedener Rauhfuttersarten aus Wirtschaften in denen Knochenbriichigkeit auf- tritt, Sachs, landwirtsch. Zeit. 42, 167-171, 227. 640 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1895 Kellner, O., A. Kohler and F. Barnstein: Untersuchungen verschiedener Rauhfutterarten aus Wirtschaften, in denen Knochenbriichigkeit auf- tritt, Biedermann's Centralbl. f. Agriculturchem. 24, 441-443. 1869a Kemmerich, E.: Untersuchungen uber die physiologische Wirkung der Fleischbruhe, des Fleischextracts und der Kalisalze des Fleisches, Arch. ges. Physiol. 2, 49-93. 1869b Kemmerich, E.: Beitrage zur physiologischen Chemie der Milch, Ibid. 2, 401-414. 1909 Kennaway, E. L., and J. B. Leathes: A Preliminary Note on the Examin- ation of the Fat in the Liver in Health and Disease, Proc. Roy. Soc. Med. 2, Part III, Path. Sect., 136-144. 1910 Kepinow, L.: Ueber den Einfluss der Blutkorperchenlipoide auf die Blut- bildung, Biochem. Zeit. 30, 160-171. 1908 Khuen, A.: Beitrage zur Kenntnis des Mineralstoffwechsels, insbesondere des Kalkes beim Wiederkauer, Diss., Bonn. 41 pp. 1909 Kida, Y. : Ueber den Einfluss der hoheren Temperatur beim Sterilisieren der Milch, Jour, of College of Agr., Imperial Univ. Tokyo 1, 141-144. 1907a Kikkoji, T.: Ueber das Vorkommen von einem Nucleinsaure spaltenden Fermente in Cortinellus edodes, Zeit. physiol. Chem. 51, 201-206. 1907b Kikkoji, T.: Ueber die Nucleinsaure aus der menschlichen Placenta, Ibid. 53, 411-414. 1909 Kikkoji, T.: Beitrage zur Kenntniss des Caseins und Paracaseins, Ibid. 61, 139-146. 1910 Kilbourne, E. D.: Food Salts in Relation to Beriberi, Philippine Jour. of Sci., B, 5, 127-135. 1911 Kimura, K., and W. Stepp: Untersuchungen iiber den Gehalt des Blut- serums an atherloslichem Phosphor bei verschiedenen Krankheiten, Deut. Arch. klin. Med. 104, 209-215. 1911 Kinberg, Georg: Beitrag zur Kenntnis des Stoffwechsels bei N- Hunger, Skand. Arch. Physiol. 25, 291-314. 1906 Kitagawa, F., and H. Thierf elder: Ueber das Cerebron. III. Mitteilung, Zeit. physiol. Chem. 49, 286-292. 1896 Klautsch, A.: Einige Mittheilungen iiber die Verwendbarkeit von H. O. Opel's Nahrzwieback als Nebenkost fur Sauglinge und an Rachitis leidende Kinder, Jahrb. Kinderheilk. 43, 190-202. 1908 Kleinertz: Die Anwendung von physiologisch reinem Lecithin in der Ther- apie, Med. Klinik 4, 191, 192. 1899 Klemperer, Georg: Zur Behandlung der Phosphaturie, Therapie der Gegenwart, N. F. 1, 351-354. 1908 Klemperer, Georg: Ueber Phosphaturie, ein Beitrag zur Prophylaxe der Nierensteine, Ibid. 49 (N. F. 10), 3-11. 1902 Klemperer, G., and F. Tritschler: Untersuchungen iiber Herkunft und Loslichkeit der im Urin ausgeschiedenen Oxalsaure, Zeit. klin. Med. 44, 337-366. 1908 Klien, G.: Ueber den Wert des Beif utters von phosphorsaurem und kohlensaurem Kalk bei verschiedenen Tiergattungen, Milch-Zeitung 1908, No. 3. (1911) Klieneberger, Otto L.: Die Behandlung der progressiven Paralyse mit Natrium nucleinicum, Berlin, klin. Wochenschr. 48, 330-333; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911).. 923. PHOSPHORUS METABOLISM 641 1906 Klimmer and Schmidt: Beitrag zur Aetiologie der Halisteresis ossiura nebst therapeutischen Bemerkungen, Monatshefte prakt. Tierheilk. 17, 481-517. 127 refs. 1882 Klinkenberg, W.: Ueber die Nucleiine, Zeit. physiol. Chem. 6, 566-571. 1909 Klotz, M.: Milchsaure und Sauglingstoffwechsel, Jahrb. Kinderheilk. 70, 1-61. 1905 Klotz, Oskar: On the Presence of Soaps in the Organism in Certain Pathological Conditions (A Preliminary Communication), Proc. Amer. Physiol. Soc, in Amer. Jour. Physiol. 13, XXI, XXII. 1893 Klug, Ferd., and Viktor Olsavszky: Einfluss der Muskelsarbeit auf die Ausscheidung der Phosphorsaure, Arch. ges. Physiol. 54, 21-26. 1908 Knapp, Paul: Experimenteller Beitrag zur Ernahrung von Ratten mit kiinstlicher Nahrung und zum Zusammenhang von Ernahrungs- storungen mit Erkrankungen der Conjunctiva, Zeit. exp. Path. u. Ther. 5, 147-169. 1898 Knauthe, Karl: Zur Kenntniss des Stoffwechsels der Fische, Arch. ges. Physiol. 73, 490-500. 1898a Knopf elmacher, Wilhelm: Verdauungsriickstande bei der Ernahrung mit Kuhmilch und ihre Bedeutung fur den Saugling, Beitrage z. klin. Med. u. Chir. 1898, 18, 7-75. 1898b Knopf elmacher, Wilhelm: Ueber Caseinverdauung, Wien. klin. Wochen- schr. 11, 1024, 1025. 1899 Knopf elmacher, Wilhelm: Neue Versuche uber Caseinausnutzung, Ibid. 12, 1308-1310. 1900 Knopf elmacher, Wilhelm: Versuche iiber die Ausnutzung des Kuhmilch- kaseins, Jahrb. Kinderheilk. 52, 545-571. 1914 Knopf, Martin: Ueber das Nucleoproteid nach Hammarsten aus Rinder- pankreas. I. Mitteilung, Zeit. physiol. Chem. 89, 170-174. 1888 Kobler, G.: Zur Kenntniss der Osteomalacie, Wien. klin. Wochenschr. 1, 459-461; 482, 483. 1900 Kobrak, Erwin: Beitrage zur Kenntniss des Caseins der Frauenmilch, Arch. ges. Physiol. 80, 69-85. 1906 Koch, Egbert: Ein Beitrag zum Phosphorstoffwechsel, St. Petersburger med. Wochenschr. 31, 400-402. 1913 Koch, Mathilde L.: Contributions to the Chemical Differentiation of the Central Nervous System. I. A Comparison of the Brain of the Albino Rat at Birth with That of the Fetal Pig, Jour. Biol. Chem. 14, 267-279. 1902a Koch, Waldemar: The Lecithans, their Function in the Life of the Cell, Univ. of Chicago Decennial Pub. 10, 1-12. 1902b Koch, Waldemar: Zur Kenntniss des Lecithins, Kephalins und Cerebrins aus Nervensubstanz, Zeit. physiol. Chem. 36, 134-140. 1903 Koch, Waldemar: Die Lecithane und ihre Bedeutung fur die lebende Zelle, Ibid. 37, 181-188. 1904 Koch, Waldemar: Methods for the Quantitative Chemical Analysis of the Brain and Cord, Amer. Jour. Physiol. 11, 303-329. 1905a Koch, Waldemar: Therapeutic Value of Lecithin in Infant Feeding, St. Louis Courier of Med., June 1905. 1905b Koch, Waldemar: Relation of Kreatinin Excretion to Variations in Diet, Amer. Jour. Physiol. 15, 15-29. 642 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1906 Koch, Waldemar: Ueber der Lecithingehalt der Milch, Zeit. physiol. Chem. 47, 327-330. 1907a Koch, Waldemar: The Relation of Electrolytes to Lecithin and Kephalin, Jour. Biol. Chem. 3, 53-56. 1907b Koch, Waldemar: Zur Kenntnis der Schwefelverbindungen des Nerven- systems, Zeit. physiol. Chem. 53, 496-507. 1909a Koch, Waldemar: Phosphorus Compounds as Brain Foods, Jour. Amer. Med. Assoc. 52, 1381-1383. 1909b Koch, Waldemar: Die Bedeutung der Phosphatide (Lecithane) fur die lebende Zelle. II. Mitteilung, Zeit. physiol. Chem. 63, 432- 442. 1910a Koch, Waldemar: Zur Kenntnis der Schwefelverbindungen des Nerven- systems. II. Mitteilung. Ueber ein Sulfatid aus Nervensubstanz, Ibid. 70, 94-97. 1910b Koch, Waldemar: Pharmacological Studies on the Phosphatids. I. Methods for the Study of their Combinations with Drugs and Other Substances, Jour. Pharmacol, and Exp. Therapeut. 2, 239-244. 1912 Koch, Waldemar: Should the Term Protagon be Retained, Proc. Soc Bi-ol. Chem., in Jour. Biol. Chem. 11, XL. 1906 Koch, Waldemar, and William H. Goodson: A Preliminary Study of the Chemistry of Nerve Tissue Degeneration, Amer. Jour. Physiol. 15, 272-279. 1913 Koch, W., and M. L. Koch: Contributions to the Chemical Differentiation of the Central Nervous System. III. The Chemical Differentiation of the Brain of the Albino Rat during Growth, Jour. Biol. Chem. 15, 423- 448. 1910 Koch, W., and F. C. McLean: Pharmacological Studies on the Phos- phatids. 3. The Relation of the Phosphatids to Overton and Meyer's Theory of Narcosis, Jour. Pharmacol, and Exp. Therapeut. 2, 249-252. 1907 Koch, Waldemar, and Sydney A. Mann: A Comparison of the Chemical Composition of Three Human Brains at Different Ages, Proc. Physiol. Soc, in Jour, of Physiol. 36, XXXVI-XXXVIII. 1909 Koch, Waldemar, and Sydney A. Mann: A Chemical Study of the Brain in Healthy and Diseased Conditions, with Especial Reference to Dementia Praecox, Archives of Neurol, and Psychiatry 4, 2-46. 1910 Koch, W., and H. T. Mostrom: Pharmacological Studies on the Phos- phatids. 5. The Function of the Brain Phosphatids in the Physiologi- cal Action of Strychnine, Jour. Pharmacol, and Exp. Therapeut. 2, 265-269. 1910 Koch, W., and F. H. Pike: Pharmacological Studies on the Phosphatids. 2. The Relation of the Phosphatids to the Sodium and Potassium of the Neuron, Ibid. 2, 245-248. 1907 Koch, W., and Howard S. Reed: The Relation of Extractives to Protein Phosphorus in Aspergillus Niger, Jour. Biol. Chem. 3, 49-52. 1910 Koch, Waldemar, and W. W. Williams: Pharmacological Studies on the Phosphatids. 4. The Relation of Brain Phosphatids to Tissue Metab- olites, Jour. Pharmacol, and Exp. Therapeut. 2, 253-264. 1905 Koch, Waldemar, and Herbert S. Woods: The Quantitative Estimation of the Lecithins, Jour. Biol. Chem. 1, 203-211. 1910 Kochmann, Martin: Zur Kenntnis des Kalkstoffwechsels unter Berttck- sichtigung des Stoffwechsels der Phosphorsaure und der Magnesia. Vorlaufige Mitteilung, Biochem. Zeit. 27, 85, 86. PHOSPHORUS METABOLISM 643 1911 Kochmann, Martin: Ueber die Abhangigkeit des Kalkstoffwechsels von den organischen Nahrungskomponenten beim erwachsenen Hunde, nebst Bemerkungen iiber den Stoffumsatz der Phosphorsaure und der Magnesia. I. Mitteilung, Ibid. 31, 361-376. 1912 Kochmann, Martin: Zur Wirkung des Phosphors auf den Kalkstoffwech- sel des Hundes, Ibid 39, 81-87. 1911 Kochmann, Martin, and Ernst Petzsch: Ueber die Abhangigkeit des Kalk- stoffwechsels von den organischen Nahrungskomponenten beim erwachsenen Hunde nebst Bemerkungen iiber den Stoffwechsel der Phosphorsaure und der Magnesia. II. & III. Mitteilungen, Ibid 32, 10-26; 27-42. 1904 Kbhler, A., F. Honcamp, M. Just, J. Volhard, M. Popp, and 0. Zahn: Ueber die Assimilation des Kalkes und der Phosphorsaure aus ver- schiedenen Kalkphosphaten durch wachsende Tiere, Verhandl. d. 76. Versamml. d. Gesell. deut. Naturforsch. u. Aerzte, 1904, II, pt. 1, 152- 154; also Landwirtsch. Versuch. Stat. 61, 458-479. 1907 Kohler, A., F. Honcamp, and P. Eisenkolbe: Weitere Untersuchungen iiber die Assimilation der Phosphorsaure und des Kalkes aus Kalkphos- phaten durch wachsenden Tiere, Landwirtsch. Versuch. Stat. 65, 349- 380. 1903 Konig, J.: Chemie der menschlichen Nahrungs- und Genussmittel. 4th Edition. Vol. I (1903); Chemische Zusammensetzung der mensch- lichen Nahrungs- und Genussmittel. 1535 pp. (By A. Bomer). Vol. II (1904) : Die menschlichen Nahrungs- und Genussmittel, ihre Herstellung, Zusammensetzung und Beschaffenheit, nebst einem Abriss iiber die Ernahrungslehre. 1557 pp. Vol. Ill, pt. 1 (1910); Unter- suchung von Nahrungs-, Genussmitteln und Gebrauchsgegenstanden. 1. Teil. Allgemeine Untersuchungsverfahren. 772 pp. 2. Teil. (Not out in 1913): Die Untersuchung und Beurteilung der einzelnen Nahrungs-, Genussmittel und Gebrauchsgegenstande. 1913a Konig, J., and J. Grossfeld: Das Fischsperma als Nahrungsmittel fur den Menschen, Biochem. Zeit. 54, 333-350. 1913b Konig, J., and J. Grossfeld: Der Fischrogen als Nahrungsmittel fur den Menschen, Ibid. 54, 351-394. 1911 Koeppe, Hans: Studien zum Mineralstoffwechsel, Jahrb. Kinderheilk. 73, 9-49. 1904 Kovesi, Geza, and Wilhelm Roth-Schulz: Pathologie und Therapie der' Niereninsuffizienz bei Nephritiden auf Grund eigener Untersuchungen, Leipzig. 284 pp. 1888 Kolpakcha, J. M.: (On the Source of N in the Urine), Phiziologicheskii Sbornik, Kharkov 1888, Vol 1, p. 53; through U. S. Dept. Agr., Office Exp. Sta., Bui. 45, revised edition 1898, pp. 320-325. 1910 Kondo, Kura: Ueber die Ausscheidung von organisch gebundenem, Phos- phor im Ham, Biochem. Zeit. 28, 200-207. 1902 von Korczyriski, L. R.: Zur Kenntnis des Stoffwechsels bei Osteomalacic, Wien. med. Presse 43, 1073-1078; 1131-1133; 1177-1180; 1228-1232. 1900 Kornauth, Karl: Fiitterungsversuche mit einem Hunde bei Verab- reichung verschiedener stickstoffhaltiger Materialien, Zeit. f. d. Land- wirtsch. Versuchswesen in Oesterreich 3, 1-25; 133-162. 1901 Kornauth, Karl: Ueber Fersan, ein neues eisen- und phosphorhaltiges Nahrpraparat, Zeit. diatet. u. physikal. Ther. 4, 480-486. 644 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1900 Kornauth, Karl, and O. von Czadek: Ueber ein neues Nahrpraparat "Fersan," Zeit. f. d. Landwirthsch. Versuchswesen in Oesterreich 3, 556-573. 1892 Kornblum, Herwarth: Ueber die Ausscheidung des Stickstoffs bei Nieren- krankheiten des Menschen im Verhaltniss zur Aufnahme desselben, Arch. path. Anat. u. Physiol. 127, 409-445. 1904 Kornf eld, F. : Ueber Protylin und seine therapeutische Verwertung, Wien. med. Presse 45, 2297-2304. 1892 Korsakov, N. : Sur la reproduction artificielle du rachitisme chez quelques animaux, Internat. zool. kongress, Moskau 1892, 261-267. 1899 Kort, A.: (Ueber den Einfluss der Trachtigkeit der Kuh auf den Gehalt der Milch an Mineralstoffen, insbesondere an Phosphorsaure und Kalk), L'Ingenieur agricole de Gembloux 1, 453-475; through Jahresb. ii. d. Fortschr. d. Thierchem. 30 (1900), 256, 257. 1879 Kossel, Albrecht: Ueber das Nuclei'n der Hefe, Zeit. physiol. Chem. 3, 284-291. 1880 Kossel, Albrecht: Ueber das Nuclein der Hefe. II. Ibid. 4, 290-295. 1881 Kossel, Albrecht: Untersuchungen iiber die Nucleine und ihre Spaltungs- producte, Strasbourg. 19 pp. 1882 Kossel, Albrecht: Zur Chemie des Zellkerns, Zeit. physiol. Chem. 7, 7-22. 1885 Kossel, Albrecht: Ueber das Nuclei'n im Dotter des Hiihnereies, Arch. Anat. u. physiol. 346, 347. 1886 Kossel, Albrecht: Weitere Beitrage zur Chemie des Zellkerns, Zeit. physiol. Chem. 10, 248-264. 1891a Kossel, Albrecht,: Ueber die chemische Zusammensetzung der Zelle, Arch. Anat. u. Physiol., physiol. Abt., 181-186. 1891b Kossel, Albrecht: Ueber einige Bestandtheile des Nervenmarks, Ibid., 359-366. 1893 "Kossel, Albrecht: Ueber die Nucleinsaure, Ibid. 157-164. 1894 Kossel, Albrecht : Weitere Beitrage zur Kenntniss der Nucleinsaure, Ibid. 194-200. 1895 Kossel, Albrecht: Ueber die Aufnahme des Phosphors in den tierischen Organismus, Monatsschr. Geburtsh. u. Gynakol. 1, 175-178. 1896 Kossel, Albrecht: Ueber die Bildung von Thymin aus Fischsperma, Zeit. physiol. Chem. 22, 188-190. 1900 Kossel, Albrecht: Bemerkungen zu der Erwiderung des Herrn Bang, Ibid. 31, 410. 1911 Kossel, Albrecht: Chemische Beschaffenheit des Zellkerns, Munch, med. Wochenschr. 58, 65-120. 1893 Kossel, A., and Fr. Freytag: Ueber einige Bestandtheile des Nerven- marks und ihre Verbreitung in den Geweben des Thierkorpers, Zeit. physiol. Chem. 17, 431-456. 1893 Kossel, A., and Albert Neumann: Ueber das Thymin, ein Spaltungs- product der Nucleinsaure, Ber. deut. chem. Gesell. 26, III, 2753-2756. 1894 Kossel, A., and Albert Neumann: Darstellung und Spaltungsprodukte der Nucleinsaure. (Adenylsaure), Ibid 27, II, 2215-2222. 1896 Kossel, A., and Albert Neumann: Ueber Nucleinsaure und Thyminsaure, Zeit. physiol. Chem. 22, 74-81. 1894 Kossel, H.: Ueber die Einwirkung der Nucleinsaure auf Bakterien, Arch. Anat. u. Physiol., physiol. Abt. 200-203. PHOSPHORUS METABOLISM 645 1903 Kostytschew, S.: Ueber Thymonucleinsaure, Zeit. physiol. Chem. 39, 545-560. 1912 Kovaliova, M. M.: Influence des preparations phosphorees sur les processus d'oxydation evoluant dans l'organisme animal, Archives des sci. biol. 17, 279-307. 1910 Kowalevsky, Katharina: Ueber die Zusammensetzung der Nucleinsaure aus Hefe, Zeit. physiol. Chem. 69, 240-264. 1903-4 von Koziczkowsky, Eugen : Beitrage zur Kenntniss des salzstoffwechsels mit besonderer Beriicksichtigung der chronischen Nephritiden, Zeit. klin. Med. 51, 287-330. 1857 Krabbe, H. : Om Phosphorsyremaengden i Urinen og om de Phosphorsure Jordarters Udfaeldning deraf ved kogning (Ueber die Menge der Phosphorsaure im Harn und iiber die Ausscheidung der Erdphosphate beim Kochen des Harns), Kjobenhavn 1857; through Arch. path. Anat. u. Physiol. 11 (1857), 478-480. 1907 Kramer, Reinhard: Ueber die Wirkung des phosphorsauren und des kohlensauren Kalkes als Beigabe bei Verabreichung von Riibenblattem an Milchkiihe, Inaug. Diss., Leipzig. 69 pp. 1900 Krchivetz, M. J. W.: De l'elimination d'azote et d'acide phosphorique au cours de l'inanition complete soit simple, soit accompagnee d'excitation douloureuse, Archives des sci. biol. 8, 37-56. 1893 Krehl, L.: Ueber die fettige Degeneration des Herzens, Deut. Arch. klin. Med. 51, 416-450; through Jahresb. u. d. Fortschr. d. Thierchem. 23 (1893), 371-373. 1895 Kriiger, Friedrich: Ueber den Schwefel- und Phosphorgehalt der Leber- und Milzzellen in verschiedenen Lebensaltern, Zeit. Biol. 31, 400-412. 1896 Kriiger, Th. Richard: Ueber die Abspaltung von Kohlensaure aus Phos- phorfleischsaure durch Hydrolyse, Zeit. physiol. Chem. 22, 95-102. 1899 Kriiger, Th. Richard: Zur Kenntniss der Nucleone, Ibid. 28, 530-534. 1895 Kiihnau, W.: Experimentelle und klinische Untersuchungen iiber das Verhaltniss der Harnsaureausscheidung zu der Leukocytose, Zeit. klin. Med. 28, 534-566. 1896-7 Kiihnau, W.: Ueber das Verhalten des Stoffwechsels und der "weissen Blutelemente bei Blutdissolution, Deut. Arch. klin. Med. 58, 339-367. 1895 Kiihns, C: Untersuchungen iiber die chemische Zusammensetzung der harten Zahnsubstanzen des Menschen in verschiedenen Altersstufen, Deut. Monatsschr. Zahnheilk. 13, 361-377; 450-465. 1908 Kiinzel, Werner, and Alfred Schittenhelm : Zur Frage des Nucleinstoff- wechsels beim Menschen. Vorl. Mitt., Zentralbl. ges. Physiol, u. Pathc des Stoffwechsels N. F. 3, 721-724. 1909 Kiinzel, Werner, and Alfred Schittenhelm: Gegenseitige Beeinflussung der Fermente des Nucleinstoffwechsels, Zeit. exp. Phys. u. Ther. 5, 393-400. 1907 Kiittner, S.: Ueber den Einfluss des Lecithins auf die Wirkung der Verdauungsfermente, Zeit. physiol. Chem. 50, 472-496. 1909 Kiittner, S.: Peptische Verdauung des Kaseins vom Standpunkte der Aciditat seiner Spaltungsprodukte, Arch. ges. Physiol. 129, 557-602. 1910 Kutanin, Michael: Beitrage zur Kenntnis der chemischen Zusammenset- zung des Gehirns bei verschiedenen Tieren, Inaug. Diss., Berlin, 1910; through Jahresb. u. d. Fortschr. d. Thierchem. 40 (1910), 445. 646 OHIO EXPEEIMENT STATION: TECHNICAL BUL. 5 1901 Kutscher, Fr.: Das proteolytische Enzym der Thymus. I. Mittheilung,, Zeit. physiol. Chem. 34, 114-118. 1903 Kutscher, Fr., and Lohmann: Die Endprodukte der Pankreas- und Hefe- selbstverdauung, Ibid. 39, 159-164; 313-317. 1906 Kutscher, Fr., and Lohmann: Der Nachweis toxischer Basen im Harn, Ibid. 48, 1-8. 1903 Labbe, Henri: La lecithine, Revue de therapeut. 70, 721-732. 1904 Labbe, Henri: Les medications reconstituentes. La medication phos- phoree, Paris. 96 pp. 1890 Laehr, H. : Versuche iiber den Einfluss des Schlaf es auf den Stoffwechsel, Allgem. Zeit. Psychiat. 46, 286-317. 1894 Lafevre: Nombreux cas de cachexie ossifrage observes chez les vaches laitieres par suite de le grande secheresse, Annales de med. veterin. 43, 504-510. 1905 Laguesse: Medication phosphoree. Essais experimentaux et chimiques d'une albumine phosphoree synthetique, These de Paris. 88 pp. 1884 Lailler, A.: Sur l'elimination de l'acide phosphorique par l'urine dans l'alienation mentale et l'epilepsie, Compt. rend. Acad, des sci. 99, 572, 573. 1901 Lancereaux and Paulesco : Note sur l'emploi therapeutique de la lecithine, Bui. Acad, de med. de Paris 45, 685-688. 1894 Landauer, Armin-tol: Adatok A Viz Szerepehez A Szervezetben. (Beitrage zur Rolle des Wassers im Organismus), Mathematikai es termeszettudomanyi ertesito, Budapest, 1894, 12, 197; also Ungar. Arch. Med. 3, 136-188; through Jahresb. ii. d. Fortschr. d. Thierchem. 24 (1894), 532-535. 1912 Landsberg, E.: Untersuchungen iiber den Stoffwechsel von Stickstoff, Phosphor und Schwefel bei Schwangeren, Z. Geburtsh. 71, 163; through Chem. Abs. 7 (1913), 2413. 1906 Landsberg, G. : Das Lecithin, seine Rolle in Organismus und seine thera- peutische Verwendung, Zentralbl. ges. Physiol, u. Path, des Stoff- wechsels 7 (N. F. 1), 193-212. 1892 Landsteiner, Karl: Ueber den Einfluss der Nahrung auf die Zusammen- setzung der Blutasche, Zeit. physiol. Chem. 16, 13-19. 1906 Lane, A. H.: Bone Diseases Amongst Horses in South Africa, Veterin. Jour. 62 (n. s. 13), 232-245. 1897 de Lange, C. C: Vergelijkende asch-analyses, Diss., Amsterdam 1897; through Jahresb. ii. d. Fortschr. d. Thierchem. 27 (1897), 260-265. 1900 de Lange, Cornelia: Die Zusammensetzung der Asche des Neugeborenen und der Muttermilch, Zeit. Biol. 40 (N. F. 22), 526-528. 1903 de Lange, Cornelia: Zur Casuistik der Phosphaturie im Kindesalter, Jahrb. Kinderheilk. 57, 93-95. 1877 Langendorff, O., and J. Mommsen: Beitrage zur Kenntniss der Osteo- malacic, Arch. path. Anat. u. Physiol. 69, 452-487. 1875 Langgaard, Alexander: Vergleichende Untersuchungen iiber Frauen-, Kuh- und Stutenmilch, Ibid 65, 1-9. 1910 Langheld, K.: Ueber Metaphosphorsaure-athylester und dessen Anwend- ung in der organischen Chemie, Ber. deut. chem. Gesell. 43, II, 1857- 1860. PHOSPHORUS METABOLISM 647 • 1911 Landheld, K.: Ueber Ester und Amide der Phosphorsauren. II. Ueber Versuche zur Darstellung den Lecithinen verwandter Korper, Ibid. . 44, II, 2076-2087. 1912 Langheld, K.: Ueber Ester und Amide der Phosphorsaure. III. Ueber dioxyaceton- und Fructose-phosphorsaure, Ibid. 45, 1125-1127. 1903 Langstein, Leo: Bemerkungen uber das Ovomukoid, Beitrage z. chem. physiol. u. Path. 3, 510-513. 1906 Langstein, Leo: Zur Klinik der Phosphaturie, Med. Klinik, Berlin, 2, 406, 407. 1910 Langstein, L., and F. Edelstein: Ueber die Einheitlichkeit des Frauen- milchkaseins, Jahrb. Kinderheilk. 72 (Erganzungsheft), 1-15. 1910 Langstein, Leo, and Albert Memann: Ein Beitrag zur Kenntnis der Stoffwechselvorgange in den ersten vierzehn Lebenstagen normaler und friihgeborener Sauglinge, Ibid. 71, 604-611. 1913 Lanzoni, O.: Modificazioni indotte da alcuni medicamenti sulla percent- uale degli elementi costitutivi del latte di vacca, Clinica veterinaria 36, 11-23, 58-69. 1910 Lapidus, Herman: Diastase und Handelslecithin, Biochem. Zeit. 30, 39-55. 1902 Larned, E. R.: Concerning a New Series of Synthetic Salts; The Nucleids of Iron, Copper, Mercury and Silver, Therapeutic Gazette, Sept. 15, 1902; through Jour. Amer. Med. Assoc. 39, II, (1902), 938. 1905 Laumonier, J.: Remineralisation phosphoree, Bui. gener. de therapeut. 150, 779-789. 1910 Laurer, G. : Ueben die Futterverhaltnisse einen Einfluss auf die Knochen- starke aus? Deut. landw. Tierzucht 14, 437-439; through Exp. Sta. Record 24 (1911), 175. 1909 Lauxen, Nikolaus: Experimentelle Untersuchungen liber die Einwirkung des Phosphors auf Knochen- und Blutbildung bei wachsenden Hunden, Inaug. Diss., Bonn. 40 pp. 1900 Laves, E.: Ueber das Eiweissnahrmittel "Roborat" und sein Verhalten im Organismus, vergleichen mit ahnlichen Praparaten, Munch, med. Wochenschr. 47, 1339-1345. 1903a Laves, E.: Ueber Farbstoff, Lecithin und Fett des Eidotters (Vorlaufige Mitteilung), Pharmazeut. Zeitung 48, 814-816. 1903b Laves, E.: Ueber Lezithin und seine Anwendungsform, Verhandl. d. 75. Versamml. d. Gesell. deut. Naturforsch. u. Aerzte, 1903, II, 86-88. 1883 Lawes, J. B., and J. H. Gilbert: Composition of the Ash of the Entire Animals, and of Certain Separated Parts, Phil. Trans, of the Royal Soc. 174, 865-890. 1908-9 Laxa, Otakar : La production et les qualites du lait de la brebis valaque, Revue gener. du lait 7, 289-300; 313-328; 337-347; 361-373; 391-399. 1909 Leach, Albert E.: Food Inspection and Analysis, New York. 1900 Lebbin, Georg: Ueber den Verteilung der Nahrstoffe in den Huhnereiern, Zeit. offentliche Chem. 6, 148,149. 1901 Lebbin, Georg: Der Nahrwert der Huhnereier, Therapeut. Monatsh. 15, 552, 553. (1910a) von Lebedew, A.: (Investigations on the Non-cellular Fermentation Process with the Help of the Ultrafilter), Biochem. Zeit. 20, 114-125; through Chem. Abs. 4 (1910), 53, 54. 648 OHIO EXPEEIMENT STATION: TECHNICAL BUL. 5 1910b von Lebedew* A.: Ueber Hexosephosphorsaureester. I. Ibid. 28, 213- 229. 1911a von Lebedew, A.: Ueber Hexosephosphorsaureester. II. Ibid. 36, 248- 260; through Zeit. Unters. d. Nahrungs- u. Genussmittel 24 (1912), 283. (1911b) von Lebedew, A.: (The Mechanism of Alcoholic Fermentation), Compt. rend. Acad, des sci. 153, 136-138; through Chem. Abs. 5 (1911), 3298. (1911c) von Lebedew, A.: Ueber den Mechanismus der alkoholischen Garung, Ber. deut. chem. Gesell. 44, 2932-2942; Annal. Inst. Pasteur 25, 847- 851; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 781. 1912 von Lebedew, A., and N. Griaznoff : Ueber den Mechanismus der alkohol- ischen Garung. II. Ibid. 45, 3256-3272. 1906 LeClerc, J. A., and F. C. Cook: Metabolism Experiments with Organic and Inorganic Phosphorus, Jour. Biol. Chem. 2, 203-216. 1909 Le Clerc, J. A., and Robert Wahl: Chemical Studies of American Barleys and Malts, U. S. Dept. Agr., Bur. Chem. Bui. 124. 75 pp. 1911 Lederer, Richard, and Karl Stolte: Die Zusammensetzung des Menschen- und des Hund-herzens, Biochem. Zeit. 35, 108-112. 1842 Lehmann, C. G.: Untersuchimgen iiber den menschlichen Harn, Jour. f. prakt. Chem.' 27, 257-274. 1893 Lehmann, Curt, Friedrich Miiller, Immanuel Munk, H. Senator, and N. Zuntz: Untersuchungen an zwei hungernden Menschen, Arch. path. Anat. u. Physiol. 131 Suppl. 1-228. 1882 Lehmann, Ernst: Zur Wirkung des kohlensauren Kalks und der kohlen- sauren Magnesia, Berlin, klin. Wochenschr. 19, 320-324. 1894 Lehmann, Ernst: Zur Wirkung des kohlensauren Kalkes, Ibid. 31, 538- 540. 1859 Lehmann, Jul.: Ueber die mineralischen Nahrstoffe, insbesondere iiber die Erdphosphate als Nahrstoff des jungen thierischen Organismus, Landwirtsch. Versuch. Stat. 1, 68-86; also Annal. der Chem. u. Pharm. 108 (1858), 357-378. 1873 Lehmann, Jul.: Ueber den Werth des Fleischfuttermehls, Zeit. d. land- wirtsch. Vereins in Bayern, Dec. 1873; through Jahresb. ii. d. Fortschr. d. Agriculturchem. 17 (1876), 183, 184. 1877 Lehmann, Jul.: Den Einfluss der Nahrung auf die Knochenbildung, Amtlicher Ber. d. 50. Versamml. deut. Naturforsch. u. Aerzte in Miinchen, 215. 1878 Lehmus, Emilie: Ueber den relativen Werth der Phosphorsaure im Urin beim Kinde, Central-Zeitung Kinderheilk. 1, 291, 292. 1903 Leichnam, Charles: Urologie comparee du rachitisme et de la scoliose des adolescents. Theorie rachitique de la scoliose. Traitement general de la scoliose, These de Paris. 49 pp. 1899 Leipziger, Richard: Ueber Stoffwechselversuche mit Edestin, Arch. ges. Physiol. 78, 402-422; also Inaug. Diss., Breslau, 1899. 1901 Leo, Hans: Zur Phosphorbehandlung der Rachitis, Verhandl. d. Versamml. d. Gesell. f. Kinderheilk. 18, 145-148. 1902 Leo, Hans: Ueber Alkalinurie, Deut. Arch. klin. Med. 73, 604-615. 1909 Leonard, V. N., and Walter Jones: On Preformed Hypoxanthin, Jour. Biol. Chem. 6, 453-460. PHOSPHORUS METABOLISM 649 1901 Lepine, R.: Sur la relation existant entre l'etat graisseux du foie (avec augmentation de la proportion de la lecithine hepatique) et le phos- phore incompletement oxyde de l'urine, Compt. rend. Soc. de biol. 53, 978,979. 1882 Lepine, R., and Eymonnet: Sur la determination quantitative de l'acide phosphoglycerique dans l'urine, a l'etat physiologique et dans diverses conditions anomales, notamment dans le cas de foie gras, Ibid. 34, 622- 625. 1884 Lepine, R.: Eymonnet, and Aubert: Sur la proportion de phosphore incompletement oxyde contenue dans l'urine, specialement dans quelques etats nerveux, Ibid. 1884, 499, 500; also Compt. rend. Acad. des sci. 98 (1884), 238-241. 1879 Lepine, R.: and Jacquin: Sur l'excretion de l'acide phosphorique par l'urine dans ses rapports avec celle de l'azote, Revue mensuelle de med. et de chir. 3, 449-456; 716-728; 959-968. 1902 Lepine, R.: and Maltet: Sur l'elimination de l'acide phosphorique dans la glycosurie experimental e, Compt. rend. Soc. de biol. 54, 921, 922. 1905 Lepski, Chaim: Phosphortherapie der Rachitis, Diss., Berlin. 34 pp. 1903 Lesem, W. W., and William J. Gies: Notes on the "Protagon" of the Brain, Amer. Jour. Physiol. 8, 183-196. 1913 Leubuscher, Paul : Therapeutische Versuche mit Phosphor bei Epilepti- kern, Deut. med. Wochenschr. 39, 494-496. 1899 Levene, P. A.: On the Nucleoproteid of the Brain, Archives of Neurol. and Psychopathol. 2, 3-14. 1910a Levene, P. A.: Ueber das Ichthulin des Kabeljau, Zeit. physiol. Chem. 32, 281-284. 1901b Levene, P. A.: Darstellung und Analyse einiger Nukleinsauren, Ibid. 32, 541-551. 1903a Levene, P. A.: Darstellung und Analyse einiger Nukleinsauren. (Zweite Mittheilung), Ibid. 37, 402-406. 1903b Levene, P. A.: Darstellung und Analyse einiger Nucleinsauren. Dritte Mitteilung, Ibid. 38, 80-83. 1903c Levene, P. A.: On the Chemistry of the Chromatin Substance of the Nerve-Cell, Jour, of Med. Research 10 (N. S. 5), 204-211. 1903d Levene, P. A.: Darstellung und Analyse einiger Nucleinsauren. (Vierte Mitteilung.) Zeit. physiol. Chem. 39, 4-8. 1903e Levene, P. A.: Darstellung und Analyse einiger Nucleinsauren. (Funfte Mitteilung.) Ibid. 39, 133-135. 1903f Levene, P. A.: Darstellung und Analyse einiger Nucleinsauren. (Sechste Mitteilung.) Ibid. 39, 479-483. 1904 Levene, P. A.: Darstellung und Analyse einiger Nucleinsauren. (Sie- bente Mitteilung.) Ibid. 43, 199-201. 1905 Levene, P. A.: Darstellung und Analyse einiger Nucleinsauren. VIII. Mitteilung. Ueber die Milznucleinsaure, Ibid 45, 370-380. 1909a Levene, P. A.: Ueber die gepaarten Phosphorsauren in Pflanzensamen, Biochem. Zeit. 16, 399-405. 1909b Levene, P. A.: Ueber die Hefenucleihsaure, Ibid. 17, 120-131. 1910 Levene, P. A.: On the Biochemistry of Nucleic Acids, Jour. Amer. Chem. Soc. 32, 231-240. 1901 Levene, P. A., and C. Alsberg: Zur Chemie der Paranucleinsaure, Zeit. physiol. Chem. 31, 543-555. 650 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1908 Levene, P. A., and W. A. Jacobs: Ueber die Inosinsaure. I. Mitt. Ber. deut. chem. Gesell. 41, II, 2703-2707. 1909a Levene, P. A., and W. A. Jacobs : Ueber Inosinsaure. II. Mitt. Ibid 42, I, 335-338. 1909b Levene, P. A.: Ueber Inosinsaure. III. Mitt. Ibid. 42, I, 1198-1203. 1909c Levene, P. A., and W. A. Jacobs: Ueber die Pentose in den Nucleinsauren, Ibid. 42, II, 2102-2106. 1909d Levene, P. A., and W. A. Jacobs: Ueber die Pentose in den Nuclein- sauren. II. Mitt. Ibid. 42, III, 3247-3251. 1909e Levene, P. A., and W. A. Jacobs: Ueber Guanylsaure. (I. Mitteilung.) Ibid. 42, II, 2469-2473. 1909f Levene, P. A., and W. A. Jacobs: Ueber die Hefe-Nucleinsaure. I. Ibid. 42, II, 2474-2478. 1909g Levene, P. A., and W. A. Jacobs: Ueber die Hefe-Nucleinsaure. II. Ibid. 42, II, 2703-2706. 1910 Levene, P. A. and W. A. Jacobs: Ueber die Hefe- Nucleinsaure. III. Ibid. 43, III, 3150-3163. 1911a Levene, P. A., and W. A. Jacobs: Ueber die Inosinsaure. IV. Ibid. 44, I, 746-753. 1911b Levene, P. A., and W. A. Jacobs: Ueber die Hefe-Nucleinsaure. IV. Ibid. 44, 1027-1032. 1912a Levene, P. A., and W. A. Jacobs: Guaninehexoside Obtained by Hydroly- sis of Thymus Nucleic Acid, Jour. Biol. Chem. 12, 377-379. 1912b Levene, P. A., and W. A. Jacobs: On the Structure of Thymus Nucleic Acid, Ibid. 12, 411-420. 1912c Levene, P. A., and W. A. Jacobs : On Guanylic Acid. Second paper. Ibid. 12, 421-426. 1910 Levene, P. A., and F. B. LaForge: Ueber die Tritico-Nucleinsaure, Ber. deut. chem. Gesell. 43, III, 3164-3167. 1912 Levene, P. A., and W. A. Jacobs: Ueber die Hefe-Nucleinsaure. V. Die Struktur der Pyrimidin-Nucleoside, Ibid. 45, I, 608-620. 1913 Levene, P. A., and W. A. Jacobs: On Nucleases. Third paper, Jour. Biol. Chem. 13, 507-509. 1906a Levene, P. A., and John A. Mandel: Ueber die Kohlehydratgruppe des Milznucleoproteids, I. Mitt. Zeit. physiol. Chem. 47, 151-153. 1906b Levene, P. A., and John A, Mandel: Darstellung und Analyse einiger Nucleinsauren. XI. Ueber die Nuclei'nkorper des Eies des Schellfisches, Ibid. 49, 262-265. 1906c Levene, P. A., and John A. Mandel: Darstellung und Analyse einiger Nucleinsauren. XII. Mitteilung. Nucleinsaure der Spermatozoen des Maifisches (Alosa) Ibid. 50, 1-9. 1908a Levene, P. A., and John A. Mandel: Ueber die Konstitution der Thymo- nucleinsaure, Ber. deut. chem. Gesell. 41, II, 1905-1909. 1908b Levene, P. A., and John A. Mandel: Zur Chemie der Lebernucleoproteide. I. Ueber die Guanylsaure, Biochem. Zeit. 10, 221-228. 1911a Levene, P. A., and F. Medigreceanu : Nuclein Metabolism in the Dog, Amer. Jour. Physiol. 27, 438-445. 1911b Levene, P. A., and F. Medigreceanu: On Nucleases, Jour. Biol. Chem. 9, 65-83. 1911c Levene, P. A., and F. Medigreceanu: The Action of Gastro-Intestinal Juices on Nucleic Acids, Ibid. 9, 375-387. PHOSPHORUS METABOLISM 651 1911d Levene, P. A., and F. Medigreceanu : On Nucleases. Second paper. Ibid. 9, 389-402. 1904 Levene, P. A., and L. B. Stookey: Notiz iiber das Pankreasnucleoproteid, Zeit. physiol. Chem. 41, 404-406. 1913 Levene, P. A., and C. J. West: The Saturated Fatty Acid of Kephalin, Jour. Biol. Chem. 16, 419-422. 1905 Levy, Fritz: Ueber den therapeutischen Wert des Lecithins und der lecithinhaltigen Nahrpraparate (Lecitogen), Berlin, klin. Wochenschr. 42, 1242-1245. 1894 Levy, Moritz : Chemische Untersuchungen iiber osteomalacische Knochen, Zeit. physiol. Chem. 19, 239-270. 1905 Lewin, Carl: Ueber das Lecithin und Bromlecithin, Med. Klinik (Berlin) 1, 857, 858. 1905-8 Lewite, Maxim : Zur Frage des Zusammenhanges zwischen Wetter, Min- eralstoffgehalt der Futterpflanzen und Knochenbruchigkeit des Rindes, Mitt. d. landwirtsch. Inst, in Leipzig 8, 69-111. 1911 Lewoniewska, S.: Schwankungen in dem Gehalt der Pflanzensamen an einzelnen Phosphorsaureverbindungen in ihrer Abhangigkeit von Vegetationsbedingungen, Anzeiger Akad. Wiss. Krakau 1911, Reihe B, 85-96; through Zeit. Unters. d. Nahrungs- u. Genussmittel 26 (1913), 252, 253. 1894 Leze, R., and E. Hilsont: Essai des laits par la presure, Compt. rend. Acad, des sci. 118, 1069-1071. 1888a Liebermann, Leo: Embryochemische Untersuchungen. 1. Ueber einige weniger bekannte Bestandtheile des Huhnereies, Arch. ges. Physiol. 43, 71-151. 1888b Liebermann, Leo: Ueber das Nuclein der Hefe und kunstliche Darstellung eines Nucleins aus Eiweiss und Metaphosphorsaure, Ber. deut. chem. Gesell. 21, I, 598-600. 1889 Liebermann, Leo: Ueber das Nuclein der Hefe und kunstliche Darstell- ung eines Nucleins aus Eiweiss und Metaphosphorsaure, Centralbl. med. Wissensch. 27, 210-212; 225-227; 447. 1890 Liebermann, Leo: Nachweis der Metaphosphorsaure im Nuclein der Hefe, Arch. ges. Physiol. 47, 155-160. 1891a Liebermann, Leo: Studien iiber die chemischen Processe in der Magen- schleimhaut, Ibid. 50, 25-54. 1891b Liebermann, Leo: Notiz iiber das chemische Verhalten des Nierenparen- chyms, Ibid. 50, 55, 56. 1891c Liebermann, Leo: Ueber den Phosphorsauregehalt des Pferdeharn unter physiologischen und pathologischen Verhaltnissen, Ibid. 50, 57-65. 1893a Liebermann, Leo: Neuere Untersuchungen iiber das Lecithalbumin, Ibid. 54, 573-585. 1893b Liebermann, Leo: Studien iiber die chemischen Vorgange bei der Ham- secretion, Ibid. 54, 585-606. 1893 Liebermann, L., and Belav v. Bitto: Ueber Nucleinsaure, Centralbl. med. Wissensch. 1893, Nos. 28 and 45. 1906 Liebermeister, G.: Ueber das Nukleoproteid des Blutserums, Beitrage z. chem. Physiol, u. Path. 8, 439-444. 1847 Liebig, Justus: Ueber die Bestandtheile der Fliissigkeiten des Fleisches, Annalen der Chem. u. Pharm. 62, 257-369. 652 OHIO EXPERIMENT STATION: TECHNICAL BTJL. 5 1851 Liebig, Justus: Letters 28 and 29, Familiar Letters on Chemistry, Third edition. 1865 Liebreich, Oscar: Ueber die chemische Beschaffenheit der Gehirnsub- stanz, Annalen der Chem. u. Pharm. 134, 29-44. 1894 Lilienfeld, Leon: Zur Chemie der Leucocyten, Zeit. physiol. Chem. 18, 473-486. 1895 Lilienfeld, Leon: Ueber Blutgerinnung, Ibid. 20, 89-165. 1893 Lilienfeld, Leon, and Achille Monti: Ueber die mikrochemische Local- isation des Phosphors in den Geweben, Ibid. 17, 410-424. 1894 von Limbeck, R.: Zur Kenntniss der Osteomalacic, Wien. med. Woch- enschr. 44, 737-739; 793-796; 844-847. 1898 Lindemann, W. : Zur Toxikologie der organischen Phosphorverbindungen, Arch. exp. Path. u. Pharm. 41, 191-217. 1912a Lindet, L.: Sur les elements mineraux contenus dans la caseine du lait, Rep. Eighth Internat. Congr. of Applied Chem. 19, 199-207. 1912b Lindet, L.: Sur les formes que le phosphore et le calcium affectent dans la caseine du lait, Compt. rend. Acad, des sci. 155, 923, 924; also Bui. Soc. chim. 11 (1912), 950-952. 1913a Lindet, L.: Solubilite des albuminoiides du lait dans les elements du serum; retrogradation de leur solubilite sous l'influence du chlorure de calcium, Bui. Soc. chim. [ser. 4] 13, 929-935. 1913b Lindet, L.: Sur les caseines solubles du lait, Ibid. [ser. 4] 13, 1001-1006. 1906 Lipschitz, Arnold: Ueber den Einfluss der Hautpflege des Milchviehs sowie tiber die Einwirkung einiger Mineralstoffbeigaben zum Kraft- futter auf Milchergiebigkeit und Beschaffenheit der Milch, Ber. d. landwirtsch. Inst. Konigsberg in Pr. 7, 1-51; through Biedermann's Centralbl. f. Agriculturchem. 35 (1906), 545-549. 1910a Lipschiitz, Alex.: Untersuchungen iiber den Phosphorhaushalt des wach- senden Hundes, Arch. exp. Path. u. Pharm. 62, 210-243. 1910b Lipschiitz, Alex.: Ueber den Phosphor des Kotes, Ibid. 62, 244-252. 1911a Lipschiitz: Alex.: Zur Physiologie des Phosphorhungers im Wachstum, Arch. ges. Physiol. 143, 91-98. 1911b Lipschiitz, Alex.: Die biologische Bedeutung des Kaseinphosphors fur den wachsenden Organismus, Ibid. 143, 99-108. (1911) Lisbonne, Marc: (Wirkung von Chloriden und von Phosphaten auf die Starkeverzuckerung durch Speichel und Pankreassaft), Compt. rend. Soc. de biol. 70, 207; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 730. 1907 Lochhead, A. C, and W. Cramer: On the Phosphorus Percentage of Various Samples of Protagon, Biochemical Jour. 2, 350-356. 1897 Locke, F. S.: Note on the Influence of "Peptone" on the Clotting of Milk by Rennet, Jour, of Exp. Med. 2, 493-499. 1910 Loeb, Adam: Ueber den Eiweissstoffwechsel des Hundes und iiber die Abscheidung der Galle bei Fiitterung mit Eiweiss und Eiweissabbau- produkten, mit besonderer Beriicksichtigung der zeitlichen Verhaltnisse, Zeit. Biol. 55 (N. F. 37), 167-235. 1911 Loeb, Adam : Ueber den Einfluss der Lecithinverabreichung auf Kalk und Magnesiaausscheidung, Intern. Beitrage Path. u. Ther. der Ernahrungs- storungen 3, 235-237. 1911 Lob, Walther: Beitrage zur Frage der Glykolyse. II. Mitteilung. Die Bedeutung der Phosphate fiir die oxydative Glykolyse, Biochem. Zeit. 32, 43-58. PHOSPHORUS METABOLISM 653 1906 Lobisch, Wilhelm: Ueber Nuclein-Eiweissverbindungen unter besonderer Berticksiehtigung der Nucleinsaure der Milchdriise und ihrer ange- blichen Beziehung zur Kase'inbildung, Beitrage z. chem. Physiol, u. Path. 8, 191-209. 1890 Lonnberg, Ingolf : Beitrage zur Kenntniss der Eiweisskorper der Nieren und der Harnblase, Skand. Arch. Physiol. 3, 1-13. 1904 Loevenhart, A. S.: Ueber die Gerinnung der Milch, Zeit. physiol. Chem. 41, 177-205. 1906-7 Loevenhart, A. S., and C. G. Souder: On the Effect of Bile upon the Hydrolysis of Esters by Pancreatic Juice, Jour. Biol. Chem. 2, 415-425. 1891 Loew, Oscar: Ueber die physiologischen Functionen der Phosphorsaure, Biol. Centralbl. 11, 269-281. 1899 Loew, Oscar: The Physiological Role of Mineral Nutrients, U. S. Dept. Agr., Div. Vegetable Physiol, and Path., Bui. 18. 60 pp. 1910 Loewe, Siegfried: Ueber den Phosphorstoffwechsel bei Psychosen und Neurosen, Zeit. ges. Neurol, u. Psychiat., Originalien 4, 250-259. 1911 Loewe, Siegfried: Ueber den Phosphorstoffwechsel bei Psychosen und Neurosen. 2. Mitteilung, Ibid. 5, 445-456. 1912a Loewe, Siegfried: Zur physikalischen Chemie der Lipoide. I. Bezie- hungen der Lipoide zu den Farbstoffen, Biochem. Zeit. 42, 150-189. 1912b Loewe, Siegfried: Zur physikalischen Chemie der Lipoide. II. Die Bezie- hungen der Lipoide zu anderen organischen Substanzen (Narkoticis, Hypnoticis, u. a.), Ibid. 42, 190-204. 1912c Loewe, Siegfried: Zur physikalischen Chemie der Lipoide. III. Diffusion in Lipoiden, Ibid. 42, 205, 206. 1912d Loewe, Siegfried: Zur physikalischen Chemie der Lipoide. IV. Die Eigenschaften von Lipoidlosungen in organischem Losungsmittel. Ibid. 42; 207-218. 1904 Loewenheim, Jul.: Physiologisch und therapeutisch Erfahrungen mit der organischen Phosphor, insbesondere mit Phytin, Berlin, klin. Woch- enschr. 41, 1220-1223. (1911) Lowenstein, Joseph: Zur Behandlung der progressiven Paralyse mit Nukle'insaureinjektionen, Ibid. 48, 714-716; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 923. 1900 Loewi, Otto: Beitrage zur Kenntniss des Nucleinstoffwechsels. I. Arch. exp. Path. u. Pharm. 44, 1-23. 1900-01 Loewi, Otto: Untersuchungen fiber den Nucleinstoffwechsel. II. Mit- theilung, Ibid. 45, 157-185. 1909 London, E. S.: Zum Chemismus der Verdauung in tierischen Korper. XXXVI. Mitteilung. Ueber das Verhalten der Nucleoproteide im Magendarmkanal, Zeit. physiol. Chem. 62, 451-454. 1910 London, E. S., and Alfred Schittenhelm : Verdauung und Resorption von Nucleinsaure im Magendarmkanal. I. Mitteilung, Ibid. 70, 10-18. 1911 London, E. S., and Alfred Schittenhelm: Verdauung und Resorption von Nucleinsaure im Magendarmkanal. II. Mitteilung, Ibid. 72, 459-462. 1912 London, E. S., Alfred Schittenhelm, and Karl Wiener: Verdauung und Resorption von Nucleinsaure im Magendarmkanal. III. Mitteilung, Ibid. 77, 86-91. 1905 Long, J. H.: On the Specific Rotation of Salts of Casein, Jour. Amer. Chem. Soc. 27, 363-366. 1906a Long, J. H.: Some Investigations on Salts of Casein, Ibid. 28, 372-384. 654 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1906b Long, J. H.: On the Extraction of Fat from Feces and the Occurrence of Lecithin, Ibid. 28, 704-706; also Science N. S. 24 (1906), 244, 245. 1907a Long, J. H.: On Some Phenomena Observed in the Peptic Digestion of Caseins, Jour. Amer. Chem. Soc. 29, 223-230. 1907b Long, J. H. : On the Combining Power of Casein with Certain Acids, Ibid. 29, 1334-1342. 1908 Long, J. H.: Observations on the Stability of Lecithin, Ibid. 30, 881-895. 1908a Long, J. H., and Frank Gephart: On the Behavior of Emulsions of Leci- thin with Metallic Salts and Certain Non-electrolytes, Ibid. 30, 895-902. 1908b Long, J. H., and Frank Gephart: On the Behavior of Lecithin with Bile Salts, and the Occurrence of Lecithin in Bile, Ibid. 30, 1312-1318. 1906 Long, J. H., and W. A. Johnson: The Phosphorus Content of Feces Fat, Ibid. 28, 1499-1503. 1907 Long, J. H., and W. A. Johnson: Further Observations on the Nature of Feces Fat, Ibid. 29, 1214-1220. 1909 Lothrop, Alfred Peirce: The Effects of Bone Ash in the Diet on the Gastro-intestinal Conditions of Dogs, Amer. Jour. Physiol. 24, 297-324. 1870 Lubavin, N. : Ueber die kiinstliche Pepsin- Verdauung des Caseins und die Einwirkung von Wasser auf Eiweisssubstanzen, Hoppe-Seyler's Med. -Chem. Unters. 463-485. 1877 Lubavin, N.: Untersuchungen iiber der chemischen Natur des Nucleins, Ber. deut. chem. Gesell. 10, 2237-2240. 1889 Luciani, Luigi: Fisiologia del digiuno. Studi sull' uomo. (Das Hun- gem.) Florence. 157 pp. German edition 1890. 1899 LUning, Q.: Die anorganische Bestandtheile des Pankreas, Inaug. Diss., Wtirzburg. 26 pp. 1903 Liithje, Hugo: Ueber die Kastration und ihre Folgen. II. Mitt.: Einfluss der Kastration auf den Phosphorsaure- und Kalkstoffwechsel, Arch, exp. Path. u. Pharm. 50, 268-272. 1904 Liithje, H., and CI. Berger: In welcher Form kommt aus der Nahrung retinierter Stickstoff im Organismus zur Verwendung, Deut. Arch. klin. Med. 81, 278-315. 1901 Lumiere, Auguste, Louis Lumiere, and F. Perrin: Sur l'acide glycero- phosphoreux et les glycerophosphorites, Compt. rend. Acad, des sci. 139, 643, 644. 1903 Lusena, Gustavo: Sul contenuto lecitinico del fegato, dei reni e del cuore nella degenerazione adiposa sperimentale, Lo Sperimentale 57, 29-46; through Jahresb. ii. d. Fortschr. d. Thierchem. 33 (1903), 90. 1909 McCollum, E. V.: Nuclein Synthesis in the Animal Body, Amer. Jour. Physiol. 25, 120-141; also Wis. Agr. Exp. Sta. Research Bui. 8 (1910). 1913a McCollum, E. V., and Marguerite Davis: The Influence of the Compo- sition and Amount of the Mineral Content of the Ration on Growth,, Proc. Soc. Biol. Chem., in Jour. Biol. Chem. 14, XL. 1913b McCollum, E. V., and Marguerite Davis: The Necessity of Certain Lipins in the Diet During Growth, Jour. Biol. Chem. 15, 167-175. 1912 McCollum, E. V., and S. G. Halpin: Synthesis of Lecithins in the Hen, Proc. Soc. Biol. Chem., in Jour. Biol. Chem. 11, XIII, XIV. 1912 McCollum, E. V., J. G. Halpin, and A. H. Drescher: Synthesis of Lecithin in the Hen and the Character of the Lecithins Produced, Jour. Biol. Chem. 13, 219-224. PHOSPHORUS METABOLISM 655 1908 McCollum, E. V., and E. B. Hart : On the Occurrence of a Phytin-splitting Enzyme in Animal Tissues, Ibid. 4, 497-500. 1906a McCrudden, Francis H.: The Composition of Bone in Osteomalacia, Amer. Jour. Physiol. 17, 32-34. 1906b McCrudden, Francis H.: The Effects of Castration on the Metabolism in Osteomalacia, Ibid. 17, 211-217. 1907 McCrudden, Francis H.: The Effect of Castration on Metabolism, Proc. Soc. Biol. Chem. 1, 134, 135; also in Jour. Biol. Chem. 4 (1908), XL, XLI. 1910a McCrudden, Francis H.: The Effect of Castration on the Metabolism, Jour. Biol. Chem. 7, 185-197. 1910b McCrudden, Francis H.: Chemical Analysis of Bone from a Case of Human Adolescent Osteomalacia, Ibid. 7, 199-200. 1912 McCrudden, F. H., and Helen L. Fales: Complete Balance Studies of Nitrogen, Sulphur, Phosphorus, Calcium and Magnesium in Intestinal Infantilism, Jour, of Exp. Med. 15, 450-456. 1908a MacLean, Hugh: Versuche fiber den Cholingehalt des Herzmuskel-leci- thins, Zeit. physiol. Chem. 57, 296-303. 1908b MacLean, Hugh: Ueber das Vorkommen eines Monaminodiphosphatids im Eigelb, Ibid. 57, 304. 1909a MacLean, Hugh: On the Nitrogen- Containing Radicle of Lecithin and Other Phosphatides, Biochemical Jour. 4, 38-58; 240-257. 1909b MacLean, Hugh : On the Occurrence of a Mon-amino-diphosphatide Leci- thin-like Body in Egg Yolk, Ibid. 4, 168-174. 1909c MacLean, Hugh: Untersuchungen liber Eigelb-Lecithin, Zeit. physiol. Chem. 59, 223-229. 1909d MacLean, Hugh: Phosphatides in the Light of Modern Research, British Med. Jour. 1909, II, 677, 678. 1912a MacLean, Hugh: The Phosphatides of the Kidney, Biochemical Jour. 6, 333-354. 1912b MacLean, Hugh: On the Purification of Phosphatides, Ibid. 6, 355-361. 1912-13 MacLean, Hugh: On Carnaubon, Proc. of the Physiol. Soc, in Jour, of Physiol. 45, XVIII. 1913 MacLean, Hugh, Die Phosphatide des Herzens und anderer Organe, Bio- chem. Zeit. 57, 132-142. 1899 MacLeod, J. J. R. : Zur Kenntniss des Phosphors im Muskel, Zeit. physiol. Chem. 28, 535-558. 1906 MacLeod, J. J. R., and H. D. Haskins: Some Observations on the Behav- ior of the Endogenous Purin Excretion in Man, Jour. Biol. Chem. 2, 231-242. 1905a Maestro, Leone: Fitina e Protilina. Contribute alio studio del ricambio fosforganico, La Clinica moderna 11, 517-522. 1905b Maestro, Leone: Sull' assimilazione della fitina, Lo Sperimentale 59, 456-458. 1898 Magnani: Le iniezioni endomuscolari di fosfoluteina (lecitina Serono) in terapia oculare, Annali di ottalmologia 27, 569-571; through Stassano and Billon, 1902d. 1898 Magnus-Levy, Adolf: Ueber die Stoffwechsel bei akuter und chroniker Leukamie, Arch. path. Anat. u. Physiol. 152, 107-130. 1909 Magnus-Levy, Adolf: Der Mineralstoffwechsel in der klinischen Patho- logie, Zentralbl. innere Med. 30, I, 503-505. 656 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1869 Mai: Ein kleiner Ffitterungsversuch mit phosphorsaurem Kalk, Jahresb. u. d. Fortschr. d. Agriculturchem. 11-12 (1868-9), 547. 1894 Maier, A.: Ueber seuchenartiges Auftreten der Knochenweiche (Ehachitis) bei Schweinen im vorigen Winter und Friihjahr 1894, Ber- lin, tierarztlicher Wochenschr. 543-546. 1908a Maillard, L. C: Contribution numerique a l'etude de l'excretion urinaire de l'azote et du phosphore. I. Choix des sujets et techniques d'analyse, Jour, de physiol. et de path, gener. 10, 985-1000. 1908b Maillard, L. C: Contribution numerique a l'etude l'excretion urinaire de de l'azote et du phosphore. II. Resultats d'ensemble, Ibid. 10, 1017- 1030. 1909 Maillard, L. C: Contribution numerique a. l'etude de l'excretion urinaire de l'azote et du phosphore. III. Discussion des resultats moyens. IV. Influence de l'exercice musculaire. V. Recherche de l'influence eventuelle du vin, Ibid. 11, 201-215. 1902 Maillon, CI.: Contribution a l'etude clinique et physiologique de la lecithine, These, Lyon. 1902 Mainzer, J.: Experimented Studien iiber die Einwirkung geistiger •Thatigkeit auf den Harnstoffwechsel, Monatsschr. Psychiat. u. Neurol. 11, 81-93. 1903 Mainzer, J.: Stoffwechselstudien iiber den Einfluss gestiger Tatigkeit und protrahierten Wachens, Ibid. 14, 442-449. 1884a Mairet, A.: Recherches sur le role biologique de l'acide phosphorique, Compt. rend. Acad, des sci. 99, 243-246. 1884b Mairet, A.: De l'influence du travail intellectuel sur l'elimination de l'acide phosphorique par les urines, Ibid. 99, 282-285. 1884c Mairet, A. : Recherches sur les modifications dans la nutrition du systeme nerveux produites par la manie, la lypemanie et l'epilepsie, Ibid. 99, 328-331. 1884d Mairet, A.: Recherches sur l'elimination de l'acide phosphorique chez l'homme sain, l'aliene, l'epileptique, et l'hysterique, Compt. rend. Soc. de biol. 1884, 438-442; 461-465. 1876 Makris, C: Studien fiber die Eiweisskorper der Frauen- und Kuhmilch, Inaug. Diss., Strassburg; through Jahresb. fi. d. Fortschr. d. Thierchem. 6, (1876), 113-115. 1902 Malcolm, John: Note on the Percentage Composition of Egg-Yolk, Jour. of Physiol. 27, 356-359. 1904 Malcolm, John: On the Influence of Pituitary Gland Substance on Metabolism, Ibid. 30, 270-280. 1900 Malengreau, Fernand: Deux Nucleoalbumines et deux Histones dans le Thymus, La Cellule, 17, 339-349. 1901 Malengreau, Fernand: Sur les nucleins du thymus. (Seconde communi- cation.) Ibid. 19, 283-309. 1911 Malengreau, F., and Georges Prigent: Ueber die Geschwindigkeit der Hydrolyse der Glycerinphosphorsaure, Zeit. physiol. Chem. 73, 68-84. 1912 Malengreau, F., and Georges Prigent: Ueber Hydrolyse und Konstitution des Lecithins, Ibid. 77, 107-120. 1905 Malerba, P.: Sul ricambio del fosforo nell' encefalo, Giornale intemaz. d. sci. med., Napoli 27, 865-867. 1906 Malerba, P.: Ueber den Stoffwechsel des Phosphors im Gehirn, Zeit. angew. Chem. 19, 917. PHOSPHORUS METABOLISM 657 1892 Malfatti, Hans: Beitrage zur Kenntniss der Nucleine, Zeit. physiol. Chem. 16, 68-86. 1893 Malfatti, Hans: Bemerkung zu meinem Aufsatze: "Beitrage zur Kennt- niss der Nucleine," Ibid. 17, 8, 9. 1874 Mallet, J. W.: Analysis of Buffalo Bones, Chem. News 30, 211. 1876 Maly, M.: Ueber die anderung der Reaction (in der Losung eines Salz- gemisches) durch Diffusion und die dar durch mogliche Erklarung beim Vorgange der Secretion von saurem Harn aus alkalischem Blute, Ber. deut. chem. Gesell. 9, 164-172. 1873 Maly, Rich., and Jul. Donath: Beitrage zur Chemie der Knochen, Jour. prakt. Chem. 115 (N. F. 7), 413-441. 1906 Manasse, Armand : Ueber den Gehalt des Eidotters an Lecithin, Biochem. Zeit. 1, 246-252. 1890 Manasse, Paul: Ueber das Lecithin und Cholesterin der rothen Blut- korperchen, Zeit. physiol. Chem. 14, 437-452. 1895 Manasse, Paul: Ueber zuckerabspaltende, phosphorhaltige Korper in Leber und Nebenniere, Ibid. 20, 478-488. 1904 Mandel, A. R., and G. Lusk: Stoffwechselbeobachtungen an einem Falle von Diabetes mellitus, mit besonderer Beriicksichtigung der Prognose, Deut. Arch. klin. Med. 81, 472-492. 1905 Mandel, John A., and P. A. Levene: Darstellung und Analyse einiger Nucleinsauren. XL Mitt. Ueber die Nucleinsaure der Kuhmilch- driise, Zeit. physiol. Chem. 46, 155-158. 1906a Mandel, John A., and P. A. Levene: Darstellung und Analyse einiger Nucleinsauren. XII. Mitt. Ueber die Nucleinsaure der Niere, Ibid. 47, 140-142. 1906b Mandel, John A., and P. A. Levene: On the Pyrimidin Bases of the Nucleic Acid Obtained from Fish Eggs, Jour. Biol. Chem. 1, 425, 426. 1902 Mandel, John A., and Horst Oertel: A Further Contribution to the Knowledge of the Excretion of Organic Phosphorus in the Urine, N. Y. Univ. Bui. of the Med. Sci. I, 4, 165-170. 1896 Marcuse, Gotthelf : Ueber den Nahrwerth des Caseins, Arch. ges. Physiol. 64, 223-248. 1897 Marcuse, Gotthelf: Ueber das Verhalten der Phosphorausscheidung bei Stoffwechselversuchen mit Casein, Ibid. 67, 373-394. 1905 Marfori, Pio: Sui composti organici del fosforo. (Acido fosfoglicerico e glicerofosfati), Archivio di fisiologia 2, 217-227; through Jahresb. ii. d. Fortschr. d. Thierchem. 35 (1905), 123. 1908a Marfori, Pio: Sui composti organici del fosforo. (Acido fosfoglicerico e lecitina per iniezioni sottocutanee.) Ibid. 5, 207-216; through Jahresb. ii. d. Fortschr. d. Thierchem. 38 (1908), 588. 1908b Marfori, Pio: Sull' assorbimento e sulla assimilazione dei composti "organici" del fosforo, Arch. exp. Path. u. Pharm., Suppl., Schmiede- berg Festschrift, 378-388. 1898 Marischler, J.: (Ueber einen Fall von iibermassiger Schweissabsonder- ung der oberen Korperhalfte mit einer Untersuchung des Stoffwech- sels.) Przeglad lekarski 37, No. 41 & 42; through Jahresb. ii. d. Fortschr. d. Thierchem. 29 (1899), 739, 740. 1907 Mark, H.: Ueber das Jekorin, Diss., Leipzig; through Jahresb. ii. d. Fortschr. d .Thierchem. 37 (1907), 465. 658 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1895 Markotum, K.: Ueber ein phosphorhaltigen Eiweisskorper in der Schild- druse im Zusammenhang mit der Frage iiber die Function derselben, St. Petersburger med. Wochenschr. 1896, Beilage No. 1; through Jahresb. ii. d. Fortschr. d. Thierchem. 26 (1896), 518, 519. 1911a Marre, Francis: Contribution a 1 'etude de l'utilisation des sels mineraux du lait par le nourrisson, Revue d'hygiene et de med. infant. 10, 37-45. (1911b) Marre, Francis: (Das Lecithin der Butter milch), Revue gener. de chim. pure et appl. 14, 56-58; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 187. 1913 Marshall, E. K., Jr.: On the Self-Digestion of the Thymus, Jour. Biol. Chem. 15, 81-84. 1911 Martin, R.: (Zusammensetzung der Schaf milch des Bezirks Roquefort), Annal. des Falsific. 4, 86-88; through Zeit. Unters. d. Nahrungs und Genussmittel 25 (1913), 62, 63. 1902a Martinet, Alfred: Indications et contre-indications de la medication phosphorique, La Presse med. Jan. 8 and 11, 1902; through Jour. Amer. Med. Assoc. 38 (1), 431. 1902b Martinet, Alfred: Les nucleines en therapeutique, Ibid. 10 (2), 1215-1218. 1903 Martinet, Alfred: La medication phosphorique dans la psychasthenic, Ibid. 11 (2), 805-807. 1905 Martinet, Alfred: Pharmacologic de l'acide phosphorique. 1: Action digestive; 2. Action nerveuse et humerale, Revue gener. des sci. 16, 568-575; 611-616. 1910 Masing, Ernst: Ueber das Verhalten der Nucleinsaure bei der Forchung des Seeigeleis, Zeit. physiol. Chem. 67, 161-173. 1911a Masing, Ernst: Chemische Beitrage zur Blutregeneration, Arch. exp. Path. u. Pharm. 66, 71-83. 1911b Masing, Ernst: Ueber eine Beziehung zwischen Kernstoffgehalt und Entwickelung, Ibid. 75, 135-140. 1913 Maslow, M. S.: Ueber die biologische Bedeutung des Phosphors fur den wachsenden Organismus und seine Einwirkung auf die intrazellularen Fermente, St. Petersburger med. Wochenschr. 38, 97, 98. 1904 Masoin, P.: Nouvelles recherches chimiques sur l'epilepsie, Archives internat. de pharmacodynamic, 13, 387. 1902 Massacin, Cornel : Ueber den Einfluss des lecithins auf den Eiweissansatz, Deut. med. Wochenschr. 28, 756, 757. 1913a Masslow, M.: Ueber die biologische Bedeutung des Phosphors fur den wachsenden Organismus. I. Untersuchungen iiber den Einfluss des Phosphors auf die Entwicklung von Tieren und auf den Phosphor- und Stickstoffumsatz, Biochem. Zeit. 55, 45-62. 1913b Masslow, M.: Ueber die biologische Bedeutung des Phosphors fur den wachsenden Organismus. II. Untersuchung der Organe auf ihren Gehalt an Phosphor und intracellularen Fermenten, Ibid. 56, 174-194. 1901 Massol and Gamel: Du role des hypophosphites dans le medication phos- phatee et hypophosphitee, Bui. Acad, de med. de Paris, Oct. 8, 1901; through Jour. Amer. Med. Assoc. 37, II, 1353. 1910 Masuda, Niro: Ein Beitrag zur Analyse des Gehirns, insbesondere iiber den Cholesterin- und Fettsaurengehalt desselben, Biochem. Zeit. 25, 161-164. 1897 Mathews, Albert: Zur Chemie der Spermatozoen, Zeit. physiol. Chem. 23, 399-411. PHOSPHORUS METABOLISM 659 1910 Mathison, G. C: The Output of Organic Phosphorus in Urine, Biochem- ical Jour. 4, 274-279. 1908 Matsushita, T.: Ueber die Aetiologie der Polyneuritis gallinarum und der Beriberi, Ber. ii. d. 14. internat. Kongr. Hygiene u. Demographie 4, 141-144. 1901 Maurel, E.: Influence des variations de ralimentation sur les quantites d'acide phosphorique et de chlorures contenus dans Purine, Compt. rend. Soc. de biol. 53, 429-431. 1904 Maurel, E. : E valuation approximative de la quantite minima d'acide phos- phorique urinaire et de la quantite minima de cette substance neces- saire a. l'organisme dans les conditions de la ration moyenne d'entretien, Ibid. 56, 751-754. 1891 Maxwell, W.: On the Behavior of the Fatty Bodies, and the Role of the Lecithines, during Normal Germination, Amer. Chem. Jour. 13, 16-24. 1893 Maxwell, W.: Movement of the Element Phosphorus in the Mineral, Vegetable, and Animal Kingdoms, and the Biological Function of the Lecithines, Ibid. 15, 185-195. 1913 Mayer, Andre, and Georges Schaeffer: Recherches sur la Constance lipocytique. Teneur des tissus en lipoides phosphores, Compt. rend. Acad, des sci. 157, 156-159. 1907 Mayer, Arthur: Beitrage zur Kenntnis des Mineralstoffwechsels der Phthisiker, Deut. Arch. klin. Med. 90, 408-424. 1907 Mayer, A., and E. F. Terroine: Sur les jecorines naturelles et arti- ncielles, Compt. rend. Soc. de biol. 62, 773-775. 1896 Mayer, Paul: Ueber den Einfluss von Nuclein und Thyreoidinfutterung auf die Harnsaureausscheidung, Deut. med. Wochenschr. 22, 186-188. 1905 Mayer, Paul: Ueber das Verhalten des Lecithins zu den Fermenten, Ber- lin, klin. Wochenschr. 42, 1102, 1103. 1906a Mayer, Paul: Ueber die Spaltung der lipoid Substanzen durch Lipase und liber die optischen Antipoden des naturlichen Lecithins, Biochem. Zeit. 1, 39-52. 1906b Mayer, Paul: Ueber Lecithinzucker und Jekorin sowie iiber das physikal- isch-chemische Verhalten des Zuckers im Blut, Ibid. 1, 81-107. 1913 Mayesima, Junichi: Ueber die Resorption der Hefenucleinsaure nach ausgedehnter Resektion des Dunndarms beim Hunde, Zeit. physiol. Chem. 87, 418-422. 1911 Medigreceanu, Florentin: Neuere Arbeiten iiber die Nuclease, Med. Klinik. Wien., 7, II, 1209, 1210. 1911 Medigreceanu, F., and L. Kristeller: General Metabolism with Special Reference to Mineral Metabolism in a Patient with Acromegaly Com- plicated with Glycosuria, Jour. Biol. Chem. 9, 109-120. 1910 Meier, F.: Ueber den Nucleinstoffwechsels des Schweines, Inaug. Diss., Giessen. 35 pp. Through Exp. Sta. Record 26 (1912), 363. 1882 Meigs, Arthur V.: Milk Analysis, Philadelphia Med. Times, July, 1882, 660-664. 1883 Meigs, Arthur V.: Proof that Human Milk Contains only About One Percent of Casein; with Remarks upon Infant Feeding, Proc. Philadel. Co. Med. Soc. 6, 92-108. 1912 Meigs, Edward B.: Contributions to the General Physiology of Smooth and Striated Muscle, Jour. Exp. Zool. 13, 497-571. 660 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1912 Meigs, Edward B., and L. A. Ryan: The Chemical Analysis of the Ash of Smooth Muscle, Jour. Biol. Chem. 11, 401-414. 1905 Meinertz, J.: Zur Kenntnis des Jecorins, Zeit. physiol. Chem. 46, 376- 382. 1909 Meisen, Jacob: Experimentelle Untersuchungen uber die Wirkung von Nucleinsaure auf Blut und Knochenmark, Inaug. Diss., Bonn. 28 pp. 1911 Meisen, Jacob : Ueber die Wirkung der Nucleinsaure, speziell auf Blut und Knochenmark, Med. Klinik. Wien., 7, II, 1946. 1912 Mellanby, J.: The Coagulation of Milk by Rennin, Jour, of Physiol.; 45, 345-362; through Chem. Abs. 7 (1913), 1726, 1727. 1872 Mendel, E.: Die Phosphorsaure im Urin von Gehirnkranken, Arch. Psychiat. u. Nervenkrankh. 3, 636-672. 1906 Mendel, Lafayette B.: The Formation of Uric Acid, Jour. Amer. Med. Assoc. 46, I, 843-846; 944-947. 1907-8 Mendel, Lafayette B., and Philip H. Mitchell: Chemical Studies on Growth. II. The Enzymes Involved in Purine Metabolism in the Embryo, Amer. Jour. Physiol. 20, 97-116. 1906 Mendel, Lafayette B., and Frank P. Underhill: Experiments on the Physiological Action and Metabolism of Anhydro-oxy-methylene- diphosphoric Acid (Phytin Acid), Ibid. 17, 75-88. 1903 Mendel, Lafayette B., Frank P. Underhill, and Benjamin White: A Physiological Study of Nucleic Acid, Ibid. 8, 377-403. 1909 Menten, Maude L.: The Distribution of Fat, Chlorides, Phosphates, Potassium and Iron in Striated Muscle, Trans. Canadian Inst. 8, III, 403-421. 1911 Merck, E.: The Glycerophosphates, Annual Report 25, 1-30. About 68 references. 1912 Merck, E.: Lecithin, Ibid. 26, 1-71. About 525 titles and references. 1907 Mesernitzy, P.: (Die quantitative Veranderung des Lecithins im Organis- mus wahrend der Entwickelung.) Russky Wratsch, 1907, 302-304; through Jahresb. ii. d. Fortschr. d. Thierchem. 37 (1907), 513. 1912 Messing, Basia: Ueber einige mineralische Bestandteile im normalen und pathologischen Gehirn, Inaug. Diss., Zurich, 1912; through Zentralbl. Biochem. u. 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PHOSPHORUS METABOLISM 661 1913 Michaelis, Leonor: Die allgemeine Bedeutung der Wasserstoffionen- konzentration fur die Biologie, Oppenheimer's Handbuch der Biochem. des Mensch. u. der Tiere, Erganzungsband, 10-62. 1908 Michaelis, Leonor, and Peter Rona: Untersuchungen liber den parenter- alen Eiweissstoffwechsel. I. Arch. ges. Physiol. 121, 163-168. 1896 Michel, Charles: Recherches sur la nutrition normale du nouveau-ne, L'Obstetrique 1, 140-158. 1897 Michel, Charles: Sur le lait de femme et l'utilization de ses materiaux nutritifs dans l'organisme du nouveau-ne sain, Ibid. 2. 518-533. 1898 Michel, Charles: Composition moyenne du lait de femme, L'Union phar- maceut. 39, 385-389. 1899 Michel, Charles: Sur la composition chimique de l'embryon et du foetus humains aux differentes periodes de la grossesse, Compt. rend. Soc. de biol. 51, 422, 423. 1900 Michel, Charles: Sur la composition organique et minerale du foetus et du nouveau-ne, L'Obstetrique 5. 252-260. 1899 Michel, Charles, and M. Perret: Etude des echanges nutritifs azotes et mineraux chez un nourrisson de 2 mois V2, Bui. Soc. d'obstetrique de Paris 2, 98-105. 1906 Michel, Charles, and M. Perret: La ration alimentaire de l'enfant depuis sa naissance jusqu' a l'age de deux ans, Rev. Soc. d'hyg. aliment, et de 1'alimentation rationnelle de l'homme 3, 209-362. 1900 Micko, K.: Vergleichende Untersuchungen iiber die bei Plasmon und Fleischnahrung ausgeschiedenen Kothe, Zeit. Biol. 39, 430-450. 1900 Micko, K., P. Mttller, H. Poda, and W. Prausnitz: Untersuchungen iiber das Verhalten animalischer Nahrungsmittel im menschlichen Organ- ismus, Ibid. 39, 277-278. 1878 Miescher, F.: Die Spermatozoen einiger Wirbeltiere. Ein Beitrag zur Histochemie, Verhandl. d. naturforsch. Gesell. z. Basel 6, 138-208. 1881 Miescher, F.: Ueber das Leben des Rheinlaches im Siisswasser, Arch. Anat. u. Physiol./ anat. Abt., 193-218. 1896 Miescher, F.: Physiologisch-chemische Untersuchungen iiber die Lachs- milch, (Edited by O. Schmiedeberg) Arch. exp. Path. u. Pharm. 37, 100-155. 1897 Miescher, F.: Die histo-chemischen und physiologischen Arbeiten von Friedrich Miescher gesammelt und herausgegeben von seinen Freund- en. Leipzig, 2 Vols. 138 and 543 pp. 1904 Migliaccio, Alessandro: La lecitina in terapia infantile. Osservazioni sull' azione del rimedio in alcune malattie dei bambini, La Pediatria 12, 753-769. 1911 Mihara, Shinji: Beitrage zur Kenntnis der Fermente der Stierhoden, Zeit. physiol. Chem. 75, 443-455. 1898 Milesi, C: Di un corpo fosforato isolato dell' albume dell' novo pres- entante i caratteri chimici d'un mucoide, Bollett. della Soc. med. chir. di Pavia, 1898, No. 3, p. 184; through Jahresb. ii. d. Fortschr. d. Thier- chem. 28 (1898), 38, 39. 1911 Milkowicz, G. S.: (Experimental Increase of Phagocytosis), Russky Wratsch, 1911, No. 22, 943-946; through Chem. Abs. 6 (1912), 252. 1909 Miller, J. R., and Walter Jones: Ueber die Fermente des Nucleinstoff- wechsels bei der Gicht, Zeit. physiol. Chm. 61, 395-404. 662 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1896 Milroy, T. H.: Ueber die Eiweiss-Verbindungen der Nucleinsaure und Thyminsaure und ihre Beziehung zu den Nuclei'nen und Paranucleinen, Zeit. physiol. Chem. 22, 307-326. 1908 Milroy, T. H. : Changes in the Chemical Composition of the Herring Dur- ing the Reproductive Period, Biochemical Jour, 3, 366-390. 1898 Milroy, T. H., and J. Malcolm: The Metabolism of the Nucleins under Physiological and Pathological Conditions. Part I. Jour, of Physiol. 23, 217-239. 1899 Milroy, T. H., and J. Malcolm: The Metabolism of the Nucleins. Part II. Further Observations on Excretion in Leucocythaemia. Intra- cellular Metabolism in the Granular Leucocytes, Ibid. 25, 105-130. 1912 Minami, D.: Ueber den Einfluss des Lecithins und der Lipoide auf die Diastase (Amylase), Biochem. Zeit. 39, 355-380. 1898 Minkowski, O.: Untersuchungen zur Physiologie und Pathologie der Harnsaure bei Saugethieren, Arch. exp. Path. u. Pharm. 41, 375-420. 1878 Miroczkowski : Ueber den Phosphorsauregehalt im Schafs-, Kalbs- und Hunde-Serum, Centralbl. med. Wissensch. 16, 353, 354. 1911 Mitra, Mariano: (Presence of Nuclease and Connectivase in the Gastric Contents of Infants), Folia clinica 3, 274-278; through Chem. Abs. 5 (1911), 3591 and 7 (1913), 640. 1902 Mitulesco, J. : Beitrage zum Studium des Stoffwechsels in der chronischen Tuberkulose, Berlin, klin. Wochenschr. 1027-1029; 1054-1056; 1073- 1075; 1096-1100. 1903a Mitulesco, J.: Die Vortheile der combinierten Behandlung bei der chron- ischen Tuberkulose. Vortrag. Deut. med. Wochenschr. 29, 367-369; 385, 386. 1903b Mitulesco, J.: Beitrage zum Studium des Stoffwechsels in der Tuber- kulose, Zeit. Tuberkulose 4, 515-537. 1909 Mochi, Alberto: L'eliminazione dell' N, P, Ca, Mg, nei conigli digiuni trattati con iniezioni di estratto di ipofisi, Atti r. Accad. d. fisiocrit. Siena 218 (1 of ser. 5), -835-856. 1910 Mochi, Alberto: II ricambio dell' N, P, e Ca nei conigli trattati con iniezioni di estratto di ipofisi, Rivista d. patol. nerv. e ment. 15, 457- 476. 1901 Mochizuki, Junichi: Ueber die Resorption von Eiweisskorpern von der Schleimhaut des Dickdarmes nach Versuchen mit Thymus-klystieren, Arch. Verdauungskrankh. 7, 221-233. 1901 Modica, O., and E. Audenino: (Einfluss des Zentralnervensystems auf den organischen Stoffwechsel), Archivio di psichiat., sci. penali ed antropol. 22, Fasc. 4-5; through Jahresb. ii. d. Fortschr. d. Thierchem. 32 (1902), 672, 673. 1869 Mors and Muck: Beitrage zur Kenntniss der Osteomalazie, Deut. Arch. klin. Med. 5, 485-504. 1903 Mohr, L. : Ueber das Ausscheidungsvermogen der kranken Niere, Zeit. klin. Med. 51, 331-348. 1905 Moll, Leopold: Beitrag zur Ernahrungstherapie der mit Phosphaturie (Calcarurie) einhergehenden Neurosen im Kindesalter, Prager med. Wochenschr. 30, 582-585. 1909 Moll, Leopold: Die klinische Bedeutung der Phosphorausscheidung im Harn beim Brustkind, Jahrb. Kinderheilk. 69 (19 of ser. 3), 129-152; 304-332; 450-478. PHOSPHORUS METABOLISM 663 1912 Molyakov, M. G.: (Administration of Sodium Nucleinate in Scarlet Fever), Russki Wratch .11 (1912), 301-303; through Chem. Abs. 7, (1913), 1921. (1911) Monrad: Kaseinklumpen im Kinderstuhl im Zusammenhang mit Roh- milchernahrung, Monatsschr. f. Kinderheilk. 10, 244-246; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 298. 1910 Monvoisin, A.: (Die Zusammensetzung krankhaft veranderter Milch, insebesondere der Milch tuberkuloser Kuhe), Jour, de Pharm. et de Chim. [7] 2, 23-30; through Zeit. Unters. d. Nahrungs- u. Genussmittel 23 (1912), 25. 1901 Moore, Benjamin, and William H. Parker: On the Functions of the Bile as a Solvent, Proc. Roy. Soc. of London 68, 64-76. 1908 Moore, Benjamin, Herbert E. Roef, Robert E. Knowles: The Effects of Variations in the Inorganic Salts and the Reactivity of the External Medium upon the Nutrition, Growth and Cell Division in Plants and Animals, Biochemical Jour. 3, 279-312. 1895a von Moraczewski, Waclaw: Verdauungsproducte des Caseins und ihr Phosphorgehalt, Zeit. physiol. Chem. 20, 28-51. 1895b von Moraczewski, Waclaw: Ueber den Chlor- und Phosphorgehalt des Blutes bei Krebskranken, Arch. path. Anat. u. Physiol. 139, 385-405. 1896 von Moraczewski, Waclaw: Ueber den Chlor- und Phosphorgehalt des Blutes bei krankhaften Zustanden, Ibid. 146, 424-452. 1897a von Moraczewski, Waclaw: Die Mineralbestandtheile der menschlichen Organe, Zeit. physiol. Chem. 23, 483-496. 1897b von Moraczewski: Stoffwechselversuch bei Diabetes mellitus, CentralbL innere Med. 18, 921-932. 1897c von Moraczewski, Waclaw: Stoffwechseluntersuchungen bei Carcinom und Chlorose, Zeit. klin. Med. 33, 385-431. 1898a von Moraczewski, Waclaw: Stoffwechselversuch bei Diabetes mellitus, Ibid. 34, 59-88. 1898b von Moraczewski, Waclaw: Stoffwechselversuche bei Leukamie und Pseudoleukamie, Arch. path. Anat. u. Physiol. 151, 22-52. 1899 von Moraczewski, Waclaw: Ueber die Ausscheidung der Harnbestand- theile bei Fieberbewegungen, Ibid. 155, 11-43. 1900a von Moraczewski, Waclaw: Stoffwechsel bei Lungenentziindung und Einfiuss der Salze auf denselben, Zeit. klin. Med. 39, 44-92. 1900b von Moraczewski, Waclaw: Stoffwechselversuche bei schweren Anamien, Arch. path. Anat. u. Physiol. 159, 221-252. 1901 von Moraczewski, Waclaw: Stoffwechsel bei Akromegalie unter der Be- handlung mit Sauerstoff, Phosphor, etc., Zeit. klin. Med. 43, 336-360j 611-616. (1902) von Moraczewski, Waclaw: (Ueber den Verlauf des Fiebers beim Men- schen mit ausgeschnittener Milz), Gazeta lekarska 22, 868; through Jahresb. ii. d. Fortschr. d. Thierchem. 32 (1902), 746. 1903-4 von Moraczewski, Waclaw : Ueber Ausscheidung von Oxalsaure, Indican und Aceton bei Diabetes unter dem Einfiuss der Nahrung, Zeit. klin. Med. 51, 475-501. 1905 von Moraczewski, Waclaw: Ein Beitrag zur Kenntnis der Phosphaturie, CentralbL innere Med. 26, 401-414. 1902 Moreigne, H.: Action du jus de raisin sur l'organisme, Archives de med. exper. et d'anat. 14, 343-377. 664 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1909 Morel, L.: Les parathyroides dans l'osteogenese. (Premiere note.) Compt. rend. Soc. de biol. 67, 780-782. 1910 Morel, L.: Les parathyroides dans l'osteogenese. (Deuxieme note.) Ibid. 68, 163-165. 1906 Morgan, A., C. Beger, and G. Fingerling: Weiterer Untersuchungen iiber die Wirkung der einzelnen Nahrstoffe auf die Milchproduction, Land- wirtsch. Versuch. Stat. 64, 93-242. 1901 Morichau-Beauchant, P. E. R,: Etude therapeutique sur la lecithine, These de Paris. 141 pp. 1885 Mosetig-Moorhof : Milchsaure als Zerstorungsmittel pathogener Gewebe, Centralbl. Chirurgie, 193-196. 1853 Mosler, Friedrich: Beitrage zur Kenntniss der Urinabsonderung bei ge- sunden, schwangeren und kranken Personen, insbesondere quantitative Bestimmung der phosphorsauren Verbindungen. Inaug. Diss., Giessen. 1899 Mosse, Prosper and Oulie: Influence de l'ovariotomie double et de l'in- gestion d'ovaires sur quelques elements de la secretion urinaire chez la chienne, Compt. rend. Soc. de biol. 51, 447-449. 1911 Moszkowski, Max: Meine Erfahrungen iiber Prophylaxe der Beriberi in Hollandisch-Neuguinea, Arch. Schiffs- u. Tropenhygiene 15, 653-661. 1899 Mott, F. W., and J. O. Wakelin Barratt: Observations on the Chemistry of Nerve Degeneration, Archives of Neurol., 1899, I, 346-358. 1904 Mott, F. W., Arthur Edmunds, and W. D. Halliburton: Regeneration of Nerves, Proc. Roy. Soc, March 1904. 1901 Mott, F. W., and W. D. Halliburton: The Chemistry of Nerve-degenera- tion, Phil. Trans, of the Roy. Soc. of London 194 B (1901), 437-466; Jour, of Physiol. 26, XXV, XXVI; through Jahresb. u. d. Fortschr. d. Thierchem. 31 (1901), 558. 1902 Mott, F. W., and W. D. Halliburton: Regeneration of Nerves, Ann. Rept. of the British Assoc. 1902. 1902a Mouneyrat, A.: De l'arsenic et du phosphore organiques dans le traite- ment de la tuberculose pulmonaire. (Extrait.) Compt. rend. Acad. des sci. 134, 667, 668. 1902b Mouneyrat, A.: Sur une nouvelle medication arsenio-phosphoree (His- togenol) dans le traitement de la tuberculose pulmonaire, Compt. rend. Soc. de biol. 54 (4 of ser 11), 314, 315. 1905 Moussu, G., and J. A. W. Dollar: Diseases of Cattle, Sheep, Goats and Swine, New York, 1905. 785 pp. 1902 Miiller, Erich: Beitrag zum Kalkstoffwechsel des Sauglings, Verhandl. d. Gesell. Kinderheilk. 19-20 (1902-3), 221-226. 1911 Miiller, Erich: TJeber Ernahrung debiler Kinder mit molkenreduzierter Milch an der Hand von Stoffwechseluntersuchungen, Jahrb. Kinder- heilk. Supplement, 73, 252-262. 1913 Miiller, Erich, and Ernst Schloss: Beitrage zur Kenntnis des Stoffwech- sels besonders der Mineralien im Sauglingsalter. I. Einleitung, Ibid. 77, 635-639. 1884 Miiller, Friedrich: Ueber den normal en Koth des Fleischfressers, Zeit. Biol. 20, 327-377. 1889 Miiller, Friedrich: Stoffwechseluntersuchungen bei Krebskranken, Zeit. klin. Med. 16, 496-549. 1873 Miiller, Jakob Worm: Zur Kenntniss der Nucleine, Arch. ges. Physiol. 8, 190-194. PHOSPHORUS METABOLISM 665 1913 Miiller, Johannes, and H. Reinbach: Ueber Maskierung des Blutfettes und der Blutlipoide sowie uber Verdauungslipamie beim Menschen, Zeit. physiol. Chem. 86, 469-483. 1897 Miiller, Martin : Ueber den Gehalt der menschlichen Muskeln an Nucleon, Zeit. physiol. Chem. 22, 561-566. 1900 Miiller, Paul: Ueber den organischen Phosphor der Frauenmilch- und der Kuhmilchfazes, Zeit. Biol. 39, 451-481. 1908 Miiller, W., and G. von Wendt: Wie futtert der Landwirt zweckmassig Rubenblatter ? Berlin. . 31 pp. 1898 Muggia, Alberto: Sulle iniezioni di lecitina e di tuorlo d'uovo nella cura dell' anemia ed atrepsia infantile, La Pediatria 6, 302-308. 1911 Mulier, Rechla: Ueber Phosphorsaureausscheidung bei Lungentuberku- lose, Inaug. Diss., Zurich. 40 pp. 1908 Munk, Fritz: Ueber "lipoid Degeneration," Arch. path. u. Anat. u. Physiol. 194 (4 of ser. 19), 527-565. 1887 Munk, Immanuel: Ueber die Ausfuhr des Stickstoffs und der Asche- bestandteile durch den Harn, Berlin, klin. Wochenschr. 430-433. 1894 Munk, Immanuel: Beitrage zur Stoffwechsel- und Ernahrungslehre, Arch. ges. Physiol. 58, 309-408. 1895 Munk, Immanuel : Ueber den Einfluss angestrengter Korperarbeit auf die Ausscheidung der Mineralstoffe und der Aetherschwefelsauren, Arch. Anat. u. Physiol., physiol. Abt., 385-387. 1904 Nakayama, M.: Ueber das Erepsin, Zeit. physiol. Chem. 41, 348-362. 1842 Nasse, H.: Ueber die Bestandtheile der Knochen in einigen Krankheiten, Jour. f. prakt. Chem. (Erdmann) 27, 274-280. 1899 Nemser, M. M. H.: Sur la question de savoir comment les nucleines se comportent dans l'inanition, Archives des sci. biol. 7, 221-232. 1908a Nerking, Joseph: Beitrage zur Kenntniss des Knochenmarks, Biochem. Zeit. 10, 167-191. 1908b Nerking, Joseph: Die Verteilung des Lecithins im tierischen Organismus, Ibid. 10, 193-203, 1909 Nerking, Joseph: Narkose und Lezithin, Munch, med. Wochenschr. 56,. II, 1475-1478. 1908 Nerking, J., and E. Haensel: Der Lecithingehalt der Milch, Biochem. Zeit. 13, 348-353. 1899a Nesbitt, Beattie: On the Presence of Cholin and Neurin in the Intes- tinal Canal during its Complete Obstruction, Proc. Amer. Physiol. Soc* in Amer. Jour. Physiol. 2, VIII. 1899b Nesbitt, Beattie: A Research on Autointoxication, Jour, of Exp. Med. 4, 1-18. 1866-71 Nessler, J.: Knochenbruchigkeit beim Rindvieh, Verhandl. d. natur- wissensch. Vereins, Karlsruhe, 3-5, 23, 24. 1873 Nessler, J.: Untersuchungen der Knochen von Knochenbruchigen Rind- vieh, Landwirtsch. Versuch. Stat. 16, 187-192. 1900 Netter, L.: E changes nutritifs dans l'allaitement artificiel, These de Paris. 79 pp. 1912 Neubauer, H., and G. Hillkowitz: Der Kalk- und Phosphor sauregehalt und Nahrwert des im Jahre 1911 geenteten Heues und Strohs, Land- wirtsch. Zeit. Rheinprovinz 13, 111, 112. 1908 Neuberg, Carl : Zur Frage der Konstitution des "Phytins," Biochem. Zeit. 9, 557-560. 666 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1909 Neuberg, Carl: Notiz iiber Phytin, Ibid. 16, 406-410. (1911) Neuberg, Carl: Versuche mit Jodocitin, Ther. d. Gegenwart 52, 359, 360; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 890. 1907 Neuberg, C, and B. Brahn: Ueber die Inosinsaure, Biochem. Zeit. 5, 438-450. 1911 Neuberg, C, and E. Kretschmer: Weiteres iiber kiinstliche Darstellung von Kohlenhydratphosphorsaureestern und Glycerinphosphorsaure, Ibid. 36, 5-14. 1910a Neuberg, C, and H. Pollak: Ueber Kohlenhydratphosphorsaureester. I. Ueber Saccharosephosphorsaure, Ibid. 23, 515-517. 1910b Neuberg, C, and H. Pollak: Ueber Kohlenhydrat-Phosphorsaureester. II. Ueber Saccharoseschwefelsaure und die Phosphorylierung von Eiweiss, Ber. deut. chem. Gesell. 43, II, 2060-2068. 1910c Neuberg, C, and H. Pollak: Ueber Phosphorsaure- und Schwefelsaure- ester von Kohlenhydraten, Biochem. Zeit. 26, 514-528. 1910d Neuberg, C, and H. Pollak: Ueber die Phosphorylierung von Eiweiss und die Bindung des Phosphors in den naturlichen Phosphorproteinen, Ibid. 26, 529-534. 1898 Neumann, Albert: Zur Kenntniss der Nucleinsubstanzen, Arch. Anat. u. Physiol., physiol. Abt., 374-378. 1899 Neumann, Albert : Verf ahren zur Darstellung der Nucleinsauren a und ft und der Nucleothyminsaure, Ibid. 1899, Suppl. 552-555. 1893a Neumann, J.: Ueber die Einwirkung des dem Futter beigegebenen phos- phorsauren Kalkes auf den Aschengehalt der Milch, Milch-Zeitung 22, 701-704. 1893b Neumann, J.: Tierphysiologische Untersuchungen. I. Beitrag zur Kenntniss des Assimilation anorganischen Nahrstoffe im Tierkorper, Jour. f. Landwirtsch. 41, 343-380. 1894 Neumann, J.: Tierphysiologische Untersuchungen. II. Ueber den Einfluss des phosphorsauren und kohlensauren Kalkes auf die Korperge- wichtszunahme, Ibid. 42, 33-67. 1906 Neumann, Rudolf Otto: Untersuchungen iiber die Einwirkung des Protylins auf die Phosphorausscheidung des Menschen, Munch, med. Wochenschr. 53, 1558-1561. 1894a Neumann, Siegfried: Quantitative Bestimmung des Calciums, Magne- siums und der Phosphorsaure im Harn und Koth bei Osteomalacic, Arch. Gynakol. 47, 202-223. 1894b Neumann, Siegfried: Ueber die Verhaltnisse der Ausscheidung von Cal- cium, Magnesium und Phosphorsaure bei Osteomalacie, Ungar. Arch. Med. 3, 276-298. 1896 Neumann, Siegfried: Weitere Untersuchungen iiber die Stoffwechselver- haltnisse des Calciums, Magnesiums, der Phosphorsaure und des Nitro- gens bei puerperaler Osteomalacie, mit besonderer Rucksicht auf die durch die Kastration und andere therapeutische Eingriffe verursachten Veranderungen des Stoffwechsels, Arch. Gynakol. 51, 130-185. 1902 Neumann, Siegfried, and Bernbard Vas: Ueber den Einfluss der Ovar- iumpraparate auf den Stoffwechsel, Monatsschr. Geburtshilfe u. Gynakol. 15, 433-451. 1912 Newman, L. F., G. W. Robinson, E. T. Hainan, and H. A. D. Neville: Some Experiments on the Relative Digestibility of White and Whole Meal Breads, Jour, of Hygiene 12, 119-143. PHOSPHORUS METABOLISM 667 1908 Nichols, J. B.: Acid Intoxication, Washington Med. Ann. 7, 133-149. 1886 Nissen, Franz: Ueber das Verhalten der Kerne in der Milchdruse wahrend der Absonderung, Arch, mikroskop. Anat. 26, 337-342. 1912 Nizzi, F.: Le bilan du phosphore, de la lecithine et des graisses, Encephale 7, II, 245. 1911 Njegovan, Vladimir: Beitrage zur Kenntniss der pflanzlichen Phos- phatide, Zeit. physiol. Chem. 76, 1-26. 1913 Njegovan, Vladimir: Enthalt die Milch Phosphatide? Biochem. Zeit. 54, 78-82. 1898 Nolf, P.: Des nucleines, Annales de l'lnst. Pasteur 12, 361-368. 1898 Noll, Alfred: Ueber die Bildung von Lavulinsaure aus Nucleinsaure, Zeit. physiol. Chem. 25, 430-433. 1899 Noll, Alfred: Ueber die quantitativen Beziehungen des Protagons zum Nervenmark, Ibid. 27, 370-397. 1907 von Noorden, Carl: Metabolism and Practical Medicine. Vol. I. The Physiology of Metabolism. Vols. II and III. The Pathology of Metabolism. Chicago. 452, 525, and 1320 pp. 1883-4 North, W.: The Influence of Bodily Labor upon the Discharge of Nitro- gen, (Abstract) Proc. Roy. Soc. of London 36, 11-17. 1904 Novi, Ivo: L'eliminazione dei fosfati durante la cura antirabica e la sua modificazione per opera della terapia fosfoglicerica, Memoire d. r» Accad. d. sci. d. Institute di Bologna [6] 1, 157-177. 1908 Novi, Ivo: Terapia della fosfaturia con preparati organici del fosforo- glicerofosfato sodico e fitina), Ibid. [6] 5, 97-108. 1909 Novi, Ivo: Fitina e glicerofosfati. Nota II. Azione sul lavoro mus- colare, Ibid. [6] 6, 301-316. 1874 Odenius, M. V., and J. Lang: Fall af lymphorrhoea, pachydermia lymphorrhagica af Dr. M. V. Odenius, i Lund, Nordiskt Medicinskt Arkiv 6, No. 13; through Jahresb. ii. d. Fortschr. d. Thierchem. 5 (1875), 128, 129. 1899 Odenius, R. : Nagra undersokningar ofver en nukleoproteid ur mjolk- korteln, Upsala Lakaref. Forh. (N. F.) 5, 582-588; through Jahresb. ii. d. Fortschr. d. Thierchem. 30 (1900), 39. 1899 Oechsner, De Coninck: Sur l'elimination de l'azote et du phosphore chez les nourrissons, Compt. rend. Acad, des sci. 129, 223, 224. 1901 von Oefele, Freih.: Trippelphosphate im Stuhlgang, Pharmazeut. Zentralhalle 42, 191-198. 1907 von Oefele, Freih.: Bioson als Phosphornahrmittel auf Grund von Kot- untersuchungen, Deut. med. Presse 11, 27, 28. 1909 Oeri, Felix: Ein Beitrag zur Kenntnis des Phosphorsaure- und Kalk- stoffwechsels beim erwachsenen gesunden Menschen, Zeit. klin. Med. 67, 288-306; 307-318. 1898-9 Oertel, Horst : Beitrag zur Kenntniss der Ausscheidung des organisch gebundenen Phosphors im Harn, Zeit. physiol. Chem. 26, 123-170. 1911-12 Ogata, Masakiyo: Ueber das Wesen der Rachitis und Osteomalacic, Beitrage z. Geburtshilfe u. Gynakol. 17, 23-54. 1912 Onodera, Nakamura and Tateno : Untersuchung iiber den Stoffwechsel bei den Kakkekranken je nach der Verabreichung von poliertem Reis Oder nicht poliertem Reis, Mitt. Med. Ges. Tokio 26, No. 23, 4, 5. 668 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1904 Orgler, Arnold: Ueber das Vorkommen eines protagonartigen Korpers in der Nebennieren, Beitrage z. wissensch. Med. u. Chem., (Festschrift f. Ernst Salkowski), 285-288. 1902 Osborne, Thomas: Bestimmung des Schwefels in den Proteinkorpern, Zeit. anal. Chem. 41, 25-35. 1900a Osborne, Thomas B., and George F. Campbell: The Nucleic Acid of the Embryo of Wheat and its Protein Compounds, Jour. Amer. Chem. Soc. 22, 379-413; also 23rd Ann. Rept. Conn. Agr. Exp. Sta., New Haven (for 1899), 305-339. 1900b Osborne, Thomas B., and George F. Campbell: The Proteids of the Egg Yolk, Jour. Amer. Chem. Soc. 22, 413-422; also 23rd Ann. Rept. Conn. Agr. Exp. Sta., New Haven, (for 1899), 339-348. 1900c Osborne, Thomas B., and George F. Campbell: The Protein Constituents of Egg White, Jour. Amer. Chem. Soc. 22, 422-450. 1911 Osborne, Thomas B., and H. H. Guest: Hydrolysis of Casein, Jour. Biol. Chem. 9, 333-353. 1902a Osborne, Thomas B., and Isaac F. Harris: The Nucleic Acid of the Wheat Embryo, Ann. Rept. Conn. Agr. Exp. Sta. for 1901, 365-430. 1902b Osborne, Thomas B., and Isaac F. Harris: Die Nucleinsaure des Weizen- embryos, Zeit. physiol. Chem. 36, 85-133. 1908 Osborne, Thomas B., and F. W. Heyl: The Pyrimidine Derivatives of Nucleic Acid, Amer. Jour. Physiol. 21, 157-161. 1909 Osborne, Thomas B., and D. Breese Jones: Hydrolysis of Vitellin from the Hen's Egg, Ibid. 24, 153-160. 1911a Osborne, T. B., and L. B. Mendel: Feeding Experiments with Isolated Food-Substances, Carnegie Inst., Pub. 156, parts 1 & 2. 1911b Osborne, T. B., and L. B. Mendel: The Role of Different Proteins in Nu- trition and Growth, Science 34, 722-732. 1912a Osborne, T. B., and L. B. Mendel: Feeding Experiments with Fat-Free Food Mixtures, Jour. Biol. Chem. 12, 81-89. 1912b Osborne, T. B., and L. B. Mendel: Ueber Futterungsversuche mit isolier- ten Nahrungssubstanzen, Zeit. physiol. Chem. 80, 307-382. 1912c Osborne, T. B., and L. B. Mendel: The Role of Gliadin in Nutrition, Jour. Biol. Chem. 12, 473-510. 1912d Osborne, T. B., and L. B. Mendel: Maintenance Experiments with Iso- lated Proteins, Ibid 13, 233-276. 1913 Osborne, T. B., and L. B. Mendel: The Relation of Growth to the Chemi- cal Constituents of the Diet, Ibid. 15, 311-326. 1901-2 Osborne, W. A.: Caseinogen and its Salts, Jour, of Physiol. 27, 398-406. 1908 Ostertag, and Zuntz: Untersuchungen iiber die Milchsecretion des Schweines und die Ernahrung der Ferkel, Landwirtsch. Jahrbiicher 37, 201-260. 1899 Oswald, Ad.: Die Eiweisskorper der Schilddriise, Zeit. physiol. Chem. 27, 14-49. 1902 Oswald, Ad. : Die Chemie und Physiologie des Kropf es, Arch. path. Anat. u. Physiol. 169, 444-479. 1912 Otolski, and Biernacki: Ueber die Phosphatide in den Organen der mit getoteten Tuberkelbacillen geimpften Kaninchen, Biochem. Zeit. 41, 375-385. 1906 Otolsky, S. W.: Das Lecithin des Knochenmarks, Ibid. 4, 124-153. PHOSPHORUS METABOLISM 669 1886 Ott, Adolf: Ueber einige die Phosphate des Harnes betreffende Ver- haltnisse, Zeit. physiol. Chem. 10, 1-10. 1903 Ott, Adolf: Zur Kenntniss des Stoffwechsels der Mineralbestandteile beim Phthisiker, Zeit. klin. Med. 50, 432-440. 1862 Pagenstecher: Ein Beitrag zur Statistik des Kaiserschnittes nebst einem Anhang: Ueber Osteomalacie, Monatsschr. Geburtzkunde 19, 111. 1894 Pages, C: Physiologie de la matiere minerale du lait. These de Paris, 1894; through Milch-Zeitung 25, 86, 87 and the following review. 1888 Paijkull, Lincoln: Ueber die Schleimsubstanz der Galle, Zeit. physiol. Chem. 12, 196-210. 1913 Paladino, Raffaele: Untersuchungen iiber einige Veranderungen des Stoffwechsels bei Tieren nach Exstirpation der Schilddruse und der Parathyroiden, Biochem. Zeit. 50, 497-507. {1900) Panek, K.: (Ueber das Verhaltniss des Phosphorsauregehaltes des Harns zum Kalk- und Magnesiagehalt bei der sogenannten Phosphaturie), Przeglqd lekarski 39, 1; through Jahresb. u. d. Fortschr. d. Thierchem. 30 (1900), 772, 773. 1902a Panella, Amilcare: L'acido fosfocarnico dei muscoli dopo la morte, Arch- ivio di farmacol. e terapeut. 10, 323-361. 1902b Panella, Amilcare: L'acido fosfocarnico nella sostanza cerebrale, Giornale d. reale Accad. di med. di Torino 65, 398-400. 1902c Panella, Amilcare: L'acido fosfocarnico del sangue, Archivio di farmacol. e terapeut. 10, 439. 1903a Panella, Amilcare: L'acide phosphocarnique dans la substance cerebrale, Archives ital. de biol. 39, 260-262. 1903b Panella, Amilcare: L'acide phosphocarnique des muscles apres la mort. Ibid. 39, 263-283. 1903c Panella, Amilcare: L'acide phosphocarnique du sang, Ibid. 39, 283-291. 1903d Panella, Amilcare: L'acide phosphocarnique dans le testicule, Ibid. 39, 441, 442. 1903e Panella, Amilcare: L'acide phosphocarnique des muscles blancs et des muscles rouge, Ibid. 39, 443-451. 1903f Panella, Amilcare: L'acido fosfocarnico dei muscoli bianchi e rossi, Archivio di farmacol. e terapeut. 11, 91-103. 1903g Panella, Amilcare: L'acide phosphocarnique dans la substance nerveuse centrale, Archives ital. de biol. 39, 452-470. 1903h Panella, Amilcare: L'acido fosfocarnico nella sostanza nervosa centrale, Giornale d. reale Accad. di med. di Torino, 66, 423-443. 1903i Panella, Amilcare: Sul contenuto di acqua e di nucleone dei testicoli di cavallo, Archivio di farmacol. e terapeut. 11, 291-302. 1904a Panella, Amilcare: Sur le contenu d'eau et de nucleone des testicules de cheval, Archives ital. de biol. 42, 289-297. 1904b Panella, Amilcare: Eau et nucleone des muscles lisses, Ibid. 42, 368-382. 1904c Panella, Amilcare: L'acqua ed il nucleone della milza, Archivio di fis- iologia 1. 539-549. 1906a Panella, Amilcare: II nucleone e l'acqua del cervello in animali a digiuno, Archivio di farmacol. speriment. 5, 70-76. 1906b Panella, Amilcare: Sul rapporto fra quantita di sostanza usata e quanti- ty di nucleone determinata, Ibid. 5, 216-219. 670 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1902 Panzer, Theodor: Ueber das Verhalten von unterphosphorigsauren Cal- cium im thierischen Korper, Zeit. Untersuch. d. Nahrungs- u. Genuss- mitt. 5, 11-14. 1906 Panzer, Theodor: Ueber das sogenannte Protagon der Niere, Zeit. physiol. Chem. 48, 519-527. 1870 Papillon, Fernand: Recherches experimentales sur les modifications de la composition immediate des os, Compt. rend. Acad, des sci. 71, 372-374. 1870-1 Papillon, Fernand: Recherches experimentales sur les modifications de la composition immediate des os, Jour, de l'anat. et de la physiol. 7* 152-163. 1873 Papillon, Fernand: Recherches experimentales sur les modifications de la composition immediate des os, Compt. rend. Acad, des sci. 76, 352-355. 1890 Pappel, A., and H. Droop Richmond: The Milk of the Gamoose, Jour, of the Chem. Soc, Trans., 57, 754-760. 1877 Paquelin and Joly: Des pyrophosphates en therapeutique; leur mode d'action, Compt. rend. Acad, des sci. 85, 410-412. 1878 Paquelin and Joly: Du role physiologique des hypophosphites, Ibid. 86, 1505, 1506. 1913 Parnas, J.: Ueber die gesattigte Fettsaure des Kephalins,.Biochem. Zeit. 56, 17-20. 1908 Parrozzani, A.: Influenza di quantito progressive di concimi fosfatici sul contenuto in sostanze organische fosforate ed azotate, e sul rapporto fra fosforo ed azote dei semi mais, Staz. sperim. agrar. ital. 41, 729- 738. 1909 Parrozzani, A.: (The Relationship of Organic Phosphorus to Amido- nitrogen and to Non-proteid Nitrogenous Compounds in Ripe Seeds), Rend. Soc. chim. Ital. 2nd ser., 1, 205-207; through Exp. Sta. Record 25 (1911), 432. 1894 Pasqualis, G.: SulP assorbimento e l'eliminazione dell' acido fosfoglicer- ico e suo ricerca nelle orine e nel sangue, Annali di chim. e farmacol., 20 Agosto, 1894; through Jahresb. ii. d. Fortschr. d. Thierchem. 24 (1894), 283-285. 1905 Passon, Max: Zur Beifutterung mit phosphorsaurem Kalk, Jour. f. Land- wirtsch. 53, 113-134. 1913 Patein, G.: Histones et nucleohistones; leur recherche dans les liquides de l'organisme, Jour, de pharm. et de chim. 7, 55-60. 1895-6 Paton, D. Noel : On the Relationship of the Liver to Fats, Jour, of Physiol. 19, 167-216. 1898 Paton, D. Noel: The Phosphorus Compounds of the Muscle and Genitalia of the Salmon, and their Exchanges, Fishery Bd. of Scotland, Rept. of Investigations on the Life Hist, of Salmon, 143-155. 1897-8 Paton, D. Noel, with Francis D. Boyd, James C. Dunlop, A. Lockhart Gillespie, G. Lovell Gulland, E. D. W. Greig, S. C. Mahalanobis, and M. I. Newbigin: The Physiology of the Salmon in Fresh Water, Jour. of Physiol. 22, 333-356. 1900 Paton, D. Noel, J. C. Dunlop, and R. S. Aitchison: Contributions to the Study of the Metabolism of Phosphorus in the Animal Body, Ibid. 25, 121-224. 1899 Paton, D. Noel, J. C. Dunlop, and Ivison Macadam: On the Modifications of the Metabolism Produced by the Administration of Diphtheria Toxine, Ibid. 24, 331-335. PHOSPHORUS METABOLISM 671 1910 Patta, Aldo: Osservazioni intorno al comportamento degli ipofosfiti nell' organismo, Archivio di farmacol. speriment. 9, 1-7; through Jahresb. ii. d. Fortschr. d. Thierchem. 40 (1910), 1385. 1912 Patta, Aldo; Nuove ricerche intorno alia influenza della lecitina sul bilancio azotato e fosforato, Ibid. 13, 515-528. 1904 Patten, A. J., and E. B. Hart: The Nature of the Principal Phosphorus Compound in Wheat Bran, Amer. Chem. Jour. 31, 564-572; also N. Y. Agr. Sta. Bui. No. 250. 1876a Pavy, F. W.: The Effect of Prolonged Muscular Exercise on the System, Lancet, 1876, I, 319, 320; 353-356; 392-394; 429-431; 466-468. 1876b Pavy, F. W. The Effect of Prolonged Muscular Exercise upon the Urine in Relation to the Source of Muscular Power, Ibid. 1876, II, 815-818; 887-889. 1877 Pavy, F. W.: The Effect of Prolonged Muscular Exercise upon the Urine in Relation to the Source of Muscular Power, Ibid. 1877, I, 42-44. 1895 Pekelharing, C. A.: Ueber die Beziehung des Fibrinferments aus dem Blutserum zum Nucleoproteid, welches aus dem Blutplasma zu er- halten ist, Centralbl. Physiol. 9, 102-111. 1896 Pekelharing, C. A.: Ueber das Vorhandensein eines Nucleoproteids in Muskeln, Zeit. physiol. Chem. 22, 245-247. 1902 Pekelharing, C. A.: Mittheilungen iiber Pepsin, Ibid. 35, 8-30. 1914 Pekelharing, C. A.: Ueber den Einfluss von Phosphatiden auf die Blut- gerinnung, Ibid. 89, 22-38. 1905 Pende, N.: II sangue nell' osteomalacia, Bullett. d. reale Accad. med. di Roma, 31, 161-198. 1882 Penzoldt, F., and R. Fleischer: Experimentelle Beitrage zur Pathologie des Stoffwechsels mit besonderer Beriicksichtigung des Einflusses von Respirationsstorungen, Arch. path. Anat. u. Physiol. 87, 210-262. 1902 Percival, A. L.: Sur les variations du phosphore mineral, conjugue et organique, dans tissus animaux, Compt. rend. Acad, des sci. 135, 1005- *007. 1908 Peritz, Georg: Lues, Tabes und Paralyse in ihren atiologischen und ther- apeutischen Beziehungen zum Lecithin, Berlin, klin. Wochenschr. 45, I, 53-56. 1908-9a Peritz, Georg: Ueber die Beziehung der Lues, Tabes und Paralyse zum Lecithin, Deut. Zeit. Nervenheilk. 36, 87-94. 1908-9b Peritz, Georg: Ueber das Verhaltniss von Lues, Tabes und Paralyse zum Lecithin, Zeit. exp. Path. u. Ther. 5, 607-621. 1910 Peritz, Georg: Zur Pathologie der Lipoide, Ibid. 8, 255-278. 1892 Petit, P.: Distribution et etat du fer dans l'orge, Compt. rend. Acad, des sci. 115, 246-248. 1893 Petit, P.: Sur une nucleine vegetale, Ibid. 116, 995-997. 1873 Petrowsky, D.: Zusammensetzung der grauen und der weissen Substanz des Gehirns, Arch. ges. Physiol. 7, 367-370. 1906 Petry, Eugen: Ueber die Einwirkung des Labferments auf Kasein, Beitrage z. chem. Physiol, u. Path. 8, 339-364. 1866 von Pettenkofer, Max, and Carl Voit: Untersuchungen iiber den Stoff- verbrauch des normalen Menschen, Zeit. Biol. 2, 459-573. 1889 Peyer, Alexander: Die Phosphaturie, Sammlung klinischer Vortrage, Volkmann, Innere Med. No. 112 (whole No. 336). 672 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1904 Pfaundler, Meinhard: Ueber die Elemente der Gewebsverkalkung und ihre Beziehung zur Rachitisfrage, Jahrb. Kinderheilk. 60, 123-177. 1899 Pfeiffer, Theodor, and Wilhelm Scholz: Ueber den Stoffwechsel bei Par- alysis agitans und in Senium uberhaupt (mit Beriicksichtigung des Einflusses von Schilddriisen-tabletten), Deut. Arch. klin. Med. 63, 368- 422. 1874 Piccard, J.: Ueber Protamin, Guanin und Sarkin als Bestandtheile des Lachssperma, Ber. deut. chem. Gesell. 7, 1714-1719. 1910 Pighini, Giacomo: Ueber die Bestimmung der enzymatischen Wirkung der Nuclease mittels "optischer Methode," Zeit. physiol. Chem. 70, 85-93. 1911 Pighini, Giacomo: Ueber die Esterase und Nuclease des Serums bei ver- schiedenen Formen von Geisteskrankheiten, Biochem. Zeit. 33, 190-217. 1912 Pighini, G., and Flaminio Nizzi: Chemische und biochemische Unter- suchungen liber das Nervensystem unter normalen und pathologischen Bedingungen. III. Mitt. Aufsuchung der Esterase und der Lecithase in der normalen und pathologischen Cerebrospinal fliissigkeit, Ibid. 42, 145-149. 1906 Pignatti, Augusto: Influenza dei composti fosforati organici sulla ferrat- ina e sul suo contenuto in fosforo, Bollett. delle sci. med. di Bologna 77 (6 of ser. 8), 230-243; through Jahresb. u. d. Fortschr. d. Thierchem. 36 (1906), 585. 1898 Pinzani: (Experimentelle Untersuchungen liber den Einfluss der Kastra- tion auf den Stoffwechsel und die Blutbeschaffenheit), Archiv. ost. e gin., 1898; through Centralbl. Gynakol. 23, 1311. 1897 Piatt, Charles: The Normal Urine, Jour. Amer. Chem. Soc. 19, 382-384. 1903 Plenge, H.: Ueber die a-nucleinsaures Natron losende Wirkung einiger Mikroorganismen, Zeit. physiol. Chem. 39, 190-198. 1908 Plimmer, R. H. Aders: The Proteins of Egg-yolk, Jour, of the Chem. Soc. 93, 1500-1506. 1913a Plimmer, R. H. Aders: The Metabolism of Organic Phosphorus Com- pounds. Their Hydrolysis by the Action of Enzymes, Biochemical Jour. 7, 43-71. 1913b Plimmer, R. H. Aders: The Hydrolysis of Organic Phosphorus Com- pounds by Dilute Acid and by Dilute Alkali, Ibid. 7, 72-80. 1906 Plimmer, R. H. A., and W. M. Bayliss: The Separation of Phosphorus from Caseinogen by the Action of Enzymes and Alkali, Jour, of Physiol. 33, 439-461. 1909-10 Plimmer, R. H. A., Maxwell Dick, and Charles C. Lieb: A Metabolism Experiment with Special Reference to the Origin of Uric Acid, Ibid. 39, 98-117. 1909 Plimmer, R. H. A., and R. Kaya: The Distribution of Phosphoproteins in Tissues. Part II, Ibid. 39, 45-51. 1913 Plimmer, R. H. A., and Harold J. Page: An Investigation of Phytin, Bio- chemical Jour. 7, 157-174. 1908 Plimmer, R. H. Aders, and F. H. Scott: A Reaction Distinguishing Phos- phoprotein from Nucleoprotein and the Distribution of Phosphopro- teins in Tissues, Jour, of the Chem. Soc, Trans., 93, 1699-1721. 1909 Plimmer, R. H. Aders, and F. H. Scott: The Transformations in the Phosphorus Compounds in the Hen's Egg during Development, Jour, of Physiol. 38, 247-253. PHOSPHORUS METABOLISM 673 1871 Plosz, P.: Ueber das chemische Verhalten der Kerne der Vogel- und Schlangenblutkorperchen, Hoppe-Seyler's Med. chem. Unters., 461-462. 1873 Plosz, P.: Ueber die eiweissartigen Substanzen der Leberzelle, Arch. ges. Physiol. 7, 371-390. 1900 Poda, H., and W. Prausnitz: Ueber Plasmon, ein neues Eiweisspraparat, Zeit. Biol. 39, 279-312. 1889 Pohl, Julius: Bemerkungen fiber kfinstlich dargestellte Eiweissnucle'ine, Zeit. physiol. Chem. 13, 292-297. 1884 Politis, Georgios: Ueber das Verhaltnis der P 2 5 zum N im Harn bei Fiitterung mit Gehirnsubstanz, Zeit. Biol. 20, 193-214. 1906 Pollak, Emil: Die Nucleoproteide in der Behandlung septischer Krank- heitsformen, Arch. Gynakol. 89, 479-521. 1885 Pommer, Gustav: Untersuchungen iiber Osteomalacie und Rachitis nebst Beitragen zur Kenntniss der Knochenresorption und -Apposition in verschiedenen Altersperioden und der durchbohrenden Gefasse, Leip- zig. 506 pp. 1894 Popoff, P. M.: Ueber die Einwirkung von eiweissverdauenden Fermenten auf die Nucleinstoffe, Zeit. physiol. Chem. 18, 533-539. 1907 Porges, O., and E. Neubauer: Physikalisch-chemische Untersuchungen iiber das Lecithin und Cholesterin, Biochem. Zeit. 7, 152-177. 1905 Posner, Edward R., and William J. Gies: Protagon a Mechanical Mixture of Substances or a Definite Chemical Compound? Jour. Biol. Chem. 1, 59-112. 1900 Posternak, S.: Contribution a. l'etude chimique de l'assimilation chloro- phylliene. Sur le premier produit d'organisation de l'acide phosphor- ique dans les plantes a, chlorophylle, avec quelques remarques sur la role physiologique de l'inosite, Revue gener. de botanique 12, 5-24; 64-73. 1903a Posternak, S.: Sur un nouveau principe phospho-organique d'origine vegetale, la phytine, Compt. rend. Soc. de biol. 55, 1190-1192. 1903b Posternak, S.: Sur la matiere phospho-organique de reserve des plantes a chlorophylle, Compt. rend. Acad, des sci. 137, 202-204. 1903c Posternak, S.: Sur les proprietes et la matiere phospho-organique de reserve des plantes a chlorophylle, Ibid. 137, 337-339. 1903d Posternak, S.: Sur la constitution de l'acide phospho-organique de reserve des plantes vertes et sur le premier produit de reduction du gaz carbonique dans l'acte de l'assimilation carbonique, Ibid. 137, 439- 441. 1905 Posternak, S.: Sur la composition chimique et la signification des grains d'aleurone, Ibid. 140, 322-324. 1905 Pouchet, G., and J. Chevalier: Action des composes phosphores sur la circulation, Bui. gener. de therapeut. 150, 915-921. 1906 Pouchet, G., and J. Chevalier: Action du phosphore et des composes phos- phores organiques sur la circulation, Ibid. 151, 307-312. 1905 Power, Frederick B., and Frank Tutin: The Relation between Natural and Synthetical Glycerylphosphoric Acids, Proc. of the Chem. Soc. 21, 72,73; also Jour, of the Chem. Soc. 87 (1905), 249-257. 1907 Preti, Luigi: Ueber die spontane Ausscheidung einer Caseinverbindung aus Milch, Zeit. physiol. Chem. 53, 419-426. 674 OHIO EXPEKIMENT STATION: TECHNICAL BUL. 5 1891 Preysz, Kornel: (Wie hat man auf die Ausscheidung der Phosphor- saure beziigliche Versuche anzustellen ? ) , Magyar orvosi archivum 1891, 50; through Jahresb. u. d. Fortschr. d. Thierchem. 21 (1891), 252, 353. 1892-3 Preysz, Kornel: Einfluss der Muskelarbeit und der geistigen thatigkeit auf die Ausscheidung der Phosphorsaure beim Menschen, Ungar. Arch. 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Ill + 68 pp. 1905-6 Rasquin, M. : Les matieres phophatees dans l'alimentation des jeunes animaux, Rapports II Congr. internat. de l'aliment. rationelle du betail, 62-68. 1912 Rather, J. B.: The Forms of Phosphorus in Cottonseed Meal, Texas Agr. Exp. Sta. Bui. 146, 3-16. 1913a Rather, J. B.: The Phosphorus Compounds of Cotton Seed Meal and Wheat Bran, Texas Agr. Exp. Sta. Bui. No. 156. 18 pp. 1913b Rather, J. B.: Phytic Acid in Cottonseed Meal and Wheat Bran, Jour. Amer. Chem. Soc. 35, 890-895. 1893 Raudnitz, R. W. : Ueber die Resorption alkalischer Erden im Ver- dauungstract, Exp. Arch. 31, 343. 1903 Raudnitz, R. W.: Bestandteile, Eigenschaften und Veranderungen der Milch, Ergebnisse d. Physiol. 2, 1. Abt. s 193-325o 669 refs. PHOSPHORUS METABOLISM 675 1858 von Recklinghausen, F.: Die mineralischen Bestandtheile junger Mensch- enknochen, Arch. path. Anat. u. Physiol. 14, 466-476. 1891 von Recklinghausen, F.: Die fibrose oder deformirende Ostitis, die Osteo- malacic und die osteoplastische Carcinose in ihren gegenseitigen Bezie- hungen, Festschr. d. Assistenten R. Virchow's, 1891. 89 pp. 1879 Regnard, P. : Sur la composition chimique des os dans l'arthropathie des ataxiques, Compt. rend. Acad, des sci. 89, 1041, 1042. 1908 Reh, Alfred: Ueber die Polypeptidphosphorsaure (Paranucleinsaure) des Kaseins, Beitrage z. chem. Physiol, u. Path. 11, 1-18. (1911) Reicher, K.: Zur Kenntnis des Fett- und Lipoidstoffwechsels, Verhandl. d. Kongr. f. innere Med. 28, 327-330; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 107. 1904 Reiss, Emil: Eine Beziehung des Lecithins zu Fermenten, Berlin, klin. Wochenschr. 41, 1169-1171. 1894 Rem-Picci, G., and G. Bernasconi: Sull eliminazione dei fosfati per le urine nelle febbri malariche, II Policlinico, Apr. 1894; through Jahresb. u. d. Fortschr. d. Thierchem. 24 (1894), 574-581. 1913 Renall, Montague H. : Ueber den stickstoffhaltigen Bestandteil des Keph- alins, Biochem. Zeit. 55, 296-300. 1911 Rengniez, P.: Le phosphore dans les farines, These de pharmacy, Paris, 1911; through Revue scient. 49 (1911), 176, 177. 1905 Renner : Kunstliche Hyperleukocytose als Mittel zur Erhohung der Wider- standskraft des Korpers gegen operative Infektionen. Wirkung subkutaner Hefenuklei'nsaureinjektionen auf den menschlichen Organ- ismus, Mittheil. aus d. Grenzgebiet. d. Med. u. Chir., Jena, 15, 89-150. 1910 Renshaw, R. R., and K. N. Atkins: Bactericidal Properties of Lecithins and Choline Salts, Jour. Amer. Chem. Soc. 32, 130-132. 1904 Renvall, G.: Zur Kenntniss des Phosphor-, Calcium- und Magnesium- umsatzes beim erwachsenen Menschen, Skand. Arch. Physiol. 16, 94- 138. 1913 Ribbert, Hugo: Beitrag zur Rachitis, Deut. med. Wochenschr. 39, I, 8-10. 1900 Richet, Charles: Du serum musculaire, Compt. rend. Acad, des sci. 131, 1314-1316. 1901 Richmond, H. Droop: The Composition of Milk, The Analyst 26, 310-318. 1912 Riedel, A. G. J. D.: Zur Kenntnis des Eigelblecithins, Riedel's Ber., Mar. 1912, 24-33; through Zentralbl. Biochem. u. Biophys. 13 (1912), 562, 563. 1868 Riesell, Albert: Ueber die Phosphorsaure-Ausscheidung im Ham bei Einnahme von kohlensaurem Kalk, Hoppe-Seyler's Med. chem. Unters. 319-324. 1910 Rising, Adolf: Bidrag till kannedomen omde i vara narungsmedel fdre- kommande fosforforeningama (Phosphorus Compounds in Food Ma- terials), Svensk. Kemisk Tidskr. 22, 143-150; through Chem. Abs. 5 (1911), 2402. 1895 Robert: Rachitis und Osteomalacia, Ber. u. d. Veterinarwesen im K. Sachsen 40, 91. 1906 Robert, H., and J. Parisot: Les phosphates urinairies dans la paralysie agitante, Compt. rend. Soc. de biol. 60, 1084, 1085. 1879 Roberts, William: Note on the Existence of a Milk-Curdling Ferment in the Pancreas, Proc. Roy. Soc, London, 29, 157. 1881 Roberts, William: On the Estimation of the Amylolytic and Proteolytic Activity of Pancreatic Extracts, Ibid. 32, 145-161. 676 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1906-7 Robertson, T. Brailsford: Studies in the Chemistry of the Ion-Proteid Compounds. IV. On Some Chemical Properties of Casein and their Possible Relation to the Chemical Behavior of Other Protein Bodies, with Especial Reference to Hydrolysis of Casein by Trypsin, Jour. Biol. Chem. 2, 317-383. 1907 Robertson, T. Brailsford: Note on the Synthesis of a Protein through the Action of Pepsin, Ibid. 3, 95-99. 1908 Robertson, T. Brailsford: On the Influence of Temperature upon the Sol- ubility of Casein in Alkaline Solutions, Ibid. 5, 147-154. 1909a Robertson, T. Brailsford: On the Synthesis of Paranuclein through the Agency of Pepsin and the Chemical Mechanism of the Hydrolysis and Synthesis of Proteins through the Agency of Enzymes, Ibid. 5, 493-523. 1909b Robertson, T. Brailsford: On the Nature of the Chemical Mechanism which Maintains the Neutrality of the Tissues and Tissue-fluids, Ibid. 6, 313-320. 1910a Robertson, T. Brailsford: Concerning the Relative Magnitude of the Parts Played by the Proteins and by the Bi-carbonates in the Main-? tenance of the Neutrality of the Blood. Ibid. 7, 351-357. 1910b Robertson, T. Brailsford: On the Refractive Indices of Solutions of Cer- tain Proteins. II. The Paranucleins, Ibid. 8, 287-295. 1911 Robertson, T. Brailsford, and H. C. Biddle: On the Composition of Cer- tain Substances Produced by the Action of Pepsin upon the Products of Complete Peptic Hydrolysis of Casein, Ibid. 9, 295-302. 1908 Robertson, T. Brailsford, and C. L. A. Schmidt: On the Part Played by the Alkali in the Hydrolysis of Proteins by Trypsin, Ibid. 5, 31-48. 1913 Robertson, T. B., and Hardolph Wasteneys: On the Changes in Lecithin Content which Accompany the Development of Sea-urchin Eggs, Arch. Entwickelungsmechanik d. Organismen 37, 485-496. 1895 Robin, Albert: Les glycerophosphates; Action sur la nutrition, indica- tions therapeutiques, mode d'administration, Bui. gener. de therapeut. 128, 385-400; 433-444. 1911 Robin, Albert: Beitrag zur -chemischen Zusammensetzung der carcino- matosen Leber, Zentralbl. f. d. Physiol, u. Path. d. Stoffw., N. F. 6, 577-583; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 344. 1913a Robin, Albert: Sur la mineralisation comparee des regions cancerisees du foie et des regions relativement saines, Compt. rend. Acad, des sci. 156, 334-336. 1913b Robin, Albert : Recherches sur les variations de l'acide phosphorique dans l'urine et le foie des cancereux, Ibid. 156, 2018-2020. 1895 Rockwood, C« W.: Ueber das Vorkommen der Fleischsaure im Harne, Arch. Anat. u. Physiol., physiol. Abt., 1-4. 1898 Rohmann, F.: Stoffwechselversuche mit phosphorhaltigen und phosphor- freien Eiweisskbrpern, Berlin, klin. Wochenschr. 35, 789-791. 1902 Rohmann, F.: Ueber kiinstliche Ernahrung. Vortrag. Klin.-ther. Woch- enschr. 9, 1306-1311. 1907 Rohmann, F.: Ueber kiinstliche Ernahrung, Deut. med. Wochenschr. 33, 1568. 1908 Rohmann, F.: Ueber kiinstliche Ernahrung von Mausen, Allgem. med. Central-Zeitung 77, 129. PHOSPHORUS METABOLISM 677 I860 Roll, M. F.; Die Knochenweiche (Rachitis), Lehrbuch der Path. u. Ther. d. Hausthiere, 2d. edit., p. 85. 1897 Roeske, Georg: Ueber den Verlauf der Phosphorsaure-Ausscheidung beim Menschen, Inaug. Diss., Greifswald, 44 pp. 1908 Roger, H.: Sur le role des phosphates dans la saccharification salivaire, Compt. rend. Soc. de biol. 65, 374, 375. 1909 Rogers, L. A.: (Gegorene Milch), Report of the Bureau of Animal Industry 1909, 133-161; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 185. 1910 Rogozinski, F.: Beitrage zur Kenntnis des Phosphorstoffwechsels im tierischen Organismus, Bui. internat. de l'Acad. des sci. de Cracovie, B, 1910, 2, 260-312. 1909 Rohde, Alice, and Walter Jones: The Purin Ferments of the Rat, Jour. Biol. Chem. 7, 237-247. 1866 Roloff, F.: Ueber Osteomalacia und Rachitis, Arch. path. Anat. u. Physiol. 37, 433-503. 1869 Roloff, F.: Ueber .Osteomalacie, Ibid. 46, 305-316. 1875a Roloff, F.: Ueber die Ursache der Rhachitis, Zeit. landwirtsch. Central- Vereins d. Prov. Sachsen 32, 261-263. 1875b Roloff, F.: Ueber Osteomalacie und Rachitis. I. Arch, prakt. Tierheilk. 1, 189-220. 1879 Roloff, F.: Ueber Osteomalacie und Rachitis. II. Ibid. 5, 152-163. 1887 Romeyn, D. : Ondersoekingen over den invloed van alcohol op den Mensch, Akad. Prolfschrift, Amsterdam, 1887; Munch, med. Wochenschr. 1887, No. 33; through Jahresb. ii. d. Fortschr. d. Thierchem. 17 (1887), 400-403. 1846 Ronalds, Edmund: Remarks on the Extractive Material of Urine, and on the Excretion of Sulphur and Phosphorus by the Kidneys in an Unox- idized State, Phil. Trans, of the Roy. Soc. of London 136 (1846), 461- 464; also Phil. Mag. [3] 30 (1847), 253; also Jour. f. prakt. Chem. 41 (1847), 185-188. 1895 Roos, E.: Ueber die Einwirkung der Schilddruse auf den Stoffwechsel nebst Vorversuchen iiber die Art der Wirksamen Substanz in derselben, Zeit. physiol. Chem. 21, 19-41. 1911 Rose, Anton Richard: The Toxicity of Phytin, Rept. Amer. Chem. Soc. for 1911, Science 35 (1912), 393. 1912a Rose, Anton Richard: A Study of the Metabolism and Physiological Ef- fects of Certain Phosphorus Compounds with Milch Cows. II. N. Y. Agr. Exp. Sta., Tech. Bui. No. 20. 1912b Rose, Anton Richard: A Resume of the Literature on Inosite-Phosphoric Acid, with Special Reference to the Relation of that Substance to Plants, Biochemical Bui. 2, 21-49. 1911 Rose, A. R., and J. T. Cusick: The Influence of Phosphorus Compounds on the Yield .and Composition of Goat's Milk, Rept. Amer. Chem. Soc. for 1911, Science 35 (1912), 393. 1849 Rose, Heinrich : Ueber die unorganischen Bestandtheile in den organisch- en Korpern, Poggendorff's Annalen d. Phys. u. Chem. 76 (16 of ser. 3), 305-404. 1912 Rosenbloom, Jacob: A Quantitative Study of the Lipins of Bile Obtained from a Patient with a Biliary Fistula, Proc. Columbia Biochem. Assoc. Biochemical Bui. 2, 182. 678 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1913a Rosenbloom, Jacob: The Biochemistry of the Female Genitalia. II. The Lipins of the Ovary and Corpus Luteum of the Pregnant and Non- pregnant Cow, Jour. Biol. Chem. 13, 511, 512. 1913b Rosenbloom, Jacob: A Quantitative Chemical Analysis of Human Bile, Ibid. 14, 241-243. 1909 Rosenheim, Otto: Proposals for the Nomenclature of the Lipoids, Bio- chemical Jour. 4, 331-336. 1907 Rosenheim, Otto, and M. Christine Tebb: The Non-existence of "Pro- tagon" as a Definite Chemical Compound, Jour, of Physiol. 36, 1-16. 1908a Rosenheim, Otto, and M. Christine Tebb: The Optical Activity of So- called "Protagon," Ibid. 37, 341-347 1908b Rosenheim, Otto, and M. Christine Tebb: On a New Physical Phenom- enon Observed in Connection with the Optical Activity of So-called "Protagon," Ibid. 37, 348-354. 1908c Rosenheim, Otto, and M. Christine Tebb : On So-called "Protagon," Quart. Jour, of Exp. Physiol. 1, 297-304. 1909a Rosenheim, Otto, and M. Christine Tebb: The Non-Existence of "Pro- tagon" as a Definite Chemical Compound, Ibid. 2, 317-333. 1909b Rosenheim, Otto, and M. Christine Tebb: On the Lipoids of the Adrenals. (Preliminary communication.) Proc. Physiolog. Soc, Jour, of Physiol. 38, LIV-LVI. 1909c Rosenheim, Otto, and M. Christine Tebb: The Lipoids of the Brain. Part I. Sphingomyelin, Ibid 38, LI-LII. 1910 Rosenheim, Otto, and M. Christine Tebb: Die Nicht-Existenz des sogen- annten "Protagons" im Gehirn, Biochem. Zeit. 25, 151-160. 1901 de Rothschild, H., and L. Netter: Apropos des quantites de lait qu'il convient de donner dans l'allaitement artificiel et de leurs rapports avec les echanges nutritifs chez le nourrisson, Compt. rend. Soc. de Biol. 53, 658-661. 1910 Rotky, Hans: Beitrage zur Pathologie des Nucleinstoffwechsels, Deut. Arch. klin. Med. 98, 540-582. 1902 Rotondi, Giorgio: Sulla digestione del caseinogeno di donna e di mucca, Lo Sperimentale 56, 68-83; through Jahresb. ii. d. Fortschr. d. Thier- chem. 32 (1902), 999-1000. 1912 Rubinraut, A.: Stoffwechselversuche bei Akromegalie, Inaug. Diss. Zurich. 19 pp. 1903 Rubow, V.: Unders0gelser over normale og fedtdegenererede Hjaerter som Bidrag til Hjaertemusklens physiologiske Kemi og til Fedtdegen- erationens (Researches on normal and fatty degenerated hearts as a contribution to the physiological chemistry of the heart muscles and to the pathogenesis of fatty degeneration), K0benh. 128 pp. 1905 Rubow, V.: Ueber den Lecithingehalt des Herzens und der Nieren unter normalen Verhaltnissen, im Hungerzustande und bei der fettigen De- generation, Arch. exp. Path. u. Pharm. 52, 173-204. 1893a Riidel, G.: Ueber die Resorption und Ausscheidung des Kalkes, Ibid. 33, 79-89. 1893b Riidel, G.: Ueber die Resorption und Ausscheidung der Kalksalzen bei rachitischen Kindern, Ibid. 33, 90-100. 1900 Rumpf, Th., and O. Schumm: Ueber den Stoffwechsel eines Vegetariers, Zeit. Biol. 39 (N. F. 21), 153-158. PHOSPHORUS METABOLISM 679 1895 Ruppel, W. G. : Zur Kenntniss des Protagons, Ibid. 31, 86-100. 1912 Ryan, L. A., and Edward B. Meigs: The Chemical Constituents of the Ash of Smooth Muscle, Proc. Amer. Soc. Biol. Chem., Jour. Biol. Chem. 11 (1912), XXV; also Proc. Amer. Physiol. Soc, Amer. Jour. Physiol. 29 (1912), XV, XVI. 1907 Saccone, Giovanni: Sul ricambio del fosforo dopo lesione od asportazione di organi importanti, Annali di med. navale 13, I, 573-598. 1905 Sachs, Fritz: Ueber die Nuklease, Zeit. physiol. Chem. 46, 337-353. 1891 Sagnier, Henri: L'industrie laitiere a Vichy, Jour, de l'agriculture, 26, 2, 470-473; through Milch-Zeitung 20 (1891), 890. 1908 Saikai, Tadasu: A Chemical Study of Nonstriated Mammalian Muscle, Jour. Biol. Chem. 4, 483-495. 1900 Saint- Aubin, Henri S.: Injections hypodermiques d'huile de vaseline et de jaune d'oeuf (Medication stimulante), These de Paris. 41 pp. (1907) St. Bondzynski and A. Gonka: (Ein Beitrag zur Kenntnis der organisch- en Grundsubstanz der Zahne), Sprawozdanie X zjardu lekarzy I przyrodnikow polskich S. 105, vorl. Mitteil.; through Jahresb. ii. d. Fortschr. d. Thierchem. 37 (1907), 478. 1912 Sakaki, C: Ueber einige Phosphatide in der Placenta, Mitt. Med. Ges. Tokio 26, No. 22, 1. 1913a Sakaki, C: Ueber einige Phosphatide aus der menschlichen Placenta. I. Biochem. Zeit. 49, 317-325. 1913b Sakaki, C: Ueber einige Phosphatide aus der menschlichen Placenta. II. Ibid. 49, 326-332. 1913c Sakaki, C: Ueber einige Phosphatide aus der menschlichen Placenta. III. Ibid. 54, 1-4. 1913d Sakaki, C: Ueber die Phosphorverteilung in der Placenta, Ibid. 54, 5-10. 1910 Salant, William, and F. C. Hinkel: The Influence of Alcohol on the Com- position of Urine. I. Jour, of Pharmacol, and Exper. Therapeut. 1, 493-517. 1885 Salkowski, E.: Zur Kenntnis des Pferdeharns, Zeit. physiol. Chem. 9, 241- 245. 1893a Salkowski, E.: Ueber den Verbleib des Phosphors bei der Verdauung des Caseins, Centralbl. med. Wissensch. 31, 385, 386. 1893b Salkowski, E.: Ueber das Verhalten des Caseins bei der Magenverdau- ung und die Verseifung der Fette, Ibid. 31, 467-469. 1896a Salkowski, E. : Ueber das Verhalten des Caseins zu Pepsinsalzsaure, Arch. ges. Physiol. 63, 401-422. 1896b Salkowski, E.: Ueber die Anwendung eines neuen Caseinpraparates "Eucasein" zu Ernahrungszwecken, Deut. med. Wochenschr., 225-229. 1899 Salkowski, E.: Kleinere Mittheilungen. 1. Ueber das erste Produkt der Verdauung des Caseins durch Pepsinsalzsaure, Zeit. physiol. Chem. 27, 297-302. 1901 Salkowski, E. : Ueber die Paranucleinsaure aus Casein. I. Ibid. 32, 245- 267. • 1904 Salkowski, E.: Zur Kenntnis des Harns und des Stoffwechsels der Her- bivoren, Ibid. 42, 212-250. 1909a Salkowski, E.: Ueber das Ferratin Schmiedebergs, Ibid. 58, 282-289. 1909b Salkowski, E.: Ueber die Bindung des Eisens im Nucleoproteid der Leber, Ibid. 59, 19-21. 680 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1913a Salkowski, E.: Ueber das Verhalten des jodparanucleinsauren Eisens im Organismus, Biochem. Zeit. 49, 152-164. 1913b Salkowski, E.: 1st es moglich, den Gehalt des Gehirns an Phosphatidyl zu steigern? Ibid. 51, 407-422. 1894-5 Salkowski, E., and Martin Hahn: Ueber das Verhalten des Phosphors im Casein bei der Pepsinverdauung, Arch. ges. Physiol. 59, 225-250. 1878 Salomon, Georg: Ueber die Verbreitung und Entstehung von Hypoxan- thin und Milchsaure im thierischen Organismus, Zeit. physiol. Chem. 2, 65-95. 1907a Sammis, J. L.: The Chemistry of Milk Curdling, Twenty-fourth Ann. Kept. Wis. Agr. Exp. Sta., 171-189. 1907b Sammis, J. L.: The Coagulation of Fresh and Alkaline Milks, Ibid. 190- 206. 1909 Sammis, J. L., and E. B. Hart: The Relation of Different Acids to the Precipitation of Casein and to the Solubility of Cheese Curds in Salt Solution, Jour. Biol. Chem. 6, 181-187. 1895 Sandmeyer, W.: Ueber die Ausnutzung des Paranucleins im tierischen Organismus, Zeit. physiol. Chem. 21, 87-89. 1912 Sani, G.: Azione del fosfato monocalcico nella conservazione dei foraggi verdi, Atti. Accad. Lincei 21, II, 108-112. 1894 Sanson, Andre: Sur l'enrichissenient du lait en phosphates, Compt. rend. Soc. de biol. 46, 154, 155. 1896 Sanson, Andre: Sur l'assimilabilite des glycerophosphates, Ibid. 48, 685- 687. 1912 Santonocetto, Vittorugo: La fitina nel ricambio organico azotato fisio- logico, Bullett. delle sci. med. di Bologna, 223-232; through Zentralbl. Biochem. u. Biophys. 14 (1912-13), 476. (1911) Sarvonat, F., and Gentz: (Ueber den Verlauf der Phosphorsaureausschei- dung im Harn), Compt. rend. Soc. de biol. 70, 629; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 468. 1910 Sarvonat, F., and Rebattu, J.: Action de la tuberculose sur la mineral- isation du cobaye, Jour, de physiol. et de pathol. gener. 12, 934-941; also Compt. rend. Soc. de biol. 69 (1910), 127-129. 1909 Sato, T.: Beitrag zur Kenntniss des Nucleoproteids der Milz, Biochem. Zeit. 22, 489-494. 1907 Satta, G.: Sulla distribuzione del fosforo negli organi e sulla decompos- izione dei composti fosforati organici. Parte prima. Archivio sci. med. Torino 31, 407-412. 1908 Satta, G.: Sur la distribution du phosphore dans les organes et sur la decomposition des composes phosphores organiques, Archives ital. d. biol. 49, 375-380. 1910 Satta, G., and G. M. Fasiani: Influenza dei lipoidi sull' autolisi del fegato, Giornale d. reale Accad. di med. di Torino 73 (16 of ser. 4), 285-288. 1913 Satta, G., and G. Gastaldi: Sul ricambio della lecitina introdotta per bocca, Archivio di farmacol. speriment. 15, 402-406. 1902 Sauerbruch: Ein Beitrag zum Stoffwechsel des Kalkes und der Phos- phorsaure bei infantiler Osteomalacic, Inaug. Diss., Leipzig. 51 pp. 1910 Sauerland, F.: Ueber den Eisengehalt der echten Nucleinsaure, Zeit. physiol. Chem. 64, 16-20. PHOSPHORUS METABOLISM 681 1911 Sawadski, J. W.: (Klinische Beobachtung iiber den Phosphor des Bultes), Russki Wratsch, Nr. 54; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 136. 1908 Scaffidi, Vittorio: Ueber die Verteilung des Eisens in der Leber, Zeit. physiol. Chem. 54, 448-460. 1909a Scaffidi, Vittorio: Ueber das Nucleoproteid der Schweinsleber, Ibid. 58, 272-281. 1909b Scaffidi, Vittorio: (Purine Excretion in Animals Treated with Nucleic Acid), Lo Sperimentale 63, 243-253; through Chem. Abs. 3 (1909), 2990. 1910-11 Scaffidi, Vittorio: Sulle modificazioni del ricambio purinico nella fatica in alta montagna, Internat. Beitrage z. Path. u. Ther. der Ernahrungs- storungen 2, 145-183. 1907 Schabad, J. A.: (Der Einfluss des Phosphors auf den Kalkstoffwechsel bei rachitischen und gesunden Kindern), Aerzte Zeitung 1907, No. 13 (Russian); through Jahresb. ii. d. Fortsch. d. Thierchem. 37 (1907), 661. 1908 Schabad, J. A.: Der Phosphorlebertran in der Therapie der Rhachitis. Sein Einfluss auf den Kalkstoffwechsel, Therapie der Gegenwart 49, 260, 261. 1909a Schabad, J. A.: Der, Phosphor in der Therapie der Rachitis. Der Ein- fluss des Phosphors auf den Kalkstoffwechsel bei rachitischen und ge- sunden Kindern, Zeit. klin. Med. 67, 454-494. 1909b Schabad, J. A.: Die Behandlung der Rachitis mit Lebertran, Phosphor und Kalk. Ihr Einfluss auf den Kalk- und Phosphorstoffwechsel bei Rachitis, Ibid. 68, 94-110. 1909c Schabad, J. A.: Zur Bedeutung des Kalkes in der Pathologie der Rach- itis. Abhandlung I. Der Mineralgehalt gesunder und rachitischer Knochen, Arch. Kinderheilk. 52, 47-67. 1909d Schabad, J. A. : Zur Bedeutung des Kalkes in der Pathologie der Rachitis. Abhandlung II. Der physiologische Kalkbedarf und Rachitis infolge von unbefriedigtem Kalkbedarf, Ibid. 52, 68-106. 1909e Schabad, J. A.: Der Kalk in der Pathologie der Rachitis, Berlin, klin. Wochenschr. 46, 823-826; 923-925. 1910a Schabad, J. A.: Die gleichzeitige Verabreichung von Phosphorlebertran mit einem Kalksalze bei Rachitis, Jahrb. Kinderheilk. 72, 1-17. 1910b Schabad, J. A.: Phosphor, Lebertran und Sesamol in der Therapie der Rachitis, Zeit. klin. Med. 69, 435-474. 1910c Schabad, J. A.: Zur Bedeutung des Kalkes in der Pathologie der Rachitis. Abhandlung III. Der Kalkstoffwechsel bei Rachitis, Arch. Kinderheilk. 53, 380-414. 1910d Schabad, J. A. : Zur Bedeutung des Kalkes in der Pathologie der Rachitis. Abhandlung IV. Der Phosphorstoffwechsel bei Rachitis, Ibid. 54, 83- 110. 1910-11 Schabad, J. A.: Der Kalkstoffwechsel bei Tetanie, Monatsschr. Kinder- heilk. 9 (Originalien 1), 25-35. 1911a Schabad, J. A.: Der Kalkgehalt der Frauenmilch. Zur Frage der un- genugenden Kalkzufuhr als Ursache der Rachitis, Jahrb. Kinderheilk. 74, 511-535. 682 OHIO EXPERIMENT STATION: TECHNICAL *BUL. 5 (1911b) Schabad, J. A.: Zwei Falle von sogenannter "Spatrachitis." Der Min- eralgehalt der Knochen und der Mineralstoffwechsel im Vergleich zu der kindlichen Rachitis, Mitt. a. d. Grenzgeb. d. Med. u. Chirurg. 23, 82-99; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 360. 1911c Schabad, J. A.: (Der Aschegehalt der Knochen und der Mineralstoffwech- sel im Vergleich mit dem bei Rachitis), Wratschebnaja Gazeta 1911, Nr. 10 u. 11; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 360. 1911a Schabad, J. A., and R. F. Sorochowitsch: Lipanin als Ersatzmittel des Lebertrans bei Rhachitis. Sein Einfiusz auf den Stoffwechsel, Monat- schr. Kinderheilk. 9 (Originalien 1), 659-671. 1911b Schabad, J. A., and R. F. Sorochowitsch: Die Behandlung des Rachitis mit Lebertranemulsionen (Emulsion Scott and Parke Davis mit Nat- rium- und Calciumhypophosphit und Emulsion mit essigsaurem Kalk) und ihre Einwirkung auf den Stoffwechsel, Ibid. 10 (Originalien 2), 12- 34. 1912 Schabad, J. A., and R. F. Sorochowitsch : Zur Frage von Wesen der giinst- igen Wirkung des Lebertrans bei Rhachitis. 1st das wirksame Agens des Lebertrans ein Ferment? Ibid. 11 (Originalien 3), 4-20. 1889 Schabanowa, Anna: Ueber Phosphorbehandlung der Rachitis, Jahrb. Kinderheilk. 29, 392-410. 1912 Schafer, E. A.: Effects upon Growth and Metabolism of the Addition of Small Amounts of Ovarian Tissue, Pituitary, and Thyroid to the Nor- mal Dietary of White Rats, Quart. Jour, of Exp. Physiol. 5, 203-228. 1887 Schaffer, F.: Ueber das Casein und die Wirkung des Labfermentes in der Kuhmilch, Landwirtsch. Jahrb. d. Schweiz. 1, 43-58. 1908 Schaumann, H.: Beriberi and Nucleinphosphorsaure in der Nahrung, Arch. Schiffs- und Tropenhygiene 12, Beiheft 5, 37-57. 1909 Schaumann, H.: Weitere Beitrage zur Aetiologie der Beriberi, Ibid. 13, Beiheft 6, 82-90. 1910 Schaumann, H.: Die Aetiologie der Beriberi unter Beriicksichtigung des gesamten Phosphorstoffwechsels, Ibid. 14, Beiheft, 8, 385 pp. 294 refs. 1911a Schaumann, H.: Further Contributions to the Etiology of Beri-beri, Trans, of the Soc. of Tropical Med. 5, 59-91. 1911b Schaumann, H.: Erwiderung auf C. Eijkman, Polyneuritis gallinarum und Beriberi, Arch. Schiffs- u. Tropenhygiene 15, 728-737. 1912a Schaumann, H.: Ueber die Darstellung und Wirkungsweise einer der in der Reiskleie enthaltenen, gegen experimentelle Polyneuritis wirk- samen Substanzen, Ibid. 16, 349-361. 1912b Schaumann, H.: Zu dem Problem der Beriberiatiologie, Ibid 16, 825- 838. 1912c Schaumann, H.: Weitere Beitrage zur Aetiologie der Beriberi, Ibid. 16, Beihefte, 137-170. 1905 Schenck, Martin: Die bei der Selbstverdauung des Pankreas auftretenden Nucleinbasen, Zeit. physiol. Chem. 43, 406-409. 1903 Schenke, V.: Untersuchungen iiber die Futtermittel des Handels, veran- lasst 1890 auf Grund Beschlusse in Bernburg und Bremen durch. den Verband landwirtschaftl. Versuchs-Stationen im Deutschen Reiche. XXXII. Phosphorsaurer Kalk als Futterbeigabe, Landwirtsch. Versuch. Stat. 58, 291-312. PHOSPHORUS METABOLISM 683 1880 Schetelig: Ueber die Herstammung und Ausscheidung des Kalkes im ge- sunden und kranken Organismus, Arch. path. Anat. u. Physiol. 82, 437-455. 1911 Scheunert, A., A. Schattke and E. Loetsch: CaO-, MgO-, P 2 5 - Gehalt von Heu und Hafer, nach deren Verfutterung Pferde an Osteomalacie erkrankten, Biochem. Zeit. 36, 240-244. 1897a Schiff, Arthur: Ueber die Beeinflussung des Stoffwechsels durch Hypo- physis und Thyreoideapraparate, Zeit. klin. Med. 32, Suppl., 284-290. 1897b Schiff, Arthur: Hypophysis und Thyroidea in ihrer Einwirkung auf den menschlichen Stoffwechsel, Wien. klin. Wochenschr. 10, 277-285. 1901a Schilling, Fr.: Die Tripelphosphate im Stuhle Ikterischer, Centralbl. Stoffwechsel- u. Verdauungskrankh. 2, 391, 392. 1903 Schittenhelm, Alfred: Die Nucleinbasen der Faeces unter dem Einfluss anhaltender Faulnis, Zeit. physiol. Chem. 39, 199-202. 1904a Schittenhelm, Alfred: Ueber die Harnsaurebildung in Gewebsausziigen, Ibid. 42, 251-258. 1904b Schittenhelm, Alfred : Ueber die Fermente des Nucleinstoffwechsels, Ibid. 43, 228-239. 1905a Schittenhelm, Alfred: Ueber die Harnsaurebildung und die Harnsaure- zersetzung in den Auszugen der Rinderorgane. Ein weiterer Beitrag zur Kenntnis der Fermente des Nuclinstoffwechsels, Ibid. 45, 121-151. 1905b Schittenhelm, Alfred: Der Nucleinstoffwechsel und seine Fermente bei Mensch und Tier, Ibid. 46, 354-370. 1907 Schittenhelm, Alfred: Natur und Wesen der Gicht, Med. Klinik, Berlin, 1907, Beiheft 4, 89-110. 1908 Schittenhelm, Alfred: Ueber die Fermente des Nucleinstoffwechsels, Zeit. physiol. Chem. 57, 21-27. 1909a Schittenhelm, Alfred: Ueber die Umsetzung verfiitterter Nucleinsaure beim Hunde unter normalen und pathologischen Bedingungen, Ibid. 62, 80-99. 1909b Schittenhelm, Alfred: Ueber die Fermente des Nucleinstoffwechsels menschlicher Organe, Ibid. 63, 248-268. 1910 Schittenhelm, Alfred: Ueber den Nucleinstoffwechsel des Schweines, Ibid. 66, 53-69. 1904 Schittenhelm, A., and Ernst Bendix: Ueber das Schicksal der in die Blutbahn eingebrachten Nucleinsaure, Deut. med. Wochenschr. 30, 1164, 1165. 1905 Schittenhelm, A., and Ernst Bendix: Vergleichende Untersuchungen liber die Wirkung verschiedener Nukleinsaure auf den thierischen Organis- mus, Zeit. exp. Path. u. Ther. 2, 166-178. 1906 Schittenhelm, Alfred, and Julius Schmidt: Ueber die Fermente des Nucleinstoffwechsels, Zeit. physiol. Chem. 50, 30-35. 1907a Schittenhelm, Alfred, and Julius Schmidt: Ablauf des Nucleinstoffwech- sels in menschlichen Organen, Zeit. exp. Path. u. Ther. 4, 424-431. 1907b Schittenhelm, Alfred, and Julius Schmidt: Ablauf des Nucleinstoffwech- sels in der Schweineleber, Ibid. 4, 432-437. 1903a Schittenhelm, A., and F. Schroter: Ueber die Spaltung der Hefenuclein- saure durch Bakterien. I. Mittheilung, Zeit. physiol. Chem. 39, 203- 207. 684 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1903b Schittenhelm, A., and F. Schroter: Ueber die Spaltung der Hefenuclein- saure durch Bakterien. II. Mittheilung and III. Mittheilung, Ibid. 40, 62-69; 70-80. 1904 Schittenhelm, A., and F. Schroter: Ueber die Spaltung der Hefenuclein- saure durch Bakterien. IV. Mittheilung, Ibid. 41, 284-292. (1911) Schittenhelm, Alfred, and Philip Seisser: Ueber die Beeinflussung des Stoffwechsels von Kaninchen und Hund durch Zufuhr von Nuklein- saure, Harnsaure und Allantoin (gleichzeitig ein Beitrag zur Frage der Nuklei'nharnsaureverbindungen), Zeit. exp. Path. u. Ther. 7, 116- 133; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 477, 478. 1909 Schittenhelm, Alfred, and Karl Wiener: Ueber die Vorkommen und die Bedeutung von Allantoin im menschlichen Urin, Zeit. physiol. Chem. 63, 283-288. 1912 Schittenhelm, Alfred, and Karl Wiener: Ueber den Abbau der Nuclein- saure durch Organfermente, Ibid. 77, 77-85. 1910 Schkarin, A. N.: Ueber den Einfluss der Nahrungsart der Mutter auf Wachstum und Entwicklung des Sauglings, Monatsschr. Kinderheilk. 9 (Originalien 1), 65-69. 1902 Schlagdenhauffen and Reeb: Sur la presence de la lecithine dans les vegetaux, Compt. rend. Acad, des sci. 135, 205-208. (1911) Schloss, Ernst: Die chemische Zusammensetzung der Frauenmilch auf Grund neuer Analysen, Monatsschr. Kinderheilk. 9, 636-640; Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 206. 1913a Schloss, Ernst: Zur Behandlung der Rachitis mit Lebertran, Kalk, und Phosphor auf Grund von Stoffwechselversuchen, Deut. med. Wochen- schr. 39, II, 1505-1508. 1913b Schloss, Ernst: Zur Therapie der Rachitis. 1. Mitteilung. Die Wirkung von Phosphorlebertran und Calcium aceticum auf den Stoffwechsel des naturlich ernahrten rachitischen Kindes, Jahrb. Kinderheilk. 78 [3rd ser. 28], 694-722. 1914a Schloss, Ernst: Zur Therapie der Rachitis. 2. Mitteilung. Die Wirk- ung von Phosphorlebertran, Calcium aceticum und organischen Kalk- phosphaten auf den Stoffwechsel des naturlich ernahrten rachitischen Kindes, Ibid. 79 [3rd ser. 29], 40-65. 1914b Schloss, Ernst: Zur Therapie der Rachitis. 3. Mitteilung. Die Wirk- ung von organischen und anorganischen Kalkphosphorpraparaten auf den Stoffwechsel des naturlich ernahrten rachitischen Kindes, Ibid. 79 [3rd ser. 29], 194-214. 1911 Schloss, Oscar M., and James M. Crawford: The Metabolism of Nitro- gen, Phosphorus, and the Purin Substances in the New-Born; with special Reference to the Causation of the Uric Acid Infarcts of the Kidney, Amer. Jour. Diseases of Children 1, 203-229. 1896 Schlossmann, Arthur : Ueber die Eiweissstoff e der Milch und die Method- en ihrer Trennung, Zeit. physiol. Chem. 22, 197-226. 1897 Schlossmann, Arthur: Ueber Eselsmilch, Ibid. 23, 258-264. 1905a Schlossmann, Arthur: Ueber die Bedeutung des Phosphors in der Milch fur den Sauglingsorganismus, Med. Klinik 1, 249-251. 1905b Schlossmann, Arthur: Ueber Menge, Art und Bedeutung des Phosphors in der Milch und iiber einige Schicksale desselben im Sauglingsorganis- mus, Arch. Kinderheilk. 40, 1-39. PHOSPHORUS METABOLISM 685 1904 Schlossmann, Arthur, and Ernst Moro: Die Ernahrung des Erwachsenen mit Kuh- und mit Frauenmilch, Zeit. Biol. 45, 261-291. 1847 Schmidt, Carl: Knochenerweichung durch Milchsaurebildung, Annalen der Chem. u. Pharm. 61, 329-335. 1882 Schmidt: (Materialien zur Erklarung der Eigenschaften von Frauen und Kuhmilch), Russian Diss.; through Centralbl. Gynakol. 6, 776. 1883 Schmidt-Miilheim : Beitrage zur Kenntniss der Milchsecretion, Arch. ges. Physiol. 30, 602-620. 1906 Schmidt-Nielsen, Sigval: Zur Kenntnis des Kaseins und der Labgerin- nung, Upsala Lakaref Forh. (N. F.) 11, Suppl. Hammarsten Fest- schrift No. XV, 1-26 (1906); through Jahresb. ii. d. Fortschr. d .Thier- chem. 36 (1906), 255, 256. 1900 Schmiedeberg, O. : Ueber die Nucle'insaure aus der Lachsmilch, Arch. exp. Path. u. Pharm. 43, 57-83. 1907 Schmiedeberg, O.: Beitrage zur Kenntniss der tierischen Nuclei'nsaure, Ibid. 57, 309-337. 1904 Schmoll, E.: The Chemical Origin of Leucocytes, Johns Hopkins Hosp. Bui. 15, 238-247. 1875 Schmuziger, F.: Zur Urinuntersuchung bei puerperaler Osteomalacie, Centralbl. med. Wissensch., 946-949. 1895 Scholz, Wilhelm : Ueber den Einfluss der Schilddriisenbehandlung auf den Stoffwechsel des Menschen, insbesondere bei Morbus Basedowii, Cen- tralbl. innere Med. 16, 1041-1058; 1069-1083. 1905 Scholz, Wilhelm : Ueber den Stoffwechsel der Cretinen, Zeit. exp. Path. u. Ther. 2, 271-384. (1911) Schottin, W. R.: Phosphorarmut der Rindenzentren Ursache der Migrane, Heilung durch Phosphorlecithin-Oel, Med. Klinik (Dresden) 9, 339- 341; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 486. 1893-4 Schoumow-Simanowsky, E. O. : Ueber den Magensaf t und das Pepsin bei Hunden, Arch. exp. Path. u. Pharm. 33, 336-352. 1901 Schreiber, E.: Ueber die Verwendung des frischen Kaseins in der Ernahrung, Centralbl. Stoffwechsel- u. Verdauungskrankh. 2, 115-118. 1897 Schreiber and Waldvogel: Ueber Sanose, ein neues Eiweisspraparat, Deut. med. Wochenschr. 23, Therapeut. Beilage, 65-67. 1876 Schrodt, Max: Vergleichende Knochenuntersuchungen, angestellt am Skelete eines Fleischfressers, Landwirtsch. Versuch. Stat. 19, 349-400. 1889 Schrodt, Max: Ueber die Zusammensetzung von Buffelmilch, Vierteljahr- esschr. ii. d. Fortschr. d. Chem. d. Nahrungs- u. Genussmittel 4, 137, 138. 1885 Schrodt, M., and H. Hansen: Ueber die Zusammensetzung der Aschen von Kuhmilch, Landwirtsch. Versuch. Stat. 31, 55-83. 1905 Schroder, G.: Ueber neuere Medikamente und Nahrmittel fur die Be- handlung der chronischen Lungentuberkulose, Zeit. Tuberkulose u. Heilstattewesen 7, 238-242. 1906 Schroder, G.: Ueber neuere Medikamente und Nahrmittel fur die Be- handlung der chronischen Lungentuberkulose, Ibid. 8, 509-518. 1887 Schroter, R. : Untersuchungen iiber die Eiweisskorper der Menschen- und Kuhmilch, Jahrb. Kinderheilk. n. s. 26, 362-371. 1913 Schryver, S. B.: Some Investigations on the Phenomena of "Clot" Form- ation. Pt. I. On the Clotting of Milk, Proc. Roy. Soc. London (B) 86, 460-481. 686 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1890 Schtscherbak, A.: Beitrage zur Lehre von der Abhangigkeit des Phos- phorumsatzes von gesteigerter oder herabgesetzter Gehirnthatigkeit, Diss., St. Petersburg; through Neurolog. Centralbl. 10 (1891), 171, 172. 1878 Schiitzenberger, P.: Faits pour servir a l'histoire de la levure de biere, Compt. rend. Acad, des sci. 78, 493. 1903 Schulte-Bauminghaus, Clemens: Ueber die Wirkung und den Verbleib einiger an Milchkuhe gefutterten Mineralstoflverbindungen. (Eisen, Calcium, Chlor, Phosphorsaure.) Mitteil. d. landwirtsch. Inst. d. kbnig. Univ. Breslau 2, 25-69. 1899 Schulz, Fr. N., and O. Falk : Phosphorsaureausscheidung nach Castration, Zeit. physiol. Chem. 27, 250-254. 1901 Schulz, Fr. N., and J. Mainzer: Ueber den Verlauf der Phosphorsaure- ausscheidung beim Hunger, Ibid. 32, 268-277. 1895 Schulze, E.: Ueber die Bestimmung des Lecithingehaltes der Pflanzen- samen, Ibid. 20, 225-232. 1897 Schulze, E.: Ueber den Lecithingehalt einiger Pflanzensamen und Oelkuchen, Landwirtsch. Versuch. Stat. 49, 203-214. 1907 Schulze, E.: Ueber den Phosphorgehalt einiger aus Pflanzensamen dar- gestellter Lecithinpraparate, Zeit. physiol. Chem. 52, 54-61. 1908a Schulze, E. : Ueber die Darstellung von Lecithin und anderen Phosphatid- en aus Pflanzensamen verwendbaren Methoden, Ibid. 55, 338-351. 1908b Schulze, E.: Ueber pflanzliche Phosphatide, Chem.-Zeitung 32, 981-983. 1904 Schulze, E., and N. Castoro: Findet man in Pflanzensamen und in Keim- pflanzen anorganische Phosphate? Zeit. physiol. Chem. 41, 477-484. 1894 Schulze, E., and S. Frankfurt : Ueber den Lecithingehalt einiger vegetab- ilischer Substanzen, Landwirtsch. Versuch. Stat. 43, 307-318. 1891a Schulze, E., and A. Likiernik: Darstellung von Lecithin aus Pflanzen- samen, Ber. deut. chem. Gesell. 24, 71-74. 1891b Schulze, E., and A. Likiernik: Ueber das Lecithin der Pflanzensamen, Zeit. physiol. Chem. 15, 405-414. 1911 Schulze, E., and U. Pfenninger: Untersuchungen iiber die in den Pflanzen vorkommenden Betaine. I. Mitteilung, Ibid. 71, 174-185. 1889 Schulze, E., and E. Steiger: Ueber den Lecithingehalt der Pflanzensamen, Ibid. 13, 365-384. 1896 Schulze, E., and E. Winterstein: Ueber einen phosphorhaltigen Bestand- theil der Pflanzensamen, Ibid. 22, 90-94. 1903 Schulze, E., and E. Winterstein: Beitrage zur Kenntnis der aus Pflanzen darstellbaren Lecithine (Erste Mittheilung.) Ibid. 40, 101-119. 1906 Schumoff-Simanowski, C, and N. Sieber: Das Verhalten des Lecithins zu fettspaltenden Fermenten, Ibid. 49, 50-63. 1895 Schwartz, Emil : Ueber die Phosphorsaurestoffwechsel bei der Pneumonle, Wien. med. Blatter 18, 775-778; 792-794; 807, 808. 1894 Schwiening, Heinrich: Ueber fermentative Prozesse in den Organen, Arch. path. Anat. u. Physiol. 136, 444-481. 1904 Scofone, L.: Sul comportamento della fitina nelP organismo, Giornale d. reale Accad. di med. di Torino 10 (ser. 4), 630, 631. 1899 Scott, F. H.: On the Structure, Microchemistry and Development of Nerve Cells, with Special Reference to their Nuclein Compounds, Trans, of the Canadian Inst. 6, 405-438. 88 refs. 1906 Scott, F. H. : On the Liberation of Phosphorus Compounds from Nucleins, British Med. Jour., 1791, 1792. PHOSPHORUS METABOLISM 687 1889 Sebelien, John: (Ueber Peptone und ahnliche Substanzen), Tidsskrift for Physik og Chemi 9 (ser. 2), 234-256; through Biedermann's Centralbl. f. Agrictilturchem. 18 (1889), 717, 718. 1894 Sebelien, John: Ueber das bei der Pepsindigestion des Caseins abge- spaltene Nuclei'n, Chemiker Zeitung 18, 2018. 1895 Sebelien, John: Ueber das Verhalten des bei der Pepsindigestion des Caseins abgespaltenen Pseudonucleins, Zeit. physiol. Chem. 20, 443-454. 1904 Secheret, Georges: Contribution a. l'etude de la medication phosphoree. Etude therapeutique et clinique de l'acide anhydro-oxy-methylene- di- phosphorique, These de Paris. 131 pp. 1899 Sedlmair, August Carl: Ueber die Abnahme der Organe, insbesondere der Knochen, beim Hunger, Zeit. Biol. 37, (n. s. 19), 25-58. 1900 von Seelhorst, C, N. Georgs and F. Fahrenholtz: Einfluss des Wassers- gehaltes und der Dungung des Bodens auf die Production und die Zusammensetzung von Futterpflanzen italienisches Raigrass und Klei, Jour. f. Landwirtsch. 48, 265-286. 1879 Seeman: Zur Pathogenese und Aetiologie der Rachitis, Arch. path. Anat. u. Physiol. 77, 299-315. 1897 Senator, H.: Zur Kenntniss der Osteomalacic und der Organotherapie, Berlin, klin. Wochenschr. 34, 109-112; 143, 144. 1888 Sendtner, J.: Zur Phosphaturie, Munch, med. Wochenschr., 671, 672. 1897a Serono, Cesare: Sur les injections de lecithine chez l'homme et chez les animaux. Note preventive. Arch. ital. de biol. 27, 349-354. 1897b Serono, Cesare: Ricerche sulle iniezioni di lecitina, La Riforma medica, Napoli, 3, 616-618; 627-630; 637-639. 1902 Serono, Cesare: Considerazioni terapeutiche sull' uso della lecitina, Gaz- zetta degli ospedali (Milan) 23, II, 1246-1248. 1911 Serono, Cesare, and Antonietta Palozzi: Sui lipoidi contenuti nel tuorlo d'uovo, Archivio di farmacol. speriment. 11, 553-570; through Chem. 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Edwin Sinclair: Calcium, Mag- nesium and Phosphorus in Food and Nutrition, U. S. Dept. Agr., Office Exp. Sta. Bui. 227. 70 pp. 1910 Shibayama, Gorosaku: Some Observations Concerning Beriberi, Philip- pine Jour, of Sci., B, 5, 123-125. 688 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1857 Sick, Paul: Versuche iiber die Abhangigkeit des Phosphorsauregehaltes des Urins von der Phosphorsaurezufuhr, Arch, physiol. Heilk. 1 (n. s.), 482-503. 1909a Sieber, Nadina: Zur Frage der Einwirkung von Alkohol auf den Gehalt der tierischen Organe an Phosphatiden, Biochem. Zeit. 23, 304-323. 1909b Sieber, Nadina: Die Lipoide der Lunge, Zeit. physiol. Chem. 62, 250-253. 1909 Sieber, N., and W. Dzierzgowski : Die Zusammensetzung der Lunge, Ibid. 62, 254-258. 1879 Siedamgrotsky and Hofmeister: Die Einwirkung andauernder Milch- saureverabreichung auf die Knochen der Pflanzenfresser, Arch, wis- sensch. u. prakt. Thierheilk. 5, 243-282. 1898 Siegert, F.: Ueber typische Osteomalacie im Kindesalter, Munch, med. Wochenschr. 45, 1401-1404. 3893 Siegfried, Max: Ueber eine neue stickstoffhaltige Saure der Muskeln, Ber. d. k. Sachs. Gesell. d. Wissensch., (1893), 484-489. 1894 Siegfried, Max: Ueber Fleischsaure, Arch. Anat. u. Physiol., physiol. Abt., 401-418. 1895 Siegfried, Max: Ueber Phosphorfleischsaure, Ber. deut. chem. Gesell. 28 r 515-519. 1896 Siegfried, Max: Zur Kenntniss der Phosphorfleischsaure, Zeit. physiol. Chem. 21, 360-379. 1897 Siegfried, Max: Zur Kenntniss des Phosphors in der Frauen- und Kuh- milch, Ibid. 22, 575-578. 1899 Siegfried, Max: Zur Kenntniss der Extractivstoffe des Muskels, Ibid. 28, 524-529. 1905 Siegfried, M., and H. Mark: Zur Kenntniss des Jecorins, Ibid. 46, 492- 496. 1904 Siegmann, J.: Ueber die Anwendung des Protylin "Roche" bei Neurosen, Aerztlichen Central-Zeitung, therapeut. Ratgeber 16, 9-11. 1868 Siewert, M.: Ueber den Stickstoffumsatz der im Korper verbrauchten Eiweisskorper, Zeit. ges. Naturwissensch. 31, 458-480. 1906 Sikes, Alfred W.: On the Phosphorus and Calcium of Human Milk, Thesis, London; also Jour, of Physiol. 34, 464-480. 1904 Silberstein, James: Ueber die Anwendung organischer Phosphorprapar- ate in Form des Hamoprotagons, Deut. Med.- Zeitung, 289- 291. 1902 Simnitski, S., and P. Rodoslawow: (Beitrag zur Urologie des Ikterus), Wratschetnaja gazetta 1902, No. 40-42; through Jahresb. u. d. Fortschr. d. Thierchem. 32 (1902), 672. 1911 Simon, Friedrich: Zur Kenntnis der Autolyse des Gehirns, Zeit. physiol. Chem. 72, 463-483. 1911 Simpson, G. C. E., and E. S. Edie: On the Relation of the Organic Phos- phorus Content of "Various Diets to Diseases of Nutrition, Particular- ly Beriberi. I., Annals of Trop. Med. and Parasitology 5, 313-345; through Chem. Abs. 6 (1912), 1458. 1901 Siven, V. A.: Zur Kenntniss des Stoffwechsels beim erwachsenen Mensch- en, mit besonderer Beriicksichtigung des Eiweissbedarfs, Skand. Arch. Physiol. 11, 308-332. 1903 Siwertzow, D. I.: (Vergleichende Untersuchungen iiber den Lecithin- gehalt bei menschlichen Embryonen und bei Kindern im fruhen Lebensalter), Diss., St. Petersburg; through Biochem. Centralbl. 2 (1904), 310, 311. PHOSPHORUS METABOLISM 689 1908 Sleeswyk, J. G.: Ueber die angebliche bakteriolytische Eigenschaft des Lecithins und iiber die Immunisierungs mittels Lecithin-Typhustoxine, Deut. med. Wochenschr. 34, II, 2263-2265. 1906a Slowtzoff, B.: Ueber die Resorption des Lecithins aus den Darmkanal, Beitrage z. chem. Physiol, u. Path. 7, 509-513. Other spellings of this author's name are: Slowtzow, Slowzow, and Slovcov. 1906b Slowtzoff, B.: Die Wirkung des Lecithins auf den Stoffwechsel, Ibid. 8, 370-388. 1909a Slowtzoff, B.: (Ueber den Einfluss der Fischnahrung auf den Phosphor-, Kalk- und Magnesiaumsatz), Verhandl. Gesell. Russ. Aerzte, St. Peters- burg 76, 220; through Zentralbl. Biochem. u. Biophys. 10 (1910), 375, 376. 1909b Slowtzoff, B.: (Der Phosphor-, Calcium- und Magnesia- Stoffwechsel bei Ersatz der Fleischnahrung durch Fisch), Russki Wratsch, 8, 1232- 1234; through Jahresb. u. d. Fortschr. d. Thierchem. 39 (1909), 606. 1911 Smetanka, Franz: Zur Herkunft der Harnsaure beim Menschen. Ein Beitrag zur Physiologie der Drusen, Arch. ges. Physiol. 138, 217-274. 1889 Smith, Fred: The Chemistry of the Urine of the Horse, Proc. of the Royal Soc. 46, 328-340. 1912 Smith, J. Lorrain, and W. Mair: The Development of Lipoids in the Brain of the Puppy, Jour, of Path, and Bacteriol. 17, 123-126. 1904 Smolenski, J.: O znaczeniu leczniczen zwaizkow glicerynofosforowych u dzieci, (Ueber der therapeutischen Wert der Glycerophosphatverbind- ungen bei Kindern), Medycyna, Warszawa, 32, 733; 756; 777; through Jahrb. Kinderheilk. 61 (1904), 680. 1909 Smolenski, K.: Zur Kenntnis der aus Weizenkeimen darstellbaren Phos- phatide. V. Mitteilung. Ueber Phosphatide. (Winterstein's series.) Zeit. physiol. Chem. 58, 522-526. 1905 Snowman, J. : The Therapeutic Action of Glycerophosphates in Combina- tion with Albumens, Birmingham Med. Review 58, 519-526. 1888 Soldner, F.: Die Salze der Milch und ihre Beziehungen zu dem Verhalten des Caseins, Landwirtsch. Versuch. Stat. 35, 351-436. 1902 Soldner, F.: Die Aschenbestandtheile des neugeborenen Menschen und der Frauenmilch, Verhandl. d. Gesell. f. Kinderheilk. 19, 154-160. 1902 Soetbeer, Franz: Ueber Phosphaturie, Jahrb. Kinderheilk. 56, 1-10. 1902 Soetbeer, Franz, and Hans Krieger: Ueber Phosphaturie, Deut. Arch. klin. Med. 72, 553-559. 1912 Sokolov, S. K.: (Assimilation of Sodium Nucleinate in Old and Young Organisms), Russki Wratch 11, 1065-1067; through Chem. Abs. 7 (1913), 1896. 1909 Soli, Ugo: Modifications du developpement des os chez les animaux prives de thymus, Archives ital. de biol. 52, 217-224; through Chem. Abs. 5 (1911), 2128. 1902 Somerfeld, Paul, and Wilhelm Caro: Zur Kenntniss der Ausniitzung von Phosphor und Stickstoff bei reiner Milchernahrung alterer Kinder, Arch. Kinderheilk. 33, 161-166. 1880 Sotnitschewsky: Glycerinphosphorsaure im normal en menschlichen Harn, Zeit. physiol. Chem. 4, 214-216. 690 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1878 Soxhlet, F.: Untersuchungen iiber den Stoffwechsel des Saugkalbes, Erster Ber. u. d. Arbeiten d. K. K. landwirtsch. Versuchsstat. z. Wien. 1878, 101-105; also Oesterreich. landwirtsch. Wochenbl. 4 (1878), 290- 292, 315, 316, 327, 328; through Jahresb. ii. d. Fortschr. d. Thierchem. 8 (1878), 333-337 and Centralbl. f. agr. Chem. 7 (1878), 748-752, 887- 891. 1893 Soxhlet, F.: Die chemischen Unterschiede zwischen Kuh- und Frauen- milch und die Mittel zu ihrer Ausgleichung, Munch, med. Wochenschr. 40, 61-65. 1903 Soxhlet, F.: Bezeichnung und Beschaffenheit des fur Futterungszwecke dienenden phosphorsauren Kalks, Landwirtsch. Versuch. 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Arch. 17, 419. 1906 Stanek, Vladimir: Ueber die quantitative Bestimmung von Cholin und Betain in pflanzlichen Stoffen und einige Bemerkungen iiber Lecithine, Zeit. physiol. Chem. 48, 334-346. 1845 Stark, James: On the Bones. Part I. Chemical Constitution of the Bones of the Vertebrated Animals, Edinburgh Med. and Surg. Jour. 63, 308- 325. 1910 Starkenstein, Emil: Die biologische Bedeutung der Inositphosphorsaure, Biochem. Zeit. 30, 56-98. 1911 Starkenstein, Emil: Ionenwirkung der Phosphorsauren, Ibid. 32, 243-265. 1902a Stassano, H., and F. Billon: Contribution a la connaissance de Taction de la lecithine sur les leucocytes, Compt. rend. Soc. de biol. 54, 167-169. 1902b Stassano, H., and F. Billon : Sur la leucocytose produite dans le peritoine par les injections de lecithine, Ibid. 54, 169, 170. 1902c Stassano, H., and F. Billon: Contribution a la connaissance de Taction de la lecithine sur les elements figures du sang, Compt. rend. Acad, des sci. 134, 318-321. 1902d Stassano, H., and F. Billon: Sur la diapedese des leucocytes charges de lecithine et sur Tabsorption de la lecithine par l'endothelium vasculaire, Ibid. 134, 430-432. 1903a Stassano, H., and F. Billon: La lecithine n'est pas dedoublee par le sue pancreatique meme kinase, Compt. rend. Soc. de biol. 55, 482, 483. 1903b Stassano, H., and F. Billon: La lecithine pure ingeree se retrouve inalteree dans la lymphe provenant des chyliferes, Ibid. 55, 924-926. PHOSPHORUS METABOLISM 691 1907a Steel, Matthew, and William J. Gies: On the Use of Bone Ash with the Diet, in Metabolism Experiments on Dogs, Amer. Jour. Physiol. 20, 343-357. 1907b Steel, Matthew, and William J. Gies: On the Chemical Nature of Para- nucleoprotagon, a New Product from Brain, Ibid. 20, 378-398. 1869 Steiner, Jacob: Ueber die pathologisch-anatomischen Veranderungen bei der Osteomalacie, Inaug. 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F. 41], 366-395. 1913b Stepp, Wilhelm: Fortgesetzte Untersuchungen liber die Unentbehrlich- keit der Lipoide fiir das Leben. Ueber das Verhalten der lebenswich- tigen Stoffe zu den Lipoidextraktionsmitteln, Ibid. 62 [N. F. 44], 405- 417. 1907 Stern, M., and H. Thierf elder: Ueber die Phosphatide des Eigelbs, Zeit. physiol. Chem. 53, 370-385. 1893 Sternberg, Maximilian: Ueber Diagnose und Therapie der Osteoinalacie, Zeit. klin. Med. 22, 265-313. 1904 Steudel, H.: Zur Kenntnis der Thymusnucleinsauren. I. Mitt., Zeit. physiol. Chem. 42, 165-170. 1905a Steudel, H. : Zur Kenntnis der Thymusnucleinsauren. II. Mitt., Ibid. 43, 402-405. 692 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1905b Steudel, H.: Zur Kenntnis der Thymusnucleinsauren. III. Mitt., Ibid. 46, 332-336. 1906a Steudel, H. : Ueber die Oxydation der Nucleinsaure. I. 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Physiol. 156, 384-394. 1901 Thudichum, John Louis William : Die chemische Konstitution des Gehirns des Menschen und der Tiere, Tubingen. 339+XII pp. 1876 Thudichum and Kingzett : Phosphorhaltige Substanzen aus dem Gehirne, Ber. deut. chem. Gesell. 9, 950. 1885 Tichomiroff, A.: Chemische Studien iiber die Entwickelung der Insekten- eier, Zeit. Physiol. Chem. 9, 518-532. 1904 Tigerstedt, Carl: Ein Beitrag zur Kenntniss des Phosphorstoffwechsels beim erwachsenen Menschen, Skand, Arch. Physiol. 16, 67-78. 1911 Tigerstedt, Robert: Zur Kenntniss der Aschenbestandteile in der frei- gewahlten Kost der Menschen, Ibid. 24, 97-112. 1912 Timoshok, Peter: Ueber den Einfluss des Nuclein-natriums (Merck) auf die fermentative Function der Organe und Gewebe bei Staphylo- kokkeninfection, Inaug. Diss., St. Petersburg, 1912; through Zentralbl. Biochem. u. Biophys. 15 (1913), 762, 763. 1906 Timpano, Pietro: La cura fosforata col "Protylin," La Clinica moderna 12, 565-568. 1905 Tobler, Ludwig: Phosphaturie und Calcariurie, Arch. exp. Path. u. Ther. 52, 116-139. 1911 Tobler, Ludwig: Ueber Veranderungen im Mineralstoffbestand des Sauglingskorpers bei akuten und chronischen Gewichtsverlusten, Jahrb. Kinderheilk. 73, 566-585. 1910 Tobler, L., and F. Noll: Zur Kenntnis des Mineralstoffwechsels beim gesunden Brustkind, Monatsschr. Kinderheilk. 9, 210-217. 1908 Togami, K.: Notiz zur Kenntnis des Phosphorstoffwechsels, Med. Klinik, Wien. 4, II, 1837, 1838. 1897 Tomlinson, Oscar R.: Nuclein Solution; a Clinical Study, Jour. Amer. Med. Assoc. 29, 478, 479. 1898 Tonelli, Cesare: Contributo clinico all' uso della fosfoluteina Serono, Gazzett. degli ospedali e delle cliniche 19, I, 710, 711. 1909 Tornani, E.: (Lecithin and other Components of Egg Yolks), Bollettino chim. farmaceut. 48, 520, 521; through Jour. Chem. Soc. 96, II (1909), 818. 1910-11 Towles, Caroline: Untersuchungen iiber den Einfluss des Phosphor- lebertrans auf den Stoffwechsel eines rachitischen Sauglings, Zeit. Kinderheilk., Originalien 1, 346-355. 1911 Toyofuku, T.: Ueber die parathyreoprive Veranderung des Rattenzahnes, Frankfurter Z. Pathol. 7, 249-294; through Chem. Abs. 5 (1911), 2397. 1911 Trier, Georg: Aminoathylalkohol, ein Produkt der Hydrolyse des "Leci- thins" (Phosphatids) der Bohnensamen, Zeit. physiol. Chem. 73, 383- 388. 1912 Trier, Georg: Ueber die Gewinnung von Aminoathylalkohol aus Eileci- thin, Ibid. 76, 496-498. 1913a Trier, Georg: Ueber die nach den Methoden der Lecithindarstellung aus Pflanzensamen erhaltlichen Verbindungen. I. Mitteilung. Einleitung- Bohnensamen, Ibid. 86, 1-32. 1913b Trier, Georg: Ueber die nach den Methoden der Lecithindarstellung aus Pflanzensamen erhaltlichen Verbindungen. I. Mitteilung. Einleitung- gleichende Hydrolyse von Eilecithin, Ibid. 86, 141-152. PHOSPHORUS METABOLISM 697 1913c Trier, Georg: Ueber die nach den Methoden der lecithindarstellung aus Pflanzensamen erhaltlichen Verbindungen. III. Mitteilung. Hafer- samen, Ibid. 86, 153-173. 1913d Trier, Georg: Ueber die nach den Methoden der Lecithindarstellung aus Pflanzensamen erhaltlichen Verbindungen. IV. Mitteilung. Erbsen, Schwarzkiefer, Reis, Ibid. 86, 407-414. 1874 Tripier, L.: Recherches sur la production artificielle du rachitisme, Archives de physiol. normale et pathol., (1874) 108. 1909 Trowbridge, P. F., and F. W. Woodman: Changes in the Composition of the Skeleton of Beef Animals. (First paper.) Jour. Ind. and Engin. Chem. 1, 725-732. 1903 Trunz, August: Ueber die mineralischen Bestandteile der Kuhmilch und ihre Schwankungen im Verlaufe einer Laktationsperiode, Zeit. physiol. Chem. 40, 263-310. 1912a Tschernorutzky, Helene: Ueber das Vorkommen von Nucleinsaure in reifen Heringseiern, Ibid. 80, 194-197. 1912b Tschernorutzky, Helene: Das Verhalten einiger Nucleinsauren zu glukosidspaltenden Fermenten, Ibid. 80, 298-306. 1911 Tschernoruzki, M.: Ueber die Wirkung der Nucleinsaure auf die ferment- ativen Prozesse im tierischen Organismus. Biochem. Zeit. 36, 363-375. 1912 Tschernoruzki, M.: Ueber die gegenseitige Wirkung von Nucleinsaure und nucleinspaltenden Ferment im tierischen Organismus, Ibid. 44, 353-391. 1911 Tschikste, Anastasia: Ueber die Wirkung des im Schilddrusenkolloid enthaltenen Nucleoproteides bei Morbus Basedowii, Deut. med. Woch- enschr. 37, 2217-2222. 1889 von Tschirwinski, N. : Zur Frage uber das Wachstum der Rohrenknochen und den muthmasslichen Zusammenhang dieses Wachstums mit dem Wechsel der Schneidezahne bei den Schafen, Landwirtsch. Jahrb. 18, 463-475. 1884 Tuczek, Franz: Mittheilungen von Stoffweehseluntersuchungen bei abtinirenden Geisteskranken, Arch. Psychiat. u. Nervenkrankh. 15, 784- 799. 1906 Tunnicliffe, F. W.: The Behavior in the Body of Certain Organic and Inorganic Phosphorus Compounds, Congres internat. de med., Lis- bonne, 15, sect. 4, 181-193 (English); also Archives internat. de pharmacodyn. et de ther. 16, 207-220 (English). 1906 Tutin, F., and A. C. O. Hann: The Relation between Natural and Synthet- ical Glyeerylphosphorie Acids. II. Jour, of the Chem. Soc. 89, 1749- 1758. 1909 Tyshnjenko, A.: (Ueber den Einfluss des Phytins und des glycerinphos- phorsauren Natriums auf den Stickstoff- und Phosphorstoffwechsel.) Diss., St. Petersburg. 117 pp. Through Jahresb. u. d. Fortschr. d. Thier- chem. 39 (1909), 598. (1911) Uffenheimer, A., and Yoshiyiro Takeno: Der Nachweis des Kasei'ns in den sogenannten Kase'inbrockeln des Sauglingsstuhls mit Hilfe der biologischen Methodik, insbesondere der Anaphylaxie, Zeit. Kinder- heilk. 2, 32-61; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 298. 1895 Uhlrich: Knochenbruchigkeit, Ber. ii. d. Veterinarwesen im Konigreiche Sachsen 40, 41. 698 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1901 Ulpiani, C: Attivita ottica della lecitina,, Atti d. reale Accad. Lincel, ser. 5., Rend. Classe di sci. fis., mathemat. e natur. 11, I, 368-375; 421- 425. 1902 Ulpiani, C, and G. Lelli: Su un nuovo proteide del cervello, Ibid. 11, II, 18-24; also Gazzetta chim. ital. 32, 1, 466-473 (taken through Jahresb. ii d. Fortschr. d. Thierchem. 32 (1902), 528, 529). 1911 Ulrich, Chr.: Beitrage zur Kenntnis des Fischfleisches, Arch, der Phar- macie 249, 68-92. 1896 Umber, F.: Ueber den Einfluss nucleinhaltiger Nahrung auf die Harn- saure-bildung, Zeit. klin. Med. 29, 174-189. 1900 Umber, F.: Das Nucleoproteid des Pankreas, Ibid. 40, 464-479. 1901 Umber, F.: Ueber die fermentative Spaltung der Nukleoproteide ira Stoffwechsel, Ibid. 43, 282-303. 1895 Umikoff: Biology of Phosphorus, Diss., St. Petersburg. 1908 Underhill, Frank P., and Tadasu Saiki: The Influence of Complete Thyroidectomy and of Thyroid Feeding upon Certain Phases of Intermediary Metabolism, Jour. Biol. Chem. 5, 225-241. 1902 Ungar, E.: Zur Phosphorbehandlung der Rachitis, Munch, med. Wochen- schr. 49, 999-1003. 1907 Urano, Fremihiko: Neue Versuche iiber die Salze des Muskels, Zeit. Biol. 50 (N. F. 32), 212-246. 1913 Ushenko, A. I.: (Metabolism in Thyroidectomized Dogs), Russki Wratch 11, 1751-1754; through Chem. Abs. 7 (1913), 3150. 1910 Usuki: Die Fettverdauung im Magen und Dunndarm und ihre Beein- flussung durch Lecithin, Arch. exp. Path. u. Pharm. 63, 270-293. 1909 Vageler, Hans: Untersuchungen iiber das Vorkommen von Phosphat- iden in vegetabilischen und tierischen Stoffen, Biochem. Zeit. 17, 189- 219. 1854 Valenciennes, A., and E. Fremy : Recherches sur la composition des oeuf s dans la serie des animaux, Compt. rend. Acad, des sci. 38, 469-484. 1905 Valenti, Adriano: II nucleone nel latte di donna nei diversi periodi di allattamento, Soc. med. -chir. di Pavia, Sitz. 19, V, 1905; through Biochem. Centralbl. 4 (1905-6), 550. 1908 Valenti, Adriano: Sul contenuto in nucleone del latte di donna durante l'allattamento, Archivio di farmacol. speriment. 7, 447-459; through Jahresb. u. d. Fortschr. d. Thierchem. 38 (1908), 284. 1890-1 Van Acker en, Friedr.: Ueber Harnsaureausscheidung bei einigen Krank- heiten, insbesondere bei Morbus Brightii, Charite-Annalen 17, 206-227. 1909a Van Dam, W.: Beitrag zur Kenntnis der Labgerinnung, Zeit. physiol. Chem. 58, 295-330. 1909b Van Dam, W.: Ueber die Wirkung des Labs auf Paracaseinkalks, Ibid. 61, 147-163. 1912 Van Dam, W.: Die Verdauung des Caseins durch Pepsin vom Kalb, Schwein und Rind, Ibid. 79, 247-273. 1907 Van Herwerden, M.: Beitrag zur Kenntnis der Labwirkung auf Casein, Ibid. 52, 184-206. 1905 Van Hoogenhuyze, C. J. C, and H. Verploegh: Beobachtungen iiber die Kreatininausscheidung beim Menschen, Ibid. 46, 415-471. 1900 Vannini, Giuseppe: II ricambio materiale nell' anchilostomoanemia, II Policlinico 7, Sez. med. 29-51. PHOSPHORUS METABOLISM 699 1904 Vannini, Giuseppe: Beitrag zum Stoffwechsel bei Chlorose, Arch. path. Anat. u. Physiol. 176, 375-413. (1911) Vannini, Giuseppe: Der Stoffwechsel bei einem Falle von Kalkablager- ungen unter der Haut (Kalkgicht), Zentralbl. f. d. Physiol, u. Path. d. Stoffw. N. F. 6, 520-523; through Jahresb. ii. d. Fortschr. d. Thierchem. 41, (1911), 484. 1914 Van Slyke, Donald D.: The Hexone Bases of Casein, Jour. Biol. Chem. 16, 531-538. 1911 Van Slyke, Donald D., and George F. White: The Relation Between the Digestibility and the Retention of Ingested Proteins, Ibid. 9, 219-229. 1907 Van Slyke, Lucius L., and Alfred W. Bosworth: I. Some of the First Chemical Changes in Cheddar Cheese. II. The Acidity of the Water- Extract of Cheddar Cheese, N. Y. Agr. Exp. Sta. (Geneva), Tech. Bui. No. 4. 22 pp. 1909 Van Slyke, Lucius L., and Alfred W. Bosworth : A Volumetric Method for the Determination of Casein in Milk, Jour. Ind. and Engin. Chem. 1, 768-771; also N. Y. Agr. Exp. Sta. (Geneva), Tech. Bui. No. 10. 20 pp. 1912 Van Slyke, Lucius L., and Alfred W. Bosworth: Composition and Proper- ties of Some Casein and Paracasein Compounds and their Relations to Cheese, N. Y. Agr. Exp. Sta., Tech. Bui. No. 26. 32 pp. 1913a Van Slyke, Lucius L., and Alfred W. Bosworth: Method of Preparing Ash-free Casein and Paracasein, Jour. Biol. Chem. 14, 203-206. •1913b Van Slyke, Lucius L., and Alfred W. Bosworth: Preparation and Com- position of Unsaturated or Acid Casemates and Paracaseinates, Ibid* 14, 211-225. 1913c Van Slyke, Lucius L., and Alfred W. Bosworth: Valency of Molecules and Molecular Weights of Casein and Paracasein, Ibid. 14, 227-230. 1913d Van Slyke, Lucius L., and Alfred W. Bosworth: Composition and Prop- erties of the Brine-Soluble Compound in Cheese, Ibid. 14, 231-236. 1902 Van Slyke, L. L., and E. B. Hart: I. A Study of Some of the Salts Formed by Casein and Paracasein with Acids: Their Relations ta American Cheddar Cheese, Amer. Chem. Jour. 28, 411-438; also N. Y. Agr. Exp. Sta. (Geneva), Bui. No. 214, pp 53-79. 1904 Van Slyke, L. L., and E. B. Hart: Chemical Changes in the Souring of Milk and their Relations to Cottage Cheese, N. Y. Agr. Exp. Sta. (Geneva), Bui. No. 245, 36 pp. 1905a Van Slyke, L. L., and E. B. Hart: X. Casein and Paracasein in Some of their Relations to Bases and Acids, Amer. Chem. Jour. 33, 461-496. 1905b Van Slyke, L. L., and E. B. Hart: The Proteids of Cream, Butter and Buttermilk in Relation to Mottled Butter, Jour, of the Amer. Chem. Soc. 27, 679-690. 1905c Van Slyke, L. L., and E. B. Hart : Some of the Relations of Casein and Paracasein to Bases and Acids, and their Application to Cheddar Cheese, N. Y. Agr. Exp. Sta. (Geneva), Bui. No. 261. 37 pp. 1906 Van Slyke, L. L., and D. D. Van Slyke: I. On the Action of Dilute Acids upon Casein when no Soluble Compounds are Formed. II. The Hydrolysis of the Sodium Salts of Casein, N. Y. Agr. Exp. Sta., Tech. Bui. No. 3, pp 75-160. 1907 Van Slyke, L. L., and D. D. Van Slyke: The Action of Dilute Acids upon Casein when no Soluble Compounds are Formed, Amer. Chem. Jour. 38, 383-456. 700 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1894a Vaudin, L.: Sur l'acide citrique et le phosphate de chaux en dissolution dans le lait, Annales de lTnst. Pasteur 8, 502-505. 1894b Vaudin, L.: Sur le phosphate de chaux en dissolution dans le lait, Ibid. 8, 856-862. 1895 Vaudin, L.: Sur le phosphate de chaux du lait, Compt. rend. Acad, des sci. 120, 785-787. 1897a Vaudin, L.: Sur la richesse du lait en elements mineraux et en phos- phates terreux, Annales de lTnst. Pasteur, 11, 541-544. 1897b Vaudin, L.: Sur le phosphate de chaux du lait, La Laiterie, 51, 52; through Vierteljahreschr. ii. d. Fortschr. a. d. Geb. d. Chem. d. Nah- rungs- u. Genussmittel 12 (1897), 167. 1893 Vaughan, Victor C, Fredk. G. Novy, and Charles T. McClintock: The Germicidal Properties of Nucleins. A Preliminary Contribution from the Laboratory of Hygiene of Michigan University, Medical News 62, 536-538. 1811 Vauquelin, M.: Historique des travaux chimiques entrepris jusqu'ici sur la matiere cerebrale, Ann. of Philos. 1, 332; Ger. trans, in Schweigger's J. 8 (1813), 430-460. 1908 Vay, F.: Ueber die immunisierende Wirkung von Lecithinausziigen aus Pestbazillen, Deut. med. Wochenschr. 34, II, 2265, 2266. 1912 Vedder, E. B.: A Fourth Contribution to the Etiology of Beriberi, Phil- ippine Jour, of Sci., B, 7, 415-422; through Exp. Sta. Record 28 (1913), 764, 765. 1912 Vedder, E. B., and E. A. Clark: A Study of Polyneuritis Gallinarum. A 5th Contribution to the Etiology of Beriberi, Ibid. 7, 423-462; through Exp. Sta. Record 29 (1913), 180. 1913 Venturi, F., and V. Massella : Influenza della Fitina sulla eliminazione dei composti azotuti in individuo normale, Arch, di Farm, sperim. e Sci. affini 16, 97-118. 1849 Verdeil, F.: Untersuchung der Blutasche verschiedener Thiere, Annalen der Chem. u. Pharm. 69, 89-99. 1900 Ver Eecke, A.: Les echanges materiels dans leurs rapports avec les phases de la vie sexuelle. Etude des lois des echanges nutritifs pen- dant la gestation, Memoires couronnes et autres memoires publies par l'Acad. roy. de med. de Belgique 15 (1902), 1-159. 1884 Vermeulen, Charles E. A.: Onderzoekingen omtrent de physiologische werking van het natrium-hypophosphiet, Doct. Diss., Amsterdam. 49 pp. Through Jahresb. ii. d. Fortschr. d. Thierchem. 14 (1884), 249- 252. 1913 Vernon, H. M. : Die Rolle der Oberflachenspannung und der Lipoide f iir die lebenden Zellen, Biochem. Zeit. 51, 1-25. 1898 Villinger, A.: Vom Phosphor-Stoffwechsel, Verhandl. d. Congr. f. innere Med. 16, 328-334. 1902 Vincent, Swale: Die Eiweisskorper der glatten Muskelfasern, Zeit. physiol. Chem. 34, 417-429. 1852 Virchow, Rud.: Ueber parenchymatous Entzundung, Arch. path. Anat. u. Physiol. 4, 261-324. 1853 Virchow, Rud: Das normale Knochenwachsthum und die rachitische Storung desselben, Ibid. 5, 409-507. 1911 Vivier, A.: (Einfluss der Fiitterung mit Rubenblattern auf die Produc- tion und Zusammensetzung der Milch), Annal. des Falsific. 4, 638-641; through Zeit. Unters. d. Nahrungs- u. Genussmittel 25 (1913), 61, 62. PHOSPHORUS METABOLISM 701 1905 Voltz, W.: Ueber den Einfluss des Lezithins auf den Eiweissumsatz ohne gleichzeitige Asparaginzufuhr und bei Gegenwart dieses Amids, Arch. ges. Physiol. 107, 415-425. 1880a Voelcker, Augustus: On the Composition of Ewe's Milk, Jour, of the Roy. Agr. Soc. of England, [2] 16, 592-594. 1880b Voelcker, Augustus: On the Composition of Goat's Milk, Ibid. [2] 16, 594, 595. 1875 Vogt: Ueber Wirkung der Milchsaure auf Knochenwachsthum, Berlin. klin. Wochenschr. 12, 473. 1901 Vogt, Hans: Ein Stoffwechselversuch bei akuter Gicht, Arch. klin. Med. 71, 21-28. 1906 Vogt, Hans: Der zeitliche Ablauf der Eiweisszersetzung bei verschieden- er Nahrung, Beitrage z. chem. Physiol, u. Path. 8, 409-430. 1877a Voit, Carl: Ueber das Verhalten der Kalkschalen der Huhnereier bei der Bebrutung, Zeit. Biol. 13, 518-526. 1877b Voit, Carl. Ueber den Einfluss kalkarmen Futters auf die Knochen, Amt- licher Ber. d. 50 Versamml. deut. Naturforsch. u. Aerzte in Miinch- en, 242-244. 1878 Voit, Carl: Ueber den Einfluss kalkarmen Futters auf die Knochen, Zeit. Tiermedizin 4, 128-131. 1881 Voit, Carl: Physiologie des allgemeinen Stoffwechsels und der Ernah- rung, L. Hermann's Handbuch 6, Thl. 1, 79. 1880 Voit, Erwin: Ueber die Bedeutung des Kalkes fur den thierischen Organ- ismus, Zeit. Biol. 16, 55-118. 1909-10 Voit, E., and J. Zisterer: Bedingt die verschiedene Zusammensetzung der Eiweisskorper auch einen Unterschied in ihren Nahrwert? II. Mitteilung. Die physiologische Wertigkeit des Kaseins und seiner Spaltungsprodukte, Ibid. 53 (N. F. 35), 457-498. 1904 Volhard, J.: Wie wirkt ein Ueberschuss von kohlensaurem Kalk im Fut- ter auf die Ausnutzung der Futterbestandteile, Landwirtsch. Versuch. Stat. 61, 305-312. 1873 Volkmann, A. W.: Ueber die naheren Bestandteile der menschlichen .Knochen, Konigl. Sachs. Gesell. d. Wissensch. zu Leipzig. Ber. u. d. Verhandl. mathemat. -phys. Classe 25, 275-305. 1913 Volter, B.: Beitrage zur Kenntnis der Chemie der Krebstumoren, Biochem. Zeit. 55, 260-265. 1910 Vorbrodt, Wladyslaw: Untersuchungen iiber die Phosphorverbindungen in den Pflanzensamen, mit besonderer Berucksichtigung des Phytins, Bui. internat. de l'Acad. des sci. de Cracovie, Serie A 1910, 414-511. 1899 Vosgien and Geroline: Recherches sur l'assimilabilite des phosphates mineraux et leur action dans l'alimentation, Compt. rend. Soc. de biol. [11] 1, 770-772. 1909 Vozarik, Am.: Aciditat, Ammoniak, Phosphorsaure und Gesamtstickstoff im Kinderharn bei eiweissarmer und eiweissreicher Ernahrung, Arch. Kinderheilk. 50, 199-242. 1911 Vuaflart, L.: Azote et acide phosphorique dans le ble et dans la farine, Jour, d'agric. pratique (n. s.) 22, 395, 396. 1912 Vuaflart, L.: Azote et acide phosphorique dans le ble et dans la farine, Rept. Eighth Internat. Congr. of Applied Chem. 15, 371-374. 1905a Wagner, J. Ph.: Le phosphate de chaux dans l'alimentation du betail de la ferme, Jour. Soc. cent. agr. Belg. 52, 268-281. 702 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1905b Wagner, J. Ph.: Le phosphate de chaux dans l'alimentation du betail S, la ferme, Second Congr. internat. de Paliment. rationelle du betail, Rappt, 103-114. 1906 Wagner, J. Ph.: Le phosphate de chaux dans l'alimentation du betail de la ferme, Bui. mensuels Soc. des sci., agric. et arts de la Basse- Alsace, 40, 100-109. 1892 Wagner, Paul A. E.: Ueber die diuretische Wirkung des Borax, Inaug. Diss., Kiel. 29 pp. 1902 Waldvogel: Der Stoffwechsel im Gichtanfall, Centralbl. Stoffwechsel- u. Verdauungskrankh. 3, 1-6, 242. 1906 Waldvogel and Tintemann : Zur Chemie des Jecorins, Zeit. physiol. Chem. 47, 129-139. 1877 Walter, F.: Untersuchungen iiber die Wirkung der Sauren auf den tier- ischen Organismus, Exper. Arch. 7, 148. 1891 Walter, G.: Zur Kenntniss des Ichthulins und seiner Spaltungsproducte, Zeit. physiol. Chem. 15, 477-494. 1912a Walters, E. H.: Studies in the Action of Trypsin. I. On the Hydroly- sis of Casein by Trypsin, Jour. Biol. Chem. 11, 267-305. 1912b Walters, E. H.: Studies in the Action of Trypsin. II. (a) On the Influ- ence of the Products of Hydrolysis upon the Rate of Hydrolysis of Casein by Trypsin; (b) The Autohydrolysis of the Caseinates, Ibid. 12, 43-54. 1910 Ward, Edgar P.: Nuclein by Intravenous Injection in the Treatment of Tuberculosis, Medical Record (N. Y.), 77, 528-531. 1890 Warschauer, Eugen: Ueber Osteomalacie und Untersuchungen des Stoff- wechsels bei derselben, Inaug. Diss., Wurzburg. 24 pp. 1908 Waters, H. J.: The Capacity of Animals to Grow under Adverse Condi- tions, Proc. Soc. Prom. Agr. Sci. 1908, XXIX. 1909 Waters, H. J.: The Influence of Nutrition upon the Animal Form, Ibid. XXX. 1909-10 Waters, H. J.: How an Animal Grows, Kansas State Board of Agr., 17th Biennial Rept., 1909-1910. 1867 Weber, Otto: Zur Kenntniss des Osteomalacie, insbesondere der senilen und iiber das Vorkommen von Milchsaure in osteomalacischen Knoch- en, Arch. path. Anat. u. Physiol. 38, 1-15. 1901 Weber, S.: Versuche iiber kunstliche Einschrankung des Eiweissumsatzes bei einem fiebernden Hammel, Arch, exper. Path. u. Pharm. 47, 19-47. 1905 Wechsler, Emil: Vorlaufige Mitteilung iiber Ernahrungsversuche mit Phytin und Protylin, Allgem. Wiener med. Zeitung 50, 110, 111. 1872 Wegner, Georg: Der Einfluss des Phosphors auf den Organismus, Arch. path. Anat. u. Physiol. 55, 11-45. 1876 Wegner, Rudolph: Experimentelle Beitrag zur Wirkung der Milchsaure- injection auf die lebenden Knochen, Inaug. Diss., Greifswald. 1895a Weintraud, W.: Ueber Harnsaurebildung beim Menschen, Arch. Anat. u. Physiol., physiol. Abt., 382-385. 1895b Weintraud, W.: Ueber den Einfluss des Nucleins der Nahrung auf die Harnsaurebildung, Berlin, klin. Wochenschr. 32, 405-409. 1900 Weintraud, W.: Ueber den Abbau des Nucleins im Stoffwechsel, Ver- handl. d. 18. Congr. f. innere Med., 232-243. PHOSPHORUS METABOLISM 703 1911 Weiser, Stephan: Ueber den Ca-, Mg-, P- und N-Umsatz des ungarischen Landschweines, Mitt, der Vers. -Anstalt Ungarns 1911, 733-746; through Jahresb. u. d. Fortschr. d. Thierchem. 41 (1911), 552. 1912 Weiser, Stephan: Ueber den Ca-, Mg-, P- und N-Umsatz des wachsenden Schweines, Biochem. Zeit. 44, 279-289. 1871a Weiske, H.: Ueber den Einfluss von kalk- und phosphorsaurearmer Nah- rung auf die Zusammensetzung der Knochen. I. and II., Zeit. Biol. 7, 179-184; 333-337. 1871b Weiske, H.: Ueber den Einfluss des als Beigabe zum Futter gereichten phosphorsauren Kalkes auf dem Aschengehalt der Milch, Annalen d. Landwirtschaft in den Konigl. Preussisch. Staaten 11, 309, 310. 1872a Weiske, H. : Ueber den Enfluss verschiedener der Nahrung beigemengter Erdphosphate auf die Zusammensetzung der Knochen, Zeit. Biol. 8, 239-245. 1872b Weiske, H.: Ueber die verschieden Zusammensetzung des Ziegenharns bei rein vegetabilischer und rein animalischer Nahrung, Ibid. 8, 246- 250. 1873 Weiske, H.: Ueber Assimilation von phosphorsaurem Calcium, Jour. f. Landwirtsch. 21, 139-154. 1874 Weiske, H.: Ueber Knochenzusammensetzung bei verschiedenartiger Ernahrung. (Vierte Abhandlung.) Zeit. Biol. 10, 410-438. 1880 Weiske, H.: Untersuchungen iiber die Ernahrungs-Vorgange des Schafes in seinen verschiedenen Altersperioden, Landwirtsch. Jahrb. 9, 205- 300. 1886 Weiske, H.: Ueber die Zusammensetzung von Blut, Leber und Fleisch unter verschieden Verhaltnissen, Jour. f. Landwirtsch. 34, 417- 424. 1888 Weiske, H.: Uebt die Beigabe von neutral em phosphorsauren Calcium zu normal beschaffenem Futter einem Einfluss auf die Korpergewichts- zunahme der Thiere und auf das Gewicht, resp. die Zusammensetzung der Knochen aus? Ibid. 36, 279-294 1889 Weiske, H.: Untersuchungen iiber Qualitat und Quantitat der Vogel- knochen und Federn in verschiedenen Altersstadien, Landwirtsch. Ver- such. Stat. 36, 81-103. 1891a Weiske, H.: Uebt anhaltende Aufnahme von sauren Mineralsalzen einen Einfluss auf die Zusammensetzung der Knochen aus? I. Ibid. 39, 17-30. 1891b Weiske, H.: Uebt anhaltende Aufnahme von sauren Mineralsalzen einen Einfluss auf das Korpergewicht der Tiere und auf die Zusammen- setzung der Knochen aus? II. Abhandlung. Ibid. 39, 241-268. 1892 Weiske, H. : Versuche iiber den Einfluss welchen die Beigabe verschieden- er Salze zum Futter auf das Korpergewicht und die Zusammensetzung der Knochen und Zahne ausiibt. III. Abhandlung. Ibid. 40, 81-108. 1893 Weiske, H.: Zur Beigabe von phosphorsauren Kalk zum Futter der Tiere, Milch-Zeitung 22, 322. 1894 Weiske, H.: Ueber die Zusammensetzung der Skelette von Tieren gleicher Art und Rasse, sowie gleichen Alters, aber verschiedener Grosse, Landwirtsch. Versuch. Stat. 43, 475-480. 1895a Weiske, H.: Weitere Beitrage zur Frage iiber die Wirkung eines Fut- ters mit sauren Eigenschaften auf den Organismus, insbesondere auf das Skelett, Zeit. physiol. Chem. 20, 595-605. 704 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1895b Weiske, H.: Zur Frage iiber die Bedeutung der Calciumphosphat-Bei- gabe zum Futter f iir den tierischen Organismus, Landwirtsch. Versuch. Stat. 45, 242-245. 1895c Weiske, H.: Versuche iiber die Wirkung einer Beigabe von Calcium-, Strontium- resp. Magnesiumcarbonate zu einem kalkarmen, aber phos- phorsaurereichen Futter auf den Thierischen Organismus, insbeson- dere auf die Zusammensetzung des Skelettes, Zeit. Biol. 31, 421-448. 1896 Weiske, H.: Vergleichende Untersuchungen iiber die chemische Zusam- mensetzung der Knochen, Zahne etc. wilder und zahmer Kaninchen, Landwirtsch. Versuch. Stat. 46, 233-238. 1897 Weiske, H. : Ueber den Einfluss der Nahrungsentziehung auf das Gewicht und die Zusammensetzung der Organe, insbesondere der Knochen und Zahne, Zeit. physiol. Chem. 22, 485-499. 1885 Weiske, H., B. Dehmel, G. Kennepohl, B. Schulze, and G. Flechsig: Ver- suche iiber etwaige Einflusse, welche die Aufnahme freier Saure auf die Verdauungsvorgange sowie auf den Stickstoff- und Mineralstoff- Umsatz im Korper der Herbivoren ansiibt, Jour. f. Landwirtsch. 33, 21-76. 1881 Weiske, H., and G. Kennepohl: Untersuchungen iiber Schaf milch unter verschiedenen Verhaltnissen, Ibid. 29, 451-472. 1873 Weiske, H., and E. Wildt: Untersuchungen iiber die Zusammensetzung der Knochen bei kalk- oder phosphorsaurearmer Nahrung. (Dritte Abhandlung.) Zeit. Biol. 9, 541-549. 1899 Weiss, K.: Ueber die Eiweissstoffe der Leguminosensamen, Inaug. Diss., Miinchen. 36 pp. Through Jahresb. u. d. Fortschr. d. Thierchem. 30 (1900), 40-42. 1907 Weiss, L.: Beitrage zur Kenntniss der im Gerste und Malz vorkommen- den Phosphorverbindungen, Diss., Miinchen. 63 pp. Through Jahresb. u. d. Fortschr. d. Thierchem. 37 (1907), 1107, 1108. 1908 Weissmann, R.: Ueber einige mit Phytin erzielte Erfolge, Therapeut. Monatsh. 22, 470-472. 1908 Wellman, O.: Untersuchungen iiber den Umsatz von Ca, Mg und P bei hungernden Tieren, Arch. ges. Physiol. 121, 508-533. 1910 Wells, H. Gideon: The Purine Metabolism of the Monkey, Jour. Biol. Chem. 7, 171-183. 1909 Wells, H. Gideon, and Harry J. Corper : The Purines and Purine Metabo- lism of the Human Fetus and Placenta, Ibid. 6, 469-482. 1905 von Wendt, Georg: Untersuchungen iiber den Eiweiss- und Salz-Stoff- wechsel beim Menschen, Skand. Arch. Physiol. 17, 211-289. 1909 von Wendt, Georg: Zur Variability der Milch. 2. Ueber den Einfluss verschiedener Salzbeigaben zum Futter auf Zusammensetzung und Menge der Kuhmilch, Ibid. 21, 89-145. 1910 von Wendt, Georg: Ueber den Einfluss des Hohenklimas auf den Stoff- wechsel des Menschen, Ibid. 24, 247-258. 1905 Werner, Richard: Zur Kenntnis und Verwertung der Rolle des Lecithins bei der biologischen Wirkung der Radium- und Rontgenstrahlen, Deut. med. Wochenschr. 31, I, 61-63. 1913 Wesselkin, N. W.: Ueber die Ablagerung vom fettartigen Stoffeu in dea Organen, Arch. path. Anat. u. Physiol. 212, 225-235. 1902 West, H. A.: Therapeutics of the Glycerophosphates, Merck's Archivwfc 4, 263-265. PHOSPHORUS METABOLISM 705 1882 Weyl, Th., and H. Zeitler: Ueber die saure Reaction des thatigen Muskela und iiber die Rolle der Phosphorsaure beim Muskeltetanus, Zeit. physiol. Chem. 6, 557-565. 1903 Wheeler, Henry L., and Treat B. Johnson: On Cytosine or 2-oxy-6-amino- pyrimidine from Tritico-Nucleic Acid, Amer. Chem. Jour. 29, 505-511. 1913 Wheeler, Ruth: Feeding Experiments with Mice, Jour. Exp. Zool. 15, 209-223. 1903 Wheeler, W. P.: The Importance of Mineral Matter and the Value of Grit for Chicks, N. Y. Agr. Exp. Sta. (Geneva), Bui. No. 242, 293-314. 1899 White, W. Hale, and F. Gowland Hopkins: On the Excretion of Phospho- rus and Nitrogen in Leukhaemia, Jour, of Physiol. 24, 42-47. 1894 Whitfield, Arthur: Note on the Chemistry of Muscle, Ibid. 16, 487-490. 1874 Wibel, F.: Die Constitution des Knochenphosphates, insbesondere die Existenz und Bildung einer basischen Verbindung; (CasPzOs+xCaO). Jour. f. prakt. Chem N. F. 9, 113-132. 1912 Wieland, Hermann: Beitrage zur Aetiologie der Beri-Beri. I. Analytische Untersuchungen iiber den Phosphorgehalt von Ernahrungskranken Tieren, Arch. exp. Path. u. Pharm. 69, 293-306. 1904 Wilbur, R. L.: Acidosis, Jour. Amer. Med. Assoc. 43, II, 1228. 1900 Wildiers, Eugene: Inutility de la lecithine comme excitant de la crois- sance, etc., La Cellule 17 (2), 385-405. 1872 Wildt, Eugen: Ueber die Zusammensetzung der Knochen der Kaninchen in den verschiedenen Altersstuf en, Landwirtsch. Versuch. Stat. 15, 404- 454. 1874 Wildt, Eugen: Ueber die Resorption und Secretion der Nahrungsbestand- theile im Verdauungskanal des Schafes, Jour. f. Landwirtsch. 22, 1-34. 1879 Wildt, Eugen: Studien iiber den Verdauungsprocess des Schafes, Ibid. 27, 177-248. 1904 Wiley, H. W., W. D. Bigelow, and Others: Influence of Food Preserva- tives and Artificial Colors on Digestion and Health. I. Boric Acid and Borax, U. S. Dept. Agr., Bur. Chem. Bui. No. 84, Part I, 477 pp. 1906 Wiley, H. W., W. D. Bigelow, F. C. Weber and Others: Influence of Food Preservatives and Artificial Colors on Digestion and Health. II. Sali- cylic Acid and Salicylates, Ibid. 84, Part II, 479-760. 1907 Wiley, H. W., W. D. Bigelow, F. C. Weber and Others: Influence of Food Preservatives and Artificial Colors on Digestion and Health. III. Sul- phurous Acid and Sulphites, Ibid. 84, Part III, 761-1041. 1908a Wiley, H. W., W. D. Bigelow, F. C. Weber and Others: Influence of Food Preservatives and Artificial Colors on Digestion and Health. IV. Ben- zoic Acid and Benzoates, Ibid. 84, Part IV, 1043-1294. 1908b Wiley, H. W., W. D. Bigelow, F. C. Weber and Others: Influence of Food Preservatives and Artificial Colors on Digestion and Health. V. Form- aldehyde, Ibid. 84, Part V, 1295-1500. 1893 Willdenow, Clara: Zur Kenntnis der peptischen Verdauung des Kaseins, Inaug. Diss., Bern. 50 pp. 1904 Willstatter, Richard, and Karl Ludecke: Zur Kenntniss des Lecithins, Ber. deut. chem. Gesell. 37, III, 3753-3758. 1914 Wilson, D. H., and P. B. Hawk: Fasting Studies. XII. The Ammonia, Phosphate, Chloride and Acid Excretion of a Fasting Man, Jour. Amer. Chem. Soc. 36, 137-146. 706 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1908 Wilson, R. A., and W. Cramer: On Protagon: Its Chemical Composition and Physical Constants, its Behaviour towards Alcohol, and its Indi- viduality, Quarterly Jour, of Exper. Physiol. 1, 97-110. 1891 Winkler: Osteoporose, Wochenschr. f. Tierheilk. u. Viehzucht 1891, No. 16, 146. 1905 Winterberg, Josef: Ueber einige mit Phj^tin am Krankenbette gesam- melte Erfahrungen, Aerztliche Central-Zeitung 17, 406-408. 1909 Winternitz, M. C, and W. Jones: Ueber den Nucleinstoffwechsel mit be- sonderer Berucksichtigung der Nucleinfermente in den menschlichen Organen, Zeit. physiol. Chem. 60, 180-190. 1897 Winterstein, E.: Ueber einen phosphorhaltigen Pflanzenbestandtheil, welcher bei der Spaltung Inosit liefert, Ber. deut. chem. Gesell. 30, II, 2299-2302. 1908 Winterstein, E.: Ein Beitrag zur Frage der Konstitution des Phytins, Zeit. physiol. Chem. 58, 118-121. 1906 Winterstein, E., and O. Hiestand: Zur Kenntnis der pflanzlichen Leci- thine, Vorlaufige Mitteilung. 47, 496-498. 1908 Winterstein, E., and O. Hiestand: Beitrage zur Kenntnis der pflanzlichen Phosphatide. II. Mitteilung. Ibid. 54, 288-330. 1909 Winterstein, E., and K. Smolenski: Beitrage zur Kenntnis der aus Cer- ealien darstellbaren Phosphatide. rV. Mitteilung. Ueber Phosphatide, Ibid. 58, 506-521. 1909a Winterstein, E., (and L. Stegmann): Beitrage zur Kenntnis pflanzlicher Phosphatide. III. Mitteilung. Ueber ein Phosphatid aus Lupinus albus. (Introduction by Winterstein.) Ibid. 58, 500-505. 1909b Winterstein, E., (and L. Stegmann) : Ueber einen eigenartigen phosphor- haltigen Bestandteil der Blatter von Ricinus. VI. Mitteilung. Ueber Phosphatide, Ibid. 58, 527, 528. 1906 Wintgen, M., and O. Keller: Ueber die Zusammensetzung von Lecithinen, Arch. f. Pharmacie 244, 3-11. 1913 Withers, W. A., and B. J. Ray: Studies in Cotton Seed Meal Intoxication. I. Pyrophpsphoric Acid, Jour. Biol. Chem. 14, 53-58; also N. Carolina Agr. Exp. Sta. Rept. 1912, 141-145. 1897 Wittmaack, Karl: Ueber den Nucleongehalt der Kuh-, Frauen- und Ziegenmilch, Zeit. physiol. Chem. 22, 567-574. 1900 Worner, Emil, and H. Thierf elder: Untersuchungen iiber die chemische Zusammensetzung des Gehirns, Ibid. 30, 542-551. 1903 Wohlgemuth, Julius: Ueber das Nucleoproteid der Leber. I. Mittheilung, Ibid. 37, 475-483. 1904a Wohlgemuth, Julius: Ueber das Nucleoproteid der Leber. II. Mittheilung, Ibid. 42, 519-523. 1904b Wohlgemuth, Julius: Ueber das Nucleoproteid der Leber. III. Mitteilung, Ber. deut. chem. Gesell. 37, 4362. 1905 Wohlgemuth, Julius: Ueber das Nucleoproteid der Leber. IV. Mitteilung, Zeit. physiol. Chem. 44, 530-539. 1907 Wohlgemuth, Julius: Untersuchungen iiber den Pankreassaft des Men- schen. IV. Mitteilung. Ueber ein in ihm enthaltenes komplexes Hemolysin und iiber die Darstellung des Lecithids, Biochem. Zeit. 4, 271-280; 5, 349. PHOSPHORUS METABOLISM 707 1911 Wolf,, Charles G. L., and Emil Oesterberg: Eiweissstoffwechsel beim Hunde. II. Stickstoff- und Schwefelstoffwechsel wahrend des Hungers und bei Unternahrung mit Eiweiss, Kohlenhydraten und Fetten, Ibid. 35, 329-362. 1870 Wolff, Emil: Die landw. -chemische Versuchsstation Hohenheim und deren Thatigkeit in den Jahren 1866-1870. Berlin. 121 pp. Autoabstract, Landwirtsch. Jahrb. 2 (1873), 244. 1871 Wolff, Emil: Aschen-Analysen von landwirthschaftlichen Producten, Fabrik-Abfallen und wildwachsenden Pflanzen, Berlin. 194 pp. About 2800 analyses. 1874 Wolff, Emil: Die rationelle Futterung der landwirtschaftlichen'Nutztiere (Landwirtschaftliche Futterungslehre), Berlin. 239 pp. 1880 Wolff, Emil: Aschen-Analysen von land- und forstwirthschaftlichen Producten, Fabrik-Abfallen und wildwachsenden Pflanzen, Berlin. 170 pp. About 1600 new analyses. 1886 Wolff, Emil: Grundlagen fur die rationelle Futterung des Pferdes, Ber- lin. 155 pp. 1873 Wolff, E., W. Lunke, M. Fleischer, and J. Skalweit: Versuche liber das Verdauungsvermogen von zweierlei Schafrassen in verschiedenen Wachsthumsperioden und bei verschiedener Futterungsweise, Land- wirtsch. Jahrb. 2, 221-308. 1912a Wolff, G.: Ueber den Kalk- und Phosphorsaurestoffwechsel des Saug- lings bei knapper und reichlicher Ernahrung mit Kuhmilch, Inaug. Diss., Berlin. 29 pp. Through Exp. Sta. Record 29 (1913), 166. 1912b Wolff, G.: Ueber den Kalk- und Phosphorstoffwechsel des Sauglings bei knapper und reichlicher Ernahrung mit Kuhmilch, Jahrb. Kinderheilk. 76 [3rd ser. 26], 180-204. (1911) Wolpe, J. M.: Ueber die Wirkung des organischen Phosphors (Phytin) beim runden Magengeschwur, Arch. Verdauungskrankh. 17, Erganz- ungsh. 135-150; through Jahresb. ii. d. Fortschr. d. Thierchem. 41 (1911), 280. 1902 Wood, Horatio C, Jr.: On the Toxic Action of the Decomposition Prod- ucts of Lecithin, Univ. of Penn. Med. Bui., May 1902, 98-101. 1903 Wood, Horatio C, Jr.: The Role of Lecithin in Pathology and Thera- peutics, Merck's Archives 5, 257-259. 1869 Wood, Luther Hodges: Researches on the Influence of Mental Activity upon the Excretion of Phosphoric Acid by the Kidneys, Proc. of the Conn. Med. Soc, Ser. 2, 3, 197-222. 1892 Wrampelmeyer, E. : Der Lecithingehalt der Butter, Landwirtsch. Versuch. Stat. 42, 437, 438. 1894a Wroblewski, Augustin: Beitrage zur Kenntniss des Frauencasei'ns und seiner Unterschiede vom Kuhcasei'n, Mittheil. aus klinik. u. med. Inst. d. Schweiz (Annales suisses des sci. med.) 2, 291-337. 1894b Wroblewski, Augustin: (Ueber das Casein in der Frauenmilch. Chem- ische Unterschiede zwischen der Frauen- und Kuhmilch), Gazeta lekar- ska, No. 36 and 37, pp 951 and 988; through Jahresb. ii. d. Fortschr. d. Thierchem. 24 (1894), 211-213. 1898 Wroblewski, Augustin: Ein neuer eiweissartiger Bestandteil der Milch, Zeit. physiol. Chem. 26, 308-313. 1901 Wroblewski, Augustin: Ueber den Buchner'schen Hefepresssaft, Jour, £, prakt. Chem. [2] 64, 1-68. 708 OHIO EXPERIMENT STATION: TECHNICAL BUL. 5 1912. Wuertz, Ad.: Versuche iiber die Verteilung der Phosphor- Saure auf Harn und Kot, Biochem. Zeit. 46, 103-111; through Chem. Abs. 7 (1913), 499. 1912 Yamigawa, R., T. Koyana, H. Midorikawa and T. Mogi: Experimentelle Studie iiber Ursache und das Wesen von Kakke, Mitt. Med. Ges. Tokio 26, No. 23, 1-4. 1884-5 Yeo, Gerald F., and E. F. Herroun: A Note on the Composition of Hu- man Bile Obtained from a Fistula, Jour, of Physiol. 5, 116-123. 1913 Ylppo, Arvo: Der isoelektrische Punkt des Menschen-, Kuh-, Ziegen-, Hunde- und Meerschweinchenmilchcaseins, Zeit. Kinderheilk. 8, Orig- inalien, 224-234. 1910 Yoshimoto, S.: Ueber den Einfluss des Lecithins auf den Stoffwechsel, Zeit. physiol. Chem. 64, 464-478. 1907 Young, William John: The Organic Phosphorus Compound Formed by Yeast- juice from Soluble Phosphates, Proc. of the Chem. Soc. 23, 65, 66. 1909 Young, William John : The Hexosephosphate Formed by Yeast- juice from Hexose and Phosphate, Proc. Of the Royal Soc. 81, B, 528-545. 1911 Young, William John: Ueber die Zusamensetzung der durch Hefepress- saft gebildeten Hexosephosphorsaure. II. Biochem. Zeit. 32, 177-188. 1912 Yushchenko, A. I.: Richesse de divers organes de l'homme et des ani- maux en nuclease, — ferment dedoublant l'acide nucleique, Archives de sci. biol. 17, 1-12; through Chem. Abs. 7 (1913), 1726; see also Jusch- tschenko, 1911. 1879 Yvon, P.: Sur le lait phosphate, Repertoire de pharmacie et jour, de chim. med. 7, 403, 404. 1899 Zadik, H.: Stoffwechselversuche mit phosphorhaltigen und phosphor- freien Eiweisskorpern, Arch. ges. Physiol. 77, 1-21. 1904 Zaitschek, Arthur: Zur Kenntniss der Pepsinsalzsaureloslichkeit der Milch und der Casei'ne, Ibid. 104, 550-563. 1901a Zaky, Aly: Influence de la lecithine sur l'elimination de l'acide urique, Compt. rend. Soc. de biol. 53, 830-832. 1901b Zaky, Aly: Sur- l'influence exercee par la lecithine sur l'elimination de l'acide urique, Bui. de l'Acad. de med. [3] 46, 215. 1886 Zaleski, St. Szcz: Studien iiber die Leber. I. Eisengehalt der Leber, Zeit. physiol. Chem. 10, 453-502. 1902 Zaleski, W.: Beitrage zur Verwandlung des Eiweissphosphors in den Pflanzen. (Vorlaufige Mitteilung.) Ber. deut. bot. Gesell. 20, 426- 433. 1907 Zaleski, W.: Ueber den Umsatz der Phosphorverbindungen in reifenden Samen, Ibid. 25, 58-66. 1866 Zaleksy: Ueber die Zusammensetzung der Knochen des Menschen und verschiedener Thiere, Hoppe-Seyler's med. -chem. Unters., 19-48. 1881 Zander: Zur Lehre von der Aetiologie, Pathogenie und Therapie der Rachitis, Arch. path. Anat. u. Physiol. 83, 377-391. 1912 Zaribnicky, Franz: Ueber die chemische Zusammensetzung der Pferde- lymphe, Zeit. physiol. Chem. 78, 327-332. 1904 Zeiz, Richard Herrmann: Ein Fall von Osteomalacic, der trotz Castra- tion und lange fortgesetzter Phosphordarreichung nicht zur Heilung kam, Diss., Miinchen. 79 pp. PHOSPHORUS METABOLISM 709 1895 von Zeynek, Rich.: Chemische Untersuchung des Inhalts zweier Lymph- cysten, Zeit. physiol. Chem. 20, 462-471. 1910 Zickgraf : Ueber die Phosphatausscheidung bei Tuberculosen und Chlor- otischen, Centralbl. innere Med. 31, 273-275. 1909 Ziveri, Alberto: Modificazioni al metodo di ricerca della colina e nuove indagini sulla presenza di essa e di lecitina nel liquido cefalo-rachideo, Rivista di patol. nerv. e ment. 14, 134-136. 1894 Zoja, Luigi: Sulla presenza di lecitina nelle cellule alveolari del polmone e sul significato semeiologico delle gocce mieliniche dello sputo, Gaz- zetta medica, Torino 45, 801-806; 821-827. 1912 Zuckmayer, F.: Beitrag zur Aufnahme und Verwertung von Kalk und Phosphorsaure durch den Darm, Arch. ges. Physiol. 148, 225-256. 1905 Zuelzer, G.: Ueber die klinische Bedeutung der anorganischen Bestand- teile des Hams, Med. Klinik, Berlin, 1, 958-960. 1876 Zuelzer, W.: Ueber das Verhaltniss der Phosphorsaure zura Stickstoff im Urin, Arch. path. Anat. u. Physiol. 66, 223-251; 282-311. 1881a Zuelzer, W.: Die klinische Bedeutung der Phosphorsaure des Harns, Trans, of the Internat. Med. Congr., London, 2, 154, 155. 1881b Zuelzer, W. : Harnuntersuchungen mit Rucksicht auf off entliche Hygiene, Ibid. 4, 568-570. 1871 Zum: Zur Knochenbriichigkeit der Rinder, Der Thierarzt 10, 13-16. 1900 Zuntz, N.: Ueber den Einfluss gewisser phosphorhal tiger Substanzen auf das Wachstum, Therapie der Gegenwart 2, 529, 530. 1913 Zunz, Leo: Stoffwechselversuche bei Osteomalacic, Arch. Gynakol. 99, 145-166. 1900 Zweifel, Paul: Aetiologie, Prophylaxis und Therapie der Rachitis, Leipzig. 188 pp. Through Congr. Internat. de Med., 1900, Sect, de Gynecol., 583-585. INDEX Absorption and elimination of phosphorus, general, 181 Absorption and secretion of phosphorus in alimentary tract, 182, 183 Absorption of calcium phosphate as affected by colloidal state, 185; colloids, 185; intestinal reaction, 185 phosphorus as affected by calcium, 184, 185; enzymes of the alimentary tract, 185, food fat-— infants, 459 Acid diet, effects on phosphorus content of liver, 136 Acid intoxication, 476 Acidosis, 476 analytical measure of, 477 from an ash-free diet, 478 in diabetes, 510 in fever, 479 influence on phosphorus metabolism, 476 in gastrointestinal disorders of children, 478 symptoms of, 478 Acids, effect on bones, 387, 388 composition of flesh, 388 phosphorus of urine, 199 phosphorus excretion, 198 Acromegaly, in relation to the hypophysis, 479 phosphorus metabolism in, 479 Adenase, definition, 28 Adenin, composition, 18 Adrenals See Suprarenal Capsules Age, influence of, metabolism of growing boys, 511, 512 on bones, 113 brain, 133 the digestion of phytin, 313 liver, 136 muscles, phosphocarnic acid content, 305 nitrogen and phosphorus ratio, 215 pancreas, 141 phosphorus intake, 410 spleen, 139, 140 urine, 407 Alcohol ingestion, allantoin in urine, 483 ferment activity of blood serum, 540 lecithin of liver, 483 nucleic acid metabolism, 240 phosphatid content of organs, 484 phosphorus metabolism, 483 rate of metabolism, 483 uric acid elimination, 483 Aleuronate, feeding experiments, 322, 550 Alfalfa, inorganic phosphorus, as affected by fertilizer, 99 phosphorus in, as affected by water supplied, 94 fertilizer, 99 hay, ether extract phosphorus, 90 inorganic phosphorus, 78 mineral analyses, 78, 94 phosphorus, 78 Allantoin in urine after alcohol ingestion, 483 "niif*! pit* spirit 24^ 2S1 of dogs, 240, 243, 247; men, 237; rabbits, 247; swine, 243 711 712 INDEX Alloxur bases, composition of, 17 Alloxuric bodies. See Purins Altitude, influence of, nitrogen and minerals — men, 429 Amniotic fluid, phosphorus content, 457 Anaemia, effects of, on blood, 496, 499, 507; cerebrospinal fluid, 537; organs, 507; phosphorus and chlorine relations in the organs, 508; urine, 499; water accumulation in the organs, 508 lecithin therapy, 501, 529, 531 nuclein therapy, 502 phosphorus metabolism, 492 protylin therapy, 301 tuberculous, nuclein-saline treatment, 586 Apples, inorganic phosphorus in, 78 mineral analyses of, 78 phosphorus in, 78 Arteriosclerosis, calcification from phosphatid cleavage, 449 effects on blood, 501 the brain, 135 Arthritis, excretion of phosphorus, 484 Artificial food preparation, insufficiency of, 357 protein-free milk, preparation of, 360 value of, 360 Ash-free diet, acidosis from, 478 Ash of milk compared with the ash of the young, 167 Atrophy, lecithin therapy, 531 Autolytic cleavage of phosphatids and of nuclein, 187 phosphorus compounds, 187 Bacteria in the alimentary tract, effects on nucleoprotein digestion, 234 Bananas, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Barley bran, lecithin phosphorus, 82 organic phosphorus, 82 phosphorus, 82, 84 grain, inorganic phosphorus, 79 lecithin content, 87 (phosphatid) content, 85, 89 phosphorus, 79, 82 organic phosphorus, 82 phosphorus, 79, 84, 87 protein phosphorus, 79 kernel, phosphorus, 84 Barlow's disease, phosphorus metabolism, 485 Basedow's disease, lecithin of blood serum, 500 Beans, lecithin content, 87 (brown), inorganic phosphorus, 79 nuclein and phosphoprotein phosphorus, 79 phosphatid phosphorus, 79 phosphorus, 79 phytin phosphorus, 79 (garden), lecithin (phosphatid) content, 85 phosphorus, 85 (kidney), lecithin (phosphatid) content, 85 lecithin phosphorus, 81 phosphorus, 81, 85 phytin phosphorus, 81 (navy), inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 (Windsor), inorganic phosphorus, 79 lecithin phosphorus, 79 phosphorus, 79 protein phosphorus, 79 INDEX 713 Beechnut cake, lecithin (phosphatid) content, 85 Beer, distribution of phosphorus, 80 Beet leaves (sugar), lecithin content, 87 molasses, favorable to phosphorus absorption, 369 pulp, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 root (sugar), lecithin content, 87 Beets (sugar), nuclein and phosphoprotein phosphorus, 88 phosphatid phosphorus, 88 phosphorus, 88 Benzoic acid, phosphorus retention after, 368 Beriberi, cause of, 484 lack of phosphatese, 491 phosphorus, 488 phosphorus metabolism disturbance, 485 Bermuda grass, mineral analyses as affected by water supply, 94 phosphorus as affected by water supply, 94 hay, ether extract phosphorus, 90 Bile, general analyses, 174 influence on lecithin absorption, 289 pancreatic juice, 282 pancreatic digestion of casein, 270 inorganic phosphorus— cattle, 174 lecithin content — hedgehog, 107; men, 174; rabbit, 107; several, 174 mineral analyses — men, 105 nucleoproteins — cattle, 174 phosphorus— men, 105 phosphorus content, 173 Bile salts and lecithin, solubility of fats and soaps, 283 Biocitin, retention of lecithin from, 295 feeding experiments, 295 lecithin content, 532 therapy, 532 Bioplastin, description, 93, 531 Bioson, composition, 93 therapy, 532 Bleeding, effect on composition of organs, 507 Blood cell count, affected by acute pneumonia, 517; lecithin ingestion, 533; lecithin injection, 528, 529, 530; nuclein therapy in blood diseases, 502; osteomalacia, 557; phytin ingestion, 533. See also Leucocytosis cells, differentiation of the phosphatids of, 153 circulation as affected by protylin, 551 coagulating action of nucleoproteins and nucleic acids, 250, 251, 255 composition, affected by acromegaly, 480; anaemia, 496; arteriosclerosis, 501; chlorosis, 496; crisis of diseases, 499; degen- eration and regeneration of severed nerves, 538; diabetes mellitus, 511; diet, 369; diseases, 496, 498; lecithin ingestion, 533; leukaemia, 495, 497; mental activity, 462; pain, 463; pneumonia and anaemia, 507 lecithin content, affected by lecithin injection or ingestion, 498; nephritis, 548; polycythaemia rubra megalosplenica, 500 of ash, affected by osteomalacia, 557 partition of phosphorus, affected by thyro-parathyroidectomy in the dog, 560 phosphatid content for normal and pregnant women and for new-born infants, 472 phosphorus content, 149 affected by endoarteriitis, 512; kidney diseases, 548 714 INDEX Blood diseases, phosphorus compound treatment, 501 metabolism and characteristics, 492 dissolution, leucocytosis and changes in metabolism during, 496 ether-alcohol soluble phosphorus — rabbit, 313 inorganic phosphorus in — cat, 151; cattle, 152; dog, 151; goat, 151; horse, 152; rabbit, 151; sheep, 152; swine, 151, 369 lecithin content — cat, 151; cattle, 152; dog, 151; goat, 151; horse, 152; men, 498, 500; rabbit, 107, 151; sheep, 152; swine, 151 mineral analyses of — cat, 151; cattle, 152; dog, 151; fowl, 153; goat, 151; horse, 152; men, 105, 497, 498, 499, 501, 507; rabbit, 151, 313; sheep, 152, 153; swine, 151, 327, 369 nuclein phosphorus — cat, 151; cattle, 152; dog, 151; goat, 151; horse, 152; rabbit, 151; sheep, 152; swine, 151 phosphocarnic acid — cattle, 150; dog, 149; rabbit, 149 phosphorus— cat, 151; cattle, 107, 152, 153; dog, 151, 560; fowl, 153; goat, 151; horse, 152; men, 104, 105, 497, 498, 499, 501, 507, 512, 557; rabbit, 151, 313; sheep, 152, 153; swine, 151, 327, 369 serum, effect of parathyroidectomy on partition of phosphorus — dog, 560 esterase content as affected by nervous diseases, 540 lecithin content — men, 542 as affected by diabetic lipaemia and cholaemia, 501; diseases, 500; epilepsy, 541; paralysis, 500, 542; tabes and paralysis, 498; tabo-paralysis, 498 normal, 498, 542 lipase content as affected by nervous diseases, 540 nuclease content as affected by nervous diseases, 540 phosphorus in — dog, 560 phosphorus content in syphilis and cancer, 500 Bluegrass, inorganic phosphorus, 78, 95 affected by fertilizer, 96 mineral analyses of, 78 affected by fertilizer, 96 phosphorus, 78, 95 affected by fertilizer, 96 Blue-joint grass, mineral analyses, affected by water supply, 94 Body analyses, effects of phytin and sodium phosphate — rabbit, 313 Body composition, affected by disease, general, 506 Bone ash, constancy of composition, 386 feeding, compared with other phosphatic supplements, 828 influence on character of bones, 328 urinary phosphorus, 206 phosphorus balances — swine, 328 experiments with dogs, 427 diet, effect in experimental osteomalacia, 394 formation, influenced by electricity, 390; parathyroids, 559; potassium salts, 390; the thyroids, 578, 579 katabolism in diabetes, 510 phosphorus starvation, 394 on calcium- or phosphorus-poor diet, 394 . marrow, lecithin and phosphorus content, 118 lecithin content — cat, 119; cattle, 119; children, 119; dog, 119; hedgehog, 107; horse, 118, 119; men, 119, 490, 542; rabbit, m 107, 119; sheep, 119; swine, 119 lecithin content as affected by dementia paralytica, 542 normal, 542 phosphorus content, affected by dementia paralytica, 542 phosphorus in — cat, 119; cattle, 119; children, 119; dog, 119 horse, 119; men, 119; rabbit, 119; sheep, 119; swine, 119 Bone meal, inorganic phosphorus, 78 lecithin phosphorus, 82 phosphorus, 78, 82 feeding, nitrogen and mineral balances — dog, 323 INDEX 715 Bone meal, influence on bones, 388, 391 composition of milk, 380, 381 parts of body, 370 development of fowl, 391 nitrogen and mineral retention, 512 phosphorus excretion, 210, 224 production of milk, 381 mineral analyses, 78 retention, 224, 324 supplement in calf feeding, 421 Bone phosphate, composition, 117 formula, 118 Bones, composition, affected by age, 113 calcium-poor diet, 535 diet, 384 fast— dog, 440; rabbit, 441, 443 lactose injection, 554 lecithin, 569; guinea pigs, rabbits, dogs, 300 osteomalacia, 554 rachitis, 575 tuberculosis, 585 mineral matter — swine, 394 after treatment with glycerin and potassium hydrate, 113 relation of calcium and magnesium, 556 See also Malnutrition of the bones; Osteomalacia; Rachitis ether-alcohol soluble phosphorus — rabbit, 313 inorganic phosphorus — dog, 300; guinea pig, 300; rabbit, 300 lecithin content — hedgehog, 107; rabbit, 107 malnutrition, 534 mineral analyses of — cattle, 112, 113; children, 572; dog, 111, 112, 387; fowl, 112, 114, 386; gander, 112; goat, 112; goose, 113; men, 105, 112, 113; other animals, 112; rabbit, 112, 114, 115, 313, 386, 389, 390, 441; sheep, 112, 386, 388; swine, 327, 394 phosphorus in— cattle, 113; children, 572; dog, 111, 387; fowl, 114, 386, 391; gander, 112; goose, 113; horse, 556; men, 104, 105, 113, 462, 556; rabbit, 114, 115, 313, 386, 389, 390, 554-555; rats, 506; sheep, 386, 388; swine, 327, 394 Boric acid, phosphorus retention, 368 Brain, composition, affected by diseases; anaemia, 507; carcinoma, 507; dementia praecox and general paralysis, 540; epilepsy, 541; paralysis, 539; pneu- monia, 507 growth, 133 lecithin— guinea pigs, rabbits, dogs, 300 phosphatids affected by age, 135, 136 alcoholism, 484 phosphocarnic acid content, 305, 446 phosphorus .compounds, affected by tuberculosis, 588 normal, 588 under various conditions, 132 content, affected by age, 135; arteriosclerosis, 135; nerve degeneration, 542; polyneuritis, 490; tubercu- losis, 588 normal, 588 Thudichum's classification of the constituents, 128 Brain and nerves, cephalin phosphorus — guinea pigs, 588 ether-alcohol soluble phosphorus — rabbit, 313 extractive phosphorus — children, 132; men, 132; dog, 132 inorganic phosphorus — dog, 134 lecithin content— cat, 107; children, 110, 111; dog, 130, 300; guinea pigs, 300; hedgehog, 107; men, 130, 539; new-born infants and fetuses, 110, 111, 130; rabbit, 107 716 INDEX Brain and nerves, lecithin phosphorus — guinea pigs, 588 lecithins and cephalins — children, 133 lipoid phosphorus — men, 132; dog, 132, 134; children, 132,133 mineral analyses — men, 105, 507; rabbit, 313; swine, 327 nuclein content — men, 130 phosphorus — dog, 132 nucleoprotein content — men, 130 nucleoproteins — dog, 130 phosphatid content— dog, 135; new-born infants and fetuses, 135 phosphatids — guinea pigs, 588 phosphocarnic acid — cat, 130; cattle, 130; dog, 130, 305; fowl, 130; guinea pig, 130; men, 130; rabbit, 130; sheep, 130; swine, 130 phosphorus in — cattle, 107, 131; children, 132, 133, 134; dog, 132, 134, 300; new-born infants and fetuses, 134, 135; guinea pigs, 300, 588; horse, 131; men, 104, 105, 129, 131, 462, 507, 539; rabbit, 300, 313; sheep, 149; swine, 131,327 protein phosphorus — children, 132, 133; dog, 132; men, 132 Bran, compared with acid-washed bran, metabolism and milk production experiments — cows, 306 phosphatic supplements, balance and slaughter experi- ments — swine, 328 washed bran and calcium phosphates, maintenance and slaughter experiments — swine, 327 washed bran and washed bran plus potassium phytate, balance and milk production experiments— cow, 312 influence on character of bones, 328 composition of parts of the body, 327, 370 phosphorus metabolism, 326, 487 nuclein and phosphoprotein phosphorus, 88 phosphatid phosphorus, 88 phosphorus, 88 Bread, inorganic phosphorus, 77 lecithin content, 87 mineral analyses, 77 nuclein and phosphoprotein phosphorus, 88 phosphatid phosphorus, 88 phosphorus, 77, 88, 366 white and whole wheat, nitrogen and phosphorus balances — men, 366 nitrogen and phosphorus content, 366 nutritive value — man, 367; pigeon, 489 Brewer's grains, lecithin content, 87 products, phosphorus compounds of, 80 Bromlecithin in anaemia, 502 chlorosis and secondary anaemia, 501 Bronchopneumonia, effects on urinary phosphorus, 516 Buckwheat grains, lecithin (phosphatid) content, 85 Buffalo grass, ether extract phosphorus, 90 Bunge's Law, 167 Burr clover, ether extract phosphorus, 90 Butter, contains a component essential to normal growth, 358, 362 phosphorus, 162 Buttermilk, lecithin, 164 Cabbage, inorganic phosphorus, 78 mineral analyses, 78 nuclein and phosphoprotein phosphorus, 88 phosphatid phosphorus, 88 phosphorus, 78, 88 Cachexia, lecithin therapy, 528 Calcareous degeneration, cause, 503 INDEX 717 Calcium carbonate, fed with hay, effect on digestion, 223 oats, 389 skim milk, mineral balances — calves, 222 influence on amount and composition of goat milk, 383 bone katabolism, 384 calcium and phosphorus retention, 376 growth, 228 phosphorus absorption, 548 phosphorus elimination, 203, 209, 222, 385 phosphorus storage, 425 Calcium in relation to rachitis, 567 loss, affected by hypophysis injection in fast, 482 in acromegaly, 480; diabetes mellitus, 511 of bone, replaced by magnesium and strontium, 386 phosphate, effects on growth of offspring, 227 precipitated, phosphorus balances — swine, 328 use of, with beet leaves, 225 See also Bone Meal and Bone Ash Calcium-poor diet, effects on bones, 554; tissues, 225 limits phosphorus storage from corn, 425 nitrogen and mineral balances — horse, 423 Calcium salts, effect on bone formation, 225 bones, 385, 386, 389, 391; tissues, 226 relation to phosphorus absorption, 548 excretion, 198, 203, 209, 211, 221, 385 metabolism. See also Phosphaturia, Rachitis and Calcium Metabolism retention, 217, 218, 220, 224, 225, 376, 425 Cancer, effect on composition of the blood, 500; liver, 504; organs, 507 phosphorus studies in connection with, 503 presence of nuclease, 504 sodium nucleate injection before operations for, 504 Carbohydrate-phosphoric esters, feeding experiments, 550 Carbonophosphates, definition, 154 estimations of — milk, 154 in milk, treatments affecting, 154 Carbonates, alkaline earth, supplements to calcium-poor, phosphorus-rich rations, 390 oats, 389 Carbohydrates, constituents of nucleic acids, 19 Caries, 504 fungosa, lactic acid treatment, 504 Carnaubon, chemistry, 71 occurrence, 142 Cartilage, phosphorus compounds, 120 Casein-calcium preparations, treatment of rachitis, 576 Casein, chemistry, 32 cleavage by colon bacillus, 269 feces, 269 products of, hydrolytic, 41 coagulation, by acids and by heat, 36 renin, 38 Bang's description, 40 Bosworth's interpretation, 40 conditions favorable to, 38 the enzyme, 38 Hammarsten's interpretation, 39 in the stomach, 261 Mellanby's interpretation, 41 Schryver's interpretation, 41 composition, elementary analysis and empirical formula, 34 relation of phosphorus to the molecule, 43, 44 718 INDEX Casein content of cream, 163, 164 fermented milks, 162 milk, affected by fat in the diet, 379 general state of nutrition, 379 pathological states, 382 relation to other constituents, 153 skimmed milk, 163, 164 whole milk, 155, 156, 157, 161, 163, 164, 166, 167 digestion, 261 feeding experiments, 272, 293, 427 See also Casein maintenance experiments in vitro, 185, 267 peptic, affected by carbohydrates, 267 degree of acidity, 268 Salkowski's description, 264 products of peptic, 263, 264 rectal feeding experiment, 404 differences in, from different species, 43, 265 different species, peptic digestion, 267 effect on ferratin of liver, 301 flow and composition of milk, 383 in feces of infants, 458 maintenance experiments, 320, 326, 331, 342, 343, 359, 360, 362, 363, 454 occurrence of, 33 physical and chemical properties, 35 products of partial cleavage, value as food, 272, 356 racemization of, 36 secretion, 260 Caseonphosphate of calcium, treatment of rachitis, 576 Castration and ovariotomy, phosphorus metabolism, 559 metabolism and composition of the body, 506, 580 Cattle, mineral analyses of, 106 phosphorus in, 106 Caudol, description, 93 Cephalin, chemistry, 67 composition, elementary analyses, empirical formulae, 68 fatty acid component, 67 nitrogenous base, 67 estimations of — organs, 588 feeding, effect on phosphorus of brain, 303 in intermediary metabolism, 278 occurrence, 67 of brain and nerves, special studies, 130 organs as influenced by tuberculosis, 588 Cereal foods, mineral metabolism — swine, 375 affected by calcium — swine, 375 Cerebrospinal fluid, changes in nerve degeneration, 536 nucleoprotein content, general paralysis, 542 ■Chestnuts, lecithin (phosphatid) content, 85 phosphorus in, 85 Chickens, ether-soluble phosphorus, 448 inorganic phosphorus, 448 phosphorus, 448 vitellin phosphorus, 448 Children, mineral analyses, 111 phosphorus, 111 Chlorosis and tuberculosis compared as to phosphorus and chlorine elimination, 584 bromlecithin treatment, 502 influence on composition of blood, 496 blood serum and erythrocytes, 498 phosphorus metabolism and other characteristics, 492 INDEX J19 Chlorosis, lecithin treatment, 530, 532 nuclein treatment, effect on blood counts, 502 Choline, constitution, 60 in nerve degeneration, 536 Chorea, lecithin therapy, 532 Chyle, lecithin content, 177 phosphorus, 177 Chymosin, identity with pepsin, 38 occurrence and action, 38 Clover, phosphorus affected by amount of water supplied, 93 hay, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Cocoanut cake, lecithin (phosphatid) content, 85 Colamin, mother substance of choline, 61 Corn bran, phosphorus, 83 Corn, effect on composition of bones, 391 germ, phosphorus, 83 gluten, phosphorus, 83 grains, inorganic phosphorus, 77, 79 lecithin (phosphatid) content, 85, 87 phosphorus, 79 mineral analyses, 77 phosphorus, 77, 79, 83, 87 protein phosphorus, 79 nature of the phytin, 53 shucks, ether extract phosphorus, 90 specific effects on swine, 370, 376, 425 stover, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Cottonseed cake, lecithin phosphorus, 91 nuclein phosphorus, 90, 91 phosphorus, 91 Cottonseed meal, inorganic phosphorus, 78 mineral analyses, 78 nature of the phytin, 53 phosphorus, 78 toxic effects of, 91, 226, 227 Cowpea hay, ether extract phosphorus, 90 inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Cowpeas, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Cream, casein content, 163, 164 glycerophosphate phosphorus, 163 lecithin phosphorus, 163 organic phosphorus, 163 m phosphorus, 163, 164 Cretinism attributed to hypophysis and thyroid glands, 481 metabolism studies, 578 phosphate treatment, 579 thyroid treatment, 546, 579 Cuorin, chemistry, 68 occurrence, 125, 126, 142 Cytosin, composition, 19 Dates, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Deaminases, definition, 28 720 INDEX Degeneration and regeneration of severed nerves, chemical changes of the blood accompanying, 538 Degenerative nervous diseases, organic phosphorus in the urine, 539 Dementia paralytica, effect on cerebrospinal fluid, 537 praecox, effects on brain, 540 effect on ferment activity of cerebrospinal fluid, 541 Demineralization, phytin treatment, 533 Dental tartar as derived from carbonophosphates of the saliva, 176 Dentine of normal and diseased teeth, composition, 504 Development of young as influenced by diet of mother, 373 Diabetes, glycerophosphate treatment, 521 mellitus, composition of blood, erythrocytes and blood plasma, 498 lecithin content of blood serum, 500 phosphorus metabolism, 510 phosphaturia in. See Phosphaturia, 561 Diabetic acidosis, calcium, magnesium and phosphorus loss, 478 lipaemia, effect on lipoids of blood serum, 501 Diamino-monophosphatids, chemistry, 69 Diastase, influence of lecithin, 285 lipoids, 286 Diet and species, effects on phosphorus elimination, 207 Dietaries, inorganic constituents, 409, 410 Digestion and absorption of lecithin and glycerophosphates, 286 influence of phosphates, 186 of fat, related to lecithin content of bile, 173 nucleic acids and nucleoproteins, animal experiments, 237 nUcleoproteins by enzymes from alimentary tract, 231 Digestive mucosa, phosphorus compounds, relation to food digestion, 146 secretions, phosphorus content, 172 _ . Diphtheria, phosphorus and nitrogen in the urine, 516 toxin, effect on composition of heart, 583 metabolism, 583 Diseases, general consideration, composition of the body, 507 Diuresis from inorganic salts, 546 Dogs, phosphorus, 581 Dwarf state due to hypofunctioning of hypophysis, 482 Dyspepsia, effects on phosphorus retention, 459 Dyspnoea, experimental, effect on phosphorus elimination, 479 Earth phosphates of the urine in pneumonia and typhoid fever, 519 Eck fistula, definition, 239 Edestin feeding experiments, 321, 322, 326, 331, 333, 431 Eggs, changes during incubation, 276, 446 feeding experiments, 323 fish, nucleic acid content, 171 nucleoalbumins, 170 phosphorus compounds, 148 fowl, ether-soluble phosphorus, 448 inorganic phosphorus, 78, 441, 448 lecithin content, 87, 447 mineral analyses, 78, 169-170, 441 nucleoalbumins, 170 phosphorus, 78, 169, 441, 448 variability in composition, 170 vitellin phosphorus, 448 frog, organic phosphorus compounds, 147 nutritive value as sole diet of men, 291 phosphorus, 169 sea-urchin, phosphorus changes during development, 449 Egg white, mineral analyses, 169 nuclein and phosphoprotein phosphorus, 88 phosphatid phosphorus content, 88 phosphorus, 88 INDEX 721 Egg yolk, differentiation of phosphatids, 171 effect on digestion of milk, 533 lipoid deposits, 302 lecithin content, 171 mineral analyses, 170 nutritive value, compared with that of milk, 290, 291 plasmon, 291 treatment in anaemia and infantile athrepsia, 501 polyneuritis, 489 Elements involved with phosphorus in katabolic processes, 213; calcium and magnesium, 203, 377, 575; nitrogen, 215; sodium and potassium, 201 Elimination of phosphorus, cutaneous, 184 • factors influencing the paths, 182; acids, 198; acids of fermentation, 459; bone dust, 512; calcium and magnesium, 203, 377, 574; diet of infants, 418; fats, 478; indigestion of breast-fed infants, 457; rickets, 573-574; salts of sodium and potas- sium/ 201; species, 221; summary, 211; type of diet and species, 207 relation to nitrogen elimination, 214 phosphates in the urine, method, 188 Endogenous and exogenous purins, definition, 246 methods of estimation, 246 purin elimination, factors influencing, 246 Endoarteriitis, nitrogen, calcium, magnesium and phosphorus balances — men, 512 phosphorus content of blood — men, 512 metabolism, 512 Enzyme activities, affected by administration of lecithin, 281 nucleic acids, 252 phosphates, 186 of blood serum, effect of nervous diseases, 540 cerebrospinal fluid in nervous diseases, 540 organs as affected by nucleic acid, 250 phosphorus of diet, 343 Enzymes, casein-coagulating, occurrence, 38 effecting cleavage of phosphorus compounds, 185 of individual organs. Nuclein ferments, 235 reversible action, 266 Epilepsy, effect on composition of brain, 541 ferment activity of blood serum, 540 cerebrospinal fluid, 541 lecithin of blood serum, 541 phosphorus content of cerebrospinal fluid, 537 metabolism, 541 choline as a cause, 537 organic phosphorus in urine after attack, 541 phosphorus treatment, 547 Erepsin, digestion of nucleic acids, 232 Esterase of blood serum in nervous diseases, 540 Etherization, delayed phosphorus elimination after, 526 Eucasein, description, 92 Exercise, influence on decomposition of bone tissue, 406 nitrogen, calcium and phosphorus balances, 413 phosphorus metabolism, 464 ratio of nitrogen to phosphorus, 469 uric acid elimination, 466, 469 urinary phosphorus, 461 nuclein katabolism of, reduced by training, 466 Exophthalmic goiter. See Morbus Basedowii 722 INDEX Fast, influence on composition of parts of body, 305, 438 phosphorus metabolism, 438, 481, 512 Summary of evidence, 446 phosphorus excretion, 214, 399, 426 protein katabolism, 217 Fats and soaps, solubility, affected by bile salts and lecithin, 283 Fatty degeneration of liver, glycerophosphoric acid of urine, 192 effect on lecithin of liver, 513 Feathers, mineral analyses, 127 phosphorus content, 126 Feces, autoputref action of nuclein, 234 lecithin, 288 mineral analyses, 105 phosphorus content, 105, 208 and compounds, 194 See also Phosphorus balances and phosphorus excretion purins, due to bacteria, 234 Fermentation of sugars, influence of phosphates, 31 Ferments. See Enzymes Ferratin, occurrence, 138 Fersan, composition, 93, 550 feeding experiments with geese and men, 550 Fertilizers, effects on composition of foods, 95 Fetuses and new-born young, phosphorus content, 104, 108, 471 Fever, acidosis, 479 experimental, gas exchange, 516 phosphorus metabolism, 514 scarlet, sodium nucleate treatment, 519 Fish, lecithin (phosphatid) content, 122 mineral analyses, 121 phosphorus, 121, 122 phosphorus, calcium and magnesium retention after, 368 Fistula, Eck, definition, 239 use in metabolism experiments, 239 London's, in nucleic acid studies, 244 Flax seeds, lecithin (phosphatid) content, 85 phosphorus, 85 Floats, balance and slaughter experiments with swine, 328 See also Rock Phosphate Flour, graham, inorganic phosphorus, 79 nuclein and phosphoprotein phosphorus, 79 phosphatid phosphorus, 79 phosphorus, 79 phytin phosphorus, 79 red dog, mineral analyses, 77 rice, inorganic phosphorus, 79 nuclein and phosphoprotein phosphorus, 79 phosphatid phosphorus, 79 phosphorus, 79 phytin phosphorus, 79 rye, inorganic phosphorus, 79 nuclein and phosphoprotein phosphorus, 79 phosphatid phosphorus, 79 phosphorus, 79 phytin phosphorus, 79 wheat, factors influencing phosphorus content, 80, 99 germ, nuclein phosphorus, 78 phosphorus, 78 phytin phosphorus, 78 inorganic phosphorus, 77 mineral analyses, 77 phosphorus, 77, 78, 80, 99 value affected by milling process, 585 INDEX 723 Food phosphorus, advantage of complexity of organization, 355 Foods, composition, affected by fertilizers, 95 in relation to phosphorus metabolism, Sbb lecithins, 84 nuclein phosphorus, 90 phosphorus compounds, 76 phytin, 81 references, total phosphorus content, 103 Formaldehyde, phosphorus retention after, 368 Gastric juice, phosphorus, 172; dog, 172; men, 199 phosphate content dependent on degree of acidity, 200 Genitalia, organic phosphorus— ass, 148; cattle, 148; fish, 147; hedgehog, 107; horse, 148; rabbit, 107 phosphorus— cattle, 149; fish, 146, 147 phosphorus changes with season, 147 content, 146 Giantism due to excessive functioning of hypophysis, 482 Glidine, components, 533 feeding experiments, 533 Glucosides, digestion, 233 \ ■ Glycerophosphate and lecithin, physiological effects, bZZ, 58Z of calcium, commercial, constituents, 74 casein (sanatogen), feeding experiments, 325 Glycerophosphates, acid and neutral salts, physiological effects, 520 effect on ferratin of liver, 301 intracellular ferments, 281 phosphorus excretion, 209, 521 power of oxidation, 280 respiration coefficient, 280 metabolism of, digestion and absorption, 286 feeding and injection experiments, 209, 324, 325, 342, 343-354, 520, 582 See also Glycerophosphoric acid compounds specific effects on swine, 343-354 synthesis, 74 urine, 190 Glycerophosphate therapy, 518, 520, 546, 582 with antirabes treatment, 521, 583 Glycerophosphoric acid compounds, balance experiments with, 290 growth and composition of animals as affected by, 296 metabolism, 276 See also Glycerophosphates cream, 163 decomposition in the organs, 520 elimination, path, 209 in the blood, 520 urine in fatty degeneration, 513 of urine, 190 skimmed milk, 163 whole milk, 163, 166 Gout, as disturbance of purin metabolism, 525 associated with phosphaturia, 565 phosphorus metabolism, 522 purin excretion on light and dark meat compared, 525 Grass (Guam), ether extract phosphorus, 90 mineral analyses, as affected by fertilizer, 95 (Para), ether extract phosphorus, 90 phosphatid phosphorus, 89 phosphorus as affected by fertilizer, 95 See also Bermuda, buffalo, blue-joint, Indian bunch, Johnson and blue grass; also meadow fescue 724 INDEX Growth and composition of animals as affected by compounds of glycerophoa- phoric acid, 296 affected by inorganic phosphates, 227 effect on phosphorus requirement, 389 Guanase, definition, 28 Guanin, composition, 18 Guanylic acid, composition, 22 digestion, 233, 236 effects, 250 Haematogen, Bunge's theory, 46 nature, 172 phosphorus content, 46 Hard water, influence on teeth, 394 Hay and grass compared in feeding experiments, 373 compared with oats in effect on bones, 389 mineral analyses, 422 phosphatid phosphorus, 89 phosphorus, 422 _ See also alfalfa, Bermuda, clover, cow-pea, oat, peanut, timothy and vetch hay Heart, cephalin phosphorus — guinea pig, 588 composition, affected by various diseases, 507 lecithin content — children, 110, 111; dog, 125, 443; men, 125; new-born infants and fetuses, 110, 111; sheep, 125 phosphorus — guinea pig, 588 mineral analyses, 507 phosphatid content — guinea pig, 588 phosphorus — cattle, 126 . phosphocarnic acid — dog, 124; horse, 124 phosphorus — guinea pig, 588; men, 507; sheep, 149 phosphorus changes effected by tuberculosis, 588 Hedgehog, lecithin content, 107 Helicoproteid, occurrence, 47 Hemaproton, composition, 531 Hemaprotagon therapy, 531 Heminucleic acid, definition, 20 Hemiplegia, effect on composition of spinal cord, 538 Hemorrhage, influence on phosphorus metabolism, 526 Hemp cake, lecithin (phosphatid) content, 85 seeds, inorganic phosphorus, 79 lecithin phosphorus, 79 phosphorus, 79 protein phosphorus, 79 Heparphosphatid, composition, 69 Herbivora, carnivora and omnivora — acidosis, 478 composition of blood, 369 excretion of phosphorus, 207 subcutaneous administration of lactic acid, 553 Histogehol, description, 92, 93 Hookworm, phosphorus metabolism, 527 Hyperleucocytosis after nucleic acids, 246, 247, 252 Hypophosphites, elimination, 206, 221, 222, 223, 226, 227 feeding experiments with, 343-354 influence on phosphates in intestine, 223 in urine, 222 with cod-liver oil in treatment for rachitis, 576 Hypophysis and thyroid glands, reciprocal functions, 481 effects of removal, 482 phosphorus content, 146 studies in relation to acromegaly, 479 INDEX 725 Hypophysis therapy, diuretic effect, 480 in acromegaly, 479 with hypoplastic dwarf, 482 Hypoxanthin, composition, 18 Hysteria, phosphorus content of cerebrospinal fluid, 537 Ichthulin, chemistry, 47 Icterus, effect on phosphorus metabolism, 527 relative phosphorus and chlorine in urine, 518 Incubation, phosphorus metabolism, 446 Indian bunch grass, mineral analyses, affected by water supplied, 94 phosphorus, affected by water supplied, 94 Infantile athrepsia, lecithin therapy, 501, 529 Infants' bodies, phosphorus content, 108 Infants, phosphorus metabolism, 449 requirements, 414 Inorganic and organic phosphorus, maintenance experiments, 357 metabolism experiments, 320 nutritive value, 318, 355, 364, 427 phosphates and nerve stimulation, 463 changes in egg during incubation, 447 function, in muscular action, 464 metabolism experiments with, 221 phosphorus in cancer of liver, 504 salts, absorption of, influenced by their physical condition, 376 as nerve tonic, 546 for progressive paralysis and for psychoses, 545 influence on amount and composition of milk, 382 Inosinic acid, composition, 22 Insanity, effect on phosphorus metabolism, 543 Intestinal extract, digestion of nucleic acids, 234 infantilism, phosphorus absorption, 459 juice, digestion of nucleic acids, 233, 245 Iodocitin, components, 533 Iodothyrin, distinction from thyroidin, 545 effect on exophthalmic goiter, 582 Iodtriferrin, metabolism, 247 Iron of the nucleoproteins, 15 * Jaundice. See Icterus Jecorin, chemistry, 65 Johnson grass, ether extract phosphorus, 90 Kaffir corn fodder, ether extract phosphorus, 90 inorganic phosphorus, 77 mineral analyses, 77 phosphorus, 77 Kidney, cephalin phosphorus — guinea pigs, 588 composition, affected by various diseases, 507 diseases, composition of blood and blood serum, 498 lecithin content — cat, 107; dog, 142; hedgehog, 107; rabbit, 107, 142 phosphorus — guinea pig, 588 mineral analyses — men, 507 nuclein content — cattle, 142 partition of phosphorus, 142 phosphatid content — guinea pig, 588 effects of alcoholism, 484 phosphocarnic acid content — dog, 142; horse, 142 phosphorus, 141; cattle, 141, 142; guinea pigs, 588; men, 141, 507; sheep, 149; swine, 141, 373, 374 content and distribution, 588 in tuberculosis, 588 'Kufeke mehl," calcium, magnesium and phosphorus balances — infants, 414 Lactation, effect on phosphorus elimination, 209 Lactic acid studies in osteomalacia, 552 726 INDEX Lead poisoning, composition of blood and urine, 499 Lecithid in rice, 80 Lecithalbumins, Liebermann's, 47, 66 as agents in urinary excretion, 189 Lecithin, beet leaves and roots, 87 chemistry, 60 cleavage, asymmetric, 287 by colon bacillus, 289 Staphylococcus, 289 succus entericus, 289 in putrefaction, 537 composition, 60 compounds, 64 dextro- and laevo- forms, 287 digestion and absorption, processes, 286 effects on composition of parts of the body, 299, 300, 301, 343-354, 370 ferment activity of organs, 281 ferratin of liver, 301 growth, 296 hydrochloric acid of stomach, 551 lipoid deposits, 302 milk production, 301 and composition, 383 phagocytic index of leucocytes, 281 power of oxidation, 280 respiration coefficient, 280 weights of parts of body, 298, 299, 300, 301 to prolong life, 298. feeding and injection experiments, 280, 281, 286, 314, 341, 342, 343, 343-354, 370, 383, 498 function as antihaemolytic agent, 280 bringing about viscosity of cells, 278 intermediary product in oxidation of fats, 276, 280 substratum for cell reactions, 277 in digestion of fat, 173, 277, 282 influence on diastase, 285 , in intermediary metabolism, 276 relation to bone formation, 568, 569 significance of the colloid nature, 278 in relation to narcosis, 278, 280 of beet leaves and roots, 87 bile, 174 in relation to digestion of fat, 173, 277 blood, 107 as affected by disease, 496, 511, 541, 542, 548 of different species, 151, 152 bodies of young animals in relation to helplessness at birth, 277 bone marrow, 107, 118, 119, 542 in dementia paralytica, 498, 542 bones, 107, 506 brain and nerves, 107, 130, 300, 539, 588 as affected by tuberculosis, 588 of human fetuses and children, 110, 111 eggs, 147, 171, 447 during incubation, 276, 341, 447 ether and benzene extracts of vegetable substances, 86 feces, 195, 196 in tabes and tabo-paralysis, 498 sources, 288 genitalia, 107, 147 heart, 107, 125, 588 as affected by starvation, 443 tuberculosis, 588 INDEX 727 Lecithin of heart, in health and disease, 125 of human fetuses and children, 110, 111 kidney, 107, 142, 588 as affected by tuberculosis, 588 liver, 107, 137, 588 as affected by destruction of leucocytes, 494 tuberculosis, 588 in relation to fatty degeneration, 300, 513 various diseases, 513 of human fetuses and children, 110, 111 lungs, 588 as affected by tuberculosis, 588 medulla, 588 as affected by tuberculosis, 588 milk at different periods, 166 effect of pasteurization, 157 identity doubted, 158 in relation to brain weight of the young, 277 the buttermilk, 157 of different species, 155, 162, 169, 450 skimmed milk and cream, 163 miscellaneous common foods, 87 muscles, 107, 122, 125, 588 as affected by nerve stimulation, 463 starvation, 443 tuberculosis, 588 of human fetuses and children, 110, 111 nitrogenous concentrates, 85, 91 pancreas, 141 pancreatic juice, 141 parts of the body — rabbits, 107 plants and in miscellaneous foods, 84 sperm, 148 spinal cord after hemiplegia, 538 spleen, 140, 588 as affected by tuberculosis, 588 suprarenal capsules, 143 thymus, 143 phosphorus of milk, 156, 157 of different species, 157 seeds, grains and cereal products, 79, 80, 82, 85, 87, 89 properties, 63, 284 »»_.#.*» synthesis in developing eggs, 341 egg production, 342 therapy, 528 after activation by Rontgen rays, 532 effect on narcosis, 546 in anaemia, 501 exophthalmic goiter, 582 lues, tabes, syphilis and paralysis, 497, 546 migraine, 547 neurasthenia, 545 tabes, 546 _ . , tuberculosis, 585 Lecitho-proteins, 66 Lecitogen, lecithin content, 501 treatment in anaemia, 501 Lentil seeds, inorganic phosphorus, 79 lecithin (phosphatid) content, 85 phosphorus, 79, 81 phosphorus, 79, 81, 85 phytin phosphorus, 81 protein phosphorus, 79 728 INDEX Lettuce, phosphatid phosphorus, 89 Leucocytosis in blood dissolution, 496 induced by bromlecithin, 501 lecithin ingestion, 533 injection, 530 nucleate ingestion, 519 injection, 547 nucleic acid injection, 258 nucleins, 247, 257, 502 pneumonia, 499 phytin, 533 See also Leukaemia Leukaemia, effects on composition of blood, 497, 498 phosphorus metabolism, 493 Linseed oil meal and corn, specific effects on swine, 376 inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Lipanin treatment in rachitis, 567, 573 Lipoid content of brain as affected by age— dog, 135 organs, 588 as affected by tuberculosis, 588 phosphorus estimations in nerve tissue, 134 spleen, 139 Lipoids, absorption by, 280 definition, 58 essential for life, 358, 362, 363 Liver, cephalin phosphorus — guinea pig, 588 composition as affected by cancer, 497, 504 various diseases, 507 ether-alcohol soluble phosphorus — rabbit, 313 ferratin, effects of organic phosphorus compounds on, 301 lecithin content — cat, 107, 137; children, 110, 111; hedgehog, 107; new- born infants and fetuses, 110, 111; rabbit, 107, 137 as influenced by alcohol poisoning, 484 destruction of leucocytes, 494 fast, 446 various diseases, 513 in fatty degeneration, 513 phosphorus — guinea pig, 588 mineral analyses — men, 105, 507; rabbit, 313; swine, 327 nuclein phosphorus — cattle, 137; dog, 137; fowl, 137; new-born infants and fetuses, 138; rabbits, 138 nucleoprotein content — rabbit, 139 pathological changes of the phosphorus content, 137 phosphatid content — guinea pig, 588 phosphatids of, differentiated, 138 phosphocarnic acid— dog, 138; horse, 138 phosphorus — cattle, 107, 136, 137, 149; dog, 137; fowl, 137; guinea pigs, 588; men, 104, 105, 507; new-born infants and fetuses, 136; rabbits, 313; sheep, 136; swine, 137, 327, 373, 378 relation to nuclein metabolism studied by means of Eck fistula, 240 Livetin, occurrence, 45 Lues, spinal, lecithin content of blood serum, 500 lecithin therapy, 531, 546 Lung diseases, phosphaturia in. See Phosphaturia Lungs, cephalin phosphorus — guinea pig, 588 composition, as affected by pneumonia, 507 lecithin content — cat, 107; hedgehog, 107; rabbit, 107 phosphorus — guinea pig, 588 mineral analyses — men, 105, 507 INDEX 729 Lungs, phosphatid content — guinea pig, 588 as affected by alcoholism, 484 phosphorus — guinea pigs, ^588; horse, 146; men, 104, 105, 507; sheep, 149 compounds, 145 Lymph, mineral analyses — men, 105 phosphorus — horse, 177; men, 105, 177 Magnesium, substitution for calcium in bone, 386, 390 Maintenance of neutrality, 178, 181 Malnutrition of bones, 534 called pseudorachitis, 573 contrasted with osteomalacia, 552 Malt, lecithin (phosphatid) content, 89 phosphorus, 89 Mange, benefits from nucleins, 257 Mangel wurzel, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Massage, effect on phosphorus excretion, 466 Meadow fescue, phosphorus as affected by water supplied, 93 Meat, ash, effects on growth, 227 inorganic phosphorus, 78, 120 lecithin content, 87, 104, 107, 125 light and dark compared as to purin excretion, 525 meal added to corn, specific effects on swine, 376 mineral analyses, 78, 121 nuclein content, 104 and phosphoprotein phosphorus, 88 phosphatid phosphorus content, 88 phosphocarnic acid content, 104 phosphorus, 78, 88, 104, 107, 120, 121 ration compared with edestin, 338, 339 Meconium, mineral analyses, 194 phosphorus, 194 Medulla, organic phosphorus compounds — guinea pigs, 588 phosphorus — guinea pigs, 588 Melancholia, lecithin therapy, 531 Mellin's Food, analysis, 451 as a milk supplement for infants, 451 compared with human milk in composition, 451 Mental and other nervous disorders, effects on phosphorus metabolism, 536 use of drugs and effects on phosphorus metabolism, 545 Mental work, influence on phosphorus metabolism, 461 Metabolism in osteomalacia, 556 of casein, 260 general summary, 274 glycerophosphoric acid compounds, 276 inorganic phosphates, 221, 318 nucleoproteins and nucleic acids, 229 phosphocarnic acid, 303 phosphorus in relation to other elements, 213 phytin, 305 See also Digestion Metacasein formation by pancreatic extract, 269 Metaphosphates, poisoning from, 226 Metaphosphoric acid, poisonous effects, 221 poisoning from, 227 Migraine, lecithin treatment, 547 Milk, calcium content, as affected by calcium salts, 571, 573 range of variation, 158 casein content, 160; ass, 155, 161; cow, 155, 161, 163, 164, 166; goat, 161; others, 161; woman, 155, 161, 167 730 INDEX Milk, casein content, affected by tuberculosis, 382 of human, factors influencing, 162 phosphorus — ass, 156; cow, 156, 157; woman, 156, 157 changes resulting from heating or standing, 164 with progress of lactation, 165 component essential to normal growth, 358, 360, 362 composition, compared with that of the young, 167 in relation to time required for young to double in weight, 168 fermented, organic phosphorus compounds, 162 glycerophosphoric acid phosphorus — cow, 163, 166 inorganic phosphorus — ass, 156; cow, 154, 156, 157, 165 lecithin content— ass 155, 162; cow, 87, 155, 162, 163, 166, 169, 450; goat, 162; others, 162, 169; woman, 87, 155, 162, 169, 450 phosphorus— ass, 156; cow, 156, 157, 163, 166, 450; woman, 156, 157 metabolism experiments, 273, 274, 322, 326, 367, 414, 417, 449, 456, 457, 518, 570 mineral analyses— ass, 159; cow, 159; goat, 159; others, 159; woman, 105, 159, 160, 418 modification of cow's, for human infants, 417, 449, 450 nuclein and phosphoprotein phosphorus — cow, 88 nucleon content-ass, 155, 163; cow, 155, 162, 163; goat, 163; woman, 155, 163 phosphorus — goat, 157; cow, 157; woman, 157 of different species compared in diet of man, 367 infant feeding, 449 phosphatid content, 162 as affected by pasteurization, 157 doubted, 158 in the brain weight of young animals, 277 phosphorus content — cow, 88, 162 phosphocarnic acid (nucleon) content, 162 phosphorus— ass, 156, 159; cow, 88, 156, 157, 159, 160, 163, 164, 165, 166, 168, 380, 381, 450; goat, 157, 159, 168, 383; others, 159, 168; woman, 105, 156, 157, 159, 160, 167, 168, 405, 418, 450 content as affected by inflammation of the udder and tuberculosis, 382 protein phosphorus — woman, 158 salts of, 160 secretion as affected by foods, 379 lecithin, 301 phytin, 307, 312, 315 effect on phosphorus requirement, 389 utilization of minerals from, 456, 457 whey feeding for infants, 455 See also Skimmed milk Millet bran, lecithin phosphorus, 82 phosphorus, 82 hay, ether extract phosphorus, 90 inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Mineral constituents of blood under normal conditions, 501 human organs after death from various diseases, 507 milk, 158 elements in the urine during fast, 400 matter of bones as obtained by removal of organic matter with glycerin and potassium hydrate, 113 retention by the fetus, 471 Monamino-diphosphatids, 68 -monophosphatids, 60 INDEX 731 Mongoloid idiocy, thyroid treatment, 546 Mononucleic acids, definition, 21 chemistry, 21 Mononucleotides, definition, 21 Morbus basedowii in relation to phosphorus metabolism, 581 Muscle phosphatids differentiated, 125, 126 , Muscles, comparisons of striated and non-striated, 123 cephalin phosphorus— guinea pig, 588 ' inorganic phosphorus— cattle, 120, 126; dogs, 468; rabbit, 463; sheep, 78 lecithin content— children, 110, 111; dog, 125, 443; hedgehog, 107; new-born infants and fetuses, 110, 111; rabbit, 107, 463; sheep, 125 phosphorus — fish, 122; guinea pig, 588 mineral analyses— cat, 121; cattle, 121; deer, 121; dog, 121; fish, 121; fowl, 121; frog, 121, 124; men, 105, 121; rabbit, 121; sheep, 78; swine, 121, 124, 327 nuclein and pseudonuclein phosphorus — fish, 122, 147 phosphorus — cattle, 122; fowl, 122 phosphatid content — guinea pig, 588 phosphorus — cattle, 126 phosphocarnic acid — dog, 305; men, 123; new-born infants and fetuses, 123; rabbit, 304 phosphorus — dog, 468 phosphorus— cat, 121; cattle, 107, 120, 121, 122, 123, 126, 149; children, 458, 572; deer, 121; dog, 121, 468; fish, 121, 122, 146-147; fowl, 121, 122; frog, 121, 122, 124 phosphorus— guinea pig, 588; men, 104, 105, 121, 462; rabbit, 121; sheep, 78; swine, 121, 124, 327, 370, 372, 373 phosphorized proteins spared during atrophy, 445 phosphorus changes effected by exercise — dogs, 468 nerve stimulation, 463 tuberculosis, 588 compounds, 120 See also Heart Muscular action, in relation to lactic acid and potassium phosphate, 464 _ formation and phosphorus elimina- tion, 465 Mushrooms, lecithin (phosphatid) content, 85, 87 Myosin, feeding experiment, 320 Myostromin formation from myosin, 293 Myxoedema attributed to hypophysis and thyroid gland, 481 thyroid treatment, 546, 578 vegetable diet and disodium phosphate treatment, 546 Neottin, chemistry, 71 Nephritis, effects on blood, 498, 500, 548 and urine, 499 phosphorus metabolism, 547 purin excretion delayed, 525 Nerve degeneration, changes in phosphorus compounds, 536 katabolism, nitrogen and phosphorus ratio, 216 Nerves, composition. See Brain and Nerves Nerve stimulation, electrical, phosphoric acid change, 463 influence on phosphorus metabolism, 463 Nervous disorders, effects of drugs on phosphorus metabolism, 545 on phosphorus metabolism, 536 disturbances as cause of phosphaturia. See Phosphaturia excitement, effect on urinary phosphorus, 462 Nervinol, description, 93 Neuralgia, chronic, glycerophosphate treatment, 521 Neurasthenia, glycerophosphate treatment, 521 lecithin treatment, 545, 585 phosphorus content of cerebrospinal fluid, 537 excretion under excessive protein feeding, 545 732 INDEX Nitrogen and phosphorus ratio in feces during pregnancy, 472 fever, explained, 519 muscle, influence of degeneration, 445 organs, influence of fast, 445 urine, influenced by cow's and human milk, 451, 456; epilepsy, 541; exercise, 469; experimental diphtheria infection, 516; fast, 401, 402, 438, 439, 440, 442, 443, 644; fever, 514, 516; hypophysis treat- ment, 481; lmyphatic leukaemia, 495; a meat diet, 215; nephritis, 548; phases of malarial fever, 516; pregnancy, 472; sleep, 472; state of nutrition, 215; sweating, 467; thyroidectomy, 580; vari- ous diseases, 563 See also Phosphaturia Normal phosphorus metabolism, 178 Nuclease content of blood serum in nervous diseases, 540 organs, effect on thyroidectomy, 580 definition, 28 influenced by administration of nucleic acids, 253, 254 in the stomach, age at which it appears, 233 occurrence, 235 Nucleic acid cleavage by organ ferments, 235 enzymes concerned, 28 partial, 20 possible paths, 29 acids, absorption, 244 a and ft, definition, 20 "biological value," 254 chemistry, 16 digestion by erepsin, 232 peptic, 231 tryptic, 231 effects, miscellaneous, 250 on action of Bacillus coli, 252 Staphylococcus infection, 250 composition of parts of body, 301 enzyme activities of the organs, 250 flow and composition of milk, 383 hydrochloric acid of the stomach, 551 leucocytosis, 248, 252 power of oxidation, 280 purin excretion, 245 respiration coefficient, 280 tubercle bacilli, 257 weights of parts of the body, 301 feeding and injection experiments, 237, 247, 343-347 See also Nucleic acids, effects, and Nucleic acids, therapy and prophylactic use functions as active agents in specific body processes, 252, 255 in fish eggs, 171 intestine, 146 metabolism, 229 methods of introduction, comparison of results, 249 prophylactic use, 258, 504 therapeutic and prophylactic use, 256 Nucleic acid therapy, abscesses, 502 paralysis, 547 scarlet fever, 519 tuberculosis, with disodium-methyl-arsenate, 586 INDEX 733 Nuclein, definition, 13 _ -free diet in gout, nitrogen and phosphorus balances — men, 524 in blood, 151, 152; bones, 506; brain and nerves, 130, 134; cereal products, 79, 88; eggs, 88; fetuses, 138; genitalia, 147; kidney, 142; liver, 137, 138; meat, 88, 104; milk, 88; muscle, 122, 147; nitrogen- ous concentrates, 90, 91; pancreas, 141; rice, 80, 88; seeds, 79; spleen, 139, 140 suprarenal capsules, 143; thymus, 143; vegetables, 88 katabolism in exercise reduced by training, 466 metabolism, 229 in gout, 523 phosphorus distribution in foods, 90 synthesis, 30, 229, 341, 354 therapy, 256 in anaemia, 502 chlorosis, 502 Nucleins, digestion, 239 ferments of individual organs, 235 peptic, 231 tryptic, 231 effects on bacteria, 252 composition of parts of the body, 301 flow and composition of milk, 383 leucocytosis, 247 weights of parts of the body, 301 feeding experiments, 238, 301, 309, 343, 383, 522 Liebermann's, feeding experiments, 322 prophylactic use, 252 therapeutic and prophylactic use, 256 Nucleinases, definition, 30, 236 occurrence, 236 Nucleoalbumins of eggs, 170 Nucleohistone, definition, 144 occurrence, 144 Nucleon. See Phosphocarnic acid Nucleoprotein digestion, accompaniments, 245 direct observations on the alimentary tract, 244 effects of bacteria in the alimentary tract, 234 peptic, 232 tryptic, 231 katabolism, methods of experimental study, 232 Nucleoproteins, chemistry, 13 cleavage products, 17 composition, 15 feeding experiments with, 237, 322 formed from phosphoproteins during incubation, 446, 447 functions as active agents in specific body processes, 255 in bile, 174; brain, nerve, spinal cord, 130; eggs, 147; genitalia, 147; liver, 139; pancreas, 141; pancreatic juice, 141, 173; spleen, 140; thymus, 143; thyroid, 144 metabolism, 229 rectal feeding, 239 Nucleosidases, definition, 30, 236 occurrence, 236 Nucleosides, definition, 21 Nucleothyminic acid, definition, 21 Nucleotidases, definition, 30, 236 occurrence, 236 Nucleotides, digestion, 233, 236 Nucleotin, definition, 21 Nutrose, composition, 92, 237, 320 feeding experiments, 237, 320 734 INDEX Oat grains, inorganic phosphorus, 77 mineral analyses, 77, 422 phosphatid phosphorus, 89 phosphorus, 77, 83, 89, 96, 97, 422 hay, ether extract phosphorus, 90 hull, phosphorus, 83 kernel, phosphorus, 83 ' plants, phosphorus as affected by water supplied, 93, 94 Oats compared with hay in effect on bones, 389 phosphorus compounds, as affected by composition of soil, 96 phytin, 53 Oedema, effect of varying water, sodium chloride and phosphorus content of the food, 549 Oil cakes, flax, lecithin (phosphatid) content, 85 Onions, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Oophorin treatment for osteomalacia, 558 Organic and inorganic phosphorus, comparative nutritive value, summary, 364 phosphorus compounds, chemistry, 13 in the urine, 190 changes in lymphatic leukaemia, 495 low requirement, 330 Organs, composition as affected by disease, 507 Orizanine an essential nutrient, 490 cures polyneuritis, 490, 491 occurrence, 491 properties, 490 Ortho-, meta- and pyrophosphates, comparison in animal tests, 226 pyrophosphoric acids, chemical comparison, 227 pyro- and metaphosphoric acids as anticoagulating agents, 256 Osteomalacia, 552 distinct from malnutrition of the bones, halisteresis of the bones, fragility of the bones and osseous cachexia, 552 effects on blood, 557 bone formation attributed to lecithin, 569 composition of blood ash, 557 bones, 554 parathyroids, 559 etiology as a phosphorus metabolism disturbance, 485 due to excessive lactic acid production, 553 disease of the thyroid gland, 558 experimental, due to low calcium diet, 394 . . effects of dicalcium phosphate and of bone, 394 metabolism, 556 of calcium, magnesium and phosphorus, 559 methods of treatment, 558 Ovarian preparations, effects on metabolism of spayed and normal dogs, 506 Ovariotomy, influence on metabolism and composition of the body, 506 in osteomalacia, 558 influence on calcium, magnesium and phosphorus metabolism, 558, 559 Ovovitellin, chemistry, 45 maintenance experiments, 360 tryptic digestion, 271 Oysters, phosphocarnic acid content, 123 Palm cake, lecithin (phosphatid) content, 85 nuclein phosphorus, 90 Pancreas, in relation to diabetes, 510, 511 organic phosphorus compounds — cattle, 141; dog, 141 partition of phosphorus, 141 phosphorus content, 140; cattle, 141; children, 141; men, 141; new- born infants, 141; sheep, 149 INDEX 735 Pancreas, removal of, effect of calcium, nitrogen and phosphorus of the urine of a fasting dog, 512 Pancreatic digestion of casein, in vitro tests, 267 fats, relation of bile to, 282 lecithin to, 282 extract, digestion of nucleic acids, 234 juice, digestion of nucleic acids, 233, 245 nuclein, 233 inorganic phosphorus — dog, 173 organic phosphorus compounds — dog, 141, 172 phosphorus, 172; cattle, 173; dog, 173 Papain digestion of casein, 271 Parachymosin, occurrence and action, 38 Paralysis agitans, thyreoidin therapy, 545 and senility, thyreoidin therapy, 545 effects on phosphorus metabolism, 541 general, changes in the brain, 540 lecithin treatment, 546 progressive, effect on ferment activity of cerebrospinal fluid, 541 lecithin content of serum, 500 phosphorus content of cerebrospinal fluid, 537 treatment with inorganic salts, 545 nucleic acid, 547 nucleins, 257 Paranucleic acids from phosphoproteins, 33 Paranucleins, definition, 14 from phosphoproteins, 33 Parathyroid treatment, effect on bone formation, 559 Parathyroidectomy, investigations of the results, 559 Parathyroids in relation to formation of dentine and enamel, 560 osteomalacia, 559 phosphorus metabolism, 559 Peanut cake, lecithin (phosphatid) content, 85 nuclein phosphorus, 90 hay, ether extract phosphorus, 90 Peanuts, inorganic phosphorus, 78 mineral analyses, 78 phosphorus, 78 Peas, lecithin content, 87 (phosphatid) content, 85 phosphorus, 81 phosphorus, 81, 85 phytin phosphorus, 81 (yellow), inorganic phosphorus, 79 nuclein and phosphoprotein phosphorus, 79 phosphatid phosphorus, 79 phosphorus, 79 phytin phosphorus, 79 Peptic digestion of nucleoproteins, 232 casein, 262 affected by carbohydrates, 267 as a reversible action, 266 comparison of casein from different species, 265, 267 phosphorus of the nuclein resulting from, 263 results, 264 Salkowski's description of the process, 264 under different conditions of acidity, 268 Phosphate of calcium, amounts to be used for different animals, 535 deposits under the skin, 503 for diabetes mellitus, 511 experimental osteomalacia, 394 influence on composition of parts of body, 327, 328, 385, 736 INDEX Phosphate of calcium with phosphorized cod-liver oil, for rickets, 576 influence on composition of parts of body, 327, 328, 385, 386, 388, 389 milk composition and production, 379, 380, 383 other feeding experiments, 222, 223, 322, 324, 326, 828 precipitated, compared with bone ash, floats and bran in feeding experiments, 328 See also Bone Meal potassium, feeding experiment, 324 produces a rachitic effect in bones, 569 sodium compared with organic phosphorus compounds in feeding experiments, 312, 314, 326, 335, 343 for cretinism, 579 myxoedema with a vegetable diet, 546 influence on composition and production of milk, 381, 383 of parts of body, 313, 370, 385, 389, 391 injection, influence on excretion as affected by species, 209 Phosphates, absorption, 182 acid sodium phosphate unfavorable to the use of oats, 389 addition to silage, 101 and lecithin content of muscle as affected by nerve stimulation, 463 effect on composition of bones, 385 milk, 380 effects on growth, 228 nausea and catharsis, 357 feeding experiments, 342 See also the individual phosphates influence on digestion, 186 enzyme action, 176, 186 fermentation, 31 in maintenance of neutrality, 178 intestinal changes, 184 method of elimination in the urine, 188 of alkaline earths, feeding experiments, 221, 222, 224, 225 bones, effects of diet, 384 sodium and calcium, feeding experiments, 326 potassium compared with lecithin and with a mix- ture of phosphorus compounds for maintenance of life, 343 urine as affected by acid in the blood, 445 pneumonia and typhoid fever, 519 time of day and increased ingestion of protein, 475 various diseases, 561 paths of elimination, 182 Phosphate therapy in psychasthenia, 545 psychatoxies, 545 See also the individual phosphates Phosphatese, definition, 32 essential in organic phosphorus synthesis, 491 in cure of polyneuritis and beriberi, 491 Phosphatid content of blood as affected by anaemia, 496 chlorosis, 496 eggs, 171 liver as affected by cancer, 504 of incubating chick, 449 milk, 162 organs as affected by alcoholism, 484 tubercle bacilli, dead, 587 tuberculosis, 587 definition, 58 deposits as a result of lecithin and egg yolk feeding, 302 INDEX 737 Phosphatid, synthesis, 74, 341 Phosphatids, chemistry, 58 classification by Bang, 59 Rosenheim, 58 differentiation in blood cells, 153; egg yolk, 171; flours, 89; heart muscle, 125, 126; kidney, 142; liver, 138; lungs, 145; spleen, 140; suprarenal capsules, 143 function as semipermeable membrane, 278 in brain, 135, 588; heart, 588; kidneys, 588; liver, 588; lungs, 588; medulla, 588; muscles, 588; spleen, 588; suprarenal capsules, 143 naming of, 58 "plant, chemistry, 73 physico-chemical reactions with inorganic and organic substances, 278 Phosphaturia, 561 following quinine treatment for fever, 515 in artificially fed infants, 459 tuberculosis, effect of arsenic-phosphorus treatment, 586 phytin treatment, 315 Phosphocarnic acid, chemistry, 49 cleavage products, 49 function as a carrier of mineral elements in the body fluids, 303 an energy-producing nutrient, 304 a source of muscular energy, 467 in blood, 150; brain and nerves, 130, 305; genitalia, 147; kidnev, 142; liver, 138; meat, 104; milk, 155, 157, 162, 163; muscle, 123, 124, 304, 305, 468; spleen, 140; urine, 191 brain, effect of fast, 305 nervous excitement, 305 muscle, factors influencing amount, 303 metabolism, 303 Phospholipins in genitalia, 148 Phosphoprotein, definition, 33 Phosphoproteins, chemistry, 32 in eggs, 147; genitalia, 147; pancreas, 141; pancreatic juice, 141, 173 occurrence, 147 of animal origin, 47 vegetable origin, 48 Phosphoric acid administration, benefits from, 224 in dyspepsia, 223 in case of hypochlorhydria, 223 results in rheumatism, 224 Phosphorized proteins spared in degenerating muscle, 188 fever, 519 Phosphorus absorption during rectal feeding, 404 in intestinal infantilism, 459 effect of low fat content of diet, 568 and calcium balances with cattle, 222; dogs, 225, 323, 435; infants, 414, 416, 417, 418, 453, 455, 570; growing children, 411, 412, 413, 453; men, 218, 219, 293, 326, 403, 408-409, 512; sheep, 221; swine, 375 phosphatid content of organs as influenced by chronic tubercu- losis, 588 vaccination with dead tubercle ba- cilli, 587 balances with calves, diet of skimmed milk and mineral supple- ments, 222 738 INDEX Phosphorus balances with children convalescent from scarlet fever on a diet of milk, 518 cows, high and low intake compared, 420 phosphorus rations compared, 420 rations of different common foods, 420 dogs as affected by external hemorrhage, 526 inorganic salts, 225 phytin, 311 diets varying in amount of fat, carbohydrate or protein, 435 increasing amounts of food, 437 normal rations supplemented by inorganic salts, 205 organic and inorganic phosphorus compared, 323 growing boys, ordinary mixed diet, 511, 512 taking different amounts of exercise, 413 horse, low calcium -rations, 423 infants, artificially fed, 414, 416 breast-fed, 418 diet of milk gruel and malt, 455 healthy^ and diseased, on natural and artificial milk diet, 452 milk diets of varying composition, 417 normal and rachitic, on milk diets, 570 organic and inorganic phosphorus com- pounds compared, 325 raw and sterilized milk, computed to kilo- gram body weight, 416, 453 men, 218 as influenced by antirabes with and without sodium glycerophosphate, 521 casein added to normal mixed diet, 293 excessive water drinking, 473 fatigue of mountain climbing, 469 lecithin, 291 added to mixed diet, 295 loss of sleep; crackers, butter, _ and milk diet, 432 nuclein-free diet in gout, 524 nucleins added to mixed diet, 238 nucleins, gout patient and con- trol, 522 phosphate of sodium added to ordinary mixed diet, 202 phytin added to simple diet, 486 overfed condition, 306 sanatogen added to normal mixed diet, 293 at different altitudes, 429 diet low in phosphorus with supplements added at intervals, 407 of crackers and milk, 409 diets composed principally of different sorts of rice, 488 containing different amounts of phosphorus, . 486 . ' varying in phosphorus and nitrogen con- tent, 433 content, 404 protein content, 433 INDEX 739 Phosphorus balances with men, endoarteriitis, 512 following a fast, 402 normal mixed diet, 403, 406 overfeeding, 219 phosphate, glycerophosphate and phytate of cal- cium compared as added to mixed diet, 324 of calcium and casein (proton) com- pared as added to bread and milk diet, 322 and sodium compared with milk in simple diet, 326 senility and paralysis agitans as affected by thyreoidin, 545 simple mixed diet, 411 whole wheat bread and white bread compared, 366 rabbits, phytin and sodium phosphates compared, 312 rats as influenced by phosphorus added to nitrogen- free rations, 340 powdered beef and edestin compared, 338, 339 without organic phosphorus, 335 with and without protein, 337 sheep as influenced by phosphates of calcium and magnesium added to a ration of meadow hay, 221 normal foods, 426 swine as influenced by nucleic acids, 242 cereal foods with and without additional calcium, 375 common food mixtures, 378 different types of phosphorus compounds, 328 young, 424 women during progress of pregnancy, 472 See also Phosphorus Requirements calcium and magnesium metabolism as influenced by ovariotomy in case of osteomalacia, 558, 559 complexes artificially prepared, investigations of physiological effects, 549 content of dietaries, 409, 410 foods, references, 103 organs, urine and feces in relation to nitrogen content. See Nitrogen and phosphorus ratio young dogs as influenced by removal of certain organs, 580 distribution in different cuts of beef, 120 muscles as affected by exercise — dogs, 468 elemental, effects on mineral metabolism in dogs, 577 excretion as influenced by acids, salts, diet and species of subject, 198 exercise, sweating and massage, 466 fast, 399, 426, 466; fat, 459; mental work, 461; oil, 576; rachitis, 573, 574; sodium carbonate in acidosis, 510; sweating, 467; wine drinking, 469 in fever, variations explained, 519 relation to chlorine in various diseases, 518 740 INDEX Phosphorus elimination in relation to urea and to sulphur, effects of exercise, 464 loss during fast; sources of the phosphorus, 438 metabolism as influenced by alcohol ingestion, 483; altitude, 429; amount of food, 430, 458; castration and ovarioto- my, 505, 559; exercise, 464; hemorrhage, 526; hypophysis therapy, 479; nerve degeneration, 536; nerve stimulation, 463; parathyroids, 559; thirst and water drinking, 473; thyroid glands, 577; time of day, 474; toxins and antitoxins, 582 during fast, 438; fever, 514; first few days of infant life, 414, 458; hypnotic sleep, 442; incubation, 446; infancy, 449; pregnancy, 470; sleep, 472 in acromegaly, 479; arthritis, 484; blood diseases, 492; cancer, 503; chlorosis and anaemia, 492; diabetes mellitus, 510; endoarteriitis, 512; epilepsy, 541; gout, 522; icterus, 527; insanity, 543; leukaemia, 493; mental and other nervous disorders, 536; mental and other nervous disorders influenced by administration of phosphorus compounds and other drugs, 545; morbus based owii, 581; nephritis, 547; paralysis, 541; pneumonia, 515; rachitis, 566; rheumatism, 484; tuberculosis, 583 See also Phosphorus balances and Phosphorus require- ments of foods, 76 lipoids, constancy of, in cells and organs, 281 milk, 153, 381 as affected by foods, 379 silage corn with and without added floats, and of silage made from the same, 102 the blood, 149 changes in disease, 496 bones as influenced by acidified hay, 387 alkaline earth carbonates and sul- phate added to oat rations, 390 diets of hay, hay and oats, oats or oats plus NaH 2 P0 4 , 389 lime- and phosphorus-poor diet with and without added phosphates, 386 lime-poor diet, 387 teeth, marrow and cartilage, 111 brain as affected by arteriosclerosis, 135 chronic tuberculosis, 588 nerves and cerebrospinal fluid, 127 diet, advantage of complexity of organization, 355 digestive secretions, 172 feces, 194 of infants, conditions affecting, 459 See also Phosphorus Balances genitalia, 146 hypophysis, 146 kidney, 141 as influenced by chronic tuberculosis, 588 liver, 136 as influenced by chronic tuberculosis, 588 muscles, 120 spleen, 139 suprarenal capsules, 143 thymus, 143 thyroid, 144 INDEX 741 Phosphorus of urine as gauge of digestive disturbances in infants, 564, 565 * F influenced by the crisis of disease, 499 in arthritis and chronic rheumatism, 484 minimum amount inferred from amniotic fluid content, 457 organic, in urine after epileptic attack, 541 w as affected by degenerative nervous diseases, 5d9 a pathological symptom in the breast-fed infant, 565 output in urine, as influenced by age of subject, 407 artificial dyspnoea, 479 exercise, 464 extirpation of pancreas, 511 fast, 399, 439, 440, 444 fever, 514 mental work and by diet, 461 rest and exercise, 461 sleep, 472 thirst and water-drinking, 473 time of day, 474 of breast-fed infants, 456, 457 horse, 422 See also Phosphorus Balances requirement as judged by excretion during fast, 399 of animals, general discussion, 397 cattle, 419; dogs, 426; horses, 422; infants, 414; adult human beings under normal conditions, 403; men, 487, 512; rats, 428; sheep, 425; swine, 424 See also Phosphorus Balances retention, agents for reducing, 480 as affected by dyspepsia, 459 during pregnancy, 472 underfeeding and overfeeding, 433 in relation to typhoid convalescence, 434 starvation, effects on tissues of the body and on phosphorus excre- tion, 436 with dogs, 394 milch cows, 394 rats, 430 therapy in diseases of the blood, 501 epilepsy, 547 malnutrition of the bones, 535 mental and other nervous diseases, 545 osteomalacia, 558, 559 rachitis, 567 tuberculosis, 585 Phytase, definition, 57 occurrence, 185, 310 Phytate of calcium, feeding experiment, 314, 324 sodium, toxic effects, 314 Phytic acid formula, 53 synthesis, 54 Phytin, chemistry, 51 cleavage by enzyme, 57, 185 constitution, 54 clinical experiments, 315 effects in polyneuritis, 486 on composition of parts of the body, 327, 370 character of bones, 328 digestion, 314 diuresis, 307 excretion of calcium, magnesium and phosphorus, 206 glycogen of the liver, 305 742 INDEX Phytin, effects on hydrochloric acid of the stomach, 551 leucocytosis, 533 milk, composition, 381 and flow, 307, 312, 315, 383 nitrogen and phosphorus metabolism, 487, 311 paths of elimination of calcium and magnesium, 207 power of oxidation, 280 feeding experiments, 305, 326, 328, 343-354, 486 in bones, 82; cereal products, 79, 83, 84 feces, 196; seeds, 79, 80, 81, 82 influence on phosphaturia, 315 under antirabes treatment, 566 occurrence, 51, 81 of corn, chemistry, 53 cottonseed meal, chemistry, 53 foods, 81 oats, chemistry, 53 laxative effects, 310, 312 metabolism, 305 pharmacodynamic value, 316 therapy, effect on erythrocytes and haemoglobin, 533 in rachitis, 569 utilization, influenced by species, 197 Phyto-vitellins, investigations, 48 Pituitary gland. See Hypophysis Plant phosphatids, chemistry, 73 Plants, distribution of phosphorus compounds, 84, 87 Plasmon, description, 92 feeding experiments, 239, 274, 290, 550 with phosphorized cod-liver oil for rachitis, 576 Pneumonia, croupous, effect on phosphorus of the urine, 193 effects on composition of urine, organs and blood, 499, 500, 507, 515, 517, 519 Polycythaemia rubra megalosplenica, lecithin of the blood, 500 Polyneuritis, cause, effects and cure, 486 Polynucleic acids, composition, 23 definition, 22 structure, 25 Polynucleotides, definition, 22 Poppy seed cake, nuclein phosphorus, 90 Potassium iodide treatment in acromegaly, increased calcium and phosphorus elimination, 482 Potato, sweet, mineral analyses, 78 white, mineral analyses, 78 Precipitated calcium phosphate, effect on character of bones, 328 Pregnancy, diet of the mother not reflected in the composition of the young, 471 effect on phospholipins of ovary and corpus luteum, 148 phosphorus requirement, 389 lipoid content of the blood of the mother and child, 472 nitrogen and phosphorus ratio in intake, urine and feces, 472 phosphorus metabolism during, 471 storage during, 408 Prophylactic use of nucleic acids, 258 Proprietary preparations of phosphorus compounds, descriptions, 92 physiological effects, 550 Protagon, chemistry, 72 Protein-free milk, characteristic effects, 359, 360 preparation, 359 Proton, feeding experiments, 322, 407 rectal feeding experiment, 404 Protylin, description, 93 effects, 301, 551 INDEX 743 Protylin, feeding experiments, 301, 324 preparation and properties, 551 use in anaemia, 301 rachitis, 301, 569 scrofula, 301 Prunes, mineral analyses, 78 Pseudonucleins, definition, 14 from phosphoproteins, 33 Pseudorachitis distinguished from true rachitis, 513 effect on phosphorus and calcium metabolism, 570, 575 Psychasthenia, phosphate therapy, 545 Psychatoxies, phosphate therapy, 545 Psychoses, treatment with inorganic salts, 545 Purin bases, composition, 17 excretion after nucleic acids, 240, 242, 245 during and after muscular activity, 469 on light and dark meat, 525 metabolism. See also Gout Purins of the feces, due to bacteria, 234 urine, origin, 246 Pyrimidin bases, composition, 19 nucleotides, digestion, 233, 236 Pyro- ortho- and meta-phosphoric acids compared as to poisonous nature, 221 Pyrophosphates, poisoning from, 226, 227 elimination of, 221 Pyrophosphoric acid in vegetable substances, 91 poisoning from, 221, 227 Rabbit, organic phosphorus compounds, 107 Rachitis and calcium metabolism, 567 distinguished from malnutrition of the bones, 573 etiology, 567 as phosphorus metabolism disturbance, 485 phosphorus metabolism and phosphorus treatment, 567 treatment, 567 lecithin, 531 protylin, 301 Rape cake, lecithin phosphorus, 82, 91 nuclein phosphorus, 90, 91 organic phosphorus, 82 phosphorus, 82, 91 seeds, inorganic phosphorus, 79 lecithin phosphorus, 79, 82 organic phosphorus, 82 phosphorus, 79 protein phosphorus, 79 Reaction of body tissues and fluids, relations of phosphates to, 178 Red dog flour, mineral analyses, 77 Rennet coagulation of casein, Bang's interpretation, 40 Bosworth's interpretation, 40 conditions favorable, 39 Hammarsten's interpretation, 39 in the stomach, 261 Mellanby's interpretation, 41 Schryver's interpretation, 41 Rennin. See Chymosin Rheumatism, chronic, forms of phosphorus in urine, 484 phosphoric acid treatment, 224, 484 Rice bran, phosphorus content and distribution, 82 diet, phosphorus balances, 486, 488 flour, phosphorus content and distribution, 79 mineral analyses, 77 744 INDEX Rice, phosphorus content and distribution, 80, 82, 491 as affected by milling processes, 78, 79, 488 polish and wheat bran, specific effects on swine, 376 (polished) diet as cause of beriberi, 485 straw, ether extract phosphorus, 90 Roborat, description, 93- feeding experiments, 550 Rock phosphate, effects on growth, 227 metabolism and slaughter experiments, 328 Roughage compared with cereals and mill feeds, 373 Rye grains, inorganic phosphorus, 79 lecithin content, 87 (phosphatid) content, 85 phosphorus, 79 phosphorus, 79, 87 protein phosphorus, 79 Sahidin, composition, 69 Saliva, composition, 175 Salicylic acid, phosphorus retention after, 368 Salts of sodium and potassium, effects on phosphorus excretion, 200 Sanatogen, composition, 92 feeding experiments, 292, 325 therapy, 532 Sanose, composition, 92, 273 feeding experiments, 273 Sciatica, glycerophosphate treatment, 521 Sclerosis multiplex, phosphorus content of cerebrospinal fluid, 537 Scrofula, nuclein therapy, 257 protylin therapy, 301 Secretion of phosphorus into alimentary tract, 182 Seeds, distribution of phosphorus compounds, 77, 83, 84 See also the individual seeds and grains Sesame cake, lecithin (phosphatid) content, 85 seeds, phosphorus content and distribution, 82 Sheep, mineral analyses of entire body, 106 Silage, addition of phosphates, 101 Skimmed milk added to corn, specific effects on swine, 376 and cream, compared with whole milk with regard to phospho- rus compounds, 163 mineral analyses, 78 Sleep, influence on phosphorus metabolism, 432, 472 Sorghum, ether extract phosphorus, 90 Soy bean cake, phosphorus content and distribution, 91 hay, mineral analyses, 78 beans added to corn, specific effects on swine, 376 mineral analyses, 78 phosphorus and lecithin content, 87 (phosphatid) content, 85 Species, effects on the paths of phosphorus excretion, 211 Sperm, fish, phosphorus content and distribution, 148 Spermatozoa, phosphorus compounds, 148 Sphingomyelin, chemistry, 70 in blood cells, occurrence, 153 Spleen, ash analyses, 105 composition as affected by various diseases, 507 lecithin content, 107, 140 mineral analyses, 507 nuclein phosphorus, 140 nucleon content, 140 nucleoprotein, 140 phosphatid content as influenced by alcoholism, 484 ■ phosphatids, 588 INDEX 745 Spleen, phosphorus, 139, 140, 149, 507, 588 phosphorus content and distribution in lipoids as influenced by chronic tuberculosis, 588 partition, 139 as influenced by age, 139 removal, effect on metabolism and composition of body, 580 Split-protein feeding for infants, 455 Springer's cereal decoction, description, 92 Sterilized and raw milk, compared, in vitro digestions, 274 metabolism experiments, 415 milk, infant feeding experiments, 414, 453, 454 Stored protein, Kolpakcha's idea, 216 Striated and non-striated muscles, comparisons of phosphorus content, 123 Strontium, substitution for calcium in bone, 386, 390 treatment in rachitis, 568 Sulphur elimination as related to nitrogen and phosphorus elimination, 220 Sunflower seeds, phosphorus and phosphatid content, 81, 85 Superphosphate, feeding experiments, 386 Suprarenal capsules, phosphorus content and partition, 107, 143 Sweating, influence on phosphorus excretion, 466, 467 Sweet-breads, feeding experiments, 324 Swine, mineral analyses of entire body, 106 Synthesis of glycerophosphates, 74 lecithin in developing eggs, 341 egg production, 342 nucleins as leucocytes, 229 disussion, 30 enzymes effecting, 230 in developing eggs, 229, 341 fish genitalia, 229 suckling animals, 229 on purin-free diet, 229 organic phosphorus compounds in fasting fish, 354 from inorganic, animal investiga- tions, 318 discussion and animal experiments, 318 phosphate-carbohydrate compounds, 31 -protein compounds, 30 phosphatids, 74 in brain development, 341 phytic acid, 54 Synthetic glycerophosphoric acid, absorption, 293 Szekely's milk, composition, 454 infant feeding experiments, 454 Tankage, mineral analyses, 78 Teeth, composition, 115 as influenced by caries, 504 fast, 441 dentine, mineral analyses, 113, 116 phosphorus, 113, 116 . ■ enamel, mineral analyses, 113, 116, 117 phosphorus, 113, 116, 117 influenced by hard water, 394 lime-poor diet less than bones, 388 mineral analyses, 113, 313, 505 organic phosphorus, 115 phosphorus, 113, 313, 505 relation of parathyroids to dentine formation, 560 Tetra-ethyl-phosphonium iodide, injection experiments, 549 Therapeutic and prophylactic use of nucleins and nucleic acids, 256, 502, 504, 519, 547, 586 use of phytin, 315, 533, 569 746 INDEX Thudichum's classification of the brain constituents, 128 Thymic acid, definition, 20 Thymin, composition, 19 Thymonucleic t acids, definition, 19 Thymus, feeding experiments, 324 nucleic acid, structure, 26, 27 phosphorus content and partition, 143, 149 Thyraden treatment for osteomalacia, 558 Thyrein. See Iodothyrin Thyreiodin. See Iodothyrin Thyreoglobulin, occurrence and significance, 145 Thyreoidin. See Thyroidin, 545 Thyroiodinin. See Iodothyrin Thyreonucleoalbumin, occurrence and significance, 144 Thyroid glands and hypophysis, reciprocal functions, 481 in relation to phosphorus metabolism, 577 investigations as to the active component, 145 preparations, descriptions and names, 545 treatment, influence on bone formation, 559 physiological effects, 577 treatment in acromegaly, 479 influence on phosphorus and calcium excre- tion, 480, 482 cretinism, 546 exophthalmic goiter, 581 leukaemia, 493 mongoloid idiocy, 546 myxoedema, 546 See also Iodothyrin, Thyraden and Thyroidin Thyroids, effects on general metabolism, 577 phosphorus and iodine content, 579 content and partition, 144, 149 Thyroidectomy, effects on nuclease content of organs and blood, 235 bone formation, 559 physiological effects, 577 Thyroidin, distinction from iodothyrin, 545 treatment after thyroidectomy, 580 effect on phosphorus excretion by a thyro-parathyroid- ectomized dog, 579 in paralysis agitans, 545 senility and paralysis agitans, phosphorus balances, 545 Thyroiodin distinguished from thyroidin, 545 Thyroiodinin. See Iodothyrin Thyro-parathyroidectomy, effect on partition of phosphorus of the blood, 560 phosphorus elimination, 561 Time of day, effect on phosphorus metabolism, 474 Timothy hay, mineral analyses, 78 Torulin as cure for beriberi, 490 composition, 490 Triamino-diphosphatids, chemistry, 69 -monophosphatids, chemistry, 71 Tricalcol, absorption, 184 Triferrin, effects on composition of liver, 139 Triticonucleate of sodium treatment in tuberculosis, 587 Triticonucleic acid, chemistry, 91 effects, 250 in wheat germ, 91 treatment in tuberculosis, 257 Tropon, feeding experiments with a man and a dog, 550 Tuberculin treatment in tuberculosis, 584 Tuberculosis and chlorosis, phosphorus and chlorine elimination, 584 chronic, changes in lipoids of organs, 587 demineralization, 584 INDEX 747 Tuberculosis, effect on lipoids and phosphorus of organs — guinea pigs, 588 phosphorus of milk, 382 lecithin therapy, 530, 531, 532 nucleic acid and disodium-methyl-arsenate therapy, 586 therapy, 257 nuclein-saline treatment, 586 phosphaturia in. See Phosphaturia phosphorus metabolism and phosphorus treatment, 583 with melancholia, phosphorus content of cerebrospinal fluid, 537 Tumor on the brain, phosphorus content of cerebrospinal fluid, 537 Turkish baths, influence on phosphate, uric acid, urea, sulphate and chloride excretion, 466 Turnips, phosphorus content and partition, 88 as affected by fertilizer, 100 Tryptic digestion of casein, influence of alkalinity, 272 in relation to ionproteid theory, 271 in vitro tests, 267 nucleoproteins, 231 ovovitellin, 271 Typhoid fever, effects on composition of blood, 500 and urine, 499 Ulcer of the stomach, phytin ingestion, 316 Uracil, composition, 19 Uric acid, composition, 18 elimination after nucleic acid ingestion, 245 See also Gout Urinary elimination of phosphorus, method, 188 Urine, composition as affected by vegetable diet — several species, 211 See also Phosphorus balances, and many feeding experiments excessive phosphorus. See Phosphaturia glycerophosphoric acid content in fatty degeneration explained, 513 organic phosphorus content, 190 as affected by croupous pneumonia, 193 degenerative nervous diseases, 539 epileptic attack, 541 a pathological symptom in the breast-fed infant, 565 of infant's, 456 phosphoric acid esters, 191 phosphorus content as affected by calcium salts, 207 a gauge of digestive disturbances in infants, 564, 565 ratio of phosphorus to nitrogen. See Nitrogen and phosphorus ratio in urine Vegetable casein, feeding experiment, 322 diet, effects on phosphorus elimination, 208 human urinary nitrogen and phosphorus, 209, 210 as influenced by mental work, 461 phosphorus and alkali phosphates as T , , , , • influenced by physical exercise, 465 Vetch hay, ether extract phosphorus, 90 Vitamine, definition, 365 Vitellin, cleavage products, 46 in eggs, 448 feeding experiments, 320, 321, 322 loss in incubating eggs, 447 Water-drinking and thirst, influence on phosphorus metabolism, 473 effects on composition of forage plants, 93 Water in the organs in disease, 509 on the brain, phosphorus content of cerebrospinal fluid, 537 748 INDEX Wheat bran and rice polish, specific effects on swine, 376 mineral analyses, 77 phosphorus content and partition, 77, 82, 85 germ flour, phosphorus content and distribution, 78 lecithin (phosphatid) content, 85 mineral analyses, 77 tritico-nucleic acid, 91 gluten, mineral analyses, 77 grains, inorganic phosphorus, 77, 79 as affected by fertilizer, 100 lecithin content, 87 (phosphatid) content, 85 phosphorus, 79, 82 mineral analyses, 77 as affected by fertilizer, 100 organic phosphorus, 82 phosphorus, 77, 79, 87, 99 as affected by fertilizer, 97, 100 protein phosphorus, 79 inadequacy for nutrition, 384, 385 middlings added to corn, specific effects on swine, 376 mineral analyses, 77 phosphorus, references, 76 straw, mineral analyses, 78 phosphorus as affected by fertilizer, 96 Whey, in infant feeding, 458 mineral analyses, 78 modification of milk; effect on mineral retention, 459 phosphorus elimination, 459 protein, definition, 39 Whole milk, skimmed milk and cream, phosphorus compounds, 163 Wine, effect on nitrogen and phosphorus elimination, 469 Xanthin bases. See Purin bases ' composition, 18 Xanthooxidase, definition, 28 Yeast cures polyneuritis, 489 endotrypsin, 234 nucleic acid, structure, 26 Zein, maintenance and slaughter experiments, 331 COLUMBIA UNIVERSITY LIBRARIES This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, as provided by the rules of the Library or by special ar- rangement with the Librarian in charge. DATE BORROWED DATE DUE DATE BORROWED DATE DUE 1955 C2BI1 140IM100 j QP535.P1 F74 Forbes A review of the literature of