Speoial Report Series. Ko. 71. ilribg (ttouncil MEDICAL RESEARCH COUNCIL The Aetiology and Pathology of Rickets from an Experimental point of view BY V. KORENCHEVSKY, M.D. LONDON PUBLISHED BY HIS MAJESTY'S STATIONERY OFFICE i 1922 ^i Price 4*. net Cornell University Library RJ 396.K84 The aetiology and Pathology ?'S± ' CORNELL UNIVERSITY THE Ifllomer Veterinary Hihrary FOUNDED BY ROSWELL P. FLOWER for the use of the N. Y. STATE VETERINARY COLLEGE 1897 ^tibg (Eouncil MEDICAL RESEARCH COUNCIL The Aetiology and Pathology of Rickets from an Experimental point of view BY V. KORENCHEVSKY, M.D. LONDON PUBLISHED BY HIS MAJESTY'S STATIONERY OFFICE 1922 MEDICAL RESEARCH COUNCIL The Viscount Goschen, C.B.E. (Chairman). William Graham, LL.B., M.P, The Rt. Hon. F. B. Mildmay {Treasv/rer). Sir Frederick W. Andbewes, D.M., F.R.S. Professor T. R. Elliott, C.B.E., D.S.O., M.D., F.R.S. Henry Head, M.D., F.R.S. Professor F. Gowland Hopkins, D.Sc, F.R.S. Major-General Sir William B. Leishman, K.C.M.G., O.B., F.R.S. Professor D. Noel Paton, M.D., F.R.S. Sir Cuthbert S. Wallace, K.C.M.G., O.B., F.R.C.S. Sir Walter M. Fletcher, K.B.E,, M.D., Sc.D., F.R.S. {Secretary). THE AETIOLOGY AND PATHOLOGY OF RICKETS FROM AN EXPERIMENTAL POINT OF VIEW. By V. KOBENCHEVSKY, M.D. yfessor of Experimental Pathology h Medical Academy at Petrograd.) From the Depa,rtment of Experi/mental Pathology, Lister Itistitwte. (Foiinerli/ Pi-ofessor of Experimental Pathology in the Military Medical Academy at Petrograd.) CONTENTS. PABE 1 Iktkoductioh ... 5 2. ExPEKIMENT.iL METHODS . . , . 6 3. Calcium Costent in the Body and Skeleton oe Eaghitic Subjects as compared with that of children with nobmal skeletons . . 10 (o) Calcium content in the body , . , . . . . . . 10 (6) The composition of the skeleton, principally its calcium content . 12 (c) Caloiiun content of the soft tissues in rachitic children . ,. 15 (d) Content of calcium, phosphorus, and magnesium in the blood in rickets and in osteomalacia 16 4. Histological Changes in the Skeleton in Rickets and Osteomalacia 19 (a) Uricalcified bone or osteoid tissue 19 (6) Osteoporosis in rickets and in osteomalacia ..... 21 (c) Changes in the epiphyses ...... 24 i. The enlargement of the epiphyseal cartilage ..... 23 ii. The deficiency or absence of calcium deposition in the zone of provisional calcification . 25 iii. ' Irregvlariiies in the penetration of the hone-mairow blood-vessels and the disorganisation of cartilage » . . " . . . . .26 (ri) Callosities after spontaneous fractures ...... 28 (e) General conclusion 28 5. Calcium Metabolism in Normal and in Rachitio Subjects ... 28 (o) Noi-mal subjects ... . . > . . 29 (b) Rachitic subjects ... 32 6. Metabolism in Osteomalacia . . ..... 35 7. Effects oh Calcium Metabolism of the Introduction of various QUANTITIES of CalCIUM INTO THE ORGANISM . . ... 37 {a) The normal organism .... .37 (6) The rachitic organism . . ... . . 40 8. Effects of Cod-Livee Oil and Elementary Phosphorus on Calcium Metabolism .42 9. The Influence of Diet oh Calciom Metabolism . . . . 43 ■10. The Minimum Demand fob Calcium by the Growing Orgahism, and THE Possibility of Calcium Starvatioh ih RicKirrs . . . 46 (a) Calcium content of human milk . . 46 (6) The possibility of calcium starvation in rickets . 49 (1379) Ps. 22688. Wt. 11476. 360/589. 2000. 12/22. o.u.P. a 2 11. Ikfluenoe on the Skeletoh of Ahimals of Calcium Stabvatiok, either ALONE OR IN combination WITH AN INTAKE OF STRONTIUM . . .53 (a) Histological changes of the skeleton 53 (6) Chemical changes of the skeleton 60 (c) Chemical changes in the soft tissues and in the blood . . 62 (d) Calcium metabolism in calcium starvation .... 63 (e) The author's experiments in feeding rats on a diet deficient in calcium alone . . ........ 64 (/) The author's experiments in feeding rats on a basal diet (rich in vitamins) but without the addition of a saline mixture . 71 (g) General summary : the points of similarity and difference between rickets and the effects of calcium starvation . . 71 12. Influence on the Skeleton of a Diet deficient in Antirachitic Factor (a) Historical . . 73 (6) The author's experiments in feeding rats on a diet deficient in antirachitic factor . .80 (c) Summary of experiments dealing with influence of special feeding of mothers during pregnancy or lactation upon the skeleton of mother rats and young 109 (d) General Summary and conclusions of Chapter 12 . . . 112 13. Influence on the Skeleton of the simultaneous Deficiency in the Diet of Antirachitic Factor and Calcium (a) Historical 116 (&) The author's experiments in feeding rats upon a diet deficient both in calcium and antirachitic factor 117 (c) Conclusions 122 14. The Author's Experiments in feeding Eats upon the Basal Diet deficient in Antirachitic Factor and without the addition of A Saline Mixture 123 15. The Effect of Elementary Phosphorus on the Skeleton of Animals ON A Diet either rich or poor in Calcium 123 16. Influence on the Skeleton of a Diet deficient in Phosphorus or in Phosphorus and Antirachitic Factor simultaneously (a) Historical . 126 (6) The author's experiments with a diet deficient in phosphorus or both in phosphorus and antirachitic factor . . . 130 17. Influence of Arsenic on the Skeleton ....... 131 18. Influence of Obganic Acids on the Skeleton 132 19. Influence of Confinement and Deprivation op Exercise on the Skeleton 134 20. Moisture and Rickets (a) Historical 138 (6) The author's experiments 138 21. Influence of Light on the Skeleton 146 22. The Theory of the Infective Oribin op Eiokets (ffl) Historical 148 (6) The author's experiments with some bacteria .... 151 23. Influence of Heredity on Incidence or of Predisposition to Rickets . 154 24. General Summary 160 25. References 162 26. Description of Photomiceogbaphs 169 1. Introduction. It is principally during the last few years that considerable pro- gress has been made in the experimental investigation of rickets. Newly discovered facts have thrown fresh light on the results of previous investigations. In the present work an attempt has there- fore been made to collect the more important experimental facts concerning the aetiology and pathology of rickets, and to compare them critically with the corresponding clinical data of human rickets. As a result of these investigations, and on the basis of the results of his own experiments, the author has attempted to draw certain definite conclusions. Thus the work falls into two sections : literary research and personal experiment ; the latter was started at the beginning of 1920, and a preliminary communication of results was published in 1931. In order to facilitate an interpretation of the results obtained, the literary review of each separate question is followed by an account of corresponding experiments made by the author. In the literary review some of the investigations had to be quoted from abstracts, owing to the impossibility of consulting the original works. This fact is always noted in the bibliography, as, unfortunately, an abstract does not always give the substance of the original quite correctly. The object of the author's own experiments was to elucidate the following questions : (1) The influence on the skeleton of the deficiency of calcium, of so-called vitamin A, or of both these factors simultaneously, in the diet ; (2) the importance of these factors in the diet of the mother during lactation and during pregnancy for the development of the skeleton of the offspring ; (3) the influence of the internal secretory glands regarded as important to the metabolism of calcium salts in the organism ; (4) among other things, the influence of confinement and of moisture was also ascertained. A few experiments were conducted with the object of determining the influence of a deficiency of phosphorus alone, or in conjunction with a deficiency of vitamin A, as also the influence of certain bacteria. In order to avoid making the present communication too lengthy, investigations concerning the influence on the skeleton of the internal secretory sexual and parathyroid glands have been published in the form of separate articles. The interpretation of the influence of diets deficient in the fat- soluble accessory factor discovered by McCoUum and Davis in 1913-15j now generally designated vitamin A, is complicated by the possible existence of more than one active principle dissolved in animal fats. From the researches of McCoUum and others, vitamin A was understood to .be an essential nutritional organic factor of unknown composition present in milk; in some animal fats, and in an especially great amount-in cod-liver oil. The absence of this factor ifci thedifet is accompanied by arrested growth, xerophthalmia, loss of appetite, deterioration in the general nutrition causing severe cachexia, and finally death. A few years later, Mellanby (1918, 1919) observed that a diet arranged so as to be poor in vitamin A occasioned rickets in puppies, but, while of opinion that the promotion of growth and the increased calcification of bones of puppies were due to the same vitamin in fats, he pointed out (1921) that in the then state of knowledge it could not be confidently asserted that these were identical substances. In 1921 McColIum and his co-workers published observations which they regarded as indicative that in the animal fats, cod-liver pilj &c., there are two different organic factors : one possessing anti- rachitic properties and the other antixerophthalmic and apparently also the other properties attributed to vitamin A, antirachitic excepted. ; This has been recently followed by a further communication (1922) in which they bring forward experiments which, in their opinion, prove the existence of a fourth or antirachitic vitamin. They conclude that this antirachitic factor is present in milk fat in less proportion that vitamin A, but predominates in cod-liver oil. In the experiments about to be described cod-liver oil was chosen as a suitable addition to the dietary of those animals to whom I wished to ensure an adequate supply of antirachitic factor on the basis of the experience of McCollum and others and because of the known variability of milk fat in vitamin A. (Drummond, Coward, and Watson, 1921 ; Butcher, 1921.) My experiments do not touch upon the question of the identity of these two factors, and for brevity of description and without ^ore- judicjtng the question, of identity, the principle concerned in iyifiuencing calciwm metabolism- and calcification of the skeleton will be (galled antirachitic factor and the diet deprived of that factor -'A diet ; - 2. EXPEBIMENTAL METHODS. _ The experiments were conducted on rats born at the Lister Insti- tute from stocks that have been under as careful observa,tion as the experimental animals themselves. Two years' experience has shown\me the necessity of the strictest control of the diet of rats bred for, experiments, as a condition essential to the success of experiments in ricjsets. Unless such conditions are observed^ there is the possibility of the, development of spontaneous rickets, and, consequently, of the grossest erroi*. The rats used for breeding wei-e kept in roomy cages, and fed oh & diet No. 1 consisting of about J5 c.cm. per diem 'per rat of fresh milk with 1-1-5 per cent, calcium lactate, fresh- cabbage leaves, oats, bran, white bread, and ;(qccasjonally) meat. ; On.soich a. dj^t; healthy rats were born and grew up, with a chemicaily , and hi^^oiogically normal skeleton^ and normal transpsu'ent yellow tejeth. p.tn'ing! the period of lactatioiii the mpther. rat§ xeeei^ved milk ad libitum. On putting their young on the rickets-producing diets employed by me, I easily succeeded in producing typical changes of the skeleton. After ascertaining the very important part played in the produc- tion of rickets by the deficiency of antirachitic factor, calcium, and phosphorus in the diet, since the end of 1921 I have introduced diet No. 2 consisting of diet No. 1 with addition of about t gm. per rat daily of paste rich in these factors, of the following composition : Commercial casein . . . . .30 gm. Starch . 60 Cod-liver oil .... 5 Yeast 6 Sodium chloride .... . 0-3 Tricalcium phosphate . . . . . 4-8 Calcium lactate .... . 0-3 Potassium phosphate . . 0-6 Water . 67 c.cm. When a rat gave birth to young and was removed to a separate cage, the amount of the above-mentioned paste was increased to about 15 gm. daily, milk being given ad libitum. The offspring born and bred under these conditions (as will be described later in detail) grew more rapidly and were less susceptible to chance infec- tions and diet deficiencies. Notwithstanding the identical conditions of growth, different litters showed (vide infra) certain fluctuations in the composition of the skeleton and its histological picture. This confirms the correctness of my invariable rule of using rats of the same litter in each experiment, as the resistance of different litters to the efiects of rickets-producing diets varied. It was only in my experiments where a deficient diet was started during pregnancy or lactation that this rule could not, of course, be observed, and the control animals had to be taken from another litter. All the rats experimented on were fed on the following basal diet No. 3 : Purified casein Starch . Fat . Marmite Grange juice . Distilled water 18 gm. 52 „ 15 „ 5 „ 5 50 c.cm. Among several hundred rats fed on this basal diet (plus the below- mentioned salt-mixture), there were four or five cases of paresis of the hind-legs or bloody urine. Therefore of late, instead of marmite I have used the same amount of dried yeast, as recommended by American authors, and increased the amount of orange juice from 5 c.cm. to 8 c.cm. Since then I have not observed the complicaiiidns above mentioned. This mixture contained about 0-04^0'045 per cent. Ca and 0-2 per cent. P. In the food mixture intended to be rich in antirachitic factor a raixtui'e of 12-5lgm. of butter ^^s 2-5 gm. of cod-liver oil 8 was used as fat. For food deficient in antirachitic factor cotton- seed oil was used. To this basal diet was added 5 gm. of saline mixture. The one used for food rich in calcium had the following composition, as recommended by McCollum (1917): (No. 1) Sodium chloride 5-19 gm. Magnesium sulphate (MgSO^ + 7 HgO) . . 16-4 „ Sodium acid phosphate (NaHgPOj+HgO) . 10-41 „ Potassium phosphate (K.HPOJ . . . 28-62 „ Calcium phosphate (CaH^(PO,)2 + H20) . 16-20 „ Calcium lactate . . .... 39-00 „ Ferric citrate . . .... 3-54 „ To the diets intended to be deficient in calcium, a saline mixture (modifi.ed by me) of the following composition was added : (No. 2) Sodium chloride Magnesium sulphate Sodium phosphate . Potassium phosphate Ferric citrate . Sodium bicarbonate . 5-19 gm. 16-4 „ 27-91 „ 28-62 „ 3-54 „ 21-2 ,. In some of my first experiments, to a diet intended to be deficient in calcium I added the following; saline mixture : (No. 3) Sodium chloride Magnesium sulphate Sodium acid phosphate Potassium phosphate Ferric citrate , 7-78 gm. 24-60 „ 15-61 „ 42-93 „ 5-31 „ I stopped using this saline mixture, as it differed in alkalinity from the normal saline mixture No. 1. After, and possibly on account of this, the rats developed cachexia with increasing rapidity. For a diet intended to be deficient in phosphorus, the following saline mixture was added to the above-mentioned basal diet No. 3 : (No. 4) Sodium chloride . 9-6 gm Magnesium sulphate . . . 16-4 „ Potassium chloride . 24-4 „ Calcium lactate . . 57-5 „ Ferric citrate - - 3-5 „ As this diet with saline mixture No. 4 contained about 0-2 per cent, phosphorus, it cannot be called especially deficient in phos- phorus ; it was merely considerably poorer in phosphorus than the (Ji^ts containing saline mixtures Nos. 1, 2, and 3. Fifteen to thirty gi'ammes of food mixture were supplied to each animal according to age and appetite. In th6 text following we shall denote the diets by the following letters : ' N ' = normal mixture, i. e. containing butter and cod-liver oil as fats, and saline mixture No. 1 (Ca = 0-356 %, P = 0-5 %) ; '_N - Ca ' = the same, but with saline mixluie No. 2 (Ca = 0-045 %, P = 0-576%); ' -A' = containing saline mixture No. 1, but having cotton-seed oil as a fat, i.e. deficient in antirachitic factor (Ca = 0-251 %,P = 0-51 %); ' _A-Ca' = a diet containing cotton- seed oil and saline mixture No. 2, i. e. deficient in antirachitic factor and calcium (Ca = 0-045 %, P = 0-555 %) ; ' - P ' = containing saline mixture No. 4, i. e. deficient in phosphorus (Ca = 0-256 %, P = 0-2 %) ; ' — SM ' = basal diet without any saline mixture (Ca = 0-045 %, P = 0-2 %). My diets before addition of water and orange juice, both the normal and that deficient in the antirachitic factor, contained about 0-4 per cent. Ca and about 0-8 per cent. P. The calcium intake was quite sufiicient fof the normal calcification of the skeleton (see Tables, Nos, 20 and 29) of rats kept on the ' N ' diet which contained a liberal amount of the antirachitic factor. The skeleton of rats fed on this diet showed a normal and strikingly constant calcium content (see Tables, Nos. 20 and 29). The histological picture was likewise normal. McCoUum (1922) considers the optimum amount of calcium to be 0-64 per cent, of the air dry materials, that is, greater than I used. His diet was, however, different and contained less antirachitic factor. Whether I should have obtained different results in my — A experi- ments had I increased the calcium in the diet, may probably be answered in the affirmative ; for all the evidence so far accumulated, and referred to in Chapters 7 and 11 below points to the fact that an abundance of calcium can to some extent compensate for a deficiency of the antirachitic factor in a diet sufficient in other respects. Unless the amount of antirachitic factor is known to be constant in a diet there is obviously room for considerable divergence of opinion as to what is the optimum amount of Ca, and accordingly, in experiments designed to test the influence of varying amounts of the anti- rachitic factor, the most striking results may be expected to arise when the content of calcium in the diet is not excessive. The rats were kept in airy cages in a well-lit and well- ventilated room main- tained at about 20-22° C. The windows faced NNE. and were kept shut. The animals were not exposed to direct sunlight. My own experivients, therefore, on the effect of varying amounts of anti- rachitic factor only refer to diets containing about 0-4 per cent. Ca in air dry diet, i. e. not an excessive amount of calcium, and to animals maintained in absence of direct sunlight. . In order to judge of the comparative effect of the different diets, each separate litter of rats was divided into groups fed on different diets, one or two rats being used as controls and fed on N diet. On the whole, notwithstanding certain physiological fluctuations, the skeletons of the normal rats were practically constant as regards chemical composition and histological, structure. The changes obtained on abnormal diets were likewise characteristic on the whole. In view of this, it was possible to class all the animals fed on one and the same diet in one large gfoup, and compare the average results obtained with the average results of other groups. An account of the detailed reports of each experiment would take 10 up far too much space, considering the amount of material I have to deal with. . In aU' the animals the chemical examinations of water and calcium content were made on the bones of the leg (femur, tibia^ and fibula) ; the muscular and connective tissues were carefully removed from the bones, but the cartilages and thin ligaments con- necting the knee-joint laterally and posteriorly were left intact. In determining the calcium content of the food I employed the usual oxalate method. In determining the calcium content of the bones I followed Aron's (1910) method, which in this case yields good results owing to the fact that up to 98 per cent, of the bone-ash is composed of calcium salts. The advantages of this method are very great, owing to the simplicity and rapidity of procedure. Histological examination of the ribs, humenis, and radius, and Sometimes of other bones of the skeleton as well, was carried out in every case. The bones, after fixation in 10 per cent, of fonnalin, were either incompletely decalcified in Miiller's fluid, or the unde- calcified bones were cut. In the first case haematoxylin-eosin staining was employed ; in the second case the material was treated with AgNOg and then stained with alum carmine and eosin. The total number of rats used in the experiments described herein was 345.^ 3. Calcium Content in the Body and Skeleton of Rachitic Subjects as compaeed with that of Children with NoEMAL Skeletons. In starting to investigate the diseases in which a disturbance in the metabolism of calcium in the body plays a prominent part, it is first of all necessary to ascertain how far the calcium content in the body, skeleton, and tissues of a rachitic child differs from that of non- rachitic children. These important data likewise serve as a starting-point in determining the minimum physiological demand for calcium by the child. (a) Calcium Content in the Body. Brubacher (1891, p. 527) found the following figures of phosphorus and calcium in. 100 gra. of fresh body of prematurely born infants : Age in weeks. CaO %. P^O, % Foetus I 28 1.04 1-J09 Foetus II 36 1-lS 1.15 Prof Aron (1908) drew up the following table (No. 1) of the con- tent of CaO in the bodies of new-born infants, on the basis of analyses made by various authors (p. 46) : 1 In tliis report I lidve summarized nearly all my experiments dealing with the influeiloe irt deficient diets. Out b'f the" total 345 rats 62 were mentioned in other cotnmunications. ' 11 Table No. 1. Percentage of ash „ , , Amount of CaO , .Authors. in the body of rercentage oj per 1,000 gm. of the the child. ^^^ '" "*'*• weight of child's hody. Hugouneng (1899) 3-55 40.48 14.39 gm. Michel (1899) - 3-37 ' 41.89 13-97 „ Giacosa (1894) 3-35 41.92 13.72 ,, Steinitz (1904) .3.04 37.90 11.52 „ De Lange (1900) 2.95 38-89 11.51 „ Soldner (1903) 2.66 38.08 10-12 „ Average 3.15 39.79 12-53 „ On the basis of these analyses, Aron (1908) and Schabad (1910, ?>, 6) take the CaO content in the body of a new-born infant at about l'2-l-25 per cent., or, according to Aron, in any case not under 1 per cent, of the child's total weight (and that only according to Soldner's analysis). Thus, for every 100 gm. of increase in the weight of a growing infant, normally from 1-0 to 1-25 gm. of CaO should be deposited. Dibbelt (1910, 2), however, disputes the accuracy of these deductions, as in calculating the calcium deposited in the growing organism it would be more correct to start from the higher figures of the calcium content in the skeleton of an adult (about 11-5 per cent.). Moreover, the growth of the skeleton is not proportionate to that of the body. According to Vierordt, the body of an adult male is 21 times as heavy as that of a new-born infant, whereas his skeleton is 27-2 times as heavy. Therefore Dibbelt, on the basis of these data and of his own calculations, thinks that the minimum demand for CaO is about 1-9 gm. per 100 gm. of increase in the weight of the child. At the present time it would be wiser to take as a basis^ the figures of Aron and Schabad, who draw their conclusions froia direct analyses of the bodies of new-born infants. This view is likewise supported by the analysis of Sommerfeld (1900), who found that the amount of ash in the body of a three-months-old infant amounted to 2-73 per cent, of its total weight (or 3-15 per 100 parts of the fat- free body). Steinitz (1904) analysed the chemical Constituents of the bodies of four infants, aged 13 days, 2|^, 3, and 3| months respec- tively. All the infants were sufiering from severe cachexia, from which they died. On comparing the composition of their bodies with that.of a new-born infant or the three-months-old' infant in Sommer- feld's case, the author found no essential difference in the content of N, ash, CaO, and P2O5. There was only a decreased amount of fat. Thus, for instance, the asb content per 100 parts of the fat^ free l)ody^ was as follows : ... Case: I. Sommerfeld . .■ . 3-15% - ■„ II. Steinitz 1- . . . , 3-36% n III. » . . . ... 3.26 % : ,, IV 3.34% Biesideg that; ^^einitz and Weigert (1905) exaiiiined the coi-pse of a four-mbliths'^ld. infant. whQ had died pf acute gastric deraiige- '-;Ip thecase of bne.infant-the anvQunt ofash was not ascertained, . . , 12 ment, and had for a long time been- fed on a diet rich in carbo- hydrates, but poor in albumen, fats, and salts. Per 100 gm. of the weight of the body, without fat, there were 3-94 gm. of ash, the calcium content in the latter being even somewhat greater than in Steinitz's IV case. As is known, rachitic subjects are sometimes emaciated; but, according to Steinitz's data, this morbid condition does not materially affect the calcium or phosphorus content in the organism. Assuming the weight of a normal new-horn infant to be approxi- mately 3,000 gm., and the average CaO content 1'25 per cent., the body should contain about 37-5 gm. of CaO (Schabad). As the soft parts contain not more than 0-6 gm. of CaO, it follows that the normal skeleton must contain about 37 gm. of CaO, or 7-7 per cent. CaO (taking the weight of tlie skeleton at Schabad's figure, i. e. 16 per cent, of the total weight). What is the calcium content in the body of a rachitic child ? A direct determination of the calcium content of a one-year-old infant, made by Steinitz and Weigert (1905, 1) proved the calcium content to be about 21-4 gm., or that the CaO was 0*83 per cent, of the total weight of the body. Schabad (1910, 6), on the basis of his analyses of the bones in cases of manifest rickets in children of 18 months to 1 year 10 months, found analogous figures, i.e. about 21 or 22 gm. CaO. Cases of congenital rickets are very rare, and therefore there is reason to think that infants are born with nor- mally constituted skeletons. Hence the highly important conclusion that a child suffering from rickets loses about one-third of the amount of calcium contained in its body at birth. Conclusions. 1. The body of a rickety child contains far less calcium than that of a normal child. 2. A child suffering from rickets may lose about one-third of the amount of calcium contained in its body at birth. (b) T/ie Composition of the Skeleton, principally its Calcium Content. In discussing the chemical composition of the bones, it is necessary to bear the following points in mind : 1. Spongiosa, as compared with corticalis of the bones, is richer in HjO and fat and poorer in ash (Friedleben (1860), Brubacher (1890), Schabad (1910, 4), and others).. 3. The various bones of the skeleton have a different com- position of organic and inorganic parts (Brubacher, Aron, and Sebauer (1908), Schabad, and others). 3. On the whole, the older the subject the richer is the skeleton in ash and the poorer in water (Wildt, Weiske (8), Brubacher, and others). 4. The conditions of nutrition may have a marked effect on the composition of the skeleton. The changes in the latter, depending on the abundance or deficiency in the diet of phosphorus, calcium, or antirachitic factor will be dealt with in the respective chapters. 13 Starvation. Here I shall touch on the question of the influence o£ starvation on the osseous system. This is a very important question, as when attempts are made to induce experimental rickets, the animals not infrequently lose their appetite as a result of the various experimental conditions. The effect of underfeeding must, of course, be taken into account in drawing conclusions from the results obtained. In their experiments, Weiske, Seldmair (1899), E. Voit (1905), Wellmann (1908), and others have investigated the effects of starvation on the skeleton likewise. The results of the experiments made by all these authors agree. For instance, according to Seldmair, 100 gm. of dry bone contain the following : Table No. 2. Star ■ving Cfflfe. Normal cat A. E ! {38 days). C {SB days). 12-56 % .35.17 % 50.95 % 26.14 % 2.84% 40.22 % 56.55 % 29-68 % 4-11% 38-68 % 55-46 % 29.20 % Fat Osseine Ash CaO Thus starvation causes marked loss of fat in the bones, such loss being almost total when starvation is unduly prolonged. In connexion with the diminution of the amount of fat in the bones during starvation, dried bones show a certain increase in the , other components, e. g. calcium. This difference is not perceptible in the case of bones deprived of fats. For instance, the cats above mentioned showed the following proportion of CaO per 100 gm. of dry, fat-free bone : Cat A — 29-9 per cent., B — 30'39 per cent., and C — 30-45 per cent. In any case, starvation cannot produce such a decrease in the percentage of calcium content of the bone as might simulate the alterations produced by rickets. On the contrary, an examination may show a certain increase in the calcium content of the skeleton. Thus experiments on starving animals corroborate the above- mentioned results of the analyses of the bodies of emaciated children (Steinitz). The water content in the bones of starving animals is somewhat increased, e. g. the cats in Seldmair's experiments show from 38-5 to 43-3 per cent, instead of the normal 32-4 per cent. Aron thinks that this increase of water is due to the increased water content of . the bone marrow, as in his experiments the bone marrow in the skeleton of calves which had died of starvation contained only a small percentage of solid matter and tenths of one per cent. of fat. ■ Nevertheless, notwithstanding the absence of marked percentage changes in certain component parts of the bones, the latter show a loss in absolute figures of all their component parts as the result of starvation. Thus Wellmann's (1908) experiments show that 176 gm. of skeleton of an ' average ' rabbit lose about 21 gm. in weight during starvation, distributed as follows (p. 530) : 14 Water Fat ... Other organic matter Ash . . . Caleium . Phosphorus MagQesium 3-5 gm. 9.3 „ 5-0 „ 3-8 „ 1.8 „ 0-6 „ 0-04 „ In accordance with this, there is, during starvation, increased excretion of calcium from the organism, the bulk of which comes from the skeleton {v. Nooi'den (1906), Cathcart (1907'), and .others). The alteration in the ratio between phosphorus and nitrogen in the urine likewise points to the production of surplus phosphorus from the bone tissue. Thus during starvation the processes of the resorption of the bone are intensified. On the other hand, starva- tion leads to a diminution in the formation of new tissues, osseous tissue among others. Therefore during starvation one may expect a priori that the combined action of these two processes will result in a more or less acute form of osteoporosis. Unfortunately I could find only one histological work, and that in a very short reference, namely, Gusmitta's (1895), who investigated the bodies of starved but other vFise healthy animals. This author mentions dilatation .of the Haversian canals, slight enlargement of bone corpuscle.s, and marked diminution in the amount of adipose cells in the bone marrow. Rickets. The chemical constitution of the skeleton in rickets has been investigated by Marchand (1842), Friedleben (1860), Brubacher (1890), Schabad (1910, 4), and others. Rachitic bones, as compared with normal, are richer in water and poorer in ash, especially in their phosphorus and calcium content. The most marked alterations were more frequently found in the long bones. Thus, for instance, according to Brubacher, the composition of the femur was altered as follows (p. 539) : Table No. 3. , Control femur of girl {aged 4) who had died r%pidl,y of diphtheria. Femur of a girl (agedS^) suffering from rickets. Water Fat Asli CaO MgO 45.29 12.29 21.59 11-00 0.26 '8.58 73-16 0-49 6-20 2-74 0-04 1-97 b The Chemical Changes in the Skeleton in Osteomalacia are analo- gous to those observable in rickets, i. e. they are chiefly characterized by an impoverishment of the bones in calcium and phosphorus (Frey (1863), Weber (1867), Huppert (1867), Moers and Muck (1869), Langendorf and Mommsen (1877), Levy (1894), Galinard and Konig (1905), Capezzuoli (1909), and others). Many analyses likewise indicate increased fat in bones of osteomalacia subjects. An example,' is theapalysis made by Langendorf and Mommsen of the head of the femur of a man (aged 38) suffering from osteoinalacia. The control 15 was the bead of the femur of a man about the same age, who had died of tuberculosis. Table No. 4. Dry normal bone. Osieomalaciiic bone. Fat Fat-free solid matter 24.3 % 75.7 % 60.4% 39.6% In 100 parts of fat-free osseous Organic matter Ash tissue : 45-8% 54.2% 62.2% 37.8% In 100 parts of ash : CaO PA 53.1% 43-9% 44-5 % 34.8 % In Weber's case of senile osteomalacia, 100 parts of fresh bone contained : fat, 23'4 per cent. ; other solid organic matter, from 13.2 to 15-8 per cent. ; ash, from 9-4 to 11-9 per cent. Conclusions. 1. In starvation, the bones lose, in absolute figures, most fat and other constituents to a lesser degree. The percentage of calcium in the bones is not decreased. 3. In rickety skeletons the water content is increased and there is a mai-ked decrease in the amount of ash, particularly calcium and phosphorus. 3. In osteomalacia the chenges differ from those in rickets only in the bones being abnormally rich in fat. (c) Calcium Content of the Soft Tissues in Rachitic Children. Brubacher, Stoeltzner (1899) found that the desiccated soft tissues of rachitic subjects might contain more calcium than those of non- rachitic. In Stoe]tzne)''s rachitic subjects, however, the cardiac muscle and, to some extent, the brain, were poorer in calcium than similar organs in control children, Ri'ubacher found that, in the fresh condition, the muscles of rachitic children sometimes contained d' normal amount of calcium, but had an increased HjO content. In Brubacher's case of two children, the chemical analysis of whose femurs I have already given, the content of CaO and PgOg per 100 gm. of muscle were as follows : Table No. 5. Control infant. Rachitic infant. CaO P2O5 CaO PjOs Fresh muscles Dry fat-free muscles 0.01 0.39 0.04 2-01 0.01 0.28 0.07 1.69 Aschenheim and Kaumheimer (1911), on the contrary, found that rachitic muscles were poorer in calcium. In three non-rachitio subjects they found from 65 to 81 mgm. CaO per 100 gm. of dry fat^ free muscle ; in manifest rickets (six childi-en) the figures were from 35 to 53 mgm. ; 100 gm. of ash of the muscle of non-rachitic subjects showed from 1-16 to 1-73 gm. of calcium, whereas rachitic subjects showed from 0-51 to 1-10 gm. Provinciali (1913) also, found a diminished calcium content in the 16 Hve. ia »eMtU Fi...., .*», »d |.«^^^^^^ Xp?Jrrt.rip°i=»'-> sa: ..<^ fo„nd n„ dift.«„«, as poinnared with the control puppies. . • . • -.r. Thus u^to the present time, the nuinbor ot mvestigations with similar 'results has not been great enough to permit of any final con- clusion being arrived at. Therefore btoeltzner is hardly correct in going so far as to assert, on the basis of the few analyses made, that the characteristic feature of human rickets is normal calcium content of the soft tissues. Schabad draws particular attention to the danger of making any deductions from the scanty data concerning the fact that the calcium content in the tissues may be influenced by the stage of rickets at which analysis is made and the varying metabolism of calcium during the course of that disease, e. g. it may either increase or remain normal during the cure of the disease. Conclusiona. The results hitherto obtained in the investigations of the calcium content of the soft tissues in rickets do not permit of any definite conclusions being drawn. (d) Content of Galcium, Phosphorus, and Magnesium in the Blood in Rickets and Osteomalacia. According to Abderhalden, the blood corpuscles contain a very small part of the total amount of calcium in the blood. Loeper and Bechamp (1909) found about 7 mgm. of CaO per 100 gm. of blood, from 13 to 14 mgm. in serum, and from 5 to 6 mgm. in the coagulum. The later investigations of Cowie and Calhoun (1919) showed that 100 c.cm. of whole human blood contain about 8-45 mgm. CaO, 100 c.cm. of serum 12'07 mgm., 100 c.cm. of washed blood cor- puscles 3-47 mgm. Analyses and calculations made by Findlay, Paton, and Sharpe (1921) show that in 100 c.cm. of whole human blood the blood corpuscles contain from 9 to 15 per cent, of the total calcium in the blood, i.e. a quantity which must be taken into account. Halverson, Mohler, and Eergeim (1917), however, very justly point out possible sources of error in investigating calcium in whole blood : (1) the bulk of the calcium is still to be foHnd in the serum, and (2) the number of red corpuscles in the blood may fluctuate in a marked degree, especially in pathological condi- tions. This latter circumstance may completely alter the figure of the calcium content of the blood. Therelbi'e the results of the investigation of calcium in whole blood can be of importance only in such cases when the volume ratio of the blood corpuscles and plasma in the blood in question are known. The fact that this is not taken into account is apparently the reason why Aschenheim's (1914) figures show such fluctuations : in healthy adults the content per 100 ccm. was from 5-9 to 12-3 mgm. CaO; in non-rachitic children from 3-16 to 12-2 mgm., and in rachitic children from 3.7 to 17-9 mgm. Nevertheless, on the basis of these %ures Aschenheim draws the conclusion that in rachitic (as opposed to non-rachitic) subjects fluctuations in the calcium content in the 17 blood both below and above the normal average figure (8 to 10 mgm.) are far more frequent. Katzenellenbogen (1913), using Wright's method, did not find any perceptible decrease in the calcium content of the blood of rachitic subjects. He suggests that perhaps his rachitic subjects were con- valescent. Findlay, Paton, and Sharpe (1921) investigated the calcium content in the blood of six normal subjects, and found that the amount of CaO in the blood of adults (11 mgm. per 100 com.) was less than in the case of children (13 to 16 mgm.). They did not discover any marked difference in the calcium content in the blood of the control puppies and those with experimental rickets. As might be expected from the foregoing explanation, the investi- gations of the calcium content in the serum of the blood yielded considei-ably more constant results. Denis and Talbot (1931) employed Lyman's method (with certain modifications) in determin- ing the calcium content of plasma. They investigated various cases (a total of 119 children) and found that the calcium content in the blood was perceptibly lower in cases of tetany (1 to T-7 mgm. CaO per 100 c.cm. of plasma), pneumonia (sometimes down to 4-4 mgm.), and rickets. The total number of rachitic subjects examined for calcium content was 28: in 7 children in the acute stage of the disease the content was from 2 to 8 mgm., and in 21 convalescent cases it was from 4-5 to 11-2 mgm. However, as in almost every case, rickets were complicated by some other disease (in 7 cases pneumonia, which likewise involved a low calcium content in the blood), the authors do not venture to draw any definite conclusions. It is necessary to add that Lyman's method of determining the calcium content in the blood has been criticized by some (Howland and Kramer). Howland and Marriot (1917-18), Halverson, Mohler and Bergeim (1917), Meysenbug and McOann (1921), find that in the majority of cases the calcium content in 100 c.cm. of the blood serum of healthy subjects fluctuates between 10 and 11 mgm.; more- over, according to Howland and Marriot, age has no perceptible effect on the calcium content. Howland and Marriot found, on investigating 21 cases, that in rickets the average calcium content was 9-4 mgm. In 7 of the above cases the calcium content was approximately normal ; in 9 cases there was a slight decline, and only in 5 cases Was the calcium content in the serum as low as 7-9 to 8-8 mgm. The magnesium con- tent showed no essential alteration. Meysenbug and McCann found that in 5 cases of rickets the calcium content per 100 c.cm. of serum was 9-8, 9-0, 8-7, 8-5, and 7-6. The percentage of diffusible calcium was normal. Howland and Kramer (1921) found no essential change in the calcium content in 5 cases of rickets, while in 12 cases it was reduced to 7-5-9'8 mgm. per 100 c.cm. of serum. The magnesium content was within normal limits. These authors were the first to di'aw attention to the following important fact discovered by them. The content of inorganic phosphorus in sixteen non-rachitic children fluctuated about 5-4 mgm. per 100 c.cm. of blood serum. In 22 cases of rickets the content was sharply decreased to 0-6- 3-2 mgm., the average being 2-0 mgm. In 12 cases of rickets the (1579) B 18 children were given cod-liver oil, whicli had very little efiect on the increase of calcium in the blood, while at the same time it caused a marked increase in the inorganic phosphorus content, e. g. from 1-9 mgm. before the cod-liver oil treatment to 5-5 mgm. after such treatment. The authors have drawn the following conclusions from the facts discovered by them. ' In rickets we believe, therefore, that there is constantly a marked, and, for the causation of the pathological lesion, an important deficiency in inorganic phosphorus. To this deficiency is to be ascribed the failure of calcium deposition ' (p. 114). ' During the period of active rickets, the calcium concentration may be normal or slightly reduced. The reduction does not seem to depend directly on the rickets. There are reasons for believing that in many instances the reduction is associated with a latent form of tetany. The inorganic phosphorus of the serum is regularly reduced in active rickets, sometimes to an extreme degree. During the process of healing, whether occurring spontaneously or as the result of the administration of cod-liver oil, the phosphorus content of the serum gradually rises to a normal figure and often somewhat above this. Relapses are accompanied by a fall in the phosphorus concentration of the serum.' All the children under 2^ years of age, in whom the authors found an inorganic phosphorus content of the serum of 3-0 mgm. or less, had been sufi'ering from active rickets (p. 119). Iversen and Lenstrup's (quoted by Howland and Kramer, p. 119) results were quite similar to those of Howland and Kramer. They also found an increase of the phosphorus after the administration of cod-liver oil. In summarizing the available data concerning calcium content of serum or blood in rickets it is necessary to note that the calcium content is in some cases normal, in others decreased. However, as the investigations have been far from numerous, and, moreover, it is not known in what stage of rickets such investigations were made, it is difficult to draw any definite conclusion. Howland and Kramer's discovery of the reduction in the phos- phorus content in the cases of rickets investigated by them is exceedingly interesting, and deserves further investigation. In the few investigated cases of osteomalacia, as opposed to rickets, all the authors found an increase in the calcium content of the blood : Marquis, 12 mgm. ; Veron and Marquis, 16-2 mgm.; Capellani (1909), 28.3 mgm. and 44-9 mgm. ; Aschenheim, 15-9 mgm. Adler (1912) alone found a nearly normal calcium content in the blood of an osteomalacia patient— 9-8 mgm. CaO. Should further investigation corroborate the fact of a high calcium content of the blood in osteomalacia, this fact might, to a certain extent, serve as an argument in favour of the existence of halisteresis in osteomalacia. As we know, the probability of this has been repeatedly pointed out. Conclusions. 1. In rickets a normal or diminished amount of calcium in the blood may be observed. 2. As the stage of rickets at which the examination of the blood was made is unknown, it is impossible to draw any conclusion as to the causes of the fluctuation of calcium in the blood in rickets. 19 3. In rickets, the amount of inorganic phosphorus in the blood was found to be diminished. 4. In osteomalacia, apparently, the calcium coatent in the blood is increased. 4. Histological Changes in the Skeleton in Rickets AND Osteomalacia. From a chemical point of view, rickets is characterized by the impoverishment of the bones in lime and phosphorus and their enrichment in water. Its pathological essence was exactly established and worked out first by Pommer (1885) and then by Schmorl (1906-13), and may be shortly defined as follows : in rickets calcium is either no longer deposited, or deposited iu an insufiicient degree in the newly forming bone or the proliferous cartilage. In order to understand the numerous variations of experimental and human rickets, as well as osteomalacia, it is necessary to examine individually the histological features which make up the pathological picture of these diseases. (a) Uncaloijied bone or osteoid tissue. Physiologically bone is formed without calcium, but the latter is very rapidly deposited therein. In consequence of this, normal bones show only a very narrow zone of osteoid tissue when examined microscopically. In rickets and in osteomalacia the thickness of the osteoid layer is considerably above the normal ; in very severe cases the trabeeulae of spongiosa and the cortical bone consist . almost exclusively of osteoid tissue. The calcification of the bone is to be observed only in the centre. In rickets and osteomalacia osteoid tissue is present in the bones of the whole skeleton, but even in typical cases of the disease the amount varies in the different bones : (a) osteoid tissue is formed more abundantly in more quick growing bones or parts of a bone (Schmorl) ; (6) the mechanical effects of weight and pressure on the bone, and the tension of the tendons and muscles likewise increase the new formation of osteoid tissue (Pommer, Schmorl); (c) any reconstruction of the skeleton, requiring increased depositions of new bone in certain sections and reabsorption in others, likewise affects the amount of osteoid tissue. Erdheim (1914) thus describes this in the gi-owth of the thorax of rachitic rats : in the growing ribs, in connexion with the changes in their curvature and form, the greatest amount of osteoid tissue is deposited in the subperiosteal portion of the front surface of the rib, and then on the endosteal side of the reverse wall of therib. Erdheim's observations on this point have been confirmed by my experiments on rats with experimental rickets. Virchow, KoUiker, and Kassowitz (quoted by Erdheim) likewise mention analogous pheno- mena in the growth of the cranium. Thus those phenomena which B 3 20 physiologically lead to an intensive formation of bone, in cases of rickets or osteomalacia lead to an intensive formation of osteoid tissue. The soft osteoid tissue, however, cannot perform the function of hard calcified bone. Therefore, to make up for this, it is fre- quently deposited to a far greater degree than would be required in the case of firm bone. Comparatively few authors (Reckling- hausen (1891, 1898), Vierordt (1896), Schmidt (1897), Ribbert (1905), Dibbelt (1910), Aschenheim) likewise explain the formation of osteoid tissue in rickets or osteomalacia by halisteresis, i. e. by means of the extraction of calcium salts from the old calcified bone (decalcification). Recklinghausen supports this view by giving his description of the ' Gitternfiguren ' in the bone. Admitting that these ' Gitternfiguren ' indeed show a diminution in the amount of lime salts, Schmorl considers that this is still no proof that decrease in calcium salts is produced by decalcification. Schmorl observes, however, that the question cannot be finally settled by histological methods. Nevertheless, he himself found places in rachitic bones where there were no active osteoblasts near the newly formed osteoid tissue to account for the presence of osteoid tissue. On the other hand, Vierordt, Dibbelt, and Aschenheim point to cases of rickets and osteomalacia where the softening of the bones proceeds with surprising rapidity, 'under the very eyes of the physician ', and where an enormous quantity of calcium is excreted by the organism, reaching 0-48 gm. CaO per diem, and in one case of osteomalacia observed by Odermatt being so high as 1-57 gm. CaO per diew,?- The above-mentioned causes of the fluctuations in the amount of osteoid tissue in the different bones may likewise serve to explain the unequal amount of osteoid tissue in skeletons of different rachitic subjects. It is quite natural that only in growing children can large deposits of osteoid tissue be formed, and contrariwise, when no new bone is formed owing to the cessation of growth, the formation of osteoid tissue ceases. That is why in the case of rachitis tarda, and of rickety children who have stopped growing, the amount of osteoid tissue may be small (see the descriptions of similar cases by Schmorl (1906, 1909), Looser (1920). Looser found that in rachitis tarda even periosteal osteophytes are not always present, and if they do develop, it is only in a moderate degree. Another condition affecting the amount of osteoid tissue depends on the state of the nutrition of the organism. According to Friedleben's (1860) observations, children suffering from severe atrophy, usually following gastro-intestinal diseases, have very hard bones, containing a normal or even excessive amount of ash. At the restitution of normal digestion the general nutrition of such children improves quickly, but simultaneously rickets develops. Schmorl found that the rachitic changes in the skeleton were always far more marked in rachitic subjects with satisfactory nutrition than in those suffering from poor nutrition. Pfeitfer (1885) observed that rickety infants nursed by their own mothers frequently grew normally, with a proper adipose layer, and were well developed 1 A more detailed account will be found in the chapter on metabolism in rickets. 31 (' prachtvoU entwickelten Kinder '). That is the reason why some- times even fairly pronounced rickets passes unnoticed by the parents. Siegert (4) distinguishes two forms of rickets : the hyperplastic, with great thickening of the epiphyses, observable in well-nourished children, and the osteoporotic form, without any great enlargement of the epiphyses, but with craniotabes, observable in cachectic children. In connexion with this, Stoeltzner remarks that such a division is not advisable, as he has observed that cachectic rickety children immediately acquire typical and, moreover, highly marked changes in the skeleton as soon as they begin growing again and their nutrition is improved. As previously mentioned, Friedleben came to the same conclusion. Naturally, poor nutrition is accom- panied by a disturbance of the functions of cellular growth, of the growth of the cartilage and bone cells among others. In addition, the chemical investigations mentioned above show that in starvation part of the osseous tissue is burnt up by the organism for the development of the energy required. Therefoi'e, these conditions have a marked retarding effect on the new formation of osteoid tissue and the enlargement of epiphyses. Of course this does not mean that such children cannot be suffering from rickets : in my opinion, they often have it in a latent form, with indefinitely manifested clinical symptoms, and the disease becomes apparent, as Friedleben and Stoeltzner have observed, as soon as children begin to put on weight. Erdheim very justly remarks that, from the point of view of skeletal statics, the cessation of growth is an advantage to the rickety child, as a small skeleton is far more advantageous to the latter than a larger but soft skeleton. (b) Osteoporosis in Rickets and in OsteoTnalacia. In connexion with slight rachitic changes in the skeleton of emaciated children it is necessary to touch upon the question of osteoporosis in rickets. Osteoporosis consists of rarification of bone next to periosteum, bone marrow, or Haversian canals. Therefore the bone becomes thin walled, has a sieved appearance from widening of Haversian canals, trabeculae of spongiosa scanty or sometimes absent. This process is produced by increased bone resorption or by diminished bone apposition or by combination of both pro- cesses. Some authors do not attach any pai-ticular significance to this phenomenon, and even in cases of mai'ked osteoporosis in experimental rickets regard it as one of the distinctions from human rickets (Schjaorl). Its presence in a small degree is ex- plained as the ' physiological osteoporosis in children ' (Schwalbe, 1877). In new-born children Friedleben (1860) often found deficient calcification in different bones of the skull, which disappeared some weeks or months after birth. Independently of this the same author noticed sometimes very pronounced osteoporosis in the back bones of the cranium. He assumes this to be physiological because nearly all children three or four months old undergo this skeletal change. Schmorl (1909, p. 413), however, has also observed — in more rare 22 cases of prolonged and severe rachitis, with a severe and general disturbance of nutrition — osteoporosis exceeding the limits of the ' physiological '. As an explanation of this phenomenon, he adduces his own and Stoeltzner's experiments in vertical suspension of the extremities in cases of fractured bones. Such suspension caused the development of osteoporosis in the bones and callus due to : (a) anaemia, preventing the new formation of bone, but not prevent- ing, and perhaps even inci'easing, the resorption of bone ; (b) in- activity of the bone. The latter is adduced by Schmorl as an explanation of osteoporosis in the bones of paralysed extremities. Stoeltzner, in bis later article on rickets (1910, p. 100), and also Dibbelt (1912), are even of the opinion that rachitic bones are usually more or less osteoporotic. Moreover, Stoeltzner points out that osteoporosis reduces the strength of the bone to a far greater degree than when the newly formed bone remains for a long time in an osteoid state. Kassowitz (1912, p. 501), on the basis of his investigations, regards osteoporosis not as a casual complication in rickets, but as one of the most important components of the picture of rickets and osteomalacia. He justly criticizes (p. 4^4 and later) Pommer, who considers the processes of the new formation and resorption of bone in rickets as normal, while at the same time he himself describes the picture of osteoporosis in the bones in the aforesaid diseases. Ziegler (1898) is still more decided, and gives the following definition of rickets in his manual (p. 214) : ' Rickets ... is a general disorder of nutrition, which appears during childhood and is characterized anatomically by increased bone resorption, by deficient calcification of the cartilage, and by the formation and persistence of imperfect uncalcified bone or osteoid tissue.' Schmorl and Stoeltzner, on the basis of their observations, consider that the cause of osteoporosis in rickets lies principally in the reduced formation of new bone, and not in the process of increased resorption of the bone. Ziegler, however, attaches great importance to the process of resorption as well (p. 215) : 'Resorption of the osseous tissue already formed always takes place during the period when the bones are growing, but it is con- fined to definite regions. In rickets the state of things, is so far altered that the amount of lacunar I'esorption is excessivp, with the result that in marked cases considerable portions of the already completed bony skeleton are again destroyed. In the long tubular bones and in the short bones, the cortical stratum is thereby rendered more or less osteoporotic, and the osseous trabeculae of the cancellous tissue become attenuated or disappear. The compact substance of the flat bones of the skull may be reduced to a*few lamellae, so that the characteristic diflferentiation of the bony structure into an outer and inner table and a diploe is entirely effaced.' Looser (1920), in his investigations of rachitis tarda in patients with arrested growth and frequently with poor nutrition, points out three characteristic symptoms of the disease : the presence of osteoid tissue, the change in the endochondrial growth of the bone, and atrophy, i.e. osteoporosis of the bones. In Looser's cases, corticalis was frequently transformed into spongiosa. In the cases 23 of rachitis tarda described by Schmorl (1906), such violent manifesta- tions of osteoporosis were rarer, and, moreover, did not affect all the bones of the patients. Shipley, Park, McCollum, and Simmonds (1931, 1) thus very justly explain the osteoporosis frequently observed in experimental rickets in rats : ' With tlie rapid decline in the nutrition of the animals occurring during the latter part of the experiments and the corresponding loss in weight, a com- plete cessation, or at least a great retardation in the rate of growth of the skeleton must have occurred. There must have come about, therefore, a diminished requirement for those substances essential to ossification and calcification, which are insufficiently supplied in the diet. Moreover, it seems probable that a supply of those substances may have been liberated from the tissues of the animals themselves, for example, as the result of the resorptive processes in operation in the bones. From a theoretical standpoint, therefore, it seems possible to think that the processes in operation in the animal leading to the development of the state of extreme malnutrition may have been instru- mental in restoring to the organism conditions under which Ca deposition in the skeleton again became possible. For the development of rickets growth is necessary. If the deficiences in the diet are of such a nature as to render growth of the skeleton impossible, rickets cannot develop. If growth were brought to an end in an animal already rendered rachitic through the administration of faulty diets, there is reason to expect that the rachitic lesion would disappear and a condition of osteoporosis develop.' In order to corroborate this proposition, McCollum, Simmonds, Shipley, and Park (1922, 1) induced severe rickets in rats by means of a diet deficient in phosphorus (0-302 per cent. P) and antirachitic factor. After 35 or 40 days, as shown by their control experiments, there was a complete disappeai-ance of calcium salts from the zone of provisional calcification of cartilage in such animals. If the animal be now totally deprived of food for 3 to 5 days longer, and then killed, fairly considerable deposits of calcium salts may be found in the zone of provisional calcification. The authors are of the opinion that in starvation the increased oxidation of the subject's own tissues liberates phosphorus, which serves to replace the lack of the latter in the diet. In my opinion, another explanation could be given similar to that which I gave as to latent rickets in atrophic children, i. e. in the experiments of the American authors rickets was not cured, but merely changed to the latent form. On all the foregoing grounds, the principal cause of osteoporosis must evidently lie in connexion with the decline in nutrition, which in some cases accompanies rickets from the very beginming of the disease (Siegert's osteoporotic form of rickets). In other cases it merely complicates prolonged and severe rachitis at the stage of its highest development. The possibility of calcium starvation playing an important part in the development of osteoporosis will be dealt with later. CoTiclusions. 1. In rickets and osteomalacia the varying amount of osteoid in the different bones depends on : (a) the varying degree of the growth of the bones ; (b) the difference in the mechanical effects of weight, pressure, and strain on the bone ; (c) the processes of the reconstruction of the skeleton. 2. The vai-ying amount of osteoid in skeletons of different rickety 24 children depends on : (a) the energy of growth, and (6) the state of nourishment of the organism. 3. Osteoporosis occurs frequently in human rickets or osteoma- lacia^ and most probably are to be explained by the cessation of growth and the decline in the nourishment of the patient. 4. My experimental results lead me to the belief that for the anatomical pathological diagnosis of rickets and osteomalacia there is only one essential, namely, retardation or absence of lime deposition in the newly produced bone, giving a picture in which osteoid tissue is evidently increased in amount. 5. Changes in the periosteal and endosteal growth of bone, which goes on throughout life, can be observed independently of any changes in the endochondral growth of bone. This can be seen most strikingly in those eases of experimental rickets in which growth of the animal has ceased, osteoid tissue being greatly increased, while no changes are observed in the caa-tilage (see also Korenchevsky, 1922, 2, Figs. 4 and 6). 6. The formation of osteoid tissue around the trabeculae of the subchondral spongiosa occurs in the same manner as that around cortical bone, namely, through the activity of the osteoblasts. Therefore in rickets the production of osteoid tissue and its calcifica- tion occurs simultaneously in both regions (see also Korenchevsky, 1922, 2, Figs. 4 and 6). (c) Changes in the Epiphyses. Normally ^ every bone marrow capillary situated opposite a column of cartilage cells in the zone of provisional calcification dissolves up the cell capsules of that column, so that the cartilage cell column is replaced by a cylinder containing ingrowing capillaries and bone marrow elements. The walls of the cylinder consist of intercellular cartilaginous tissue, impregnated (in this zone) with calcium salts. Thus primary cartilaginous spongiosa is formed, on the trabeculae of which bone is deposited by the osteoblasts, and the cartilaginous spongiosa becomes osseous spongiosa. As soon as part of the zone of provisional calcification has been utilized for this purpose, a fresh zone is formed by the multiplication of cartilage cells and the deposition of calcium salts in the intercellular cartilage between the cells. Schmorl and others think that the regular and straight gi-owth of bone marrow capillaries is due to this intercellular cartilage cylinder, whose walls, hardened by calcium, prevent lateral growth of capillaries. In the classical picture of rickets, the following abnormalities in the epiphyseal cartilages may be observed :" enlarge- ment of the proliferous cartilage, principally in the zone of hyper- trophic cartilage cells ; disorganization of the disposition of cartilage cells ; the abnormally deep and irregular growing of bone marrow ' A good description of the normal process of bone formation with figures is given in Schafer's Text-book of Microscopic Anatomy. London, 1912, p. 142, or in Ziegler's Text-book of Pathological Anatomy. English transl. New York-London 1897, i, p. 196. 25 blood-vessels into the cartilage ; insufficiency or absence of deposits of calcium salts in the zone of provisional calcification. (i) The enlargement of the epiphyseal cartilage. According to Pommer, Schmorl, Heubner (1911), and others the enlargement of the epiphyseal cartilage proceeds from the blood-vessels of the bone marrow preventing or retarding the dissolution of the capsules of the cartilage cells in the zone of provisional calcification. Owing to this these cells are retained in the cartilage for an abnormally long time, and thus, with the normally continued formation of new cartilage cells, increase the area of proliferous cartilage. There is no question, however, of any increase in the number of cartilage cells formed. On the contrary, Erdheim (1914) justly assumes that the formation of new cartilage cells is likewise retarded, but to a less degree than the dissolution of the cells in the zone of pro- visional calcification. This is the only explanation of rachitic bones being shorter than normal bones, notwithstanding the enlargement of the epiphyseal cartilages. Only a few authors (Kassowitz, 1913) explain the increase in the size of proliferous cartilage by an abnormal multiplication of cartilage cells due to a special rickets- producing irritation. (ii) The deficiency or absence of calcium deposition in the zone of provisional calcification. In rickets the zone of provisional calcifi- cation is either partially or almost totally deprived of calcium salts. Calcium salts in rickets are not infrequently deposited in the cellular cartilaginous tissue under the perichondrium. Wieland and Stoeltzner observed this in infantile rachitis. Erdheim observed it in spontaneous rickets in rats. I have likewise seen this picture very often in cases of experimental rickets. Erdheim thinks that this deposition of a calcareous cylinder round the enlarged cartilage is nature's attempt to strengthen the latter. Apart from the classical decrease or even total disappearance of lime salts fi-om the zone of provisional calcification, there have been cases of rickets where not only has the calcification of this zone been preserved, but there has even been an increase in length. Schmorl explains this as due to repeated short attacks of rickets followed by remissions : during the short attacks of rickets the zone of provisional calcifica- tion of cartilage has no time to lose all its calcium, and with the new deposition of calcium during the subsequent remissions the successive layers of this zone are imperceptibly connected with the preceding ones. Thus there is an apparent extension of the zone. Erdheim does not accept this explanation in the case of spontaneous rickets in rats, as in none of his cases has he observed interrupted layers with calcium. Erdheim suggests another explana- tion, which, on the grounds of my own experiments, seems more pro- bable. If the dissolution by the blood-vessels of the zone of pro- visioTial calcification proceeds more slowly than the deposition of calci/wm in that zone, conditions may very easily arise under which, in rickets, this zone may remain calcified or may even increase. In my experiments on rats, whose skeletal growth had been arrested in length (a) physiologically by advanced age, (b) pathologically by insufficient diet, I not infrequently observed a marked retardation 36 in the new formations of primary spongiosa, and a more or less normal saturation with lime of the zone of provisional calcification. In such cases, the whole zone of proliferous cartilage was most frequently even smaller than the normal, and consisted of a decreased number of layers of small cartilage cells. ^ If, on the one hand, there is hardly any new formation of cells in the proliferous cartilage, and, on the other hand, their destruction by the blood- vessels of the bone marrow is likewise almost completely arrested, then, even when the deposition of calcium in the zone of provisional calcification is greatly retarded, the latter may be sufficiently calcified by the deposition therein of very small quantities of lime. As soon as the endochondral growth of bone is resumed, the deficiency of calcium becomes immediately and characteristically apparent. Grandis and Mainini (1900) ^ made a micro-chemical investiga- tion of the process of ossification of cartilage both in normal and rachitic bones. For the micro-chemical detection of phos- phoric acid they employed ammonium molybdate dissolved in nitric acid, and'purpurin for staining the inorganic calcium. In the normal epipliyseal cartilages they discovered a phosphoric acid reaction only in the cell nuclei of resting cartilage : in the pro- liferous cartilage the reaction was very marked not only in the nuclei but also in the protoplasm of the cartilage cells. Towards the periphery of the zone of provisional calcification the reaction in the cells grew weaker and finally disappeared, but was very marked in the intercellular tissue. The authors are of the opinion that lime deposition takes place in the intercellular cartilage area containing inorganic phosphorus. They also think that the source of phosphoric acid lies in the cartilage cells, whereas lime is directly deposited from the blood. In rickets the deposition of phosphoric acid in the intercellular tissue of the zone of provisional calcification is very irregular, and in many places it is scanty. Pacchioni (1903), on the whole, corroborates the observations of Grandis and Mainini. The authors consider that the cause of the inadequate deposition of calcium and phosphorus in the proliferous cartilage lies in the pathological changes in cartilage-cell metabolism. (iii) Irregularities in the penetration of the bone marrow blood- vessels and the disorganization of cartilage. The fact that, there is irregularly deep penetration of the bone marrow blood-vessels into the zone of proliferous cartilage depends upon alterations. in the rate of penetration, the process being in some cases retarded, whilst in others it almost ceases. It is difficult to explain this altered activity of the bone marrow blood-vessels, and an explanation of this point is of importance in solving the rickets problem. The irregularly penetrating blood-vessels and the bone marrow which accompanies them dissolve and penetrate deeply into the cartilage in some places, leaving sections of undissolved cartilage in other parts. According to Schmorl, owing to the absence of calcium in the cartilage cylinders, the growth of the blood-vessels is not ' See PI. 15, Pig. 88, and PI. 16, Fig. 40. 2 Unfortunately I could not obtain the original work of the authors in Italian, and am quoting it from Stoeltzner's communication. 37 regulated, and the latter grow and branch off in diflferent directions, utterly disorganizing the normal disposition of the layers of cartilage cells. On the basis of my own experiments I think that this generally accepted explanation does not completely explain the changes in question. I have often observed that in animals fed on a diet deficient only in calcium there has been a completely regru^ar though tardy penetration of blood-vessels into cartilage, associated with a great increase in proliferous cartilage and an absence of lime salts in the zone of provisional calcification. Increased disorganization of the cartilage likewise depends upon whether the blood-vessels which have grown into the cartilage ramify and form irregular marrow cavities, transforming the cartilaginous walls of the cavi- ties into osteoid tissue. The effect of the weight of the body on the softened cartilage, and the rachitic curvature of the bones at the osteo-chondral junctions further increase the disorganization of the cartilage and give a totally incorrect direction to the line of osteo- chondral junction. In connexion with the extent of the changes in the epiphyseal cartilages of different bones and in different rachitic subjects, we see the influence of many of the factors which affect the amount of osteoid tissue. According to Schmorl, a very important factor is the energy of endochondral growth, which sometimes differs in different bones, being greatest in the ribs, less in the lower epiphyses of the femur, the upper epiphyses of the humerus, the epiphyses of the tibia and fibula, the lower epiphyses of the radius and ulna and ^ of the femur, and less again in the other bones. In association with the less energetic longitudinal growth of the skeleton in more adult organisms, the changes of endochondral ossification in rachitis tarda may be manifested very faintly, and, moreover, only in the more energetically growing bones, i. e. the ribs (Schmorl). From a pathological point of view, the transition from typical rickets to rachitis tarda, and from the latter to osteomalacia, takes place imperceptibly, -and there is no essential difference between them (Pommer, Schmorl, Erdheim, Looser, and others). Rickets is a disease of the growing, osteomalacia of the adult, organism. The results of my experiments lead me to take the same view (Korenchevsky, 1932, 2). The slightness of the changes in epiphyseal cartilage must be due to cessation of the growth of the organism owing to various patho- logical causes, including cachexia. That is why Siegert, Stoeltzner, Schmorl, Erdheim, and others observed that the changes in the skeleton of well-nourished rachitic children were more decided than in cases of malnutrition. This must always be borne in mind in explaining the picture of experimental rickets, according to whether the animal experimented upon is well nourished or exhausted, whether it is growing or has stopped growing. Conchisions. 1. Although the changes in the proliferating carti- lage aid the diagnosis of rickets, yet they are not an essential factor : the increase and disorganization of proliferating cartilage depend directly upon the rate of growth of the bones. 3. The amount of calcium deposited in the zone of provisional 28 calcification depends, on the one hand, upon the retardation or absence of the process of lime deposition, and, on the other hand, upon the rate of growth of the proliferating cartilage. Therefore there may even be cases of rickets with a deposition of calcium salts in the zone of provisional calcification. (d) Callosities after Spontaneous Fractures. Spontaneous fractures in rickets and osteomalacia naturally bear the impress of these diseases, manifested principally by the amount of osteoid tissue in the callus, instead of the fractures being united by callus of normal calcified bone. As the soft osteoid tissue is not sufficiently strong to consolidate the fracture, by way of compensa- tion the callosities of rachitic bones are much larger than those of normal. (e) General Conclusion. One must agree with Looser that in rickets and osteomalacia the active processes of the formation and growth of bone are mostly decreased : the deposition of lime salts in the bone and cartilage is delayed, as well as the dissolution of the cartilage cells in the zone of provisional calcification ; the longitudinal growth of the bones is retarded, and frequently there is a delay in the formation of new bone, conducive to osteoporosis. An increase of the activity of the bone-forming processes may be observed only in those cases of rickets where the osteoid tissue grows out under the periosteum and in the spongiosa in greatly increased quantities, sometimes trans- forming the spongiosa into compacta, and increasing the thickness of the corticalis several times. Thus both a chemical and a histo- logical analysis of the skeleton in rickets and osteomalacia yield completely analogous results, the principal being a decreased depo- sition of calcium salts in newly formed bone. There is no doubt that the key to the problem of rickets is to be found in the discovery of the cause which prevents the deposition of calcium salts into the bone and bone-forming^ro^i/erous cartilage. The following chapters are devoted to an attempt to elucidate the question. 5. Calcium Metabolism iisr Normal and in Rachitic Subjects. Notwithstanding the great number of investigations made, the question of the metabolism of calcium in the organism has not been adequately elucidated even in normal conditions, and even less so in rickets. The reasons for this are as follows : 1. A great proportion of calcium is excreted from the organism through the intestines. In adults it is only in certain cases that about one-half of the calcium can be excreted through the urine (Bertram (1878), Ren vail (1904)), but usually the quantity is 29 far less, from a small amount to 30 or 40 per cent, of the total cal- cium excreted. In childhood only a small proportion (up to 9 per cent.) of calcium is excreted through the urine. The excretion of calcium is especially small in the case of rickety children. This allows certain authors to ignore the calcium in the urine when investigating the metabolism of calcium in children (Findlay, Paton, and Sharpe (1921), Holt, Courtney, and Fales (1920)). But the amount of calcium excreted through the intestines cannot be ascer- tained, because it is mixed with the non-absorbed calcium of the food. 2. Findlay, Paton, and Sharpe rightly point out that the amount of excreta per diem is subject to great fluctuations, but the percentage of calcium contained in the excreta is more or less constant. There- fore, in order to obtain a more or less exact figure of the calcium excreted through the bowelj experiments should cover a longer period of time when the separation of the faeces of the experimental period is not effected by exact methods (for example, by means of carmine). Owing to the difficulties connected with experiments on metabolism, many of the investigations of children have been of very short duration, and were conducted without separating the faeces. This makes it necessary to regard the results with a certain degree of caution. (a) Normal Subjects. Nevertheless, in spite of the above-mentioned difficulties, it is already possible to form a rough idea of the metabolism of calcium in the organism under normal conditions and in rickets. The literature, up to 1921 on the subject of the metabolism of calcium has been collected and properly criticized by Findlay, Paton, and Sharpe, who added a great many of their own valuable observations. I have tried to collect and present the most important data obtained in the appended tables, Nos. 6, 7, and 8. Table No. 6. Human Milk. Age. Number of children. CaO retained or assimilated per cent, of intake. Minimum. Maximum. Average. 1-6 months Normal children 14 21.6 82.8 59 Rickety children 8 1 negat. 58 31 7-13| months Rickety- children .5 2 negat. 30-9 15-3 Authors, and number' of children examined.^ Birk [1910] (2), Michel [1896] (4), Schlossman [1904] (3), Tobler and Noll [1910] (1), Michel and Ferret [1899] (1), Klotz [1909] (1), Schabad [1910, 5] (1), Blauberg [1900] (1) Birk and Orgler [1910] (1), Orgler [1911] (1), Schabad [1910, 6](2), Schloss [1914] (2), Grosser [1920] (2) Schabad [1910, 6] (2), Grosser [1920] (3) 1 In tables Nos, 6, 7, and 8 the figures in square brackets denote the date of publication by authors. The figures in round brackets the number of children examined. 30 s-ti ri. s I agp s,"^^ ^S^-i^i^ wis I S'SF^I- -Ufa "-'-'« i'-Ji Si S ^-SJa ^ -322 a 12 2.2 2 m2" ">S Ss~a ^"moJ -^^s -aS-s S,a ^ Sim » ■« S OS OS >*S W SM f" — ^ 1— I « I I Jrt *-* ' — ' ' — ' S S S.s" § g .bI 1^ t^'-^ja S ^ w I — I I — I S'-'Os fc,SSG'^-2 "-1-3 ■S -^i S a g,o -o ■=>£ a 3 « O ■8 w to -Sg OO 00-«> O-S'O) O I -^ -^ O fe; H H l-H s 1 — r a, si w. A 02 -d ^ ^ o s n h oa i 03 ■a m d g (^ 5S,-~4iU3 loop t6t01> l-j Ji"*- tj g to lo xs-^tDioio •< «a ^ ■ 5 i EH I o »1 1-1 1-1 a 9 ■«- • S fflrt a -a aa§ M,2b0a,- cuts i "?■ £ 'Sa'So" lis lit II • -^4-^^^ 1.1:5 ^1:1 ^i III II 2^^ wtef^ZSl^ floe P."3 I ■::; s 33 Some bighly important conclusions can be drawn from these not very numerous data. These conclusions cannot, however, be accepted as quite indisputable, in view of the above-mentioned remarks, and also of certain reasons which will be brought forward later. 1. The calcium contained in human milk is assimilated and retained much better than that in cow's milk or in mixed diet. 2. Apparently, in artificial feeding, older children (from 2 years) assimilate and retain calcium better than infants under one year. It is, however, necessary to bear in mind the following facts. Rickets is most frequently developed during the first year of the infant's life ; it may, therefore, be assumed that the lower average percentage figures found for calcium retention during the first year of life are due to the fact that many infants with latent rickets are classed as normal. In my opinion this explains the negative, or only slightly positive, balance of calcium in some eases. Therefore I think that (1) the average figures of calciv/m retention in normal children should he much higher than they are represented in Tables Nos. 6, 7, and 8, approaching to the figures ofTnaadmum retention; (2) the question of the influence of age must as yet be an open one. (3) Holt, Courtney, and Fales, in cases of feeding on cow's milk or mixed diet, attempt to determine the absolute minimum of CaO introduced, for the purpose of obtaining the normal figure of reten- tion. In the cases observed by the authors, with a very low intake of calcium with the food (equal to or under 0-09 gm. GaO per kilo per diem), the retention of calcium in the organism declined, on an average, from 40-4 per cent, to 20-3 per cent. Therefore, from a comparison of the average figures of CaO introduced into and retained by children fed on milk, the authors consider that the minimum physiological requirement is 0-13 gm. CaO per diem per kilo of the child's weight, while in the case of children on a mixed diet the figure is 0-09 gm. CaO. (b) Rachitic Subjects. Taking 0-09 gm. as the minimum CaO required, of the 90 cases of calcium metabolism in normal children fed on cow's milk inves- tigated by various authors, in 79 cases the intake of CaO was over 0-09 gm., and in 11 cases it was equal to or below that figure. In rickets, of 50 cases where calcium metabolism was investigated, only in 3 was the figure less than or equal to 0-09 gm. CaO, being above that in all the other cases. That is to say, taking the authors' minimum measure of the physiological requirement in calcium, it . would appear that, both in normal and in rachitic cases, a sufficient amount of calcium was ingested. I shall return to this question later. In rickets the average figure of the retention or assimilation is sharply reduced in infants fed on human milk, viz. to 15-31 per cent, instead of the normal 59 per cent. ; a negative balance was observed only in 2 of 13 cases. In rick'ety infants fed on cow's milk or its modifications, the average figures of the positive balance of 33 the calcium percentage retained showed a decline below the normal average only in the case of infants of from 7 to 24 months. But whereas at the age of 1 to 6 months only 13 per cent. (3 out of 33) of the normal infants showed a negative balance, in rickety infants the percentage rose to 55 per cent. (6 out of 11). Similarly, rickety infants of 7 to 13 months showed a negative balance in 29 per cent, (in 6 cases out of 31), and rickety infants of 13 to 24 months in 33 per cent, (in 3 cases out of 13). The respective control groups showed no negative balance. On mixed diet no marked difference between rickety and normal children was observed. This should, perhaps, be explained by the more advanced age of children on such diet, when rickets begins to undergo spontaneous cure. (5) Finally, in investigating convalescent rickets, a very high average retention of calcium was observed, i. e. 59 per cent., the minimum being likewise high. No negative balance was observed. All the children of this group had cod-liver oil. It has already been mentioned that a child suffering from rickets can lose about one-third of the calcium contained in its body at birth (Steinitz and Weigert, Schabad). Therefore even theoretically we should expect a negative balance in the metabolism of calcium in rickets. The far from numerous experiments already made show that in rickets a negative balance is of frequent occurrence with arti- ficial feeding and in infants fed on human milk. The daily loss of calcium may be very great : in Dibbelt's case 0-339 gm. CaO, in Orgler's 0-367 gm., and Klotz's 0-481 gm. The question arises, why is this negative balance not observed in most cases of rickets, if not in all ? This may be explained in the following way : (1) Even if in the growing child the deposition of calcium in the skeleton continues, but is less than normal, the newly formed bones will still be deficient in calcium, and rickets will develop. More- over, the disease may develop so slowly that the difference in the metabolism of the rickety and the normal child may not be apparent, and yet be quite sufficient to cause changes in the skeleton. That is to say, the rachitic changes of the skeleton may appear without any negative balance of calcium, though with a low positive one. (3) The pathological investigations of Schabad and the observa- tions of Looser and others have shown the frequency of short remissions in rickets. In view of the small number of investigations made of the metabolism in rickety children, and the short periods covered by such investigations, it is quite possible that some of the experiments coincided with periods of remission. In general, the course of rickets is frequently irregular, and periods in which the growth of the organism (and therefore of the skeleton) is arrested are followed by periods of increased growth. The picture of the disease, and therefore of the insufficient metabolism, is especially marked in the latter case. (3) Schabad (1910, 4) has pointed out a fact of enormous impor- tance, which may be observed on the basis of the data in Tables (1S79) c 34 Nos. 6-8, and which has now been corroborated by the accumula- tion of new evidence. It gives a good explanation not only of the pathogenesis of rickets, but also of the paradox of the negative calcium balance in outwardly health}' children. He thinks — and the majority of investigators now agree with him — that rickets is originally latent. During this stage, when the child appears to be normal and healthy, rickets can be diag- nosed only by one symptom, namely, a negative calcium balance. As a result of the disturbance in calcium metabolism, the disease enters its second stage, with changes in the skeleton that can he diagnosed clinically. In this early stage of definite and progressive rickets calcium metabolism remains negative. The next stage is characterized by very pronounced clinical features of the disease, with the important exception that the bone softening ceases. Simultaneously the calcium balance is equal to zero or slightly positive. Then comes the last stage of change, with an increased deposition of calcium salts in the osteoid tissue of the skeleton. This stage is characterized by an abnormally high positive balance, and is especially perceptible when the process of the cure of rickets is forced by the introduction of phosphorus with cod-liver oil {vide infra). To illustrate this, Bii-k and Orgler (1910) describe a case of negative calcium balance ( — 0-177 gm. CsbO per diem) in a child without any manifestations of rickets. The disease developed three months later, and an investigation of metabolism then showed a positive balance ( + 0-079 CaO). Dibbelt's (1910) child had a negative balance ( — 0-339 gm. CaO) at the height of the disease. Some time after, a fresh investigation of metabolism showed an abnormally increased positive balance ( + 0-756). Only a week after this second investigation, there was a perceptible clinical improvement in the disease. A similar case is described by Find- lay, Paton, and Sharpe (1921). They chose a child whom, in view of its normal condition and healthy skeleton, they proposed to use as a control in an investigation of metabolism in rickets. Quite unexpectedly they found it had a very low positive balance (0-05 gm. CaO per kilo per diem, 0-283 gm. CaO having been introduced). Several months after the child developed ' a most severe degree of rickets '. Having this fact before him, Dibbelt insists on the necessity of making repeated systematic investigation of metabolism in children from the earliest age, for only then can rickets be diagnosed in its latent stage, as showing a negative Ca balance. He himself found a negative balance in two infants 21 and 25 days old respectively. Grosser (1920) advises that special attention should be paid to prematurely born children, in one of whom he had found a negative balance at the age of 4 weeks. Comparing the above-mentioned facts with the conclusions which may be drawn from Tables Nos. 6-8, there is reason to think that Schabad has rightly described the metabolic picture in difierent stages of rickets. After what has been said, it is easy to understand even those cases of negative or abnormally low positive balance which various investigators have found in outwardly ' normal ' children 35 who were really suffering from latent rickets. It is likewise very difficult to determine clinically when the cure of rickets has begun : as a result, many authors speak of the normal or even supernormal calcium retention in rachitis florida. Apart from the two other causes mentioned in the beginning of the present chapter (see pp. 28 and 29), a third factor has greatly retarded the exact determination of the disturbance of calcium metabolism in this disease, namely the contradictory fluctuations in calcium meta- bolism at different stages of rickets. Conclusions. 1. Schabad's views on the different character of the calcium metabolism in rickets at different periods of the disease have been corroborated by the majority of investigators. 2. In latent or incipient rickets the calcium metabolism is nega- tive ; during the period of manifest and progressive rickets the calcium balance remains negative ; during the period of markedly manifested rickets, but with the cessation of the process of softening of the bones, the balance of calcium metabolism is at zero or only slightly positive ; during the period of convalescence or remission of rickets the calcium metabolism is markedly positive. 6. Metabolism in Osteomalacia. All the data obtained by different authors I have summarized in Table No. 9 on p. 36. Of the 13 cases of osteomalacia quoted in Table No. 9, in 8 cases the balance of calcium was negative, in 1 case it was negative or positive, and in 4 cases it was positive. To this must be added a negative calcium balance in one case of Capellani (1909) and Loeper and Bechamp's cases (1909), where the authors found that in osteomalacia the calcium excreted in faeces forms 183 per cent, of the intake. Thus in a considerable majority of cases of osteo- malacia the calcium balance was negative. Hotz attributes the high positive balance in Neumann's cases to the beginning of con- valescence. In Odermatt's second patient also convalescence was quite definite. In general, what was said concerning the difficulties of investigation of calcium metabolism in rickets is applicable also to osteomalacia. In the cases observed by Sauerbruch, Hotz, and His, the phos- phorus and cod-liver oil treatment had a favourable effect on the retention of calcium in the organism. Castration had a similar effect in one of Neumann's cases and in one case of Goldthwait, Painter, Osgood, and McCrudden. In two of Neumann's cases it had no effect at all. Phosphorus metabolism in osteomalacia does not correspond with that of calcium, and in the majority of cases the phosphorus balance is positive. Gonclusions. 1. In most cases of osteomalacia the calcium balance was found negative. 2 36 Table No. 9. Metabolism of Ca aTid P in Osteomalacia. Authors and Cases. Limbeck (1894). Early osteomalacia, aged 56 Neumann (1896) (1) Puerperal osteomalacia, aged 36 (as) Before castration (6) After castration (2) Very seTcre osteomalacia, aged 36 (a) Before castration (6) After chloroform anaesthetic (c) After castration (3) Severe osteomalacia, aged 84 (o) Before operation (average from 3 experi- ments) (6) After hysterectomy Korozynsky (1902) (1) Puerperal osteomalacia, aged 41 (o) Mixed diet (&) Vegetable diet (c) Mixed diet (d) Mixed diet (e) Tablets of ovaries (2) Nullipara, aged 20 (a) Mixed diet (6) Vegetable diet (c) Mixed diet (d) Tablets of ovaries Sauerbru(ih (1902). Osteomalacia juvenilis, boy, aged 8 (a) Before treatment (6) Cod-liver oil and P-treatment (c) After treatment His (1902). Osteomalacia, patient aged 17 (ffl) Before treatment (6) Treatment with phosphorus, apparently in cod-liver oil (c) After treatment Goldthwait, Painter, Osgood, and McCrudden (1905). Girl, aged 16 (a) Before castration (6) After castration Hotz (1906) (1) Patient aged 40 (a) Before treatment (6) P + Kassovyitz emulsion treatment (c) After treatment {d) Treatment with thyroid tablets (2) Patient aged 61 (ffl) Before treatment (6) P + Kassowitz emulsion treatment (c) After treatment (d) Treatment with thyroid tablets Odermatt (1910) (1) Osteomalacia, patient aged 64 (2) Osteomalacia, patient aged 30 (convalescence) Length of experiment s). 6 5 5 4 5 5 19 Balance : in gm. per diem. CaO. -o.a -0.07 + 1.16 + 0.12 H-0.09 + 0.10 + 0.61 + 0.40 -0.17 P2O5. -1.20 + 1.26 + 0.82 + 0.64 + 0.81 + 0.02 + 0.68 5 5 5 5 4 + 0.83 + 0.11 0.01 -0.16 -0.37 + 1.26 + 0.36 + 1.20 + 0.81 + 0.14 5 5 5 4 -0.23 -0.43 + 0.06 -0.09 + 0.83 + 0.14 + 0.95 + 0.83 11 10 7 -0.04 + 0.21 -0.17 + 0.25 + 0.18 + 0.29 11 9 -0.07 + 0.8 + 0.40 + 0.25 + 0.20 8 14 -0.14 + 0.20 -0.04 10 11 10 8 + 0.06 + 0.21 -0-05 + 0.01 + 0.13 + 0.26 + 0.36 + 0.52 8 12 7 8 -0.24 + 0.02 0.00 -0.15 + 0.10 -0.06 + 0.04 + 0.21 80 30 -1.57 + 0.24 + 0.18 -0.40 37 .2. The metabolism of phosphorus in osteomalacia does not correspond with the metabolism of calcium, the phosphorus balance in the majority of cases being positive. 7. Effects on Calcium Metabolism of the Introduction of VARIOUS QUANTITIES OF CaLCIUM INTO THE ORGANISM. (a) The Normal Orgaivism. Findlay, Paton, and Sharpe, and Holt, Courtney, and Fales have demonstrated on children that the absorption of calcium from the intestines has a tendency to increase with increased intake in the food, and to decrease with its diminution. We have already given the average figures of the absoi-ption of calcium introduced in quantities greater and less than 0-09 gm.CaO per diem per kgm. body-weight by the last-mentioned authors. Renvall (1904) found that a healthy adult may have a negative calcium balance if less than a certain total minimum (according to Renvall, 0-7 to 0-86 gm. CaO i) is introduced. In Bertram's (1878) experiment, when 0-385 gm. CaO was introduced, there was a balance of — 0-014 gm. With an intake of 5-985 gm. CaO the balance was + 0-273, the bulk of CaO (5-414 gm.) being voided in the faeces and 0-298 gm.' in the urine. That is to say, of the enormous quantity of calcium introduced, only a comparatively small portion was retained, while all the rest was excreted, principally in the ' faeces and, to a considerably smaller extent, in the urine. The experiments of other authors confirm these observations (Herz- heimer (1897), Eenvall, Oeri (1909), and others). Even this increased amount of calcium deposited, however, is subsequently excreted by the organism, as the latter, apparently, can retain only a definite quantity of calcium required by it. Notwithstanding the continued increased introduction of calcium, the latter either begins to be retained in far smaller quantities, or the balance may even become negative. In this respect Renvall's experiment, carried on for thirty-two days without a break, is especially instructive (pp. 111-17). Table No. 10 gives the results. Table No. 10. riod. Length of period {days). Amount of CaO introduced per diem. Balance. I II III IV V 8 7 6 5 6 0.86 gm. 0.909 „ 1.197 „ 1.486 ,. 1.470 „ + 0.028 gm, -0.017 „ + 0.164 „ + 0.173 „ + 0.011 „ Neurath (1911) and Katzenellenbogen (1913) did not, in general, find any increase of calcium in the blood with an increased intake in the case of children. Voorhoeve (1911) gave about 2-7 gm. CaO 'per diem, for several 1 Renvall is of the opinion that when growth has been completely arrested, this minimum requirement of calcium may be still less. 38 days to patients suffering from different diseases and noticed that the calcium content of the blood increased. This enrichment of the blood with calcium continued several weeks after the calcium feeding ceased. 0-55 gm. CaO per diem, however, caused no increase or sometimes a slight one. The negative results of the other authors (Neurath) he explains by the fact that the doses of calcium given to patients were too small. Eey (1895) introduced 0-8 gm. of CaO, in the form of calcium acetate, into the blood of dogs, and thirty minutes after found only 0-31 gm. present in the blood. That is to say, during that time the blood had managed to eliminate about 0-5 gm. CaO. Nevertheless, four days after the injection there was still a perceptible increase in the calcium content of the blood. Experimenting on animals, Neurath found that a very great increase of calcium in the food was needed to cause a very slight increase of calcium in the blood. Fenyversy and Freund (1913) injected l'3-0-78 gm. calcium chloride intravenously into rabbits. Two to seven minutes after- wards, some 80 or 90 per cent, of the calcium introduced had dis- appeared from the blood. Clark (1920) likewise could obtain only a transient increase in the calcium content of the blood of rabbits by introducing calcium either subcutaneously or intravenously. He could not, however, produce this effect by increased intro- duction of calcium with the food. Meigs, Blatherwick, and Gary (1919, 1) were likewise unable to observe any consideralDle increase in the calcium content in the blood of cows after increase of Ca in their food. Heubner and Eona (1919) determined the influence of calcium chloride, introduced either intravenously, subcutaneously, per rectum, or by inhalation, on the calcium content in the blood of normal cats. Immediately after intravenous injection the calcium content of blood showed an increase of 100-200 per cent., but it returned to normal in two hours' time. Subcutaneous injection increased the calcium content in the blood 1| times within the first hour, this level being maintained for hours. The absorption of calcium pe"^ rectum, was very bad. From the trachea, by inhalation, on the contrary, the absorption was very good. In the latter case it was found possible to raise the calcium content in the blood by 33 per cent, of the normal. After prolonged calcium starvation, when the calcium content of the blood is low, it is apparently possible to increase it even by the increased introduction of calcium per os. Thus Boggs (1908) succeeded in increasing the calcium content of the blood by 33 to 36 per cent, in the course of three to five days, by the introduction of 4-8 gm. of calcium lactate or acetate, in the food of two dogs who had been fed on a diet deficient in calcium (meat and bread). Kochmann and Petzch (1911), experimenting on dogs, confirmed these results. The negative calcium balance observed in dogs in calcium starvation was transformed by the authors into a positive balance by the increased intake of lime salts. In order to obtain this result, they were obliged to introduce calcium in quantities greatly in excess of the calcium deficit. Denis and Minot (1920), in their feeding experimeots on cats and 39 rabbits, likewise observed that they could only increase considerably the calcium content of the blood in the case of animals whose calcium content had been low at the beginning of the experiment. The experiment made by R. Berg (1911) on himself is of great importance, as it shows the metabolism of calcium, phosphorus, and magnesium in calcium starvation in humans. The author was interested principally in the metabolism of phosphorus. The experiment was conducted for a period of 92 days. The diet con- sisted principally of meat, white bread, biscuits, potatoes, rice, macaroni, sugar, honey, beer, and wine. Besides this, it included from 11 to 16 c.cm. milk and 13 to 15 gm. buttex-. The diet con- tained an average of 3,555 ' net ' calories per diem. With this diet the average intake by the organism was 0-237 gm. Ca, 3-042 gm. HPO^, and 0-124 gm. Mg. Taking Renvall's figures (0-7-0-86 gm. CaO) of the normal calcium requirement of an adult as a standard, Berg's intake was less than half the requisite amount. The small quantity of milk and butter, in my opinion, made the diet deficient in fat soluble factor. Naturally the author lost weight on such a diet ; at the beginning of the experiment he weighed 82 kgm., and by the end of the experiment he had lost 9-| kgm. The author explains his loss of weight by the fact that the pre- dominance of meat in his diet poisoned his organism by the exces- sive intake of HPO^ and SO^. As a result, towards the end of the experiment and after its termination the author developed anaemia and neurasthenia. In any case, the negative balance of phosphorus was a great detect in this experiment. I think that the deficiency of the diet in fat soluble factor may also have been very important in causing the abnox'mal metabolism and loss of weight. I have summarized the results obtained by Berg in the following table. No. 11: Table No. 11. Ca in gm. per HPO. in gm. per Nun iber and igth of Diet. pe. riod. ^' ^ Loss ( — ) period. lei ' Loss ( - ) or reten- periods. Intake. or reten- Intake. tion ( + ). tion ( + ). I. (8 days) Diet alone 1.800 -2.0^8 24-191 -5.229 II. (7 days) Diet + 6-2 gm. Ca3(POi)2 per diem 17.022 t 3.192 44-199 + 4-491 III.i (7 days) Diet alone 1.580 -8-965 22-748 -6.322 IV. (7 days) Diet + 10.3 gm. CaHPO, per diem 19-416 + 0-645 64-114 + 3.264 V. (7 days) Diet alone 1-816 -3.510 22-128 -9.512 VI. (7 days) Diet+10.2gm.Ca(H2PO4)2 per diem 18-720 + 3.237 98-066 - 3.949 VII. (7 days) Diet alone 1-646 -4-799 21-310 -5-617 VIII. (7 days) Diet + 5-35 gm. lecithin per diem 1-649 - 0-368 27-038 + 1-808 IX. (7 days) Diet alone 1-583 -1-590 19-919 -5-849 X. (7 days) Diet + 5-36 gm. lecithin -1 3 gm. calcium lactate per diem 6.167 + 0-510 24-777 -6.621 XI. (7 days) Diet alone 1-732 -0-652 24-630 + 1.960 XII. (U days) Diet + 3 gm. calcium lac- tate per diem 12.472 + 2-591 42-554 -10.872 * Periods III, V, VII, IX, and XI immediately follow the foregoing periods. 40 As may be seen from the table above, in calcium starvation (accompanied by deficiency of fat soluble factor in the diet) there is a considerable loss of calcium and phosphorus in the organism. The . introduction of calcium salts caused a temporary improvement in the balance of calcium, but as soon as the extra intake of calcium ceased, in the period following there was an excretion of the calcium retained in the organism during the preceding period. Lecithin, apparently, had a favourable effect on the calcium balance. On the basis of the effects produced by lime salts not only on the metabolism of calcium, but also on the relation of the latter to phosphorus, Berg considers that in the case of a deficiency of calcium in the organism (e. g. in rickets), calcium phosphates are useless, whereas calcium lactate is worthy of attention. His conclusions drawn from many of the above-mentioned investigations will possibly be corrected in the future because the presence of antirachitic factor was not taken into consideration. (b) The Rachitic Organism. Schabad (1910), and likewise Orgler (1911), investigated the effects of the introduction per os of various calcium salts (phosphate, acetate, and citrate) on the calcium metabolism in seven experiments on five rachitic subjects. Orgler critically analysed these experi- ments, and came to the following conclusions. In only two cases was there any increased depositiou of calcium after the introduction of lime salts per os. But it was just these two rickety children who, prior to the introduction of extra calcium per os, had retained it in their organism to a degree above the normal, i. e. they were recovering from rickets. No indications of the influence of increased calcium intake were found in the three other children, two of whom were at the stage of progressive rickets, with an abnormally low calcium balance, while the third was recovering, with an exceedingly high calcium retention which did not give any hope of any great deposition of the latter. Oil the grounds of these experiments Orgler thinks that an increased intake of* calcium salts in rickets can only be of use at the recovery stage, and then only provided that, prior to the introduc- tion of the salts, the retention of calcium had not reached the maximum. In progressive rickets, in Orgler's opinion, the increased intro- duction of calcium is useless, as at that stage the organism of a rickety child has lost its capacity for depositing calcium. This question has again been thoroughly investigated on ten rickety children by Grosser (1920). His experiments were made under unquestionably good conditions, each rachitic subject being observed for several weeks, e. g. for 64 days in Case No. 7. The results of these observa- tions are, shortly, as follows : Of the different calcium salts (acetate, glycero-phosphate, and chloride) examined by the author, the most efficacious proved to be glycero-phosphate, introduced subcutaneously. In the majority of cases, after the introduction of the other salts, both per os and sub- 41 cutaneously, there was only a temporary retention of calcium (' Scheinretention '), for during the period following introduction the calcium balance was almost always negative, and the calcium which had been retained was voided. There was no subsequent excretion of the retained calcium after a subcutaneous injection of calcium glycero-phosphate. In the case of one five-months-old rickety infant with a slightly positive balance, the subcutaneous injection of calcium chloride and simultaneous administration of sodium glycero-phosphate per os had a positive effect. Similarly, when the same patient had a subcutaneous injec- tion of calcium glycero-phosphate, there was a marked improvement in the balance of calcium (from 0-09 gm. to 0-34 gm. per diem). In the case of rickety children with a highly positive balance, the introduction of calcium produced no perceptible results. As an example of Grosser's excellent work, we may take the following experiment on a rickety child of 7 months. The diet was sterilized human milk in a feeding-bottle. The experiment lasted 64 days, and was divided into sixteen periods of 4 days each. Table No. 13. Average introduction Average retention riods. Remarks. of CaO per diem. 0/ CaO per diem. gm. gm. 1 Normal 0.234 0-011 2 Injection 4.7 gm. Ca glyc. phos- phorioum - 0.471 0-240 3 Normal 0.257 0-008 i Normal 0.280 0-031 5 Injection 2 gm. CaClj 0.418 0-105 6 Normal 0.263 -0.072 7 5-75 gm. Ca glyc. phosph. per os. 0.511 0.118 8 Normal 0.246 -0-002 9 3 gm. calc. acet. per os. 0-523 0-135 10 Normal 0.224 0-035 H Normal 0.248 0-019 12 Normal 0.284 0-027 13 Injection 2-5 gm. calc. glyc. phosph. 0-372 0-153 14 Injection 3 gm. calc. glyc, phosph. 0-359 0-249 15 1 Normal 0-237 0-02 In one observation made by Schloss (1914) on a 2^-months-old rickety infant fed on human milk, and with a moderate positive balance of calcium, no change in calcium metabolism was observed after the introduction of l-S gm. calcium acetate per os. Summarizing the results of all the above-mentioned experiments, it must be acknowledged that the subcutaneous injection of certain calcium salts may have a favourable effect on calcium metabolism in rickets. After Grosser's experiments, the sceptical attitude towards the calcium treatment of rickets must give way to further investi- gation and the discovery of the most easily assimilated combinations of calcium for subcutaneous injection. Moreover, it is apparently necessary for successful action of the calcium compound that it should include the phosphoric acid group. At the same time, these experiments to a certain degree may throw doubt on the statement that one of the essential distinctions between rickets and calcium 42 starvation is that in the former the introduction of calcium salts has no effect on the disease, whereas in calcium starvation it is invariably successful. Attempts to produce a more powerful effect on calcium meta- bolism in rickets by the introduction of calcium salts together with cod-liver oil have not been very numerous, but have been made by Schabad (1910, 7, 8), Schabad and Soroohowitch (1911), Schloss (1914), Grosser, and Brown, McLachlan, and Simpson (1920). The only convincing experiment was one made by Schloss, in which the separate introduction of calcium acetate or cod-liver oil had no effect whatever on the metabolism, whereas the simultaneous intro- duction of these preparations considerably increased the retention of calcium by the organism. Grosser's experiment yielded negative results. Conclusions. 1. The view that the introduction of calcium salts has no effect on the metabolism of calcium in rickety children must now be reconsidered in consequence of recent investigations on calcium metabolism in men and animals. 2. Apparently certain calcium salts, when introduced in a parti- cular manner, e. g. calcium glycero-phosphate subcutaneously, may have a favourable effect on the metabolism of calcium in rickets. 8. Effects of Cod-Liver Oil and Elementary Phosphorus ON Calcium Metabolism. The effects of both these substances have to be dealt with at one and the same time, as in the majority of cases their action has been investigated simultaneously. The fii-st accurately conducted experi- ments on the metabolism of calcium, which showed the advantage of employing cod-liver oil in rickets, were undoubtedly Schabad's (1909, 1910, 3). That author investigated the action of an emulsion of phosphorus in cod-liver oil, and ascribes the favourable effects of his first experiment to the phosphorus. Schabad's later experiments, however, showed him that the principal effects were due to the cod-liver • oil. The addition of phosphorus merely increased the action of the cod-liver oil. In Schabad's experiments, phosphorus alpne had no very perceptible effect on calcium metabolism. Cod-liver oil had no effect on calcium metabolism in healthy children while sharply increasing its retention in rickety children. The following observa- tion of Schabad (1910, 3) on Timofeiev, a rachitic 'subject, shows the effects of cod-liver oil : Treatment. Pm-centage of ingested CaO retained m organism. No treatment 4.79 Phosphorus +0-1 olive oil lO.ll Sesame oil, 2 teaspoonf uls 0.97 Cod-liver oil, 2 teaspoonfuls 43.35 Cod-liver oil, 2 teaspoonfuls + phosphorus 72.49. Schabad also observed that the cod-liver oil and phosphorus treatment in rickets not only improved calcium metabolism, but 43 \ likewise had a favourable eflect on the absorption of nitrogen and fat, and the retention of phosphorus in the organism. Birk (1908), Meyer (1913), Holt, Courtney, and Fales corroborated the conclusions drawn by Schabad concerning the favourable effects of cod-liver oil. Moreover, Schloss and Franck (1914) found, in three cases of rickets, that cod-liver oil had a cumulative action. For instance, in the case of Nobbe, a rachitic child, CaO intake was constant during the whole period of the experiment (19 days). Before the administration of cod-liver oil the balance was negative (— 0-014 gm.); during treatment, in the course of the following 14 days (divided into three periods) the balance gradually in- creased : + 0-143 gm., + 0-238 gm., + 0-519 gm. The experiment made by these authors failed to show any special advantage from the addition of -phosphorus to the cod-liver oil. In observations on two rickety subjects fed on human milk Schloss did not discover any favourable effect of cod-liver oil. Brown, McLachlan, and Simpson (1920), in 14 cases of rickets accompanied by tetany, found a low value in the calcium content in the blood. The administration of cod-liver oil plus phosphorus increased the amount of calcium in the blood in 14 days, e.g. from 6-5-7-5 mgm. per 100 c.cm. of whole blood before treatment, to 8-9-7 mgm. after treatment. The simultaneous introduction of calcium lactate had merely a slight accelerating effect, without any independent action. Howland and Kramer (1921) investigated 13 rachitic cases for calcium and phosphorus content in the blood before and after treat- ment with cod-liver oil, but found no marked effect of the latter on the calcium content. On the other hand, the inorganic phosphorus ' content, reduced in rickets, was greatly increased after the treatment, the average rise being from 1-9 mgm. per 100 c.cm. of serum before treatment to 5-5 mgm. after treatment. Lehman (1921) could not find any influence of cod-liver oil on the calcium content of the blood of healthy rabbits. In juvenile and adult osteomalacia cod-liver oil produces the same favourable effect on calcium metabolism as it does in rickets (Sauerbruch, His, Hotz). Conclusions. 1. Cod-liver oil causes a marked increase in the retention of calcium and phosphorus in the organism of rickety children, and may cure rickets and osteomalacia. 3. The addition of phosphorus to cod-liver oil apparently improved the favourable action of the latter in some cases. 9. The Influestce of Diet on Calcium Metabolism. The effects of both human and cow's milk have already been mentioned. Kochmann and Petzsch (1911) caused a considerable loss of calcium from the organism of healthy dogs in previous calcium equilibrium by adding fats, carbohydrates, or albumin to their previous diet. The authors ascribe this not so much to the altered conditions of digestion as to the action of the mechanism which neutralizes the poisonous metabolic products produced by over- feeding. 44 Kothberg (1907), Steinitz (1903), Orgler and Meyer (1908) observed a reduced retention of calcium in children, following increased in- take of fats. The majority of the authors explain this action of fats by the increased formation of calcium soaps in the intestines. As has already been mentioned, cod-liver oil does not act in this way (Schabad (1910, 3)). Telfer (1931, 1), however, could find no diflFerence in the absorption and retention of calcium after the addition of 6 gm. of butter to a milk diet of a healthy eight-months-old child. Neither did he observe in the same child any particular difference between butter and cod-liver oil as regards any effect on the retention of calcium.^ It was only in cases of a marked disturbance in the absorption of fat from the intestines (e. g. when the flow of bile into the intestines is retarded) that Telfer (1921, 2) found an abnormally large quantity of calcium in the excrement. Schlesinger's (1904) experiments on dogs, of removing the pancreas or ligaturing the bile duct, showed that, together with the marked decrease in the absorption of fat observable in such cases, calcium is excreted from the organism through the intestines in increasing quantities. At the same time, the amount of calcium in the urine decreases. The diminished absorption of calcium is. likewise ex- plained by Schlesinger as being caused by the increased formation of calcium soaps : e. g. before the operation one of the dogs excreted 0-69 gm. of such soaps, whereas after the operation the latter was increased to 3'24 gm. Rothberg observed in children that an increase of carbohydrates in the food may likewise cause a decreased retention of calcium in the organism, but the effect produced by carbohydrates is less than that produced by fats. Freund (1909) observed that malt extract improved the retention of calcium in rickets, and Mellanby (1919), on puppies with experi- mental rickets, confirmed the usefulness of malt extract. Dubois and Stolte (1913) managed to improve the calcium balance in the case of three children by the addition of alkali to their food. These authors explain the action of the alkali in terms of a test-tube experi- ment : an emulsion of calcium palmitate in water at once begins to froth on the addition of several drops of alkali, and part of the insoluble calcium soap is converted into soluble alkali soap. The authors then think that the addition of alkali dissolves insoluble calcium soaps already formed in the intestine and prevents their excretion. Seemann (1879) and Zander (1881) drew attention to the impoi-tant part played by sodium chloride in the absorption of calcium salts by the organism. In their investigations, they started from the experiments of Bunge (1873), who proved that the organism becomes deficient in chloride of sodium when there is a surplus intake of potassium salts with the food. As, according to Bunge, the most usual food, especially among the poor, contains a super- abundance of potassium salts, this fact explains the actually in- dispensable addition of common salt to human food. ^ Schabad also showed no effect from administration of cod-liver oil to healthy children. 45 Seemann thinks that the impoverishment of the blood in chlorides, from the causes indicated by Bunge, leads to the insufficient forma- tion of hydrochloric acid in the stomach. An insufficient quantity of this acid prevents the transformation of the insoluble calcium salts in the food into soluble calcium chloride. That is why the absorption of calcium is disturbed. In connexion with this, See- mann explains the frequency of rickets among the poorer classes, who feed principally on vegetable food, rich in potassium. See- mann's own experience showed him that the enrichment of the food in common salt or the introduction of hydrochloric acid alleviates rickets. The conclusion drawn by Seemann is very categorical (p. 312) : ' The impoverishment of rickety bones in calcium salts is the result of a specific digestive disturbance, consisting in the insufficient formation of hydrochloric acid in the stomach.' Zander is in complete agreement with Seemann's theory, on the basis of the analyses of the milk of the mothers of rickety children and those of normal children. The results of such analyses are as follows : Ratio of Ratio q/ Na:K a:P Normal milk 1:2.5 1:1-2 ' Rachitic ' milk 1 : 4-4-5 1 : 3-5-4-5 The amount of potassium and phosphoric acid in the milk of ■ women suckling rickety infants thus turns out to be greater than in normal milk. This must lead to an increased loss of sodium and chlorine, followed by the decreased foi-mation of HCl in the stomach of the rickety infant and the decreased absorption of calcium from the intestine. On the basis of indirect investigations, Zweifel (1900) accepts this theory. Some corroboration of the view held by Seemann and Zander may be found in the experiments of Allers and Bondi (1907). These authors increased the calcium content of the blood of rabbits by approximately 100 per cent, by introducing hydrochloric acid into the stomach. At the same time all the other bases of the blood were increased only by 11 per cent. This theory is contradicted by the experiment of Hunaeus (1909), who administered 1 gm. hydrochloric acid per diefn to a nursing mother and failed to obtain any increase in the calcium content of her milk. Dibbelt (1910) obtained a negative balance in the case of a dog, which was in a state ofapproximate equilibrium as regards calcium, by the addition of common salt to its food. Denis, Sisson, and Aldrich (1923) failed to obtain an increase in the calcium con- tent of milk by the introduction of calcium chloride, either per os or intravenously, in the case of she-goats. Thus, though Seemann's theory is interesting, as yet there are no indisputable data in favour of its importance in the aetiology of rickets. Conclusions. The action of fats (except cod-hver oil) on the retention of calcium in the organism, as also of alkalis, common salt, and hydrochloric acid, has not as yet been sufficiently investigated. 46 Apparently the excessive introduction of fats (except cod-liver oil) in the food or a marked disturbance in the absorption of fats from the intestine causes a decrease in the retention of calcium in the organism. 10. The Minimum Demand for Calcium by the Growing Organism, and the Possibility of Calcium Starvation IN Rickets. The facts collected in the preceding chapters clearly show the impoverishment in calcium salts of the rachitic skeleton as well as the abnormally low retention, or even loss, of calcium from the organism in the early stages of rickets. Some cause or other, according to Pommer and Schmorl, prevents the deposition of calcium in the bone formed during the disease. Probably this cause accounts for the abnormal calcium metabolism. Theoretically, the following possibilities may be brought forward in explanation : 1. Calcium starvation is developed in the organism owing to insufficient amount of lime salts introduced with the food ; 2. A sufficiency of calcium salts is introduced, but they are absorbed from the alimentary canal in insufficient quantities, i. e. there is a secondary calcium starvation ; 3. The bones lose their capacity for taking the lime salts from the blood and depositing them even when there is a normal intake and absorption of calcium ; 4- The bones lose their capacity for firmly retaining calcium salts, in consequence of which the latter are thi'own out into the blood, and the osseous tissue is transformed into osteoid tissue (hali- steresis, decalcification). In considering the first and second possibilities, i. e. of calcium starvation in children, it is necessary to determine what is the usual calcium intake of the child at the age most susceptible to rickets, and what is the minimum amount of calcium which it requires. As at this age the child feeds on milk, the first question is, what is the calcium content of milk, both human and cow's ? The assimilation of calcium from both these species of milk has already been dealt with above. (a) Calcium, Content of Huvian Milk. At the present time there are on record 88 analyses of the milk of mothers of children whose skeletons have been found normal by clinical examination, and 36 analyses of the milk of mothers of rickety children (Friedleben (1860), Bunge (1874), Seemann (1879), E. Voit (1880), Zander (1881), Pfeiflfer (1885), Babeau (1898), Zweifel (1900), Blauberg (1900), Soldner (1903), Dibbelt (1908, 1909, 1910, 3), Hunaeus (1909), Mauro-Greco (1909), Schabad (1910, 47 5), Bahrdt and Edelstein (1910)). The work of the last-named authors and Schabad contains a table summarizing the 88 analyses of ' normal ' human milk at all periods of lactation. Of this number, only two analyses (Volt's, Bahrdt and Edelstein's) yielded very high figures, i. e. 0-08 per cent, of CaO, and 0-0707 per cent. The other analyses have been tabulated by me as follows (Table No. 13) : Table No. 13. Oalciv/m Content of Milk from Mothers of Healthy Children. Percentage of CaO in Milk. Number of cases. 0-063-0.064 2 0.051-0-058 7 0.045-0.049 16 0-040-0.044 24 0-034-0.039 22 0-030-0.033 13 0.029 2 It is first of all necessary to ma.ke some observations with regard to the above figures. Bahrdt and Edelstein estimated the calcium content of milk in the form of CaSO^ by Aron's (1910) method. This method yields good results only when calcium, salts are practically alone in the ash (e.g. as in the bones). With a com- paratively considerable content of other salts in the ash, as is the case in milk, by Aron's method there is always the possibility of a varying quantity of other than calcium salts being precipitated* This, apparently, must be the explanation of the fluctuations in calcium content of the various portions of milk taken during short periods, observed by Bahrdt and Edelstein. Similarly, the same causes probably account for the figures in their analyses being above the actual. The special object of Mauro-Greco's work was to determine the relation between various quantities of calcium in the mothei"'s milk and the blood formation of the child. There- fore he did not pay sufficient attention to the condition of the skeleton. For this reason all the figures of milk analyses where Mauro-Greco indicates anaemia in the child or mother, have been placed by Bahrdt and Edelstein in a separate class, containing 13 cases in all, with fluctuations of CaO from 0-027 to 0-0103 per cent., the average being 0-0153 gin. CaO per 100 c.cm. of milk (a strikingly low calcium content). Taking all the above-mentioned stipulations into consideration, it is probable, on the basis of the above table, that the most frequent calcium content of milk is from 0-030 to 0-049 per cent. Bahrdt and Edelstein, taking account of literally all the analyses, estimate the average calcium content in the milk of mothers of rickety children at 0-0426 per cent. CaO. Schabad in his calculations uses the extreme figures observed by him, i. e. from 0-036 per cent, to 0-047 per cent. The analyses of the milk of mothers of rickety children are far less numerous. I have summarized in the following table results compiled principally by Schabad and Bahrdt-Edelstein (Table No. 14). 48 Table No. 14. Calcium Content of Milk from Mothers of Rcwhitic Children. Percentage of CaO in milk. Number of cases. 0.040-0.046 3 0.034-0.038 6 0.030-0.083 5 0.025-0.029 8 0.019-0.023 4 Average 0.0315 CaO % 26 In ' rachitic ' human milk the' amount of calcium is undoubtedly less than in normal milk, and the largest group shows a calcium content of 0-035 to 0-029 per cent. This decline in the calcium content in the milk of mothers of rickety infants is of a con- stant character. For instance, in one case Schabad repeated his investigation five times in three months, and found from 0-0209 to 0-0269 per cent. CaO. Taking into consideration the analyses of the milk of mothers of anaemic children (very probably suffering from rickets likewise) in Mauro-Greco's cases, with an _ average content of 0-0153 per cent. CaO, the question of the frequency of an abnormally low calcium content in human milk, and especially in the milk of mothers of rickety children, will undoubtedly be answered positively. The amount of ash in the milk during the first months of lactation is larger than during the last months (Pfeiffer, Camerer, and Soldner). The same fact was observed by Hunaeus with regard to calcium, though this is not apparent from the analyses of Pfeiffer, Soldner, and Schabad. Bahrdt and Edelstein calculated the average figures of the above-mentioned 88 analyses of the milk of mothers of non-rachitic children at various periods of lactation, and obtained the following results : 1-3 month of lactation . . . 0-044 per cent. 4-6 ,, „ ... 0-036 per cent. After 6th month 0-040 per cent. A considerable number of the cases were examined only in the first and second periods. That is to say, apparently in the later months of lactation the amount of calcium in the milk is somewhat decreased, which of course might be a factor in the origination of calcium starvation in the period in question. Dibbelt, Hunaeus, as well as Bahrdt and Edelstein, added calcium salts to the food of nursing mothers for the purpose of determining the possibility of thus increasing the calcium content in their milk. Dibbelt (1910, 3) obtained positive results : in one case the calcium content rose from 0-058 per cent, to 0-085 per cent., and in another from 0-047 per cent, to 0-060 per cent. Bahrdt and Edelstein, and Hunaeus could not discover any marked enrichment of the milk in calcium aiter the introduction of an increased amount of CaO into the food. Investigations were likewise made upon the effect on the calcium content in the milk of animals, e. g. cows, of the introduction of calcium salt into the food. Weiske (1871) and Jensen (1904) were 49 unable to obtain constant and marked results in this respect. Schulte-Bauminghaus, Neumann (1893), Wendt and Muller (1908), and Wendt (1909) sometimes observed an increase of calcium in tlie milk on the introduction of certain lime salts. For instance, in the last-named author's experiment this was obtained with diacid calcium phosphate, but not with calcium carbonate or glycero- phosphate. Pfeiffer drew attention to another abnormality in the milk of mothers of rickety children, namely, the low phosphoric acid content. He examined the milk of three mothers of rickety children for phosphoric acid, and found in ash of the milk : 15-6 per cent., 24-99 per cent., and 18'26 per cent, (the average 19-6 per cent.) of phosphoric acid ; the ash of the milk of three mothers of normal children yielded the following: 32'64 per cent., 27-55 per cent., and 23-77 per cent., the average being 24-7 per cent. The author points out that when the milk is poor in phosphorus even an excess of calcium cannot be utilized by the organism. (b) The Possibility of Calcium Starvation in Rickets. Breast-fed infants. In Chapter 3 it was pointed out that in genera], for every 100 gm. of increase in the child's weight, according to Aron or Schabad about 1-25 gm. CaO should be deposited. Aron even admits that 1-0 gm. CaO may be sufficient. In my calculations for Tables Nos. 15 and 16,1 take the minimum reqixire- ment at a figure based on Sijldner's analyses, viz. 1-0 gm. CaO. Knowing the daily increase in the child's weight, the calcium content of the milk, and the intake of calcium, it is possible to make an approximate comparison between the actual retention of calcium from the milk and the theoretically calculated demand.^ In my calculations I have taken the CaO content in human milk at 0-043 per cent., and the calcium retention from human milk at 70 per cent. (Schabad). Although the figures used by me in my calculations are somewhat different from those of Aron and Schabad, on the whole I have been obliged to come to more or less the same conclusions, namely : (1) During the first year the calcium requirement is greater than in succeeding years ; (2) During the first year the greatest calcium requirement is in the first six months ; (3) Even taking Bahrdt and Edelstein's high average for the calcium content of milk (0-043 per cent.), as also Schabad's high calcium retention fi'om human milk at 70 per cent, (the average being 59 per cent., see Table No. 6), during the first two months the demand for calcium is not covered by intake. From the third month the ' These calculations were made by Schabad (1910, 5), Ai-on (1908), and Dibbelfc (1910, 3). In view of the criticism of their high figures in calculating the require- ment, and somewhat low ones for the calcium content of milk (Aron), I have drawn up the Tables Nos. 15 and 16 in accordance with the latest observations and critical remarks of different authors. (1579) D 60 11 -^^ = Q "? "=? O O T-H o o o + + + I I I I Cm «5 s o = :* '^ C^ t> O '^l M m 05 -* i 6 6 o o I I O !2i m 3 HO Op 5- 111? s h "S S -s ^^ .15 O o o o o o O o U3 ^ W O O X) 00 «) 05 o s« ss. 00 CO O o o o -eH CO Cq O O O o o o d N S :2 9 9 *? 9 li s O « 1^ S s .6 ■» -^ SO s'« S 'H^«o-^THGO»ai£icoos(Mdd odddd-OodddoSS d d W.2 o'S ^ s CO lO ^^ o o o o "2" 2 t s C3 o § o o d o QO l> "s o o ^ St. B O M 0.5 > *■ £>■ inT o o o o o "i. o cT cq" 00 T-( r-f 00~ i i o o lO o oT oq- eq ffl o Ml 00 (M 00 o!) ci .i 4 oi ^ T-H cq ©q cq CO CO - C8 • ffq ^ cq CO ill .2 3 eq 51 balance is established, and then there is an excess over tlfe require- ment ; (4) But, as the figures in Table No. 13 show, tlie calcium content of the milk of mothers of normal children is frequently below the figure 0-043, and in rachitic milk this is almost invariably the case. Moreover, the retention of calcium from human milk is always less in rickety children, and is very often much less than 70 per cent, (see Table No. 6). Therefore the possibility of calcium starvation in the case of children fed on human milk, and especially of rickety children, is undoubtedly a real one ; for such low calcium contents as 0-019 and 0-033 per cent, in the milk of mothers of rickety children have been recorded. As early as 1885 Pfeiffer expressed the opinion that the mothers of rickety children should be advised not to nurse them. If we take several of the cases used by Aron, and amend the figures of CaO introduced in accordance with the average calcium content of milk according to Bahrdt and Edelstein (0-043 per cent.), we get the following results (Table No. 16) : Table No . 16. CaO Cmisump- Intake 70% Yptention required Child Age Increase in weight {gm.). iion of o/CaO (1 % of \/ftlLlbt (weeks). milk with milk 1 ObL" ULLKJ tU {gm.). increase in From. To. Increase. {kgm.). {gm.). weight) {gm.)^ Haehner I 1-22 3,039 6,670 3,631 112.6 48-4 33-9 36-8 Haehner II 1-10 2,880 5,045 2,165 43.6 18.8 13.2 21-7 Kleiber . 1-10 2,870 4,870 2,000 45.5 19.6 13-7 20 Laui-e 3-9 4,140 5,920 1,780 46.0 19-8 13-9 17.8 Maohill I 2-13 ( 14-27 3,470 5,640 2,170 53.6 23-1 16.2 21.7 5,640 7,350 1,740 68-5 29-5 20-7 17-4 That is to say, only in one case, in Machill's infant observed between the 14th and 27th weeks, did the retention of calcium exceed the requirement. In all the other cases the calcium retention was below the theoretically calculated requirement. The calcium requirement was approximately satisfied in the case of Haehner I. An average child with an average appetite, according to Czernj'- Keller, quoted from Aron (pp. 56-7), will drink about 144-9 kgm. of milk during the time its weight increases from 3,000 gm. to 8,346 gm. Dui-ing this period the child will therefore require to deposit about 53-5 gm. CaO. For this purpose the milk should contain about 0-053 per cent. CaO, with 70 per cent, of retention — figures that are above the average ! Apparently matters are better as regards a diet of cow's milk at the same age, for whole milk freely covers the calcium requirement of the child, but this is not always the case with diluted milk. According to Heubner, during the second and third month of its life the child should be given cow's milk equally diluted with water, to the approximate amount of 900 ccm. per diem,. The calcium content of whole milk at 0-158 per cent,, 900 ccm. of 'half-milk' will contain 6-71 gm. CaO, and assuming a calcium retention of 30 per cent. (i. e. slightly above the average), this works out at D 2 52 about 0'2r gm. of retained CaO. The calcium requiremeDt at the age above mentioned ia from 0-26 to 0-29 gm. CaO, which might, however, be covered by a quite possible retention, e. g. 40 per cent. (0-28 gm. CaO). Aron indicates some other factors which may conduce to calcium starvation. The quantity of milk consumed by a child depends on its state of nutrition. Therefore the normal calcium content in milk rich in organic matter may prove insufl5cient for the child, as the latter will consume a smaller quantity of such milk. Having enumerated the figures of PfeifFer's analyses, Aron found that the milk of mothers of rickety children contained less ash and calcium per 100 calories than the milk of mothers of normal children. Finally, Aron points out that over-feeding, by causing excessive growth, may make the normal amount of calcium in the milk insufficient for the rapid and abundant new formation of osseous tissue. Aron and Sebauer (1908), in their experiments on animals, observed this phenomenon in over-feeding the latter with food containing calcium to an amount corresponding to the minimum requirement. Esser (1907) — from clinical experience of increased leucocy tosis of the blood found both in rickets and over- feeding and also on the basis of certain experiments — ascribes great importance to over-feeding in the aetiology of rickets. Unfortunately, it is difficult to judge of these experiments, owing to the absence of a detailed report thereon. In concluding the present chapter, it is necessary to point out the possible coincidence, during the first six months of life, of calcium starvation and the beginning of the development of rickets at the same age. Further, Schabad (1910, 4, 5) points out the following coincidences : (1) He has found a decrease in the calcium content of the bones of a child towards the end of the first six months of life, as compared with that of a new-born infant ; (2) Friedleben (1860) observed in children of this age a physio- logical intensification of the processes of resorption in the back of the cranium (osteoporosis) ; (3) Schwalbe (1877) found physiological osteoporosis of the long bones in normal children from this age up to the end of the second year. A comparison of the above-mentioned facts with thepseudo-rachitic osteoporosis found in animals suffering from calcium starvation (see below) necessarily leads to the conclusion that conditions may arise —principally during the first six months of a child's life — under which calcium starvation is developed in a more or less severe form. Whether the deficiency in calcium is merely an incidental pheno- menon accompanying rickets, or whether it predisposes to the latter disease, or, perhaps, even causes it, is a question which the clinician has not been able to answer categorically. Clinical observations have only been able to establish the fact that rickets apparently cannot be cured by the mere introduction of calcium salts into the organism of the patient. Nevertheless, this may merely indicate that the aetiology of rickets does not consist exclusively of calcium starvation. In that case, it is quite conceivable that the deficiency of calcium, combined 53 with some other cause or causes inducing rickets, may play a very important part in the pathogenesis of rickets. Attempts have been made to solve this problem definitely by means of experiments on animals. Conclusions. 1. Normal human breast milk contains no excess above the minimum amount of calcium which the child requires during the first few months of life. 2. The milk from mothers of rickety children seems to be poorer in calcium than that of mothers of normal children, especially if the percentage is calculated with relation to caloric content of the milk. 3. A normal amount of calcium in the milk may prove insufficient for the child if its growth is abnormally great on account of over- feeding. 4. Cow's milk contains a greater amount of calcium than human, but dilution of the milk and the often-observed bad absorption of calcium from it combine to make calcium starvation possible when feeding with cow's milk. 5. Digestive disturbances may also affect the absorption of calcium. 6. Therefore calcium starvation may play an important r61e in the aetiology of human rickets or of a special form of this disease. 11. Influence on the Skeleton of Animals of Calcium Staevation, either alone ce in combination with an INTAKE OF STEONTIUM. The question of the effects of calcium starvation on the skeleton has been experimentally investigated from two points of view : the histological and the chemical. Closely connected with the question of calcium starvation are the experiments made to ascertain the effects of strontium on the skeleton, first because they were nearly always on animals fed on a diet deficient in calcium, and secondly because the results of such experiments explain much in the patho- genesis of skeletal changes in calcium starvation. (a) Histological Changes of the Skeleton. Of the investigations carried on in this, direction it is necessary to dwell chiefly on those made after the publication of Pommer's work. All previous work is of little value, because at that time nothing was known of the importance of osteoid tissue in diagnosing rickets. Therefore I shall only mention the pre-Pommer period works that are useful in the light of present-day knowledge. In 1842 Chossat, by feeding pigeons on a diet poor in calcium (grains of wheat), produced a most severe form of bone fragility as a result of osteoporosis. The pigeons died in a state of constant diarrhoea. Chossat drew attention to this new form of diarrhoea as 54 'diarrh^e par insuffisance des principes calcaires'. Hitherto too little attention has been paid to this form of gastric disorder, which has been mentioned by all authors who have experimented with a diet deficient in calcium. The experiments of Weiske and Wildt (1873), Edwards (1861), Roloff (1866-79), E. Voit (1880), Baginsky (1882), and Chedle were mainly on various animals (pigeons, dog's; pigs, sheep, &c.). On the basis of these experiments in feeding animals on. a diet deficient in calcium, the majority of these authors thought that they had produced rickets. It is true that, macro- scopically, in many cases the changes in the bones were completely similar to those in rickets : softness and bending, spontaneous fractures, and thickening of the epiphyses. Microscopically, the changes in the cartilage described by Voit corresponded to those in rickets. In Volt's experiments the puppies were fed on fresh meat, hog's lard, and distilled water, while the control animals had bone ash added to their food. Baginsky's histological investigations, in which attention was paid likewise to the deposition of calcium in the zone of provisional calcification and in the osteoid tissue, have not been described vfiih sufficient fullness. Nevertheless the histo- logical picture described by that author corresponds, more or less, to rickets, both as regards the great disorganization of the pro- liferous cartilage and the deficiency or absence of calcium in the zone of provisional calcification, as well as in the trabeculae spongiosae (osteoid tissue). Baginsky gaye his puppies boiled meat, hog's lard, and distilled water, i. e. diet poor also in vitamins. One of the puppies had, in addition to this, 2 gm. of lactic acid, while the control puppy had 2 gm. of calcium phosphate. Stilling and Mehring (1889) fed a pregnant bitch on a daily ration of 600 gm. of boiled horseflesh, 40 gm. of melted fat (not stated of what kind), and distilled water. The bitch had a litter of six pups, of which one was killed immediately alter birth, and the rest soon died of weakness, and autopsy showed no macroscopical indications of any rachitic changes. The bitch was killed on the 126th day after the beginning of feeding. Autopsy showed a softening of the bones of the spine and the pelvis, and microscopic examination showed both osteoporosis and a great quantity of osteoid tissue. The trabeculae were covered with well-defined osteoblasts. The disease was localized principally in the bones of the pelvis and spine, as is the case in osteomalacia, and the authors took it for the latter disease. Unfortunately, the histological changes are only described very shortly. In puppies fed on a diet deficient in calcium, Korsakoff (1883) found macroscopically changes in the bones characteristic of rickets, but microscopically osteoporosis with thin layers of osteoid tissue on the trabeculae. The changes in the epiphyseal cartilages were those of typical early rickets. Korsakoff finds that the changes obtained would have been exactly similar to rickets, if the amount of osteoid tissue and the changes in cartilage had not been so small. The addition of strontium to the above diet deficient in calcium produced changes very similar to rickets. In connexion with this, the author admits the possibility of the existence of a rickets- producing irritant. 55 Cremer (1891) also said that he produced rickets in dog.j when fed on a diet poor in calcium, whether it contained strontium or not. Troitzky (1897) fed puppies, a young pig, a kid, and guinea-pigs on a diet deficient in calcium. The puppies and pig were fed principally on potatoes, the kid having a small quantity of hay and grass in addition. Cultures of intestinal bacteria were added to the food of the guinea-pigs. Clinically some of the animals showed changes similar to "rickets, but microscopically thei-e was not the slightest indication of even a mild form of rickets. Miwa and Stoeltzner (1898) experimented on a puppy fed on fresh horseflesh, hog's lard, and distilled water. The puppy's gi'owth was satisfactory, and in about a month it developed rachitic symptoms in the bones. It was killed about two months after the dieting was begun. The authors' histological methods were un- questionably good and they gave descriptions of the changes produced. Microscopic examination showed a thickening and disorganization of the proliferous cartilage, a normal deposition of calcium in the zone of provisional calcification, osteoporosis of the bone, thin layers of osteoid tissue on the trabeculae, observed physiologically in rapidly growing puppies of the larger breeds, and thickening of the peri- osteum. This picture they termed pseudo-rachitic osteoporosis, and consider the following features as characteristic of the malady : (1) Changes similar to rickets in the proliferous cartilage ; (2) but with a normal deposition of calcium in the zone of provisional calcification ; (3) Osteoporosis (even the coi'ticalis consisting of a coarse reticula- tion of trabeculae). The authors ascribe this osteopoi-osis to the' insufficient formation of new bone ; (4) Very narrow borders of osteoid ; (5) Periosteal overgrowth. In true rickets, as distinct from this pseudo-rickets (according to the majority of authors), osteoid tissue covers the bone in thick layers ; in severe cases the osteoid tissue is the material of which the bulk of the trabeculae are formed; there is an absence of calcium in the zone of provisional calcification, and a development of periosteal osteophytes ; finally, the osteoporosis is not so severe. Aron and Sebauer (1908) experimented on twelve puppies. The diet deficient in calcium consisted of horseflesh, maize, lard, potassium chloride. The control puppies were fed on the same diet, supplemented by 3 gm. of bone ash. Macroscopically, the resulting picture was very similar to rickets. Getting (1909) microscopically examined the bones of the dogs experimented on by Aron and Sebauer. He found osteoporosis, extension of the zone of proliferous cartilage, an almost total absence of deposition of calcium in the zone of provisional calcification, and a broader layer of osteoid than is normally the case. Nevertheless, nowhere was the latter so broad as is tound in rickets even of moderate severity. The osteoporosis was due to increased resorption of bone by very numerous osteoclasts. The compacta of the bone consisted of a network of rarified trabeculae. The osteoblasts were likewise well defined in many places : they formed one or more laj'^ers on one side of the trabeculae, forming osteoid tissue, while 56 the other lime-impregnated side of the trabeculae was eroded by numerous osteoclasts. Getting sees the following differences between this picture and rickets : Whereas in rickets the deficiency of lime in the bone is due to lack of calcium, deposition in the newly formed bone, in the case of diet deficient in lime this decrease of the calcium content of the bone depends oil the increased resorption of the bone con- taining lime. It is only in some places that* abnormal, though slight, extension of the layers of osteoid tissue indicates that the calcification of the bone has been retarded. The author explains the almost normal deposition of calcium in the bone on a diet deficient in calcium by the auto-regulation of the organism, which takes lime from the parts where it is least wanted, and deposits it where it is most needed. The necessary lime is extracted from the skeleton by the action of osteoclasts. Dibbelt (1-6) experimented on puppies and adult dogs, and like- wise investigated the effects of calcium starvation on the skeleton of bitches during pregnancy and lactation and the skeletons of the puppies they had suckled. In his experiments the diet consisted chiefly of meat, with the addition either of lard or 'fat',^ carbo- hydrates, or rice, with addition of common salt. The control animals received calcium phosphate in addition. The resulting histological changes were not the same in all Dibbelt's animals. These changes were most like rickets in the animals fed on flesh and carbohydrates, and least like in those fed on flesh and fat. Dibbelt explains the various degrees of change by the difference in diet, and considers the addition of fat to the meat formed the least suitable diet for the production of rickets, the addition of carbo- hydrates giving the most suitable combination. Lehnerdt, criticizing the possibility of diet having any eft'ect, points to the difference in the age of the dogs and in the experimental periods as an explanation ' of the results obtained. Admitting that there is some justice in Lehnerdt's objections, yet, on the basis of the modern teaching on vitamins, one must of course take Dibbelt's point of view, since fat can always contain a certain amount of antirachitic factor. The change in the skeleton more commonly met with in all the dogs was osteoporosis caused by the increased activity of osteoclasts. The changes were especially marked in dogs fed on meat and carbo- hydrates, and ended in the extension and disorganization of the zone of proliferous cartilage, penetration of the blood-vessels into the car- tilage, insufiicient calcification of the zone of provisional calcification, and enlargement of the cartilage canals, a considerable amount of osteoid tissue, well-developed osteoblasts accompanied by numerous osteoclasts,newformations of periosteal osteophytes, and fibro-ceUular transformation of the bone marrow. Schmorl, who himself investi- gated one of the bones of Dibbelt's dogs, regards the resulting picture as pseudo-rickets, because (1) the processes of the new formation and resorption of bone were very marked, which (according to Schmorl) is not the case in human rickets ; (2) the osteoporosis was strongly ' Being unable to obtain Dibbelt's fundamental work, I have quoted it from Lehnerdt's (1910). It is not clear what 'fat' Dibbelt xised, but at any rate it was pot hog's lard (except when specially indicated). 57 indicated, and (3) the presence of osteoid tissue was regarded as ' incidental ' (?). Osteoporosis and an increased amount of osteoid tissue were found in the skeleton of a bitch fed on a diet deficient in calcium during pregnancy and lactation. Uibbelt calculated that during pregnancy the bitch ga,ve up about 4- 23 gm. CaO out of her skeleton for the calcification of skeletons of the foetus, owing to which the skeletons of the four new-born puppies were normal. The two puppies left alive were very backward in their development ; one had calcium phosphate administered to it towards the end of the experiment. Owing to this the puppy recovered, and the skeleton was noi-mal. The skeleton of the other showed the usual changes observed in calcium starvation, without any increase in the amount of osteoid tissue, but with a great development of fibrous tissue (' hypoplasia of the skeleton '). Dibbelt, who at first regarded the changes obtained in some cases as rickets, subsequently was more careful in characterizing these changes, recognizing that there were some diflTerences in the histological picture. Dibbelt also points to the difference between the growth and development of a child and that of a puppy; the former doubles its weight in 180 days, and the latter in 9 days, and in accordance with this all the processes of resorption and new formation of bone must be far more rapid in the case of the puppy. McCollum, Simmonds, Shipley, and Park (1921, 3) conducted experiments in feeding rats on mixed diets' sufficient (in the authors' opinion) in all respects except in calcium. The changes obtained by them were macros copically and microscopically completely analogous to rickets : the condition was characterized by increased persistence of the epiphyseal cartilage, its invasion by blood-vessels from the shaftj failure of lime salt deposition, the formation of a mixed zone between the cartilage and shaft (the rachitic metaphysis) and the over-produc- tion of osteoid tissue. The authors think that the above-described pathological condition differs from human rickets in that the arrangement of the proliferative zone of cartilage cells is maintained and that the evidences of bone resorption in the diaphysis are excessive. The authors' microphotograph No. 1 corroborates this description. The differences as compared with human rickets, pointed out by the authors, are, in my opinion, too slight for a diagnosis of rickets to be excluded. The addition of cod-liver oil to the diet prevented the development of the changes above mentioned and gave a picture of osteoporosis. Butter, in the quantities used by the authors (2-5 per cent.), did not possess such properties. Elliot, Crichton, and Orr (1922) also induced' rickets in pigs on a diet deficient in calcium, but containing all the vitamins. Prof. Schmorl's laboratory published Dr. Oehme's (1910) work, also on the question of calcium starvation, but in connexion with the effects of strontium. The experiments were made on 4 puppies fed on horseflesh (raw or boiled), fat, rice starch, grape sugar, and 5 gm. NaCl per diem. Apparently Oehme's dogs received a cer- tain amount of antirachitic factor in the fat, which made a difference between that author's experiments and some of Dibbelt's. The first 58 puppy was fed exclusively on this diet, and was killed in 6 weeks; the second had SrHPO^ with his food, and was killed iu 8 weeks ; the third puppy, like the second, was fed for 6 weeks on a diet con- taining strontium, after which for 15 days the amount of strontium salt was reduced, but about 2*9 gm. Ca3(P04\ per diem was added for the purpose of observing the curative action of calcium on the changes produced. The fourth puppy was used as a control and received 2-4 gm. C&^(P0^2 P^^ diem with its food. As compared with the control puppy, the others were very backward in gaining weight. The first dog fed on a diet deficient in calcium developed rachitic changes of the proliferous cartilage, absence of calcium in the zone of provisional calcification, osteoporosis, and a slight increase in the amount of endosteal and periosteal osteoid, but less than even in slight cases of rickets. Unlike the cases observed by Stoeltzner, the osteoporosis was due to the increased resoi-ption of the bone by very numerous osteoclasts. As in Gotting's experi- ments, in certain portions of the bone the osteoblasts were also numerous and well developed. The dog fed on a diet deficient in calcium, but with the addition of strontium, showed microscopic changes typical of severe rickets, with enormous new formations of endosteal and periosteal osteoid. In the case of the third dog, which for two weeks before the end of the experiment had received calcium phosphate together with a decreased amount of strontium, all the rachitic changes caused by strontium were reduced as a result of the deposition of calcium in the cartilage and osteoid tissue. Summing up the results of his experiments, Oehme finds that the dog fed on a diet deficient in calcium showed, on the whole, the same differences from rickets as had been observed by previous authors, viz. osteoporosis (very seldom occurring in rickets, in his opinion), too small a quantity of osteoid, and comparatively good impregnation of new bone formations with calcium. The author says, however (p. 256) : ' If, with Stoeltzner, Getting, and Dibbelt, I find that on a diet deficient in calcium dogs develop changes in the skeleton difiering from human rickets, of course that does not imply that I consider it irrefutably proved that a diet deficient in calcium plays no part in the pathogenesis of rickets.' At the same time, the author remarks on the difference between the organism of the dog and that of a human being, as well as the difference in the ages at which rickets was produced in animals on the one hand, and observed in children on the other. With a diet deficient in calcium, but with the addition of stron- tium, the author sees onlj' the following unessential differences from rickets in the bones : (1) The bones give up their calcium somewhat more easily on decalcification in Miiller's fluid. (2) On a diet — Ca + Sr the processes of the resorption of bone are retarded, and the deposition of fresh osteoid tissue is increased. According to the author, this is not the case in rickets. (3) On a diet — Ca + Sr, the introduction of calcium |;er os causes a rapid penetration of calcium into the osteoid, and thus acts 59 favourably on the disease. Stoeltzner thinks that in rickets the osteoid is quite incapable of taking in lime. Oehme does not con- sider Stoeltzner's assertion proved, and therefore cannot assign any special importance to this distinction. (4) Moreover, these experiments are arguments against Stoeltz- ner's assertion that calcium stimulates growth of bone, because the addition of calcium to the diet — Ca + Sr caused a disappearance of the osseous tissue formed in excess, and not a more active new formation of the latter. Lehnerdt (1909-10) studied the effects of strontium on animals fed on a diet deficient in calcium. In the first series of experiments made conjointly with H. Stoeltzner (who undertook the chemical examinations), puppies were used. In one case, not described in detail, an almost complete closure of the bone-marrow cavity by osteoid tissue was observed. In the second series of experiments strontium was introduced in the food of pregnant doe rabbits, and the changes in the skeletons of the offspring (to the number of 32) were observed. The third series of experiments was undertaken to investigate the changes in the ofispring (4 puppies and 8 rabbits) of mothers fed during lactation on a diet deficient in calcium, but con- taining strontium. The dogs were fed on raw horseflesh, 50 gm. fat (nature not stated), and distilled water. The rabbits were fed on a diet which, for normal conditions, contained apparently sufficient calcium, i. e. oats, hay, various greens, beetroot, potatoes. The changes in the puppies and the rabbits in both series were essentially the same, and were manifested chiefly in vigorous new • formations of ujQcalcified bone, osteoid. The new bone was formed so rapidly and in such quantities that the milk of doe rabbits, although on normal diet, was found to be relatively poor in cal- cium. There was not enough of the latter for the complete impregnation of the bones, but as soon as the animals began to be fed on a diet richer in calcium (cow's milk), the osteoid rapidly began to be impregnated with lime salts. According to Lehnerdt, it is this ability of ' strontium osteoid ' to absorb calcium as soon as the latter enters the organism in sufficient quantities that principally distinguishes this disease from rickets. What is especially characteristic in rickets is the loss by the osteoid of the capacity to absorb lime salts. Against the diagnosis of rickets in Lehnerdt's experiments is the absence of marked changes iu the proliferous cartilage, which in some animals only showed slightly defective deposition of lime in the zone of provisional calcification. The line of costo-chondral junction was straight. A marked increase in the new formation of subchondral spongiosa was observable only along the bone, form- ing, as it were, narrow ' cartridges ' of osteoid. With the increase of new formations of bone the resorption of the latter was retarded : in some cases the remains of the primary cartilage bone might be observed. Lehnerdt's experiments are of great importance as showing the possibility of producing very serious changes in the skeleton of the ofispring through the blood and the milk of the mother. There is likewise the most interesting discovery that strontium is an irritant 60 inducing the new formation of enormous masses of osteoid tissue. Some authors (Virchow, Kassowitz, &c.) admit also the formation of osteoid by similar means in human rickets. (b) Chemical Changes of the Skeleton. The chemical composition of the skeleton in calcium starvation has been investigated by many authors, and in nearly all cases there was an increase in the water and a decrease in the calcium and phosphorus content. Of the analyses of various authors I refer chiefly to Weiske's (1895) excellent experiments. Papillon (1870) and Konig (1874) had already proved that in animals fed on a diet deficient in calcium the deposition of strontium in the skeleton is small (5 to 8-5 per cent, of the total bone ash). Weiske made his first experiment on five growing rabbits of one litter, aged 3f months. They were fed on oats, a diet comparatively deficient in calcium. The first had no other food, the second had CaCOg in addition, the third had CaSO^, the fourth had SrCOj, and the fifth MgCOg. In 100 gm. of dry and fat-free matter the component parts of the bones showed the following percentage composition (see Table No. 17): Table No. 17. Percentage composition of skeleton of adult rabbits fed on oats with various additions. Oa.'s+ CaCO,. Oats + CaSOt. Oais + StCO,. OWs + Mg003. Oats. Organic matter 38-66 39.83 44.53 43.02 43.38 Ash 61.34 60.17 55.47 56.98 56.62 OaO 31.48 30.72 23.02 28.36 28-85 SrO — — 4-09 — — MgO 0-73 1.11 0.61 1-39 0-63 As this experiment was made on comparatively adult rabbits, requiring far less calcium than very young ones, Weiske made his second experiment (extending over a month) on four rabbits of one litter, aged 5 weeks. Of these one was killed at the beginning of the experiment ; of the remaining three, one was fed on oats alone, the second on oats + CaOOg, and the third on oats + SrCOg. The rabbit which had SrCOg in its food had a good appetite all the time^ and its growth was satisfactory ; the two others consumed less food, stopped growing, and became thinner. The composition of their dry, fat-free skeleton is shown in the following table, No. 18 : Tablk No. 18. Percentage coinpositio7i of skeleton of young rabbits. Oo(s + SrC03. Oats. 61-10 % 47-88 % 38-90 „ 52-12 „ 16-28 ,, 25-50 „ 2-76 „ - 0-67 „ 0-87 „ 15-16 ,, 20-27 „ In the bones : Oafe + CaCO; Organic matter 39-95 % Ash 60-05 „ CaO 30-41 „ SrO MgO 0-77 „ P^Or, 23.41 „ 61 In order to form a clear idea of the chemical changes in the bones due to the eflfects of a diet deficient in calcium, both with and with- out the addition of strontium, it is necessary to consult the following- table of absolute quantities of the component parts of the skeleton of the rabbits in the above-mentioned experiment of Weiske (see Table No. 19). Table No. 19. Composition of the skeleton of the same young rabbits as in Table No. 18 {absolute amounts). 0a(s-|-CaCO3. Oate + SrCOj. gm. gm. Weight of dry, 42.44 29.16 fat-free skeleton Organic matter 16.94 17.82 As)i 25.47 11.34 CaO' J 2.90 4.75 SrO 0.81 MgO 0.33 0.19 PaOj 9.98 4.42 Oats, gm. Raibit killed at beginning of experiment. gm. 27.88 26.04 13.35 14.53 7.11 11.64 14.40 7.16 0.24 5-65 0.26 5.74 Thus the figures obtained in Weiske's experiments clearlj' lead in my opinion to the following conclusions : (1) In young animals when, owing to the great rapidity of growth, the organism requires a great deal of calcium, the chemical deterio-. ration in the composition of the skeleton, under the influence of a diet deficient in calcium, is of a much graver character than at a later age. (2) On the above-mentioned diet of oats the changes in the skeleton are characterized by a relative decrease in ash content, particularly calcium and phosphorus. As the absolute figures show, the content of ash, calcium, and phosphorus in the rabbit fed on oats alone and in the rabbit killed before the commencement of the experiment were equal, i. e. from the beginning to the end of the experiment there was absolutely no increase in the inorganic components in the rabbit on a diet deficient in calcium. The only increase was in the organic bone tissue (osteoid). (3) Under the influence of strontium, all these- changes were still more marked, but with certain peculiarities : (a) As shown by the table of absolute figures, the calcium content in the skeleton of the ' strontium ' rabbit was decreased to a far greater extent than before the beginning of the experiment, i.e. strontium caused decalcification of the skeleton. As the histologi- cal investigations made by Lehnerdt and also Oehme showed a very marked decline in the processes of I'esorption of bone, this decalcifi- cation cannot be explained by the increased resorption of old bone. Apparently, in the ' strontium ' skeleton we have the only known case of very probable halisteresis, i. e. the elimination of lime by its extraction from the old bone, without the resorption of the organic matter of the bone. (6) The content of organic bone-tissue, i.e. of osteoid, in the 62 skeleton of the ' strontium ' rabbit was, both absolutely ard rela- tively, far greater than that of the rabbits fed on a diet either rich or poor in calcium. That is to say, strontium forms osteoid tissue not only by halisteresis, but chiefly by increased new formations of non-calcified bone. The above-mentioned histological examinations favour this view. Thus strontium clearly possesses the properties of an irritant increasing the growth of bone.^ As is known, some authors insist on the existence in rickets of ' irritants ' causing an increase of new formations of osteoid. The investigations of Voit (1880), Baginsky (1882), Aron and Sebauer (1908), who examined the chemical com- position of the skeleton in calcium starvation, yielded the same results as Weiske's experiments. The only exceptions are the pigeons in Edwards's experiments (1861), the she-goat in Weiske's experiment (1871, 1), and the kid experimented on by Weiske and Wildt (1873). Voit, criticizing these experiments of the latter authors, points out that the usual changes which occur in the skeleton on a diet deficient in calcium may have been prevented ty (1) the fact that the aforesaid authors' animals were starving in general, and (2) the calcium content in the food was apparently sufiBcient for the needs of the animals. Weiske (1874) likewise obtained negative results in the case of rabbits 5 months old fed on barley extracted by means of hydro- chloric acid. The animals at once began to lose greatly in weight and died in 28 to 60 days, having lost from 32 to 47 per cent, of their original weight. The control rabbits, kept absolutely without food, died in approximately the same time and lost nearly as much in weight as the rabbits fed on extracted barley. (c) Chemical Changes in the Soft Tissues and in the Blood. The chemical composition of the muscles in calcium starvation has been investigated by diS'erent authors, but not with the same results : Weiske, Aron and Sebauer did not find any diflference in the calcium content as eompai'ed with the muscles of the control animals. Voit (1880) and Forster (1876) found a reduction of 36 to 44 per cent. Besides that, Voit found a slight decrease in iron (7-5 per cent.), and a slight increase of magnesium (6 per cent.). On a diet deficient in calcium animals show increased nervous excitability. Many authors have found a connexion between disturbances in calcium metabolism and tetany. The investigators have therefore tried to explain the increased nervous excitability of dogs in calcium starvation by fluctuations in the calcium content of the brain. Voit, Aron and Sebauer, and Pexa (1910) actually found a more or less marked impoverishment of the brain in calcium, e. g. ' ' Note. — (After completion of the book.) Shipley, Park, McCoUum, Simmonds, and Kinney (1922) produced an exaggerated form of rickets in rats on a diet con- taining 0-008 per cent. Ca and 0-4 per cent. P with addition 2-2 per cent. SrCOs. Except calcium deficiency and the presence of strontium the authors consider their diet satisfactory in all other respects. Tlie administration of eod-livor oil did not cause any change in the pathological picture produced. 63 Pexa's dogs, fed on a diet deficient in calcium, had 0-01 per cent. CaO, as against 0-04 per cent, in the control. Quest (1906), on the contrary, found no deviation from the normal in two experimental dogs. Voit found that the brain had lost 49-3 per cent, of iron and gained 6-9 per cent, of magnesium. In the blood, a decrease of calcium in calcium starvation was found by Voit (19'2 per cent.), Forster (0-034 gm., as against the normal 0-057 gm. per 100 gm. of di-y blood), Aron and Sebauer (0-041 gm. as against 0-045 gm. CaO per 100 gm. of dry blood), and Quest (0-010 per cent. CaO as against the normal 0-014 per cent, in whole blood). Weiske found a decreased calcium content (from 20 to 27 per cent.) in the blood of rabbits receiving an addition of SrCOg or MgCOg, while rabbits on calcium starvation showed no changes. Patterson (1908) could not discover such a decrease, but rather the contrary ; Patterson, however, experimented on rabbits of an unknown age. Moreover, Voit found a slight increase in magnesium in the blood (9-1 per cent.) and a considerable diminution of iron (25-4 per cent.). In the liver Voit, and Baginsky also, found a decrease in calcium, the former only 6-5 per cent., and the latter a very coni=iderable decrease, i. e. 0-029 to 0-036 per cent., instead of the normal 0-081 per cent. Weiske, on the contrary, found an increased calcium content. Besides that, Voit found a decrease of 19-2 per cent, in iron and a 26 per cent, increase in magnesium in the liver. Dibbelt found irregular fluctuations in the calcium content of the soft tissues of animals undergoing calcium starvation. - (d) Calcium Metabolism in Calcium Starvution. A negative calcium balance in calcium starvation was found in the above-mentioned (p. 39) experiment on himself by Berg (1911), by Weiske in a she-goat, by Forster in dogs, by Perl, Dibbelt, Schlesiger in dogs, and Goitein in rabbits. In some animals the loss of calcium by the organism at times reached an enormous figure, e. g. Weiske's she-goat lost 63-8 gm. Ca from its tissues in the course of the 50 days' experiment ; in 26 days Forster's dog excreted 15-5 gm. in the faeces alone, the intake for the same period being 2-29 gm. ; Perl's (1878) dog lost about 0-24 gm. CaO per diem from its tissues; one of Schlesinger's dogs lost 0-472 gm., and another 0-457 gm. Goitein (1906) found that if rabbits took in less than 0-16 gm. Ca per dievrb and per kilo in their food (maize diet), they began to lose calcium, notwithstanding an equilibrium in nitrogenous metabolism. In contrast to these experiments, Dibbelt found that most of his growing puppies undergoing calcium starvation showed a positive balance, though sometimes it was below the normal. In conclusion, it is necessary to point out two highly important facts in connexion with nearly all the experiments in calcium starva- tion. In these experiments the animals were fed principally on flesh, carbohydrates, oats, and maize, which are all deficient in \itamins. Some authors added animal fat to the diet. Fat might be a source of antii-achitic factor, provided it was not lard. In some experiments it was lard that was given, but in the great majority of experi- ments the kind of fat is not mentioned. Therefore, some experiments were essentially experiments on a diet deficient only in calcium, while in others there was a deficiency in calcium and vitamins, including antirachitic factor (e. g. Dibbelt's experiment, where the diet consisted of .meat and carbohydrates or hog's lard). I think that the difference in the results obtained by the authors may be attributed, to a considerable extent, to the factors above mentioned. (e) The Author's Experiments in feeding Rats on a Diet deficient in Gcdcium alone. This diet contained about 0-045 per cent, calcium and about 0-576 per cent, phosphorus.^ The amount of paste consumed daily fluctuated between 10 gm. and 12-5 gm. ; only rats which began to be fed on it at a more advanced age consumed a greater quantity — up to 25 gm. Therefore the average intake per diem per rat was about 4-11 mgm. Ca and 58-144 mgm. P. Three types of experiment were carried out upon 46 animals in all. In the first series 20 rats were put on N — Ca diet after being weaned : 13 rats at the age of about 18-34 days, 5 rats at the age of 58-69 days, and 2 rats at the age of 83-100 days. The second series of experiments was conducted upon 9 young rats whose mother had been on N — Ca diet during lactation. After being weaned they were kept on N — Ca diet. The third series of experiments on 17 rats, kept on N — Ca diet, but containing as the source of antirachitic factor butter only (without cod-liver oil), are described on p. 105 (rats 610, 610 C, D, E) and on p. 143 .(litters 612 and 613). Mothers of these, rats were also kept on the same diet during lactation. First Series of Experiments. The outward appearance and behaviour of these rats did not differ materially from that of normal animals. Only a certain shyness and increased nervous excitability was observable. When rapidly lifted by the skin at the nape of the neck, the rats showed symptoms which I mentioned in my investigation of the influence of parathyroidectomy on the skeleton of rats: spasmodic contraction of the two hind-paws against the abdomen, the extension of one fore-paw across thorax and abdomen whilst the other is flexed against the chest wall. Sometimes both fore-paws are flexed against the chest, with the claws clenched. Both in parathyroidectomy and in calcium starvation this complex sj'mptom apparently points to increased nervous excitability. In the control rats it was far less frequently met with, and in a far lesser degree. Sometimes the rats suffered from diarrhoea, a fact observed by other authors (Ghossat, 1842), likewise in animals fed on calcium-deficient diet. The mechanism of the development of this diarrhoea is not clear, and yet it is undoubtedly worthy of special investigation. In my opinion, this may be of special importance in elucidating the aetiology and therapy of some forms of infantile 1 The details of the diets used will be found on pp. 7-9. 65 diarrhoea, of the origin of which too little is known : it is possible that there is a connexion between calcium starvation and some digestive disturbances in children. The weight of the rats on Ca deficient diet was less than that of their normal controls, as may be seen from the comparison of the respective figures in Tables Nos. 20 and 21. When rats under Table No. No. of rat. 136 136 A" 136 B 2 14 42 42 42 45 20. Average 131 132 128 129 180 Chemical Gom,position of the Slceleton of Normal Rats.^ 56 (50-60) 60 60 70 70 78 In bones. Weight, gm. HjO %• Fresh Ca %. Dry Ca %. 70 77 75 76 533 55-9 66.3 50.2 7.9 7.5 7.1 9.0 16.9 17.0 16.2 18.2 Average 43 (40-50) 74.5 53.9 7.9 17.1 2 5 56 56 139 139 49.4 49.3 10.4 10.0 20.6 19-7 139 198 209 195 200 169 49.4 44.7 44.4 43.0 43.4 43.5 10.2 11.4 11.5 11.7 11.6 11.5 20.2 20.6 20-7 20.6 20.2 20.3 Average 68 (60-80) 194 43.8 11.5 20.5 178 84 152 39.9 12.6 21.0 130 85 187 38.2 13.7 22.1 181 87 165 39.6 13.0 21.5 177 90 192 38.5 13.5 21.9 179 90 177 40.5 12.7 21.4 162 91 195 386 13.7 223 148 101 250 37.4 13.9 22.2 149 101 215 39.9 12.4 20.5 436 105 239 37-9 14.2 22.7 501 109 158 38.9 13.0 21.2 544 109 178 39.7 12.9 21-3 430 118 192 39.4 13.0 21.5 216 118 155 38.8 13.0 21.2 215 119 175 38.0 12.8 20.7 Average 100 (80-120) 188 39.0 13.2 21.5 213 125 159 37.1 14.2 22.5 214 127 159 36.7 13.3 21.1 218 127 194 87.9 15.1 24.4 433 130 238 38.6 13.9 22.G 482 130 216 37.7 14.2 22.7 62 135 263 34.9 14.3 22.1 493 139 180 38.1 13.3 21.5 169 175 200 33.0 15.0 22.4 Average 136 (120-180) 201 36.8 14.2 22.i • Chemical composition of the skeleton of norma,! i-ats given in Table No. 20, age 60-80 days, coiTesponds well with that of normal rats, of litter 606 (see p. 91) and 610 A and B (see p. 107). 2 Rats 136 A and 136 B were kept on breeding diet No. 1. (1579) E 66 Table No. 21. Chemical Composition of the Slceleton of Rats fed on N — Ca diet.^ First Series. No. 0/ rat. Age in days. At the Fined, beginnin of diet. In bones. Final ^ weight. HaO 0/ /o- Fresh Ca%. Dry Ca %. 15 45 18 29 60.9 3.6 9.1 16 45 18 42 52.6 4.1 8.5 17 45 18 33 59.1 8.5 8.6 Average 45 (40-50) 18 35 (-56) 57.5 (+7; 3.7 (-53) 8.7 (. -50) 6 56 18 75 63.9 2.8 7.8 9 56 18 55 61.2 3.8 9.8 Average 56 (50-60) 18 65 (-53) 62.6 ( + 27) 3-3 (-68) 8.8 (- -56) 189 78 (60-80) 30 92 (-53) ,53.0 (+21) 5.2 (-55) 11.4 (- -44) 187 84 34 106 52.2 6.4 13.4 186 90 32 177 47.7 6.2 11.8 188 90 32 140 49.1 6.2 12.1 Average 88 (80-120) 33 139 (-24) 49.7 ( + 27) 6.3 (-53) 12.4 (- -42) 220 125 58 154 41.1 12.0 20.3 243 ab. 125 83 205 39.9 11.4 19.1 221 127 59 138 37-4 11.0 17.5 66 134 62 207 351 13.6 -20.9 67 134 62 262 35.0 18.4 20.7 63 134 62 287 33.8 13.7 20.5 Average 130 (120-180) 64 209 (-4) 37.1 ( + 0.8) 12.5 (-13) 19.8 (- -12) Second Series. Eats whose mothers were fed on N— Ca diet during lactation.'' 243 A 60 44' 42 65.6 3.9 11.4 : Dead 138 70 243 63 63.3 2-8 7.6 139 70 24 = 77 61.4 2.8 7.3 Average 67 (60-80) 31 61 (-68) 63.4 ( + 45) 3.2 (-72) 8.8 (- -58) 141 81 243 43 62.8 2.7 7.2 243 B 84 443 87 56.2 4.8 11.0 243 C 84 443 80 57.1 4.0 9.3 243 D 84 442 123 54.7 4.5 10.1 243 E 84 44' 84 57.2 4.2 9.9 140 85 24' 95 58.6 3.1 7.5 Average 84 (80-120) 37 85 (-55) 57.8 ( + 48) 3.9 (-71) 9.2.( -62) one month of age were put on a N — Ca diet, and killed about 2 or 2| months after the beginning of the dieting, their weight was, on an average, 53-56 per cent, below that of the controls. When the N — Ca dieting was begun at the age of about 2 months, and ' The figures in brackets show the percentage difference from the normal : above normal + and below normal — . * Compare the chemical composition of rats of second series with that of rats 610 C, D, E (see p. 107) and of litters 612 and 613 (see p. 144). ' Age on the day when separated from mother. S7 continued for about 3 months, it was not observed that the. above- mentioned deficient diet had any marked influence on the weight of the animals (see Tables No. 20 and No. 21). By the end of the experiment the teeth of the rats kept on N — Ca diet change from yellowish and semi-transparent to opaque white. Autopsy does not, as a rule, reveal any marked changes in the skeleton, with the exception of the more or less distinct hyperaemia of the bones, which is always observed. Separate cases of fracture or infraction were observed only in a few of the ribs of three rats which had been put on the diet at an early age (18-30 days). Histological examination showed a marked change in the skeleton, depending upon the age at which feeding had been begun. When begun before 35 days of age, the following picture was observed : The proliferous cartilage was normal or slightly enlarged. Its zone of provisional calcification was, in most cases, impregnated with calcium or, more seldom (only in two cases), with defects. In no case was the ingrowing of the bone marrow blood-vessels into the cartilage observed. The spongiosa and corticalis were slightly or moderately osteoporotic. The Haversian canals were considerably dilated and contained fibro-cellular bone marrow. The same kind of bone-marrow was observed among the trabeculae of the spongiosa. The latter were interlaced, narrow and few. There were especially few of them in the epiphyses of the long bones. TJie bone tissue was covered very unequally with osteoid, the smallest quantity being on the trabeculae of the spongiosa, generally not' in excess of the normal, while the largest amount was on the inner surface of the diaphyses of the ribs. The thickness of osteoid on the ribs was equal to -^ or J, or — in certain parts of the diaphysis — even equal to the thickness of the calcified bone. In the other bones it never attained such a considerable thickness. Frequently the osteoid was not separated by a marked line from the calcified portion of the bone, as is the case in rickets on a —A or — A — Ca diet, and, becoming evenly impregnated with calcium salts, imperceptibly merged into calcified bone: the latter was thus separated from the -pure osteoid by a wide area of osteoid incompletely impregnated with calcium salts. On the other hand, the calcified bone did not usually acquire a saturated blue colour (even when the undecalcified bone was dyed with haema- toxylin), but was always pale. This fact still further toned down the sharpness of the boundary between the osteoid and calcified portions of the bone. Often a number of well-developed osteoblasts in the region of the spongiosa and near the osteoid layers were normal, but not infrequently the osteoblasts were badly defined and few in number. The number of osteoclasts was always increased. Thus the increased activity of the osteoclasts and the frequently lessened activity of the osteoblasts played a part in the production of osteo- porosis. Often the cambium layer of the periosteum was thickened. The bone marrow was usually hyperaemic. In rats which had been put on a Nr^Ca diet at the age of about 2 months or later, the changes in the bones, were characterized only by slight osteoporosis, principally due to the diminished activity of the osteoblasts and by hyperaemia of the bone maiTow. E 3 68 Second series of experiments. This series of experiments was conducted upon two litters of rats, 9 in all, originating from two mothers. The latter were put on N — Ca diet immediately after giving birth to their young, and kept on it during the whole period of lactation. On the termination of lactation 4 young rats of the first litter were separated from their mother, at the age of 24 days, and 4 young rats of the second litter at the age of 44 days. The young rats continued on a N — Ca diet and were destroyed : 2 at the age of 70 days, and 6 at the age of about 84 days, i. e. after being weaned they were fed on a N — Ca diet for a period of 40 to 61 days. Most of these rats, both when alive and when examined post nrwrtem, showed a picture similar to rickets, more marked in the case of the young rats weaned at the earlier age of 24 days. The animals were thin, but not emaciated. As in the case of the rats of the first series, they sufiered from shyness, increased nervous excitability, and tendency to diarrhoea, had opaque and sometimes fractured teeth, and were 55-68 per cent, lighter in weight than the control animals. The bones of the limbs, and in some cases the spine, were more or less distorted ; in the rats of the first litter the thorax was depressed on both sides, owing to the angular curvature of the ribs. With the exception of one rat, all had spontaneous fractures of the ribs, and one had fracture of the tibiae and fibulae. The bones were hyperaemic. Microscopical examination showed that the changes in the bones of this series were far more analogous to rickets than in the case of the rats of the first series. The proliferous cartilage was enlarged ; in some rats the zone of hypertrophic cells contained from 20 to 27 layers, but without their being markedly disorganized. The zone of provisional calcification either showed considerable defects in de- position of lime, or was almost entirely deficient in lime salts. As in true rickets, the presence of calcium salts was observed mostly between the cartilage cells situated directly under the perichondrium. The line of costochondral junction was bent towards the bone marrow, and only in the case of a few rats was its regularity broken by a slight ingrowing of blood-vessels from the bone marrow. The spongiosa was either osteoporotic, or, on the contrary, consisted of a thick network of distorted trabeculae. The trabeculae varied in thickness and length, and over a considerable area the cartilage cells were unabsorbed. The corticalis was osteoporotic, and the cambium layer of the periosteum was thickened, and consisted of juicy cells, in some places connected with the bone marrow. The amount of osteoid in the subchondral spongiosa was, on the whole, less in volume than in the other portions of the bone. The thick- ness of the osteoid varied in different portions of the bone, being either equal to that of the calcified .portion of the bone oi", more frequently, less than that. In some of the rats in certain portions of the bone the osteoid was even thicker than the calcified bone. As in the bones of the rats of the first series, the osteoid frequently passed imperceptibly, without marked boundaries, into calcified, pale-coloured bone. In most of the rats the number of osteoblasts and osteoclasts was increased, and frequently on one side of the trabecula the osteoblasts (sometimes arranged in sever.al rows) were 69 depositing new bone, while on the other side it was being destroyed by osteoclasts. The areas of callus after fracture or infraction were composed chiefly of osteoid, sometimes in great quantities, the centre of the trabeculae of the callus being more or less impregnated with lime salts. The bonu marrow in the. region of the spongiosa and inner surface of the corticalis was mostly of a fibro-cellular charac- ter and was hyperaemic everywhere. (See PI. 3, Figs. 5, 6, and 7.) Chemical examination of the skeleton. As may be seen from a compai'ison of the figures in Table No. 20 with those of Table No. 21, the common features of the chemical changes of the skeleton of rats on a N — Ca diet are as follows: increase in water and diminution in calcium. As has akeady been pointed out, this change was observed by the majority of other investigators. The calcium deficiency is far more perceptible in the fresh bone than in the dry, i. e. this phenomenon partly depends on the greater water content of the skeleton of animals fed on a N — Ca diet. And indeed, in those cases where no considerable increase in the water content of the bones is observable, the diflference in the percentage decrease of the calcium content in fresh and dry bone is very small (see Table No. 21, figures corresponding to the average age of rats of 45 and 130 days). The most marked changes in the skeleton were observed in the case of rats whose mothers had also been fed during lactation on a N — Ca diet : as compared with their controls, the water content of the bones of such rats showed an average increase of 45 to 48 per cent, above the nonnal, the calcium (JOntent of the fresh bone being decreased by 71-72 per cent., and that of the dry bone by 58-62 per cent, below the normal. In rats put on a N — Ca diet after separation from their mother, the greatest changes were obtained when feeding was begun not later than the age of one month. Under such conditions, the water content of the skeleton was increased by 7-27 per cent., while the calcium content of the fresh bone was decreased, on an average, by 53-68 per cent., and that of the dry bone by 42-56 per cent. When the above- mentioned diet was begun at the age of about 2 months and later, in accordance with the absence of considerable alterations in the weight of the animal and in the histological picture of the bones, the chemical changes were not great, viz. the water content was fairly normal, while the calcium content was, on an average, from 12 to 13 per cent, below the normal. This difference in the degree of the skeletal changes depending upon whether animals are put on a N — Ca diet s\.t an earlier or later period is, of course, explained simply by the diflference in the reserves of calcium in the organism of the animal at diflferent ages : not only does a young, animal con- tain less calcium, but the demand for calcium by a rapidly growing animal is far greater than in the case of an adult. In my ex- periments it was quite clear that the results of macroscopical, microscopical, and chemical examinations were in complete corre- spondence. Is it possible to agree with Gotling and Lehnerdt that the impoverishment of the bones in calcium on a N— Ca diet is chiefly caused by the decrease in the amount of osseous tissue in the skeleton, due to osteoporosis 1 70 I have already noted that the decrease in the calcium content is always observed also in the dry bone. On comparing the percentage decrease in the calcium content in the fresh and dry bone (Table No. 31) it will be found that there is a relatively small differ- ence between the two figures. Besides that, in all my preparations stained with haematoxylin, the bone containing lime was very pale in colour. This latter circumstance, as well as the rapid decalcifi- cation of bones in Miiller's fluid, points likewise to a decrease in the calcium content of the bones. Thus the decrease in the amount of bone tissue due to osteoporosis is only a minor cause of the deficiency of calcium in the bones. The principal cause is the decreased deposition of lime salts in the newly forming bone. The cause of the decreased deposition lies only in calcium starva- tion, on the removal of which the bone again becomes normal. I think that, histologically, the calcium starvation is indicated by the indefinite and broad area of the gradual transformation of calcified bone into osteoid, frequently observed in the bones of I'ats on a N-Ca diet. Summarizing the results of my experiments upon a diet deficient only in calcium, I may draw the following conclusions : I. In animals fed after weaning on a diet deficient in calcium : (1) there was an increase in the water content of the skeleton, and a decrease in its lime content ; (2) the latter depended chiefly on the decreased deposition of calcium in tbfe newly forming bone, and to a lesser degree, on the decreased amount of osseous tissue in the skeleton ; .. (3) histologically, either osteoporosis or a picture analogous to mild rickets was observed ; • , (4) the intensity of the above-mentioned changes depended on the age at which the N — Ca dieting was begun : the greatest changes were observed when the dieting was begun at the age of not more than 35 days, and the least changes were observed when the dieting was begun at the age of 2 months or more. II. In rats whose mothers were on N — Ca diet during the whole period of lactation : (1) the macroscopical, microscopical, and chemical changes in the skeleton were most severe, being very similar to, if not identical with, the picture of rickets of moderate severity ; (2) thus the normal calcium content in the food of the mother during lactation is one of the most important factors in the normal development of the skeleton and, in all probability, in the prevention of rickets in the offspring. III. Considerable quantities of cod-liver oil, up to 170-200 mgm. per diem fed to the young as well as to the mother, did not prevent the development of the above-mentioned changes in the skeleton of rats, whose mothers were fed on a diet deficient in calcium during lactation. IV. The other abnormal phenomena observed in rats on a diet deficient only ia calcium were : the opacity and fragility of the teeth, reduced weight, increased nervous excitability, and frequent diarrhoea. ' ' ~ : 71 (f) The author's experiments in feeding rats on a basal diet rich in antirachitic factor but without the addition of a saline mixture. Eight rats were fed on the above-mentioned basal diet, without the, addition of the saline mixture.* The diet contained about 0-045 per cent, of calcium and about 0-22 per cent, of phosphorus. The I'ats consumed on an average about 10 to 14 gm. of food per diem.. The dieting was begun after weaning when they were 21-30 days old. The average duration of the dieting was 57 days. The outward appearance and behaviour of the rats was exactly similar to that of the corresponding group on the N — Ca diet, i.e. there was the same retarded growth, shyness, increased nervous excitability, and ten- dency to diarrhoea. In weight the rats were 20-50 per cent, below their controls. The teeth were more or less opaque. The macro- scopical changes in the skeleton of the animals were not very marked. No fractures of the bones were observed. The skeletons of the animals of this series were not chemically examined. Micro- scopical examination revealed the following particulars: The proliferous cartilage was either normal or slightly enlarged. The zone of provisional calcification was impregnated with lime salts, except in one case, where it contained defects of calcium deposition. No abnormal ingrowing of blood-vessels into the cartilage was observed. The subchondral spongiosa was osteoporotic, consisting of narrow and not numerous trabeculae. The corticalis was like- wise narrower than the normal, and in some rats, owing to the ingrowing of blood-vessels with fibrous bone-marrow elements, was transformed into a bony network consisting of thin trabeculae. ' When stained with haematoxylin the bone containing calcium was pale in colour, even when decalcification was very short (one day). The amount of osteoid either did not exceed the normal (3 rats) or was slightly or moderately increased (5 rats). Frequently the osteoid merged into calcified bone without any marked boundary line. The number of osteoclasts was slightly increased in all cases ; the number and appearance of the osteoblasts was either normal (over the layers of osteoid) or diminished. The bone marrow in the area of the spongiosa, as well as in the rarified corticalis, was of a fibro- cellular character. In some cases the cambium layer of the periosteum was thickened. From the above experiments I am able to draw the following conclusion : Feeding rats on a basal diet, but without the saline mixture, and deficient not only in calcium but also phosphorus, resulted in the same skeletal changes as were observed by me in my experiments in feeding rats on a diet deficient in calcium only. (g) General Sv/mmary. Taking into consideration the results of my experiments and those of the authors above mentioned, let us compare the points of similaiity and difference between rickets and the marked effects of calcium starvation. ' I'he details of the diet used will be found, on pp. 7-9. 72 Points of similarity. The clinical picture of the two is almost identical. Frequently the histological picture likewise shows all the features of rickets: enlargement and in some cases dis- organization of the proliferous cartilage, penetration of blood- vessels into the latter, defects in, or sometimes the total absence of, lime deposition in the zone of provisional calcification of cartilage, and excessive amount of osteoid tissue. I think that many of the objections brought forward by authors on the grounds of histological analysis are not convincing. For some critics, osteoporosis and increased bone resorption found in calcium starvation are among the chief reasons for not considering this process identical with rickets. In Chapter 4 (b), however, I have collected a whole series of cases in which severe osteoporosis is met with in rickets. Furthermore, the cases of latent infantile rickets in cachectic children have not as yet been sufficiently examined from a pathological point of view. It has been pointed out that the marked increase both in formation and destruction of bone occurring in calcium starvation are not observed in infantile rickets. But, in bringing forward this argument, it must not be forgotten that in experimental animals all the processes of the formation and destruction of tissues are about twenty times as rapid as in children, e.g. a child doubles its weight in 180 days, a dog in 9 days, and a rabbit in 6 days. Schmorl has described cases of the most energetic resorption of osseous tissue of certain bones, observed in human rachitis tarda. Finally, when an increased amount of osteoid — that typical pathological indication of rickets — was found, it was regarded as ' something incidental ', even in the presence of othei" symptoms of rickets. After a critical review of all the experiments in calcium starvation, Lehnerdt brings forward the (to him) irrefutable argu- ment that in calcium starvation the bone is normal in quality, and normally calcified, but the skeleton merely contains too small a quantity of bone tissue, i. e. is osteoporotic. In my opinion, this is not the case. Weiske's experiments, referred to above, have shown that, the increase in organic matter of the bone is due to new formation of osteoid and diminished impregnation of the bone with calcium. My own experiments corroborate this view. The small amount of osteoid in the skeleton can hardly be brought forward as an argument against the identity of experi- mental and human rickets. For if it were, the cases of incipient rickets or rachitis tarda (Looser's cases) would have to be excluded from the group of human rickets. Chemical analysis of the skeleton likewise shows the same identity of the changes in rickets with those in calcium starvation. Stoelzner from the beginning put forward as an irrefutable fact that in rickets the soft tissues contain a normal amount of lime, while in calcium starvation the content is diminished. In Chapters 3 (c) and 11 (c) I have pointed out that, whilst some authors found a diminished calcium content in the soft tissues in some cases of rickets, others, on the other hand, in cases of acute calcium starvation, did not find the soft tissues invariably deficient in their calcium content. Furthermore, both, in calcium starvation and in rickets, the 73 metabolism may show a marked negative calcium balance. There- fore, I think that a careful examination of the question must lead to the conclusion that in some cases it may be possible, by calcium starvation, to produce a disease which clinically, chemically, macro- scopically, and even histologically, may simulate rickets. In spite of this similarity I am convinced that there is an essential difference between the two, and I consider the introduction of the term ' rickets from calcium starvation ' (but not ' pseudo-rachitic osteo- porosis ') as most desirable. In this group of rickets I propose to include the skeletal changes caused by calcium starvation or those caused by the introduction of strontium into the organism of animals in a state of calcium starvation. In the latter case there is no osteoporosis, and therefore the term ' pseudo-rachitic osteoporosis ' is inapplicable. Points of difference. What is the essential difference between rickets from calcium starvation and other forms of rickets ? Only this : deficiency of diet in calcium alone is necessary for the de- velopment of rickets from calcium starvation. Other forms of rickets may be developed even on a diet containing a normal amount of calcium. The difference between the two diseases lies more in aetiology than in a morphological or chemical difference in the composition of the skeleton. Totally different causes produce clinical, chemical, and histological pictures which in certain cases are very similar, if not identical. I wish to point out the enormous importance of calcium starva-* tion in the development of certain forvis of human ricJcets. Even by themselves the facts collected by Schabad, Aron, Dibbelt, and others, as well as by myself, show that calcium starvation may be, and indeed is, very easily iand frequently observed in a child at the age when rickets develops. I shall speak more fully of the im- portance of calcium starvation in the development of rickets in discussing my experiments on the effects of a diet deficient in calcium and antirachitic factor. In conclusion, I shall quote the opinion of such an experienced clinician and investigator of rickets as Schabad (1910, 5) on the connexion between the deficiency in calcium and rickets : ' It is possible that, together with true rickets, a person may be suffering from pseudo-rickets produced by an insuflicient intake of calcium ; clinically pseudo-rickets cannot be distinguished from rickets, although the former has the peculiarities which are charac- teristic of experimental rickets.' 13. The Influence of a Diet deficient in Antirachitic Factor ON THE Skeleton. « (a) Historical. In 1906 Hopkins suggested the idea that rickets can be caused by a nutritive error consisting of the deficiency in the diet of some special organic factors. 74 Prof. Mellanby was the first to approach experimentally the question of the existence of an antirachitic vitamin in food. As early as 1918, on the basis of experiments on puppies, he suggested that rickets may be produced by the absence in the diet of a special accessory food factor. In his last communications (1920, 1921), as a result of very numerous experiments, he came to the conclusion that the antirachitic factor is apparently identical with vitamin A : the quantitative fluctuations of both factors in various substances are identical, and whatever weakens or destroys the action of vitamin A has a similar effect on the antirachitic qualities of a given sub- stance. Mellanby considers that the most suitable diet for produc- ing rickets in puppies is the following : white bread ad libitum, skim-milk — 175 to 250 c.cm., yeast — 5 to 10 gm., orange juice — 5 c.cm., linseed oil — 10 c.cm., sodium chloi'ide — 1 to 2 gm. This diet contains a sufficient quantity of vitamins B and C, but is very poor in vitamin A. I think that for rapidly growing puppies it may likewise be deficient in calcium salts : according to Aron's calculations the a,mount of CaO required by a growing animal is not less than 1 per cent, of its increase in weight. Taking puppy No. 175 (Mellanby (1921), p. 37) as an example, the increase in weight in 22 weeks was about 4j600 gm., i. e. under normal conditions there should have been a deposit of about 46 gm. CaO in the organism. Taking the CaO content of milk at 0-17 per cent., and the average amount of bread consumed daily at 400 gm. (containing 0-014 per cent. CaO), then in 22 weeks the puppy obtained about 66 gm. CaO from its food, that is to say, its calcium requirements would have been satisfied only provided that 70 per cent, of the calcium taken with the food was retained. Apparently, however, in Mellanby 's experiments the deficiency in calcium was not the factor involved, as shown by one experiment (p. 54) on -a six- weeks old puppy. Notwithstanding the daily addition of 5 gm. Gsi^{T0^)2 to its food, it developed acute rickets, with an impoverishment of the bones in calcium (from about the normal 13 per cent, to 9-8 per cent. CaO). Mellanby arrived at the following conclusions : When to the basal diet food-stufis with a lai"ge content of antirachitic factor wei:e added, rickets did not develop, while rickets which had developed on the basal diet began to improve. Among fats, cod-liver oil has the strongest antirachitic action, then come butter and suet. Lard contains, a very small quantity of antirachitic factor. Vegetable oils contain smaller quantities, or hardly any at all, and may be arranged as follows according to their antirachitic properties : pea- nut oil, coco-nut oil (these being the most active), rape-seed oil, cotton-seed oil, palm-kernel oil, oiive oil, linseed oil, and babassu oil (the weakest). Hydrogenated fats are poor in antirachitic factor. Milk and the yolks of eggs contain a large amount of antirachitic factor. Meat has a small preventive action (partly due to the residue of not easily removable fat), and so has malt extract in doses over!80 c.cm. An excess of bread, carbohydrates, cereals, and casein deficient in calcium prevent the normal deposition of calcium in the bone and conduce to the development of acute forms of rickets. When the above-mentioned substances are consumed in excess, the animals grow very fast, and apparently there is not 75 enough calcium to keep up with the too rapid and excessive forma- tion of bone. In the puppies used in these experiments of Mellanby, Mrs. M. Mellanby (1918) found the following changes in the teeth : softening, falling out, delayed eruption, irregularity in position, partial absence or defective enamel, low calcium content. In his experiments, Mellanby used radiographic and histological methods of diagnosis. Further, by the chemical determination of calcium in the bones the results of microscopic examination were frequently checked. In rickets it was always found that there was a decrease in the calcium content which was sometimes very considerable, e. g. as low as 6-6 per cent. CaO, instead of the normal about 14-4 per cent. In order to determine the influence of a deficiency in vitamin A in the diet, other experiments have been made, namely those of the following authors : Hai'den and Zilva (1919) on monkeys, Tozer (1921) on guinea-pigs, Mackay (1931) on kittens, Shipley, Park, McCollum, and Simmonds (1921) on rats, Hess, McCann, and. Pap- penheimer (1931) on rats, Zilva, Golding, Drummond, and Coward (1921) on pigs, Elliot, Crichton, and Orr (1922) on pigs. All these investigations showed no rachitic changes in the skeleton of animals on a diet deficient only in vitamin A. In most cases osteo- porosis of the skeleton was observed. In experiments on pigs and possibly on other animals some authors have not paid enough attention to the influence of light. Possibly some results obtained are due to this. In all these experiments, with the exception of those on pigs, the animals were killed at the period of loss of weight and more or less manifested cachexia. These results depend not only on the deficiency in vitamin A, but also on the reduced consumption of food owing to loss of appetite. Mellanby's experimental animals grew and did not suffer from cachexia. This group of experiments includes the investigations made by Herter (1898), who at that time knew nothing of vitamins, but made several interesting observations. He experimented on 4 young pigs about two months old, for the purpose of determining whether fat starvation was not the cause of rickets. The diet deficient in fats consisted of skim-milk, containing aboui 0-05 per cent, of fat, i.e. was a diet deficient in antirachitic factor. One of the pigs had first Mellin's food, and then unrefined sugar in addition. This diet was continued for about one year. The pigs fed on skim-milk showed delayed growth, general disturbance of nutrition, weakness and paralysis of the limbs, and disappearance .of fat from the subcutaneous tissue. The appearance of fat atrophy was analogous to the 'serous fat atrophy ' described by Flemming in the case of humans as a result of wasting disease. The subcutaneous fat turned into a gelatinous mucoid mass, but containing no mucus; the fat cells showed a marked reduction in size, were totally devoid of fat, and were filled with a peculiar, granular, albuminous substance. These changes were not obsei"ved when carbohydrates were added to the food in considerable quantities^ A very important fact observed by Heirter is the reduced absorp- tion of phosphates from the intestines on a diet deficient in fat. 76 The addition of 100 gm. of suet to skim-milk very, rapidlj' increased the excretion of phosphate in the urine, and reduced the amount of PgOj and Ca in the faeces, i. e. improved the absorption of these salts. The author thinks that his pigs did not develop rickets. He omits, however, to describe the microscopical appearance of the skeleton, and thus to show the real absence of the rachitic changes. Findlay, Paton, and Watson, from Professor Paton's laboratory (1918-1921), published several papers against the ' evidence that milk fat (butter) contains any accessory factor pi'otecting against the development of rickets '. These papers will be dealt with in detail in the chapter on the influence of confinement in the aetiology of rickets. Here it is only necessary to mention that the authors' conclusions are based on the fact of the development of rickets in puppies ffed on a diet containing an ample amount of butter (up to 14-5 gm.), and the non-development of rickets in other puppies, fed on a diet containing little milk fat, but 'kept in the country and freely exercised in the open air '. Strictly speaking, there is nothing unexpected in this, because rickets is just the disease which is developed on a milk diet. The question therefore is merely, why in some cases milk or butter prevents rickets or cures it, and in others it has no such effect. The experiments of Hopkins (1920, l),Drummond and Coward (1920), and Zilva (1919, 1920) have shown that vitamin A is comparatively easily destroyed in butter by oxidation. Dutcher (1921) found that 5 per cent, of butter from cows on a diet satisfactory in every respect was quite sufficient for the normal growth of rats. This growth could not be attained on a diet of 20 per cent, of butter if the cow was fed on a diet deficient in vitamin A. Whereas Hopkins in twice repeated investigations (1912 and 1920) managed to maintain normal growth in rats by adding only 2 com. of milk to their non-vitamin diet, Osborne and Mendel (1920) could obtain very fair, though still not normal growth, only by the addition of 15 ccm. of milk. Dutcher (1921) found considerable fluctuations in the vitamin A content of milk, depending upon the time of year and diet of the cow. Drummond, Coward, and Watson (1921) found (1) that butter is usually twice or three times a less potent source of vitamin A than the amount of milk which would yield that butter ; (2) butter shows wide variations as a source of vitamin A. Some samples of butter, investigated by these authors, possessed a growth-promoting power little or no better than the average refined vegetable oils which are usually classified low down in the list of food-stuffs containing vitamin A. Most variations arise from difference in the diet of the cow, depending chiefly on whether the cow has been fed on a diet rich in vitamin A or not. The low vitamin value of butter is produced during the winter months from stall-fed cattle on dry feeds of hay, roots, and cake. The milk used in Vienna by Chick, Dalyell, Hume, Mackay, and Smith (1922) for their observations during the winter showed low value in content of vitamin A as tested by experiments on rats. Forbes and his collaborators (1916-18) have shown that milking cows kept on diet rich in calcium but probably deficient in. organic It factor may have a negative calcium balance. Even the addition of calcium salts to a ration of grain, dry alfalfa hay, and corn silage did not lead in their experience to the establishment of a positive calcium balance. Meigs, Blatherwick, and Gary (1919) also observed in some experiments a negative or slightly positive calcium balance in a dry pregnant cow on a diet rich in calcium but apparently deficient in organic factor (dry alfalfa hay, com silage, grain). The authors explained this abnormality in calcium balance by the disturbance of the cow during the experiment. Hart, Steenbock, and Hoppert (1931) experimented upon 5 goats, both milking and dry, kept on a mixed diet deficient in calcium and the organic factor. When fresh green oats were added instead of dry oats straw the amount of calcium assimilated was increased. The freshly dried oat straw seemed to retain the properties of the fresh green oats. Cod-liver oil changed in some experiments negative calcium balance to a positive. Therefore the authors think that the same factor affecting calcium metabolism and present in cod-liver oil is also found in green oats and grasses. Thus it has been shown that fluctuations in the vitamin A con- tent of milk and butter are very marked, and this may well prove to be the explanation of the contradictory results obtained in different experiments. Furthermore, butter cannot be considered as containing a very large amount of vitamin A : it may be 250 times poorer in it than cod-liver oil (Zilva and Miura, 1931). Shipley, Park, McCoUum, Simmonds, and Parsons (1921) likewise found that a fresh deposition of lime salts was observable in the zone of the provisional calcification of the cartilage as early as 3-8 days after the addition of 1-3 per cent, cod-liver oil to rickets-producing diets. Professor Mellanby has always cautiously expressed his opinion on the identity of vitamin A and an antirachitic factor. Shipley, Park, McCoUum, and Simmons (1931, 4, 1933, 3) in their latest communications are inclined to consider that the two factors are not identical. On compound diets deficient in calcium they failed to prevent the development of very severe rickets-like diseases of the skeleton by the addition of butter, and, on the contrary, 1 to 3 per cent, cod-liver oil possessed that property. Even after the addition of cod-liver oil, however, the skeleton and general health of the rats were still far from normal. The nutrition of the rats, their fertility, success in rearing young, and length of life were far better on the aforesaid deficient diets on the addition of 1 per cent, cod-liver oil than of 10 to 30 per cent, butter. But when the amount of calcium in the food was sufficient, even about 3 per cent, of butter was enough to give the animal the requisite amount of antirachitic factor necessary for the normal development of the skeleton. In another diet, deficient in vitamin A and phos- phorus (0'3 per cent.), .causing severe rickets, the addition of 10 per cent, butter did not prevent the development of rickets, while, on the contrary, the addition of 2 per cent, cod-liver oil brought about a normal development of the skeleton. On the basis of all the above-mentioned facts McCoUum and co-workers are of the opinion that there are two distinct organic factors opei'ating in the nutria tion of a mammal, which are associated with certain fats : one is 78 vitamin. A, indispensable to the growth of animals and for the prevention of the development of keratoiiialacia ;. the second is necessary for the normal development of the skelfetoh. The latter factor is found in slight amounts in butter and in great amounts in cod-liver oil. As shown by Zilva and Miura's very important experiments cod-liver oil may contain over 200 times more vitamin A than butter does, and this enormous difference in the vitamin A content may be itself partly sufficient to explain the difference in their action. My own expeiiments have shown that pronounced rickets can be produced in rats on a diet deficient in calcium, con- taining 13-5 per cent, butter plus 2-5 per cent, cod-liver oil, pro- vided that the mothers of the rats were fed on the same diet during lactation. In human rickets the striking effect of the- cod-liver oil treat- ment was observed long ago. I have quoted experiments showing the favourable effects of cod-liver oil on the metabolism of rickety children (Schabad, Birk, Schloss, &c.). Hess and Unger (1917) made experiments on negro children in New York, over 90 per cent, of whom suffer from rickets. The experiments consisted in the preventive administration of varying doses of cod-liver oil to the children, beginning from the age of 4 months to 1 year. The following tables (Nos. 22 and 23) are the best illustration of the results obtained : Table No. 22. Number of cases of rickets among children receiving and not receiving cod-liver oil prophylactically. Duration of therapy in months. Number of infants. Infants not developing rickets. Infants developing rickets. Percentage non- rachitic. 6 6 i 32 5 12 16 30 4 7 1 •2 1 5 15 93 80 58 6 Oil given (average total 54 oz.) Oil given (average total 23 02.) Oil given (average total 21 oz.) Oil not given Table No. 23. Relationship of breast and artificial feeding to cod-liver oil prophylaotyis. Duration of therapy in months. Oil given (average total 54 oz.) 6 Oil given (average total 23 oz. ) 6 Oil given (average total 21 oz.) 4 Oil not given The second table shows that human milk does not necessarily prevent rickets, and that cod-liver oil is a stronger antirachitic remedy. Miss Ferguson (1920), under the direction of Dr. Findlay, repeated Hess and Unger's experiments on infants aged 1-| to 12 months who showed no signs of rickets prior to the experiment. Cod-liver oil was administered to the amount of 2 oz. per week, i.e. approxi- mately the same amount as in Hess and Unger's experiments. In Breast Feeding. Infants not Infants evdoping developing rickets. rickets. Artificial Feeding. Infants not Infants developing developing rickets. rickets. 22 1 3 1 4 4 1 12 8 1 3 1 1 3 79 six months from the beginning of the cod-liver oil treatment, the children -were again examined by Dr. Findlay, with the following results (Table No. 24) : Table No. 24. Diagnosis. Oil cases. Control cases. Non-rachitie 23 = 85 per cent. 10 =33 per cent. Rachitic 3 = 11 „ „ 18 = 60 „ ,, Doubtful 1=4,,,, 2 = 7 „ „ Total 27 children 30 children That is to say, the energetic antirachitic prophylactic action of cod-liver oil was proved in a marked degree by Hess and Unger, and to a lesser degree by Fergusson. The small number of cases observed by Mackay (1920) of the influence of cod-liver oil on rickety children does not permit of any definite conclusions being drawn. Hume and Nirenstein (IS 21), on the basis of the treatment of 130 cases of hunger osteomalacia, have been convinced of the advantage of cod-liver oil as compared with vegetable oils of the rape group. Similar conclusions have been arrived at by Dalyell and Chick (1921). Park and Howland (1921, 2) investigated radibgraphically, and in some cases microscopically, the deposition of calcium salts in the skeleton of rickety children before and after the cod-liver oil treatment. ' In two or three months so much infiltration with salts has taken place that the extremities of the bones, except for deformities, were practically normal. . . . The authors look upon cod-liver oil as a specific for rickets. They have not seen it fail in any single instance, and they have known it to cure the rickets even though the children were dying of some other disease. . . . They know of liardly another drug that in disease exerts so regular, certain and specific an effect as does cod-liver oil in rickets ' (p. 443). Cod-liver oil, however, requires some time to take effect. ' One child died of an intercurrent disease 6 days after, and another 12 days after the beginning of cod-liver oil medication. Tn the bones of neither of these children was there any deposition of calcium salts appreciable oh micro- scopical examination ' (p. 342). Chick, Dalyell, Hume, Mackay, and Smith (1922) were able to prevent rickets at a time when rickets is most prevalent (winter and spring) by the addition of cod-liver oil to the diet, when the omission of this in control children produced I'ickets. Of 24 of these children on a diet of milk, sugar, cereals, 14 developed rickets. Of 27 on another diet containing cod-liver oil, no case of rickets developed-. Mori (1904) and Bloch (1917) have both described a.disease in children completely analogous to the xerophthalmia in rats on a — A diet. This disease was cured by cod-liver oil treatment with extraordinary rapidity. H. Chick and E. Dalyell (1921) showed that in backward children suffering from rickets, when the introduction of cod-liver oil alone had no effect, good results were obtained by a combined treatment of cod-liver oil with antiscorbutic vitamin Ci 80 (b) The authors experiments in feeding rats on a diet deficient in antirachitic factor. The diet employed ^ contained a sufficient quantity of all the food components required by the organism with the exception of antirachitic factor ; the amount of the latter must have been almost negligible. This diet contained about 0-351 per cent, calcium (0-3514 per cent. CaO) and about O-Sl per cent, phosphorus. At first the rats consumed about 12-15 gm. of the food mixture per diem, then, towards the end of the experiment, the rats lost weight, especially if the experiment was prolonged, and consumed as little as 9 or even 6 gm. Young and small rats of litter 225 (see p. 86, Table No. 27 (2)) whose mother had been on —A diet during pregnancy or lactation sometimes consumed only from 2 to 3 gm. towards the end of the experiment. Thus towards the end of the experiment a diminished intake of food was observed in the case of many rats, and to this their cessation of growth and lowered nutrition must be partly attributed. In all 175 rats were kept on this diet, being divided into nine groups : Group I. 41 rats begun on — A diet after weaning. Group II. A litter of 6 rats of whom the mother was fed on — A diet during lactation. Group III. 18 rats, of whom both parents had received — A diet before conception anJ the mother during pregnancy and lactation. Groups IV-VII. 91 rats used to investigate the influence of the diet received before weaning upon the development of, or resistance to, rickets. Group VIII. 13 rats used to investigate the influence of moisture upon the skeleton. For the description of this experiment see Chapter 20, p. 138. * Group IX. 6 rats were experimented upon to elucidate the influence of Micrococcus candicans on the skeleton (see p. 152). In all my experiments with special feeding during lactation' (Groups II-IX), by the term ' diet during lactation ' I mean the food of the mother and of the offspring during this period. As far as young are concerned, during the first 15-18 days they receive exclusively the milk of the mother, but after that period and up to the time of weaning they consume in addition some amounts of the mother's diet. Group I. Rats whose —A diet was begun after weaning. The outward appearance, activity, and weight of the rats on a diet defi- cient in vitamin A has frequently been described, and therefore I shall not dwell in detail on these points. It is, however, necessary to remark that the decline in weight, emaciation or cachexia, and xerophthalmia were not invariably observed in my rats. As is known, these phenomena develop with exceptional ease and most frequently when the dieting is prolonged. Manj' of my rats were killed at the stage of arrested growth, and some of them were even gi'owing still, though very slowly. At this stage neither emaciation nor xerophthalmia were observed. On the contrary, these rats con- ' The details of the diet used will be found on pp. 7-9. 81 tained a considerable amount of fat, and were fully active. Four of them gave birth to young. Post-mortem results showed that the rats of this group could be divided into three categories : the first included only three individuals in which a picture of severe rickets was observed macroscopically, viz. compressed thorax with ribs bent at an angle, curvature of the spine or extremities^ numerous fractures of the ribs and in some cases of the extremities likewise, with thickened costo'-chondral junctions. In 8 rats the picture of rickets was shown only by the more or less numerous fractures of the ribs, with large calluses. The remaining 30 rats could not easily be diagnosed macroscopically, with the exception, of course, of cases of severe osteoporosis. The teeth of all were opaque and of some broken. Microscopically osteoporosis was present in 16 cases, slight rickets in 13 cases, moderate in 11 cases, and rather severe in one case only. In the osteoporotic bones the line of the provisional calcification of the cartilage was impregnated with lime, and the proliferous cartilage was not enlarged and often was even diminished. In the older rats (about 3 months) the whole zone of provisional calcification frequently took the form of a bone plate containing one or two rows of atrophic cartilage cells, pointing to the complete arrest of longitudinal growth of the bone (see PI. 6, Fig. 13). The spongiosa was represented by a very few, sometimes isolated, thin trabeculae (see PI. 6, Fig. 13). The cortical bone was likewise very thin. The number of osteoblasts was decreased. The slightly increased resorption of bone by osteoclasts was observed in only about one-quarter of the cases, and then* chiefly only in the region of the spongiosa. Therefore the osteo- porosis in such cases must be attributed principally to the decline or even cessation of active osteogenesis. No special changes could be observed in the periosteum or bone marrow. In the above 18 rats showing slight rickets changes in the skeleton took the form of a slight increase in the proliferous cartilage (to 9 or 10 rows of its hypertrophic cells). In addition slight defects in the deposition of calcium were sometimes present in the zone of pro; visional calcification. In most cases the latter was not even enlarged and was normally impregnated with calcium salts. Only in one case of slight rickets was the zone of hypertrophic cartilage cells enlarged to 32 rows. The ingrowth of blood-vessels into the cartilage (and that not very deeply) was observed only in 3 of the 13 cases. The perios- teum and bone marrow showed no special changes. There was one indication that the changes were rachitic, the presence of an increased amount of osteoid. As I have already explained, this symptom alone suffices for a diagnosis of rickets. Therefore in all those cases in which the amount of osteoid was obviously increased, but was less than the thickness of the calcified bone, slight rickets was diagnosed. In all cases of slight rickets, with the exception of two, the activity of the osteoblasts was decreased to a more or less marked degree. The number of osteoclasts was normal or slightly increased. In accordance with the retardation of osteogenesis owing to the lowered activity of the osteoblasts in all cases of mild rickets, various degrees of osteoporosis were observed, from the mildest to the fairly severe form. Osteoporosis, however, did not reach the stage of fractured bones. (1679) F 82 In 12 rats microscopical examination showed a picture of moderate rickets The number of layers of cartilage cells ot the prohterous cartilage was increased in various degrees : in most cases the increase was moderate, up to 9-18, and in one case to 50. The cai-tilage cells mostly retained their arrangement in columns and only in two cases was it considerably disorganized. The zone of provisional calcifica- tion was either entirely deprived of calcium salts or the deposition of the latter was insufficient. On the side of the bone marrow the ingrowing of blood-vessels was observed in the case of six rats (i. e. half the number in this group). In one case in the area of the osseochondral junctions of the radius expansions of the blood-vessels in the form of fairly large cavities might be observed. The proli- ferous cartilage was sometimes directly transformed into osteoid, and stained faintly with eosin. The spongiosa consisted of a more or less dense network of trabeculae calcified internally and contain- ing columns of cartilage cells. The trabeculae were enclosed in a layer of osteoid, of a thickness that was most frequently less than that of the calcified bone, more rarely being equal or thicker than the latter. Occasionally trabeculae consisting entirely of osteoid tissue were observed. In most cases the trabeculae, and more rarely the corticalis, were insufficiently calcified, taking the form of granu- losities among which osteoid tissue might be observed. The trabeculae varied in thickness, in dependence on the absence or presence of osteoporosis. In the former case, especially in the ribs bent at an angle, the trabeculae were thicker than, or as thick as, the normal. The corticalis, as well as the spongiosa, depending on the presence or absence of osteoporosis, was of normal thickness or thinner. The osteoid on the corticalis was, in most cases, as thick as the calcified bone, covering the latter in layers of varying thick- ness on the side of the periosteum and endosteum. Very often the thickness of osteoid in different sections and sides of the corticalis of the rib fluctuated in the manner described by Erdheim (see p. 9), depending on the degree of mechanical reaction on the particular side of the bone and the rapidity of the growth of the bone in that part. My preparations thus corroborate Erdheim's observations. The activity of the osteoblasts in most sections of the bone was de- creased, but there were sections of bone covered with numerous and well-developed osteoblasts, under which there were thick layers of osteoid. In most cases the number of osteoclasts was normal, being slightly increased only on those sides of the spongiosa trabe- culae which were not covered with osteoid. Thus, in this group of rats likewise the skeleton was osteoporotic, though in most cases only in a slight degree ; the cause was the same, i. e. principally the depressed activity of the osteoblasts and, to a lesser degree, the increased activity of the osteoclasts. No marked changes were observed in the periosteum. The bone marrow in the ai'ea of spongiosa, and sometimes on the inner surface of the corticalis, was fibro-cellular, and in the case of some rats, likewise hyperaemic. All the above-mentioned changes were more marked in the ribs, and less so in the other bones of the skeleton. The calluses formed as a result of spontaneous fractures likewise bore traces of the delayed process of calcification, Their size, and the amount of osteoid in 83 them, varied with the degree of osteoporosis. In cases of slight or no osteoporosis they were very large and consisted of solid masses of osteoid, cartilage, and, to a lesser degree, of fibrous tissue. The more defined the osteoporosis, the less did the callus represent a solid mass, and the more was its tissue divided into trabeculae. In the latter case calcification of the trabeculae was more con- siderable. Moreover, the callus formed arcade-like growths of osteoid under the periosteum. The bone-marrow of the calluses was, in most cases, of a fibro-cellular character. The marked increase in the amount of osteoid in the calluses likewise pointed to rachitic afiections of the skeleton; Figs. 14, 15, and 16 (Plates 6 and 7) are examples of a more pronounced rickets produced by — A diet. Table No. 25. Chemical composition of \ the skeleton of rats fed on —A diet. ^ Age in days. In bones. Final No. Of Attheie- , ginning weight in __j^,^ . HjO Fresh Dry Histological results rat. Final. of diet gm. 0/ /o Ca% Ca% 166 50 30 75 53.1 8.7 18.5 Osteoporosis. Pneumonia 165 52 30 59 54-4 8.3 18.1 II 11 Average — 51 30 67 (- 55) 53-8 ( + 9) 8-5 (- .17) 18-3 (- 9) 195 84 34 103 43-5 10.3 18.3 Slight rickets 196 89 32 125 52.7 7.8 16-5 Moderate rickets ^ 194 89 32 110 45.9 8.6 15.9 I) II 167 91 25 105 48-1 9.1 17.7 Slight rickets 478 93 35 87 51-3 7-5 15-4 II II 509 104 45 128 45.7 8.4 15.5 Moderate rickets 505 109 22 81 49.5 7.6 15.0 II 11 238 118 50 133 41.1 8.2 13.8 „ osteomalacia 226 119 51 150 38.0 10.3 16-7 Slight osteomalacia 242 119 51 131 39.0 9.9 16.3 Moderate osteomalacia Average — 102 38 115 (- ■ 40) 45-5 ( + 14) 8.8 (- -33) 16.1 (- -25) 228 125 57 148 40.0 12.1 20-2 Slight osteomalacia 225 125 57 132 41.8 10.2 17.6 Moderate osteomalacia 461 138 70 189 42-1 10.9 18.9 „ rickets 487 139 41 82 50.9 6.4 13.1 Pronounced rickets 495 139 41 117 47.6 7-3 14-0 Moderate rickets 496 139 41 122 49.9 6-9 13.9 11 II 446 144 40 99 47.2 8.8 16-6 Slight rickets 55 147 61 123 41.9 11.0 18.9 Osteoporosis 498 173 70 90 47.1 9.2 17.5 Moderate rickets Average- — 141 53 122 (- ■ 39) 45.3 ( + 23; 1 9.6 (- -32) 16.7 (- -25) 173 175 85 186 32.7 14.3 22.2 Osteoporosis 174 175 85 145 36-5 14.6 21.8 ,; 69 195 153 192 42.5 12.9 22.4 ,1 71 195 153 180 44.1 11-1 19.8 73 195 153 150 33.1 12.6 18.8 11 Average — 187 126 171 (- -14) 37.8 ( + 3; 18.1 (- -8) 21.0 (- .6) 227 120 57 100 37.9 13.9 22.3 Osteoporosis > The figures in brackets show the percentage difference from the normal : above normal + and below normal — . f2 84 Chemical examination (see Table No. 25) showed the same general results as histological. Of the 41 rats 37 were chemically examined, including 9 cases of osteoporosis, 6 cases of mild rickets, and 12 cases of moderate rickets. In the table the rats are divided into groups according to age, and the resulting average figures must be compared with average figures of normal rats of corresponding ages (see Table No. 20). The 50-60 days' group includes only two rats, which died three weeks after the beginning of the feeding from pneumonia accompanied by a rapid loss of weight and osteoporosis. The inconsiderable changes in chemical composition of the skeleton of these animals must apparently be attributed to the shortness of the feeding period. In the second group, of 10 rats aged 80 to 120 days, the changes in the chemical composition of the skeleton were marked : with a comparatively moderate increase of water content of the bones (an average of 14 per cent.) their calcium content showed an average decrease, viz. of 33 per cent, in the fresh bone and 25 per cent, in the dry, as compared with the normal. Approximately the same figures were found in the next group (9 rats) aged from 120 to 180 days. In the last group (5 rats), aged 175 to 195 days, in which the feeding was begun at the age of about 3 months, on the whole no great deviations from the normal were observed, but even in this group two rats (Nos. 71 and 73) had a decreased calcium content: 16 per cent, in the fresh bone and 14 per cent, in the dry. RatNo. 227 stands quite apart. The two months' dieting was begun at the age of about 2 months, and its skeleton showed no difierence whatever from that of its control rat of the same litter, kept on N diet (see rat No. 213, Table No. 20). Two other rats of the same litter kept on the same diet (Nos. 225 and 228, Table No. 25) showed a slight but perceptible decline in the calcium content of the skeleton. Rat No. 227 may therefore serve as a rare example of insusceptibility to the harmful effect of lack of antirachitic factor, such as has been observed by other authors. Thus chemical examination showed that after" the rats had been fed on — A diet for a period of not less than 40 days, and at the age of not more than 70 days at the beginning of the feeding, the water content of the skeleton was increased and the calcium content was considerably diminished. Only in one case (out of 20 rats) were these changes not observed. On comparing the chemical composition of the skeleton of the rats with slight changes (osteoporosis or mild rickets) with that showing moderate rickets, the following conclusion is inevitable : in osteo- porosis the changes are least marked. But even in mild rickets there may be cases in which the deterioration in the skeletal com- position is similar to, or even more pronounced than, that observed in more severe rickets (e.g. compare, in Table No. 25, the composition of the skeleton of rats Nos. 478 and 446 with that of Nos. 196, 242, 461, 498). In some eases this may be explained by the feeding on —A diet having been begun at a more advanced age. There is no doubt, however, that on the whole the greatest chemical changes of the skeleton have always been observed in more severe rickets. Thus one and the same pathological factor — deficiency of the food 85 in antirachitic factor — caused different changes in rats, namely, either osteoporosis alone or mild or moderate rickets in most cases complicated by osteoporosis of varying severity. On the basis of the more detailed investigation of my experiments described above, some idea may be formed of the causes of these variations in the pathological pictures obtained. In Table No. 26 the rats fed on — A diet are divided into three groups, according to the state of their skeleton, and for each of these groups are given data enabling the reader to form an idea of the state of nutrition of the rat, i.e. its weight and manner of death, as well as the age at which it began to be fed on the deficient diet. Table No. 26. 16 rats 13 rats 12 rats with with with osteoporosis only . slight rickets. moderate rickets. Died 6 Killed 10 13 12 Weight declined „ arrested 15 1 7 8 2 5 „ increased 3 5 Feeding begun at the age of about 1 month 4 7 5 Feeding begun at the age of about 2 months 8 6 7 Feeding begun at the age of about 3 months and more i An examination of this table shows that osteoporosis was deve- , loped in rats with disturbed nutrition — indeed it was only in this group that one-third of the rats died from exhaustion or chance infection combined with exhaustion — and that nearly all decreased in weight. On the other hand, there was not a single case of death among the rachitic rats. The more severe the rickets, the fewer rats showed loss of weight and the more continued to gi'ow, or at any rate did not decrease in weight. Moreover, both histo- logical and chemical examination showed that when feeding was begun at the age of over 3 months, only slight changes in the skeleton were observed, chiefly of an osteoporotic character. Thus my results fully coincide with the conclusions regarding the appearance of osteoporosis in human and experimental rickets which I had arrived at in Chapter 4 on the grounds of the analysis of literary data. They likewise explain the slight changes in the proliferous cartilage and the occasionally observed more or less marked calcification of the zone of provisional calcification, I will not repeat the explanation of this already given (see p. 35). As is shown by the results of both chemical and histological examinations, antirachitic factor is indispensable to normal calcium metabolism in the organism: when there is a deficiency of anti- rachitic factor the deposition of calcium in the skeleton is decreased and retarded. Perhaps the mechanism of its action may be roughly compared to that of amboceptor, a view which I suggest as possi- ble. Another explanation suggests the necessity of the presence of antirachitic factor for ;the normal absorption .of calcium from the intestines, and the diminished absorption of calcium whien there is a 86 deficiency of antirachitic factor in the diet. This question can be finally settled only by an investigation of metabolism, which has as yet not been done. Group II, of rats on —A diet whose- mother had also been fed on that diet during lactation. In one experiment a ratj after giving birth to six young, was placed on — A diet during the whole period of suckling of the young rats. The mother rat was kept on the diet No. 1 before and during the pregnancy. The young rats, after having been weaned at the expiration of about a month, remained on —A diet. Three of them were killed at the age of 70 days, and three at the age of 85 days. After separation from the mother the young rats gained about 10 gm. on an average in weighty and then the increase in weight was arrested. Of the rats killed at 85 days of age one (No. Ill, see Table No. 27), with symptoms of osteoporosis. Table No. 27. Chemical composition of the skeleton of rats on — A diet whose mothers were fed on the same —A diet during lactation or during fregnancy and lactation. No. of rat. 106 107 108 Final. At the day when separated from mother. Histological Results. „. , Intones. Final . weight " ^ fresh, dry. »'« 0/ Ca Ca gm. /o o^ o/^ (1) Eats whose mother was fed on —A diet during lactation. 70 25 46 50-1 5-7 11.6 Osteoporosis 70 25 48 53.9 5-4 11.6 Moderate rickets. 70 25 43 54.3 4.9 10.8 Slight rickets. Osteoporosis Average 70 25 46 52.8 5.3 11.3 (-76) (^21) (-54) (-45) per cent, above ( + ) or below (—) normal. 109 85 26 46 59.1 5.4 18.4 Slight rickets. Osteoporosis 110 85 25 46 54.6 6.1 13.5 tt J) iy TT 111 85 25 37 62.5 6.6 14.9 Osteoporosis Average 85 25 43 58.7 6.7 13.9 (-77) ( + 25) (-56) (-35) per cent, above ( + ) or below ( — ) normal. (2) Rats, whose mothers were fed on —A diet during pregnancy and lactation. 226 b 37 36 20.5 70.2 2.3 7.6 Moderate rickets 225 c 37 36 23 64.5 3.0 8.6 Slight 225 D 37 36 17 70.4 2.1 7.0 Severe ,, . Osteoporo- 226 E 37 36 14 66.0 3.2 9.5 Osteoporosis sis 225 F 37 36 19 704 2.5 8.1 Moderate rickets Average 37 36 18-7 68.3 2.6 8-2 (-73) ( + 23) (-64) (-60) per cent, above ( + ) or below (— ) normal. 406 64 30 34 56.7 6.5 15-1 Slight rickets. Osteoporosis 413 68 30 61.5 5.7 14.7 Moderate rickets. Osteoporosis A 68 25 42 68.9 5.5 136 ii ij If n ' 408 75 30 38 56.4 7.2 16.6 Slight rickets. Osteoporosis 414 76 30 54.6 7-3 16.2 Osteoporosis 411 77 30 46 57.4 6.6 18.7 Severe rickets. Osteoporosis D 79 26 46 55.9 6.9 16.7 Osteoporosis Average 72 29 41 67.8 6.5 15-4 (-79) ( + 30) (-43) (-25)pereent. above ( + ) or below (-) normal. 87 died when considerably reduced in weight ; the others had not lost much weight (some 2 gm.), but were killed upon the same date to afford a comparative observation. Notwithstanding the slight loss in weight, all the rats were exceedingly cachectic, their fur being covered with bald patches (see Pi. I, Fig, 2). Their teeth were quite opaque. Xerophthalmia and anaemia were very marked in all. Spontaneous fractures of the ribs were observed. Microscopical examination showed moderate rickets in one case (see Fig. 18), thi-ee rats had mild rickets, and two had osteo- porosis. The bones of the rachitic rats were also osteoporotic to a considerable degree, which may be explained by their cachectic condition. The proliferous cartilage was only slightly enlarged (up to 8 or 10 rows). In the zone of provisional calcification in- sufficient deposition of lime was observed in one case only. The abnormal ingrowth of blood-vessels into the cartilage was observed in a slight degree only in ribs of three of the rats. The sub- chondral spongiosa consisted of a small number of thin trabeculae containing unabsorbed cartilage cells in the upper portion. The amount of osteoid was abnormally increased in four cases. The osteoid covered both the inner and outer surface of the bone in a layer of varying thickness : in two cases in most sections of the bone the layer was equal to, and in one rat was in some places even thicker than, the layer of calcified bone. In two other cases of rickets, though most of the layers of osteoid were abnormally thick, they were thinner than the calcified bone. The number of osteo- blasts was diminished, while that of osteoclasts was increased. In the rachitic rats the callus of the spontaneous fractures consisted of cartilaginous and osteoid tissue with a comparatively small amount of fibrous tissue. On the other hand, in the case of rats with osteo- porosis alone, the callus consisted chiefly of fibrous tissue. The chemical composition of bone showed a moderate increase in water content (21 to 25 per cent.) and a great impoverishment in calcium — 54 to 56 per cent, below the normal in fresh bone, and 35 to 45 per cent, below in dry bone. Thus in this group of rats, in connexion with marked cachexia, there was a much lowered activity in the formation of new bone, and a more marked disintegration of bone. These phenomena, of course, prevented the development of severe rachitic changes in the bones. Nevertheless, the rachitic picture was clear fi.-om histological examination in four cases. Chemical analysis pointed to a very profound disturbance of the process of bone calcification in aU cases. Group III, of rats on —A diet whose mothers were fed on the same diet during pregnancy and lactation. As is known, it is a rule that rats kept on —A diet have no offspring, and therefore the males are usually left together with the females. Occasionally, however, they do bear young. This was recently observed among my rats in one case, and amongst the rats of Dr. Zilva ^ at the Lister Institute in three cases. I availed myself of this material in order to determine whether the feeding of parents on —A diet during the periods of conception, pregnancy, and lactation has any dete- riorating effect on the condition of the skeleton of their ofispring. ' I wish to thank Dr. S. Zilva for allowing me to use these rats. 88 After separation from the mother the offspring continued to be kept on —A diet. In all, I had at my disposal 18 rats from 4 mothers. Unfortu- nately, at the beginning of my investigation I tried to keep the rats as long as possible on —A diet, and, as a consequence, all the in- vestigated rats were in a state of severe cachexia. Thirteen of them died, and only five were killed, in a very poor condition. The final age of 5 rats was 37 days, the rest being from 45 to 79 days old. As in the other experiments, the cachexia of the rats was the result of a deficiency of the food in antirachitic factor, of the marked decrease of appetite towards the end of the experiment, and of the frequently occurring infection — pneumonia or enteritis. All the rats had opaque teeth. On macroscopical examination the only in- dications of rickets were the spontaneous fractures of ribs, and their occasional angular curvature. Microscopical examination yielded analogous results in the rats of all four litters. In general, of the eighteen rats of this series, in four cases there was only osteoporosis, in five mild rickets, in five rickets of medium severity, and in four severe rickets. In all the rats rickets was complicated by severe osteoporosis, an idea of which may be gathered from Figs. 19 and 21. Osteoporosis was to have been expected, as a result of cachexia and an insufficient intake of food. In the rats suffering from osteoporosis alone, the picture of the skeleton was that usual in that disease, as already described in the preceding groups of rats on —A diet. It is, however, necessary to observe that in one case the proliferous cartilage was enlarged sixteen times, and the zone of provisional calcification showed defects in the deposition of lime salts. In these Cases likewise osteoporosis was caused chiefly by the decreased number and lowered activity of the osteoblasts. The osteoclasts were increased in number only in some places, principally in the region of the spongiosa. In slight rickets there was a slight enlargement of the proliferous cartilage with defects in the zone of provisional calcification. The line of costochondral junction was irregular, owing to the slightly irregular ingrowth of blood-vessels. The spongiosa was composed of a very few thin trabeculae, consisting chiefly of a row of cartilage cells, either completely deprived of osteoid or covered only with a thin layer of the latter. The amount of osteoid both in the spon- giosa and the corticalis was only in a few parts equal in thickness to the calcified bone ; in most parts of the bone the amount of osteoid was equal to half the latter, or even less. In the cases of moderate rickets the enlargement of the zone of hypertrophic cartilage cells in the ribs reached from 25 to 30 rows, and even more. The zone of provisional calcification was either almost entirely deprived of lime salts, or the deposition of the latter was very defecti ve. The bone-marrow blood-vessels had grown deeply into the cartilage, branching out widely and irregularly. Near the blood-vessels the cartilage had been transformed into osteoid. Most portions of the bones were covered by the latter in layers approxi- mately as thick as the calcified bones. Some of the trabeculae of the spongiosa consisted of osteoid alone, others contained a con- siderable portion of calcified bone. In the callus resulting from 89 spontaneous fractures osteoid was likewise observed in great quantities. In sevei'e rickets the osteoid tissue was in excess of the calci- fied bone (see Fig. 17). In all stages of lickets the number of osteoblasts was decreased, but in varying degrees : in severe rickets in some cases it was nearly normal, in slight rickets the de- crease was greatest. The number of osteoclasts was somewhat increased. In all cases the most marked changes were observed in the ribs, next in the radius, and least in the humerus. Chemical examination was made of the skeleton of 13 out of 18 rats (see Table No. 37) and showed a moderate increase in the water content (33-30 per cent.) and a great deficiency in calcium, the content of the latter being 43-64 per cent, below the normal in fresh bone and 25-50 per cent, below in dry bone. Thus certain changes are observable in the skeleton of rats whose mothers were kept on — A diet as compared with those of rats whose mothers were kept on a normal diet. In the former the retardation or arrest of growth takes place earlier and to a greater extent; there is a greater deficiency in the skeleton of calcium salts, and cases of manifest rickets, even with a preponderance of osteoid tissue over calcified, are more frequent. It is true that, as shown by Fig. 16, even among the rats of the second group there may be cases of pronounced rickets (rat No. 487). This rat No. 487, as well as rats 495 and 496, belonged to a litter whose mother during pregnancy and first five days of lactation was on a diet similar to No. 1, but without addition of milk and calcium salts. The diet of the mother was therefore to some extent deficient in calcium and antirachitic factor. Such marked changes were only once observed by me among the rats of the second group. Thus, feeding the mother on a diet deficient in antirachitic factor during pregnancy or lactation causes a marked deterioration in the general condition and composition of the skeleton of the offspring (provided that offspring continues to be fed on a deficient diet after separation from the mother). In order to elucidate further the influence of the diet received before weaning upon the production of, or protection from, rickets in the young offspring, experiments were made upon the following groups IV-VII of rats. Group IV. Rats whose suckling inothers were kept on different special diets during lactation. This experiment was conducted on 5 mother rats and 31 offspring whose dietary at different periods of the experiment was as follows : Table No. 38. No. ofsticMing Diet. motheT and of rats suckled by During During Ofyowng her. pregnaney. lactation. after weaning. 606 N N N (control) 606 N N -A 602 N — A + 2C.C. of -A milk per diem 608 N -A -A 604 N — 90 The littel-s of four of these mothers (602, 603, 605, and 606) were mixed, so that each suckled some of her own and some of another's. The litter of a fifth mother (604) was equally distributed amongst these four. By this means it is possible to see the influence of the period of lactation only. After being weaned, the young rats of group 606 on N diet consumed on an average about 18 gm. of food per diem (towards the end of the experiment — up to 30 gm.). The young rats of gi'oup 605 consumed on an average 16 gm., and sometimes a little more (17-18 gm.). Moreover, in spite of their being on —A diet, there was no loss of appetite towards the end of the experiment. The young rats of group 602 and 603 consumed an average of 8 gm. (from 13 to 6 gm.). The consumption of —A diet in litters 602 and 603 decreased towards the end of the experiment, as has been usually observed. Fig. 42 shows the curves of the weights of average young from each suckling mother. 12 RAT'S [malc) 9 RATS frerM ale) Fig. 42. Weight curves of average rats (male and female) from litters 602, 603, 605, and 606. The weight curves of the average I'ats from litters 602 and 603 are very similar. That of litter 605 approaches the curve of the normal rats in litter 606. The diflerence between their curves is due to difference in the diets of suckling mothers and offspring during lactation, normal for rat series 605, deficient in antirachitic factor for rat series 602 and 603. Mother 602 received in addition to her diet 2 c.cm. of milk per diem ; the young suckled by her, however, received no cow's milk. The following Tables (Nos. 29, 30, 31, and 32) show the chemical composition of the skeleton of young : 91 o as •3 s> ■3 ? I o ° 2 fi S ^6 odsodsdioTHoo ft-"^ d e1 ^1 ^9< ^^ ■•jf ^1 ^3^ "'j^ ^fl* ^^ III ' St I I I I I I I I I " » tQ a, g >> i"^l cq lO ^ t* ^ -^ us I CO Gfl *^ t* *"• -< 1-1 i~* T-t 1-t : -^co«oojgcoc2ooI>OOI>00 ^^ 00l>0000000000> O '-<^'-Hi-1i-H!Mt-1i-1 i3 -to o fi .§ § I CO Cq • oi ds o A r-( oi O U3Q0CD00'<^^r-(-^ ill Ut !Si§S!3!3Si3^ 1 •« «, I ■§ ■« 8 = 5. ^ §1 ^ g =i O CO 6 1-5 m < S-l S Q0t"OTC0l0C00SO fiO r-l 00 U5 O < lO CO (M CQ CO I I I I I I I I •^ '\3 '^ O*-"^ O+l^ =><• iHO^CO-^kOCOt^QO v-x ^-^ v_y *_• — /• — ^ ^^ >^^ u3>ou3Udiau3ur)Ud oooooooo CO CO CO 50 CO CD CO CO 92 i-Sj I >.§ It i - -S g 1^ g o '^i^ ^ CO d N H ~ 1; M O 03 o S- e s I ^1' Oft Co 00 A CO iO ^ ^ eO "5 (N '<* CT :^ ^ tH iH »-( jH T-l iH O CO U5 SO "^ o w t> ^ kO 03 CD -^ U3 Cq (M OS OS t> tD *? CO ^ -^ 00 "jH I> CO lO U5 ?0 tH lO ?0 50 lO lO -^ lO "3 «0 «0 -^ii -^ OT -«* '* CO C<1 ifi U5 -^ lO lO 5D «0 O t* lO t> I> (O *o O O O O O O o CO CO CO CO CO CO CO O 0> l>- '<* N O W O 00 CO V 00 -^ lO ** ^S la W3 (M CO CO CO CO I I I I I I I r^ ©I CO -^ lO eo t^ \_^ \_^ ^.-'^^ — ^ ^^ — ^ CO CO eq (M eq iM eq o o o o o o o <0 CO (0 ^ QO CO eo U5 CO 5- g5 5. ■S <= •11 1 S s M IB '^ ^ to -S ■§ T" © 'r* .0 ^ pS ^ ^ ' '1 l-§l ■1 • 1 iM s ==1 = ■^ '-T S CQ 3 OT iS5 «o -^ M 5D l> CO -^ "^ ■* W W CO q s So ■ W ^ -^ -i* 5D U5 > t> «D to ui 16 cb Oi t> CO CO tH O OS ."fe.g'^. 11 I lig^s.g'e 0000000 W CO CO W CO CO M .8 6 CO d §=61 I . ''^ §'"fc ^ OOOIO-HICOCO-* ^2^S ■*^eococococo 'f^ I I I I I I I iS'fe.'! , Ot CH Crt-Ot Ot CH 1-1 GO CO -^ kO CO t« CO CO CO CO CO CO CO 0000000 CO CO CO CO CO CO CO to CO 93 The average chemical composition of the skeleton of the young in these four groups was as follows : Table No. 33. Diet. No. of litter. of mother during lactation. of young after weaning. H.0% Fresh bone. Ca% Dry bone. Ca% 606 605 603 602 N N -A —A + 2 com. milk N -A -A -A 43.7 46.4 62-8 52.7 11.4 10.1 6.4 6.6 20.3 18.8 18.5 14.0 The macroscopical examination of the rats also showed marked differences : the rats of group 605 were very fat, had no abnoi-mali- ties of the skeleton, and differed from the control group 606 only in having slightly opaque teeth. However, the i-ats of groups 602 and 603 had completely opaque teeth, no fat, and about one-third of them were emaciated. The only macroscopical changes in the skeleton were fractured ribs : this was found in 2 rats of group 602 and in 5 rats of group 603. Macroscopical examination of the rats, the chemical composition of the skeleton, and weight curves of the young on — A diet whose mother was on N diet during lactation (No. 605) show a strikingly increased resistance to the unfavourable influence of —A diet. Chemical investigation shows in litter No. 605 a skeletal composi- tion not far from, normal, whilst litters No. 603 and No. 602 show a marked deficiency in calcium content and high percentage of water in their bones. The addition of 2 c.cm. of fresh milk daily to the diet of mother rat 602 showed no marked influence on the young when compared with those of rats 603. Finally, it can be seen that the chemical composition of the skeleton of rats of group 606 corresponds well with that given elsewhere for normal rats in Table No. 20 age groups 60-80 days (see p. 65) and in Table No. 45 rats 610 A and b (p. 107). At the same time the composition of the skeleton of rats of litter 605 corresponds well with analysis given for similarly fed rats in Table No. 35, p. 96. The results of microscopical examination coincided well with those of macroscopical and chemical examination. In group 606 the structure of the skeleton of the young rats was normal (see Figs. 22 and 28). As may be seen in Fig. 23, the ribs of some of the rats show a slight concave flexure of the cost.o-chondral junctions, which I have sometimes observed in the case of some rapidly growing normal young rats. Group 605 did not differ greatly from the normal rats of group 606, and the skeleton of most of the rats of this group was nearly normal (see Figs. 23, 24, and 29, Pis. 9, 10, and 1 1). Thef oUowing abnormalities of the histological structure of the bones were, however, noted in some of the rats of this group : slight osteoporosis, a greater concavity of the costo-chondral June- 94 tions, and sometimes in rapidly growing bones (ribs) a slight deficiency of lime deposition in the zone of provisional calcification. The amount of osteoid was within normal limits (see Figs. 23, 24, and 29). In contrast to this the skeleton of the rats of group 603 in most cases showed a picture of manifest rickets of the nature of late rickets, i.e. without a.ny great increase in the proliferous cartilage. The number of layers of hypertrophic cells did not exceed 12. The zone of provisional calcification was defective. There was an irregular growth of blood-vessels into the cartilage. In some rats th6 spongiosa under the costo- chondral junction formed a rachitic metaphysis consisting of osteoid and cartilage, which was being directly trans- formed into osteoid tissue. Most frequently the amount of osteoid equalled that of calcified bone, and less frequently was smaller or in a few cases even exceeded it. The number of osteoblasts in the region of the spongiosa was normal, and they were clearly defined, while in the region of the diaphyses it was most frequently insuffi- cient. The number of osteoclasts was normal or slightly increased. The most marked rachitic changes were observed in the ribs. On the whole, the changes corresponded to moderate rickets, and less often to mild rickets (see Figs. 25, 26, 27, 30, and 31). In group 602 the skeletal changes were completely analogous in character to those in the bones of group 603, but were not quite so marked : in no case was rickets of moderate degree found, whilst in group 603 this was present in 4 out of 7 cases. Apparently, the addition of 2 c.cm. of milk per diem to the diet of the mother during lactation hindered the development of more severe rickets. In three rats of this group which had died before the tei-mination of the experiment (602 (3), 602 (6), 602 (7)) the skeleton showed, not rickets, but osteoporosis and great hyperaemia of the bone-man-ow, i. e. this experiment likewise affiDrded a fi-esh confirmation of the correctness of the above-mentioned view as to the causes producing osteoporosis on a — A diet. On the whole the results of the experiment with Group IV point to the fact that the feeding of the offspring before weaning on a diet rich in antirachitic factor makes them strikingly insusceptible to the unfavourable effects of a diet deficient in animal fat. Never- theless it is true that the unfavourable effects of the deficiency of antirachitic factor were clearly observable, even though only in a slight degree, principally in the weight curves and the chemical composition of the. skeleton. Group V. Changes in the skeleton of rats on ~A diet whose mothers were fed on different diets during the period of lactation. The experiment was conducted upon a group of rats consisting of 8 mothers and 59 young i-ats. The results of the experiment ai-e shown in PI. 8, Fig. 20, and PL 17, Figs. 43 and 44, and Tables Nos. 34-43. The young rats were killed at the age of 65 days, the feeding on — A diet extending over a period of 41 days after wean- ing in the case of those which survived until the end of the experiment. Four mothers (618, 619, 620, and 621) had their litters mixed as in the preceding experiment, i. e. each mother rat suckled some of her own offspring and some of another rat's. They had a total of 38 young rats, 11 of which died during the experiment, so 95 that 27 remained to be killed at the end of it. Each of the remain- ing four mothers was left to suckle her own young, the total number being 21, of which 3 died before the end of the experiment. The diets of the mothers during pregnancy and lactation, as that of the young rats, are shown in Table No. 43, and partly in Tables Nos. 35-42. N-Ca diet of the mother rat 619 during lactation contained as the source of antirachitic factor butter only (without cod-liver oil). The amount of food consumed per diem by the young rats after weaning fluctuated as follows (Table No. 34) : Table No. 34. Rat of litter 618 about 13-20 gm. „ 619 „ 7-8 „ , 620 „ 5-7 „ „ 621 „ 7-9 „ „ 622 „ 9-11 „ „ 623 „ 8-12 „ „ 624 ,, 10 „ „ 625 „ 5-8 „ As may be seen from Table No. 34 the smallest amount was consumed by litters 620 and 625, whose mothers had been fed on — A — Ca diet during lactation. The consumption was greatest, and frequently almost normal, only in litter 618, whose mother had been fed on N diet during lactation. The lower figure stated for groups 619-625 refers to the amount consumed at the end of experiment. The weight curves of the average rats of different litters (see PI. 17, Figs. 43, 44), both male and female, show a marked difference between the growth of rats who had been fed on N diet during lactation, and those who had been on a deficient diet. There was no very substantial difference between the weight curves of the rats kept on different deficient diets during lactation. Rats of litter 618 continued to grow approximately at the same rate as the analogous rats of group IV (of. the weight curves in Fig. 42, p. 90, with those in Figs. 43 and 44). Of all the rats of litter 618, only one (618 (9)) was weak and slow of growth (see Table No. 35) from the very beginning of the experiment, even while suckled by its mother, on a diet sufficient in every respect. Therefore this should be regarded as accidental, and, strictly speaking, this young rat should not be taken into account at all. This case on histological examination revealed no signs of rickets, although the chemical analysis showed considerable decrease in the calcium content of the skeleton, i.e. 21-6 per cent, below the normal (in dry bone). The autopsies of the rats also showed a marked difference between litter 618 and the rest. The rats of litter 618 were fat, with well- developed muscles, and without any pathological indications. The autopsies of the rats of litter 619, whose mother was fed during lacta- tion on N — Ca diet, containing no cod-liver oil, showed severest indications of rickets : macroscopically opaque teeth, indented thorax, angular curvature, and numerous spontaneous fractures of the ribs, &c. 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Of the 10 rats of this litter, seven died before the end of the experi- ment from cachexia and gastro-enteritis. The only macroscopic indications of rickets were opaque teeth, and, in some cases, frac- tures of the ribs and thickening of the costo-chrondral junctions. The rats of litters 621, 622, 623, 624, and 625 were nearly all more or less emaciated. They had opaque teeth, fractures of the ribs, and in some cases a very marked thickening of the costo- chondral junctions. Some rats of these litters also showed indented thorax and angulation of the ribs. The most marked rachitic changes in the above-mentioned litters were observed in litter 623. Two rats of this litter (623 B and 623 C) showed not only curvature of the ribs, but of the spine likewise, as well as of some of the bones of the extremities. The chemical changes (see Tables Nos. 35-42) corresponded, on the whole, to those observed in the rats of Group IV, i. e. first of all, they showed the same striking difference between the composition of the skeleton of rats whose mother was on N diet during lacta- tion (618) and the skeleton of rats whose mothers were on deficient diets (619-625) during lactation. As I have mentioned on the basis of the figures of Table No. 20 and also No. 29, the chemical composition of the skeleton of normal rats at the age of 60 to 80 days is practically constant. It is noteworthy that the figures of the chemical composition of the skeletons of litters 618 (Table No. 35, p. 96) and 605 (see Table No. 30, p. 91) are also nearly identicaL Thus cod-liver oil, P, and Ca contained in N diet and administered in sufficient quantities to the mother during lactation and to the young in addition to mother's milk before weaning preserve the offspring from the rickets-producing effects of —A diet given subsequently. A somewhat more marked deterioration of the chemical composition of the skeleton was obtained in rats whose mothers had not received sufficient calcium during lactation (619, 625) or during pregnancy (623). In this respect litter 620 formed an exception. The histological examination was completely in accordance with the results of the examination of the weight of the rats and of the macroscopical and chemical data. The histological diagnosis of the affections of the skeleton is shown for each rat in Tables Nos. 35-43. In general, it is necessary to mention that in the rats of litter 618 the skeleton differed from the normal only in being slightly osteoporotic. This was caused by a small increase in the number of osteoclasts. The osteoblasts were well developed and normal in amount. As in group 605 (p. 93) some of the rats showed a slightly greater convexity of the line of costo-chondral junctions in the direction of the bone marrow. In some rats the histological structure of the whole skeleton was nearly normal (e.g. 618(2)). Among the rats in groups 621, 622, 624, kept on —A diet during lactation, the most prevalent picture was that of moderate rickets, 104 jusfc as in the corresponding group 603 of the preceding experiment. As in the preceding experiment this must be explained partly by the presence of considerable quantities of cod-liver oil, calcium, and phosphorus in the diet No. 3 of the mothers during pregnancy. To a certain extent this view is supported by litter 623, whose mother had no cod-liver oil or separately administered calcium and phosphorus during pregnancy. The skeletal changes of the rats of this litter, as compai-ed with others, were more markedly rachitic. Apparently the deficiency of the mother's diet in calcium during lactation, with the presence in the diet of a "normal, but moderate amount of antirachitic factor ^ (litter 619), may be a specially suitable combination for producing severe rickets in the offspring, fed on a diet deficient only in antirachitic factor after weaning. It was in this litter (619), and also in the following experiments on litters 610 and 314 (pp. 106-7), that severe rickets was observed. The deficiency of the diet during lactation in antirachitic factor and calcium simultaneously is frequently too great a disturbance, leading to emaciation of the offspring (litter 620) and large mortality from infectious diseases. It is necessary to mention that with this diet the appetite of the offspring was at its lowest. Naturally, the osteoporotic effect on the skeleton of an insufficient intake of food is very marked in litter 620 and 625. I have obtained the same results from feeding rats on a — A — Ca diet during the period of lactation in another experiment (see p. 121). Among the rats of litters 620 and 625 the most prevalent picture was that of moderate rickets with osteoporosis. Group VI. Rats fed on —A. diet, whose vnother was kept on Diet No. 1 (but without milk and additional calcium salts) during pregnancy and lactation. This experiment was made on 3 rats of litter 314 and was to a certain extent a repetition of the experiment on litter 623. The diet No. 1, but without milk and additional calcium salts, was moderately deficient in calcium and antirachitic factor. After weaning the offspring were kept on —A diet. The results of this experiment are shown in Table No. 44. The young rats after separation from their mother consumed about 9-10 gm. of food per diem. This experiment was carried on for a much longer period than experiments in Groups II— V : the young rats were kept on — A diet for a period of 78 days and were killed at the age of 118 days. On autopsy rat 314a was not emaciated and had a small amount of fat, perfectly opaque teeth, and skeletal changes characteristic of severe rickets. Rats 314b and 314c were emaciated, and the only indications of rickets were spontaneous fractures of the ribs. The chemical composition of the skeleton showed a considerable increase in water content, about 35'4 per cent, above the normal, and a great impoverishment in calcium — 46-2 percent, below the normal in fresh bone and 30-3 per cent, below normal in dry bone. Histologically, in litter 314 there was a difference between rat A (pronounced' rickets, see Fig. 11, PJ. 5), which grew steadily though 1 Diet containing as the source of antirachitic factor butter only, but no cod- liver oil. 105 slowly and was fairly well nourished, and rats B and C (slight rickets and osteoporosis) with aiTCsted growth and emaciation. Thus this experiment showed the same results as the somewhat similar experiments with rats 487, 495, and 496 (see p. 89) and with litter 623, namely the appearance of pronounced rickets in the better nourished rats of the series when a diet moderately deficient both in calcium and antirachitic factor was given during pregnancy or lactation. Group VII. Skeletal changes in young rats on N, N — Ca or — A diet. N — Ca diet during lactation. Litter 610. In this experiment the mother-rat was fed on diet No. 2 until the birth of the 10 young rats. Diu-ing lactation, which continued 28 days, the mother-rat was on N — Ca diet, in which butter was the only fat; therefore neither the mother-rat during lactation nor the young rats received any cod-liver oil. After the young rats were separated from their mother (at the age of 28 days) they were divided into 3 groups : A and B were put on N diet, C, D, and E were left on N — Ca diet, and F, G, H, K, and L were put on —A diet. After 48 days, at the age of 76 days, the young rats were killed. Those on N diet consumed about 12 to 20 gm. of food per diem, those on N — Ca or —A diet about 6 to 8 gm. The weight of the rats is shown in curves (Fig. 45, PI. 17). The results of the chemical and histological analysis are given in Table No. 45, p. 107. The weight-curves show that the retardation in weight as com- pared with the rats on N diet was approximately the same in the « case of the rats fed on either of the deficient diets. Only one rat (610 h) died before the end of the experiment (in the group on —A diet). On autopsy the young rats on N diet were found to be fat and showed no abnormalities. It is necessary to mention the striking recovery on that diet of one of the rats (610b), separated from the mother when weighing 13 gm. only and in condition of severe cachexia. In the rats on deficient diets, the macroscopical indications of rickets were very marked in the case of all three on N — Ca diet, and in a lesser degree in 2 out of 5 rats on — A diet (610 K and 610l). These two latter stiU had a little fat, and their muscles were well developed. Rat 610 F had no fat, but its muscles were well developed. Eats 610 G and H were emaciated, especially the latter, which died before the end of the experiment. The rachitic changes were of the usual character : curvature of the ribs, spine, and sometimes of the bones of the extremities ; com- pression of the thorax, numerous fractures of the ribs and fibula, and thickening of the costo-chondral junctions. These changes were most marked in rat 610 E. The chemical composition of the skeleton of rats on N diet showed normal water and calcium content (com- pare with analogous figures of rats at the age 60-80 days in Table No. 20, p. 65). The chemical examination of the rats fed on N — Ca diet yielded the same results as in the above-mentioned analogous experiments (see Table No. 21, second series, rats aged 67 days), i.e. great impoverishment of the skeleton in calcium and increase in .^ 106 r-O S o o CO ^ I 6 so ^ ^ CO S i 05 p .M qoo^ ^ -* «o C CD Tj4 CO CD ^ 06 ■^ 00 CO CO CD 06 liO IC '^ I >^ a -I ■ e -^-e i^ = ■S ^ rg 9d la CO ^ I (M CO ^ CO ^ sis l> CT i-l a s >-i CO 00 s- S K o 0> CO g A. ^ 10 ^ 10 ■S I 5- o O /€ ^1^^ 9,^ : ^ ^ • OS o >> CO OT "^ 00 00 Oi 00 W 00 lO CO CO CO N CO iO CO o '-' CO ^ T^ M M (^ CO CO t^ CD 00 50 «D W »0 ^ CO CO CO I> ?D 00 -^ O -* w -i!ti -^ la ITS ic o e o o o base's oooocx) 8 S LSi \!JX g S s ^ GO 00 00 -^ -^ -^ |> I> o ^ 00 CO W 00 00 '^ •^ ''H ^ ^ ^ fl CO o o to ■ l> O l> t> CO 03 I> X CO '^ w C5! 00 rH d JO 6 m n ry. Histological Results. of- Final. separated from weight HjO Ca Ca rat.' mother. ingm. % % % 117 40 26 30 64.1 3.7 9.5 Osteoporosis 119. 40 26 30 63-3 3-3 8.6 Slight rickets 115 42 26 29 65-1 2.7 7.6 U }f 244 a 46 23 593 3-0 7.3 JJ JJ 244 b 46 .24 58.6 2.9 6;9 Moderate rickets 244 c 46 31 58-7 3.1 7.6 jt . It 2-1 4 D 46 — 23 59.4 2.9 7.2 ij n Avera ,ge 44 27 61.2 3-1 7-8 (-64) .( + li) (-61) (-54) per cent, above ( + ) or below (— ) noi-mal 121 51 28 45 54-6 3-8 83 Moderate rickets (-68) (+11) (-63) (-59) per cent, above (+) or below ( — ) normal 122 70 28 47 53.9 4.1 8-8 Severe rickets 125 70 28 43 61.4 3.8 9.8 Moderate rickets Average 70 28 45 57-7 4-0 9.3 (-77) ( + 32) (-65) (-55) per cent, above ( + ) or below ( — ) normal. (c) Conclusions. The conclusions which may be drawn from the above experiments in feeding rats on a diet deficient both in antirachitic factor and calcium are as follows : (1) The changes typical of rickets occur, in my experiments, most readily in rats kept on a diet deficient both in antirachitic factor and calcium. (2) The changes in the skeleton are the result of (a) elimination of the influence of antirachitic factor on calcium metalaolism, which causes rachitic disorder's ; (h) the effects of calcium starvation, intensifying the rachitic changes in the bones ; (c) the osteoporosis-producing effects of eliminating the func- tions of fat-soluble factor and calcium, elements which conduce to the normal gi'owth of tissues and to the maintenance of a normal appetite. (3) The skeletal changes on — A — Ca diet differ from those on a diet deficient in antirachitic factor alone in that the newly formed bone remains unimpregnated with calcium salt (osteoid) to a con- siderably greater extent. (4) The above experiments confinn the observations on human rickets (Schabad, Aron, Dibbelt, &c.), showing the importance of calcium starvation as one of the possible factors in its development. (5) Experiments in feeding the mother on — A — Ca diet, as well as on diets —A and N — Ca, during lactation, have confirmed the con- clusion as to the importance of the normal nutrition of the mother for the normal development of the skeleton of the offspring.^ 1 (After completion of the book.) McCollum, Simmonds, and Kinney in 1922 produced in rats the changes, described by me (1921), on a complicated diet. 123 14. The Author's Expeeiments in feeding Rats on the Basal Diet, beficient in Antirachitic Factor, and without THE addition OF A SaLINE MIXTURE ( — A — SM). Seven rats were fed on the basal diet deficient in antirachitic factor, but without the addition of the usual amount (5 per cent,) of the saline mixture employed by me.^ As compared with the normal, such a diet was poor' in antirachitic factor, calcium, phosphorus, mag- nesium, potassium, sodium, and iron. It contained about 0-045 per cent, calcium and 0'2 per cent, phosphorus. At the beginning of the experiment the rats consumed about 15-12 gm. of food per diem, then about 9 gm., and towards the end of the experiment about 6 gm. The skeletons of these rats were not chemically examined. The outward appeai-ance and behaviour of the rats, macro- and microscopical examination of the skeleton showed no signs of any difference in the results as compared with the results obtained with the — A — Ca diet. The dieting was begun at the age of 21-27 days, and continued for 1 or 2 months. Four rats died from cachexia, and three were killed in a very bad condition. According to the intensity of the changes the rats may be classed as follows : Osteoporosis only Mild rickets 3 Moderate rickets 1 ' Severe „ 3 They all suffered from osteoporosis to a greater or less degree, owing to the causes which have already been detailed in the chapters on —A and — A — Ca diets. The changes obtained may be easily seen from Fig. 39, representing the costochondral junc- tion of the lib of the rab suffering from severe rickets. Thus, this group of experiments permits of the conclusion that when the normal saline mixture is excluded from a diet deficient in anti- rachitic factor, the chief effect on the skeleton is due to loss of calcium in the mixture. 15. The effect of Elementary Phosphorus on the Skeleton op Animals on a Diet either rich or poor in Calcium. The toxic effect of phosphorus on the osseous tissue, taking the form of so-called 'phosphoric ' necrosis of the bones, has been known for a long time. Offer (1899) compared the chemical composition of a normal jaw with that of one affected with phosphoric necrosis, and obtained the following results. (Table No. 51.) deficient in antirachitic factor and calcium (containing about 0-053 % Ca and 0.365 % P), 1 For the composition of the diet — A— SM see pp. 7-9. 134 Table No, 51. Normal Jaw with phos- jaw, phono necrosis. 0/ 0/ /o /o Matter soluble in ether 0.53 1.19 Organic matter . . . 29.68 30-17 In ash : CaO 53-02 54-61 ,, „ MgO 1-14 1-16 „ „ PjOs 82-4 40-56 Kochmann (1907) experimented on rabbits with acute and chronic phosphorus poisoning. Four rabbits served as controls, four were suffering from acute poisoning, and two from chronic. The sub- joined Table No. 53 shows the changes in the composition of the bones. Table No. 52, Ash content in . , t ■ j /n/\ dry bones ^'^ contained (%) : % Calcium. Phosphorus. Normal bones 54-7-58-9 87-8 18-0 Bones in acute poisoning 57-0 — 59-4 37-1 17-4 Bones in chronic poisoning 64-0-64-8 39-0 20-3 Thus the investigation of human subjects and experiments on animals showed that in chronic phosphorus poisoning there is an increase of ash, phosphorus, and calcium in the bones. Even on the grounds of chemical investigations alone, this influence of phos- phorus suggested the possibility of its being useful in rickets. On the basis of histological investigations, Wegner (1872), whose ex- periments are regarded as classical, came to the same conclusion. He experimented on gi'owing and on adult rabbits, cocks, cats, calves, and dogs. On administering small doses of phosphorus to the animals (about 0-00015 gm. per diem in pillules), new forma- tions of compact bone were observed in the zones of endochondral bone, instead of the normal spongiosa. The bone-marrow cavities were reduced to the size of the Haversian canals. The cortical bone likewise became more compact because of reduction of Haversian canals and of the size of bone-marrow cavity. In some of the animals (cocks), owing to the vigorous new formation of bone, the marrow cavity was almost completely occluded. That is to say, there was the picture of sclerosis of the bone. With a sufficient calcium intake in the food all the new-formed bone was normally impregnated with lime. This was confii-med not only by the histological picture of the skeleton, but also by chemical analysis of the bones : the latter were found to contain a normal or even increased amount of calcium phosphate. Thus Wegner showed that in inconsiderable and non-toxic doses phosphorus merely produces greater activity of the normal processes of new bone formation in the skeleton. Phosphoric acid or its salts gave less marked results in doses from 800. to 1,000 times greater than the effective dose of phosphorus. The property possessed by phosphorus of acting in insignificant doses supports the view that it acts on the skeleton ^er se, and not in the form of products of its oxidation. 125 When Wegner fed his animals on a diet deficient in calcium, the increased new formation of bone continued as before, but with this difference, that the newly formed bone was not impregnated with lime and remained osteoid. Moreover, the proliferous cartilage underwent rachitic changes: Not only Wegner, but Virchow also, regarded the whole picture of the changes in the skeleton of animals on a diet deficient in calcium but rich in phosphorus as typical of rickets. Maas (1872) confirmed Wegner's conclusions, which Korsakoff and Kissel (1896)^ could not do. Kassowitz (1884), on administering small doses of phosphorus (0-1-0-3 mgm. P) to rabbits and fowls, found in their bones merely an increased deposition of lime in the proliferous cartilage, with an enlargement of the layer of primary spongiosa. When phosphorus was administered in doses of 0-3-0-5 mgm. per diem, the rabbits and fowls, even when fed on a diet which the authors did not consider deficient in calcium (what diet ?), showed changes very similar to rickets. Kassowitz also produced in cocks a closure of the bone-marrow cavity with rough osseous tissue with indications of osteoid, i. e. a picture analogous to that obtained by Wegner. In one of the fowls Kassowitz produced acute osteoporosis with a separation of the epiphyses. Unfortunately, Kassowitz's histological technique did not reach the standard required in the investigation of bone by Pommer's method. According to Kassowitz small doses of phosphorus stimulate the growth of the bone. At the same time there is a contraction of the, bone-marrow blood-vessels, resulting in a diminished resorption of the bone and an increased deposition of calcium therein. Large doses of phosphorus produce changes of an inflammatory character similar to rickets in the bones; these are accompanied by dilatation and increase in the number of bone blood-vessels, a marked increase in the process of resorption of bone, and a slight increase in the new bone formations. Small doses iiTitate the bone, whilst large doses poison it. Many chemicals have a directly opposite effect on the organism, which varies with the size of the dose. Kassowitz thinks that in rickets the changes in the skeleton may be due to the toxic effect of various metabolites on the blood-vessels of the more rapidly grow- ing parts of the bone. In support of this view Kassowitz instances syphilis, which produces in the bones a picture similar to rickets. As has already been mentioned Wegner attributes the property of improving the calcification of bones not only to phosphorus but likewise to phosphoiuc acid or its salts, though in a lesser degree. Lehman (1921), aftei- one injection of large doses of phosphates into the blood of rabbits, did not observe in the latter any improvement in the processes of calcification of the skeleton. The increased amount of phosphates in the blood after their injection disappeared rapidly. Thus chemical and histological research has shown that in non- toxic doses phosphorus may possess the property of stimulating the ' Lehnerdt very truly explains the negative results obtained by the last-named authors by the fact that the dosage of phosphorus was unsuitable, and that they did not follow Wegner's experimental methods. 126 growth of bone and deposition in it of calcium phosphates. At the same time these experiments show that when the diet is deficient in calcium this increased growth of bone may be manifested in skeletal changes similar to rickets. This fact points to the enm^nious iinportance of keeping a child on a diet rich in calci/u/m salts token advninistering phosphorus therapeutically. Therapeutically, phosphorus has been used in rickets for a very long time. It is most highly recommended, especially by Kassowitz, who frequently administered phosphorus in almond oil. Unlike the great majority of authors Kassowitz sees no special advantage in combining phosphorus with cod-liver oil. The advantage of administering phosphorus in rickets and osteomalacia has been mentioned by many clinicians. There are, however, very few accurate investigations of the effects of phosphorus in rickets, owing to the fact that phosphorus is generally administered in cod-liver oil, a powerful factor in itself. In one case of hunger osteomalacia care- fully observed by Dalyell and Chick (1921), when phosphorus pills had been given without effect cod-liver oil produced a rapid cure. On the basis of experiments in metabolism in rickets Schloss questions the utility of adding phosphorus to cod-liver oil. Schabad, on the contrary, found it beneficial while at the same time observing no metabolic effects of phosphorus alone in rickets. Phemister (1918) for a long time used phosphorus in two bone diseases, Perthe's disease and dyschondroplasia. "With the first there were no favourable results ; with the second Phemister considers that the results were favourable, but the first signs of improvement appeared so long after treatment was begun that it is hardly a convincing proof of the action of phosphorus. Conclusions. 1. Phosphorus, in non-toxic doses, may apparently stimulate the growth of bone and conduce to the deposition of calcium salts therein. 2. When phosphorus is therapeutically administered a sufiicient amount of calcium salts must be introduced with the food, because in experiments on animals, if the salts in the diet were insuflicient, even though the phosphorus produced increased bone formation, the bone remained uncalcified and rachitic. 16. Influence on the Skeleton of a Diet deficient in Phosphoeus, oil IN Phosphoeus and Antirachitic Factor SIMULTANEOUSLY. (a) Historical. Weiske (1871, 1) fed an adult she-goat on a diet deficient in phosphorus, consisting of straw treated with hydrochloric smd, casein, sugar, starch, sodium chloride, and carbonate of calciufei,'* JHiis 127 diet is likewise deficient in all vitamins. (The goat took in a small amount of vitamin A in the casein. — Auth.) During the 42 days of the experiment the goat had an intake of about 52-5 gm. phosphoric acid and excreted about 62'6 gm. A chemical analysis of the bones did not show any essential difference as compared with the bones of the control goat, fed on hay and bran. There was a difference, however, in the milk : the milk of the goat on a diet deficient in phosphorus was poor in ash and phosphorus, while nearly normal as regards the calcium content. r Forster (1873) very justly remarks that the loss of phosphoric , acid from Weiske's goat was too small to affect the skeleton per- ■ceptibly. Weiske and Wildt (1873) in another experiment fed a kid of 2^ months old on a diet deficient in phosphorus, but found no changes in the chemical composition of the skeleton. Lipschiitz (1910) made experiments in feeding 2 puppies on a diet deficient in phosphorus. The controls were 3 puppies fed on a diet deficient in phosphorus to which phosphorus or casein was added. The sixth puppy was fed on a mixed diet of meat, rice, potatoes, bread, and milk. The basal diet deficient in phosphorus had the following com- position : Eiei-albumin Merjk 50 gm. Eioe 100 „ Sugar . . . 40 „ Palmin 50 „ • Saline mixture . . 6.25 gm. The composition of the saline mixture was approximately equal to' that of dog's milk (exclusive of phosphates), and was as follows : KCl 24.0 NaCl. 16-0 CaClj(dry); 50-0 MgCIj 4.0 Ferrum oxydat. saoeharatum 6.0 Total 100.0 This diet contained about 0-07 per cent, of phosphorus, and in my opinion was also deficient in vitamins. The diet rich in phosphorus consisted of : Eieralbumin 20.0 Casein . . 30-0 Kiee . . . 100-0 Sugar. . . 40.0 Palmin . . 50.0 The saline mixture for the above diet had the following com- position : Potassium phosphate . . . 20-0 Sodium phosphate . . . 50-0 Oalcium chloride (dry) . . 25.0 Magnesium chloride . . . 2.0 Ferrum oxyd. saoeharatum 3.0 The experimental animals, on phosphorus deficient diet, showed weaker growth and curvature and weakness of the extremities ; they were not, however, far behind the control animals in weight. 128 An examination of the ribs made by Schmorl showed an enlarge- ment of the proliferous cartilage, and absence or deficiency of deposition of lime in the zone of provisional calcification, a certain degree of osteoporosis, and extravasation of blood into the bone- marrow. Osteoid tissue was not found in excess of normal. Schmorl thinks that this picture is not typical of rickets, but sugges- tive of Barlow's disease. The following experiments were conducted by Heubner upon puppies of one litter. The puppies' diet was the same as in Lipschiltz's experiment. Thus Heubner's experiments, like Lip- schiitz's, deal with diets deficient not only in phosphoras but also in vitamins. A detailed examination of the skeleton was made by Prof. Schmorl (1913), and described by him in a separate com- munication. The experiments in dieting were begun on three puppies at the age of about six weeks, and continued for 35 to 51 days. Towards the end of the experiment it was observed that the animals were losing weight. Macroscopically, no marked changes in the skeleton were observed, with the exception of a slight thickening of the costochondral junctions of the ribs. Microscopi- cally, the changes in the bones were considered to be similar to those in Barlow's disease, and consisted principally of — (1) A slight enlargement of the proliferous cartilage. (2) Defects in the deposition of lime in the zone of provisional calcification. (3) The primary spongiosa sometimes consisted of cartilaginous trabeculae impregnated with lime and frequently intertwined. New bone was being deposited on these abnormally persistent remains of proliferous cartilage. (4) The bone-marrow in some places was composed of so-called ' Gerlistmark ', i; e. very thin fibres with a limited number of spindle- shaped cells. The blood-vessels were few in number but hyperaemic. Slight extravasations of blood were observed near the blood-vessels. (5) Osteoporosis, caused by (a) retarded activity and reduced number of osteoblasts ; (b) increased number of osteoclasts. Nowhere was an increase of osteoid tissue observed. However, in the absence of the haemorrhagic diathesis typical of the disease in childi-en, the resultant picture could not be identified with. Barlow's disease. Moore (1914, 1916) experimented on pigs, feeding them exclusively on I'ice-flour, or on a considerable quantity of rice-flour with an addition of dried blood, barley, wheat shorts, and also greenstuff or skimmed milk. On this diet the animals developed disorders of nutrition similar to the symptoms of beri-beri. The bristles became coarse, dirty in colour, long, and curly. Simultaneously the animals developed skeletal changes described by S. Hadwen, the pathologist, but unfortunately only macroscopically and, moreover, very briefly : ' Bone formation proceeds irregularly, and the bones become very soft.' The addition of about 20 gm. of phosphates to the food, chiefly in the form of ground phosphate rock, almost always (48 pigs) prevented the development of morbid changes, and the animals grew up healthy. Calcium phosphate in small quantities (about 3 gm. per diem) 'fed to four pigs, failed to countei"act completely the injurious eflfects of a ration of rice meal '. 129 In 1921 Shipley, Park, McGollum, and Simhionds published several palpers devoted to the study of skeletal changes in rats fed on a diet simultaneously deficient in fat soluble vitamin and in phosphorus. The diet consisted principally of rolled oats, linseed meal, and dextrine, or of rolled oats, gelatine, and wheat gluten and dextrine, or of wheat, maize, gelatine, and wheat gluten. To each of the above diets salts were added, always including a sufficiency of CaCOg. Thus the authors considered that there could be no question of calcium starvation. For instance, the amount of calcium in some of these diets was about 0-67 and even more per 100 gm. of food mixture. The changes obtained by the authors with such diets were not always the same. Some of the animals presented a typical picture of very severe rickets, while others that of osteoporosis with satisfactory calcification of the cartilage and bones ; in others again the changes were intermediate and showed rickets complicated by osteoporosis at one stage or another. The authors emphasize the following facts : (1) A diet rich in calcium does not prevent the development of rickets. (2) In their opinion it is just the richness of the diet in calcium and deficiency in phosphorus, i.e. the disproportion of the opntentof these elements in the food, together with the absence of annrachitic factor that causes rickets. The authors support their opinion by an experiment in which calcium salt was eliminated from the diet, already deficient in vitamin A and phosphorus, and then ' a patho- logical condition developed in the skeleton, which, however, was not rickets '. The article, however, gives no information as to the number of rats experimented on, nor the character of the changes. (3) If in a diet deficient in P and fat soluble vitamin the P content be increased to the requisite amount, so that there is a deficiency only in antirachitic factor, then also no rickets was developed. Apparently, as follows from the experiments on 11 rats in one of the communications of the author, this produces a picture of osteoporosis. (4) If to the diet substances are added (e.g. 0-5 per cent, butter) which aid growth to some extent, and retard the onset of marasmus, these rachitic changes are more clearly defined. (5) If about 2 per cent, of cod-liver oil be added to a diet deficient in phosphates and antirachitic factor, no pathological changes in the skeleton are developed. Sherman and Pappenheimer (1921) describe an experiment on 30 rats, in which rickets was produced by a diet consisting of:, patent flour 95 per cent., calcium lactate 3 per cent., and sodium chloride 2 per cent. The authors hold that the addition to this diet of 0-4 per cent, of potassium phosphate, while at the same time decreasing the calcium salts content to 2-5 per cent., prevented the development of rickets. Nevertheless, the microscopical picture of the skeleton of these rats can in no way be considered normal, and, as is shown, e.g. by the microphotograph of the rib of rat No. 2606, there was an abnormal amount of osteoid even after the addition of phosphate. The rats did not grow, and died even after calcium phosphate was added to their diet. This diet was deficient not only in phosphorus but in all three vitamins, protein, fat, and salts.. (lS79) I 130 Experiments on this diet were continued by Pappenheimer, McCann, Zucker, and Hess (1921) on 31 more rats, and showed that 0-3 gm. of pasteurized butter did not prevent rickets. 'Harris yeast vitamin ', and to a lesser extent casein, and especially these two administered simultaneously, prevent the development of rickets. The authors point out the high phosphorus content in yeast. Prof. Paton (1933), on the basis of theoretical arguments and supported by indirect proofs, brings forward an interesting theory of a rickets-producing disturbance in phosphorus metabolism. He points to a limitation in the supply of phosphorus or of phosphoric acid, or a supply in unsuitable form, as the primary disturbance in the metabolism of rickets. ' An error in the metabolism of lecithin, probably in the liver, may be a causal factor in the failure of bone formation in rickets ; this faulty metabolism may, in some cases, be accompanied by an increased conversion of cholin into guanidin compounds, thus explaining the association of tetany with rickets.' (b) The author's experiments with diets deficient in antirachitic factor, in phosphorus, or in both phosphorus and antirachitic factor. In order to solve the question whetVier a reduced phosphate con- tent in the diet — SM used by me could have had any influence on skeletal changes, three experiments were conducted upon three litters of rats, a total number of 20 rats. The rats of each litter were divided into four groups : the first was fed on a normal diet (3 rats) ; the second on a normal diet but with the addition of saline mixture No. 4 instead of saline mixture No. 1 (6 rats). Phosphates were excluded from mixture No. 1 and replaced by a corresponding quan- tity of other salts (see description of technique of experiments, p. 8). The third group (8 rats) received — A diet. The fourth group (8 rats) received — A diet, but with saline mixture No. 4 substituted for saline mixture No. 1. Thus the diets of the second and fourth groups contained less phosphorus than the amount in the normal saline mixture (No. 1) ; on an average these diets contained about 0-33 per cent. P, with a calcium content of about 0-256 per cent. The feeding was begun at the age of about 35-39 days, and was continued for about 3 months or, in the case of one litter, a little longer (72 days). Of the rats on a diet rich in antii-achitic factor and deficient only in phosphorus, two died of pneumonia and one of enteritis. Of the three rats on — A diet two died of cachexia, and one of complicated pneumonia. Of eight rats on —A diet deficient also in phosphorus, four rats died of cachexia. The skeleton was in each case examined microscopically and chemically. Neither the macroscopical, histo- logical, nor chemical examination showed any essential difference between the rats fed on a diet deficient in phosphorus and those on the respective control diets. It was observed that in two of the three rats on a diet deficient in phosphorus, but otherwise normal, the skeleton was very slightly osteoporotic. Such a degree ■of osteoporosis may occasionally also be observed in rats on a 131 normal diet. I do not venture to draw any definite conclusions from these few experiments, but I point out that there was no indi- cation of the rickets-producing effect of the diet deficient both in antirachitic factor and in phosphorus. It is true that the diet I used was still fairly rich in phosphorus, but McCollum and his co-workers produced rickets on diets containing as much as 0-3 per cent, phosphorus. My experiments cannot, however, refute the numerous and excellent experiments of these American authors, if only because the composition of the diets in my experiments was entirely different. Moreover, the ratio of phosphorus to calcium in our experiments was different, and the American authors hold that this ratio is the most important condition for the production of experimental rickets. Conclusions. A summary of the results of the experiments quoted in this chapter leads to the following conclusions : 1. Apparentlj' a deficiency of phosphorus alone in a diet abun- dant in antirachitic factor produces no considerable changes in the skeleton, and in no case rickets. , 3. The experiments of Lipschiltz, as also those of Heubner and Schmorl, made with a diet deficient not only in phosphorus but in all vitamins likewise, have shown the development of osteoporosis in the bones and changes in the skeleton of puppies, similar to those observed in Barlow's disease. 3. McCollum and his co-workers produce severe rickets in rats ' by a diet poor in antirachitic factor, but very rich in calcium salts, and with a reduced or even suiScient amount of phosphorus (' a ration insufficiently supplied with the organic factor and only slightly or not at all deficient in its content of phosphorus'), {J. Biol. Ghem., 1921, 47. 522). Therefore they think that the 'physio- logical relation in the diet between the calcium and phosphate is of infinite!}' greater importance in insuring normal calcification than the absolute amount of the salts themselves ' (Ibid., 525). 17. Influence of Arsenic on the Skeleton. Gies (1878) introduced about 0-5 mgm. of acidi arsenicosi per diem into rabbits, cocks, and young pigs, for the purpose of studying the effects of arsenic on the skeleton. The changes produced were analogous to those obtained by Wegner in his experiments with phosphorus. The animals receiving arsenic weighed more than their controls, were fatter, and the bones of the skeleton were larger. The subchondral spongiosa was far more strongly developed and sclerosed, especially in the upper epiphysis of the humerus and lower one of the femur. The walls of the diaphyses were more compact and thicker. Microscopical examination showed a thicken- ing of the trabeculae, owing to the increased new formation of bone ; there was likewise a reduction of the bone-marrow cavities. The histological investigation was unfortunately made on decalcified i2 132 bones. These chaoges were very marked in the young rabbits and very slight in the adults (chiefly in the diaphyses). The offspring of rabbits receiving arsenic were born with skeletal changes on the whole similar. The rabbits developed changes in the skeleton even when arsenic was put under their bedding in the hutch and merely inhaled, not eaten. Conclusions. 1. Apparently arsenic has an action on the skeleton similar to that of phosphorus, but further investigations are necessary before indisputable conclusions can be drawn concerning this property. 2. The litters of parents who had taken arsenic were born with skeletal changes characteristic of the action of arsenic. 18. Influence of Okgaxic Acids on the Skeleton. As early as 1857 Piotrowsky and Budheim showed that 12 per cent, of oxalic acid or 2-5 per cent, of tartaric acid taken in the human alimentary canal are ^creted in urine ; citric and malic acid cannot be detected at all in the urine. Lactic acid, introduced into the organism in large quantities, could not be detected in the urine by Liebig (1844, 1847). It was only when lactic acid was introduced intravenously that Zuntz and Mering (1883) found a certain amount of it in the urine. Heiss (1876), who for 308 days gave a dog an average daily dose of about 7-4 gm, of lactic acid, could find none in the urine. Thus the majority of organic acids, when introduced through the alimentary canal, undergo complete combustion in the organism, and it is only oxalic acid that is excreted in the urine in any considerable quantity. Nevei-theless, a theory has arisen that the accumulation of organic acids in the blood extracts calcium from the skeleton and produces either rickets or osteomalacia. It is considered that the source of increased new formation of lactic acid is the increased process of fermentation in the alimentary canal in diseases of the latter (Marchand, 1842). This theory found support in the following investigations. Lactic acid was found by Marchand (1842) in rickets, in the bones and urine, by, Schmidt (1847) and Weber (1867) in the bones in osteomalacia (the latter finding 1-3 gm. of lactic acid in 100 gm. of fresh bone!), by Moers and Muck (1869) in osteomalacia, in the urine. These results wei-e not, however, confirmed by the investigations made by Frey (1863), Schmutziger (1875), Hoxter (1880), Heuss (1889), Warschauer(1890) and others. Nencky and Sieber explained the positive results of the fii'st authors as due to faulty experimental technique. Renzi (1885), Jaksch (1887), Winkel (1889), Issmer (1889), Eisenhoudt (1892), found a lowered alkalinity of the blood in osteomalacia. Fehling (1891), Limbeck (1894), having examined the blood of osteo- malacia patients, and Stoelzner (1897) the blood of rickety children, absolutely deny this. Heitzmann (1873) tried to prove experimentally the importance of lactic acid in the aetiology of rickets, on the basis of his experiments 133 on 5 dogs, 2 rabbits, 7 cats, and 1 hedgehog. The animals were fed on a diet which was, however, also deficient in calcium, lactic acid being introduced either in the food or subcutaneously. Most of the animals developed a disease similar to rickets or osteomalacia. Baginsky (1883), by adding 2 gm. of lactic acid per diem to a dog's diet, deficient in calcium, obtained more marked rachitic changes of the skeleton than without the addition of the acid. All later authors, however, regard the experiments of Heitzmann and Baginsky as inconclusive, on the grounds that the diet deficient in calcium used in the experiments can of itself, without the addition of lactic acid, produce analogous changes in the skeleton. By the introduction of lactic acid into a diet containing calcium phosphate, Rolofi" (1875) could not pi'oduce any changes in the skeleton of animals, but he obtained more severe rickets in animals fed on calcium poor diet with lactic acid than on the same diet without lactic acid. Toussaint and Tripier (1875), Heiss (1876), Siedamgrotsky and Hoffmeister (1879), and Delcourt (1899) were also unable to corroborate the rickets-producing in- fluence of lactic acid on dogs, cats, and rabbits. Heiss's chemical examination of the dog above mentioned showed a normal content of calcium and magnesium in the blood, muscles, and bones. Not- withstanding the prolonged introduction of lactic acid, the calcium metabolism was likewise normal. Both macroscopical and micro- scopical examination of the skeleton showed no deviations from the normal. Weiske (1895) investigated the influence of a diet with acid ash on the chemical composition of the skeleton of rabbits. * This diet consisted of oats, and was at the same time also com- paratively poor in calcium salts. In adult rabbits it produced very slight chemical changes in the skeleton, whereas in young rabbits the changes were considerable (see chapter on 'Calcium Starva- tion'). The deficiency of calcium in the diet does not, however, justify any definite conclusions being made as to the significance of the alkalinity of the ash, as on adding CaCOj the ash of the food not only acquired an alkaline character, but was likewise greatly enriched in calcium. The fact that Ca3(POj2 did not act as well as CaCOg may be explained by the greater difficulty of assimilating that salt. Weiske explains the favourable effects of adding hay to the diet of oats by the fact that hay has an alkaline reaction. Stoeltzner (1897) likewise investigated the effects of an exclusive diet of oats and tap water on the skeleton of rabbits. A histological examination showed no signs of rickets, but merely a diminution in bone formation. Gottihg (1909) examined the skeletons of rabbits experimented on by Caspari, who had fed them on beet-leaves or food with an addition of oxalic acid. The skeleton of a young pig experimented on by Zuntz was also examined. It had likewise been fed on beet-leaves. The macro- scopical changes were similar to those in rickets. Microscopical examination showed an enlargement of the zone of proliferous cartilage, and an insufficiency or total absence of deposition of calcium in the zone of provisional calcification. The deposition of calcium in the osseous tissue was normal. Recently Hodgson (1921) again raised the question of the possi- 134 bility of the existence of acidosis in rickets, on the grounds that the alkaline reserve in the organism of rickety children is reduced, according to her investigations, Hodgson's observations were made on 36 rickety children, and present certain difficulties in their interpretation, e. g. ' in the most acute cases one finds a lowering of the alkaline reserve associated with a comparatively small ammonia ratio in urine'. Summarizing the results of the investigations which hayebeen interpreted as supporting the acidosis theory of the origin of rickets, it must be admitted that the data in favour of the theory are unconvincing. 19. Influence of Confinement and Deprivation of Exercise on the Skeleton. Hansemann (1906) expressed the opinion that rickets originates in children under the same conditions as those which cause its development in wild animals kept in zoological gardens, that is to say, he considers it the result of deprivation of exercise and lack of fresh air. Further, Hansemann (quoted by Lehnerdt, 1910, 2) points to the following facts. Autopsies of children born in autumn and dying in spring usually show definite or strongly marked signs of rickets ; in children bom in spring and dying in autumn signs of the disease are rarely discovered. The severest forms of rickets, however, are found in children born in spring and dying about a year after. Schmorl (1909) likewise found, on the basis of the statistical data from his own post-mortem examinations, that the incipient and more advanced forms of rickets occur in the cold season, while the greater number of recovering convalescent forms occur in the end of summer and beginning of autumn. Clinical observations corroborate this. Thus rickets is especially prevalent in the months when children spend most time indoors. Findlay (1908, 1918) cites the following facts in confirmation of the significance of confinement in the aetiology of rickets : Rickets is a disease of the temperate zone, seldom met with in tropical or subtropical countries. A warm climate permits of children being brought up more in the open air, and of houses being used merely as sleeping places. Negroes and Italians, free from rickets in their native land, sufier a great deal from rickets in New York. Rickets is a disease more prevalent among the poorer classes than the rich, the children of the latter being able to take outdoor exercise, and lead a more active life than children in poor families. In the latter case the children are frequently left to themselves, and must lie still for man}' hours at a time, owing to their mothers being too busy to attend to them. Ferguson, under the direction of Paton and Findlay (1918), investigating the causes of rickets in Glasgow, noted a series of facts which serve to support the opinion that confinement and lack of exercise are conducive to rickets. Their data include 200 cases of severe rickets, 150 mild cases, and 200 non-rickety children. 135 (1) ' Inadequate air and exercise seem to be potent factors in deter- mining the onset of rickets. Over 40 per cent, of the rachitic children had not been taken out, while only 4 per cent, of the non-rachitic had been confined indoors.' Only 33 to 42' 5 per cent, of the rachitic children, as against 86-5 per cent, of the non-rachitic, had sufficient outdoor exercise. (3) The probability of the appearance of rickets increased with the number of children in the family, several children in the same fauiily sufiering from it. (See also following chapter on heredity.) (3) ' The cubic space per person was 33 per cent, less in families with cases of marked rickets than in families who were free from the disease. (4) The cleanliness of the house was distinctly better in the non-rachitic than in the rachitic family.' (5) In the two garden cities examined rickets was very un- common. Findlay was the first to make experiments to determine the effects of deprivation of exercise on the structure of the skeleton. Sixteen puppies were used for the experiment. Of these, nine were kept in a cage and deprived of exercise, while the rest served as controls and ran at large under supervision or in the same room with the puppies in the cage. All were fed on milk and oatmeal porridge. The most convincing was the experiment on three puppies of one litter. ' These animals were kept in one house, were fed ia exactly the same manner and were subjected to the same atmospheric con-, ditions as regards amount of sunlight, warmth, and purity of air. Two, however, were confined in a small cage closed on either side but open above, and only closed in front with wire netting, while the other was allowed to run about the j-oom at large and play with a cat.' One of the caged puppies died of diarrhoea without any mani- festations of rickets, and the other, at the end of the experiment, showed all the symptoms of severe rickets, while the puppy running about at large had a normal skeleton. On the grounds of the experiments the author came to the following conclusion : that ' By confining young dogs and depriving them of exercise, rickets has been invariably induced, and that although their diet was beyond suspicion, the air which they breathed pure, and their kennels were kept scrupulously clean, whereas control animals allowed exercise, but otherwise similarly treated, did not become afiected. . . . Confinement, with consequent lack of exercise, is the main factor in causing the disease' (p. 17). Paton, Findlay, and Watson (1918) repeated the experiments, with certain modifications, on two litters of puppies (8 and 9 puppies). Part of the litter remained in the laboratory and was fed on oatmeal porridge and a ration of 100-300 c.cm. of whole milk. Some of the puppies ]"eceived butter in addition, so that the amount of milk fat in the ration of some puppies reached 14"5 gm. The rest of the puppies were sent off to the country, where they were fed on oatmeal porridge and about 380 c.cm. of skim milk, i.e. a diet containing only about 2-8 gm. of milk fat. 136 In the country the dogs ran about the yard and garden, under the personal observation of one of the authors. The authors came to the following conclusion : ' Pups kept in the country and freely exercised in the open air, although they had actually a smaller amount of milk fat than those kept in the laboratory, remained free from rickets, while the animals kept in the laboratory all became rickety.' In a third communication, Paton and Watson (1931) experi- mented on four litters, with a total number of 26 puppies. Strictly speaking, these experiments tend to some extent to undermine the importance attributed by Findlay, Paton, and Watson, in their previous investigations, to confinement and lack of exercise : the authors succeeded in avoiding the development of rickets in puppies confined in the laboratorj', by paying the greatest attention to cleanliness.^ Litters were fed on the following- diet : I series of puppies — new whole milk + bread II „ „ dried whole milk + bread III „ „ dried skim milk + bread] only about + lard [ 0-3-0-6 gm. IV „ „ skim milk + bread J of milk fat. The. amount of calories in the food of all the puppies was the same, the amount of the separate constituents being adjusted for that purpose, and waa in accordance with the age and increase in weight of the pups. Of all the 17 puppies in this experiment, only 1 developed rickets. In the experiment on 9 puppies, in which the amount of calories was not equalized in the two groups, rickets was developed sooner by the puppies which received only 0-2-0-7 gm. of milk fat per kilo of body weight, and later in those receiving 7-5-11 gm. of milk fat (the diet being oatmeal porridge and whole or skim milk). The authors came to the following conclusions (p. 84) : (1) In young dogs under ordinary laboratory coixditions a liberal allowance of milk fat, up to even 14 gin. per kilo of body weight, neither prevents the onset of rickets nor cures it when it has developed. (2) Puppies kept largely in the open air may escape the develop- ment of rickets on an intake of less than 1 gm. of milk fat per day per kilo of body weight. (3) With scrupulous care as to cleanliness it is, at least some- times, possible to rear puppies iree from rickets in the laboratory on an' intake of only about 0-5 gm. of milk fat per kilo of body weight, along with bread, provided that the diet affords an adequate supply of energy. (4) The energy value of the diet, however supplied, quite apart from the presence of any hypothetical antirachitic factor in milk fat, would seem to play a pai-t in controlling the development of rickets, but that it is only a contributary part is shown by the development of rickets in pups with a high energy intake, if confined in the laboratory without scrupulous care as to cleanliness. » Although in the first experiments Findlay stated that the most careful attention had. been paid to cleanliness. 137 (5) Milk fat may be reduced to about 0-3 gm. per kilo of body •weight, if its place is taken by an equal amount of lard, without the onset of rickets. (6) The results of these observations do not support the conclu- sion . . , that rickets is a deficiency disease due to a lack of an antirachitic factor associated with milk fat. Lehnerdt (1910) likewise conducted experiments on 6 pups, 5 months old, but on autopsy no difference was found between the skeletons of the pups which had run about at large and of those confined in a small cage. Macroscopically there were likewise no signs of rickets, and the bones were hard. No microscopic examina- tion was made. The pups were fed on kitchen scraps, milk, meat, and 1 gm. calcium phosphate per day. Mellanby (1921) likewise attributed, as a result of his own experi- ments, some significance to confinement in the aetiology of rickets in puppies. This author thinks that (pp. 68-9) : Exercise has an antirachitic effect on puppies, but this action is not only subsidiary to diet, but even the possibility of exercise is dii-ectly dependent on the quality of the diet. Puppies during absolute confinement will not develop rickets if the diet is good. Puppies allowed unlimited freedom will develop rickets on rickets- producing diet. Eachitic puppies in confinement can be cured by the addition to the diet of whole milk, cod-liver oil, and egg yolk, and more especially if meat is also present in the food. Therefore the rickets-producing influence of confinement is of secondary importance to the effect of defective diet. Besides this, Mellanby gives several other reasons for not attri- buting great importance to confinement and deprivation of exercise. An infant passes a considerable part of its first year of life in sleep, and is able to make only a few limited motions. Therefore, if the factors above mentioned play a part in the development of rickets, one might expect that as a result of such a great limitation of movement, all children must develop rickets, which is not the case. Furthermore, Mellanby (1920) describes the conditions of life of the inhabitants of the Island of Lewis, in the Hebrides. The islanders live in so-called ' black houses ' built of turf and stones, with a thatched roof. There is often no chimney to the house, and as the peat fires are kept constantly burning, and there is no exit for the smoke except through the door, the condition of the atmosphere of the house can be well imagined. Cattle often live under the same roof, the byre adjoining the house, and it is some- times necessary to pass through the byre to enter or leave the building. The children are not taken out until they can walk, except possibly for a few minutes on a fine day in the summer-time. It is remarked that rickets is almost unknown in the island, and the most striking fact in the adult population is their beautiful teeth — a testimony to the absence of rickets in infancy. Under the direction of Prof. Paton (1922) two students, C. B. and A. J. McLeod, investigated the conditions on the Island of Lewis, without confirming the description given above. In their opinion, with regard to air space per person and ventilation, these Lewis 138 houses compare favourably with the slum dwellings of English cities. It was found that from the time the children can walk they are constantly in the open air, and that in 12 families it is distinctly stated that the babies had been taken out freely in fine weather. Paton adds that, in his experience, in the Outer Hebrides it is con- sidered fine weather unless it is raining and blowing hard. Sixteen of 18 families kept a cow. Stoeltzer quotes Lange's- (1877) investigations. The latter found that of 176 rickety children 60, who had the disease in a very acute form, were very well housed. Stoeltzner (1904), on the basis of his own experiment, agrees with Lange that severe cases of rickets are not infrequently met with in well-to-do families, where the greatest attention is paid to the size and proper ventilation of the nursery. In all my experiments I have found that confinement has no influence on the skeleton of normally nourished rats. Conclusions. 1. On general physiological grounds it might be expected a priori that the development of the organism, and therefore of the skeleton, should be afiected by confinement, i.e. by all the unhygienic con- ditions of life of the organism which deprive it of the possibility of enjoying the normal amount of light, fresh air, and muscular exercise. 2. The results of the experiments made up to date do not fully agree with each other, and do not explain finally the probable effects of confinement on the organism, particularly on the skeleton. 20. MOTSTDRE AND KiCKETS. (a) Historical. Hagen-Torn (1896) observed the great frequency of rickets in those provinces of Russia in which the moisture exceeds 80 per cent. Shukowsky (1900) explains this by the fact that in a more humid climate children are kept indoors much more. Pavlov observed in dogs with bilious or pancreatic fistulae a marked softening of the bones, especially those of the thorax. If measures were taken to keep the cages of these animals dry, the disease of the skeleton passed away in spite of the existence of fistulae. The bones of the sick dogs were examined histologically by Professor Moiseev, who discovered changes similar to those in osteomalacia. (b) The Author's Experiments. I conducted two experiments on 28 rats belonging to 4 families, i.e. 4 mothers and their 24 young, in order to investigate the possible significance of increased moisture in the aetiology of rickets. Experiment 1. In the first experiment I took two litters of 139 young rats (594 and 595), the diflPerence in their ages being 7 days. Eight days after the birth of one litter and one day after the birth of the other, the young rats were mixed, so that each mother fed part of her own litter and part of the other rat's litter. This neutralized the influence on the experiment of the young rats being of different litters. In Tables Nos. 53, 54 the young rats bear their mother's number, being further distinguished by a letter of their own. The mother rat 594, with 6 young rats, was placed in an ordinary cage, 25 X 35 X 11 cm. large. Rat 595, with 7 young, was placed in a cage 30 x 30 x 40 cm. large, with glass sides, glass roof and wire floor. Under the wire floor of the cage was a box full of water, which was changed only once every ten days. The cage was cleaned only once a fortnight. The urine and excreta of the rats fell into the water and decomposed. Therefore the air in the cage was not -only saturated with moisture, but with the gases of the urine and excreta decomposing in the water. Thus in this experiment, as well as in the following (litters 612 and 613), an investigation was made of the eflfect on the skeleton of rats of air that was not only very humid, but also very badly ventilated and saturated with putrid gases. I did this for the purpose of placing the rats in the worst possible conditions as regards the air they breathed and also want of cleanliness. As has been mentioned above, Haubner thinks that keeping goats and pigs in stalls contaminated with excrement is one of the most important factors in the pro- duction of rickets in these animals. ♦ In both the cages the mother rats were fed on —A diet during lactation. During pregnancy, rats 594 and 595 were fed on Diet No. 2, i.e. containing a considerable quantity of Ca, P, and cod-liver oil. After being weaned (some at 30 days of age, and others at 37 days) 9 out of 13 young rats were likewise fed on — A diet. Four young rats (Nos. 594 c, 595 h from the ' dry ' cage, and 595 f, 595 c from the ' wet ' cage) were put on N diet. The last 2 rats from the ' wet ' cage were each put into a separate glass cylinder, 40 x 12 cm. large, the lower quarter of which was separated by netting. Water was poured into the bottom of the cylinder. The ventilation in the glass cylinder was considerably worse than in the large glass cage, owing to the narrowness of the cylinder and the more closely fitting cover. This explains the loss of weight of the young rats in these cylinders, and even the death of one of them (594 f). The two young rats from the ' dry ' cage put on N diet were kept in an ordinary wire cage (25 x 25 x U cm.) and consumed from 20 to 27 gms. of food ; on — A diet, at the beginning of the experiment, the rats in the ' dry ' cage consumed about 15 gms., and at the end of the experiment — about 11 gms. In the ' wet ' cage, on N diet, one rat consumed the same amount of food as the corresponding i-at in the ' dry ' cage ; the other (594 f), which died before the end of the experiment, consumed less, about 15 gms. per diem. In the 'wet' cage, on —A diet, as in the ' dry ' cage, the rats consumed about 15 gms. per diem, the amount decreasing to 9-10 gms. towards the end of the experiment. 140 8 >5 'TS l?5 05 5. o '^^ .|.|'^.s . ^ TS I " » 1^1 ^ cq -^ o o so 06 «o !0 ^ cq 00 cj tH ^ O 00 1> ^ CO t> o 00 c^ W -i 10 »o 3 'H -* -* -* i^ s s 1 § § i 10 01 a ^ C-- i> o o CO CO CO CO o 10 ^ CO eo o CO !0 o as CO CO &0 g lO ■:-i o CO OS tH CM T-t oq -^ IM 1— ( 1-1 1— I T-( ^ 10 -^ a £ s- J 1 •il 1 1 K 1 s 1 •1 1 y 1 S -S Ji ,4 12; IB '■ si " o 1^ 3" . « eq 1 15 5? i- 5D 05 ■^ w T-t lO OS 00 tH 1-1 05 s o 1 1- |« 5? T-t M OS to CO o6 cq 00 1 2 1 53 l« O O ^ «? to t* OS 00 1 ■? o ^ aj 5? 50 'O ^ d us i to -* CO g ^ ^^ *'-> ■f g 5 « OS "> 50 to to ?o 3] 1 o to 1 1 1^ ?fl 1 cq ^ W to to CD 60 CO to a a -^ o ■^ >) >. -K) §. 1 || 1 CO o t-H s § ? lO § 2 o 1 ■1 ^ ^ ■ea So S 1 » I> l> o o c~ o rt 5" 1"^ 1 m CO CO 55 CO CO CO b 1" ^ 1 1 o ■u f««i ^ M 2 r-( o M o 00 OS o e g o eq ^ T-t OS 00 la 05 ■l i ^ s 3 i^ ■e iiC 13 +3 h-i 1 1 1 « 1 "S ■H o o» to ■<# Os' OS -O 0) m >o US iO la Td ^ fl oq 03 a 1 «ij o lO lO w ■« e la .. t-H ,-< ij- k OS m P o n ■^ -I a -fe ■< 9 S H,i ^ ^ SP S 8 ■^ s Is, O 1 --. o-§ OS OS 142 The results of the experiment are shown in Tables Nos. 53 and 54. Fig. 46, PI. 18, shows the weight curves of the average young rat (both male and female) from the ' dry ' cage and from the ' wet ' cage. No difference was observed — either in the appearance, behaviour, or at the autopsy — between the mother rats kept in the ' dry ' and the ' wet ' cage respectively. They were about the same weight and age, though the latter could not be accurately ascertained. As may be seen in Fig. 46 the young rats in the 'wet' cage were behind their controls (in the 'dry' cage) in weight. More- over, those which were in the worst ventilated cages were very much behind the controls in weight, lost weight, and even died, in spite of their being on normal diet. There was no marked difference between the behaviour and appearance of the rats in the 'dry' cage and that of the rats in the 'wet' cage, (both on —A diet). On the other hand, in the case of rats on normal diet the difference was great : the young rats in the ' wet ' cages suffered from enteritis, and were considerably more emaciated, their fur was ruffled and they were somewhat less active than the controls. Autopsy revealed nothing more than semi-opacity of the teeth, general emaciation, and gastro-enteritis. The bones of the skeleton were thinner than those of the controls. The two rats in the ' dry ' cage on normal diet were fat, with a normal skeleton and trans- parent teeth. On autopsy, rat 595 H was found to be pregnant with 8 foetuses. No great difference was observed on autopsy among the two groups of rats on —A diet. In the 'diy' cage the rats had a small, or even moderate amount of fat, which was not the case with the rats from the ' wet' cage. In the latter, however, in spite of the absence of fat, the rats were not emaciated. As regards skeletal changes, macroscopical examination revealed a distinct thickening of the costochondral junctions and several spontaneous fractures of the ribs in one of the four young rats in the ' diy ' cage, and in the ' wet ' cage in two out of five young rats. Such an absence of marked changes in the young rats born of mothers fed on —A diet during lactation is to be explained by the fact that the mothers had been fed on diet No. 2, i. e. rich in cod-liver oil, calcium, and phosphorus, from the time they were weaned and during pregnancy. Gheonical changes in the skeleton. Apparently the mother-rat's being kept in a ' wet' cage for 39 days had no effect on the chemical composition of her skeleton : the calcium content of the fresh bone was even slightly greater, and the water content less. In the rats kept on N diet in the ' wet ' cage, the composition of the skeleton as regards water content was approximately the same as that of the rats in the ' dry ' cage. The calcium content was diminished by about 12-7 per cent, below normal (in dry bone). As regards the rats on — A diet, while the water content of the skeleton was also approximately the same, the calcium content of the skeleton of those from the ' wet ' cage was also slightly decreased, by about 9 per cent, in fresh bone and 6 per cent, in dry bone. Microscopical changes. There was no difference in the histo- logical structure of the skeleton of the mother rats and their 143 offspring either in the ' dry ' and the ' wet ' cage. The changes were slight, owing to the above-mentioned fact that, until the experiment, the mother rats had always been fed on a diet rich in cod-liver oil, phosphorus, and calcium. It is very significant that of rats 594 A, 594 b, 594 d, 594 E, and 594 H, only one had moderate rickets, all the rest having it in a mild form, as the m.other of this very litter had been fed during the first week of lactation on a diet rich in cod-liver oil, phosphorus, and calcium, and had thus been able, in the course of one week, to transmit to her offspring with her milk a considerable stock of the aforesaid antirachitic factors. This experiment once more confirms what I said in chapter 12 on the influence of a diet deficient in anti- rachitic factor. The young rat 594 f, on N diet, which had died rapidly with symptoms of cachexia in the ' wet ' cage, showed the severe osteoporosis characteristic of such cases, but without any indications of rickets. In both the mother rats nothing but slight osteoporosis was observed. The epiphyseal costo-chondral junctions took the form of ' bone plates ' impregnated, like the rest of the bone, with calcium, and indicated that the epiphyseal growth of bone had been arrested. Experiment 2. The second experiment was conducted upon two mother rats (612 and 613) and their 11 young. Both litters were born within a day of each other. On the 16th day after birth the young rats were re-distributed between the two mothers as in the preceding experiment and, together with their suckling mothers^ were placed in a ' dry ' cage (mother rat 613 and 6 young) and a 'wet' cage (mother rat 612 and 5 young). In these cages the mother rats were fed on N — Ca diet for 14 days, i.e. the second half of the period of laptation. During the first half of the period of lactation the mother rats were fed on No. 1 diet, but with a considerable amount of tricalcium phosphate, and two or three times during this period they were fed on No. 2 diet with cod-liver oil. At the age of 30 days the young rats were separated from their mothers and likewise fed on the same N — Ca diet. In this experi- ment in the N — Ca diet all the fat was in the form of butter, i. e. it contained no cod-liver oil. Thus, although this diet contained a suflBcient quantity of antirachitic factor, it was less than in the case of the rats referred to in Table No. 21. Before the end of the experi- ment two rats (612 B and 612 c) died in the ' wet ' cage. For purposes of comparison, two rats from the ' dry ' cage (612 d and 612 e) were killed at the age of 30 days. These 4 rats will be separately compared below. The rats which survived to the end of the experiment — (613 A, 613 B, 613 c, and 612 F in the ' dry ' cage, and 612 A, 613 AA, and 613 bb in the ' wet') — were killed at the age of 65 days. Thus the results of the experiment upon these last rats may likewise be compared. In the ' drj' ' cage the rats con- sumed about 12 to 15 gm. of food per diem, and less in the 'wet' cage — about 7 to 10 gm. The result of the experiments are given in Fig. 47, PI. 18, and Tables Nos. 55, 56. There was no pai-ticular difference between the mother rats either as regards appearance and behaviour during life or 144 as ? P O I o B ^ d d m ►J o ?2 i ® - -^ &D 03 CO „ ^ m eg SO o ^ *'- = oj in "o ^1 '7 (5i ^ -* -* CC O ^ ,« ' S I III i rH 00 m (N 22 -^ OT W CO 5*3 >o eq >> «? o g g § § i r« s -* lo 2 S »H CO W 03 U3 W5 iO O O ;C fO 03 o o o o o o 55 03,03 03 03 03 Ol 00 50 O 00 OO g tH 00 O O 03 03 OS 00 og 03 03 S6366 66 I I ' I ' J. J ;z! !z !zi !2; iz; ^1^ ^ • « O fl (a (*< M CO CO i> tq oil te »] oods'-'Offlo »M OS -^ 00 M ia -^ O "i 03 03 M iQ Q SO ffl -^ 00 ■<*' «0 iJ'SS c5 03 ■> si i-l ffi 03 ,o O SO ■*o6 lO so i2 •« 03 03 ) 00 CI *o »o I !M (M SO so N . "t- Oi ^ !0 ^ 03 03 iO Gd r^ W3 W3 3g MsO flOf^ ^ a ^ so M W 03 «S! 03 05 « « "S rt O O 53 o O o I I I I I I Iz; fc !? !zi I? t?i CHCH-\, q+'^'H < H o -< B 03 03 CT 03 03 -H i-H I— I rH *-< so so eo O 50 O) 50 ffi 145 on autopsy. As may be seen from the weight curves (Fig. 47) the young rats in the ' wet ' cage were considerably behind those in the ' dry ' cage in weight. Their appearance and behaviour were like- wise different : in the ' wet ' cage the rats were more languid, with dirty, ruffled fur, and more emaciated ; on autopsy no fat was found in their bodies, whereas the ' dry ' cage rats which had sur- vived to the end of the experiment had a moderate amount of fat. There was no marked difference in the macroscopical changes in the skeleton between the rats of either cages, these changes being of the same nature as in the analogous rats described above (see pp. 68, 105). In all the rats killed at the age of 65 days, there was a very marked picture analogous to bad rickets. The rats which died before the end of the experiment showed severe cachexia, gastro-enteritis, and in the skeleton macroscopical examination revealed osteoporosis but no rickets. The chemical changes in the skeleton (see Tables Nos. 55 and 56) of both mother rats (whose age, though not known exactly, must have been not less than three months) undoubtedly indicated the impoverishment of the skeleton in lime (compare the corresponding figux'es in Table No. 20). This points to ■ the mother rats on a diet deficient in calcium having supplied their young with calcium from their own skeleton. No greater degree of change was observed in the chemical composition of the skeleton of the mother rat in the ' wet ' cage, as compared with that of the mother rat in the ' dry ' cage ; on the contrary, the skeleton of the former had a larger lime content. It is difficult to attach any importance to the slight differences in the chemical composition of the skeleton observed in comparing young rats of the same age in the ' dry ' and ' wet ' cage respectively (see Tables Nos. 55 and 56). The microscopical changes in the skeleton were more of the nature of severe rickets from Ca starvation in the rats in the ' diy ' cage, as compared with those of the rats in the ' wet ' cage (see tables). In the latter rats the bone tissue contained less osteoid and were more osteoporotic than in the rats in the ' dry ' cage. This may apparently, be partly connected with the decreased intake of food by the rats in the ' wet ' cage. In the rats which perished, microscopical examination showed the osteoporosis and hyperaemia of the bone marrow usually found in such cases. In the rats which had survived to the end of the experiinent, the changes were similar to those mentioned above (pp. 68, 105). In comparison with these, the only slight peculiarity was the presence of a larger amount of osteoid in the skeleton of the rats with severe rickets from Ca starvation, i.e. the microscopical picture was more like that represented, not by Fig. 6, but by Fig. 9. Gondusions. .1. The available statistical data concerning the influence of moisture in producing human rickets are insufficient for any definite conclusions to be drawn from them. (1S79) K 146 c ™ ii,« VvqH pffpfit on tlie organism as poUutiofof the air by the products of putrefaction without demonstrating that thei factors play an important part m the aetiology of rickets. , , , e . i i • < ^ , 3 The calcium content of the skeleton of rats kept m 'wef cages showed a tendency to slight decrease as compared with the skeleton of rats kept in ' dry ' cages. 21. Influence of Light on the Skeleton. Of late years interest has been reawakened in the curative effect of light on rickets, which was first observed by Buchholz in 1904. Buchholz used the ' Gliihlicht ' treatment. In 1918 Winkler discovered that Rontgen rays likewise had a favourable effect on rickets. Huldschinsky (1919, 1920, 1920 2) Putzig (1920), Karger (1920), Riedel (1920), Erlacher (1921), Men- gert (19§1), and Chick, Dalyell, Hume, Mackay, and Smith (1922), successfully employed the ultra-violet rays of mercury vapour quartz lamps in the cure of rickets. An improvement, or even a complete cure, of rickets took place after a certain number of exposures, e.g. approximately after 26 exposures in Huldschinsky's cases, and in 40 or 60 in Erlacher's cases. Some of the authors controlled the effects of therapy by means of Rontgenographical examination. Not all of these clinical observations are convincing: for example, Huld- schinsky (1920) noticed the beneficial effect of treatment with ultra- violet rays in 6 cases of rickets with tetany. But five of them were convalescent and three even received cod-liver oil before treatment with light. Hess and Unger (1920) could not observe any useful action of the ultra-violet rays in their 6 cases of rickets. In their communication they say (p. 219) : ' Violet ray treatment cannot be considered the equivalent of heliotherapy. But the fact that rickets is exceptional in the arctic region, where there is a lack of sunlight for the greater part of the year, is a strong argument against its predominant influence. . . . It was found that rickets can develop notwithstanding an abun- dance of fresh air. It occurred in the larger and in the smaller rooms of the institution, and developed in infants ai ike tuberculosis preventorium, an institution in the country where particular attention is paid to outdoor treatment. It was found likewise that a liberal allowance of light could not prevent the development of this disorder. Infants in glass cubicles were not spared more than those in the regular wards.' To these considerations of Hess and Unger we may add the tact mentioned above of the absence of rickets among children in ^se unTbt°L r'' '"^ ?^' Hebrides, Ferguson (1918) was like- frqurncvofcLesofrl'^ ^^^^ow any influence of light on the no diflference in the exposurroF JL hoti.o J 'Vf' P''^^'"*"^ rachitic children ' (p. 80). ^^ °^ rachitic and non- 147 A year later (1931) Hess and Uoger came to opposite conclusions as a result of the favourable effect of heliotherapy on 5 rickety children. Feer (1921) likewise draws attention to the improvement in rickets produced by heliotherapy in the Swiss Alps. Chick, Dalyell, Hume, Mackay, and Smith (1923) also cured rickets by exposure to sunlight. Raczynsky (1912) experimentally investigated the importance of the absence of light as an aetiological factor in the production of rickets. The experiment was conducted upon two puppies suckled by their mother. One of them enjoyed the benefits of sunlight, while the other was kept in a large, well-ventilated room in absolute darkness. At the age of 6 weeks both puppies were killed, and their bodies chemically examined. The results (per 100 gm. of body weight) were as follows : Per 100 gm. of body weight. Puppy kept in Puppy kept in the light. darlmess. CaO 1.58 gm. 0-98 gm. PjOj 1-19 ., 0.86 „ MgO 0.05 „ 0.04 „ CI 0-16 „ 0.35 „ Pe 0.02 „ 0.02 „ On the grounds of the decrease in the calcium and phosphorus content in the organism of the puppy kept in the dark, Raczynsky presumes that it has rickets. In 1922 Powers, Park, Shipley, McCoUum, and Simmonds ' made an experiment on 18 rats aged about 6 weeks, and weighing from 40 to 50 gm. All the rats were fed on the same rickets-producing food, deficient in fat soluble vitamin and phosphorus. Six control rats were left in cages under ordinary conditions, in a lighted room. Twelve rats were sent off to another town, where for a period of about two months they were subjected to the sun's rays for an aggregate period of about 243 hours. The first group of rats all developed rickets, while there was not a single case of rickets among the second group, and the calcification of their skeletons was normal. The rats under sunlight treatment consumed far more food, were more active, had a good appearance, and were even capable of conception, but were lighter in weight and smaller in size than normal rats. The authors think that ' the effect of sunlight and of cod-liver oil on the growth and calcification of the skeleton and on the animal as a whole seem to be similar, if not identical '. Hess, Unger, and Pappenheimer (1922) were likewise able to prevent the development of experimental rickets in rats produced by the diet above mentioned, by exposure to the sunlight for a period of 15 to 30 minutes. Tiie addition to the diet of 75 mgm. phosphorus in the form of potassium phosphate likewise prevented the development of rickets in rats even when kept in utter dark- ness. However, when the authors replaced 10 per cent, of the flour ^. In another communication (1922, 2) the authors could pr'event tlie develop- ment of rickets in rats kept on the same rickets-producing diet, but exposed to radiation from a mercury vapour quartz lamp. K 2 148 in the food by a corresponding amount of egg albumen, some of the rats developed rickets in spite of the sunlight treatment. Thus the increase of the cases of rickets during the winter months, and the tendency of rickets to spontaneous auto-cure m the summer months, may be explained by the theory of the j*e- ventive influence of sunlight. Conclusions. 1. The clinical and experimental investigations made up to date are in favour of the possible importance of the absence of ultra- violet rays in the aetiology of rickets. 2. Ultra-violet rays, artificially produced, and sunlight may be of great importance in the therapy of rickets. 3. The chief factor in confinement which affects the skeleton is probably the absence of light. , 32. The Theoey of the Infective Origin of Rickets. (a) Historical. This theory is supported by the incidence of cases of rickets continued for many years in certain houses and afiecting almost all the children. The endemic or epidemic character of the spread of rickets has likewise been pointed out (Edlefsen, 1901, 1902), and attention has been drawn to the frequency of cases of enlargement of the spleen in rickets, and to the fever sometimes associated with it (Mircoli, 1898, 1908). Charrin and Gley (1896) injected various toxins (diphtheric, B. pyocyaneus) into rabbits. The ofispring of such rabbits were dwarfed ; their skeletons were deformed or showed changes macro- scopically very similar to rickets. No microscopical examinations were made. Mircoli (1898) isolated staphylococci and streptococci from the cerebro-spinal fluid, the brain, and bones of a rachitic child suffering from fever and affection of the nervous system. An injection of these bacteria into rabbits produced chronic osteomyelitis, with an enlargement of the epiphyses of the bones, and affections of the nervous system— a picture which he regarded as analogous to rickets. Mircoli thinks that when children are suffering from disorders of the "digestive system, or sometimes when skin abrasions are present (e.g. in eczema, Jjoils), the common pyogenes cocci, becoming especially virulent, may penetrate into the organism. If so, they attack the parts of greatest functional activity, among others, the epiphyses of the bones, and the nervous system. Stoelzner briefly characterizes Mircoli's work as logically deficient and show- ing an ignorance of the nature of bone changes in rickets. Smaniotto (quoted by Fishl, 1901) likewise isolated various microbes (B. pyogenes, B. coli, Pneumococcus, Pneumobacillus) from the bone marrow of rickety children. To all these microbes he attributes great importance in the production of rickets. 149 Spillman (1900) isolated these microbes not only from rachitic subjects, but also from children with a normal skeleton. On injecting both the microbes and their toxins, he was unable to produce rickets in animals. He likewise obtained on the whole negative results from injecting into animals alcoholic or aqueous extracts of contents or walls of intestines of rickety children suffering from gastric disorders, or from normal children. Of 21 animals, only one showed changes which might be regarded as similar to mild rickets. Torane and Forte (1907), on the other hand, produced rickets in rabbits by injecting aqueous or alcoholic solutions of the faeces of rickety children with diarrhoea. The experiments made by Morpurgo (1900, 190.2) are considered to be far more convincing, principally because they are confirmed by Schmorl. The author isolated diplocoeci from the spinal marrow, less frequently from the liver, kidneys, spleen, and bones, of rats affected with spontaneous rickets. Sometimes he likewise found other bacteria (B. coli. Staphylococcus, &c.) associated with the diplocoeci. By subcutaneous injection he produced, sometimes after several months, rickets or osteomalacia in 27 of the 42 rats experi- mented on. In his first communication, Morpurgo regarded the disease thus produced as not completely identical with osteomalacia, but rather as something half-way between the latter and ostitis fibrosa. In most cases osteoporosis was observed in the skeleton, caused principally by the increased activity of osteoclasts. The reabsorbed bone was, replaced by fibrous tissue. In the bone maiTow there was a multitude of pigment cells, situated along the ' blood-vessels. Grave changes were likewise observed in the spinal marrow Unfortunately, the value of Morpurgo's experiments is discounted by his inattention to the question of diet. J. Koch (1909, 1911), on examining the bones of children who had died from various infectious diseases, found in the bone marrow of epiphyses various bacteria, such as staphylococci, diplocoeci, streptococci, B. coli, and other micro-organisms. B. typhosus had already been found in the bone marrow by Quincke (1894) and Busch (1898) in cases of typhoid. Bacteria have frequently been found in the bone marrow even when the blood was sterile. In some cases rachitic changes in the bones were likewise present. The author thinks that the epiphyses of the bones are a favourite domicile and breeding-place of bacteria. In his opinion, the bacterial poisons may cause hyperaemia of the bone marrow blood-vessels, and chronic inflammation of the epiphyses, resulting in rachitic changes of the skeleton. The author tried to confirm his theoretical and pathologico- anatomical conclusions by experiments on rabbits, by intravenous injections of streptococci, pneumococci, and the bacteria of Siberian plague. The bones were investigated after decalcification with nitric acid. The principal changes were characterized by osteo- porosis and certain abnormalities in the line of costochondral junctions. In any case, the changes produced in the rabbits were in no way analogous to rickets. In another investigation Koch (1912) injected cultures of various 150 bacteria intravenously into puppies aged from 5 to 12 weeks. Streptococci alone produced affections of an inflammatory character in the epiphyses and the joints, after a general infection of the whole organism. After the teirmination of the acute process, some of the dogs developed affections of the skeleton, macroscopically very similar to rickets, i. e. enlargement of the epiphyses, rosary, marked curvature of the bones of the extremities. These changes were more easily produced in puppies of a large, smooth-haired breed. The ' rachitoid ' bones were not investigated microscopically. A histological examination was made only of the bones of the puppies which had died at the period of acute infection. Un- fortunately, in this experiment likewise the bones were com- pletely decalcified with nitric acid, and the changes obtained were described very briefly ; they consisted principally in the contraction of the proliferous cartilage in the epiphyses, osteoporosis, and hyperaemia. Kubo (1912) injected staphylococci into the blood of young i-abbits, and investigated the changes in the bones from 24 hours to 6 weeks after injection. He also could not produce any changes even faintly analogous to rickets : there were only a slight inequality in the line of costochondral junctions, and inflammatory symptoms in the bone marrow, with an increased accumulation of giant cells. In one of his latest communications Paton (1922) speaks in favour of the infectious theory of rickets, his views being brought into agreement with the results of the latest investigations con- cerning the part played by vitamin A and .unhygienic surround- ings in the production of rickets. In view of the special interest of his opinion, I shall quote his conclusion in full : ^ From my study of lichets I have come to the conclusion that it is highly im- probable that a disease with such marked clinical features and pathological changes can he directly caused either by unhygienic surroundings or hy defective feeding. As is well known, both of them predispose to infection by the tubercle bacillus, but no one considers that they cause the disease ; both predispose to typhus, but, here again, an infection is the direct cause. The close association of rickets tvith the conditions of slum life, and the way in which it is apt to affect mare than one member of the family, suggest the possibility of an infective element in its causation. It is not necessary to assume that the infection is a specific one. It might be of the nature of the intestinal infection which McCarrison has shown to be associated with the onset of goitre. Bull's account of an outbreak of rickets among foxhound puppies, and the prevention of its onset by removing some of them to fresh ground, seems to point in the same direction. The onset of xerophthalmia as an accompaniment of arrested growth in the absence of the fat soluble A factor is an illustration of this predisposition to infection. The condition cannot be considered in any ofJier light than as of microbial origin rendered possible by the depressed activity of metabolism. The fact that cod-liver oil plays, at least, some part in the prophylaxis and in the cure of rickets is no argument that it does so in virtue of its containing' some factor in the absence of which rickets will develop. It is now considered a specific in rickets, as it was formerly considered a specific in phthisis, although no one ever contended that it contained an antitubercular vitamin. Its action is probably in both cases an indirect one upon the metabolism, so that resistance to the onset of disease is increased, or the morbid process, if established, is overcome. If the action of unhygienic conditions and of defective diet as simply predis- 1 The italics ai-e not in the original. 151 posing to the onset of rickets is accepted, the practical question which has to be faced in each place in which the children are largely affected with rickets is whether the preponderant factor is the former or the latter. (b) The author's experiments with bacteria. Experiment I. Intestinal bacteria. The experiment was made upon 10 rats, one of which soon died. Of the remaining 9 rats, 8 belonged to one litter; and 1 was taken from another litter. Of the intestinal bacteria I chose the three to which Metchnikov attributes great importance in the production of gastro-intestinal autointoxication, viz. : B. welchi/i, B. sporogenes, and B. bifermentans. The bacteria were obtained from the collection at the Lister Institute. The cultures of these bacteria were prepared in Metchnikov's meat medium, special atten- tion being paid to the absence of fat from the beef which served as a basis for the preparation. The cultures were mixed in equal pro- portions and given to the rats instead of water. The rats drank 10-15 c.cm. of these cultures daily very willingly in spite of the very disgusting smell. Besides this, two rats had subcutaneous injections of live cultures of B. sporogenes and B. bifermentans (0-6-2'0 c.cm.) at intervals of 1 or 2 days. The rats were divided into the following groups: (i) on normal diet (one rat) ; (ii) one rat on diet N — Ca ; (iii) three rats on N — Ca diet with the addition of cultures of the three above-mentioned bacteria to their food ; two of these rats had subcutaneous injections* of the same bacteria. Group iv (two rats) was on — A — Ca diet; group V (two rats) was likewise fed on — A— Ca diet, with the addition of bacterial cultures to the food, and one of these rats had, over and above that, injections of bacteria subcutaneously. The rats were 33 days old when put on the special diets, and were fed on them for 99 days. They were killed when 133 days oldj except one which died at the same age. This rat was kept on — A — Ca diet without introduction of bacteria. Intake of bacteria with the food or introduction subcutaneously was started when the rats were 62 days old (one month after being put on the special diets) and was continued for 70 days. At autopsy, the rats which had bacterial cultures injected subcutaneously showed, at the points of injection, under the skin inflammatory foci in various stages of resorption, containing solid caseous masses in the centre. Notwithstanding the enormous amount of subcutaneously injected cultures of B. bifermentans and B. sporogenes, the rats were not very backward in weight as com- pared with their controls, and in general, readily tolerated the injec- tiojw in spite of the local inflammatory reaction. Histological examination showed no essential difference between the skeletal changes of the rats receiving cultures of gastro-intestinal bacteria and, those of their respective controls. The results of the chemical analysis are given in the following table (No. 57) showing the average percentage (in figures) of each of the groups above mentioned. Owing to there being no difference between the results, the rats 152 subcutaneously injected with bacteria are not separated from the rats which received bacteria only in their food. Table No. 57, Content in hone (%). -an ^^ ^^ Groups. Diet. titU (jreshhone). {dry bone). I. N diet 34-9 14.3 22.1 II. N-Cadiet 33-8 13-6 20-5 III. N— Ca diet with the addition of intestinal bacteria cultures in the food . . . 35-0 13-5 20-8 IV. -A-Cadiet 50-5 6.0 12-2 V. -A— Ca diet with the addition of intestinal bacteria cultures 44-8 8.5 15-36 That is to say, the results of the chemical analysis gave the same results as th e histological , namely, the absence of any rickets-producing action of the intestinal bacteria used on the skeleton. In the group of rats on diet — A-Ca with the addition of intestinal bacteria the composition of the skeleton was even less abnormal than that of the control group. This last circumstance may be explained by casual fluctuations in the composition of the skeleton of different rats on abnormal diets. Thus my experiments, like those of Spillmann, showed no significance of the intestinal bacteria used, in the production of rickets. Experiment II. Micrococcus candicans. The micrococcus described by Morpurgo in his experiments is apparently similar to Micrococcus candicans Fliigge. This micro- coccus is usually found in air, water, or milk (Lehmann and Neumann, 1920). On bacteriological examination of the blood, bone marrow, spleen, and liver of 51 rickety rats this micrococcus was found by me on two occasions, being an accidental contamination from the air. In broth it grows in the form of diplococcus or staphylococcus. Owing to the similarity of this micrococcus with the coccus of Morpurgo, experiments were made upon 10 rats. Group I. Four rats, about 2 months old, were injected intra- peritoneally with an emulsion of a pure culture of the micrococcus : 2 rats received the whole of one agar slope each and 2 others f of an agar slope. The rats were kept on N diet. For about 16 days after the injections all the rats were very depressed, with no appetite and showing roughened fur. One of these rats died 3 days after injection with peritonitis and pleurisy. The 3 remaining rats were killed 55 days after the injection. Two of these rats grew very slowly and one at normal rate. Histological examination of the skeletons ofall three showed normal calcification of the bone and of the zone of provisional calcification. Group II. Two rats, 35 days old, weighing about 75 gm. were put on —A diet. On the same day one of them was injected intraperitoneally with ^^ of an agar slope of a culture of Micro- coccus candicans. Fifty-five days after this both rats were killed at the age of 90 days. Macroscopical and histological examinations o 153 V, o o I I g I o : § 'S o "5^ o g (5« \ J; « o fip CO M3 ^ 1-1 _j rH rH IN iH OS OS U^ CO ^ ^ t; OJ rH OJ OS O CO 00 tH lO WS ■<* M a ID u § .3 ^1 ^* TP ^91 (M 01 (M (M ^ SO sO ^ 154 of the skeleton showed no difference between them. Histologically moderate rickets was present in both. Group III. Four rats, all males belonging to litter 624, described on p. 101, were used. They were borne by a mother kept on —A diet during lactation. After weaning, the young animals were fed on — A diet. At the age of 42 days (18 days after weaning), 3 rats (A, B, and H) out of 4 were injected intraperitoneally with an emulsion of -^ of an agar slope of Micrococcus candicans, the fourth rat (D) remaining as control. Forty-five days after injection, i. e. at the age of 87 days, all rats were killed. At autopsy control rat D and two infected rats (B and H) were emaciated. Infected rat A was thin, but not emaciated. They differed in no other respect from one another. All had opaque teeth and some curved ribs showing spontaneous fractures. The results of chemical and histo- logical examinations of the skeleton of these rats are given in Table No. 58 (p. 153). No difference is apparent between the skeletons of the infected rats and the control one. Conclusions. 1. Evidence in favour of the theory of infection in the aetiology of rickets is afforded by Morpurgo's experiments, corroborated by Pi'of. Schmorl. The value of Morpurgo's experiments is, however, discounted by his inattention to the question of diet. 2. The intestinal bacteria and Micrococcus candicans have pro- duced no rachitic changes in the skeleton. 3. The validity of the theory of infection in the aetiology of rickets has not yet been proved. 23. Influence of Heredity on Incidence, or of Pre- disposition TO Rickets. A great obstacle to the elucidation of the part played by heredity in the aetiology of rickets is the fact that in the countries where rickets occurs it is so prevalent that it is often impossible to dis- tinguish between cases of mere coincidence and those which might be explained in terms of heredity. Fischl (1901) and Stoeltzner (1904) draw special attention to this difficulty. Nevertheless, these authors acknowledge, on the basis of data given below, that heredity or an hereditary predisposition deserves serious attention in the problem of the origin of rickets. Hitter von Eittershain (1863) and Pfeiffer (1885) draw attention to the fact that in families where the elder children suffered from rickets all the following children are likewise rickety. Moreover, very often the mother, or her sisters, show signs of having suffered from very acute rickets in childhood, On these grounds Pfeiffer draws the categorical conclusion that rickets is an hereditary disease, or at least one which develops on the soil of an hereditary tendency. Siegert (1903, 1905), on the basis of numerous statistical data. 155 comes first of all to the conclusion that rickets occurs more fre- quently with artificial feeding than when the child is nursed by its mother. Of the 845 artificially fed infants examined by him, 81 per cent, were rickety, whereas of 923 breast-fed infants only 31-5 per cent, were rickety. Of 31 ' rachitic ' families, where breast-fed infants developed rickets, in 29 cases it was proved that the mothers had been rickety, and 2 cases were doubtful. Of 14 families where breast-fed children did not develop rickets, the mothers had likewise been non-rachitic. Siegert draws special attention to the fact that these 1 4 families ivere living in the tuorst possible conditions. Of 14 ' rachitic ' families, where the children were brought up on artificial food, the mothers, and frequently the fathers,^ had suffered from rickets. In two families the author makes no mention of mothers being rachitic (an oversight ?). A non-rachitic child in a ' rachitic ' family is explained away by Siegert as the illegitimate offspring of a strong and healthy father. In eight cases Siegert managed to establish the fact that all the legitimate children were born rickety, while the illegitimate were free from the disease. Thus, according to Siegert, the father may have as much influence as the mother on the transference of rickets or predisposition to the disease. Siegert compares the cases above mentioned with the fact that children of healthy parents are generally free from rickets under the worst hygienic conditions, and, on the contrary, rickets occuiis under the most ideal conditions of life and nourishment in children of rachitic parents. This leads Siegert to conclude that heredity is one of the most important aetiological factors in this disease. According to this author, the predisposing factors in rickets are : improper nourishment, disoi'ders of the gastric and thoracic organs, and the general debility of the parents. Fischl (1901) cites a case of Elgood's, analogous to those observed by Siegert : a woman first married a healthy man and bore healthy children. The offspring of her second marriage to a weakly man were all rickety. Finally, during her second marriage she gave birth to an illegitimate child by a healthy father, and this child was healthy too. Professor Heubner (1911), on the basis of his extensive clinical experience, likewise recognizes the influence of heredity in the fairly frequent cases of rickets in infants at the breast, well looked after and living in excellent conditions as regards food and housing. Heubner draws attention to the fact that in such cases rickets fre- quently develops after some gastric disturbance or slight infection, e.g. alter vaccination. Kassowitz (1909) likewise thinks that some cases of rickets can be explained only by an hereditary predisposition. He repudiates direct heredity, on the grounds that at the time of conception the parents are free from the disease. He regards rickets as a congenital disease, not necessarily inherited. 1 The question of the fathers being rachitic or not was not investigated by Siegert with sufficient thoroughness, on account of technical difficulties. 156 Under the direction of N. Paton and Findlay (1918) Miss Ferguson made some highly interesting investigations in Glasgow as to the part played by social and economic factors in producing rickets. On the basis of these investigations, the following con- clusions were arrived at concerning the state of health of the mothers and fathers of i-achitic and non-rachitic children : ' A greater number of the mothers of the non-rachitic children were found to be in good health than was the case with the rachitic. Many of the latter looked anaemic and nervous, and seemed to have quite lost heart in their families. Although, owing to the indefinite nature of such information, this was not studied with care, it was ascertained that some of them had been rachitic as children, although only a few were noticeably deformed by the disease. The mothers of the non-rachitic children generally looked fresher, healthier, and more intelligent.' The following table. No. 59, compiled by me from the highly interesting figures of the authors (pp. 50-51), may serve as an illustration of the statement quoted : Table No. 59. o/ 0/ j • , ^ non-rachitic state of health. ,ZT^'nf\ ,Tnlt\ "hUdrm (SOOchMren). {150 cMldren). ^^00 chiUrm). 'ZA Good 51 68 80.5 gS Pair 20.5 19 14 ®_g Poor 28.5 18 5-5 :S g> g Good 60.5 71 87 ■3-s g Fair 18 12 i j3.g g> Poor 26.5 17 9 ^ „ ^ Good 59 li. 84 "i'si Fair 12 16 9 5o Poor 29 10 7 As in Siegert's statistical investigation, in the Glasgow investiga- tions the influence of the father's health could not be clearly elucidated for technical reasons, i.e. owing to the difloiculty of finding the father at home, the information was in most cases supplied by the wife. On the basis of this material, the authors have drawn the following conclusions (p. 53) : ' Although a slightlj^ greater number of the fathers of the rachitic than of the healthy families appeared to be under the normal in health, the difference was not so marked as in the case of the mothers.' On the basis of these results the authors draw the very cautious conclusion that ' the state of the health of the mother can at most be regarded as a contributory factor in the development of rickets, and may very possibly be merely an index of the social conditions ' (p. 51). 157 There is no doubt that the question of the heredity of rickets, or of a predisposition to the disease, might be solved more indisput- ably, if it were possible to determine the frequency of congenital rickets. It is very difficult to decide the question of congenital rickets clinically, as the earlier clinical symptoms of rickets, sufficient for the diagnosis of the disease, are still in dispute. The majority of authors regard craniotabes as the most valuable symptom for an early diagnosis. On the basis of the frequency of this symptom in new-born infants, as well as of certain others (rosary), Kassowitz (1884), Schwarz (1887), Cohn (1894), Feer (1897), Spietschka (1904), Zappert (1905), and others recognize that rickets is frequently congenital. Cohn has investigated 115 children during the first month of their lives and found that approximately half had softened cranial bones. Other rachitic features followed later. That proves to his mind (1) the rachitic character of the cranial bone softening, and (3) the existence of ' congenital ' rickets. Spietschka found craniotabes in 50 per cent, of the 1,468 new-born infants examined. Only in 7 per cent, of the cases did it fail to develop into obvious rickets. The rachitic character of this malady is further proved by its successful cure by the cod-liver oil and phosphorus treatment.. Wieland (1908), on the basis of careful clinical investigation of about 1,000 new-born infants and 500 infants of one month, came to the following conclusions : (1) Con- genital softening of the cranium occurs approximately in 30 per cent, of the cases, being characterized by localization on the apex of the cranium (' Kuppenerweiehung '), and by a tendency to spontaneous recovery. Eecovery took place at various periods, from several weeks to six months. From 70 to 83 per cent, of such new-born infants subsequently developed rickets, and Wieland regards this figure as usual. Wieland denies the rachitic origin of this congenital craniotabes, on the grounds that (1) it affects the apex of the cranium, whereas rachitic craniotabes occurs below the back of the cranium; (3) typical rachitic craniotabes develops much later, when congenital craniotabes is already cured. Kassowitz (1913), on the basis of local examination of 1,376 new-born infants, questioned the indisputability of ' local ' and ' temporary ' distinctions indicated by Wieland. He only admits that the apex of the cranium is affected more frequently in new- born infants than at a later development of rickets. Like Spietschka, he cannot draw a definite time limit : Wieland himself observed the cure of this congenital craniotabes, not only a few weeks, but even many months after birth. In general, for the same reason, i. e. the difficulty of determining the indisputable first symptoms, histological investigations of the foetus, prematurely born or new-born infants, could not solve the question of congenital rickets. Kassowitz (1885, 1913) histologically examined the skeletons of 36 aborted foetuses, 38 still-born, and 48 infants either new-born or only a few days old. In the first two groups he found about 11 per cent, of normal skeletons, aiid about 18 per cent, in the third group. In the remaining cases 158 Kassowitz diagnosed rickets. Nau (1905) also found an identical picture in the case of new-born infants with acquired rickets : an enlargement of the cartilage, ingrowing of blood-vessels into the latter, irregular costochondral junctions, and great vascularization of osseous tissue. Tschistowitsch (1897) and Wieland (1909) did not agree with Kassowitz, holding that the symptoms indicated by Kassowitz were insufficient for a diagnosis of rickets. Some of the changes found by Kassowitz were considered as more characteristic of inherited syphilis. In his last communication, however, Kassowitz (1913) gave drawings of the changes in the skeleton which seem to prove the probability of his having really found cases of congenital rickets (e.g. Fig. 8, p. 305; Fig. 13, p. 312). On the basis of their pathologico-anatomical investigations, Fede and Finizio (1901), Escher (1902), Tschistowitsch, and Wieland could not find any indisputable cases of congenital rickets. Wieland found that in new-born infants osteoid is physiologically prevalent in the skele£on over a far larger area of bone than in older infants, its width attaining 10 fi or even 15 fi (more frequently from 3 to 10 fi). Schmorl (1909), from a full and exhaustive investigation of pathologico-anatomical material, comes to the conclusion that the earliest cases of rickets occur only from the middle of the second month of an infant's life, and that from the nineteenth month of life the frequency of cases of rickets already begins to decrease. In view of the special value of Schmorl's investigations, I give the following table, No. 60, in which the author summarizes the results obtained by him (p, 439) : Table No. 60. ge of infant Incipient Rachitis Convalescent Number of cases rickets Jlorida rickets Oured rickets. in Schmorl's I tnufbi/ttij . (%)• (%)• (%)• materials. 2 100 4 3 75 25 — 16 4-6 57.6 21.2 2] .2 — 33 7-9 34 46 20-0 — 50 10-12 9.5 61.7 27.4 1.4 73 13-18 6.9 65-2 25-8 2.1 58 19-24 6.6 33.4 36-6 23.4 3» 25-36 — 22.9 24.5 52.6 57 37-48 — 8.3 12.5 79.2 24 345 Thus Schmorl's investigations emphasize the astonishingly early appearance of rickets after birth (in the first quarter of the first year of life). If a histological diagnosis of rickets can be made as early as the second or third month of the child's life, there can be no doubt that the development of the process must have begun at a still earlier age, i. e. at least in the first month. This fact is an argument for the possibility of a congenital rickets or a congenital predisposition to rickets. 159 A striking instance of the effect of ante-natal conditions upon development of rickets observed in Vienna in 1930 was personally communicated to me by Drs. Dalyell and Mackay. In a foundling institution in that city two infants were observed, the first born of a healthy mother and the second of a mother suffering fi-om osteo- malacia. The mother of the' first acted as foster mother to the second. With the exception of the earlier weeks of life when both infants were completely nourished by their own mothers, they received identical diets, consisting of breast milk from the healthy woman, supplemented by one or two bottles of cow's milk daily. The children were born in July and August respectively, and by the middle of December severe rickets was observed in the second child at 4-| months old, and in spite of treatment with cod-liver oil, stigmata of rickets persisted until the following Mai'ch when the child was about 9 months old. The first child remained healthy and developed normally. There is no doubt, however, that clinical and pathologico- anatomical investigations cannot decide the question of the possible importance of heredity in rickets. What is required is many- sided experimental research, and as yet very little has been done in that respect. The investigations which I have already quoted show that when females are fed during lactation or preg- nancy on a diet deficient in calcium (Dibbelt, 1910), and especially with the addition of strontium (Lehnerdt, 1909, 1910), or when arsenic is introduced into the organism (Gies, 1878), changes take place in the skeleton of the offspring. As the methods employed by the authors above mentioned were not, in themselves, rickets- producing, these experiments may serve only as proofs that the health of the mother is an important factor in the normal develop- ment of the skeleton. In Stilling and Mehring's experiment (1889) the skeleton of puppies borne by a bitch fed on a diet deficient in calcium was not examined histologically. I have already described (Chapters 11 and 12) the following ex- periments : (1) feeding mothers diiring lactation on diets N, N — Ca, — A, or — A — Ca, their young, after weaning, being either killed at once or kept for some time on the same deficient diet as their mother or on —A diet; (2) feeding the parents on —A diet during con- ception, pregnancy, and lactation, the young, after weaning, being kept for a certain time on the same deficient diet. All these experiments have shown that with the deficient feed- ing of the mother during pregnancy or lactation the general dis- turbances of nourishment in the organism of the offspring are far greater, and the skeletal changes are far more severe, than when the mothers were normally fed. Under these conditions, with both mother and young on a N — Ca diet, even tvhen it contained cod-liver ail, skeletal 'changes were obtained in the young (both macroscopically and microscopically) analogous to rickets. On — A diet most frequently there was the picture of typical and severe rickets, independently of whether the young- were killed imme- diately after weaning or some time after. I think that the experi- ments with —A diet especially may serve as an argument in favour of the possibility of therexistence of congenital rickets. Their chief 160 value, however, lies in the fact that they have estahlished the enormous importance of the mother's diet during lactation. Another point in favour of this is the great resistance to unfavour- able influence of —A diet, shown by the offspring whose mother was fed during lactation on a diet rich in cod-liver oil and calcium salts. The results obtained by me explain, from a new point of view, the appearance of healthy children in rickety families, and rickety children of apparently healthy parents. A diet rich in antirachitic factor and calcium phosphate during pregnancy and lactation results in healthy oflspring. On the other hand, if during another pregnancy or lactation by some chance the diet of the same mother should be deficient in these factors, her offspring will be predisposed to rickets, or may even be born with those indefinite symptoms of it mentioned at the beginning of this chapter. Of course I fully admit the possibility of an analogous action of other aetiological causes (e.g. light) predisposing to rickets or, on the contrary, preventing it. Conclusions. On the basis of the foregoing, I think that it is possible to draw the following conclusions : 1. The question of the heredity of rickets cannot be finally settled at present. 2. On the basis of clinical, pathologico-anatomical, and experi- mental investigations, it is impossible to deny the existence of a congenital disease of the skeleton — if not of a rachitic character, at any rate predisposing to rickets. 3. The presence or deficiency of antirachitic factor and calcium salts in the mother's diet during conception, pregnancy, and lacta- tion control, to a considerable extent, not only the general nutrition of the offspring, but particularly its skeletal development and the eventuality of the appearance of rickets. 4. Some clinical observations point to the possible significance of the state of the father's health for the normal development of the skeleton of the offspring. 5. The attention of mothers should be specially drawn to the necessity for the presence of a sufficient amount of antirachitic factor, phosphates, and calcium salts in their diet during pregnancy and lactation. 24. General Summary. In the present work the author has referred to, and analysed as critically as possible, about 400 investigations, the results of which have been compared with one another, as well as with the results of his own experiments. In drawing conclusions, attention has been paid chiefly to how far the various theories are supported or refuted by experimental results. The facts ascertained from the study of, rickets and osteomalacia up to the present gtime have thrown con- 161 siderable light on the pathology, aetiology, and to some extent the treatment of these diseases. There is no doubt that in order to place on a sure foundation many of the conclusions mentioned below, further and more numerous experiments and clinical observations will be requii-ed. Perhaps totally new factors may be discovered, but it is hardly likely that the significance of the fundamental conclusions obtained will be questioned. In the pathology of rickets and osteomalacia, the pathologico- anatomical and, to some extent, the chemical essence of these diseases has been elucidated, namely : the overwhelming significance in the picture of these diseases of the osteoid as compared with other * changes in the skeleton ; the causes producing the fluctuation in the amount of rachitic changes in the proliferous cartilages and bones ; the causes of the appearance of .osteoporosis ; the establishment of Schabad's periods in the metabolism of calcium in rickets. Rats kept on — A or — A — Ca diets, in which osteoporosis with or with- out slight rickets was developed, often showed the same chemical changes of the skeleton as animals of the same litter in which pronounced rickets was found on bistological examination. This i fact confirms the view already emphasized of the existence of ' latent ' forms of rickets, and indicates the importance of chemical analysis in diagnosis. In the aetiology of rickets and osteomalacia factors undoubtedly playing a great part in the origin of these diseases are deficiency in i the diet of antirachitic factor, calcium, and phosphorus. Confines ment, in the sense of insufficiency of light, fresh air, and muscular exercise, apparently also plays its part, the majority of the data in agreement with each other being in favour of the importance of light, probably of ultra-violet rays. Therefore, apparently, rickets could be caused by any one of these aetiological factors or by any combina- tion of them. From this point of view there are several forms of rickets which have to be classified from an aetiological stand- point. One of the forms of osteomalacia can be induced by deficiency in antirachitic factor or calcium in the diet of adult animals and^ especially of the mother during pregnancy or lactation. The nourishment of the mother during pregnancy, and particularly during lactation, may be of primary importance in producing or preventing rickets in the offspring. The increased incidence of rickets during the winter months and the tendency to spontaneous cure in the summer months might be explained by three theories, each on its own basis : 1. The theory that rickets is caused by a deficiency in diet of antirachitic factor may explain seasonal fluctuations in incidence of rickets on the ground that these fluctuations coincide with " seasonal variations of vitamin content in food. 2. The confinement and lack of exercises theory fits in with the seasonal incidence of the disease on the assumption that the > child gets more exercise and less confinement in summer than in winter. 3. Similarly the influence of light on cause and cure of rickets may explain the winter incidence and summer's spontaneous cure on (1S79) L / 1. 163 the assumption that the child is exposed to more sunlight in the spring and summer than in winter. In the treatment of rickets the enormous therapeutic value of cod-liver oil (owing to its richness in antirachitic factor) and the advantage of ultra-violet ray treatment, have been firmly established, both clinically and experimentally. Useful results may be expected from the attempts to find suitable methods of applying therapeuti- cally calcium salts, phosphorus, and perhaps arsenic. Sufficient attention has not been paid, clinically, to the latter. Finally, for the prevention of rickets the following factors may be taken as. most important: adequate amount in the mother's diet of antirachitic factor, calcium, and phosphates duiing pregnancy,' and especially during lactation ; a similar diet in the case of the 'father may possibly be also important ; the richness of the infant's diet in the same substances, and its. abundant use of light, fresh air, and muscular exercise. In conclusion it is ray pleasant duty to record my gratitude to the Medical Research Council for affording me a grant which has enabled me to cai-ry out this work, and to the Lister Institute for the hospitality of its laboratories. I wish also to express my sincere gratitude to Professor C. J. Martin for his helpful advice, criticism, and continuous support in this investigation. For criticism of my present work I am also indebted to Professor D. Noel Paton and Miss H. Chick. In the experimental work I have been assisted by Miss C. Rutherfurd and Miss M. 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Proliferous cartilage. zee. Zone of columns of proliferous cartilage cells. ZHC. Zone of hypertrophic cartilage cells. ZPC. Zone of provisional calcification of proliferous cartilage. Sp. Spongiosa. Co. Calcified cortical bone. CI. Callus. BM. Bone-marrow. P. Periosteum. O. Osteoid tissue. L. Lime deposition. ' BV. Blood-vessels. PLATE 1 Pig. 1. Two rats of the same litter : White — control rat No. 62, kept on N diet. Age, 132 days. Weight, 260 gm. Black— -rat No. 68, kept on -A-Cadie't. Age, 132 days. Weight, 85 gm. Pig. 2. A— control rat on N diet. Age, 70 days. Weight, 200 gm. E — rat No. 107, kept on —A diet, whose mother was fed during lactation on the same diet. Age, 70 days. Weight, 48 gm. PLATE 2 Pig. 3. Normal rat No. 79, kept on N diet. Age, 76 days. Weight, 209 gm. Kib junction. Normal calcification of the zone of provisional calcification of the proliferous cartilage and of bone tissue. Physiological osteoid tissue is seen only as a very thin layer on inner side right rib wall. Haem.-eosin. Magn. x 36. Pig. 4. Normal quickly growing young rat No. 136b, kept on breeding diet No. I. Age, 42 days. Weight, 75 gm. Normal calcification. Practically physio- logical osteoid tissue is not seen at this magnification. In this rat the histological picture of the bones was somewhat similar to that found in slight physiological osteoporosis of children (see pp, 21, 52). EP = Epiphyseal cartilage. Haem.-eosin. Magn. x 28. PLATE 3 Pig. 5. Eat No. 139, kept on N — Ca diet, whose mother was kept on the same diet during lactation. Age, 70 days. Weight, 77 gm. Kib junction. Rib is curved. Typical rickets from calcium starvation. Small enlargement of zone of proliferous cartilage. Calcification of the zone of provisional calcification beneath the peri- chondrium only. Curved and slightly disorganized line of costochondral junction by irregular ingrowth of abnormally wide prolongations of vascular bone-marrow. Spongiosa and cortical bone consisting of numerous interlaced trabeculae. Bone tissue of trabeculae and cortical bone in most places imperfectly calcified. Note imperceptibility of transition of calcified hone tissue into osteoid as compared with sharp outlines in Figs. 17, 18, 32, &c. Haem.-eosin. Hagn. x 36. Pig. 6. Rat No. 140, from the same litter as rat No. 139 and kept on the same diet, but killed at the age of 85 days. Weight, 95 gm. The same picture. Cut without decalcification and stained by silver method, showing definite calcification of most of the newly produced bone tissue. Thin layers of osteoid surrounding the 17,0: trabeculae of spongiosa and cortical bone except those of the subchondral spongiosa. Magn. X 28. Pig. 7. Eat No. 141, from the same litter as 139 and 140, and on the same diet. Age, 81 days. Weight, 43 gm. Proliferous cartilage greatly Increased. The ZPC is impregnated with lime salts (black) only near perichondrium. Subchondral spongiosa consisting nearly completely of thick curved trabeculae of osteoid. In the other, the same picture as in Nos. 139 and 140. Cut without decalcification. Silver + carmin + eosin. Magn. x 36. PLATE 4 Fig. 8. Control rat No. 610b, kept on N diet after weaning, whose mother was on N - Ca diet during lactation. Age, 76 days. Weight, 122 gm. Rib junction. Normal calcification of the zone of provisional calcification and of bone tissue. Haem.-eosin. Magn. x 36. (See page 105.) Fig. 9. Rat No. 610e, kepc on N — Ca diet, whose mother was on the same diet during lactation. From the same litter as Eat No. 61 Ob. Age 76 days. Weight, 41 gm. Rib junction. Severe rickets from calcium starvation. Rib curved at an angle. Proliferous cartilage increased and disorganized by irregular ingrowth, of highly vascular bone-marrow. Calcification of the ZPC beneath the perichondrium only. Considerable amount of osteoid tissue, especially in the angular portion (02) of the rib. In many places note the imperceptibility of transition of calcified bone tissTie into osteoid. Haem.-eosin. Magn. x 36. PLATE 5 Pig. 10. Rat 610k, kept on —A diet, whose mother during lactation was on N— Ca diet. From the same litter as rats Nos. 610b and 610e, with which compare rat 610k. Age, 76 days. Weight, 63 gm. Rib junction. Severe rickets. Eib bent at an angle. The picture is similar to that of rat 610e, but typical of rickets. Lower part of proliferous cartilage becoming directly transformed into osteoid tissue and showing rachitic metapliysis. The line of demarcation between calcified bone tissue and osteoid is better than in rat 610e. Note that the diffuse lime deposition in osteoid tissue is less than in the rat 610e. Haem.-eosin. Magn. x 36. Pig. II. Eat 314a, kept 78 days on —A diet. Still growing all the time of feeding on deficient diet. Age, 108 days. Weight, 117 gm. . Epiphysis of radiu,s. Pronounced rickets. Note the deep staining (black in figure) of all the cartilage, which indicates special chemical changes in cartilage, but not deposition of lime. This deep staining of cartilage occurs in some rachitic rats (e. g. Fig. 35, PI. 14). Haem.-eosin. Magn. x 30. PLATE 6 Pig. 12. Eat 88a, kept on —A diet 73 days. Died in stage Of cachexia, and after loss of weight. Age, 94 days. Weight, 60 gm. Kib junction. Very marked osteoporosis without any signs of rickets. No osteoid tissue. Proliferous cartilage growths completely arrested showing typical ' bone-plate ' (BP). Complete calcifi- cation of the ZPC and of bone tissue. This picture shows the influence of cachexia on the rachitic skeleton, Haem.-eosin. Magn. x 36. Pig. 13. Rat 41a, kept on — A diet 140 days. Killed in the stage of very pro- nounced cachexia. Age, 202 days. Weight, 1 18 gm. Eib junction. Very marked osteoporosis. The changes not so marked as in the case of Rat 88a. • There is some growth of proliferous cartilage, some short and thin trabeculae of spongiosa, and very thin layers of osteoid tissue, which unfortunately is not visible in the print. This case shows also the influence of cachexia on the rachitic skeleton. Haem.-eosin. Magn. x 86. Pig. 14. Rat 496, kept on — A diet 98 days. Killed in stage of sufficiently good nutrition with some fat in body. Age, 139 days. Weight, 122 gm. Modei ate rickets. Moderate increase of pi-oliferous cartilage with deficient calcification of ZPC. Rachitic metaphysis consisting almost entirely of osteoid. Trabeculae of spongiosa and coi'tical bone surrounded by considerable amount of osteoid tissue. Haem.-eosin. Magn. x 40. PLATE 7 : . . Pig. 15. Eat 498, kept on —A diet 103 days, killed in the stage of arrested growth. Not emaciated, but without fat. Age, 173 days. Weight, 90 gm. Moderate rickets. Disorganization of cartil.age and of line of costochondral junction. Deficiency 171 of lime deposition into the ZPC. Uneven amounts of osteoid tissue covering the trabeculae of spongiosa and of cortical bone. Met = metapliysis. Haem.-eosin. Magn. x 40. Pig. 16. Eat 487, kept on -A diet 98 days. Age, 139 days. Weighty 82 gm. Bib junction. Bib curved at an angle. Pronounced rickets with typical changes in the proliferous cartilage and bone tissue. Haem.-eosin. Magn. x 23. Pig. 17. Eat B, kept on —A diet, whose mother was on the same diet during pregnancy and lactation. Age, 71 days. Weight, 49 gm. Severe rickets. Haem.-eosin. Magn. x 86. PLATES Pig. 18. Eat 107, kept on —A diet, whose mother was on the same diet during lactation. Age, 70 days. Weight 48 gm. Moderate rickets with osteoporosis. Note few and thin trabeculae of spongiosa and thin rib walls, surrounded by comparatively considerable amount of osteoid tissue, as compared with calcified parts of bone. Below typical rachitic spontaneous fracture with callus consisting of osteoid and cartilage. Haem.-eosin. Magn. x 36. Pig. 19. Eat 409, kept on —A diet, whose mother was on the same diet during pregnancy and lactation. Died at stage of very pronounced cachexia with enteritis and pneumonia. Age, 78 days. Weight, 45 gm. Rickets with osteoporosis. This is one of the pictures, also typical of the influence of cachexia on the rachitic skeleton. Proliferous cartilage gi-eatly increased with deep ingrowth of blood vessels. Ca deposition in ZPC near the perichondrium only. Few and thin trabeculae of spongiosa. Osteoid covers trabeculae and cortical bone in layers thinner than calcified bone and in a few places only in layers equal to or thicker than the latter. Haem.-eosin. Magn. x 40. Pig. 20. Eat 623o, kept on —A diet, whose mother was on the same diet during last 15 days of lactation. Diet of mother during pregnancy and first 9 days of lactation was slightly deficient in antirachitic factor and calcium salts (Diet No. 1 without milk and addition of calcium salts). Age, 65 days. Weight, 57 gm. Bib junction. Severe rickets. Bib curved at an angle. Enormous increase of proli- ferous cartilage and of osteoid tissue. Lack of lime deposition in the ZPC. Haem.-eosin. Magn. x 28. , PLATE 9 Pig. 21. Bat 411, kept on —A diet, whose mother was on the same diet during pregnancy and lactation. Died at stage of cachexia. Age, 77 days. Weight, 46 gm. Bib junction. Rickets and osteoporosis. Picture similar to that of rat 409, but with greater amount of osteoid tissue. Haem.-eosin. Magn. x 40. Pig. 22. Control normal rat No. 606 (4) of the experirhent on the effect of different diets of .mothers during lactation (see page 91). Diet of mother normal during pregnancy and lactation and of this rat after weaning. Age, 75 days. Weight, 177 gm. Normal rib junction. Haem.-eosin. Magn. x 40. Pig. 23. Eat 605 (8) of the same experiment as rat 606 (4). Diet of the mother rat and of this offspring normal during lactation. Offspring on — A diet after period of weaning. Age 75 days. Weight, 120 gm. Bib junction of the rat with most marked changes of litter 605. Very slight increase of cartilage with slight deficiency in calcification of ZPC. Spongiosa more irregular than in normal rat. Osteoid tissue not exceeding the normal limits. Slight osteoporosis. Haem.-eosin. Magn. x 40. PLATE 10 Pig. 24. Bat 605 (4) of the same group as rat 605 (8), but showing nearly normal rib junction. Age, 75 days. Weight, 118 gm. Haem.-eosin. Magn. x 40. Pig. 25. Bat N 603 (3). Age, 75 days. Weight, 50 gm. Pig. 26. Bat 603 (4). Age 75 days. Weight, 45 gm. Pig. 27. Eat 603 (5). Age, 75 days. Weight, 63 gm. All these three rats are of the same experiment as rats 606 (4), 605 (8), and 605 (4). Diet oi the mother normal during pregnancy, — A diet during lactation. — A diet of the offspring after weaning. AH three rats show moderate rickets. In rat 603 (3) marked disorganization of proliferous cartilage. In rats 603 (4) and 603 (6) the stage of cartilage is more typical of late rickets. Osteoid is equ^ to, or thicker than, calcified bone tissue in most places. Haem.-eosin. Magn. x 40. 173 PLATE 11 . Fig. 28. Control normal rat 606 (9) of the same group as rat 606 (4) (see Fig. 22). Age, 75 days. Weight, 136 gm. Epiphysis of radius. Normal bone. Haem.-eosin. Magn. x 38. Pig. 29. Kat 605 (3) of the same group as rats 605 (8) and 605 (i). (See Figs. 23 and 24.) Age, 75 days. Weight, 123 gm. Badial epiphysis practically normal. Haem.-eosin. Magn. x 36. PLATE 12 Fig. 30. Rat 608 (1) of the same group 603 as the rats whose ribs are shown in Figs. 25, 26, and 27 (Plate 10). Age, 75 days. Weight, 63 gm. Epiphysis of radius. Rickets. Haem.-eosin. Magn. x 40. Fig. 31. Eat 603 (3) ; for the microphotograph of rib of same rat see Fig. 25. Epiphysis of radius. Moderate rickets. Haem.-eosin. Magn. x 40. PLATE 13 Fig. 32. Eat 204, kept on —A -Ca diet 48 days. Age, 78 days. Weight, 46 gm. Very severe rickets. Rib bent at an angle. Increased proliferous cartilage. Great amount of osteoid tissue throughout. Spontaneous fracture with callus consisting of osteoid and cartilage. Haem.-eosin. Magn. x 40. Fig. 33. Epiphysis of humerus of the same rat 204. Note the direct transforma- tion of cartilage into osteoid in the subchondral spongiosa. Disorganized cartilage, osteoid tissue and irregular ingrowth of blood-vessels constitute under the epiphyseal cartilage the large rachitic metaphysis (Met). Lime deposition in the ZPC is very defective. Some osteoporosis. Haem.-eosin. Magn. x 21. PLATE 14 Fig. 34. Epiphysis of radius of the same rat 204. The same picture. Haem.-eosin. Magn. x 22. Fig. 35. Eat 202, kept on -A -Ca diet 49 days. Age, 83 days. Weight, 84 gm. Rib junction. Severe rickets with moderately increased proliferous cartilage, deeply stained with haematoxylin (see explanation of Fig. 11, Plate 5). Below the cartilage the rachitic metaphysis, consisting nearly entirely of osteoid tissue and cartilage being transformed directly into osteoid tissue. Great amount of osteoid throughout. A spontaneous infraction in the right side of the rib wall. Haem.-eosin. Magn. x 36. Fig. 36. Epiphysis of humerus of the same rat 202. Picture typical of late rickets (without increase of proliferous cartilage (E Car)). Haem.-eosin. Magn. x 24. PLATE 15 Fig. 37. Rat 201, kept on -A —Ca diet 57 days. Age, 89 days. Weight, 43 gm. Severe rickets. Rib junction. Proliferous cartilage moderately increased. Lime deposition only near perichondrium. Considerable amount of osteoid tissue. Cut without decalcification. Silver nitrate + carmin + eosin . Magn. x 36. Fig. 38. Eat 68, kept on — A-Ca diet 99 days. Age, 132 days. Weight 85 gm. Eib junction. Osteomalacia with some osteoporosis. Narrowed epiphyseal cartilage and zone of PC (—EC). Considerable increase in amount of osteoid tissue. Huge callus after spontaneous fracture, consisting almost entirely of osteoid, is seen in the lower part of the photomicrograph. Haem.-eosin. Magn. x 28. Fig. 39. Rat 102, kept 31 days on a diet deficient in antirachitic factor and without addition of salt mixture. Age, 52 days. Weight, 40 gm. Severe rickets. Eib bent at an angle. Great increase of proliferous cartilage and of osteoid tissue (see p. 123). Haem.-eosin. Magn. x 36. PLATE 16 Fig. 40. Mother rat 625, kept 24 days during lactation on — A— Ca diet. Weight, 187 gm. Eib junction. Changes similar to those of osteomalacia seen in women during pregnancy or lactation (see pp. 109, 110). Haem.-eosin. Magn. x 42. Fig. 41 Rat 205, kept 49 days on — A— Ca diet. Age, 81 days. Weight, 70 gm. Epiphysis of humerus. Severe rickets. Increased and disorganized proliferous car- tilage with absence of calcification in the ZPC. Presence of an osteoid metaphysis beneath the epiphyseal cartilage. Great amount of osteoid tissue throughout. Haem.-eosin. Magn. x 25. Plate I (lATO) Plate, Plate 3 Plate 4 Plate 5 -2HC Plate 6 i (* Plate 7 Plate 8. L — 3V- 21 Plate 9 (1579) Plate 10 25 26 27 ■•;i ' 4 /■'-.•i' V iw M ! !B a»t^<-''- ■'■•■' Plate 1 1 30 Plate 12 31 Plate 13 ZttC 7^1- 33 ^ ,- Plate 14 36 Plate 15 Plate 16 41 Met Plate 17 MAL£S Fio. 43. Weight curves of average rats (males) from litters 618-625, kept on — A diet after weaning. Letters above the curves show the maternal diet during lactation. The figures refer to the number of the litter (see p. 95). FEMALES Fig. 44. Weight curves of average rats (females) from litters 618-625, kept on —A diet after weaning Letters above the curves show the maternal diet during lactation. Tlie figures refer to the number of the litter (see p. 95). Fio. 45. Weight curves of average rats (male and female) from litter 610. The diet on which each was kept is indicated by the letters above the correspond- ing curve. Mother rat was fed on N — Ca diet during lactation (see p. 105). (1579) Plate 18 Pig. 46. Weight curves of average rats (male and female) on N and —A diet, from 'wet' and 'dry' cages (see p. 142). Fia. 47. Weight curves of average rats (male and female) on N — Ca diet, from 'wet' and 'dry' cages (see p. 143). ^tibg Council MEDICAL RESEARCH COUNCIL (Formvrly Medical Research OommiUee, National Health TnmranceJ. LIST OF PUBLICATIONS January 1923 The following publications relating to the worlc of tlie Medical Hesearch Council can be purchased Qirough any bookseller, or directly from H.M. Stationery OfiSce, at the following addresses : Imperial House, Kingsway, London, W.C. 2, and 38 Abingdon Street, London, S.W. 1 ; 37 Peter Street, Manchester; 1 St. Andrew's Crescent, Cardiff; 23 Forth Street, Edinburgh. 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