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There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924012287243 WORKS BY J. L W. THUDICHUM, M.D. A TREATISE ON THE PATHOLOGY OF THE UEINE ; INCLUDING A COMPLETE GUIDE TO ITS ANALYSIS. By J. L. W. THUDICHUM, M.D. Second Edition. Illustrated. London : J. & A. Chxtechili.. 1877. A TREATISE ON GALL-STONES : THEIE CHEMISTRY, PATHOLOGY, AND TREATMENT. By J. L. W. THUDICHUM, M.D. Illustrated with Plates. London: J. & A. Chubchixl. A MANUAL OF CHEMICAL PHYSIOLOGY; INCLUDING ITS POINTS OF CONTACT "WITH PATHOLOGY. By J. L. W. THUDICHUM, M.D. London : Longmans, Green, & Co. 1872. ON POLYPUS IN THE NOSE, AND OTHER AFFECTIONS OF THE NASAL CAVITY. THEIE SUCCESSFUL TREATMENT BY THE ELECTRO-CAUSTIC AND OTHER NEW METHODS. By J. L. W. THUDICHUM, M.D. Third Edition. Illustrated •vrith Woodcuts. London: Longmans, Geeen, & Co. 1877. BY DK. thttdichitm: and de. dxjpee. A TREATISE ON THE ORIGIN, NATURE, AND YARIETIES OF WINE. BEING A COMPLETE MANUAL OF VITICULTURE AND CENOLOGY. By J. L. W. THUDICHUM, M.D., and A. DUPEE, Ph.D. With many Illustrations, London and New York: Macmtlian & Co. 1872. ANNALS OF CHEMICAL MEDICINE VOL. I. LOSDON : PRINTED BY SPOTTISWOODE AND CO.,' HEW-STREET SQUARE ASD PARLIAMENT STREET ANNALS OF CHEMICAL MEDICINE INCLUDING THE APPLICATION OF CHEMISTRY TO PHYSIOLOGY, PATHOLOGY, THERAPEUTICS, PHARMACY, TOXICOLOGY, AND HYGIENE VOL. I. EDITED BY jfifw:^ T H U D I C H U M, M. D. LONDON LONGMANS, GEEEN, AND CO. 1879 All rights reserved /I. O/o UMiVEESITY MJBRARY^ PEEFACE. I HAVE the honour of laying before the Medical Profession and the general scientific public the first volume of a new periodical, the principal object of which will be the advancement of the sciences named on its title by means more especially of the chemical method. The several means by which it is hoped to contribute to the attainment of this object will be the publica- tion of original researches to be carried out in physiological, pathological, chemical, and pharmaceutical laboratories in Great Britain and abroad, and the communication and difiusion of the results of the progress of chemistry as far as it relates to medical objects which has been made on fields, and communi- cated through channels, not commonly accessible to medical readers. In this endeavour it had to be borne in mind that some of these results might in their original form be too tech- nical to be readily appreciated, and might therefore admit of some interpretation and adaptation to particular wants. On the other hand it was felt that the lasting value of the publi- cation would be increased by a thoroughly practical treatment of the principal scientific questions of our time, including the communication of technical details which may be necessary to enable the reader to control, or the student and inquirer to repeat and extend, the operations of which the data furnished are the result. vi PREFACE. Medical science is at present more particularly interested in the solution of the problems concerning the nature of the causes of infectious diseases, and the nature of the processes of diseases engendered in the organism by these causes. These latter act after the manner of ferments, and while some of them are undoubtedly organised self-reproducing parasitical beings, others are supposed to be unorganised or shapeless, and in this respect to resemble the normal ferments of par- ticular organs of living beings. The proximate and final effects of both kinds of disease-causes upon the body are always massively chemical ; the ferments decompose materials of the body into substances which either engender increased consumption of oxygen and excessive production of organic heat, or act as poisons upon the nervous and muscular systems ; some are eliminated, causing loss of power and material, others are left as useless or hurtful hindrances in organs and tissues. All infectious diseases have therefore at one period or another chemical results, and these are amongst the principal objects of the inquiries proposed to" be expounded in these pages. Although physiology has its own rights as a science quite independently of practical applications, its development thus far is, and in the future probably will be mainly due to its obvious connection with the healing art. It is in this relation that we shall mainly endeavour to illustrate its progress, but without excluding concise philosophical views or instructive generalisations extending to fields of natural history. The chemical processes of disease can only be measured with the aid of physiological methods, but the attempt to unfold their quality requires also the application of a kind of chemical method which goes beyond that ordinarily employed by natural historians. The application of this method promises to be of great use in the study of disease, as it has taught us already that several zymotic processes have great analogy to forced decompositions of organoplastic substances by merely chemical agencies. PEEFACE. vii It is intended to give to the pathological experiment due scope, even though it should in some eases not directly or at once lead to any chemical development. The pathic process shows the chemical share of its composition when it has to be counteracted, be it by prevention or antidosis. Even the most modern treatment of wounds relies to a large extent upon chemical agents, and what is commonly termed antiseptic, might in a wider and perhaps better sense be termed chemical surgery. The practice of the healing art in all its branches is therefore literally interwoven with chemical principles and problems. To assert these principles, and aid in the solution of these problems, and subsidiarily, to effect the union in a focus of data which by distribution would be weak and inert, are parts of a programme which it is hoped may be found not im- worthy of the kind attention and generous support of the Medical Profession. While hoping to merit this countenance mainly by present- ■ ing the researches and observations of others, whose results I shall endeavour to give with due completeness and perspicuity, I shall venture to direct attention from time to time to work more particularly my own, and that in parts where progress has been made possible only by the support and countenance which the Lords of the Privy Council and the Local Grovernment Board have given to my researches by connecting them with the Medical Department of their service. During a long series of years the results of my work on the field of chemical medicine have been published in various reper- tories of original information, such as ' Eeports of the Medical Officer of the Privy Council ' 1866 to 1876; 'Eeports of the Medical Officer of the Local Government Board' 1876 to 1879; 'Journal of the Chemical Society' 1860 to 1876; 'British Medical Journal ' 1864 ; ' Transactions of the Medical Society of London ' ; Erdmann's ' Journal of Practical Chemistry ' ; Lie- big's ' Annals of Chemistry ' ; ' Proceedings of the Eoyal Society ' ; ' Moniteur Scientifique ' ; ' Proceedings of the Eoyal Bavarian viii PREFACE. Academy ' ; ' The Lancet,' etc. The fragmentary mode of publication, with the circumstance that I have never hitherto published my results in a collected form, has probably been of disadvantage to me with many readers. And it has facilitated a very imworthy use of my work by certain authors ; who being fully aware of the work in the places of original publication, have observed complete silence regarding it, or have sought to set it aside with some frivolous objections, while perhaps at the same time appropriating more or less of its fruits. Both in justice to myself, and in regard for the honest literary interests of science, I have at last felt myself obliged to make distinct public reference to some such proceedings, and the protests which I have- had to make in some glaring cases will be recorded in Article XXIII. of this volume. The explanation of the case is not far to seek. There are professed physiologists in this country and abroad, who though very imperfectly acquainted with either the theory or the prac- tice of chemistry, have nevertheless affected to speak as persons- having well-founded convictions on the subject : with their pre- tended convictions, however, resting on no better basis than the haphazard opinion (obiter dicta) of some professed chemist or other, or the conceited and sensational papers which, in chemistry as elsewhere, are written for mere claptrap and without research. I could quote instances by the score, where physiologists acting on that system have reproduced as their own the errors of others while omitting the better knowledge which they ought to have given to their readers. Among the professed chemists there has sometimes been equal absence of knowledge, but, in such eases, as a rule, more discre- tion. The acquaintance of the chemists with the physiological, pathological, and medical applications of chemistry has often been most slender ; but the chemists have appreciated, better than physiologists could, that discussion in chemistry is eventu- ally an affair of The Balance, and they have known that on such discussion they could not openly enter without having first PREFACE. IX made actual exact researclies of their own. Such researches, — researches which do not pay — the gentlemen to whom I refer have not undertaken. Some, nevertheless, men of externally high position, have persistently done all they could, sometimes by silence, sometimes by innuendo, sometimes by obstruction, to thwart the endeavours which others have made to advance medical science by original chemical researches. These men, however challenged, have avoided the light of publicity, and have thus impressed upon their action a character of intrigue and unfair rivalry. With the progress of time this poor policy has reacted upon its authors, whose universal incompetence to judge in medical chemistry is now either notorious or of easy demonstration. "While I have thus been obliged to forewarn the Medical Profession and the general scientific public regarding the origin of sundry kinds of opposition which the present undertaking may perchance have to face, I all the more hope that the true scientific chemist may derive from the perusal of the ' Annals of Chemical Medicine ' an assured experience to this effect, namely — that the accurate methods of which he holds the formula in his analytical training, and in the talisman of the atomic theory, are essential in medical no less than in abstract chemistry, and that inaccurate methods must not be propounded to the Faculty under the excuse, which hardly admits of serious examination, that they are ' good enough for clinical purposes.' In conclusion I venture to hope that the ' Annals of Chemical Medicine ' will be useful to several classes of readers. To the medical practitioner they will afford information on the chemical aspects of the most important questions of the science of which he represents the executive authority ; collaterally they will afford information on the^most trustworthy methods of diagnosing morbid chemical conditions, and on the significance of these conditions with regard to prognosis and treatment. To the scientific inquirer they will present in turns all the latest data from which he will have to start as a basis, if seeking to X PREFACE. enlarge the present information on any one of the subjects within their range. All contributions of original information, be such the result of observation or experiment, will be as welcome as deductive or inductive meditations, provided only they are based upon the data and principles of actual science. A predilection for a ipaximum of fact and a minimum of speculation does not ex- clude the sense of the attractions of theory, in its true meaning, as the very flower and fruit of the slow growth of the body of data. Experience, however, shows that no interpretation of natural phenomena has any lasting value, unless all the essential factors entering into their production are ascertained and appre- ciated. The accumulation of these data in a form readily acces- sible to all interested in their use will therefore promote the philosophical development of' our science much more than pre- mature generalisations. With the aid of the list of articles at the beginning, and the alphabetical indices, one of matters, the other of authors, at the end of the volume, the inquirer after information will be fully able to find any general subject or any matter of detail. To facilitate the use of any new terms and symbols, which may be found convenient abbreviations of ideas, I have added a page for their interpretation and definition. The same page will state some synonyms, and thus aid in obviating a confusion which should gradually be abated by the adoption of a imiform nomen- clature. I believe the chemical method to possess vast capabilities for enlarging the domain of pathology and medicine, and for advancing the art of healing disease ; and I trust that all who share that hope wiU aid in its realisation either as contributors, as readers, or as friends. J. L. W. THUDICHXJM, M.D. 11 Pembeoke Gardens, Kensington, AV September 1879. CONTENTS. I. The Chemical Constitution of the Oeganoplastic Sub- stances, CONSIDBEBD WITH THE AID OF THE HYPOTHESIS OF THBIE AMTLONIDB NATUEB . . . 1 II. Ultimate Ceystalloid Products of thb Chbmolysis of Oeganoplastic Substances, paeticulaelt by Caustic Baeyta. {Summm~y cmd Additions.) . . . .20 III. On the Action and Peoducts of the Staech-Teansfoem- iNG Fbements, Diastas, Ptyalin, Pancbbatin, Acids,, and vaeious Mattbes dbeivbd peom Albuminous Sub- stances. (iStoramory.) . . . . . .45 rv. The Life and Philosophy of Eobbet Julius Maybe, Phy- sician to the Town of Heilbeonn . . .65 V. On the Coloueing Mattees in the Shells of the Eggs OF BlEDS ; AN ILLUSTEATION OF CHEMICAL DIAGNOSIS AT A GLANCE, AND OF THE VALUE OF AbSTBACT ChBMICAL Kbsbaeches. (_From the Pathological Institute.) . . 93 VI. On the Oeganio Acids op the Beain, with special ebfbeencb to the Natueb of the Lactic Acid contained in it. (JPro^i the Pathological Institute.) . 104 VII. On the Chemical Decomposition op Bile and its Ingee- dients, with sepeeencb to the Theoey of the Function of the LivEE. (^Summary.) . . .117 VIII. Chitin, the simplest Niteogbnisbd Oeganoplastic Amy- lonidb. (^Summary and Additions.) .... 129 IX. On Hbmisymmbtey in the Chemical Constitution op Oeganoplastic Substances. — Hbmipeotein and Hbmi- ALBUMIN, {Smtvmcury amd Additions.) . , . .143 ■ X. Chbmolysis of Albumin by fusing Caustic Potash ; Production of Indol, Skatol, Pyeeol, Phenol, and BuTTBic Acid. (^Summary.) . . . . .148 153 Xll CONTENTS. XI. Process and Peoducts op the Puteefaction op Elastis-. Mucin, Glutin, Albumin, Hemochrome, and Blood. {Summwry^ ,.....• XII. On the Alkaloids of the Human TJeine. {CemoUdated Aocount of ResearoTies. From the Pathohgioallnstitute.') . 160 Xin. The Peopeeties and Metamoephoses of Soluble Al- bumin. (^Sarmna/ry and Additions.) . , . .175 Xrv. On Infection and the various States of Aggeegation of Infectious Matter ..... 204 XV. Conflicting Views of Contagionists and Anti-Conta- GIONISTS CONCERNING THE ORIGIN OF INFECTION. Diphtheria IN Animals. {Summary and Additiotis.') . 217 XVI. Eecent Data and Arguments in favour op and AGAINST THE HYPOTHESIS OF THE LIVING CONTAGIUM. QSummary.') ....... 228 XVII. On THE Specific Alvine Flux oe Cholera, with SPECIAL RBFBEENCB TO ITS INOEGANIC CONSTITUENTS. With Hints' concerning the Theory op Cholera. {From, the Patlwlogioal Institute.') .... 236 XVIH. Experiments on Animals fob ascbetaining the state op geeatest Infectiousness of Fermenting Cholera Poison. {From tJie Pathological Institute.) . . \ 242 XIX. Note and Experiments on the alleged existence in THE Beain of a Body termed 'Peotagon.' {From the Pathological Institute.) ..... 254 .XX. On the colouring Matters of Bile and of Gall- stones, THEiE Compounds, Derivatbs, Chemical and Spectroscopical Phenomena. {Consolidated Accmmt of Researches. From the PatheUgieal Institute.) . .264 XXI. Spermatin, a new Organic Base in Animal Organisms. {Swmmary.) ....... goi XXn. Chemical Surgery, with special reference to the Antiseptic Pharmacopcbia. {RevieTV.) , . . 309 XXIII. Historical Retrospect on eaeliee, and Ceitical Con- sideration of Contemporaneous, Researches on Biliary Pigments ...,,. 315 NAMES AND SYNONYMS . . . .335 INDEX ..... .337 LIST OF AUTHORS 34; AMALS OF CHEMICAL MEDICINE. THE CSEMICAL CONSTITUTION OF THE OUGANO- PZASTIC SUBSTANCES CONSIDERED WITS THE AID OF THE HYPOTHESIS OF THEIR AMYLONIDE NATURE. The material which forms the solid and rigid parts of the structure of plants is cellulin. This name has loeea selected because microscopical science has found that the simplest shaped elements of vegetable structure are cells, and that all more complicated structures arise out of aggregated cells by processes of peculiar union and fusion ; that the rigid woody part of cells is also the principal part when its quantity is compared with that of other ingredients, and tliat there are no cells discernible made of any other material. The youngest cells, such as those in the first rootlets and leaves of germinating seed, contain the purest cellulin ; with advancing growth and age this becomes covered by new homogeneous layers ; other additions are heterogeneous, and varying in chemical composi- tion, but one kind of matter is observed to be closely associated in some form or other with growing cellulin in all its varying forms and positions, namely a nitrogenised matter resembling the radicle which has been supposed to be the nucleus of the albuminous substances (protein). With advancing age and ceasing increase the cells assume a final rigid shape, which they maintain unaltered during the remainder of the existence of the organism of which they form part. The nitrogenised matter at 1. = 2 ANNALS OF CHEMICAL MEDICINE. the same time leaves them almost entirely, and they become ■wood. Not rarely this wood, at the time of involution of the cell-life, receives an addition in the form of deposit or incrusta- tion of mineral matter, mainly carbonate, and sometimes phos- phate of calcium, silica, and potash and other salts. The chemical composition of cellulin finds its most simple expression in the formula CgHjoOg, but it is supposed that its molecle is a multiple of this formula, and therefore it is expedient to express its formula by ^(CeHjoOg). This formula also has the advantage of including the objects of those theories according to which there are several varieties of cellulin, some being re- presented as isomers, some as polymers of what might be termed the type. In the growing parts of plants already developed cellulin is probably produced directly from and in the protoplasm, which, with its nucleus, serves as the centre of nutrition and multipli- cation, and as the mould, so to say, over which the actual cell of cellulin is formed. The cell is, in other words, the shell which protoplasm forms on its outside. But in seeds and tubers, and other organs effecting the propagation of the species by whatever means, a material for the formation of cellulin is ready for use deposited inside the cells, namely starch, or amylon. This material is not used by the very cells in which it is deposited, but is only stored there in a form insoluble for the time, but capable of being liquefied and carried to and employed in newly growing parts at the call of heat and light. Amjlon also has the empirical formula of CgHigOg, but most inquirers agree that this formula has at least to be doubled to express the considerations which arise from the simplest reaction. We are, however, justified in going further, and admitting, though hypothetically, that amylon, like cellulin, is best ex- pressed by the formula ^(CgHjgOj), in which 71 is a factor not less perhaps than 5, and in given conditions probably more. In considering amylon as a chemical individual it must be borne in mind that it is perhaps not identical with the whole of the product termed starch, but that a portion of the granules constituting this substance may be, as some maintain it is, actually cellulin. While another is amylon, and that in the concentric arrangement of the starch granules layers of these ORGANOPLASTIC AMYLONIDES, 3 substances alternate with each other. This discussion, however, is immaterial to our present purpose, because the fact remains, however the discussion may be decided, that the whole starch granule is available for fluidification by a ferment, and for transfer to parts newly formed or in course of growth. In other parts of vegetables we find carbohydrates which are closely resembling cellulin and starch, and some of them are formed out of starch either by natural or artificial processes. These are the dextrins, gums, and sugars. Dextrin from wheat starch has, like this, the composition n{Cgtt^fi^) ; gum arable or arable acid, however, is at least CijHjjOn, while bas- sorin, the gum of tragacanth, remains, like dextrin, n{Cgli^fi^), and vegetable mucilage the same. In dextrin and gums the factor n may possibly be =1 or —2, probably not more. The sugars have a great variety of formulae, and are many in number. Finite, or pine-sugar, is n(CgII,j05) ; quercite, the sugar of acorns, has the same formula. Milk sugar, or lactose, is O^JI.^fi^^ ; cane- sugar has the same formula. Passing over a, number of peculiar saccharoids occurring in rarer plants, we pause at the glucoses, the one which turns the polarised ray of light to the right being dextro-glucose, CgHjgOg, and the one which turns it to the left, or Isevo-glucose, being perhaps of the same formula, possibly, however, C,j,Hj^0i2. Closely related to these is maltose, CijHjjOj,, which will have to be considered more fully else- where. Next we notice mannitose, then mannite, CgH,^Og, an hydrogenised sugar, which can be produced by inserting hydrogen in a sugar with twelve atoms of hydrogen. Next follow some sugars which are common to plants and animals, namely inosite (phaseomannite) occurring in the muscles and brain of verte- brates, and dextro-glucose occurring in the blood and parts of animals ; and scyllite in organs of the ray, shark, and prickly dogfish. Lastly we cast a glance at the glucosides, so termed because by chemolysis they yield glucose and other products. These are present in many plants under remarkable circum- stances. Bodies like them are known to occur in animals, but are considered as rare and peculiar principles. The carbohydrates of the order of gums and sugars do not as such serve for purposes of structure, but as vast as are the structure-functions of cellulose, so are the power-producing B 2 4 ANNALS OF CHEMICAL MEDICINE. functions of the sugars. The sugars are stores of matter or power for the accomplishment of the propagation of the species amongst plants. Eoots and rhizomes, bulbs, stalks, leaves, and buds, but above all fruit abound in sugar. Lastly, in tlie living body of animals there is always sugar formed and present, whatever may be the jnode of nutrition. Sugar seems at present incapable of being artificially metamorphosed backward into cellulin ; neither does the metamorphosis appear to be effected in animals or plants. The discovery by Schwann in 1840, that the elements of structure were the same in plants and animals, was followed in 1845 by the discovery of C. Schmidt {Zur vergl. Physiologie der wirbellosen Thiere, 1845, p. 62, Ann. Pharm. 54, 318; J. Pr. Chevi. 38, 433) that some of the lower animals ' were provided with parts consisting of cellulin. These were the tuni- cata (Lamark) ; in one species of these, and more particularly in the mantle of Phallusia mamillaris, cellulin, as it was believed to be, was met with. The discovery was extended by Lowig and Kolliker {Ann. d. Sc Nat. 3 ser. v. pp. 193-232 ; J. Pr. Chem. 37, 439; Compt. rend. 22, 38, 1846), who, confirming Schmidt as to the, presence of cellulin in Phallusia mamillaris, met with the same constituent in Phallusia intes- tinalis, Phallusia monachus, Cynthia papillata, Clavelina lepadi- formis, Diazona violaceum, Botryllus polycyclus, Pyrosoma giganteum, and Salpa maxima, Didemnum candidum, Aplidium gibbulosum. They found the middle layer of the mantle of Phallusia monachus and sulcata, of Clavelina lepadiformis and Aplidium gibbulosum, to be composed of a glasslike transparent structureless substance in which many nuclei and crystals arranged in stars were imbedded ; the external layer of the mantle they observed to be crowded with very large, elegant, round cells, with thin walls, enclosing a matter clear like water, but no nucleus. In Clavelina lepadiformis the entire mantle on the stalk as well as the sprouts is permeated by such roundish and oval cells without nucleus, to such a degree that the homo- ' Schmidt also believed to have found an infusorium, Prustulia salina to contain cellulin. It was, however, afterwards shown that this creature is not an infusorium, but a plant. — Siebold, Vergl. Anat. d. mriellosen T/iirre 1 848, p. 238. OEGANOPLASTIC AMYLONIDES. 5 geneous substance, in which otherwise they are imhedded, has disappeared. Such layers of the mantle have then entirely the aspect of a vegetable tissue. In Aplidium gibbulosum, and Botryllus violaceus, the tender cells situated in the more ex- ternal layer of the mantle contain calcic carbonate, which increases towards the periphery, so that the cells of the outer- most layer appear completely calcified. In Didemnum candi- dum these calcified cells, which are here moreover provided externally with rays of calcic carbonate appear in such numbers that the entire stock of this compound ascidia appears to be penetrated by white starlike bodies. Similar to this appears, according to Milne Edwards (Observ. sur lea Asoidiea com,- posees, p. 81, pi. 8, fig. 2, b), Leptoclinum maculosum. In the mantle of Diazona violaceum, Pyrosoma giganteum, Botryllus polycyclus, Salpa maxima, and bicaudata, these tender delicate cells are not seen, but the mainly homogeneous substance contains disseminated granules and nuclei, in Pyrosoma also cells with peculiar branches, and in Diazona coloured granules, and needle-shaped crystals or concretions of calcic carbonate. Such crystalline concretions in the shape of balls or stars^ are also present in Salpa maxima, and others in branched masses in Salpa bicaudata, but they are not soluble in hydrochloric acid, and are therefore probably not chalk but silicic acid. In Botryllus the homogeneous substance of the mantle is in certain places penetrated by peculiar serpent-like fibres, which twist. in all directions, and on treatment with caustic potash are recog- nised to be wood fibres free from nitrogen. In Cynthia papillata the middle layer of the mantle consists of wavy wood-fibres, arranged longitudinally and peripherically, with interspersed nuclei, granules, crystals, and cells ; the latter contain either brown pigment granules and a nucleus, or several endogenous, cells, by which they recall the appearance of cartilage cells. The fibres of the middle layer of the mantle aid in the produc- tion of the thorny projections which appear on the otherwise horny-looking surface. In the mantle of Cynthia pomaria longitudinal fibres prevail, between which crystals, round pig- ment cells, and peculiar cells filled with yellow bodies are situated. With these cells are mixed a quite peculiar kind of cells, which are developed from pigment cells, and form thick 6 ANNALS OF CHEMICAL MEDICINE. walls during their growth; these walls then break up into fibres, which latter now surround the cavity of the former cell in concentric layers. These fibrillated cell membranes react with potash as little as the principal substance ; they are wood-fibre free from nitrogen ; all other elements of the tissue disappear in the caustic alkali (Siebold, Vergl. Anat. p. 238, Anm. 4). No such matter free from nitrogen and resembling cellulin was found by these observers in other molluscs, in annulates, helminths, echinoderms, discophores, polyps, and infusoria. In publishing their researches they expressed the apprehension that this discovery would be used by those who deny the exist- ence of any precise demarcation between animals and plants, for the purpose of supporting the opinion that as regards chemical composition also there was no fixed difference between plants and animals. They therefore exerted themselves to prevent such a hasty conclusion, as it was termed, and pointed out particularly that the woody fibre of the tunicata was never contained in the mantle of these animals in a quite pure state, but was always yet mixed with animal elements, and that there existed no lower animal, which consisted in all its parts of cellulin (Sie- bold. I.e. p. 238, Anm. 1). The researches of Lowig and Kolliker formed the subject of a report to the French Academy by Dumas, Edwards, Bous- singault, and Payen, published in the Gomp. rend. 22, 581 ; Ann. Sg. Nat. 1846, 238. Thus the matter rested for about twelve years, when Ber- thelot published some new researches on the peculiar substance of the mantle of Cynthia papillata {Gompt. rend. 47, 227 ; N. Ann. Ghvm. Fhys. 56, 149 ; J. Pr. Ghem. 76, 371 ). As the result of these he maintained that this matter was not identical but only isomeric with cellulin. On chemolysis with sulphuric acid it yielded fermentable sugar and another matter not examined. He declared it to be rather analogous to chitin, termed it tunicin, and gave it the formula, CgHioOj, or The chemical details concerning tunicin will be summarised in a future article, where also will be discussed the supposed analogy to chitin, which Berthelot maintained on the basis of his own researches on that substance also. OEGANOPLASTIC AMYLONIDES. t For about the same time Berthelot found that chitia gave by chemolysis a fermentable sugar free from nitrogen. This was at first referred to the presence with chitin of some such body as tunicin, and Peligot (see references in the summary on ehitin) actually endeavoured to find and extract the amylum- like body which was supposed to yield this sugar. It is by no means decided whether such a body does not exist mixed with chitin. But chitin itself is nitrogenous, and therefore not at first sight so like cellulose as it would appear if it were free from nitrogen. Schmidt, the discoverer of animal ceUulin, had studied chitin also, and found that by thermolysis it gave out acetic acid and ammonia. But this observation was not appreciated by Stadeler in his research on chitin ; he occupied himself with constructing a new formula for this body on the basis of its empirical composition ; he did not suspect that the sugar obtained by its chemolysis in a certain way, contained nitrogen. That under certain conditions it did contain nitrogen was only found in 1876. The chapter on ehitin to be given below will explain how closely chitin is related to tunicin or animal cel- lulin, and to the amylonides of the vegetable world, which form the permanent structure of plants. It is indeed somewhat more complicated in chemical structure-, inasmuch as it contains nitrogen ; but even that is present in the rather simple form of amide ; beyond this amide there are some simple radicles present which appear as fatty acids after chemolysis ; but the- radicle- stock determining the nature of chitin is a body like ceUulin^ an amylonide. Though Lowig and KoUiker had not found any cellulin in helminths, a body was soon discovered in the peculiar cystic form of the taenia echinococcus, which resembled in these respects the cartilaginous covering of some of the lower animals, that by chemolysis with sulphuric acid it yielded dextro-glucose. The bladders of echinococci were found by Liicke {Archiv Patholog. 19, 189) to be made of a nitrogenous substance, which dissolved in hot water under pressure, like one of the permanent cartilaginous substances. The membranes of old bladders were in part insoluble in caustic alkali even on long boiling. The fully developed helminths contain probably no such matter in their cuticle, though they are, like the bladder, 8 ANNAiS OF CHEMICAL MEDICINE. covered with calcic carbonate. But they are covered, probably, with an amylonide, and at all events their parenchynaa (in intes- tinal helminths) is pervaded by large quantities of so-called (hepatic) glycogen. In the moUusea, provided with shells, a peculiar substance exists, mainly as the organic ingredient of the shells. This was for some time believed to be identical with chitin, but was at last separated by Fremy, and later by Schlossberger, and distin- guished under the name of conchiolin. It is at once distinguished from chitin and hyalin by the amount of nitrogen, which is similar to that of the albuminous substances. The only crystal- HyaUn Chitin Byssin Spongin Conchiolin . . 44-55 45-69 _ 47-44 50-0 H . . 6-64 6-43 6-80 6-9 N . . 4-81 7-00 13-5 16-15 17-5 . . — — — — 26-6 Usable product as yet obtained from it is leucin ; this would range it under the animal matter to be considered later on. The byssus of the acephala was also supposed to contain chitin (Leuckart), but apart from its reactions, which are peculiar, it contains from 12 "2 to 13' 9 per cent, of nitrogen, and therefore differs from both chitin and conchiolin. The byssus of the mussel, when freed from impurity and a little calcic carbonate, is a mass of fibres shining like silk, and elastic like caoutchouc, and seems at first sight to have little physical similarity to other animal structures, except elastin. Spongin has also yielded leucin and glycin, but not a trace of tyrosin. It has not yet been examined for an amylonide, but as some declare it to be identical with fibroin, which has been found to yield sugar, further examination may furnish informa- tion on this point. The nitrogen in spongin is 16-15 per cent, and this ranges the substance between byssin and conchiolin. Fibroin, the substance of silk, has yielded on chemolysis tyrosin (in the large quantity of 5 to 8 per cent.), leucin and other amide-acids, ammonia, sugar recognisable by an alkaline solu- tion of cupric oxide, and especially, on long boiling, glycin. The composition of fibroin is similar to that of conchiolin, C, ^H NO 15 23 6 6 ORaANOPLASTIC AMYL0NIDE3. 9 expressing its empirical formula. Heated with caustic potash it easily forms oxalic acid. It is well intermediate in chemical structure between the cellulins on the one, and the proteins on the other extremity of the scale. Silk gelatin has yielded ty rosin, leucin, and serin, CgHjNOg (amido-oxypropionic acid) which yields glyceric acid by treat- ment with nitrous acid. Silk gelatin is the outer covering of the silk fibroin, which forms the entire fibre ; its composition is expressed by the empirical formula, CigHjjNgOg ; it thus appears to contain a molecle of water and one of oxygen more than fibroin. The difference is, however, greater than that indicated by the one in hydrogen and oxygen ; inasmuch as the radicle represented by serin has not yet been observed amongst the products of the cheraolysis of fibroin. Mucin is the substance characterising the mucous secretions and excretions of a great number of animals. Snails secrete it in large quantities to pre- vent adhesion to objects in their progress; they lubricate their way with it ; inversely almost all animals form it in their interior parts to prevent adhesion to them of foreign bodies, like food, or adhesion to each other in parts exposed to friction, or to the drying effects of the air. It exists in quantity in the glands forming the lubricating materials of the digestive canal, notably the salivary glands; but it is still more singular that the lubrication of tendons in their sheaths should be attained by it ; and that the tough material of the tendons themselves, and of all connective tissue whatever, should contain a large amount of mucin. Its formula has not yet been attempted ; it is separated from the albuminous matters by its insolubility in strong acetic acid. By chemolysis with mineral acids, or even by prolonged boiling with moderately strong acetic acid, it gives a kind of acid-albumin and grape-sugar, and probably other substances. The grape-sugar has been isolated and identified with alkaline copper solution, and with potash ley (Eichwald). The analyses of mucin differ as yet too much from each other to allow the attempt of constructing a formula. Nitrogen is found by some at 8'5, by others at intermediate figures, 9-42, 10-01, 12-64, 13-22, 14-54, 17-69 ; not all of these mucins have been shown to form sugar. The mucin from the sub- maxillary gland dried and pulverised, and then mixed with very 10 ANNA.LS OF CHEMICAL MEDICINE. dilute sulphuric acid, and heated over steam, gives a solutioUj even after 25 minutes' heating, and still more after 30 minutes, which, though a considerable amount of the mucin employed is yet undissolved, after removal of the sulphuric acid and con- centration, reduces potassio-cupric tartrate and causes cuprous oxide to be deposited. When warmed with excess of soda ley alone the liquid turns brown; it likewise reduces bismuth oxide in alkaline solution as well as sulphindigotic acid. If, off the other hand, the heating of the mucin with dilute sulphuric acid be continued for a longer time, the quantity of the reducing substance which passes into solution decreases, and seems ultimately to disappear altogether. This reducing substance is not dissolved by ether, when its acid solution is agitated there- with ; it is also insoluble in absolute alcohol, and is therefore different from grape-sugar and milk-sugar, from which it is likewise distinguished by its property of disappearing on pro- longed heating with very dilute sulphuric acid (Obolenski, Med. Chem. Unter. 1871, 590.) But enough has been shown to make mucin a factor in the argument here proposed ; it is an element of structure, and an agent of function, and from its qualities eminently fitted for both performances. Mucin is also a product of diseased action, as it occurs in the fluid contents of colloid sacs, in sarcomatous, and in fibroid tumors. Chondrinogen, or cartilage, is by itself insoluble in water. It constitutes the permanent cartilages, the bone cartilages before ossification, and the cornea of the eye. In growing stag's horn it is contained together with ossein, a mixture present in the bones of young individuals of all vertebrates. Eib-eartilage purified with dilute hydrochloric acid, when boiled with fuming hydrochloric acid, yields fermentable sugar (Gr. Fischer and Bodeker, Ann. Pharvn. 117, 111; see also Schiff, Ann. Pharm. 119, 256). This sugar (chondrose) which Bodeker had formerly described as chondroitic acid (Schmidt's Jahrbjicher, 90, 150), was found by De Bary to be laevorotatory and to differ from both dextro-glucose and Isevo-glucose. Chondrin yields by fusion with potash oxalic acid, leucin, glycin, and an acid, which is fixed and gives a crystalline lime-salt. It some- times forms a jelly with water, on cooling, like gelatin, therefore OEGANOPLASTIC AMYLOxMDES. ' 11 like starch. The products of the chemolysis of chondriri imder varying conditions are very imperfectly known, but nevertheless it is clearly marked as an amylonide. Ossein, the organic basis of bones and teeth, is also in part the organic basis of reindeer's feet, stag's horn, and whalebone. In boiling water it is slowly converted into an equal weight of gelatin, more rapidly when prepared from the bones of young animals, and very speedily after addition of small quantities of acid. It contains — C 50-4; H 6-5; N 16-9; 26-2. The bones of fish and of aquatic birds contain besides ossein a substance insoluble in boiling water, yet apparently of the same composition as ossein. When these bones are freed from lime- salts by cold dilute hydrochloric acid, and the residue is washed free from acid, and then boiled with water, the ossein dissolves as gelatin, while a transparent elastic residue, having the form of the bones, remains undissolved (Fremy, N. Ann. Ghim. Pkys. 43, 51 ; W. J. Pharm. 27, 1). Gelatin is also obtained from the gelatigenous tissues, the intercellular or so-called connective tissue, from tendons, liga- ments, fasciae, and lining membranes of sacs and cavities in the animal organism. Elastic tissue and fibro-cartilage produce a somewhat pecu- liar glutin, and wiU be considered separately. Already, thirty years ago. Hunt {Amer. Journ. of Sc, Jan. 1848, p. 74, 109) perceived that when he added the elements of ammonia to those of cellulin or of amylon, and then deducted the elements of water, he obtained a product which had very nearly the formula of gelatin — C£^+ 2NH3= C^H^oNA + 3H,0. Cellulin Gelatin The percentic composition of a body n (CgHj^NjOj) would be — C . . 50-70 H . , ■ 7-04 N . . 19-71 . . 22-55 100-00 12 AI^NALS OF CHEMICAL MEDICINE. The calculated N is higher than the quantity found by analysis. Should gelatin be found to really contain some S, then this element would have to be substituted for some oxygen in the formula. Gerhardt, who is perhaps the only author who has noticed this speculation of Hunt {TraiiS de Chim. 4, 508) put it to a test, and made an interesting discovery. He caused isinglass to boil, during some days, with dilute sulphuric acid, and obtained ammonic sulphate, and a considerable quantity of sugar (gelatose or ichthyocollose), which by fermentation was transformed into alcohol and carbonic acid {comp. Gerhardt, Ghiin. organ, appliq. a la Physiologie v6gStale de M. Liebig, p. 287.) In the case of gelatin as in the cases of the albuminous sub- stances the products of chemolysis, which are crystalloids, have been well studied ; but those which are colloids, at least in the first instance, have been neglected. We shall study them closer hereafter ; here it is important to remember hat Scheele already, ill attacking gelatin with nitric acid and heat, obtained oxalic acid, malic (saccharic) acid (questioned by Gerhardt, but with- out his giving groimds), a greasy and an astringent matter (Berzelius, Lehrb. d. Ghem. 3rd edit. 9, 800.) We shall see, in reviewing late researches on glutin, that it is proposed to distinguish and separate isinglass from ossein proper, and that therefore it is perhaps not safe to conclude from Gerhardt's experience that glutin from bones and other tissues of higher animals will also yield a fermentable sugar. Gelatin (like pure cellulin), when purified by dialysis, is imputrescible in the ordinary sense, perhaps because, being free from salts, it affords no food to the organic putrescence ferments. It combines with colloid acids, and therefore Graham termed it a colloid base. Keratin, or horn substance, the constituent of all kinds of cover- ings of the higher animals, exists in epidermis, epithelium, in hair, horns, naUs, claws, hoofs, and feathers, mixed with a very great variety of colouring ingredients, as great perhaps as that of the colouring ingredients of the chitin of the articulates. The crystal- loids obtained by its chemolysis which are best known are leucin, tyrosin, aspartic acid. The apergus of similarity to amylonides in the bearing of keratin are numerous, but not yet sufficiently OEGANOPLASTIC AMYL0NIDE3. 13 precise to be used. But most remarkable is the bearing of the serpent's skin. This organ, when treated alternately with cold oil of vitriol and cold potash ley, whereby nitrogenous substances are dissolved, leaves a substance which is isomeric with cellulin, and yields fermentable sugar on boiling with dilute acids (De Luca, Gompt. rend. 57,437). Now this cellulin might be there as such, and not a product of decomposition of keratin, as is supposed by treating it in this place. It would then be ana- logous to the occurrence of cellulin in the skin (chitin) of the silkworm. For the same author found (Gompt. rend. 53, 102) that the skin of the silkworm, and the brown coverings of the pupas which remain in the cocoons when the butterflies escape, are capable of yielding a substance isomeric with cellulin, which may- be converted into glucose. When the caterpillars are boiled for several hours with strong hydrochloric acid, and this treat- ment is repeated three times with the residue, and the residue from these processes is washed with strong potash ley, then with water, and dried between 100° and tlO", a white light substance nearly free from nitrogen is obtained, which gradually diffuses in oil of vitriol, forming a colourless gummy liquid. This solution, added by small quantities to boiling water and boiled for an hour or two, yields fermentable sugar, which further re- acts like glucose with common salt and potassio-cupric tartrate. These forms of eeUulin then might be admixtures of the keratin and chitin tissue respectively, and then they would show nothing as regards the chemical structure of the keratin. But even so the occurrence of cellulin in the skin of the serpent is a powerful argument in aid of the hypothesis of the amylo- nide structure of organised tissues. But it is of little consequence if we possess at present no direct evidence of the presence of an amylonide radicle in keratin, and in glutin from ossein, and other substances. For as they are derived from the higher albumin, and as albumin does indubitably contain an amylonide radicle, it is fair to assume that the derivates also may contain it, unless indeed they have lost the amylonide radicle in the course of their being changed into structure-elements. But this is not likely, as their composition and bearing is too much like that of albumin. We now come to the class of albuminous substances in which li ANNALS OF CHEMICAL MEDICINE. the presence of an amylonide radicle can be proved 'physiologi- cally by biolysis much better than by chemolysis. Of these albuminous bodies we have the white of egg, or albumin ; the albumin of blood-serum, or serin ; the albumin of flesh ; the contractile matter of flesh, or syntonin, and the curdling matter or myosin, then paraglobulin and fibrin as natural educts from blood and other serous liquids; then casein from milk (with which some declare paraglobulin to be identical), then globulin from the crystalline lens, and globulin from hematoglobulin, or hematocrystallin, and some abnormal products of disease not yet sufficiently studied. All these by chemolysis yield products of which the greater number are crystalloids and well understood, while another part is not yet fully interpreted. Bopp had found that when any of the albuminous substances is boiled with strong mineral acid, it yields a mixture of products containing tyrosin, leucin, leucimide, and some other crystalloids in smaller quantity. The non-crystallisable portion tastes sweet, does not undergo fermentation with yeast, evolves hydrothion when boiled with potash, and afterwards acidified with acetic acid, and forms a violet solution with potash and cupric sulphate or potassio-cupric tartrate. These inquiries were subsequently extended, and compound volatile alkalies, fixed alkaloids, such as fluorescentin, and fluopittin, a substance recalling the bearing of biliary matters by its spectrum, and others in smaller quantity were discovered. In these chemolyses the process did probably go beyond the stage at which the amylonides are, or one of them is, separated, but not destroyed. For Schiitzenberger {Chem. Centr.bl. 1875, p. 614 et seq.) decomposed, albumin by boiling it for one hour only with very dilute sulphuric acid, and found amongst the soluble products a nitrogenous body, which strongly reduced Fehling's fluid, was precipitated by ammoniacal lead acetate, gave no precipitate with mercuric nitrate, and seemed to_him to be glucose or some such body. He fmrther decomposed albumin with barytic hydrate (L c. p. 631), under pressure at a temperature of from 150° to 200°, and obtained by various manipulations, to be described elsewhere, a considerable quantity of matter, 3 to 4 grms. from 70 grms. dry coagulated albumin, OEGANOPLASTIC AMYLONIDES. 15 ■which had a clearly sugar-like taste, did not evolve ammonia when heated with potash, was soluble in water, insoluble in absolute alcohol ; when heated on platinum foil, it swelled up, and charred, without giving any sublimate, and smelt like charred bread. This product, fully exhausted with alcohol, contained Calculated for theory below. a- C . . 44*2 per cent. 44*7 per cent. H . .7-6 „ 7-7 „ N . .14-8 „ 15-0 „ Another similar product obtained in the same manner from hemialbumin contained 6. C . . 42-4 per cent. H . .8-0 „ N . . 14-5 „ From this statement it appears that one such reducing body was obtained from albumin, by the side of hemialbumin, under the influence of sulphuric acid, another from hemialbumin it- self by secondary chemolysis with baryta ; therefore two such nitrogenous copper-reducing nuclei seem to be present in albumin. Schiitzenberger considers the body a to be a compound of CijHjgNjD^ with SC^HgNOj (glykoprotein a)— H^O. The compound Cj2H2gNg04 or CgHj^NjO^ he explains as an amide of cellulin ; and he thinks that from this body might be derived the small amount of dextrin which was found in a third ex- periment, chemolysis of hemiprotein by baryta. In this experi- ment a mercmic nitrate precipitate yielded on decomposition matters from which absolute alcohol extracted a sugar-like amorphous body, which contained Exp. in. a. 1 2 3 4 C . . 49-51 48-79 — — H . . 6-78 6-86 — — N . . — — 13-45 12-8 — 14-23 Besides this nitrogenised body the baryta-salt (in experiments like Exp. III.) always contains a certain quantity of a non- IQ ANNALS OF CHEMICAL MEDICINE. nitrogenised body whicli is insoluble in alcohol, is precipitated by ammoniacal lead acetate, and by boiling with dilute sul- phuric acid is metamorphosed into a body which reduces Fehling's fluid ; the original body does not reduce Fehling's fluid, but gives oxalic acid on treatment with nitric acid. The body gave on analysis C . - 42-8 per cent. H . . 6-56 „ These figures are near those which would be yielded by dex- trin. Schiitzenberger surmised that the occurrence of this cellulin-like body in the baryta chemolysis stood in connection with the occurrence of a body reducing Fehling's fluid which was found in the sulphuric acid chemolysis. Bat although he thus recognised that ' the molecle of albumin contains a small quantity of a cellulin amide,' he neglected this ingredient in all his following calculations and theories, and came to the result that albumin was a complex ureide ; the further details of structure he explained upon this basis, and in consequence did not arrive at the explanation of the structure of albumin as we shall have to formulate it. The amylonide nature of the albuminous substances is proved by the so-called glycogenetie function of the liver, and the lactopoetie function of the mammary gland of the carnivora. It is further proved by the growth and development from eggs to the adult state of maggots reared on boiled white of egg exclusively. These remarkable proofs science owes mainly to Bernard. But he did not draw the conclusion which is here propounded, and, like other inquirers in his time, considered the formation of sugar from albumin an accidental vicarious act, performed when no carbohydrates were at hand to suit the wants of the body. The paralbumin of Scherer, obtained from ovarian cysts, contains, besides albumin, a body resembling liver-starch, in this that it dissolves in water to a turbid fluid, is precipitated by alcohol, coloured brown by alkalies and yellow by iodine, and when boiled with dilute sulphuric acid yields a product which reduces cupric and bismuthic oxide. At the same time there is an albuminous body present which can be precipitated by OEGANOPLASTIC AMYLONIDES. 17 neutralisation and boiling ; and then the liquid contains another probably albuminous body, which on boiling with mineral acids yields a flocculent brown precipitate and a solution reducing, after neutralisation, copper, bismuth, and indigo. The original substance gave by analysis 49*7 per cent. C, 7"6 H, and 7'4 to 8"8 N. This body is therefore an amylonide, similar to the lower forms of mucin ; but as a pathological product it deserves special study. The following list of organoplastic substances is so arranged that it begins with the simplest, namely, cellulin, and ends with the most complicated, i.e. albumin. As they are considered here principally in their hypothetical character of substances formed upon the type of amylon, they are termed amylonides, and the sugars to which they give rise, or are supposed to give rise, by chemical or physiological agencies are named in connec- tion with them on the right of each term. Organoplastic anvmal amylonides and sugars to which they gvoe nse. Cellulin . • • Dextrose. Glycogen or hepatic amylon Dextrose. Tuuicin . Tunicose. Chitin . Chitose. Conchiolin Conchiolose» Hyalin , Hyalose. Fibroin . Fibrose. Serin Serinose. Spongin . Spongose. Keratin . Keratose. Chondrin Chondrose. Cerebrin Phrenosin Cerebrose, - Kerasin Gelatin . Gelatose. IchthyocolUn IchthyocoUose. Mucin ■ Mucinose. Paralbumin Paralbuminose. Albumin Albuminose (lactin), or albumino-dextrose 18 ANNALS OF CHEMICAL MEDICINE. These names seem convenient as designating the origin of each variety of sugar ; they or any of them will have to be changed as soon as it can be shown that the sugar corresponds to a known variety of more general occurrence. Some terms may be doubtful, e.g. the reducing body, supposed to have been a sugar, from serin, or silk gelatin, may have been sericin, which is not a true saccharoid but a hemi-saccharoid or glyceronide, and thus resembles the fats and phosphorised substances of the brain and other tissues. Again in the case of the sugar obtained from gelatin it might be possible that one of the reducing amido- acids (glutamic and asparaginic) had simulated sugar, a doubt which requires further attention. This doubt could, however, not apply to the sugar obtained from isinglass, which did not only reduce but by fermentation gave alcohol. Hepatic amylon or glycogen occurs twice in the list, namely, as an organoplastic substance by itself and giving rise to dextrose, and as a product of the decomposition of albumin, as albuminose, or albuminous dextrose. Even if these bodies should be iden- tical, which is not very likely, the repetition will be a concession to caution, as they might be formed in different places by different processes (lactin, in the mammary gland, glycogen in the liver). . Some of the sugars differ greatly from each other at first sight. Supposing the properties of dextrose to be well known, we can compare the other bodies to it. The sugar from chon- drin, chondrose, turns the plane of polarised light to the left ; lactin turns to the right, but is a disaccharonide, one molecle of it yielding by hydration two molecles of lactose. Cerebrose is probably isomeric with dextrose, reduces copper solution, turns the ray of polarised light to the right, and crystallises from water in crystals which do not lose water at 100°, while dextrose crystals at that temperature give up a molecle of water. All these differences and their causes will be discussed or investigated in detail hereafter. The chemical constitution of the fats and of the phosphorised substances is determined by a radicle which by chemolysis to ultimate crystalloids appears as glycerin. This radicle receives additional importance in relation to our present argument, by the fact that it can be considered as a half-sugar, so to say, a ORGANOPLASTIC AMYLONIDES. 19 hemisaccharon ; its formula is about half that of sugar, its func- tion is that of a tridynamic alcohol, while the functionally mos( developed sugar is a hexadynamic alcohol. Moreover, twc molecles of glycerin (so it has been stated) may combine and form sugar. The substitution of three molecles of fatty acid for three molecles of hydroxyl in glycerin produces the vege- table and animal fats. In the phosphorised substances, however, only two molecles of hydroxyl are substituted by fatty acids ; the third hydroxyl is substituted by phosphoric acid, and to this is attached a collateral chain of smallest radicles, the complex of which is termed neurin or choUn. Fats and phosphorised substances may, therefore, be classified as hemisaccharonides, or glyceronides, or as ethers of the alcohol glycerin with simple or compound acids. To this class of bodies serin or silk gelatin may possibly belong : it contains and yields by chemolysis a glyceritic amide (sericin) serosamide, which is analogous to chitosamide thus far, that we might signalise it by the term hemichitosamide, were not the amide wanting in the ideal second half of the ideally severed saccharoid radicle. c 2 20 ANNAI5 OF CHEMICAL MEDICINE. II. ULTIMATE CSYSTALLOID PRODUCTS OF THE CSEMO- LYSIS OF TSE OROANOPLASTIC SUBSTANCES, PAR- TICULARLY BY CAUSTIC BARYTA. (Summary ana Additions.') 1. Introduction. Researches of 0. Nasse. Researches of P. Schutzenberger. Description of his process. — The forcible decomposition of albuminous substances by caustic baryta with a view of obtaining information concerning their constitution and differences was initiated by 0. Nasse {Arch, Physiol, vols, vi. vii. viii.) and led to the information that the organoplastic substances under the influence of this reagent at the ordinary pressure of the air and a temperature of 100° lose a definite portion of their nitrogen in the form of ammonia, and that the quantities thus evolved vary with different albuminous sub- stances. Nasse obtained the following relations between the nitrogen evolved and that not evolved, expressed by the amount of the former divided by the latter : — or casein . . 0-033 „ serum albumin . . . 0-0889 „ gluten . . 0-275 Nasse treated the albuminous substances first with fuming nitric acid, and then boiled them with excess of baryta during 2^ hours. This did not enable him to obtain definite products either intermediate or final, of the cleavage of the substances operated upon. When Schiitzenberger operated in this way, and continued the boiling for 120 hours, he obtained from 100 grms. dry coagulated albumin 1-4 to 2 grms. nitrogen, in the form of ammonia. The 100 grms. albumin contain, however, 15*8 grms. nitrogen ; there remained, therefore, from 14-4 to CHEMOLYSIS OF OEGANOPLASTIC BODIES. 21 l3*8 grms. nitrogen in the matters mixed with the baryta. 1'4 The proportion rj-r"^"^^'' approaches the proportion found 2 by Nasse, but is somewhat higher; the proportion —- -=0"145 is, however, far removed from it. From this it follows that the results obtainable by boiling an albuminous substance with caustic baryta are not of great importance. They point, however, unmistakably to a great difference in the constitution of the bodies above enumerated. Schiitzenberger {Bull. Soc. Chim. Paris [N.S.] 23, 161 ; and Ann. Chi/m. Phys. 16, 1879, 289) attacks the albuminous bodies by baryta in a more forcible manner. He mixes one part of coagulated substance (corresponding in most of his experiments to 100 grms. of dry substance) with from three to six parts (therefore corresponding to 300-600 grms.) of crystal-. Used baryta hydrate, and three to eight parts {i.e. 300 to 800 c.c.) of water, encloses the whole in a cylinder of cast steel lined with silver, and heats it dmring periods varying from six hours to six days, to a temperature of from 160° to 250°. The most complete chemolysis is obtained with the largest quantities of baryta at the highest temperature in the shortest time. The perfectly cool contents of the cylinder are distilled until all ammonia has been expelled and condensed in two successive receivers, the last one containing excess of hydro- chloric acid. On the top of the alkaline liquid in the first condenser floats a small quantity of a colourless oil {alhum/moV) containing, as will be shown more in detail lower down, pyrrol, an oxygenated body, free from nitrogen, and a sulphur com- pound. This oil is separated mechanically from the ammonia before the latter is mixed with the acid contents of the second receiver. For the albuminol is soluble in dilute hydrochloric acid, and quickly changed by it, reddish brown flocks of pyrrol red being deposited. The contents of the two receivers are mixed, care being taken that the mixture be acid. One-tenth of the hydrochloric acid solution, corresponding to ten grms. of albuminous substance, is used for a quantation of the am- monia as platino-chloride. Of this, on an average, under the foregoing conditions six grms. are obtained. 22 ANNALS OF CHEMICAL MEDICINE. The barytic mixture, from which all oil and volatile alkali has been expelled, is now filtered through a weighed filter, and the insoluble precipitate, consisting of baryum carbonate, oxalate, suVphite, is washed with hot water and weighed. It may contain some baryum soap in case the albuminous sub- stance had not been completely freed from fat. It may there- fore be purified by solution in hot hydrochloric acid, filtered to remove some brown flakes and any fatty acids, and then be made alkaline with caustic ammonia. On standing in a place free from carbonic acid during twelve hours the solution deposits crystallised oxalate of baryum, which is dried and weighed. But even this purified deposit may contain some phosphates of earths, from which the oxalate can be separated by boiling the precipitate (crude or purified) with sodic carbonate, filtering, acidifying the solution with acetic acid, and precipitating with calcic chloride, and treating the precipitate further in the usual manner for the quantation of oxalic acid. The clear alkaline barytic filtrate from the above-named baryum salts is now completely, precipitated by a current of carbonic acid gas, filtered, heated to boiling to destroy a little baryta bicarbonate, filtered again, and accurately precipitated with dilute sulphuric acid, so that it contains neither baryum nor sulphuric acid. The barytic sulphate is collected on a filter, washed and weighed. The filtrate, which contains no mineral substances, is distilled in a vacuum to dryness. The distillate is neutralised, and, after concentration, distilled a second time, and in this distillate the acetic acid is estimated by neutralisation with normal soda solution. The solid residue which contains aU fixed organic prod/acts of the cleavage of the albuminous substance, is a mixture of amidated com- pounds, which for the sake of brevity is termed the amido- mixture. It is completely dried in a current of air at 100° and weighed. Before proceeding to the consideration of the analytical operations to which this amido-mixture is subjected, it is requisite to record, that the baryta carbonate, obtained by passing carbonic anhydride into the solution filtered from the carbonate, oxalate, &c., after the volatile alkalies and oils have been dis- tilled off, retains with pertinacity some quantity, amounting to CKKM0LYSI3 OF OEGANOPLASTIC BODIES. 23 from 5 to 8 per cent, of the amido-mixtTire, all washing with boiling water notwithstanding. This necessitates that the baryum carbonate should be decomposed with exactly its equivalent of sulphuric acid, that the sulphate formed should be edulcorated, that the filtrates, free from baryum as well as sulphuric acid, should be evaporated in vacuo, and that the dry residue thus obtained should be added to that of the amido- mixture. The whole of the amido-mixture thus obtained is intimately mixed by powdering, and subjected to the following analytical operations : a. The C, H, and N are estimated by elementary analysis. b. The proximate constituents are isolated as far as possible, and subjected to elementary analysis. c. The quantity of tyrosin and, as far as possible, that of leucin (these bodies being the least soluble and most crystal- lisable ones) is estimated. In this manner a series of data are obtained, which may serve to explain the phenomena of the chemolysis, and aid in the attempt to ascertain the constitution of the organoplastic substances. These data are discussed in the following, under the relative headings. 2. Nitrogen separated in the form of Ammonia from, different organoplastic substances. a. Coagulated albumin, produced by dissolving mercantile scale albumin in water, adding acetic acid, and precipi- tating by heating on the steam-bath. N=maximiun 4-41 per cent. ; minimum 3-46 per cent. ; 6. Serum albumin c. Casein .... d. Fibrin from horse's blood e. Muscle fibrin, veal /. Grluten exhausted by boiling alcohol g. HemiproteiQ . A. Ossein .... i. Gelatin From the foregoing it is evident that the amount of nitrogen ejected from different albuminous substances varies ; mean 3-93 per cent. 15 3-96 55 J5 3-54 55 ?5 4-83 55 5? 4-30 55 55 4-44 55 55 3-60 59 55 • 55 3-01 2-55 55 55 24 ANNALS OF CHEMICAL MEDICINE. within certain limits. These limits and the conditions of the variations are not yet determined. It is, however, already certain that the maximum of nitrogen is obtained only by the agency of much baryta, at very high temperatures ; and that the nitrogen evolved rises with the quantity of reagent, the time of its action, and the temperature at which it acts, in a certain ratio, which is almost the same ratio as that in which, the quantity of carbonic acid evolved at the same time rises, but is different from the ratio in which oxalic and acetic acid rise. 3. Weight of the mixed haryuw, precipitate, and separata weights of the baryum carbonate and baryum oxalate. — The precipitate also contains sulphurous acid, which has not been estimated ; further, possibly fatty acid and phosphates of earths under the conditions already alluded to above ; further, it may contain some products of the decomposition of glass, if the boiling has been effected in glass vessels. From this composite nature of the precipitate, it follows that no very safe conclusions can be drawn from the comparison of its weight with that of the evolved ammonia. Coagulated albumin gave in eight experiments (200°) figures which varied between 28 and 30 grms. Fibrin from horse's blood gave 32 grms., albumin from horse's blood 30 grms., gluten exhausted with boiling alcohol 25 grms. The composition of the baryta precipitate obtained at 16Q° to 200° is constant for one and the same specimen of prganoplastic matter, but changes with the nature of the sub- stance. a. Coagulated Albumin. — Sojne sorts of mercantile white of egg gave only traces of oxalate ; another white of egg, with which five experiments were made, gave — Nitrogen mean . , = 3'84 Total bar. precipitate, mean = 30'4 The mixed baryum precipitate contained for 30 grms. — Barytic carbonate . . 20 grms. „ oxalate ... 5*7 „ Total ,, 25'7 A third variety of albumin yielded — : 3-9 grins. 30-0 )5 14-5 55 12-5 55 CHEMOLYSIS OF OEflANOPLASTIC BODIES. 25 Nitrogen .... Crude bar. precipitate Containing — Barytic carbonate . „ oxalate . . i Total . 27-0 „ It is therefore clear that the relation between, the nitrogen evolved and the crude precipitate is nearly constant, but that the latter may contain very different proportions of oxalate and carbonate. These phenomena can be explained if it is assumed that the oxamide group th-tt^ y^2^2 '^^^ replace par- tially the urea group -^^^^ \C0 just as in felspar minerals the alkaline metals can replace each other in very different propor- tions. Or the existence of two kinds of albumin may be assumed, the one deriving from urea, the other from oxamide, which are mixed iii ordinary white of egg in different proportions. The proportion between the evolved nitrogen and the total weight of the carbonate and oxalate approaches very closely that which should be obtained if it is assumed that the evolved nitrogen is derived from the decomposition of the urea or of the oxamide. Thus the experiments given above, concerning the second specimen of albumin, gave — Mean weight of nitrogen . „ baryum carbonate ,, ,, oxalate . Nitrogen calculated from carbonate „ oxalate . 3-8 20-0 5-7 2-84 0-71 3-55 Total of calculated nitrogen a figure which differs from the_ quantity foimd (3'8) only by 0-25. The third variety of albumin gave — Nitrogen calculated from the carbonate . 2-05 J, „ „ oxalate . 1*55 Total of nitrogen calculated . . 3*60 a figure which differs from the quantity found, 3-90, only by 0*3. 26 ANNALS OF CHEMICAL MEDICINE. b. Casein. Casein easily retains a little fat, which appears as baryum soap in the baryum precipitate. Oxalate and car- bonate have therefore to be estimated after purification. 100 grms. of casein, dry, gave — Baryum oxalate „ carbonate . 17-5 grms. . 7-6 „ 25-1 „ Nitrogen calculated from oxalate „ „ „ carbonate 2- 17 grms. . 1-08 „ Total nitrogen calculated Nitrogen found . 3-25 „ 3-54 „ These figures differ by only 0-3. c. Serum albumin from horse's blood. Crude precipitate 30 grms., containing — Barynm oxalate „ carbonate 16 "5 grms. 10-5 ,. 27-0 „ Nitrogen calculated from oxalate . 2*05 grms. 5} „ carbonate . 1*50 „ Total nitrogen calculated . 3-55 Nitrogen foimd . . 3-95 Difference 0*4. d. Fibrin from horse's blood. Crude precipitate 30 grms., containing — Baryum oxalate „ carbonate Nitrogen calculated from oxalate 55 55 carbonate Total nitrogen calculated Nitrogen found Difference 0-46. 11-5 grms. 20-5 55 32-0 55 1-43 55 2-91 55 4-34 J5 4-80 •^ CHEMOLYSIS OF OKGANOPLASTIC BODIES. 27 e. Hemiprotei/n. Crude precipitate 30 grms., containing — Baryiim oxalate „ carbonate . 14*7 grms, 11-5 „ 26-2 „ Nitrogen calculated from oxalate . „ „ carbonate 1-82 „ 1-63. „ Total nitrogen calculated . Nitrogen found . 3-45 „ 3-60 „ Difference 0-15. /. Vegetable fibrin (gluten exhausted with boiling alcohol). Crude precipitate 25 grms., containing — Baryum oxalate . . . 8"0 grms. The precipitate showed an unexpected peculiarity in this that it evolved only little carbonic acid on treatment with hy- drochloric, but set free a considerable amount of a liquid fatty acid. It is necessary to make further experiments to see whether the fatty acid found originates with the gluten itself or with a fatty body present as an impurity only. g. Ossein (bone-dust from the manufacture of buttons, exhausted with dilute hydrochloric acid). Crude precipitate from 100 parts dry, 26 grms. contain- ing— Baryum oxalate . . . 5*0 grms. „ carbonate . . . 15"0 „ 20-0 „ There was a baryum soap present, which explains the differ- ence of 6* grms. Nitrogen calculated from oxalate . 0"62 grms. „ „ carbonate 2-13 „ Nitrogen found . . . 3*01 „ Difference 0-36, 28 ANNALS OF CHEMICAL MEDICINE. From these experiments the conclusion seems admissible that generally the nitrogen evolved in the form of ammonia and estimated directly exceeds by 0*3 to 0*4 per cent, of the organoplastic substance, or by one-tenth of its own quantity, the amount which is calculated from the oxalate and carbonate. Bat at higher temperatures, i.e. 250°, and with an increased amount . of baryta (say 100 grms. albumin with 600 grms. baryta, 400 grms. water, heated to 250° during six hours) the quantity of nitrogen in ammonia rises to 4"41 per cent., the baryta carbonate to 12"5 per cent., and the oxalate to 24*2 per cent. These conditions, as regards albumin, will be stated below in detail under the section 5 relating to acetic acid. 4. Weight of the haryutn which is not predpitable by carbonic add, correapond'i/ng to the strong adds contained in the amido-mixture. This datum is one of great constancy for the different organoplastic substances. There was found baryum sulphate — For 100 grms . egg albumin . . 22-5-24-0 J5 serum albumin . 25 M casein . . 24 55 fibrin . . 24 55 hemiprotein . 24 55 muscle fibrin . 22-2 55 gluten . 30 55 ossein . . 13-2 Gluten is therefore distinguished by an excess of 6 grms., and ossein by its giving only about half the amount of sulphate which the albuminous substances yield in this reaction. 5. Acetic add evolved. — With regard to this datum the different albuminous substances show peculiarities. The quan- tities of the acetic acid estimated directly by neutralisation of the distillate with normal soda solution (containing one equi- valent of sodic hydrate [NaH0 = 40] in the litre) were the following : — CHEMOLYSIS OF OEGANOPLASTIC BODIES. 29 Parts from 100 Albumin, coagulated (1 50° and 3 parts of baryta) 3'4 n » (250° and 6 55 ) 4-2 » » (250° and 6 J5 )5-I Casein . (150° and 3 55 ) 3-36 Blood fibrin n 55 3-6 Serum albumin n 55 3-48 Vegetal fibrin . J5 55 2-00 Ossein . 55 55 1-44 IchthyocoUin . 55 55 1-3 Chondrin 55 55 4-7 Fibroin 55 55 2-0 Goats' hair 55 55 2-1 Human hair 55 55 4-2 Wool . 55 95 3-12 The quantity of oxalic and acetic acid (containing a trace of formic) evolved from albumin (and probably from the other substances) rises with the amount of baryta and the temperature employed. 100 grms. albumin chemolysed under the following conditions gave — Baryta Water Temperatnre Time NinNH, BaCO. Oxalate Acetic acid grms. gnns. Centigrade hrs. 200 250 140-150° 12 3-1 10-5' 5-0 2-7 300 250 175 -180° 48 3-95-4-03 10-8 8'0 3-48 300 250 200° 120 3-95 11-0 8-6 3-78 200 250 160° 28 4-1 10-8 8-6 3-66 500 300 f 175° 1260° 12 2 4-36 F 17-6 4-20 600 400 175° 48 4-03 10-7 17-6 4-92 500 400 175-180° 120 4-02 10-9 17-3 4-65 600 400 250° 6 4-41 12'5 24-2 6-4 6. EUmenta/ry analysis of the amido-mixture, being the albuminous substance minus carbonic, oxalic, and acetic addf ammonia, suVphur, plus water. — The product obtained by evaporating the solution in vacuo was always feebly coloured, friable, hygroscopic, and free from mineral substances. a. Coagulated albumin : — C=48-69 H= 7-95 to 7-82 N = ll-8 to 11-9 30 ANNALS OF CHEMICAL MEDICINE, b. Coagulated serum albumin : — C =48-83 H= 7-76 c. Casein from cow's milk : — C =49-84 H= 7-89 d. Hemiprotein : — C =49-1 H= 8-11 e. Fibrin from horse's blood : — C=48-9 to 48-99 H= 7-90 to 7-84 N = 12-l /. Gluten (vegetal fibrin) : — C=47-4 H= 7-6 g. Muscle fibrin from veal : — C =45-6 H= 7-86 C=43-7 to 44-3 H= 7-42 to 7-67 N=]3-2 The results are nearly identical as regards egg albumin, serum albumin, casein, blood fibrin, and hemiprotein. The small differences arise from a greater, or lesser proportion of tyrosin in the amido-mixture. In fact, casein yields more tyrosin than albiunin, and blood fibrin yields an amount intermediate between the two. The amido-mixture of ossein possesses a different com- position. The muscle fibrin of veal was more a mixture of different tissues than a unitary albuminous matter, and contained an uncertain amount of gelatigenous tissue, by which the low amount of carbon is explained. The numbers which have been found for the amido-residue from albuminous bodies proper (albumin, casein^ &c.), can be expressed by the following sjrnopsis : — h. Ossein : — CHEMOLYSIS OF OEGANOPLASTIC BODIES, 31 c H Foimd . 48-4 8-0 Theory 48-35 8-00 N . 12-5 . 31-1 13-50 30-15 leading to the formula G^j H54 Ng Ojg or to a multiple of that formula. 7. Quantation of Ty rosin and Leucvn. — The quantation of tyrosin may be made by treating the dry amido- mixture with a mixture of 4 parts of water and one part of alcohol in the cold. The insoluble residue is dissolved in ammoniacal water, and the ammonia expelled by boiling, whereupon on cooling tyrosin crystallises almost entirely in long white needles, which are col- lected on a filter, dried, and weighed. In this manner 1 00 parts of the following bodies yielded tyrosin Albumin . . . 2-03 to 2-4 parts Casein. . . . 4-12 Hemiprotein . . . 2-2 Fibrin from horse's blood . 3-2 to 3-5 Vegetal fibrin . . 2-0 Another mode of extracting tyrosin and leucin from the amido-mixture is the following. The solution, after removal of all baryta precipitable by carbonic anhydride, and before the application to it of any sulphuric acid, is evaporated until a crystalline pellicle forms on its surface, and allowed to cool. The crystals which form are a mixture of tyrosin and leucin, amounting to from 25 to 30 per cent, of the albuminous sub- stance employed. They are separated by the ordinary well- known methods. Schiitzenberger at first thought that as tyrosin is present in the molecle of albumin, &e., in only small proportion, it might be left out of the calculation when the general character of the reaction is considered. In this, however, those would not agree who also take the physiological and pathological significance of tyrosin as a cleavage product of albumin into account, and remem- ber that in the healthy body tyrosin is further destroyed, and yields probably aromatic products of peculiar significance as regards the composition of the excretions. We shall see at the conclusion 32 ANNALS OF CHEMICAL MEDICINE. of this article how Schiitzenberger has lately adapted his theory to this requirement. The weight of leucin is obtained approxi- mately, though less accurately than that of tyrosin, by exhaust- ing the dry amido-mixture with hot alcohol of 90 per cent, strength, and collecting the crystals which form on cooling of the sufficiently concentrated liquid. The same quantity is ob- tained when the crystals are weighed which separate when a sufficiently concentrated solution of amido-mixture is allowed to cool. In this manner there are found for albumin 24 to 26 grms. of a mixture of leucin and leucein. 8. Further consideration of the amido-mixture and its iro- gredienta. — In view of the almost complete identity of com- position of the amido-mixture from the principal albuminous substances, and leaving out of consideration vegetal fibrin, which shows certain peculiarities to be further inquired into, it may be assumed as highly probable that these mixtures are qualitatively as well as quantitatively identical. This leads to the assumption that the albuminoiis substances have a common nucleus, which has the same constitution in all. The differences between the albuminous substances would in that case depend upon the nature and quantity of the secondary substances which are combined with these nuclei. This view has the value of a good working hypothesis, and effects a revival of the protein theory of Mulder, though with the difference that the constitution of the common nucleus is accounted for. More or less complete substitution of the urea group by the oxamide group, varying quantities of tyrosin, the intervention of certain peculiar secondary groups, such as that of glutamic acid, the occurrence of a fatty acid in gluten, are sufficient to account for at least some of the differences of the albuminous substances. Ossein and gelatin belong to a different type of compounds, the nuclei of which, though they may have some analogy to those of the albumins proper, are yet different. All the funda- mental data of these bodies differ from those of the albuminous substances. The analysis of the amido-mixture shows, moreover, that lower members of the homologous series CJS.^^ ^ jNO^ enter into the molecle. The quantity of water which combines with an albuminous CHEMOLYSIS OF OKGANOPLASTIC BODIES. 33 substance previous to its cleavage is determined by the number of nitrogen atoms contaiued in the molecle. If the decomposi- tion of urea is expressed by the equation CON^H, + 2{lifi) = COjHjO + 2NH3 then it is permitted to say that for every atom of nitrogen con- tained in the albumin a molecle of water enters at some time of the entire reaction. The amido compoimds, therefore, which form exclusively the mixture which has remained in solution, exist in albumin and its congeners as imides. The amido-acids contained in the amido-mixture belong to three series : 1. CnH^n+iNOj, series of leucin. 2. CnH2n_,N02, series of the amido-acids of the acrylic series. 3. CnH2„_iN04, series of asparaginic acid. The first series tnay for the purpose of brevity be termed that of the leucins, the second one that of the leueeins. In the albuminous substances the leucins and leueeins are present in such relative quantities that the nitrogen of their sum is equally distributed between both classes of bodies ; there- fore the general formula of the amido-mixture can, leaving out of sight the bodies of the asparaginic acid series, be expressed by CnHj^NOj. The leucins and leueeins form the principal bulk of the amido-mixture (obtained by baryta chernolysis ; by hydro- chloric acid chemolysis a much greater proportion of the mem- bers of the asparaginic acid series is obtained). The amount of the matters belonging to the asparaginic acid series can be approximately estimated by a quantation of the oxy_gen which is present in excess of the proportion N : Oj. We do not propose here to give the details of the operations required to separate the various bodies or groups of bodies from each other, as they are most complicated, and yet not very effective in all their parts, as shown by the uncertainty concerning the individuality of leucein, to be referred to more at length below under tyro- leucin. Fractional crystallisation from water and alcohol is the principal means employed to separate the leucins and leueeins. Amido-acids which are close to each other in a series, can be distinguished only by elementary analysis. Microscopic exa- 34 ANNAIS OF CHEMICAL MEDICINE. mination and proportions of solubility also give some slight assistance. By various processes there were isolated the following todies ; or when they were not isolated their presence in mixtures was shown by the results of elementary analyses. Tyrosin, CgHiiNOj ; amido caproic acid, or common leucin CsHiaNO^: amido-valerianic acid CgHgNOg ; these latter two crystallise together or occur in mixtures in equivalent propor- tions ; they are deposited from boiling solutions of alcohol of 90 per cent. The matter insoluble in such alcohol may be dissolved in water, and precipitated with mercuric nitrate, at first by itself, later on with the addition of some sodic carbonate to neutralise excess of acid. The mercuric compounds are washed with boiling water, decomposed by hydrothion, decolorised by aninial charcoal, and set to crystallise. The crystals are CjH^NOg, the anhydride of glutaminic acid, termed here glutimic acid ; this acid is monobasic. The second crystals, obtained by the aid of mercuric nitrate and alkali, are a mixture of glutamic (three molecles) with asparaginic acid (one molecle), thus: 3C5H9NO4 + C4H,N04. In another operation a mixture in equivalent proportions of caproic leucein and glutaminic acid, C6H„NO„ + CbH9N04=C,iH2oNj06 is obtained. 9. The volatile body, or essential oil {alhuminol). — This product, obtained in the chemolysis of albumin with baryta, was examined further by Schiitzenberger and Bourgois {Bull. Soc. Chim. Par. [N. S.] 25, 289 ; and Ann. Ghim. Phys. 16, 1879, 325). It appears on the top of the ammoniacal dis- tillate in the first condenser, in the shape of a few drops ; its quantity is from five to six per mille of the albumin used ; it dissolves in the hydrochloric acid when the contents of the condensers are mixed and the mixture is acid. It contains pyrrol, as the following reactions show. With perchloride of iron it gives a dark green reaction and later on a black deposit ; with dilute hydrochloric acid and potassic bichromate, it gives a black precipitate ; it dissolves in dilute hydrochloric acid and the solution left to itself deposits red brown flocks, which have the composition oi pyrrol-red. CHEMOLYSIS OF ORGANOPLASTIC BODIES. 35 Found Tl decry of pyn-ol-red c . . 71-75 71-2 H . . 7-50 6-93 N . . 12-02 13-0 . . 8-88 8-87 There is probably a small quantity of a higher homologue of pyrrol-red present in the flocks, and a body containing sulphur. The red flocks gave 3-1 per cent, of sulphur, while the oil con- tained from 2-1 to 2-6 per cent, of sulphur. By fractional distillation, albuminol freed from all ammonia by agitation with a slight excess of dilute sulphuric acid (50 to 60 grms.), can be divided into portions boiling at difl^erent temperatures. One boils over between 113° and 120°, a second portion between 120° and 140°, a third between 140° and 180°, and a fourth above 180°. The first two portions contain from 18 to 17 per cent, of oxygen and 4-5 to 4-1 per cent, of nitrogen, while in the last two portions the oxygen with the sulphur amounts to from 4*5 to 3-7 per cent. only. From these and the other data furnished by elementary analysis it is probable that albuminol is a mixture of two kinds of products; the most volatile part is probably oxygenated, but free from nitrogen ; the less volatile part is nitrogenised, but free from oxygen. Now as pyrrol is present (which boils at 133°) it may be allowed that the 4-5 to 4-1 per cent, of nitrogen which are found in the portions of liquid which boil over between 113° and 140° are derived from pyrrol which is mixed with an oxygenated non-nitrogenised substance. The analyses of the first two distillates lead to an empirical formula of CigHajNOj, which requires = 69-3; H = 8'3; N=5-0; 0=17-4. From this view the sulphur found is entirely omitted. If from the foregoing formula the nitro- gen is deducted in the form of pyrrol 041X5^, there remains CijHigOj, or 3(C4Hs0). The portions boiling between 140° and •180° show a composition similar to that of the higher homo- logues of pyrrol, methyl- and ethyl-pyrrol. They rapidly yield pyrrol-red with dilute hydrochloric acid when exposed to air. The sulphur compound has not been isolated. No trace of indol has been found in this reaction, which is the more to be noted, i> 2 36 ANNALS OF CHEMICAL MEDICINE. as indol is easily obtained from albuminous matters by che- molysis with fusing potash and by putrefaction with the aid of pancreatic ferment. 10. Tyroleucin. — Among the amido acids which Schiitzen- berger {Gompt. rend. 84, 124) succeeded in isolating when he decomposed as many as ten kilogrammes of albumin with baryta, is one to which he has given the name of tyroleucin. The liquid obtained by the action of baryta upon albumin at 150° is precipitated by carbonic acid, filtered and concentrated ; ..he crystals which separate are, in the main, a mixture of leucin, tyrosin, and butalanin. The syrupy mother-liquor is diluted anew with water, and treated with an appropriate amount of sulphuric acid, to remove the baryta which is not felled by carbonic acid. After filtration and concentration a second crop of crystals is obtained, and after their removal a third. From these second and third crystals tyroleucin is obtained by a great number of fractional crystallisations (from 10 kilos of albumin about 60 or 70 grms.), aided by animal charcoal and basic lead acetate, etc. It has a chalk-like appearance, crystallises in more or less voluminous masses, is soluble in water of 16° in the proportion of 5*3 : 100, more soluble in hot water, very little soluble in cold alcohol of 90 per cent., more soluble in hot alcohol, insoluble in ether. Heated while air is excluded, it begins to fuse at 245° to 250°, and is decomposed, yielding a distillate consisting of water, of the carbonate of a volatile base in a liquid state, and some few crystals ; the fluid possesses a strongly alkaline reaction, forms clouds with strong hydrochloric acid, and has a peculiar odour and taste reminding of radish. The walls of the retort in which the distillation was performed are covered with a white snow-like sublimate, and in the bottom of the retort there remains a yellow fluid, which sets into a solid on cooling. When heated with a few drops of nitric acid on platinum, and evaporated, tyroleucin leaves a yellow residue, which becomes orange- coloured with caustic potash. Nitroso-nitrate of mercury, and microscopic examination, prove the complete absence of tyrosin. The elementary composition of tyroleucin is CjHjjNOg. The carbonated alkali, contained in the pyrogenetic distillate on neutralisation with hydrochlor, forms a compound, which with CHEMOLYSIS OP OEGANOPLASTIC BODIES. 37 platinic chloride forms a yellow crystalline double-salt of the formula PtCl4 + 2(ClHCgHj,N). From this an oily base can be isolated by distillation with caustic lime ; it smells and tastes of radish, and has the composition of coUidin. The white sublimate has the properties of butalanin, and the non-volatile residue, which solidifies on cooling, and is soluble in alcohol, has the formula Ci4H,gN202. The pyrolytic reaction is there- fore probably also synthetic or polymerising, and produces the ascertained results, according to the following equations : 2(C,H„NO,)=2(H,0) + C.,H,3N,0, 2CC,H„NO,)=CO,+C8H„N + C,H„N-+C,H„NO, In the crystalline deposits which furnish tyroleucin much leucein and butalanin are contained. Leucein may therefore possibly be a compound in equivalents of tyroleucin and butalanin. C,H„NO,+ C,H„NO,=2C8H„NO, Leucein is indeed stated to behave like tyroleucin on pyrolysis, but to yield more butalanin. On the othe:? hand, tyroleucin may be considered as a compound of butalanin CjHjjNOj with a body of the formula C9H,,N02, which differs from tyrosin only by an atom of oxygen which it contains less. When tyrosin is heated under the same conditions as those effecting the changes of tyroleucin above described, it splits up into carbonic acid and an oxygenated base of the formula CgH,,NO, which differs from collidin only by containing an atom of oxygen pot present in collidin. 11. Protooonia. — Closely rela,ted to the bodies described in the foregoing is probably a volatile ammonium base from animal albuminous matter, discovered by Thudichum in 1869 (' Eep. on Eesearches, etc.,' in the Twelfth Report of the Medical Officer of the Privy Council, 1869, p. 257). It was obtained by chemolysis in dilute sulphuric acid of albuminous constituents of brains, lungs, livers, kidneys, and muscles from human subjects. The products were distilled with excess of caustic lime in water, and yielded a distillate containing much ammonia and a compound ammonium base. The bases were first crystallised as chlorides, then redistilled, and transformed a second time into chlorides ; again distilled over potash, they were transformed into sulphates, and from these the compound 38 ANNALS OF CHEMICAL MEDICINE. base was extracted by absolute alcohol, while the great bulk of the product, ammonic sulphate, remained undissolved. The sulphate of the new base, after distillation of the alcohol, was distilled with soda, the distillate transformed into chloride, and set to crystallise. Several crystallisations were isolated, and the crystals combined with platinic chloride; the double- salts were again subjected to fractional crystallisation, and the purest products analysed. The quantations led to PtCl4 + 2(HClCjH„N). The hydrochlorate was C^HiiNHCl, and the free base CjHuN. Thudichum named the base jproto- conia, from its similarity in composition, smell, and other pro- perties to conia (or coniine), the volatile base from hemlock. Horn, which yields other products of the chemolysis of albuminous substances with facility, did not give any protoconia when treated, even in quantities up to 20 kilogrammes, like the albuminous substances mentioned. Protoconia differs from tyroleucin only by 0^, but it could not be derived from it by the same process as that which takes place in the pyrogenetic process of Schiitzenberger. It is more probably a direct product of the first sulphuric acid chemolysis, and has not as yet been obtained from other than human albuminous matters. 12. Chemolysis of the collagenous substances by baryta hydrate. — This subject was further investigated by Schiitzen- berger and Bourgois (CoTnpt. rend, 82, 262) upon isinglass, ossein, gelatin, and chondrin from the cartilages of the calf. There were obtained from 100 parts — 3£inglass Ossein Gelatin CHiondrin Nitrogen as ammo Oxalic acid . Cartonic acid Acetic acid . Elements of • amido-mixture nia . fC . H . N . lo . 3-48 4-1 2-7 1-7 44-83 7-37 14-44 33 36 3-35 3-62 3-1 1-44 46-48 7-45 14-10 32-23 2-8 3-3 2-72 1-5 45-16 7-36 14-30 33-18 3-88 4-2 2-45 4-69 46-7 7-07 11-7 34-63 From these analyses the following formulae are derived, ■ nitrogen assumed as unit : — CHEMOLYSIS OF OEGANOPLASTIC BODIES. 39 Isinglass = Cj.gj ^7-146 N 'J 2220 Ossein = C^-^^ ■"■785 N 0, Gelatin = Cj-gg ^7-20 N "208 Ch6ndrin= C4gjg HgM N 02=7 As with the albumins proper and with fibroin, so with the above substances, the nitrogen evolved (by baryta chemolysis) in the form of ammonia on the one hand, and the carbonic and oxalic acid on the other hand, stand in such proportions to each other that the simultaneous formation of these three bodies can be referred to hydration of urea and oxamide. For gelatin and chondrin the harmony is nearly perfect ; isinglass and ossein give an excess of nitrogen found amounting to 0*3 to 0"25 over the quantity calculated from the weight of carbonic and oxalic acids. The analysis of the amido-mixture shows that its composi- tion is identical, or nearly so, for isinglass, ossein, and gelatin. The proportion between the atoms of nitrogen and oxygen is nearly as 1 : 2 ; it may therefore be expected that only mem- bers of the two series C„H2n+,N02, and CnHjn_,N02 are present. The proportion between the atoms of carbon and hydrogen is as 1 : 2 ; the nitrogen is therefore almost accurately divided between members of the series CnErj„+jN02, and CaH2n_iN02. Chondrin shows peculiarities in the amount of acetic acid which it yields, which is threefold that of the other sub- stances ; further in this that the number of atoms of nitrogen are to those of oxygen in the proportion of 1 : 2*57, which in- dicates the probable presence of compounds of the series CDHjn_jN04 in the amido-mixture ; further, that the proportion between the atoms of carbon and hydrogen is nearly as n '. 2n — 1, from which it follows that the members of the two series CnHjn-iNOj, and CnIl2„_jN04 prevail. The probable composi- tion of the amido-mixture from isinglass, ossein, and gelatin is therefore : glycosin 20 ±0 25 per cent. ; alanin, CgH^NOj ; amidobutyricacid C4H9NO2; traces of glutaminic acid; members of the series CnHjn.jNOj, with n=4, 5, and 6 more than 50 per cent. When these results ai-e compared with the original com- position of isinglass, ossein, and gelatin, the following formula for the reaction which they undergo with baryta hydrate may be written : — 40 ANNALS OP CHEMICAL BTEDICINE. C,eH^,,N,,0,a + 1 8H,0 = Cfifi, + CO, Gelatin Oxalic acid Carbon, acid + 0-5(C2H,O2) + 4NH3 + C„H,,,N2o04o Acetic acid Amido-mixture The amido-mixture decomposes in the following manner : — C7.H,, Ao04o= 6C ANO, + 2C3H,N0, Glycosin Alanin + 2C,H9N02 + 6C,H ,N0, + 2CsH 3 NOa + 2C6H„yO, Amidobutyrio acid Members of the acrylic series The amido-mixture of chondrin is almost destitute of glycosin ; it contains the acids of the series CnH2n_,N'04, alanin, amide- butyric acid, and the members of the acrylic series C4HyN02 and C5H9NO2. For its decomposition the following formula may be written : — C99H15A.O42+ 18H30=C,H,0, + CO, Chondrin + 1 •5{C,Rfi,) + 4NH3 + fi3H.„N,„0,,. Amido-mixture In these compounds the number of molecles of water fixed during chemolysis is smaller than the number of atoms of nitrogen in the products. 1 3. Chemolysis of Fihrovn {Silk). — This body was subjected to chemolysis by baryta hydrate by Schiitzenberger and Bourgois {Bull. Soc. Ohim. Par. [N.S.] 25, 1 ), with the result that it yielded ammonia, oxalic and acetic acid, and a mixture of ami do compounds. 100 parts of fibroin gave nitrogen in form of ammonia 2 per cent. ; mixture of barytic carbonate and oxalate 1 8 per cent. ; acetic acid in very small quantity ; mixture of glycosin and alanin in equivalents 60 per cent. ; amido-benzoic acid 10 per cent. ; tyrosin 9*5 to 10 per cent. ; amides of the series C„H2„_jN02, C4HyN02 preponderating, about 20 per cent. The elementary composition of the amido- mixture compared with that of the fibroin shows that the amount of water taken up during the reaction stands to the nitrogen of the fibroin in the proportion of Hfi '. N. CHEMOLYSIS OF OE&ANOPLASTIC BODIES. 4J. The havr of the alpaca is of a similar chemical constitu- tion. The fixed residue contains C = 40*6, H=7-3j N=15"0. Ammonia and non-nitrogenised acids are obtained in much lesser quantities than from hair of other animals or sheep's wool. 14. Chemolysis of sheep's wool and humcm hair. — P. Schutzenberger {Gompt. rend. 86, 1878, 767) subjected sheep's "wool to the influence of an equal weight of baryta hydrate, mixed with water amounting to three or four times the weight of the wool. It yielded the general products of the chemolysis of the albuminous substances in the following proportions. 100 grms. gave — Nitrogen in the form of ammonia . 5'2 to 5'3 Carbonic acid, separated as baryum salt . 4*24 to 4*3 Oxalic acid .... 5'68 to 5-77 Acetic acid, estimated volumetrically in the distillate, obtained from the mix- ture after elimination of the baryta by carbonic and sulphuric acid . 3*18 to 3*20 Pyrrol and other volatile products . 1 to 1*5 Composition of the fixed residue or mix- ture of the amidated substances pro- duced by cleavage : C=47-85-, H = 7-67; N=12-63. A specimen of Australian wool, freed from fat, gave the same quantities of ammonia, acetic, oxalic, and carbonic acid ; the fixed residue gave on elementary analysis C= 48*03 ; H = 8*24 ; 'N'= 12"9. It seems therefore that specimens of wool of different origin differ a little in composition. The fixed residue from Australian wool has the same composition as that derived from albumin ; the fixed residue from the first specimen of wool is a little different. The fixed residue consists exclusively of the following ami- dated bodies : caproic leucin, CjHijNOj ; caproic leucein, CgHiiNOj, =12 to 15 per cent.; tyrosin, C9H,iN03=3*2 per cent. ; butyric leucin, C4H9NO2, and valeric leucin, CgHjjNOj, the former prevailing ; propionic leucin, CgHjNOj ; butyric and valeric . leuceins, C^HjNOj or 2(C4HjNOj), C^H^NOj, or 42 . ANNALS OF CHEMICAL MEDICINE. 2(C5HgN02); glucoprotein intermediate between the leucins and the leuceins, CjH,gN204 Human hair gives the same kind of fixed residue as wool, but it yields larger quantities of carbonic, oxalic, and acetic acid. A small quantity of a syrupy acid {lanophanic acid) was also obtained, which had a freely acid reaction and gave a crystallisable silversalt. This acid remains in the barytic car- bonate, when the first liquid is treated with carbonic acid. The barytic carbonate is decomposed with sulphuric acid, and the filtered liquid is concentrated. Analyses of the free acid and of its silversalt lead to the formula CjoHjgNjOg, the silversalt being C,oH,4Ag2N205. This acid is also found amongst the decompo- sition products of albumin. The acid is almost identical in composition with paraphanic acid, CiiHigNjOg, the silversalt of which is CuHi^AgjNjOg ; it differs from kryptophanic acid, CjoHjgNjOjg, by the latter acid containing Sfi^ more. There is evidently a very close rela- tionship between the ' lanophanic ' acid from wool and albumin, and the extractive acids contained in urine. (Compare Report for 1878, App. B, No. 2, pp. 325 and 331.) 15. Probable Formulae of the higher Organoplastic Sub- stances. — On attempting to give a formula for the decompo- sition of wool or of albumen; which should contain an entire molecle of the body formed by chemolysis in the smallest quan- tity, one finds the atomic weight of wool or of albumin to be very high. Wool contains, according to the published analyses, C = 50*0 ; H = 7-0; ]Sr=17-7; = 22-0; S = 3-l. This leads to the smallest formula of C3.295H5.45NjO,.,Sj.(,g. Now as 100 parts of wool give at the utmost 3"2 parts of tyrosin, wool must have a molecular weight of at least 5,500 in order to give the opportu- nity for the formation of a single molecle of tyrosin ( = 181) ; for \%\''^ = 3-2. The smallest formula derived fi:om the per- centic composition of wool just given, when multiplied by 70, leads to Cjgo.gHgjjN^gOyjSg, and this formula is decomposed by the chemolytic process in the sense of the equation Cj3o-5H38iN7oO,7Sg + 5711 fi = 3' 5(0, 11,0,) + 5-5(C02) + 21 (NH3) f KC.Tifi,) + C,H,N + C,„,H,„3N,,0,„3. CHEMOLYSIS OF OBGAJSfOPLASTIC BODIES. 43 If from the last formula, which expresses the composition of the amidated mixtm-ejWe deduct a molecle of tyrosin C9H11NO3, the rest Ci99H3ggN4jOi5o is, as in the case of albumin, a value of the formula CnH2nN204, with a slight excess of oxygen ; 71= 8 "46 for wool ; ■n,=8'8 for albumin. The expressions are therefore homologous for wool and for albumin, and the difference is due to one in the quantity of leucin. The equation for the decomposition of albumin (of which the empirical formula is multiplied by a convenient factor to attain a molecle of tyrosin) is the following : — C,4.H387N650r5S3 + 60H,0 = 4(C,H,0,) + 3(C0,) + U{NH:,) + 4(C,H,0,) + C,,,H43,N,,0,„, + S3 In both cases the free ammonia is a function of the three non-nitrogenised acids, every molecle of carbonic and oxalic acid corresponding to 2NH3, and every molecle of acetic acid corresponding to NH3. The partition of the rest of the nitrogen is effected equally between the bodies of the general formula CnHjn+jNOj on the one, and the bodies of the formulae CnH^n-, NO2 and CnHjjn.iNlQ^I on the other hand. The above formula for albumin gives a molecular weight of 5743, and the theory of its elementary composition accords with experience as follows. Theory Found c . . 52-62 52-57 H . 7-07 7-16 N . 16-62 16-6 . 21-94 21-8 s . 1-75 1-8 1 6. List of termn/nal cleavage products, mostly crystalloids obtained by the chemolysis of albuminous and collagenous substances — Carbon tetrahydride (marsh gas) . CH4 Ammonia • NH3 Carbonic acid . CO, Oxalic acid . • CjH^O Sulphiurous acid . SO^ 44 ANNALS OF CHEMICAL MEDICINE. Indol • CigHj^Nj Skatol • ClioHiiN Pyrrol . C.H^N Epipyrrol . . C,H,N(?) Acetic acid • . C,H,0, Tyrosin . C,H„NO, Amido-oenantliic acid . c,n,,m. Tyroleucin . . C,H,,NO, Protoconia . . C,H„N Amido-benzoic acid . C^HjNO^ Amido-caproic acid (leucin) . CeH.3N0, Amido-valerianic acid . C,H„NO, Amido-butyric acid . . C^H^NOa Amido-propionic acid (alanip) . CgHyNOg Ainido-oxypropionic acid (serin) . CjHjNOg Amido-acetic acid (glycosin^ . CaHgNOj Caprono-leucein . C„H„NO, (? Valeriano-leucein • C,H,NO, Butyro-leucein . C.HyNO^ Glutamic acid . C,H,NO, Glutimic acid . C,H,N03 Asparaginic acid . C,H,NO, Hypoxanthin . C^H^N^O Dextrin • «(C6H.,0,) Cellulinamide . TiCCfiH.aNO,) Phenol • C.Kfi Lanophanic acid • CjoHigNaOg Lactic acid . . CaHgOg Succinic acid . C,H,0,H,0 45 III. OJV THE ACTION AND PR0DVCT8 OF THE STAJiCH- TRANSFORMING FERMENTS, DIASTAS, PTYALIN, PAN- CREATIN, ACIDS, AND VARIOUS MATTERS DERIVED FROM ALBUMINOUS SUBSTANCES. {Summary.) The scientific consideration of the malting of barley dates from the end of last century, and may he said to have begun with the observation of Cruikshank (Scherer's Allg. Journ. d. Chem,, Leipzig, 1798, vol. 1), according to which the sprouting of moistened barley, and consequently the formation within its substance of sugar, do only take place in the presence ©f an ample supply of oxygen. This observation induced Cruikshank to attribute the formation of sugar to an oxidation of the vegetable mucilage and the starch-like components of the seed. In 1811, however, it was discovered by Kirchhof (Schweigger's Journ. 14, 389) that the transformation of starch within sprouting grain is efifected by an albuminous body contained in the seeds; he imitated the process by placing glutin, from wheat-flour, washed free from starch, in contact with potato- starch, made into paste with hot water, and observed that in a few hours the paste became fluid, and contained sugar. On applying powdered malt to a paste made from potato-starch, Kirchhof found that it effected the transformation in a shorter time than glutin ; but he continued to attribute the action to glutin in both cases, while admitting that its peculiar power was much increased in the growing seeds by the process of germination. At the same time this chemist discovered that boiling dilute mineral acids had a power similar to that of malt or glutin, of transforming starch into sugar. The mode in which starch is digested in the animal body had long been an object of sterile inquiry on the part of physio- 46 ANNALS OF CHEMICAL MEDICINE. legists, when it was discovered by Leuchs (Kastner's Archiv, 1831) that saliva had the same power as malt or glutin, of producing sugar and dextrin in starch-paste made with the aid of water and heat. This was confirmed by Schwann (Poggend. Ann. 38, 358). The attention of chemists now became more generally directed npon the subject, and many endeavours were made to define and isolate the body to which this remarkable metamorphosis was due. Th. de Saussure {Poggend. Ann. 32, 1834, 194) extracted three different bodies from crude glutin, which were distinguished from each other by differing solubilities in alcohol, and termed them respectively (vegetal) albumin, glutin, and mucin ; of these the last, mucin, possessed in the highest degree the faculty to dissolve starch-paste, and transform it into sugar, while the first two possessed it in a much lesser degree. Pay en and Persoz now described a method by which the starch-transforming substance could be isolated ; malt was digested with water, and the solution mixed with alcohol, whereby a precipitate containing the active body was produced. The greater part of the precipitate was found to be again soluble in water, and then to exhibit the starch- transforming power in an eminent degree. The part of the alcohol precipitate insoluble in water was found to be albumin. The soluble ferment, when dried at a low temperature, was amorphous, yellow and translucent ; soluble in water and weak spirit ; when heated in the moist state or in solution to 75° C. it lost its starch-transforming power ; it also became inert when kept for a longer period in the moist state or in solution. In the fresh state its power was so great that one part effected the solution of 2,000 parts of potato-starch in a few minutes. Payen and Persoz termed this substance, which they considered as a chemical individual endowed with specific properties, diastas. This name was chosen to express the hypothesis which they had formed concerning the manner in which the starch granules are transformed, namely, by bursting of their shells, and pour- ing out of their supposed gumlike contents. The name, still generally used, has survived the hypothesis, now generally abandoned, to which it owed its origin. Payen and Persoz found diastas to be present not only in malt, but also in growing seeds of oat and wheat {Ann. Chim. 53, 73 ; 56, 337), of maize and STAECH-TEANSFOEMING FEEMENTS. 47 rice {Ann. Chvm. 60, 1835, 441), in the sprouts of potatoes, and in the buds of ailanthus glandulosa. The theory of ferments thus becoming more and more de- veloped, Mialhe {Mim. sur la Digestion et I'AssiTnilat. des Mat. amyloides, 1 845 ), following up the discovery of Leuchs, separated a body from saliva which was the bearer of the sugar-forming action upon starch ; it was termed, after Berzelius, ptyalin, and its analogy to, or even identity with, diastas, was allowed or maintained. Bouchardat {Ann. Ghim. [3,] 14, 1485, 60) found that various matters of animal origin, albumin, gelatin, fresh fibrin, still more albuminous matters in a state of decom- position, such as putrid flesh, putrid glutin from plants, and others, were able to dissolve starch and transform it partially into sugar much like diastas. Sandras and Bouchardat ((7ow/p^. rend. 20, 143) demonstrated the presence in the pancreas of a ferment possessing a power similar to that of ptyalin. The natural secretion of this gland, or even an infusion of the minced organ, was found to produce sugar in starch-paste with great rapidity. These data, with the occasional inquiries of Magendie, Lassaigne, Barreswill, Bernard, Hensen, Wright, Sehiff and others, showing that bile, decomposed urine, blood serum, blood, brain matter, heart, muscle, lung, liver, kidney, were all able to exert a function upon starch more or less like that of diastas, led to the doctrine of the universal presence in vegetal and animal parts of starch-transforming ferments, a doctrine of which Mulder became the principal systematic expositor {Chem. des Bieres, p. 215). He was of opinion that there would hardly be found a vegetal juice destitute of the power to transform starch into dextrin and sugar, and believed the same power to be inherent in all animal fluids and solids, when placed under the necessary conditions. After Von Wittich had discovered a new method for con- centrating certain shapeless animal ferments he also applied it for the purpose of isolating a diastatic ferment from the animal matters known to exhibit the function {Arch. Physiol., 3, 1870, 339). He obtained active matters in small quantity from blood, blood serum, kidneys, brain, mucous membrane of stomach and intestines, but arrived incidentally, so to say, at the belief that this ferment was not exclusively a 48 ANNALS OF CHEMICAL MEDICINE. specific product of the life of certain cells in the parenchyma of plants, but also took its origin in the general chemistry of the body. Lepine {Ber. K. Sachs. Ges. d. Wissensch., October 1870) found a diastatic action in nearly all tissues of the body, but not in the crystalline lens. After four days' keeping 'the lens also exhibited the action, from which he drew the conclu- sion that the fermenting action is developed in the organs by post-mortem change ; as already observed by Bouchardat as quoted above and by Bernard upon washed fibrin (Lemons de Physiol, expir. 2, 1856. Seegen and Kratschmer (Arch. Physiol. 14, 1877, 593) have made similar observations. Even when they boiled and washed brain or muscles, these tissues regained a starch -transforming action already after a few hours ; pure serum albumin, egg albumin, casein, and fibrin acted in the same manner. But the action was slow and feeble as compared with that of ptyalin and pancreatin. They be- lieved that the liver contains a specific diastatic ferment, but were unable to extract it by the alcohol and glycerin method of Von Wittich {Arch. Physiol. 1, 1873, 28). In a later publi- cation Seegen seems to have abandoned this opinion [Arch. Physiol. 19, 1879, 106). The ground-up Uver, after having been hardened with alcohol, yielded to glycerin a mixture of glycogen and diastatic ferment. In this sohition the ferment remained inactive, as long as water was not added ; when this was added the formation of sugar began at once. This mixture had already been extracted by Epstein and Miiller (Ber. Deutsch. Chem. Ges. 8, Heft 4)., The return of the diastatic action to boiled liver magma on standing had been already observed by Abeles {Med. Jahrb. 1876, Heft 2). Concerning the general characters of these ferments Von Wittich found (after Schonbein, Zeitsch. f. Biologie, 4, 1868, Journ. Pract. Chem., 106, 1869, 257) that they show an energetic action upon hydrogen peroxide, and retain this as well as their starch-transforming action at temperatures from 60° to 80° C, at which the albuminous substances coagulate., Von Grorup Besanez {Ber. Deutsch. Chem. Ges., 1874, p. 1478, and 1875, p. Iol0)in extracting diastatic ferment by Von Wittich's process from vetches, hemp and linseed obtained mixed with this a ferment which had the power to dissolve and STARCH-TEANSFOEMING FERMENTS. 49 transform into peptones fibrin and coagulated white of egg. This observation was likely to shake the belief in the peculiarity of many of the so-called specific ferments, and made the digest- ing power of the so-called insectivorous plants a particular case of a widely diffused faculty of vegetals. The distribution of starch-transforming ferments in plants has quite lately been studied by J. Baranetzky {Die Stdrkewnv- bildenden Fermente in den Pflanzen, Leipzig, 1878, 8vo. pp. 64 and plate). He made concentrated watery extracts, of which he added from 0r5 to 1 c.c. to 3 to 4 c.c. of a solution of potato-starch prepared by boiling, and containing not above 1 per cent, of dry starch. The presence of the ferment was recognised by the liquefaction of the paste and its transforma- tion into a perfectly clear liquid. This criterion is more certain according to this author, than the appearance of sugar, because as he says, the starch may be dissolved without, or at all events before, any sugar is formed. He did not operate with pure ferments, nor did he investigate the processes on any larger scale than in testrtubes ; he made no analyses of the products, and no quantitative tests except some reductions with standard copper solution, so that his results have no stoichiometric back- bone. But they are conceived in a philosophical spirit, and extend our knowledge of the distribution of the diastatic power in the vegetal world. The following is a list of parts of vegetals in which he found the diastatic or starch-transforming power. Seeds containing starch. — Phaseolus multiflorus (germi- nating). Vicia faba, sprouting in the dark, etiolated ; cotyle- dons. Pisum sativum : the crushed hard seeds dissolve starch in 20 hours ; seeds germinating in light effect the same work in 30 minutes. Polygonum fagopyrum : growing seeds. Mira- bilis Jalapa i the seeds dissolve paste ; the germinated seeds have no greater action. Aesculus HippocastanuTn : slow action, whether germinating or not. Quercus fedunculata : no action, whether germinating or not. (Diastas forms an insoluble compoxmd with tannin or tannic acid.^Dubrunfaut 1868.) Tubers contdi/niTig starch. — Potatoes, growing shoots; Oesnena barbata, sprouting ; Dioscorea Batatas, tubers with pronts more than a metre in length : the extract, which was E 50 ANNAXS OF CHEMICAL MEDICINE. mucilaginous, dissolved starch in 30 minutes. Iris germanica, with green leaves 10 to 15 c. long, same efifect. Stalks and leaves. — Phaseolus multiflorus ; Pisum sativum, ; Vicia faba : the extracts dissolve the starch in from 3 to 20 hours. Daucus carota and Brassica Rapa ; shoots from roots dissolve the starch in from 30 to 90 minutes. Eriobotrya japonica (leaves) ; Acanthus cordifolia (leaves) ; Echvum giganteum (leaves) ; Tradescantia zebrina (leaves) ; Veltheimia viridiflora (leaves) dissolved the paste in from 20 to 48 hours. Rhizomes free from, starch. Daucus carota contains much copper-reducing sugar, and cane-sugar; transforms starch in 30 minutes. Brassica Rapa contains also a reducing sugar ; transforms starch in 20 minutes. The difference in the time required for full action is ascribed to differences in the concentration only. Few inquirers were induced by this observation to dis- tinguish between a ferment, which transforms the starch in plants into a soluble nutriment (not necessarily sugar or dextrin) without requiring the aid of heat for. the fluidification of the starch, and another which does not act upon starch in granules as obtained from vegetable parts, but requires the granules to be previously hydrated and disintegrated by water and heat. In an article on the process of digestion in animals and vegetals {Revue Scientif. 1873, 515) Bernard compared the action of the starch-transforming ferments with each other. He impressed upon his readers the fact that their action appears in seeds of cereals with the earliest symptoms of germination, in potatoes in spring even before they have been committed to earth for regeneration. In this as in most other discussions of the subject it was assumed that the ferments which liquefy starch in living plants, and maybe transform it into nutriment for new growth, or even into sugar, are the same as diastas, the ferment extracted from malt, which transforms boiled starch into maltose and dextrin, but has either no effect, or only a very slow action upon unboiled or unhydrated amylon. This assumed identity is however not only not proved but directly contradicted by the data. The data indeed lead us to believe, that if starch is transformed into sugar during the germinating process of barley, it is probably not by the agency of diastas STAECH-TRANSFOEMING FERMENTS. 51 alone that this change is effected. And it further follows as a result of the consideration, that there must he a difference be- tween the starch-transforming ferments in animals, which fluidify, hydrate and split up starch without the previous application to it of heat, and those in malt, which can only, or at least mainly (time being a condition of the comparison) transform starch previously so to say opened up by heat. Gruerin-Varry {Ann. Chim. 60 (1835) 32) observed that potato-starch sus- pended in malt extract, and allowed to stand during 63 days at 20° to 26° underwent no change that could be observed either chemically or microscopically. At 54° to 55° only did any diastatic action begin to manifest itself, a temperature at which the starch granules began to swell and burst. Schlossberger (Organ. Ghem. 1857, 121) made a similar experience and came to the conclusion, that the solution of starch in growing seeds is effected by a ferment different' from the diastas which can be isolated from the seeds. Mulder was of an opposite opinion, and explained {Ghem. des Bieres, p. 222) the difference as caused by difference in the quantity or concentration of the ferment. Baranetzky (I.e. 38) explains that these results, which opposed the idea of the unity of the starch-transforming ferment, were due to the acci- dent that the experiments were all made upon potato -starch, which is one of the most resistant to the influence of ferments. Wheat or buckwheat-starch however is attacked by ferments at the ordinary temperature in the same manner as by ferments in the seeds. The experiments were made each with 2 to 3 centigrammes of pure starch in watch-glasses : the ferments consisted of the watery solution of precipitates produced bv alcohol in vegetal extracts. The starches examined came from Polygonum fagopyrum, Phaseolus multiflorus, Mirabilis Jalapa, Quercus pedunculata, Aesculus Hippocastanum, pota- toes, wheat and rice. The microscopically visible changes which the granules undergo have been represented by Baranetzky on a plate with many figures. In now passing to a more par- ticular examination of the products which starch yields under the influence of various ferments akin to diastas or of diastas itself, it must be stated at once that our main attention will be directed upon the sugar-like products, and that the con- E 2 5-2 ANNALS OF CHEMICAL MEDICINE. sideration of the dextrin-like products, and of the question whether they are always, or in fact, transition-products, will be treated of only to such an extent as may be necessary to define its present position. Most sweet-tasting substances occurring in natural edible vegetal products were believed nearly to the end of the last century to be cane-sugar. The distinction between cane-sugar and grape-sugar and the identification of the latter as a peculiar body were not effected iintil 1792, by Lowitz (Crell's Chem. Ann. 1, 218 and 345) and Proust {Jov/rn. de Phys, et de Chim. 63, 257 ; 69, 428 ; Ann. Chim. 57, 131 and 225). Many years later Thenard and Dupuytren {Ann. Chim. 44, 45) discovered the identity of diabetic and grape-sugar. Many authors after- wards enlarged our knowledge of these transformations, particu- larly after Biot had taught a new means of diagnosing the new products from each other by their varying influence upon polarised light. But no author contributed during almost forty years, beginning from 1823, more or better new knowledge concerning glucose, and particularly the transformation-products of starch by diastas, than Dubrunfaut. In 1847 (Ann. Chim. 21, 178) he prepared the crystallised sugar obtained by the malt ferment, and observing it to show reactions differing from those of ordinary grape-sugar or glucose he submitted it to Biot, who found that its influence upon polarised liglit was much greater than that of an equal weight of grape-sugar (jCoyrupt, rend. 15, 710 ; 42, 351). Forthwith Dubrunfaut announced its peculiarity, showed that it was transformed into grape-sugar by boiling with dilute acids, that it fermented with yeast like grape-sugar, and termed it maltose. This most important discovery was either altogether ignored or doubted, or even strongly opposed, by almost all chemical authors and inquirers on the subject. This misguidance has affected a vast number of statistical researches in which the copper-solution test or the polarisation test have been relied upon as quantitative indices for grape-sugar. O'SuUivan {Journ. Chem. Soc. 25, 1872, 579) repeated the experiments of Musculus {Ann. Chim. {3] 55, 203), of Payen (Ann. Chem. [4] 4, 286), and of Schwarzer (Journ. pract. Chem. [2] 1, 212), in none of which any regard had been had STAECH-TRAKSFOEMING FEEMENTS. 53 to the discovery of Dubrunfaut, and obtained results which, while agreeing only partially with those of the chemists named and quoted, entirely confirmed those of Dubrunfaut as regards maltose. Mode of 'preparing maltose or barley sugar, — 100 grms. of air-dried starch are mixed with 300 c.c. of water at 40°, and the mixture is well stirred to diffuse as completely as possible the starch granules through the liquid, and then poured with con- tinual stirring into 2 litres of boiling water. The paste is cooled to 40°, the extract, prepared with cold water, from 20 grms. of pale malt added to it, and the mixture kept at a tem- perature of from 40° to 45° during three hours, or until the iodine test shows that all starch has disappeared. It is then boiled for some time, cooled, filtered, and the filtrate evaporated at 80° to 300 c.c. The solid matter in solution, which is a mixture calculated from the specific gravity (taking 10 grms. in 100 c.c. to be represented by 1-0385) has a specific rotatory power [a]= + 170°, and is not changed either by boiling or evaporation. The syrup is boiled for a short time with two litres of alcohol, sp. gr. 0"820 ; on cooling the clear solution is decanted from the undissolved syrup, and put aside in a stop- pered flask. At the end of about a week the sides of the vessel are found covered with a crystalline crust of pure barley sugar or maltose. Musculus and Gruber have altered the foregoing procedure a little by adding ether to the alcoholic solution of the maltose at intervals, and removing several successive crystaUisations. In this way four litres of alcoholic solution receive successively one litre, two litres, two litres, two litres — altogether seven litres of ether. Each addition produces a precipitate or crystallisation on long standing, but only the precipitates after the third and fourth addition are pure maltose. Properties of maltose. — This sugar forms white crystals, which are very soluble in water, but less soluble in alcohol than grape-sugar. Dried at 100° in a current of dry air, it has a specific rotatory power of [a] = from +149 to +150°. (Du- brunfaut had given +150°6, or three times the rotatory power of dextrose, + 65°2 x 3). The rotatory power of a recently pre- pared solution is not stronger than that of the game solution, 54 ANNALS OF CHEMICAL MEDICINE. after the lapse of several hours. Maltose does not therefore exhibit the so-called bi-rotation of dextro-glucose, that is to say, the peculiarity of rotating the ray of polarised light about twice as much immediately after solution as after some hours of standing. Maltose expands in crystallising, and in this respect resembles cerebrose. By boiling with dilute sulphuric acid it is converted into dextrose. (Starch on being boiled with dilute sulphuric acid also yields at first maltose, and the formation of dextrose is only a secondary reaction between the acid and the maltose.) Maltose is not so easily altered by watery solutions of caustic alkalies as dextrose. Under the influence of yeast it is decomposed into alcohol and carbonic acid, like dextrose, but without transforming into that body (as cane-sugar does previously to fermenting). According to Dubrunfaut, when a mixture of dextrose and maltose is fermented, both kinds of sugar are decomposed simultaneously, not one before the other; according to O'Sullivan, however, the whole of the dextrose dis- appears before the maltose is touched. Maltose is less oxydisable than dextrose, so that while a given weight of dextrose will re- duce 1 00 parts of Fehling's fluid, the same weight of maltose will only reduce 65 to 67 parts. This reaction, although always the same apparently, does not admit of any stoichiometric in- terpretation. Two molecles of dextrose take up 5 molecles of oxygen from an alkaline copper solution, but two molecles of maltose take up 3*25 molecles of oxygen, a number which does not admit of representation by a simple equation. By elemen- tary analysis of maltose the following data have been ob- tained : — Theory O'Sullivan found Theory Hans Meyer found 12 C 42-10 41-97 42-02 12 C 40-00 39-93 22 H 6-43 6-48 6-50 24 H 6-67 6-72 11 51-47 — — 12 53-33 — 100-00 These lead to the formula of a sugar isomeric with cane- sugar or milk-sugar, Ci^Hj^O,,. The needle-shaped crystals of this body, before drying at 100, contain a molecle of water of STARCH-TEANSFOEMING FEEMENTS. 55 crystallisation, and are therefore C,2H220ii + H20. O'SuUivan analysed barley sugar dried at 100° in a current of air ; Hans Meyer analysed crystallised sugar, produced by Musculus and von Mering, which had stood during four days over phosphoric acid, been heated during 10 hours at 80° C, and then been kept for several days over sulphuric acid. This body heated for a longer time to 115°, lost 4*7 1 per cent. HjO, while the theore- tical loss should have been 5 per cent. Maltose does not reduce a boiling solution of neutral cupric acetate to which a very little acetic acid has been added. (Barfoed's reagent.) This reagent is also not affected by sugar of milk, but is reduced by glucose after short warming. {Zeitschr. Analyt. Ghem. 12, 27 ; Ghem. Gentr.hl. 1873, 357.) When maltose is dissolved in strong alcohol, and alcoholic solution of potash is added as long as a precipitate is produced, maltose-potash is obtained as a precipitate insoluble in alcohol. After drying in vacuo maltose-potash is white and chalky, and very light, and therefore differs greatly in physical appearance from the glucose-potash, which is a yellow varnish-like solid. Maltose in alkaline solution reduces the salts of bismuth, gold, silver, and mercury. Second 'product of the diastatic zymolysis of starch; dextrin. — ^The precipitate obtained by the addition of alcohol to the concentrated solution of starch decomposed by diastas, may be extracted by alcohol, in which it is insoluble and remains as a white waxy mass. It is purified from sugar by dissolving it in water, and reprecipitating it with- alcohol, and repeating this process ten to fifteen times ; in this state it has a reducing power equal to (in different specimens) from 8 to 12 per cent, of glucose, and a specific rotatory^^ower of [a] — + 204° to 205°. The same substance is obtained, whether starch is treated with diastas or sulphuric or oxalic acidv When this substance is dissolved in water, so as to give a scpjion of sp. gr. 1"090, and at 20° C. treated with yeast amounting to 2 per cent, of the solid matter in solution, it is changed so as to loselts reducing power over cupric solution almost entirely, only from 0*80 to 2*20 per cent, of its weight of glucose being represented thereby, and to increase its specific rotatory power from +204° to +212°, or even +214°. Froin these data O'Sullivan came to the conclusion 56 ANNALS OF CHEMICAI, MEDICINE. that the pure dextrin would not reduce cupric oxyde ; but in this opinion he is opposed by Musculus and von Mering, who say that the dextrin obtained from any amylum under the influence of any starch-transforming ferment always reduces cupric solution, and that this reducing power can be destroyed neither by boiling the dextrin ten times with alcohol, nor by subjecting it repeatedly to the influence of yeast. These authors further admit that the reducing power of the substance obtained by the side of maltose varies between considerable limits, owing to the fact of the body not being always the same, but a mixture of several different bodies closely resembling each other, as of dextrins, similar in composition, but dissimUai- in their power of reduction and rotation. The power of reduc- tion, according to them, is the greater, the farther towards complete transformation into final products the zymolysis of the starch-molecle has proceeded. According to O'Sullivan the zymolytic dextrin is a brittle white powder, without crystalline structure. It dissolves easily in water, and, if it has previously been dried at 100°, the solution is accompanied with a rise of temperature. In cold alcohol of sp. gr. 0-82 it is not perceptibly soluble, and alcohol which has been for three days in contact with the dry substance exerts no action on a ray of polarised light passed through a layer of it, 220 mm. in length. An aqueous solution contain- ing in 100 c.c. 10 grms. dry substance, has a sp. gr. = 1-03845. Its specific rotatory power may be taken to be [a] = +213°. It is not coloured by iodine. Dried over sulphuric acid, its weight becomes constant when it contains 9*5 to 10 per cent, of water. This it completely loses in a current of dry air at 100°. The formula CgHj^Oj + HjO corresponds to 10 per cent, of water. On elementary analysis it gives data corresponding closely with the theoretical 44*44 per cent. C, and 6"17 per cent. H, required by CgH^Oj. When a solution of this dextrin is treated with malt extract, its reducing power gradually increases, and becomes constant when the cuprous oxyde precipitated (weighed as cupric oxyde) is equal to 66 per cent, glucose, calculated on the dextrin employed. The specific rotatory power of the matter in solution is then [a] = +150°, having fallen from +213°. STAECH-TEANSFOEMTNG FEEMENTS. 57 A solution of this dextrin can be boiled with cupric solution during from 12 to 14 minutes, without giving any reduction, but if the boiling (in presence of an excess of copper solution) is continued, cuprous oxyde is gradually formed, showing that the dextrin is changed into a reducing body by prolonged boiling with alkali. The action is so slow that any maltose or glucose, which may be present by the side of the dextrin, will have been destroyed some time before the dextrin commences to transform into the reducing body. Proportions between maltose and dextrin obtained under different conditions of zymolysis. — Schwartzer (Journ. Pract. Chem. 1, 1870, 212) observed that the action of the ferment, and the nature of the products of its action upon starch, depended to a large extent upon the temperature at which the fermentation was conducted. At temperatures varying from the lowest up to 60° he found an amoimt of reducing action produced corresponding, when considered as due to glucose, to from 50 to 53 per cent, of the liquefied starch. When the reaction was forced at a temperature of from 65° to 70°, an amount of reducing power was produced corresponding to only about 27 per cent, glucose. At temperatures varying between 60° and 65° he foimd amounts of reducing power corresponding to from 27 to 53 per cent, glucose. O'Sullivan extended these observations, and made them much more accurate. He found that malt extract dissolves gelatinised starch at temperatures not below 10° and not above 63° almost completely, provided the gelatinisation was perfect. The solution which, if it was hot, must be cooled after from- 5 to 10 minutes of action, and filtered, invariably contains maltose and dextrin, in proportions agreeing closely with 67*85 percent, of the former, and 32 "15 per cent, of the latter, the cupric oxyde reducing power being = 44*], and the (specific) rotatory power [a j] = + 170°6. These numbers show that the reaction probably takes place according to the formula Cl8H3oOi5 + HgO = C,aH3aOn + CgHio03 starch Maltose Dextrin O'Sullivan further found that when starch is dissolved by malt extract at any temperature between 64° and 70°, and the 58 AKNAXS OF CHEMICAL MEDICINE. solution is immediately cooled and filtered, the product invari- ably contains maltose and dextrin in proportions agreeing closely with 34-54 per cent, of the former and 65-46 per cent, of the latter, the power of reducing cupric oxide being = 22-4 per cent, of glucose, and the (sp. ?) rot. power [a j] = + 191°8. These numbers make it probable that the reaction under these circumstances proceeds according to the formula 2(C^^H3^,) + H,0=C^^H^ + 4(C£I^) starch Maltose Dextrin In this case, as in the preceding reaction, if the malt extract be not in excess the relation of the maltose to the dextrin is not materially altered by three or four hours' digestion at the temperature of decomposition ; but if, on the contrary, the ex- tract be in excess, or strongly acid, the dextrin yields maltose, and the maltose is partially converted into glucose. If the solution be boiled previous to digestion, the dextrin is not con- verted into maltose to any extent. If the unboiled solution be digested for a sufficient time with excess of malt extract, the greater part of the dextrin may be made to yield maltose, and by varying the time, and the quantity of extract, any proportion of dextrin and maltose may be obtained. From these data it follows almost with necessity, either that dextrin has a much higher atomic weight than that assigned to it above, or that the maltose formed from it owes its origin to a syntbetical reaction. When starch is dissolved by malt extract at temperatures between from 68° to 73° and the point at which the activity of the transforming agent is destroyed, if the solution be cooled and filtered at the end of five to ten minutes, the product con- tains maltose and dextrin in proportions agreeing closely with 17-4 per cent, of the former and 82-6 per cent, of the latter, the (sp. ?) rot. power of the mixture being [a j]= +202°8 and the reducing power over cupric oxide = 11-3. These data are explicable by the following equation : — 4(C^sH3oO,5) + H,0 = C^I^ + 10(C££^) starch Maltose Dextrin A quantity of malt extract containing solid matter equal to STAECH-TEANSFOEMING FEEMENTS. 59 5 per cent, of the starch taken is in almost all cases sufficient to effect the transformation. O'Sullivan is of opinion that the various decompositions expressed by the foregoing formulae are due to some change brought about by heat in the cliaracter of the decomposing agent or agents. He expressly excludes, though by obscure language (' the various decompositions are not due to any difference in the size of the starch molecule '), the consideration of a molecle of starch containing more than 18 C, although the last reaction admits of the assurhption of a molecle with 72 C, and all assumed minor molecles are obtainable by division of the largest one by 2 or by 4. He terms the transformation of starch into maltose and dextrin a decomposition, says that this decomposition is molecular, and does not take place according to one equation, but to three; he ultimately speaks of the conversion of dextrin into maltose as of a slow and gradual act of hydration. Of this he gives no formula, but he probably assumes it to take place according to the following one : 2(C,H^,)+H,0 = C^£I^, Dextrin Maltose It is at once evident, that if the existence of this reaction be admitted, all maltose obtainable from starch may be assumed to be formed by the same reaction, as was practically maintained by most authors after Payen, and lastly again by Bondonneau {Compt. r&>id. 81, 1212). The consideration is not altered by their ignoring the existence of maltose, and speaking of the product as glucose. It is not difficult to explain that dextrin once formed, is less easily transformed into maltose, than gela- tinised starch ; inasmuch as most substances originating in a chemical reaction are more inclined to suffer or cause change in the nascent state, than after having assumed an independent free existence. We may agree with O'SuUivan in the opinion that Bon- donneau's dextrins were mixtures of the dextrin which colours iodine red, and the dextrin which does not colour iodine, with sugar under certain conditions, and torrefaction products ; the objects having been extracted from commercial dextrin obtained by the action of" heat on starch. But we must bear in mind 60 ANNALS OF CHEMICAL MEDICENE. that O'Sullivan himself obtained by the action of malt extract on dextrin prepared by malt extract, a secondary dextrin, the specific rotation of -which was only = + 150°, as against the + 213° of the dextrin from which it had been produced. Musculus and Gruber obtained, by the action of diastas upon starch, three different kinds of dextrins which seemed to rotate the ray of polarised light the less the longer the diastas had acted upon the starch, and the larger was the amount of diastas which acted upon it or upon the primary dextrin sepa- rated from the maltose by fermentation or by alcohol. They all had a reducing power of 12, when glucose has a hundred, and differed only in rotation. After diastas (second) Bed. P. Hot. P. Bed. P. Eot. P. N'--l . . 12 +210° 36-5 +159° N'--2 . . 12 +199° 20 +168° N-'-S . . 12 +190° 12 +190° They gave no colour with iodine. With diastas the N""' 1 and 2 had their rotation power diminished, but their reducing power increased ; N"^- 3 remained in both respects unchanged ; N""' 1 fermented strongly with yeast, N''- 2 slowly ; N'- 3 did not ferment at all. From these data these authors conclude that there are at least two, perhaps three dextrins, which do not react with iodine, and are therefore termed (after Briicke, 'Proceedings of the Vienna Academy of Sc' 65, part 2, p. 35 of the separate impression), achroodextrins, and distinguished from each other by prefixing the first three letters of the Greek alphabet, as a, /3, and 7 achroodextrin. The W' 1 is a-achroodextrin, the N''- 3 is ;8-achroodextrin ; the N"^' 2 they seem to admit to be a mixture. The 7-achroodextrin they obtain by allowing diastas to act upon starch in the presence of some alcohol during a whole year. This body had a rotating power of + 150° (the same as O'Sullivan's secondary dextrin obtained by the action of much malt extract upon primary dextrin) and a reducing power of 28. This dextrin does not ferment with yeast, but on being boiled with dilute sulphuric acid is transformed without residue into glucose. For this reason they propose that it should be termed end-dextrin. It can also be prepared by heating starch with. dilute sulphuric acid containing 2 per cent, oil of vitriol, SXABCH-TRANSFOEMING FERMENTS. 61 until alcohol does not produce any longer any precipitate in the fluid. After removal of the acid by barytic carbonate, and destruction of the sugar by fermentation, the 7-achroodextrin alone remains in the solution. The same dextrin is obtained by tlie simultaneous action of diastas and yeast upon boiled starch ; but in this case it is necessary that the amount of diastas be small, otherwise hardly any residue is obtained. The a-achroodextrin is contained in beer brewed from strongly roasted malt, the /3-achroodextrin in beer brewed from slightly roasted malt. From so-called soluble stored (compare Musculus, Compi. rend. 1869, p. 1267, and Ann. Ghim. 1874) these dextrins are distinguished by their solubility in cold water, in which soluble starch is insoluble ; the latter acquires its right to the adjective soluble only at 50° to 60° ; its solution assumes a wine-red colour with iodine ; in the dry state it is coloured blue by iodine ; with an excess of this reagent it gradually becomes violet, yellow, and brown. Soluble starch has a sp. rot. power of [a]= +218°, and a reducing power of 6. The erythrodextrvn of Briicke becomes red with iodine, whether dry or in solution ; it is soluble in cold water. Both ' soluble starch ' and ' erythrodextrin ' are easily changed by diastas. Neither "substance seems to have been obtained in a state of purity. The theory of Musculus and Grruber concerning the zymo- lysis of starch under the influence of starch-transforming ferments (including warm dilute sulphuric acid) is about the following. Starch has the formula n{G^^^^O^^, in which n is not less than 5 or 6. The hypothesis of % = 6 leads to the same number of atoms in the molecle of starch as O'Sullivan's last formula, namely to G^^nfi^a- "^^^ effect of diastas or dilute acid upon this molecle is to split off a molecle of maltose leaving a dextrin (a) of the formula CgoHjggOg,, the one molecle of water taken up remaining with the maltose. The hydration and splitting off of maltose is now repeated, when (;S) dextria, C4gHjo04j is left ; this by another loss of maltose leads to (7) dextrin Gsfi^fi^a- This now is surmised to transform into maltose (three molecles) directly, without any formation of dextrin corresponding to the terms with G^^ or G^y 62 ANNALS OF CHEMICAL MEDICINE. It will be seen that O'Sullivan's data and theories offer great advantages over this hypothesis of Musculus and G^ruber, which is very unsatisfactory when considered with relation to the quantities of products formed by different reactions and at different stages. These quantitative relations have been ob- served by O'SuUivan, and his results are therefore entitled to the greater consideration. But even so the question of the dextrin or dextrins formed by zymolytic agents is yet open, though that concerning the sugar or sugars formed by these agents is finally determined. ZyTtiolysia of starch by saliva. — The discovery by Leuchs, confirmed by Schwann (mentioned in the Introduction) that saliva had the power to transform starch into a kind of sugar, was generally formulated in physiological works to the effect that the sugar formed was dextroglucose. It is the merit of Seegen, and after him of 0. Nasse {Pfiiiger's Archiv, 14, 1877, 473), to have shown that the sugar produced from starch by saliva is not glucose, but a sugar whose reducing power is nearly doubled by boiling with sulphuric acid. Seegen termed it ferment sugar, Xasse ptyalose ; the latter also found that achroodextrin was formed at the same time, and that, as he thought, from impurity, it retained a certain reducing power. Musculus and v. Mering find that the ptyalose of Nasse is maltose, and that in fact the decomposition of starch under the influence of saliva (half a litre of mixed human saliva to a solution of 100 grms. of starch in 1200 c.c. of water digested during 6 hours at from 30° to 40°) is the same as that which it undergoes under the influence of diastas. About 70 per cent, of the starch employed are obtained as maltose ; 1 per cent, passes into glucose by fission of a little maltose ; the rest is obtained as dextrin, which always reduces copper solution ; this dextrin is considered by them to answer to the description given of that substance by Musculus and Ghruber in the paper above referred to. Zymolysis of starch by pancreatic ferment. — The pancreatic ferment is employed as obtained in solution by extracting fresh minced pancreas with cold water. It is added to the hydrated starch, the mixture is heated for some hours to 40°, and then allowed to stand for some hours longer. The filtered liquid STAECH-TEANSFOEMING FEEMENTS. 63 is evaporated to a syrup, and this is treated with a large volume of alcohol, and to the filtered alcoholic solution ether is added. In this way dextrin remains insoluble in alcohol ; the ether added at intervals produces several successive precipitates of maltose ; the ultimate mother-liquor contains a little dextrose corresponding to ahout 2*6 per cent, of the starch employed. Zymolysis of hepatic glycogen by saliva and diastas. — 100 grms. of glycogen from the liver may be digested with 250 c.c. of saliva and 1 grm. of ptyalin or with 5 grms. diastas during some hours. By the processes above described Musculus and von Mering obtained from the digested fluid maltose and a little dextrose. The dextrin obtained from glycogen was different from that prepared from starch by the same ferments, for it formed a white powder, unchangeable by air, whereas the dextrin from starch appeared as a hygrophiHc, easily dissolving mass. The dextrin from glycogen reduced cupric solution, and the power varied between 3 and 1 9, when the same weight of glucose reduces 100. The amount of total reducing power which is developed in solutions of starch and glycogen under the influence of diastas, saliva, and pancreatic ferment varies between wide limits : it is after some hours, on an average, about 50, but increases on standing for days and weeks to 65. Grlycogenis less energeti- cally transformed by diastas than by saliva. Grlycogen with saliva yields not rarely only a reducing power of from 34 to 41, while with pancreatin it yields a reducing power of from 45 to 48 per cent, of its weight of dextrose. (Compare Seegen, Gentralbl. Med. Wiaa. 1876, N. 48, also Nasse, loc. dt.) The liver after death contains, according to Nasse, a sugar, the reducing power of which cannot be increased by boiling with dilute sulphuric acid : consequently dextrose. This is confirmed by Seegen {Arohiv. Physiol. 19, 1879, 123). This author pressed about a kilo of liver substance, subjected the pressed out juice to dialysis, evaporated the dialysate, treated the residue with absolute alcohol, and added to the solution freshly prepared solution of caustic potash in alcohol. The sugar-potash was deposited as a delicate yellow varnish-like mass. This was washed with alcohol, dried over sulphuric acid, dissolved in a little water, and tested by the polariscope, the 64 ANNALS OF CHEMICAL MEDICINE. copper reduction, and the fermentation tests. It was dextrose- potash, as formerly described by Seegen {Sitzh. Acad. Wiss Wien, vol. 64). Musculus and von Mering had also found dextrose in the liver, though they add that maltose was also certainly present. The presence of dextrin in the liver they were unable to prove with certainty. The opinion formerly entertained by Seegen {Diabetes Mellitus, 1870) and Schtscherbakofif (5er. Deutsch. Chem. Ges. 1870) that there might be two different kinds of glycogen pro- duced, the one after amylaceous, the other after albuminous diet — an hypothesis already controverted by von Mering {Arch, Physiol. 14, 1877, 283), has been finally abandoned by Seegen. The transformation of starch and glycogen into glucose by whatever intermediate stages is accompanied with the loss of the colloid, and the assumption of the crystalloid state. The change is therefore away from the organoplastic direction, and towards the formation of products which are unfit for the formation of organs, but fit for the production of power by oxydation. LIFE AND PHILOSOPHY OF MAYER. 65 IV. THE LIFE AND PHILOSOPHY OF ROBERT JULIUS MATER, PHYSICIAN TO THE TOWN OF HEIL- BRONN.": Egbert Julius Mayer was born November 25, 1814, and died March 20, 1878. His father was a pharmaceutical chemist and owned the establishment known as ' Apotheke zur Eose ' at Heilbronn on the Neckar in Wiirtemberg. He was a man of high attainments and entirely devoted to his profession and the sciences collateral to it. He was rarely seen in company, but spent nearly all his free hours in the large rooms of his house which were filled with chemical and physical apparatus, botanical and mineralogical collections, books and pictures ; he was a short, stout man with a large head and large eyes. His wife, the mother of the object of this notice, was a busy house- wife of great goodness, but no other prominent qualities, except a faculty which she shared with her husband of being easily excited to great eruptions of anger by comparatively trivial circumstances capable of producing displeasure. This latter peculiarity was inherited by their three sons, more particularly by the eldest and youngest. The eldest brother, Fritz by name, was about eight years older than his youngest brother Eobert: he was talented, of high character, and full of knowledge ; he was even better in- ' We take from a description of his life by his friend G. Eiimelin entitled ' Erinnerungen an Eobert Mayer ' in the Augsburger Allgem. Zeitung of April 30, 1878, the data concerning the course of his life, his development, and his mode of inquiry, but regarding his philosophy we shall be guided by a necro- logue in the SohwdUsche Chronik of April 6, and by Mayer's collected works, second edition, published at Stuttgart in 1874 under the title : Die MechaniA dur Wa/rme. 66 ANNALS OF CHEMICAL MEDICINE. formed than the father, more animated and given to conversa- tion, and devoted to the cultivation of natural science ; he is supposed to have exercised much influence upon the youngest brother Eobert. Eobert therefore grew up in an atmosphere of . scientific views, occupations and conversations, and developed iu it his inborn talent with the eagerness springing frona, interest. As a boy he was familiar with the use of the air pump, of several varieties of electric apparatus, with elementary chemical experiments and processes of production; he could diagnose most plants according to the system of Linne, and knew the contents of all cm-rent books used in the pharmacy of his father ; from his brother Fritz he had acquired a knowledge of algebra and the use of logarithms, but his instruction in these branches of knowledge was not methodical, and no great assi- duity was given to it ; this explains much that remained im- perfect in the performances of his productive years. Robert was an indifferent pupil at school. His otherwise excellent memory could not be exercised upon all ordinary subjects of instruction, but it could only be-used for those in which he took an interest. To these latter the ancient lan- guages did not belong. Now as in the gymnasium success mainly depended upon proficiency in the classical languages, and as his proficiency in other sciences had no opportunities of showing itself, or if it did, was not observed, he was looked upon as a mediocrity. The boy was physically neither strong nor dexterous, but he had an extraordinary amount of perseve- rance and staying quality, particularly in walking; this he cultivated, and when a student he on one occasion made the way from Tiibingen to Heilbronn, a distance of 77 kilometres, in between 14 and 15 hours. Robert had a great liking for all kinds of games, and ac- quired gi'eat proficiency at chess, several varieties of games at caxds, billiards, and ninepins ; it is related that he carried all rules to excess, but played more for the enjoyment of the theory of the game than for the sake of winning. RiJmelin was intended for the church, and in 1828 left Heilbronn to enter the theological seminary at Schonthal, a former monastery some twenty English miles to the north-east of Heilbronn upon the river Jaxt. Robert severely felt the loss LIFE AND PHILOSOPHY OF MAYER. 67 of his friend, and prevailed upon his father to send him as a private hoarder to one of the professors of the seminary ; there he participated in the instruction given in the seminary, but as the foundation scholars had all been selected by competition from a number of applicants three times as great as the number which could be received, they were well instructed and Mayer's deficiencies in classics were the more apparent, and his place was always. amongst the lowest in his form. But it began now to be felt that he had to be measured with a different measure, as he knew many things of which the seminarists had no idea, and he began to be esteenied by his schoolfellows as well as by the masters ; for he was always open-hearted and truthful ; all he said bore the character of originality ; his mode of expression became often aphoristic ; humom- and poetic quotation gave to his conversation a rare, attraction, and when he grew lively most boys looked at him with expectation ; to some the fireworks of his changing ideas were not agreeable. The professor with whom Mayer boarded was Wilhelm Klaiber, who had married a sister of Wilhelm Hauff the novelist. To this lady he was much attached, and grateful for admonitions which from others he did not so easily tolerate. Some entertainments with the aid of the magic lantern to which he here treated his schoolfellows, at this period obtained him a sobriquet, which he retained, through life : he had caused ' spirits ' to appear on the wall, and accompanied their apparition with recitations and improvisations of his own ; from this his schoolfellows termed him ' der Greist ' which literally means ' the spirit,' but has the additional significance, no doubt intended to apply sarcastically to Mayer, of ' the genius ' ; this had the effect of singling him out for ever from the great number of Mayers, which in Grermany are as difficult to distinguish from each other as in England are the Smiths, Browns, and Eobinsons. In this seminary Mayer spent three years, and as he learnt only a moderate amount of classics, but was not at all advanced in the branches of science for which he had talent, it may be said that these three years were entirely lost to him. In 1832 he passed the examination called of maturity, corresponding to the matriculation examination of English imiversities, and then entered at the age of 17^ years the university of Tubingen to F 2 68 ANNALS OF CHEMICAL MEDICINE. study medicine. He did not attract the attention of his teachers, amongst whom were Autenrieth and the two brothers Gmelia (Ferdinand and Christian), by any prominent quality. He attended the purely medical lectures, no philosophical ones, none in history or philology, although Strauss and Vischer then were great attractions ; physics he attended, but at a time when the course was given by a private teacher (privat-docent) who only filled up the vacancy until a new professor should be ap- pointed. Norrenberg, well known by his researches on polarised light, was appointed, but Mayer did not attend his lectures. On the whole he paid attention only to medical studies, and heard the lectures on systematic anatomy several times over. The higher mathematics Mayer acquired only when he was already a practising physician at Heilbronn, from Professor Bauer, now at the polytechnic school at Stuttgardt. On the whole, although Mayer followed the medical lectures with interest and regularity, he did not study in his free hours and burnt no midnight oil except for cards and social parties. He made many friends whose attachment he preserved through life ; of these several like Wunderlich and Grriesinger passed away before him. In his later university years he was induced to become a member and office bearer of one of the peculiar societies of students termed ' corps ' ; he fought some duels, learned riding on horseback, and the' art of singing, though in this latter he had shown no aptitude. But all this did violence to his entire character, which was not made for either representing or direct^ ing. As the Frankfort Diet prepared to suppress the society, like so many others all over Grermany, the society or corps de- termined to dissolve spontaneously, and in 1836 carried out and announced this resolution. But notwithstanding this spontaneous action, and because some social cohesion continued to exist between the former associates, the university authorities instituted proceediags against them, and as the result of these the founders and office bearers of the society, amongst them Grriesinger and Mayer,, were compelled to leave the university for some time Cconsi- lium abeundi). During the investigation Mayer like others was in confinement (career), and it was here that the first LIFE AND PHILOSOPHY OF MAYEE. 69 traces of that disposition to mental aberration showed them- selves, which destroyed the happiness and usefulness of the later years of his life. He refused all food, and drank water only ; the physician who examinfed him. upon the report of the pedell found him without objective symptoms, but upon Mayer's special demand, bled him twice in the arm, to remove conges- tion, of which he complained ; on the sixth day of confinement the physician reported to the university judge that 'Mayer could not be considered as completely deranged, but that he was in a state which could easily pass into madness.' Upon this report his confinement was altered to arrest upon parole at his own residence. The 'advice to leave the rmiversity' in his case was given on the ground above stated, coupled however with the collateral reasons that he had been at a ball of the casino in a frock-coat instead of the obligatory dress- coat. This latter imputation, which was actually untrue, casts a vivid light upon the proceedings resulting from the participation in the society. However, the consilium in Mayer's case was of no practical consequence, as he had completed his course of studies of five years, and therefore left Tiibingen as a matter of course. During the summer and autumn of 1837 he visited the hospitals of Munich and Vienna, and attended the clinical lectures delivered in them. In January 1838 he was admitted on petition to present himself for the first medical examination at Tiibingen, and passed it ; in the summer of the same year he also passed the major examination at Stuttgardt, in the second class, first division. In chemistry he gained honours. His examination papers were described by the examiners as ' showing thorough knowledge and independent judgment.' He returned to Heilbronn and began the trials of a young doctor. But his impatient desire for activity and a sight of distant lands caused him to aspire to an appointment in the medical service of the Dutch Colonial office. With a view to this he studied the Dutch language. He also acquired some knowledge of French, and went to Paris. In the latter town he lived for some time together with Griesinger and Wunder- lich. Thence he went to Holland, passed the examination, and received the diploma as ' Officer of health.' Impatient as he 70 ANNALS OF CHEMICAL BIEDlCINE, was, he accepted the first post which offered itself, and sailed for Java as surgeon on board a merchant-ship. The voyage was accomplished without any incident worthy of note ; but during it was developed 'to the state of conscious- ness that train of ideas by which he has made his name known to philosophers, and ranged himself as one of the foremost amongst them. In him there lived the desire to know things through his own contemplation, and find their connection by meditation ; to make out the causes of phenomena, and in search of them to travel to the most distant regions of that which could be recognised by the understanding ; he had, says his friend and biographer Eiimelin, the fateful gift to live always entirely for and in one subject, and to press the whole store of his ideas into the service of that subject ; he was purely truthful without pre- sumption or prejudice. Mayer had provided himself well with medical works and surgical instruments, and by the side of them he carried meteorological, physical, and astronomical apparatus. To the study and use of these almost his entire time was devoted, for during the eight months of the actual sea- voyage, not a single person on board became ill, and his conversation was confined to moderate intercourse with a few officers, the low-Dutch of the common sailors being unintelligible to -him. He was therefore practically in solitude, and reduced for entertainment to the resources of his own spirit. He had never been a quick or continuous reader, particularly because what interested him always reduced him again to play with his own ideas. But he could for long periods fix his entire attention, his eyes, and his mind, upon any feature of nature, upon clouds, wind, or water ; he. loved to watch a thunderstorm through all its phases from beginning to end. This inclination he could now satisfy as never before upon the grandest subjects : upon the ocean with the phenomena of flood and ebb tide, the passate-winds (trades), the calms and storms ; upon the starry sky of both hemispheres, particularly the southern one with the new signs; upon the results of the powerful action of the tropical sun ; upon the organic life of the eastern islands, particularly Java; upon a new vegetable world ; upon a series of great volcanoes, partly extinct. LIFE AND PHILOSOPHY OF MAYEE. 71 partly in action. All this he saw, not with the eyes of the painter or poet, but with those of the meditative inquirer, who wants to get a glimpse of the kind and cohesion of the most elementary powers of nature. Mayer relates that relatively small incidents directed his thoughts into particular channels. Thus the pilot had one day told him, that the waves which had been much agitated by a storm were sensibly warmer than the waters of the same sea in a state of quietness. When at Java he had to bleed some of the sailors, and was surprised to find that the venous blood was of a ligtt colour, much resembling, as he believed, arterial blood. When he mentioned this observation to some of the local physicians with whom he came in contact, they confirmed his observation, and informed him that the phenomenon was observed upon natives as well as Europeans. The Dutch doctors, however, warned him against bleeding his patients, as the practice was, in tropical climates, condemned by experience. Mayer then began to consider how the phenomenon could be explained, and recognised at once that the explanation ofifered, namely, that the great redness of the venous blood was an effect of .the greater heat of the sun, was in fact no explanation at all. This led him to consider the relation between mechanical labour and organic heat, and the wants of the body as regards food. Men had been satisfied with the enunciation that friction produced heat, but Mayer was not satisfied with it. He searched for a law of causation of these phenomena, which he felt darkly were in connection with each other. And this search now occupied his mind entirely. The single steps wliich led him to his goal, the links of the chain of his thoughts, the history of his gestation so to say, has not been preserved by anything which he either wrote or said. But he stated repeatedly that it had been necessary for him to overwhelm various errors and half-truths, and that he had been obliged to make many artificial conces- sions to existing doctrines, before he could formulate his early conceptions as a presentable hypothesis. It is however certain that he brought the fundamental idea of his subsequent writings home with himself from this sea-voyage, and that the origin of this idea was in his genius and in a ceaseless penetrating energy of thought. Tljat motion could be transformed into heat, heat -72 ANNALS OJ? CHEMICAL MEDICINK. into motion, in proportions capable of being ascertained by experiment, was one of his earliest convictions ; then came the further prospect : not only matter is indestructible, but force also; the forces of nature do not perish during or in their action and effect, but they are only changed, and continue to exist in the changed form. In February of the year 1841 Mayer returned to his native town, mainly occupied with the wish to elaborate his idea into all its details, to provide it with the support to be derived from literatui-e and experiment, and to submit it to the learned. He knew that the principal point which he had to prove, before his idea could be called a discovery, was the finding and ascer- taining the so-called equivalents of the forces which he considered to be related to each other. For it would be of no use merely to assert in general that heat and motion were mutually convertible into each other, unless it could be stated and proved that a constant relation obtained, that to a certain measure of heat always corresponded a certain measure of motion. He now began a series of experiments, the results of deduction clearly ; these he has himself described in his works ; they led him at first to the result that by the fall of a body of a certain weight from a height of about 365 metres an amount of force was produced, which by the sudden arrest of the body, would be let loose in the shape of heat, and that the amount of heat BO prodjiced was capable of raising the temperature of a weight of water equal to that of the falling body, from 0° to 1°. He subsequently found this relation still incorrect, and the height of fall required to be greater.- But this detail was insignificant compared with the enunciation of the equivalents of heat and motion as an existing law. Open-minded and coUoquious as Mayer was, he set about to make converts for his new doctrine. He spoke to his colleagues of the medical profession, to the teachers of the higher schools, and in the summer of 1841 made a journey to Stuttgardt, Tiibingen, and Heidelberg, in order to communicate his ideas to philosophers of note, of to any who would listen to them, and hear their opinions. But these attempts were not encouraging. The matter being new, and Mayer's manner of speaking being not of the kind which quietly develops, but striking and aphoristic, LtPE AND PHILOSOPHY OF MAYEE. 73 and contrasting strangely with the prevalent diction of hand- books, he was looked upon with distrust ; the young man of 27 years of age, who though known to his friends as imbued with original ideas, was unknown in the world of learning, and wanted at once to change the entire aspect of the science of physics, was listened to with impatience as a hawker of paradoxes ; he got for answers shrugs of the shoulders and shakings of the head, or he was asked whether he had read certain chapters of certain books ; and when he had to reply in the negative, the conversation ended with the recommendation to do so, and then to again examine his ideas. In later years he excused his early interlocutors with the admission that he himself had then not been quite clear on all the bearings of his theory, and that he had not yet recog- nised the necessity of rejecting the old doctrines, which his theory superseded, entirely, but had been entangled in the belief that they could be reconciled by artificial hypotheses. One man however received him well, listened to him with an open mind, and encouraged and aided him ; this was Professor Jolly of Heidelberg ; and of him Mayer has in the note given to his biographer spoken in the highest terms of grateful recognition. Mayer never read a so-called philosophical work ; Eiimelin once gave him Hegel's Logic, and the volume of the Encyclo- paedia which contained the so-called ' Naturphilosophie,' but Mayer returned them after a few days, with the statement, that he had not comprehended anything of what he had read in them, and that he could not understand these works were he to read them for a hundred years. He had at that time some axiom^ like aphorisms, which he repeated frequently, uttering them to his friends on meeting and on leaving thera, sometimes shouting them after them, when they had just left his presence. Ex nihilo nihil fit ; nihil fit ad nihilum. Causa aequat effec- tum. He challenged all comers to state what could be said against these propositions. Eiimelin has preserved a note of an argument which he had with Mayer concerning these theses ; he had objected that indestructibility was not an attribute of the phenomena of organic and psychic life, and the thesis Nihil fit ad nihilum was not generally valid. In consequence of this Mayer excluded ad interim organic and psychic phenomena from his hypothesis, and confined it to the physical phenomena. 74 ANNALS OF CHEMICAL MEDICINE. those termed in physics ' forces.' He took however on one occa- sion an illustration from the transformation of force which takes place when four horses draw a stage-coach from one place to another, say from Heilbronn to Oehringen (he was walking with Eumelin on this road and the stage-coach passed by during the discussion), and his then interlocutor ingenuously states that from this illustration he learned for the first time. what were Mayer's real intimate thoughts and philosophic objects. Towards the end of 1841 Mayer wrote the small paper, modestly entitled 'Eemarks on the Powers of Inorganic Nature.' It may be supposed that the apposition of the adjective ' inorganic ' was the result of his conversation with Eumelin, above alluded to ; this adjective, he in later years judged to be not quite appropriate; it was certainly unnecessary, as the rest of the title was a sufiScient limitation of his purpose. He forwarded the manuscript to Poggendorfif for insertion in the Annalen der Physilc und Chemie, which certainly would have been the proper medium for the publication of the essay. But Poggendorff declined it as unsuitable, and returned the manuscript to the author. The latter now sent it to Liebig for insertion in the Annalen der Chemie und Pharmacie, and here it was accepted. How great was the satisfaction with which Mayer received this information is shown by a sentence in his short biographical notes, which also introduces the reader to a new development in his life. 'In the month of May (1842), in the same hour in which I introduced to my aged parents my affianced bride, who was to found the happiness of my domestic life, and who has since stood by my side as my faithful wife, I received a letter from Griessen in Liebig's handwriting, in which I was informed that my first essay on the mechanical theory of heat had been accepted for insertion in the Anjialen der Chemie und Pharmacie.'' The name of the bride was Wilhelmine Closz, the daughter of a respected and wealthy merchant of Winnenden. Another daughter of the same house being engaged to a brother of Eumelin, the biographer, from whom we quote, it was desired that both marriages should be solemnized simultaneously, which was accordingly done, Eumelin, who was in holy orders, officia- ting on the occasion (August 14, 1842). ' LIFE AND PBILOSOPHY OF MAYEE. 15 Mayer now entered into that period of his life which, though short, was the most productive of philosophical work of value to the world, and heartfelt unclouded happiness to himself. His scientific publication received neither notice nor recognition, but he had a rapid success in medical practice, which made his circumstances, already easy and almost independent of profes- sional exertions, afHuent and secure, at an age at which others have to sustain their hardest struggle. Young as he was, he re- ceived the appointment of county surgeon {Oheramtswundarzt), and soon was able to exchange it for that of physician to the town of Heilbronn and to the poor of the town {Stadt- und Stddtischer Armenarzt). In his character of physician he was cautious, circumspect, and observant : heroics were not in his list of remedies ; the experiments which he had in younger years sometimes made upon himself, he never attempted upon his patients. Medicine he said, is not a science, but an art, the ars medendi. Every case, he averred, had to be considered by itself, and had to be treated according to the rules of an eclectic empiricism, in which the experience ex nocentibus et juvantibus must be the guiding principle. He was fond of quoting a saying of his teacher Autenrieth, that every medical system stood in the same relation to nature as the tangent stood to the circle : that it touched only in one point, and (con- sidered as a progressing phenomenon, we suppose) would again go to a distance from it, unless it was modified and by a new breaking force turned again towards the periphery. (Vain endeavour of the writer to make a tropus logical in its fiurther progress.) The biographer continues by stating that Mayer was not entirely absorbed by medical practice, but that the practice went alongside of the continuation of his inquiries in natural science, and had an intimate connection with them. The es- sential result of his mental activity during these years was the final elaboration of the fundamental idea of the indestructibility of the forces, and of the relation between motion and heat, the final exclusion of the residues of former scientific theories which were supplanted by his own, and the following out of the results of the application of his theory to all approachable problems. The relation of motion to heat now became only an instance, au 76 ANNALS OF CHEMICAL MEDICINE. example of a general law of imiversal dominaDce ; ' there are not many forces which accidentally exist and act by the side of each other, but there is only one living force, -which circulates eter- nally through changes of form.' Physics became the teaching of the metamorphoses of the forces. Astronomy was no less fructified ; chemistry, physiology, and pathology participated in the impetus which the recognition of this law gave to all philo- sophical studies. Mayer now wrote the two works upon which reposes his claim to the scientific distinction which is now ac- corded to him by philosophers all over the world. The one published in 1845 bore the title,' Organic Motion in its Rela- tion to the Change of Matter {im, the Living Body). A Contri- bution to Natural Science. In this case the title did not cover the contents. The second work, published in 1848, was entitled ^ Contributions to the Dynamics of Heaven. Both essays are now considered as classical works in German natural science, not only on accoimt of the novelty of the subject and the amount of new information which they offered at the time, but also on account of the imquestionably masterly mode, and the lucid and terse style in which the subject was presented. These works are now translated into many languages ; but at first Mayer could hardly find a publisher, and of the first work he had himself to pay the expenses of printing and publishing. The year 1848 disturbed this period of happiness and pro- ductiveness. Participating for a short time in the enthusiasm of the so-called ' Days of March,' Mayer soon placed himself decidedly on the side of authority, and became a conservative in word and action. His brother Fritz, on the contrary, became a leader of the most advanced radicals ; and as both were rigid in their convictions and carried them into effect, they were soon opposed to each other as extreme types of opposed factions. In 1 849 Fritz Mayer became a leader of insurgents who went in aid of the Baden insurgents. The wife of Fritz requested Eobert to exercise his influence to cause her husband to return, and Eobert travelled after his brother. But he was recognised, arrested, and ran great peril of being shot as a spy or enemy of ' ' Die organisohe- Bewegung in ihrem Zusammenhang mit dem Stoffi- weohsel. EinBeitrag zur Natnrkunde.' ? ' BeitrUge znr Dynamik des Himmels.' LIFE AND PHILOSOPHY OF MAYEB. 77 the radical cause. The leader Sigel however set him at liberty at once, and he returned to Heilbronn. He had not shown either fear or feebleness, but the commotion of his mind had been extreme, particularly on finding that his second yoimger brother had also become an insurgent leader. About the same time, namely, in spring 1849, he was deeply afflicted by the death of two of his children. Mayer had lived in the hope that his scientific publications would find, if not assent, yet some attention and recognition amongst physicists. But seven years had elapsed since the publication of his first paper, four years since that of his first work, and a year and a half since that of his third, without his having received a single friendly word of encouragement from anyone. Attacks however and derogating judgments there had been, but the bulk of the learned in physics took no notice whatever of his philosophy. Some disputed the priority of his discovery, others its value. The English physicist Joule had, quite independently of Mayer, and about a year later, found the equivalents of heat and motion, by a method of his own, and determined the distance through which a body must fall in order to produce the unit of heat, more correctly. He had however treated the question as a special problem in physics, and not drawn the general conclusions as to the indestructibility of the forces, and the other important results attached thereto. This led to a dispute regarding the priority of the discovery, which was subsequently decided in favour of Mayer, without prejudice to the independence of Joule. Mayer put forth his claim to priority in a letter to the French Academy of Sciences, and the Comptes rendus, 1845, reported on the relative claims of Mayer and Joule very impartially. Four years after these discussions, and when Mayer's mind had suffered much from the events which we have described above, he was induced by the desire to secure priority, to pub- lish in the extra sheet of the Allgemeine Zeitung of May 14, 1849, a short notice, entitled ' Important Physical Discovery,' with his name attached, in which he announced that he had succeeded in finding a simple means to show experimentally thfe transformation of motion into heat, described the apparatus he had constructed to that end, and claimed the priority of the 78 ANNALS OF CHEMICAL MEDICINE. discovery of the principle of the transformation against an article in the Journal des Debats which had disputed it. A week later there appeared in the extra sheet of the same Journal of May 21, an article by Dr. Otto Seyffer,then a privat- docent of physics at Tiibingen, in which Mayer's publications were spoken of in an almost contemptuous manner. Among other things the writer said, that for the professional man no discussion of Mayer's work was requisite, but laymen might find an explanation ' according to the position of science ' desirable. He continued by denoimcing Mayer's article in the Annalen der Ghemie as a mass of imtenable views concerning the forces of nature. The confusion which reigned in this article between the ideas of force, cause, effect, etc., and the deductions there- from had been sufficiently exposed in scientific joiunals. Mayer's theory, the writer continued, was a completely unscien- tific paradox and in contradiction to all clear views on the action of nature. To the apparatus described by Mayer was denied the character of novelty, and it was added that it did not prove what it was intended to prove. In this tone the entire article was conceived ; it sounded more like the correction of a fool by an indignant master, than the efilux of fair controversy. Mayer, who was not in the least aggressive, but got into great excitement when any attack was made upon him, felt quite heartbroken by this as he termed it ' public insult in so respected a newspaper.' To a reply, which he sent to the editor, - admission was refused, and all further, steps to obtain redress remained without result. Finding himself thus treated with rancour and contempt, without a word of comfort or approbation from any of those to whom his naturally afifable mind looked for sympathy, unable by the habit and nature of his mind to liberate himself even for a time from oppressive thoughts by diversions such as constitute so common a resource of most mortals, he became a prey to wounded pride and unsatisfied ambition. All arguments and entreaties of his family and friends were in vain, the excitement rose to a real frenzy, an inflammation of the brain ; this having already abated, he was, as he wrote himself, in the early morning of May 28, 1830, the heat of the season being unusually great, and he having partly in consequence of the heat spent a LIFE AND PHILOSOPHY OF MAYER. 79 sleepless night, seized by a new access of delirium, during wticli he jumped out of the window on the pavement nine metres below the window. He alighted on his legs,' which were greatly injured, though not broken. During the days following this misfortune he battled with death, but gradually improved, and after a long and painful sickness he could resume his ordinary avocations and his practice. The right leg remained weak during the rest of his life ; he had to drag it after him, and to walk with theaid of a stick. After this outburst he became more quiet, and, comforting himself with religious sentiments, based on the hypothesis of earthly punishment for moral faults, returned to his labours, and wrote Bema/rhs on the Mechanical Equivalent of Heat,^ 1850. This added nothing essential to his former publications, and was claimed in fact to be only an appendix and supplement to them. But it is important as showing that his power of writing was unim- paired; nobody could discover in it evidence of a deranged mind. However Mayer had now entered upon the dark period of his life ; he lived yet 28 years after the first attack of mania, and these years, says Eiimelin, must be considered as princi- pally occupied by repeated outbursts of mental derangement. These had the character of mania, without delusions or fixed ideas, and made it necessary, in view of what had occurred, that he should be removed to an asylum ; when he was somewhat more quiet he went to the asylum of his own free will. It appears that he was unsuitably treated at the lunatic asylum at Winnenden, and during the remainder of his life expressed him- self as greatly offended by what he had there experienced. In later years he visited the asylum at Kennenburg, where he was treated as a volunteer and guest, and not subjected to any restraint whatever. A few weeks of residence in this place had the effect of making him quiet for longer periods, during which he could return to his family. In such quiet periods he wrote yet several essays, such as the lectm-e which he gave in 1869 at the Scientific Congress at Innsbruck, ' On necessary Consequences and Inconsequences of Heat-mechanics.' ' On the Significance of Unchangeable ' ' BemerkiiDgen fiber das mechanische Aequivalent der Warme.'' 80 ANJJALS OF CHEMICAL MEDICINE. Magnitudes ; ' ' On Nutrition ;' «0n Fever; ' ' On Earthquakes;' ' On the Empty Space of Toricelli ; ' ' On the full Exchange of Forces.' (The Grerman word for this latter idea is Ausloaung, and means literally the redeeming of a pledge ; the expression is now very frequently used, particularly in experimental physio- logy, and not always appropriately). He invented a dynamo- meter, for which the Board of Trade of Wiirtemberg caused a golden medal to be given to him. Thus we see that his powerful mind was yet active and clear in the intervals from the greatest of misfortunes which can hefal a human being. Having always been opposed to materialistic views he now turned decidedly towards a positive religious belief ; it formed itself into, what Eiiraelin terms, a subjective theology, meaning a theology of Mayer's own invention ; this at one time took the shape of a proposition for the fusion of the Eoman and Pro- testant Churches. His ideas became freaks {SchruUen) which his friends discussed with leniency, and which did no harm. Politically he always had been an adherent of the principle of authority, of what may be termed extreme conservatism. This in 1866 made him sympathise with Austria, but in 1870 the victories conquered him too, and after the battle of Worth, he came to his friend Bishop Lang, and said to him the words of Job, cap. 42, 3 : 'I confess that I have spoken unwisely.' After that he was a good imperial German. In these later years he also experienced much pleasure and satisfaction by seeing his work recognised ; by feeling the glory which was to surround the recollection of his name ; by finding himself honoured as the first philosopher of his time ; by find- ing it said and admitted that his discovery had efifected an im- mense progress in the natural sciences, and had opened new iields of thought and action. Schonbein was the first to recognise the importance of his writings, to visit him and befriend him ; and the first public manifestation of recognition which he received was the diploma of corresponding member of the Natural Science Association at Basle. (185 8). After this, attention was directed to him by Liebig, in an oration delivered at Munich on a festive occasion, and then a wide circle of savants began to take notice of his researches. The university of Tiibingen, his alma mater, where LIFE AND PHILOSOPHY OF MAYEE. 81 he had taken his degree of doctor of medicine, also nominated him honoris causa doctor of philosophy and natural sciences. Diplomas arrived from Munich, Vienna, Turin, Halle, Frankfort, and from other learned societies too numerous to mention. The French Academy appointed him a corresponding member, and awarded him the prize Poncelet. The Eoyal Society gave him the Copley medal. In England he was mainly introduced to the notice of scientific circles by Tyndall, in France by "Verdet,'in Italy by Count St. Eobert. Tyndall established closer friendly relations with Mayer, and it was for Tyndall and at his request that Mayer wrote a short autobiography, from the draft of which Eiimelin has quoted and we have repeated above some passages and data. In 1 867 Mayer received the order of the Crown, and with it the title of personal nobility. These late successes helped to give to the evening of his life a happier aspect. But on the whole, says Eiimelin, Mayer must be added to the number of those men who pay for the benefits which they render to mankind with the happiness of their lives. No doubt the principal reason for his misfortune was an unfavourable disposition of the brain and mind, a very great excitability, and long endurance in the excited state ; inability to divert his thoughts from an indifferent object, from an adverse impression, or from an injury received ; and a rigid inflexibility of his will. But Eiimelin points out that it was not to the credit of Grerman science — which was possessed of an immense apparatus for the attainment of its objects, numerous general and special journals, in which the most insigaificant performances yet find room, and praise by accident or agree- ment — that a scientific discovery of the first order, one opening a new era in philosophy, should be liable to be kept out of sight by premeditated silence, or to be treated with contempt. This is mainly caused by the cohesion of cliques, and their trade protective principles ; and in this enlightened age there still happens what is commonly imputed to the dark ages, that the innovations of genius are referred to the gratitude of later generatioas. Mayer's features in younger, happier years had been florid, serene, and friendly ; in later years they bore the expression of grave or sullen seriousness. In stature he was of medium height, G 82 ANNALS OF CHEMICAL MEDICINE. and well made, but his carriage was negligent and leaning forward ; when walking he stared before himself on the ground, betraying immediately his meditating preoccupation. His senses were refined and attentive, but he had no disposition for art. In later years the attacks of excitement became less frequent, and were very short. His children developed and gave him satisfaction. But his health began to fail, and in January, 1878, he was attacked with a serious affection of the lungs. To this he succumbed on the evening of March 20, when a quiet death, as his friend says, ' brought to this storm-tossed heart the desired rest.' He was buried on the birthday festival of the German Em- peror, a great concourse of people following him to the grave 5 the town of Heilbronn was in mourning, and the flags, so plenti- fully exhibited in honour of the day, were by request of the authorities removed during the hour of the funeral ; his friend Gr. Eiimelin spoke a funeral oration as delegate of the university of Tiibingen, and could truly describe him as one of the most honoured masters of Grerman science. Mayer's mode of working was in the main deductive, and only to a small extent experimental. In this respect he was Aristotelic almost in the extreme. This limitation in one sense protected him no doubt from a fault, which has been committed by, or perhaps not rarely been imposed upon, experimental inquirers, namely that of being drawn into laborious and costly details by the want of, or desire for, more and better scientific data. He had a large body of generally admitted scientific data at his disposal ; they were no doubt a disconnected, unorganised mass, but they were truths, and for these he found the connections by thought. When he had well observed a phenomenon he considered it as a riddle to be solved, and his energetic and passionate, abiding nature left him no rest until he had ' scientifically penetrated the phenomenon, and found the principle, of which the process was the simple effect.' He started with a deep conviction, one without alternative, a kind of instinct, that there was complete harmony between the laws of thought and the objective world, and to prove this harmony by the results of experience he conceived and declared to be the LIFE AND PHILOSOPHY OF MAYER. 83 most interesting and the most comprehensive problem which can be imagined. That motion was the result of power, that this power could be shifted without transformation from body to body, was clear ; it followed that motion was a form of power. But motion disappears under varying conditions, and apparently without any effect ; but in many cases on closer observation an effect of disappearing motion is perceived: that effect is heat ; on further observation it is perceived that heat is the only effect of dis- appearing motion, that there is no other effect but heat to be made out. Upon this datum of experience Mayer formulated the rejection of the teaching of the branch of science called physics, which involved the assumption that there could be a cause without an effect, and an effect without a cause, and sub- stituted the hypothesis that heat arises from motion, that heat is transformed motion, that as motion is transferable power, heat is power, heat is force, and as originating from motion is transformed force. But in order to make of this hypothesis a scientific truth, there was wanted a numerical expression for a quantitative estimate (quantation) of the amount of heat produced by the transformation of a given amount of motion. Of motion the best known example was that which results from gravity, from the falling of bodies, previously lifted, towards the point of attraction. Mayer therefore set about to ascertain what amount of heat was produced by the fall of a body of given weight through a given distance, or taking the amount of heat as the unit to which the other data of the experience were to be formulated, he expressed the problem thus : To find the distance through which a body must fall, in order to be able to produce by the transformation of its falling power an amount of heat which shall be capable of raising the temperature of a weight of water equal to the weight of the falling body from zero to one degree of the Centigrade thermometer, Mayer approached the solution of this problem by estima- ting the distance through which the body must fall as 365 metres. It was subsequently found and recognised by Mayer himself that this number is too smallj and that 425 metres, as ascertained by the experiments of Joule, and confirmed by those of Eegnault concerning the heat and relations of pressure of Q 2 84 ANNALS OF CHEMICAL MEDICINE. gases, which have to he taken into account in the experience, is the more correct number. But this is a mere detail, an error which detracts nothing from the achievement of his relating a mechanical act, fall, to what was believed an independent potential effect or even a matter, to heat namely, and showed that they stand in proportions to each other which can be numerically expressed ; that he established the existence of the law of the mechanical equivalent of heat. This result was not obtained with the aid of the higher form of mathematical operations termed analysis (a knowledge of this branch of science Mayer acquired only in later years), but with the . simplest means of mind and matter. The one aid was the sharp discrimination of his understanding. Since the time of Newton it had been taught in philosophy that gravity was the cause which kept the universe together. Mayer recognised that in the word ' gravity,' as well as in the word ' force,' there was implied an inadmissible confusion of cause with effect. ' If we call gravity a power,' said Mayer, ' then we represent to ourselves a cause, which produces effects, without becoming itself diminished ; we therefore conceive incorrect notions about the cohesion of the causes of things. Gravity is not a force,' he continued, 'but a property. Forces however are causes. Causes however are objects, which as regards quantity are indestructible, as regards quality are changeable. As soon as the idea of force is defined in this manner, it follows as a matter of course that no force can end in nothing ; on the contrary the cause must be equal to the effect ; there must be between the sum of the effects and the quantity of the cause a mathe- matical equation ; and as every equation can be reversed, it follows that under suitable circumstances the cause can be reproduced from the effects.' Such were the principles upon which Mayer based the argument of his second publication. Besides this process of thinking, Mayer employed observation and experiment to support his hypothesis. He observed, thermometer in hand, the rise.in temperature which takes place in the rotatory machines which tear the rags in paper-mills, and which in Germany, from their origin, are called ' Dutchmen.' In his consulting-room he shook a bottle containing a litre of water until its temperature had risen from 12° to 13°, excluding LIFE AND PHILOSOPHY OF MAYEE. 85 the influence of the warmth of his hands by thick felt-gloves. This experiment made it to his mind a broad practical truth, that work could be transformed into heat. It has been said that the logical distinction between power and property, as re- gards the definition of gravity, was derived from Kant's critical inquiries. But the writer in the Schwabische Kronik asserts that there was no indication in Mayer's writings of his ever having studied this author. He had no good opinion of what was then termed philosophy, particularly ' Naturphilosophie ' in Germany, and avowed that the endeavom: to penetrate into the mystery of the order of the world by means of hypotheses was a parallel to the endeavours of the adepts. From 1842 to 1845 Mayer was incessantly engaged in the study of organic chemistry, particularly of the work of Liebig in which this science was for the first time systematically con- sidered as to its applicability to physiology and pathology. The finiit of this study was the Essay ' On Organic Motion in its connection with the Chemical Change of Matter in the Body.' It is considered that this essay bears the stamp of the maturity of his genius ; it is remarkable by the consciousness and sim- plicity of its enunciation. It contains such imperishable sayings as the following: 'There is but one force' 'In everlasting change it circulates in living and inanimate nature ; in neither is there a process without a change of the form of force.' ' The science of physics has to investigate the metamorphoses of force as that of chemistry has to consider the changes of matter.' ' The creation or annihilation of a force can neither be effected nor conceived by man.' 'From nothing, nothing can come.' ' Nothing can come to nothing.' ' Chemistry teaches the fixity of the nature of the ultimate forms of matter ; physics teach the fixity of the quantity of force, and the changeability of its forms.' ' Gravity, motion, heat, magnetism, electricity, chemical difference, are all only different forms in which appears one and the same force which governs in the universe ; for every one of them can by proper arrangements be transformed into every other.' Elated by the view of nature which he has thus obtained he exclaims : ' We feel quite well that we are about to do battle with the most firmly rooted hypotheses which are canonised by the greatest authorities ; that we desire to remove from natural 86 ANNALS OF CHEMICAL MEDICINE. science in the shape of the so-called imponderables the last remnants of Grecian gods : but we know also that nature in its simple truth is greater and more glorious than any image made by man's hands, than any illusion of the created spirit.' This sentence is no doubt somewhat obscured by inappropriate adjectives, and therefore has been considered as tainted with mysticism. The writer in the Kronik supposes that under the category of the last remnants of the gods of Greece, Mayer had meant to include in the first pla<;e the so-called vital force. Against this no doubt he declared ' war ' most directly. ' We must enter our protest,' said he, ' against the putting up of a par- ticular vital force.' ' Bring into the juices of the body of the strongest and healthiest man a grain of putrid matter, and a decomposition wiU soon ensue to which neither nature nor art can put a Kmit, and which soon becomes fatal. Where in such a case is the vital force ? where the power to make resistance to external causes of disturbances? hie Rhodus, hie salta.' Vital power with him resides in the sun. ' The sim is the con- stantly wound-up spring, which keeps the wheelwork of terres- trial action going. It is the Ught of the sun which, changed into heat, causes the motions in our atmosphere, lifts the waters up to the clouds, produces the streaming of rivers. Light, the most mobile of all forces, caught by the earth in the swiftness of its motion, is by the plants transformed into rigid shapes : for the plants on the earth produce constantly a quantity of chemical difference, they form a store in which the volatile rays of the sun are fixed and deposited so as to be fit for the subsequent use of man and aaimal. The plants take in a power, light, and produce a power, chemical difference. During the process of life there takes place only a transformation of matter as well as of force, but there is never any creation either of the one or of the other.' The truths contained in these sentences Mayer now proves more in detail. The sun-power stored in the plants is consumed by men and animals, passes into their blood, and is then changed by oxidation into heat ; the heat thus produced in the body, serves in part to maintain the body at the tempera- ture necessary for its existence, while another part is trans- formed into work. He then shows that the equivalence of heat to work obtains in the organic world as well as in the inorganic, LIFE AND PHILOSOPHY OF MAYER. 87 in which latter it has been first discerned. In the organic world also, the unit of heat is equal to the mechanical effect which a mass of one kilo produces when falling through a height of 425 metres. Starting from the results of Gay-Lussac con- cerning the expansion of a given quantity of air by heat, he arrives at certain conclusions concerning organic life. If air is warmed while its volume is constant, less heat is required than when it is heated to the same temperatiure under a constant pressure. In the first case the air is enclosed in a rigid sphere; in the second case it is enclosed in a vessel, in which on expan- sion it has to lift a weight ; the excess of heat required in the second case as compared with the first (the degree of heat to which the air is raised being equal in both cases), is in the second case used to perform work, while in the first case no work is performed. These data he applied to the consideration of the results of the combustion of coal in a steam engine, and found that the work produced is only from 5 to 6 per cent, of the amount which the coal would be capable of producing if no effect was lost in the shape of heat : and further that the work produced in a cannon by the gunpowder averaged about 9 per cent, of the theoretical amount. The temperature (amount of heat units) which 95 parts of coal will produce when burned under an open boiler filled with a certain quantity of water, can be produced in a steam boiler doing work only by the burning of 100 parts of coal. The heat produced in a living organism is governed by the same law. A part of the heat, which is supplied by the combustion of the blood, is used up by the work which the organism does ; from the amount of the work done by the organism, the amount of heat which is equivalent to it can be calculated according to the formula given. The working muscle requires more heat than the resting muscle ; the parts of the body which do the least work in a mechanical operation secrete the most sweat; the forehead overflows with sweat, while the arms consume the heat produced by an excited cir- culation in the shape of work.. For the production of a given mechanical efi'ect (in or by the human body) a certain amount of heat has to be expended, which can be estimated theoretically beforehand, and this estimate is found correct by experience. Man and animals are subjected to the same laws as the steam- 88 ANNALS OF CHKMICAL MEDICINE. engine, but man is the more perfect machine of the three, in- asmuch as under the most favourable conditions, he is able to transform 20 per cent, of heat produced by the neutralisation of chemical difference into work. Mayer uses some illustrations which, if they are correct, are very interesting ; such as that a smith, who could hammer the head of a nail until it became red-hot, effected this by transferring the heat of his arms to the head of the nail. Other illustrations given by Mayer we fear are difficult to prove as facts, though they probably assisted his argumentation in a measure. The muscle is the tool by means of which the metamor- phosis of heat into work is effected ; but this metamorphosis is not effected at the expense of the muscle. This proposition Mayer illustrates by considering quantitatively the muscle of the heart and the work which it performs. And in the course of the considerations following upon this, he attempts an appreciation of the phenomenon, already mentioned as having been observed by him at Batavia, namely that the venous blood of the sailors whom he bled there, had such a remarkably light red colour. He says, from the established laws (of the meta- morphosis of force) it follows with necessity, that the difference between the heat of the organism and the heat of the sur- rounding medium stands in a quantitative relation to the difference in the colours of the arterial and venous blood re- spectively. The greater the difference of temperature, or the production of force, the greater must be the difference in the colours ; the smaller the difference in the temperature, the smaller must also be that in the colours. This difference in the colours is an expression for the amount of the consumption of oxygen, or for the strength of the process of combustion in the body. In 1 851 Mayer published a paper ' On the Power of the Heart ' in Vierordt's Archiv.; and in 1862 a paper 'On Fever' in WuTvderlicKa Archiv. ; the latter he termed an iatrophysical essay. It cannot be said that these papers have had any particular effect upon the consideration of the relative questions, but they aided in producing the knowledge, that organic life, as far as it is open to physical consideration, is amenable to the law of the mechanical equivalent of heat. He said that ' the LIFE AND PHILOSOPHY OF MAYEE. 89 existence of an unchangeable quantitative relation between heat and work was a postulate of the physiological theory of com- bustion.' It cannot be doubted that he proved his proposition. The Dynamics of Heaven date from the year 1848. In this essay he applied his principles to the motions of celestial bodies, and came to results which constituted new prospects, new questions and answers, to astronomers ; the beating of the pulse of the earth, ebb and flood-tide, shooting stars, the heat of the sun, were here subjected to investigation, and their explanation was more closely approached than had been done before. Many of Mayer's views have become obsolete by the rapid progress of science during the last twenty years, e.g. by spectrum analysis, of which he then knew nothing ; other of his views, such as that the tides must delay the velocity of rotation of the earth and make the days longer has remained an admitted fact to this day. This result was not obtained by calculation but by de- duction from his principle. Mayer opposed the hypothesis of Herschel corceming the existence of a photosphere of the sun, a hypothesis which at the time enjoyed almost universal acceptance. He contemplated the number of rays which the sun constantly sends out, and put the question by what means the sun could be able to replace this enormous loss of power. He answered it by his principle, namely that some kind of motion must be converted into heat. ' In the entire space of the solar system there rotate an incalculable number of smaller bodies ; the comets, of which there are in space, according to Kepler's estimate, a greater number than of fishes in the sea ; the shooting stars, fireballs or meteors, the sidereal dust in the zodiacal light ; then there are all the planets ; all these move in a resisting medium, the ether, and consequently their circuits become constantly more contracted, until they at last necessarily unite with the sun. Here their motion comes to an end, and, according to the law of the mechanical equivalent of heat, their motion must be changed into heat. The fall of a mass of asteroids into the sun produces an amoimt of heat which is from 4,000 to 8,000 times greater than the heat which an equal mass of coal would be able to produce by combustion. From this circumstance the heat of the sun is greater than any heat which can be imagined on earth, and the diathermic properties of the 90 ANNALS OF CHEMICAL MEDICINE. sunbeam are greater than those of any heat beam produced on the earth. The objection which has been made to this theory, namely that if it were correct the volume of the sun must be constantly on the increase, is as to the fact correct ; but the accession is so small that 28,500 years would have to pass, before the diameter of the sun increases by as much as a second of an arc. The heat of the sun is therefore maintained by the fall of inter-planetary matter upon the sun ; all bodies which circulate round the sun have their cradle in the periphery, their grave in the centre^ Even the solar spots, and the faculae, or protuberances, receive an explanation by this hypothesis ; the fiery ocean of the sun is excited down to low depths- by the meteors which plunge into it, and its liquid and gases are projected upwards in the shape of glowing mountains.' This theory is nowadays not admitted as exclusively true, but the probability of the phenomenon cannot be denied ; it must be admitted that heat of all degrees, to white heat, may be pro- duced by the means referred to ; Mayer's explanation is on the other hand strictly true of the fiery nature of meteors, shooting stars, and similar bodies. Their motion is in the resisting medium of the air transformed into heat. The phenomena of the tides Mayer correctly estimated as regards one of their principal mechanical efiects. He termed them an arrangement for breaking and retarding the velocity of the rotation of the earth round its axis. The earth rotates from west to east, the tides from east to west. Now if the proportions of land to water are estimated according to certain data, the tidal effect would be to prolong the day by one- sixteenth part of a second in 2,500 years. La Place has how- ever proved that the length of the day has during the last 2,500 years not changed even to the extent of one five-hundredth part of a second. This does not prove, according to Mayer, that the retardation of the rotation of the earth does not take place, but only that there must be an action opposed to this retardation, which in its turn again accelerates the rotation, and this accelerating action he assumes, with other philosophers, to be caused by the slow constant contraction of the earth in conse- quence of its gradual cooling. These considerations led him to LIFE AND PHILOSOPHY OF MAYEB. 91 the hypothesis that in the existence of the earth there must have been thi-ee distinct great periods ; the first period charac- terised by a decrease of the length of the day in consequence of cooling and contraction of the earth; the second period in which this accelerating action of the contraction by cooling is counterbalanced by the retarding action of the tides ; a period therefore in which the length of the day remains constant ; and lastly the third period, in which the influence of the tides prevails over that of contraction and cooling, and in which therefore the length of the day increases. In 1870 Mayer gave a lecture on earthquakes, and in this he adverted to his theory of the effect of the tides, and stated, that with the aid of some observations of Adams of Cambridge he had come to believe, that the earth was already in the third period, in which after a time of equilibrium its age had begun to manifest itself. The Essay which Mayer published in December, 1850, 'Re- marks on the Mechanical Equivalent of Heat,' contains no new de- velopments, but is a more complete and in some respects more correct account of his previous discoveries. He did not record any progress in this publication, but on the contrary began to be- come stationary while others progressed beyond him. For some had already then begun to look upon heat itself as a form or mode of motion. Against this idea he spoke decidedly, ' for,' said he, ' in order to become heat, motion, be it a simple one, or a vibrating one like light, or radiating heat, must cease to be motion.' His quasi fear to come to this result, is explained by some as arising from his want of acquaintance with the higher branches of mathematics. We may however be quite sure that the sense of solitude in which he found himself when apparently nobody took any heed of his work and Ms endeavours and ideas, and the depression which overpowered his intellect for long periods, had more to do with the arrest of his farther develop- ment than the want of higher mathematical knowledge. It is perhaps good advice, that those who desire to study the science of physics as it is in our days, cannot do better than begin their studies with Mayer's works. They are written with a deep enthusiasm which engages one's sympathy, and with a poetical vigour which carries the heart as well as the intellect 92 ANNALS OP CHEMICAL MEDICINE. with it. He carried his theory, says a writer, which he had formed from the heliocentric standpoiat, to the widest spheres of application. He laid, through the secrets of the processes of the life of organised beings as well as through the wonders of the starry world, the even road which leads to the river of know- ledge, and to its great fountain, thought. PIGMENTS OF THE EGaSHELLS OF BIRDS. 93 ON THE COLOURING MATTERS IN THE SHELLS OF THE EGGS OF BIRDS; AN ILLUSTRATION OF CHEMICAL DIAGNOSIS AT A GLANCE, AND OF THE VALUE OF ABSTRACT CHEMICAL RESEARCHES. {From the Patho- logical Institute.) In 1858 W. Wicke, Professor of Chemistry to the Agricultural Institute at Grottingen, presented through Prof. Wohler to the Academy of Sciences of that University a paper on the colouring matters of birds' eggs, which was probably the first chemical essay on that subject ever written, and was printed in the Gottinger Nachrichten, etc., 1858, p. 314. The author treated the shells with hydrochloric acid, water, and boiling alcohol in succession, and obtained a brown colouring matter, which he declared to be biliphsein, or brown colouring matter of bile, and a green pigment, which he declared to be identical with biliverdin, the green modification of the colouring principle of bile. He found also that the green colour of his extracts had a tinge of blue, or that some extracts might be more blue than green ; and the brown extracts might appear yellow by dilution or other causes. The pigments were insoluble in water. They were deposited, according to him, on the outer layers of the shells, and he suggested that they were deposited in the cloacee of the birds upon shells ready formed of white calcic carbonate in the oviducts. In support of that surmise he quoted a communication made to him by a sportsman, who had shot several birds, which ordinarily bring forth coloured eggs, in the oviducts or cloacsB of which there were contained eggs, either quite colourless or only faintly spotted, as if they were in pro- cess of being spotted with the pigment or pigments. The subject was next treated by Sorby, in a paper which was 94 ANNALS OF CHEMICAL MEDICINE. printed in the Proceed. Zool. Soc. Lond. for 1875, p. 351. In this research the spectroscope was applied to the matters in question, and peculiar spectra were discovered, of which the author soon found the meaning. But this he explained away by a laborious argument. He distinguished a red, a blue, and a green pigment, and other little defined products, and added little to their chemical description or diagnosis. Three years later C. Liebermann {Ber. Deutsoh. Chem. Ges. 11, 1878,606) again studied the subject, being acquainted with the essay of Wicke, but evidently not with that of Sorby. He found the colours of birds' eggs referable to two matters, one blue or green, which he defined with confidence as a biliary pigment, and another not recognised as -to its origin, but distinguished by the characteristic spectrum described by Sorby. He treated the eggs with acid, and separated flakes which were mostly coloured green. Alcohol produced coloured solu- tions, which were rarely sky-blue {Turdua musicus, Stumus vulgaris, Sylvia phoenicurus, Ardea argentSa), more fre- quently green (Corvus corone), without fluorescence ; very frequently bluish-green, with strong blood-red fluorescence {Larus canus, and t^ibundua, Sterna hirundo, Soolopax, HoBmatopus, Stringa) ; in the smallest number of cases faintly reddish, fluorescent, with a feeble touch of green {Falca tinnun- culus, Sylvia hypolaris, Tetrao islandicus and cotumix, FuUca atra). He used the shells of peewit's and gull's eggs to produce pure pigments, but obtained only dark green smeaiy masses. He then described the peculiar absorption spectra of the colouring matters, as observed by Sorby, and found that they differed according to whether they were contained in a strongly acid, or in a feebly acid, or in an alkaline solution. He gave long details concerning these transformations. He found some of the pigments soluble in chloroform. As he obtained GmeHn's reaction (the changes of colours with nitroso- nitric acid) with the green (not with the red) pigment, he believed the green to be biliary, but he was not certain that it was biliverdin. He doubted Wicke's opinion of the brown pigment being biliphsein or bilirubin, but got entangled in a net of errors when he endeavoured to attack the problem by means of the supposed oxidising action upon bilirubin of PIGMENTS OF THE EGGSHELLS OF BIRDS. 95 bromine. He adopted the opinion of Wicke, to the effect that the colours were imparted to the eggs in the cloacae, and not in the oviducts. I recognised at a glance the identity of the spectra described and figured by Sorby and by Liebermann with those charac- terising cruentin, a derivate of the colouring matter of blood (hemochrome), discovered by me before 1866, and first described and figured in the Tenth Report of the Medical Officer of the Privy Gmi.ncil, Appendix 7, pp. 227-232. From this research I give the following extract, with the diagrams of the spectra belonging thereto, as the data apply, mutatis mutandis, exactly to the phenomena exhibited by solutions of the red colouring matter of birds' eggs, ovocruentin, as I propose here- after to name it. Cruentin, a new derivate of hemato-crystallin, and of hematin. — When human or animal hemato-crystallin (hemo- chrome) is boiled with sulphuric acid, it becomes chemolysed, the albumen dissolves and yields its particular products, a portion of tlie hematin formed also dissolves and colours the fluid ruby red, while a brownish red grumous matter remains suspended in the fluid in an insoluble state. This is a mixture of neutral cruentin with its sulphate. By washing with water this matter loses sulphuric acid and becomes ultimately free from it. Treated with sulphuric acid it dissolves completely, and is now sulphate of cruentin. Hematin treated with sul- phuric acid also yields cruentin. Cruentin sulphate. — When a concentrated solution of this body is examined with the spectroscope, it shows one broad black band in red to orange. The visible blue is about as extensive as the visible green. On dilution this band splits up into two, and a third very feeble band in green becomes visible just to disappear on the slightest dilution. We have thus a spectrum of three bands represented by the following diagram, in which the absorptions, carefully measured by the spectrometer, are shown in their positions relatively to the sun-lines, and the intensities of absorption are expressed by curves enclosing the black spaces indicating the absorptions. The order of intensity of these bands is not the same in all dilutiotis. There is a point, when 7 is just visible, intensity 1 96 ANNALS OF CHEMICAL MEDICINE. (I distinguish ten different degrees of intensity, expressed in diagrams by ten different lengths of the band shadows beginning AaB c Diagram, of Speetrum of Sulphate of Crueidiii. always at the upper margin of the diagram and reaching perpendicularly downwards), at which a and ^ are of equal intensity, while just before the dilution at which 7 appears, ^ is decidedly most intense. On continued dilution, however, ^ disappears before a. The absorption is at all times equal to that of a solution of blood in water of equal intensity of colour as seen by the eye, so that when the fluid shows the merest tinge of pink, the bands are yet clearly visible. Neutral fl/woreacent cruentin. — When the insoluble residue from the sulphuric acid treatment is washed to neutrality and dried, a portion of it is soluble in ether and chloroform. The ether solution has four bands which are a little paler than, but in situation as nearly as can be identical with the bands of the chloroform solution. This latter, prepared by boiling, is dark red, and shows the following spectrum. AaB C Eb HH' y 5 a li Diayra/ni of Spectnivi of Central Fluorescent Cmentin. The saturated chloroform solution was one ctr. thick. The spectrum was measured with the aid of gaslight only. It fluoresced with a splendid blood-red colour in the cone of sun- rays produced by a lens. This is the first body which is known PIGMENTS OF THE EGGSHELLS OF BIRDS. 97 to fluoresce with homogeneous light, that is to say, the same kind of light or coloms which it transmits. The fluorescent cone has an intensity of colour equal to that of a solution about ten times more concentrated. Considering its beauty alone the phenomenon may be placed by the side of those exhibited by quinine, chlorophyll, or cudbear. But by its peculiarity it is perfectly unique. We shall see below how this description applies word for word to ovocruentin. Alkaline four-banded cruentin. — The spectrum of this form is represented by the following diagram : AaB C HH' Diagram of Spectrum of AVtaline Oruentin, Neutral Jive-banded cruentin in alcohol. — When the pre- paration from which chloroform extracts the neutral fluorescent cruentin is treated with alcohol, it dissolves easily and almost entirely. The concentrated solution allows a little red to pass ; on further dilution three bands appear, ultimately five, one in red feeble, one in yellow also feeble, both narrow, and three dense and dark bands in green, the last one situated at the point of transition into blue. A aB C e j3 7 o JKagratn of Spectrxtm of five^ianded Cruentin. When this solution is made alkaline with ammonia, the band e disappears, and the others arrange themselves as stated above under alkaline cruentin. 98 ANNALS OF CHEMICAL MEDICINE. The five and four-banded cruentin are both transformed into the three-banded acid cruentin by solution in sulphuric acid. On addition of water the five-banded cruentin is again obtained, which by ammonia becomes four-banded. The five-banded cruentin was obtained by extracting with ether and much glacial acetic acid, cruentin many times dissolved in sulphuric acid and reprecipitated by water. Immediately on the acid being washed out by water one band in orange dis- appeared, the red was more intense, and the ether solution showed the four-barided cruentin spectrum. Reduced cruentin. — Cruentin dissolved in ammonia shows the four bands. When an ammoniacal tartrate solution of iron sub-oxyde is added to it, it changes its spectrum in the following manner. There is a pale band 7 in red, there is a prominently Diagram of Speetrwm of Hedrwed Cmentin. dark a in green, and a broader but paler one /3 in green to- wards blue. On being shaken with air the solution becomes much lighter, and presents again the spectrum of four-banded cruentin. When the solution of reduced cruentin is acidified with sulphuric acid, it yields a precipitate ; after filtration it is much lighter and shows the spectrum of cruentin in the dilution in whicli 7 has completely disappeared. Cruentin therefore exhibits the peculiar property shown by hemochrome and hematin, that it can be oxydised (in alkaline solution) and reoxydised (in alkaline or acid solution). During this process however much colouring matter is lost by changes not yet scrutinised. In its alkaline solution it is deoxydised and reoxydised as easily as hemochrome (hemato-crystallin). It is most remarkable, physiologically, and particularly with regard to the occurrence of this matter in eggshells, that a decomposition product of PiaMENTS OF THE EGGSHELLS OF BIRDS. 99 hemochrome of the second order retains what I will term the breathing power of the blood-corpuscle. By treatment with aqueous hydrochloric acid and warming, cruentin undergoes a permanent change, which does not permit the four bands of the alkaline solution to be reproduced by alkali. This change is described on pp. 231 and 232 of the essay quoted, and further followed out in a subsequent essay. For our present purpose the data given above will sufBce, for they will enable us fully to diagnose and identify the red or brown colouring matter of the shells of birds' eggs. Eaypenments and observations on the shells of birds' eggs. — Some, commonly termed plover's eggs, were freed from their contents, washed with water and treated with dilute hydrochloric acid. When the effervescence had subsided and after the mixture had stood many hours, the shells were trans- formed into brownish green membranes, and the solution was light violet red, indicating that an excess of hydrochloric acid had been used. Placed in the spectroscope it showed the spectrum of acid cruentin in a beautifully clear manner, blue and violet being very transparent. (With the sulphate blue and violet are much more absorbed.) The addition of ammonia to this solu- tion caused a rust-coloured precipitate of five-banded cruentin. The precipitate could be passed through all the phases of the reactions mentioned above. It was soluble in ether and chloro- form; and the latter agent even extracted the acid cruentin hydrochlorate from the acid watery solution. The coloured membranes of the eggs, after complete washing out of all hydrochloric acid by means of water, were treated wet with alcohol (drying was found to be destructive of a great part of the pigments) and gave a deep green solution. This before the spectroscope showed a considerable continuous absorption of the red, effected no doubt mainly by the green ingredients of the solution, but in part also by the absorption in red of five-banded cruentin, of which the three principal bands, /8, 7, a, occupied the same positions as the three bands produced by the suspended precipitate of five-banded cruentin from the acid watery solution just described. The alcoholic solution was therefore a mixture of the solutions of at least two bodies, the green pigment and neutral five-banded cruentin. The process n 2 100 ANNALS OF CHEMICAL MEDICINE. by which this result had been obtained indicated a way in which the green and red colours could be separated, namely by exhaustion of the shells with very dilute hydrochloric acid, in which only the calcic carbonate dissolved, while the green and the red pigment remained insoluble. After removal of all acid by water, the green pigment, ovoviridin, is soluble in alcohol ; the red pigment still remains insoluble in neutral alcohol, and dissolves only in the presence of an excess of hydrochloric acid. When the two pigments are once together in alcoholic solution, a separation of one from the other becomes extremely difficult. I now treated a large number of birds' eggs (peewit, moor- hen, thrush, blackbird, starling, fieldfare, hedge-sparrow, and others) in the same manner as those of the peewit above described. The acid watery filtrates from the shells of eggs of thrushes and blackbirds were slightly green. The alcoholic extracts of moat membranes were deep green, those from the shells of the moor-hen alone were greenish yellow. The alco- holic solution on addition of ammonia gave a precipitate. Four eggs of fieldfares (which are buff coloured with reddish spots) gave as much ovocruentin in solution as sixty thrushes' eggs (which are blue with dark spots). Blackbirds' eggs also gave a deeper coloured hydrochloric acid solution than thrushes' eggs. An experiment was made in which it was attempted to extract the lime from the eggshells by acetic acid. But this miscarried entirely, all the colouring matters being altered or transformed into widely different products. The hydrochloric acid solutions from many eggshells were evaporated, and formed a precipitate during concentration of neutral five-banded cruentin. The solution or filtrate was dark brown, and exhibited the spectrum of acid two-banded cruentin. It was precipitated with ammonia and the precipitate was filtered off; this precipitate gave a portion to concentrated hydrochloric acid in the cold, which was isolated ; the residue insoluble in hydrocliloric acid was treated with slightly acidified boiling alcohol and dissolved, forming a splendid red coloured solution of fluorescent cruentin, with all features of spectrum and reaction described. A small residue insoluble in alcohol dis- solved in Concentrated hydrochloric acid, and was cruentin hydrochlorate. PIGMENTS OP THE EGGSHELLS OF BIRDS, lOl There cannot therefore be the slightest doubt of the red colouring matter being identical with cruentin as described by me. The spectra of analogous solutions were compared by pro- jection through the same slit and the same prisms, and found to coincide completely with each other. The green pigment. — Its alcoholic solutions are at first diffi- cult to filter. They can be evaporated and leave the pigment as a green mass, which is however now no longer entirely soluble in alcohol. The residue is insoluble in hydrochloric acid ; it is partly soluble in sodic carbonate, partly insoluble. When the sodic carbonate solution is evaporated to dryness, and the pig- ment set free by acid, it is now insoluble in alcohol. In short the green pigment undergoes a continuous change by almost every kind of treatment. The green pigment is therefore much less soluble than bili- verdin ; it resembles however biliverdin, not only by its solubility in alcohol, and green colour, but also by giving Gmelin's reaction, the transformation colours, blue, green, red, orange, yellow with nitroso-nitric acid, whether in alcohoKc solution or in the dry When the green pigment in alcohol exhibits a blood-red fluorescence (in which state it resembles closely a solution of chlorophyll so called), it is mixed with fluorescent cruentin, to which latter the fluorescence is due. It then gives the cruentin spectrum, while when pure it gives no detached bands, but only a contiauous absorption on the side of the red. I have sometimes obtained a sky-blue or blue solution from eggshells directly, by the simultaneous action of hydrochloric acid and alcohol combined; but I cannot assert the presence in many eggs of a blue colouring matter. From peewit's eggs I once obtained a bluish-green extract by ether and acetic acid ; the ether solution lost all green or blue colour on standing, and retained only a slight pink colour; it had however a great power of rose-red fluorescence. The green pigment is not soluble in chloroform, or in ether. Some eggshells after extraction with hydrochloric acid, were ex- tracted with alcohol, and the solution was evaporated. During condensation the solution, as is always the case, lost its primitive brilliant colour and became cloudy and formed a deposit. The 102 ANNALS OF CHEMICAL MEDICINE. latter was removed by filtration, and the filtrate evaporated to dryness. The residue was again extracted with absolute alcohol, and the filtrate again evaporated to dryness. (What the alcohol left undissolved was partly primary impurity from the membranes, partly changed pigment.) The residue was now exhausted with ether, which dissolved a brownish red matter, mainly cruentin, and left a dark green matter undissolved, which was now entirely soluble in alcohol. The green pigment is therefore similar to, but not identical with biliverdin. It is, like the ovocruentin, a product of the villous appendages of the inner surface of the oviducts of the birds which produce coloured eggs. The idea that it might be deposited on the eggs in the cloaca, from the intestinal contents carried that way, that it might in fact be the result of a mere Boiling with fsecal biliary matter, is too improbable in itself, and is contradicted by too many data of a very certain nature. In the first instance the cloaca never contains faeces when an egg passes through it; white eggs aie never soiled with fseces in the cloaca even partially. Secondly, the pigments are imbedded in the layers of chalk which form the shell, and not only so to say painted on the outside. Further, the pigments are several, and of these ovocruentin does not occur in the fseces ; further, some eggshells are uniformly impregnated with a red or brown, while others are uniformly impregnated with a green or blue pigment ; others again are uniformly blue, but spotted with red or brown patches, and so on. These varieties show that the pigmentation of eggshells is the work of specific organs, and these organs must needs be glands distributed in various orders in the oviduct ; the oviduct is the organ in which the shell is made from bicarbonate of lime secreted by the glands in its limiting membrane ; in this chemical laboratory is the natural place for glands, which have the power to split up hemochrome, and deposit the pigment products on the structure which is so rapidly built up within its cavity. Although more than a thousand eggshells were used to extract the pigments which I have described in the foregoing, the material obtained was insufficient to enable me to ascertain the chemical composition of the products by elementary analysis. It is therefore evident, that the question of the composition of PIGMENTS OF THE EGGSHELLS OF BIEDS, 103 these pigments could not easily be decided with the aid of materials derived from them directly. Under these circum- stances we may therefore, at least as regards ovocruentin, fall back upon cruentin made from blood by chemolysis, and use it for the purpose indicated. It is at all events clear, that an abstract research, like that on cruentin, has produced results, which have enabled us at a glance to determine the nature and physiological origin of an interesting phenomenon of the animal world, and to eliminate several serious errors with which the physiological question had been surrounded. The pigmentation of eggshells is perhaps not to be considered as an important biological problem, but, as has been shown by an author in the German periodical devoted exclusively to the promotion of the philosophy of evolution, it is one of the conditions of existence and continuance of living beings, and as such is deserving of consideration. In the placenta of several kinds of mammals colouring matters are deposited, e.g. in that of the dog ; these too are of two kinds — green and red, and the green has ere this been held to be identical with biliverdin. In any case the analogy of this fact to that observed upon eggshells cannot escape attention. It involves no great stretch of the imagination to suppose that both occurrences have the same evolutional orig^. In other words, the pigment glands of the oviducts are remnants (or beginnings) of the villous coat of the uterus, the pigments are direct decomposition products of blood; the blood does not serve for the nutrition of a fcetus, but only furnishes a tribute of respect, an ornamental or useful covering to the outside of the kind of sequestered being, with which it had in anterior stages of development more intimate relations, or with which it may have them in the future if the bird is in process of evolution towards a mammal. 104 ANNALS OF CHEMICAL MEDICINE. VI. ON THE ORGANIC ACIDS OF THE BEAIN, WITH SPECIAL REFERENCE TO THE NATURE OF THE LACTIC ACID CONTAINED IN IT. {From the Pathological Institute.') Introduction. — The organic acids of the brain are obtained as ingredients of the so-called water- extract, that is to say, the watery solution of alkaloids, carbohydrates, acids, organic and inorganic salts, which is obtained when the spirit-extract of brain is gradually freed from spirit by distillation, and from the phos- phorised, nitrogen ised, and cholesterous principles by deposition and filtration. When a general analysis of these extracts is intended, it is perhaps most convenient to remove the alkaloids in the first instance by special precipitants such as phospho- molybdic acid, phosphotungstic acid, or mercuric acetate. After removal of the excess of the reagent employed, inosite may be precipitated in the usual manner, and after the removal of the excess of lead, the fixed acids may be extracted by ether. When it is however intended to extract the fixed organic acids only, they may be extracted from the original water-extract by ether after addition of an excess of sulphuric acid. In the following processes alcohol was employed at a certain stage to isolate amido-acids and nitriles (leucin and tyrosin, etc.). Anyone who will examine the water-extract of brain according to this method will not leave much in the mother liquor to be accounted for. When pure phosphomolybdic acid is employed, the ultimate mother liquor may be incinerated and used for the analysis of the fixed alkalies and earths. Treatment of the water extracts of the Human Brain after removal of Alkaloids. 1. Extraction of Formic, Lactic, and Succinic Acid. — After the bulk of the alkaloids had been removed from the extracts by mercuric acetate the filtrates OKGANIC ACIDS OP BEAIN. 105 from the mercuric precipitates were freed from mercury by hydrothion, and evaporated to a syrupy consistence ; the syrup was treated with absolute alcohol, and thereby separated into a portion soluble, and another insoluble in alcohol. Portion soluble in alcohol. — The alcohol was evaporated, the residue treated with neutral plumbic acetate. The preci- pitate consisted mainly of phosphate and chloride, and was removed. After this, basic plumbic acetate was added to the filtrate, and the precipitate collected and decomposed with hydrothion : the concentrated filtrate from the plumbic sul- phide on treatment with absolute alcohol gave a crystallisation of inoaite. The mother liquor of this inosite, freed from alcohol by evaporation and acidified, gave a small precipitate with phos- phomolybdic acid, thus proving that the mercuric acetate had not removed all the alkaloid, but left some, which was preci- pitated by basic lead acetate {third alkaloid). The lead was now removed from the mother liquor by an excess of sulphuric acid, and the acid liquid extracted with large quantities of ether at intervals. The ethereal extracts, after removal of the ether by distillation, left a mixture of acids (see below, § 4) ; of these, acetic acid, introduced with the mercury and lead salts, was evaporated on the water-bath ; the remaining syrup consisted mainly of lactic acid, biit also contained some succinic acid, as will be described more fully lower down. 2. Leucin and Allied Bodies; Tyrosin. Portion in- soluble in alcohol. — This on solution in a minimum of water retained a quantity of solid matter in suspension which was separated by filtration ; the residue on the filter was pressed, and on treatment with cold water gave to this solvent a matter recognised as leucin and allied bodies ; the matter insoluble in cold but easily soluble in hot water, was tyrosin. The leucin was purified by precipitating the coloured impurity out of its watery solution by means of mercuric nitrate, removing the excess of mercury by hydrothion, eva- porating the solution, precipitating the acid liquid by ammonia, collecting and pressing the precipitate in bibulous paper, and recrystallising it from spirit. The first crystals were pure leucin ; 106 ANNALS OF CHEMICAL MEDICINE. the second crystals were however a different body, more soluble than leucin, and not easily separated from leucin on account of the similarity of properties ; the last mother liquor dried up leaving a trace of matter. The tyrosin was purified by hydi'ochloric acid and charcoal, and precipitation with ammonia. The mercuric nitrate precipitate from leucin was also decomposed, and seemed to contain only a small amount of alkaloid No. 2. 3. Peculiwr Potassium, Salt. — The liquid part of the portion insoluble in alcohol was diluted with water, and treated with neutral lead acetate; the precipitate, decomposed by hydrothion, gave a syrup, which, treated with absolute alcohol, deposited a peculiar viscous colourless potassium salt ; this was isolated, and on account of its peculiar nature the presence of the potassium was specially proved by combustion and platinic chloride. The part of the syrup soluble in alcohol was not further examined. The filtrate from the neutral lead acetate precipitate was treated with basic lead acetate, and the new precipitate decomposed with hydrothion ; the concentrated product was treated with absolute alcohol, and gave white inosite, partly anhydrous, partly hydra ted. In the mother liquor of this inosite nothing apparently but some alkaloid No. 2 remained. The mother liquors from which the foregoing bodies had been extracted and precipitated were freed from the impurities introduced as reagents as far as possible and evapo- rated, and then formed a nearly colourless, viscid, uncrystal- lisable mass ; this was distilled in superheated steam, but yielded no glycerine. It gave a precipitate with phosphomolybdic acid, from which a syrupy alkaloid, smelling like sperma, was obtained (see alkaloid No. 3). 4. Proceedings for the sepa/ration of the acids isolated by means of Ether. Lactic Acid. — The mixed acids were heated on the water-bath until acetic acid was expelled, redissolved in water, and neutralised while hot with freshly-prepared zinc carbonate. The zinc salt was crystallised and recrystalUsed an indefinite number of times until perfectly white, crystallised throughout, and homogeneous. During these operations a coloured matter became insoluble, and had to be removed by OEGANIC ACIDS OF BEAIN. 107 repeated filti-ation. The crystallised salt was found to be pure zinc lactate, containing the variety of lactic acid known as lactic acid from flesh, or sarkolactic acid.' Not only did the salts yield the particxilar amount of water of crystallisation which distinguishes them from the zinc salts of the fermentation lactic acid, but the free acid itself showed the power of polarising light, which is not possessed by the product of the fermentation of milk. 5. Summary of analyses of Zinc Lactate from, Human Br am. Water of crystallisation. — Preparation I. (1) 0*6002 grm. dried in vacuo, on drying at 103° to 110° lost 0"0730 grm. water =12*82 per cent. Preparation II. (2) 1*6540 grm. which lost nothing in vacuo over H^SO^, dried at 105° to 108° (9 hrs.) lost 0*2135 = 12*90 per cent. HjO. (Theory 12*82 per cent. H2O.) ZnO in anhydrous salt estimated by ignition. — Prepara- tion I. (1) 0*2378 grm. left 0*0785 grm. ZnO, equal to 33*01 per cent. ZnO. (33*38 per cent ZnO required.) [Zinc-stains in crucible explain loss. The mode adopted by Wislicenus of determining Zn in lactate by combustion in platinum crucible, as he expressly says, is thus shovm not to be very good.] Zn in hyd/rated salt, dried over H^SO^ in vacuo, by pre- cipitation with carbonate, &c. — Preparation II. (1) 0*4:897 grm. gave 0*1418 ZnO, equal to 28*9 per cent. ZnO, or 23*24 per cent. Zn metallic. ' Professor Gsoheidlenof Breslauhas published a paper in ArcJiiv Physiol. 8 (1873) 171, entitled, ' On the Chemical Eeaction of the Nervous Central Organs ' ; p. 178 he examines the acid which causes the acid reaction of the grey matter, and finds it to be lactic acid. Muller had already stated that the lactic acid in the brain was zymo- and not sarko-lactic acid. Gscheidlen supports this view on the basis of a single quantation of water of crystallisation on one decigramme of calcic lactate. He operated on the brain of dogs, and of a horse. He might therefore maintain that dog and horse had differently from man and ox ordinary lactic acid, and not flesh-lactic acid in their brain, if he had really proved that his one decigramme of salt was ordinary lactate. But one quantation of water of crystallisation on a decigramme of salt can prove nothing, particularly in so delicate a question. And further, the calcium salt of lactic acid is the least suited to decide this matter, as its hydration (in different preparations of sarkolactic acid) changes apparently capriciotisly, as will be shown below. In fact the statements of Miiller, Gorup-Besanez, and others, and lastly of Gscheidlen, concerning the nature of the lactic acid from brain are proved to be erroneous. 108 ANNALS OF CHEMICAL MEDICINE. (2) 0-5415 grm. gave 0-1571 ZnO, equal to 23-28 per cent. Zn. Theory requires 23-35 per cent. Zn. 6. Summary of analyses of Zvnc Lactate from Ox Brain. Water of crystallisation. — Preparation I. (1) 0-3546 lost 0-0454, equal to 12-80 per cent. H^O. ZnO in anhydrous salt estimated by ignition. — Prepara- tion I. (1) 0-1591 gave 0-0524 ZnO, equal to 32-93 per cent. Theory required 33-38 per cent. The loss explained by method as in case of human lactate above, Zn in anhydrous salt estimated by precipitation ^cmd ignition. — Preparation II. (1) Dried at 105°, 0-2174 gave 0-0723 ZnO, equal to 26-69 per cent. Zn. (2) 0-1440 gave 0-0479 ZnO = 26-69 per cent. Zn. Theory requires 26-79 per cent. Zn, in the anhydrous salt CjHjoOgZn ; the hydrated salt Zn (CgHgOg), (HjjO)^ contains 23-35 per cent. Zn and 12-82 per cent. H^O. 7. Physical peculiarities of the Lactic Add from Brain and its Zinc-salt. — When the lactic acid as obtained from the ether extract, a state in which it was yet yellowish and gave out an odour, was decolourised by animal charcoal, and a somewhat concentrated solution of it was placed in a tube of 220 m.m. in length, and containing about 26 c.c. of fluid, and subjected to the influence of the polarised ray of yellow light in a Wild's polaristrobometer, it was found to turn the plane of polarisation to the left (to the measured extent, in the particular instance of an acid of uncertain strength, of 1° 20'). The acid was next transformed into zinc salt by boiling with zinc oxyde, and the solution of salt was evaporated to the same volume as that occupied by the free acid. It now turned the plane of polarisation still to the left, but to the extent of 3° 15'. Thus the rotation from to the left had been much increased, more than doubled, by the introduction of the zinc and the attendant thermal and hydric operations. The zinc salts, which had been obtained in a state of purity, as proved in the previous paragraph, were dissolved in water and decomposed with hydrothion ; the free acid was concentrated and became a colourless syrupy liquid ; in this state it was not perfectly brilliant, but had a slight haze, probably from a trace ORGANIC ACIDS OF BRAIN; 109 of finely divided sulphur. It was therefore allowed to stand for two months in a quiet place, and when the trace of particles had completely deposited, the clear part was isolated by decantation. This acid, on being placed in a tube of 100 m.m. in length into the polaristrobometer, now turned the plane of polarisation .to the right, in the particular instance of an acid of uncertain concentration to the extent of 2° 17' (average of seven obser- vations). It will be observed that I have made no attempt to determine the specific rotating power of any of the products obtained in these researches on lactic acid from the brain. The reason for this is the circumstance first observed by Wislicenus, that the rotating power of sarkolactic acid changes under a great number of influences, such as heat, water, and time. Thus free sarko- lactic acid when dried over sulphuric acid in vacuo during twenty-one months is transformed into a mixture of anhydride, CgHipOg (84 per cent.) first with lactic acid, CjHgOj (16 per cent.), afterwards with lactide, C3H4O2 (16 per cent.). The solution of this mixture turns the plane of polarised light to the left, (a)=— 85"93°. It is probable that all three products on treatment with water are transformed back into sarkolactic acid. The watery solution of sarkolactic acid as ordinarily obtained, directly after extraction and concentration, shows a considerable polarisation to the left. This power is suddenly and greatly diminished after every addition of water or of spirit, but on standing it rises again, without however reaching its former value. The diminution of specific rotatory power by dilution is the greater, the more concentrated was the solution used for dilution, that is to say, the greater was the dilution in propor- tion to the strength of the original solution. These changes are due to the presence of anhydrides and lactide. And as every preparation of sarkolactic acid contains these anhydrides, ac- cording to Wislicenus, pure sarkolactic acid, as a preparation, does not exist, and therefore its specific rotatory power, which this author surmised to be to the right, cannot be accurately determined. I have shown above that pure sarkolactic acid prepared from the zinc salt turns the plane of polarised light freely to the nght. But the solution of the zinc salt from which this acid was pro- no ANNALS OF CHEMICAL MEDICINE. duced, turned energetically to the l&Jt. The saturated normal solution of zinc salt turns the plane of polarised light steadily 7° 7' to the left. In oversaturated solutions the turning faculty is not increased, as might he supposed, hut on. the contrary is diminished. The polarising faculties of sarkolactic acid, its hydrate, zinc salt, and anhydride may be described as follows : Turning farthest to the riglit . CjHgOg. Turning less far to the riglit . C3H5O3 + H^O. Turning least to the Uft . . 2(C3H503)Zn. Turning more to the ie/i{ . , 2(C3H503)Zn + 2H2O. Turning farthest to the left . CgH^Os. 8. Peculiarities of the Calcium Salt of Lactic Add from Human Brain. — This salt was made from pure lactic acid ob- tained as above described. Its solubility in water was so great that attempts at its crystallisation from this solvent were foiled by the solution setting to a solid mass. It was consequently re- crystallised from strong spirit. When dried in air it was a light voluminous spongy mass of crystals. Analyses : (a) 0-7210, on drying at 105°, lost 0-1531 HjjO, leaving 0-5679 of anhydrous salt ; consequently water of crystallisation = 21-2 per cent. (6) 0-7010 gave 0-3385 CaSO4 = 0-0995 Ca, or 14-20 per cent. Ca. (c) 0-4678 gave 0-2240 CaS04= 0-0658 Ca, or 14-08 per cent. Ca. These data do not correspond to any of the recorded data conceming this salt, which recorded data themselves differ from each other, or on the assumption that there was only one hydrate, contradict each other. It is now assumed by some that the sarkolactate of calcium as commonly obtained has the formula : 2 Ca, 4(C3Hg03) + 9 H^O, water . . = 27-09 per cent. Formerly however a hydrate was mostly described containing water . ~ . = 24*83 per cent. The hydrate just described contains , = 21-2 per cent. ORGANIC ACIDS OF BRAIN. Ill Now the second salt corresponds to one with four molecles of water of crystallisation, while the last leads to no even pro- portion between salt and water -of crystallisation, but is inter- mediate between the salt containing four molecles and a hypo- thetical salt containing three molecles of water, which requires = 19"89 per cent. The new salt must therefore be considered either as a com- pound or mixture in nearly molecular proportion of the salt, containing 24'83 per cent., with the hypothetical salt containing 19*89 per cent, of water, or in more simple terms as a salt con- sisting of two molecles of anhydrous lactate and seven molecles of water of crystallisation, requiring 22'3o per cent. H^O ; pos- sibly a lower homologue of the salt with nine molecles of water. The depression of the water by mere admixture of anhydride was improbable owing to the imiform character of the crystal- lisation. I am therefore of opinion that there are at least three, if not four, different crystallised hydrates of calcic lactate, and that the amount of the hydration is probably dependent upon the con- centration of the solution, if it be a watery one, or upon the aquosity of the solvent, if it be spirit. 9. Note on the Theones concerning the Chemical Constitu- tion of the, Isomeric Lactic Adds. — The lactic acid specimens obtained from different sources behave as if they were one and the same body. But on further inquiry, particularly by means of decomposition and the analysis of calcium and zinc salts in their hydrated state, it is found that the lactic acid obtained by the fermentation of different sugars differs from the lactic acid contained in the flesh and brain ; it is further maintained that the acid obtained from flesh consists actually of at least two lactic acids ; and that in addition there ig a fourth lactic acid obtained by chemical synthesis. The lactic acid of fermentation can be produced from the lactic acid from flesh. We have therefore here to deal with an important case of isomerism, of which at least two terms were believed to be well understood. The fermentation lactic acid was said to contain the radical ethylene, and was therefore also termed ethylene-lactic acid ; whereas the flesh or sarkolactic acid was said to contain the radical ethylidene, and was termed ethylidene-lactic acid. The 112^ ANNALS OF CHEMICAL MEDICINE. acid CgHgOg, obtained from glycerin-iodopropionic acid, termed by Heintz ethylene lactic acid, although isomeric with lactic, had better be termed hydracrylic acid because on thermolysis it decomposes, yielding water and acrylic acid. (Wislicenus, Ann. Chem. 166, 1873, 3.) By oxydation with silver oxyde it yields carbonic acid, probably carbacetoxylic, glykolic, and oxalic acid, but no glyceric or acetic ; by fusion with caustic alkali, formic and acetic acid. According to Wislicenus, the synthetical ethylene lactic acid, produced from ethylene cyan- hydrine, can be obtained pure with very great difficulty only, as its salts scarcely crystallise ; and according to him also, this acid is identical with the second lactic acid, occurring in smaller quantity in the mixture of two lactic acids hitherto termed sarkolactic acid. The lactic acid which occurs in flesh in the largest quan- tity, and yields a zinc salt with two molecles and a calcium salt with 4-J molecles of water of crystallisation, is, according to Wislicenus, ethylidene lactic acid. Heintz agrees in this conception in so far that he admits a part of the sarkolactic to be ethylidene lactic acid ; but another part he assumes to be (what he terms ethylene lactic acid, namely) the above-described hydracrylic acid. (Ann. Chem. 157, 1871, 314.) The four assumed isomers of the formula CjHgOj are therefore (1) fermentation lactic acid, also termed ethylene lactic acid ; (2) principal flesh lactic acid, also termed ethy- lidene lactic acid ; (3) second flesh lactic acid, or synthetical ethylene lactic acid; (4) hydracrylic acid, termed by Heintz ethylene lactic acid. Erlenmeyer {Ann. Chem. 158, 262) could not confirm Wislicenus and Heintz as to the invariable existence of two lactic acids in the sarkolactic acid, and explained the results of these authors as possibly caused by an oversaturation of the solution of the zinc salt, to which he shows the compound to be prone. He never obtained malonic acid by oxydation of sarko- lactic acid (which had been so obtained by Dossios), and there- fore was inclined to admit that flesh may contain sometimes, but not always, two different isomers of lactic acid. Wislicenus believes that the preparation of either the synthetical ethylene lactic acid, or of the second sarkolactic acid (both of which he OEGANIC ACIDS OF BBAIN. 113 believes to be identical) in the pure state is so difficult a matter as to be almost impossible. He gives certain proofs of their identity, but they are mostly negative, such as the amorphous and syrupy condition of salts, and the solubility in almost absolute alcohol, and leaves it to be surmised that the malonic acid,- which was formed by his pupil Dossios {Ann. Chem. 146, 1868, 168) under his eyes, from crude sarkolactic acid, was derived from this ethylene sarkolactic acid. But as the acid was only obtained in small quantity, and from a crude product, its use for structure formulae is still less justified than it was for the purpose for which it has been used so many years. The preparations of lactic acid from the brain of man and the ox, which I have described above, leave no room for doubt regarding their nature ; they are specimens of the one optically active sarkolactic acid, yielding the precisely characteristic zinc salt ; that they did not yield the ordinary calcium salt is of little consequence as the question of the composition of the calcium salts of sarkolactic acid is not exhaustively answered. Whatever may be the issue of the discussion regarding the con- stitution of the dififerent lactic acids, the facts now ascertained regarding cerebral lactic acid cannot thereby be affected. I have stated the opinion above quoted regarding the alleged second acid in sarkolactic acid already in 1877 (com- pare my Pathology of the Urine, 1877, p. 461 et seq.) I now prove for the brain, what Erlenmeyer has proved for the flesh, namely, that it contains only one lactic acid. In an article in Ann. Chem. 191, 1878, 261, Erlenmeyer has subjected the question of ethylene lactic acid to an historical and experimental critical review. He had formerly not found ethylene lactic acid in flesh-lactic acid prepared by Liebig's method, and now endeavoured to find it by the method of Wislicenus; but he again failed, and obtained only some fermentation lactic acid. A similarly negative result has been, obtained by Klimenko {Ber. d. Gh. G. 9, 1604). The result of this part of the review is that Wislicenus had obtained a lactic acid containing nitrogenous matters as impurity. Erlenmeyer now produced the lactic isomer from ethylene chlorhydrine by transforming this into the nitrile CjHjNO, and the latter into the lactic acid isomer by two methods ; one, I 114 ANNALS Of CHEMICAL MEDICINE. that of Wislicenus, namely, heating with caustic soda ; another' of his own, namely, heating the nitrile with fuming hydrochloric acid. Both processes gave him an acid which was shown to be identical with the hydracrylic acid of Wislicenus ; for it gave, on heating with hydriodic acid in a sealed tube /8-iodopropionio acid. This last transformation had been vainly tried by Wis- licenus {Ann. Chem. 167, 352), and it was mainly on the basis of this failure that he declared his acid from ethylene chlorhydrine to be ethylene-lactic acid, and denied that it was hydracrylic acid. Erlenmeyer next produced ethylene-cyanhydrine CgHgNO by direct combination of ethylene-oxide and prussic acid (a synthesis already unsuccessfully attempted by Wislicenus, Ann. Chem. 167, 346), and from this ethylene-cyanhydrine he pro- duced the ethylene-lactic isomer, according to the following formulae : H,C . H HjCOH I. I \o + I = I H,C /^ CN H^C CN. H,GOH\ ir. H^C CN H^COH V+2H„0 +HC1 = H„C C00H-|-NH,C1. The reaction 11. is, however, not the only one which ethylene^ cyanhydrine gives on boiling with hydrochloric acid, acrylic acid, CjH^Oj, being fonned at the same time, which can be sepa- rated by distillation, under diminished air-pressure, with the aid of fractional saturation. And this . also yielded the y8-iodopro- pionic acid by the process above described. The lactic isomer also behaved like the hydracrylic acid of Wislicenus ( = Beil- steia's hydroxyacid from iodopropionic acid) in this, that when a concentrated solution of its zinc salt was mixed with a concen- trated solution of its calcium salt, the mixture yielded the double salt described by Heintz {Ann. Chem. 157, 291) in characteristic very little soluble crystals. (Dried ia the exsiccator they are free from water of crystallisation, and contain 8-68 per cent. Ca and 14'10 per cent. Zn.) ORGANIC ACIDS OF BEAIN. 115 From the foregoing it is evident (1) that the appellation of ' ethylene lactic acid ' cannot be applied either to the zymo- or to the sarko-lactic acid ; (2) that it is uncertain whether one or both of these isomers are ethyUdene lactic acid, and whether consequently they really dififer in their chemical structure ; (3) that the acid termed by Heintz ethylene lactic acid is really such, and is also hydracrylic, as claimed by Wislicenus ; (4) that this hydracrylic acid has not yet been shown to be present, either in the muscles or the brain of animals. The four assumed isomers of the formula C3H5O3 are therefore reduced to three, and the question regarding the constitution of the zymo- and sarko- lactic acid remains open. 10. Forvnic Acid from the Water Extracts. — ^The acids extracted by ether after precipitation of the alkaloids were in one experiment placed in a retort, and subjected to distillation. The main part of the distillate consisted necessarily of acetic acid. The acids were neutralised by barytic carbonate and eva- porated to crystallisation ; the first crystals gave 53-09 per cent. Ba (acetate requires 53*72 per cent. Ba) ; the second crystals gave 55*53 per cent. Ba, and gave a strong formiate reaction with nitrate of mercurous oxide. It is, therefore, probable, that the water extract of the brain contains a small amount oi formic acid, as has already been stated by Von Bibra and Miiller. (See Report, New Series, Ko. III. 1874, p. 206.) The amount was, perhaps, not greater than that of succinic acid to be described. 11. Succinic Acid as a Normal Ingredient of the Brain of the Ox, — The mother liquors of the zinc lactate gave with ferric chloride, not in the cold, but on boiHng, a rust-coloured precipitate, soluble in excess of chloride, forming a dark red solution. The united mother liquors were cautiously precipi- tated while boiling, arid the compound filtered off. They were next boiled with barytic carbonate, and the precipitate filtered off. The latter was extracted with hot dilute acetic acid, in which the ferric precipitate was insoluble. The ferric precipi- tates, were dissolved in water, and H2SO4, and extracted with ether ; the ether solution left the acid free. The acid, easily soluble in water, crystallised on evapora- tion. Fused, and sublimed in white vapours. Sublimate white ; no charcoal. Pungent smell of vapours. Sublimate all crystals. I 2 116 ANNALS OF CHEMICAL MEDICINE. In a mixture of BaClj, NH^HO and spirit it produced an immediate thick white precipitate. The sublimated crystals dissolved in water, and gave a clear and colourless solution. This was cautiously (litmus added) neutralised by sodic carbo- nate. The solution gave rusty precipitate with ferric chloride, soluble in excess of chloride. It gave a white precipitate with mercurous nitrate, not altered by boiling ; gold chloride gave no reaction which excludes malonic acid; uranic nitrate gave no re- action. Precipitate (after boiling and concentration) with BaClj immediately. This was soluble in HNO3, and reprecipitated by NH4HO ; still more by a little spirit. BaClj also gave preci- pitate with NH4HO and spirit, difiBcultly soluble in acetic acid. This acid is consequently succinic, C4Hg04. 12. Succinic Acid in the Human Brai/ti. — The process described in the foregoing note regarding the brain of the ox was repeated on the mother liquor of zinc lactate from the' brain of man, and exactly identical results were obtained. The ferric salt was decomposed and the acid extracted by ether. It remained in a crystalline state and was sublimed. The reac- tions were then made upon the sublimate, and found to agree exactly with those of the succinic acid. . This experiment was repeated on a second quantity with identical results. Succinic acid is thus shown to be a normal ingredient in small quantity in the brain of man and of the ox. Miiller, when extracting, lactic acid from brain, had searched for succinic, but not obtained any. This is explicable on several grounds : firstly, his method was not calculated to obtain it (he waited for crystals to form in the concentrated lactic acid) ; ai^d, secondly, the quantities, of brain matter employed by him were probably too small. (See Report, New Series, No. III. for 1874, p. 232.) The significance of succinic acid in nerve marrow is probably connected with that of the disintegration of the albuminous substances. But the possibility of merely accidental presence must not be lost sight of, as succinic acid is present in many kinds and parts of vegetables used for food of man and animals, and in wine and other fermented liquids, in which it is produced by fermentation from sugar. CHEMOLYSIS OF BILE. 117 VIL ON TSE CHEMICAL DECOMPOSITION OF BILE AND ITS INGMEDIENTS, WITS REFERENCE TO THE THEORY OF THE FUNCTION OF THE LIVER. (JSmnmary). Preparation of glylcocholic add from ox-bile; Variation of the quantity of this acid in the bile from different aniTnals. — This subject has given rise to some controversy, which is now in a fair way of being explained. Hiifner {Joum^ Pract. O^em, 10, 1874, 267) had published a new mode of obtaining this acid, which was quite unsuccessful in other hands and other places than Tubingen. It was the following : To fresh bilfe contained in a high cylinder some ether is added, so that it floats on the surface ; hydrochloric acid is now added until the bile shows a milky turbidity ; crystals soon form in the liquid, under certain conditions so rapidly that after some minutes the liquid is transformed into a solid mass of crystals. As soon as the crystallisation is completed, the ether is poured off, the crystal- lised mass is stirred up with a large quantity of water, placed on a large filter and washed with cold water until the filtrate, green at first, has become colourless. The crystals remaining on the filter are dissolved in boiling water, the solution is filtered hot ; the filtrate on cooling deposits pure glykocholic acid. The filtrate from the first crystals contains the taurocholic acid and On extending his observations, Hiifner (Journ. Pract. Chem, 19, 1879, 302), found that of a hundred specimens of ox-bile collected at Tiibingen, forty gave this crystallisation in a few minutes, another forty gave it slower, sometimes only after the lapse of hours, and in these there was also less crystallised matter, while twenty per cent, gave no crystals at all. The inquiry was now extended to the animals from which the bile 118 ANNALS OF CHEMICAL MEDICINE, came, and it was ascertained that the bile of bulls always gave the crystals, that of cows yielded them as a rule, with exceptions, that of oxen (castrates) never. The bile of calves never gave crystals. Whether the animals were fat or lean did not seem to exercise any influence upon the bile in respect of its crystal- lisability under the circumstances here considered ; green bile never gave crystals, light brown bile nearly always gave them. In respect of the causes of these variations, no reliable data have been ascertained, but it is probable that the nature of the fopd is one of their principal conditions. Kolbe treated many dozen specimens of bile at Leipzig after Hiifner's prescription, and never, not even with brown bile, obtained any crystals. But he observed a deficiency of sodium, as well as taurocholic acid, and is inclined to attribute the deficiency of the biliary acids to a primary "deficiency of common salt in the food, and then in the blood and liver of the animals. JExtraction of taurocholic acid and cholin from the first add filtrate from the crystals of glyhocholic add. — ^The united filtrate and washings are neutralised with sodic carbonate, evapo- rated to a syrupy consistency, mixed with animal charcoal, dried on the water bath, and the residue is powdered ; this is now ex- hausted with boiling alcohol, the alcoholic solution is freed from alcohol by distillation, the residue is. dissolved in water, and mixed with excess of basic lead acetate. After some standing, a lead salt settles in the shape of a glistening faintly-yellowish brown mass, lead taurocholate, from which the supernatant liquid can be easily decanted. From this precipitate tauro- cholate of soda is easily obtained and transformed into crystals by the addition of ether to the alcoholic solution. The liquid decanted from the lead taurocholate is freed from lead by hydrothion ; the filtrate is evaporated to suitable con- centration, and on addition of platinic chloride and alcohol yields the double salt of cholin. It is better to isolate the cholin by phosphomolybdic acid, and decompose the compoimd with baryta before applying the platinic chloride, as without that precaution the resulting platinum salt is liable to be con- taminated with potassium and volatile alkalies, Chemolysis of bile by baryta. Production of cholic add. — Tappeiner produces cholic acid by dissolving one part of CHEMOLYSIS OF BILE, 119 inspissated ox-bile in from ten to twenty parts of water, adding five parts of baryta water saturated boiling, and boiling the mixture during from five to seven days without interruption. The mixture is allowed to stand during a day to deposit baryum salts of fatty acids, filtered into large flasks, mixed with some ether, and then with hydrochloric acid, with the precaution of keeping most of the colouring matter in solution. After from one to three days crystals begin to appear on the surface of the precipitated cholic acid, and after from three to four weeks the entire amount of the acid is crystallised. It is filtered from the brown mother liquor, washed, and obtained pure by two crystal- lisations from alcohol. If the acid is deposited in hard cakes instead of loose masses of crystals, it must be triturated in a mortar and washed with small quantities of extremely dilute soda to dissolve all colouring matter, and after filtration washed with water. If the cholic acid after addition of the hydrochloric acid i^ allowed to stand too long in the mixture, it is liable to become mixed with a blue ferruginous body which forms gradually in the mixture ; this is soluble in alkalies with a brown colour, but again precipitated from them with a blue colour ; it is amor- phous, insoluble in alcohol, changed by this agent into a green colouring matter, and is difficult to collect, as it either passes through the filter or stops its pores. Preparation of cholic acid from pv/re glyhocholic acid. — Fifty grms. of glykocholic acid are mixed with 200 grms. of caustic baryta and six litres of water, and kept boiling during sixteen hours. The solution is filtered hot, but mixed with hydrochloric acid only after it has become cold. The cholic acid then falls in a sand-like state. It is washed repeatedly with warm water, and then dissolved in hot alcohol. From this it crystallises soon in octohedra and tetrahedra of the usual small dimensions. The crystals amount to 80 per cent, of the calcu- lated quantity. Ckolate of ethyl. — Twenty parts of cholic acid are dissolved in 140 parts of alcohol of 90 per cent, strength, and into this solution a current of. dry hydrochloric acid gas is passed. (It is necessary to prevent the mixture from getting warm, for in the case of its. temperature rising during the reaction it becomes 120 ANNALS OF CHEMICAL MEDICINE, turbid or reddish, and an uncrystallisable product is separated.) The mixture saturated with hydrochloric gas is at once, and again without allowing its temperature to rise, mixed with an equal bulk of strong alcohol. Of this mixture every 100 c.c. are allowed to flow in a thin stream into a litre of cold water. The mixture is milky at first, but after some hours shows needles, and after some days the crystallisation of the cholic ether is completed. They are washed with water, redissolved in alcohol, and reprecipitated in water, whereupon they are pure. Cholamide. — Purest, cholic ether is heated with highly concentrated ammonia in a sealed tube during six days to 1 30°, the product is diluted with nine parts- of water, boiled up and filtered. On cooling silky needles of cholamide, Cj,4H4,N04, are deposited. It is very hygroscopic, easily soluble in alcohol, less easily in ether ; it is little soluble in water, even on boiling, and is deposited from this in needles. The solutions have a neutral reaction. When dried at 115° in a current of hydrogen it fuses at 130°. Oxydation of cholic acid by vneans of ddchromate and sulphuric add. — This subject has been further studied by Tappeiner {Lieh. Ann. 194, 1878, 211). The mixture consists of ten parts of potassic dichromate and fifteen parts of oil of vitriol, the latter diluted by three times its volume of water, or "even more ; and with this quantity of liquid one part of cholic acid is treated. The warm mixture is poured upon the powdered cholic acid contained in a flask ; carbonic and acetic acid are immediately set free, and the rest of the cholic acid forms a fused layer on the top of the liquid ; the mixture is agitated until the fused acid is again hard and granular. If a volume of more than a litre of mixture in all is treated at once, no external heat is required to effect the first stage of oxydation ; this first stage consists in the formation of cholesteric, stearic, and lauric acid, besides the carbonic and acetic already mentioned ; and of an acid which is not very well defined, but passes, during the second stage of the oxydation, with the aid of external heat, into an acid termed cholanic, Cholesteric add. — By this name Eedtenhacher {Lid). Ann. 57, 162) and after him Schlieper (Lieb. Ann. 58, 377) and Gundelach and Streckejr {Lieb. Ann. 62, 205) signalised an CHEMOLYSIS OF BILE. 121 acid which they ohtained from cholesterin, choloidic, cholic and hyocholinic acids by oxydation with nitric acid. The silver salt, which was apparently crystallised, led to the formula for the acid of CjHj^Og (C = 6) ; when this had to be doubled, the acid was declared to be two-basic. This acid is now shown to be a mixture of two acids, one crystalline, to which the name of cholesteric acid is continued, CijHjgOj, and another, the pyro- acid of the first, amorphous pyro-cholesteric acid, C,,H,gOg Cholesteric acid is ol)tained at the end of the first period of oxydation by filtering the liquid from the resin through glass wool, and evaporating at a gentle heat; it deposits in fine crystals and membranes ; but when the acid is too concentrated, or the heat too great, it is transformed into the pyro-acid under effervescence. To avoid this with certainty it is useful to neutralise the greater bulk, of the free sulphuric acid before evaporation. The crystallised cholesteric acid is collected by filtration through glass wool, washed with a little cold water, and dissolved in ether, from which it crystallises in needles. Properties. — Much more soluble in hot water than in cold ; crystallises from the hot saturated solution on cooling in small needles, which form spherical masses. From alcohol it crystal- lises also in needles. It is obtained in long prisms without water of crystallisation by abandoning to spontaneous evapora- tion its dilute watery solution covered with some ether. The crystals then form principally at the plane of contact between water and ether. The acid is not volatile with steam. Its alcoholic solution turns the plane of polarisation a little to the right, but its specific rotation is probably not more than from + 8 to + 10°. It does not give any colour with sulphuric acid and sugar, and its sodium salt, in doses of from 0'5 to 1'5 grms. injected into the jugular vein of dogs of from 6 to 8 kUos in weight, or of rabbits, produces no toxic effects. Theory of C,2H,gO, : C . . 52-94 H . . 5-88 Salts. — Cholesteric acid is tribasic, and yields salts of the composition CijHiaO^M'g, CuHjgOjM', and mixtures of C,3H,^0, 122 ANNAIS OF CHEMICAL MEDICINE. M'j and C,2H,30,M'3. They are all amorphous with the excep-^ tion of the silver salt, CjjHjgOjAg. The salts with alkalies are little soluble in alcohol, so that when an alcoholic solution of the acid is mixed with an alcoholic solution of potash or soda, a syrupy precipitate is at once obtained which dries over sul- phuric acid to a white amorphous hard mass. Such a salt made by potash was mainly the tribasic mixed with a little dibasic, and yielded 27*3 per cent. K instead of the 30*58 K required by the tribasic salt. The calcium and baryum salts are distin- guished by their lesser solubility in hot than in cold water, their cold saturated solutions become turbid by heat, and clear up again on cooling. The acid cannot be saturated by boiling with carbonate of calcium or baryum ; the quantities of base taken up remain between those required by the tri- and the dibasic salts. At 100° these salts lose constantly in weight, and are trans- formed into salts of the pyro-acid. The tribasic saturated baryum salt is obtained by boiling the acid with baryta water, precipitating the excess of baryum by carbonic acid, filtering hot, and evaporating the solution over sulphuric acid. It is an amorphous white powder, and more stable at 100° than the non-saturated salts ; its solution has an alkaline reaction ; it is insoluble in alcohol. Its formula is CuHjgOjbaj, and-the amount of baryum contained in it 43*22 per cent. The silver-salt CjjHjjOjAgj, is obtained by oversaturating the watery solution of the acid with ammonia and adding silver nitrate, as a white flaky precipitate, insoluble in alcohol and water, somewhat soluble in hot ammonie nitrate, reprecipitated from this on cooling in the amorphous state. The salt bears heating to 140° without blackening much. This is the salt obtained by Eedten- bacher and other inquirers mentioned above, as is evident from a comparison of the analytical data. C,2 . . 24*22 H,3 . . 2*19 Agg . . 54*63 Another silver salt, CijHjgOjAg + H^O is obtained by adding to a moderately concentrated solution of the acid in water nitrate or acetate of silver ; the isolated precipitate is mainly soluble in CHEMOLYSIS OF BILE. 123 alcohol with a strongly acid reaction, and after evaporation of the solvent remains in crystals, probably rhombohedric forms of the hexagonal system ; the salt is much less soluble in water than the free acid ; in the amorphous state it fuses at tempera- tures below 100°, but crystallised it remains unchanged at 110°. Calculated in 100° Ag 28-49 H^O .4-7 Transformation of choleateric into pyro-cholesteric acid. — At a temperature above 100° cholesteric acid gives out carbonic acid, and is transformed into pyro-cholesteric acid, according to the equation CiaHigO,=C^H,505 + COjj. The transformation can be effected quickly by heating the acid to 1 98° in a paraffin bath ; it fuses to a brownish mass with strong effervescence, and the new product is soluble in water, alcohol and ether with acid reaction ; the solution decomposes sodio carbonate with effervescence ; the acid is not volatile with steam, and fuses at 108°. Theory: Cjj = 57*9 per cent.; Hjg=6"8 per cent. When cholesteric acid is boiled with sulphuric acid of 25 per cent, strength it is at first transformed into the pyro-acid ; but later more carbonic acid is given out, and the liquid becomes dark brown. After five hours' boiling the mixture yields to ether several acids, of which one can by distillation be isolated and recognised as similar to acetic acid, another remains behind, soluble in hot water, and deposited on cooling in membranes and flakes. The latter contains 63-0 per cent. C and 6-3 per cent. H. Cholesteric acid, when mixed with even small quantities of pyrocholesteric, loses its readiness to crystallise from water, remains on evaporation as a syrup, on drying as a gum ; from ether it is crystalUsable even when mised with somewhat more pyro-acid. Influence of nitric upon cholesteric acid. — Cholesteric acid dissolves easily in hot nitric acid, diluted with one-third part of water. The solution may be boiled for twenty hours, and after 124 ANNALS OF CHEMICAL MEDICINE. the nitric acid has been distilled off, the residue in water ex- tracted with ether. The residue from the ether solution is an uncrystallisable syrup, and seems, from its silver salts, to be a mixture of much cholesteric and little pyrocholesteric acid. Such was probably the cholesteric acid of Eedtenbacher ; his acid moreover contained perhaps small quantities of nitro- compounds. But the impurity was not so great as to affect the silver salt essentially. From cholesterin, cholesteric acid cannot easily be obtained by the process applicable to cholic acid. Experiments made in that direction have hitherto failed. Fatty acids. — A great number of fatty acids are produced during the oxydation of cholic acid, which form a complicated mixture. This is collected on a funnel, over glass wool, through which the liquid filters ; the insoluble fatty acids are treated with dilute hot soda ley, filtered hot from the chromic oxyde, and allowed to cool. The filtrate gelatinises. The addition of dilute hydrochloric acid precipitates the fatty acids, and cbolanic acid, while chromic oxyde now remains in solution. The precipitated acids are washed, and digested with dilute baryta water. The cholanate of baryum goes into solution, while the fatty acid salts remain insoluble. The latter, on decomposition by hydrochloric acid, yield the white fatty acids. The mixture of these fuses at 53 to 55°, which indicates a mixture of myristic and stearic acid. Stea/ric acid, fusing at 68'5°, was isolated irom the mixture by repeated fractional precipitation with barytic acetate, and identified by analysis of the acid and its baryum soap. Laurie acid, CJ2H24O2, was obtained by oxydising a mix- ture of fatty acids obtained like the stearic, with dichromate mixture, until the fusing point had fallen to 43"6° and did not alter any further. This acid and the acids just above are very resistant to the influence of the oxydising mixtures ; yet much lauric acid must again decompose before the higher acids are oxydised down to lauric acid. Cholanic acid, CgoHjgOg. — When the mixture of fatty acids is treated by baryta water as above described, the baryta salt of this acid goes into solution. The excess of baryta is removed by carbonic acid and boiling, and the filtrate is evaporated at CHEMOLYSIS OF BILE. 125 a low heal untft it begins to crystallise ; the mixture is then rapidly heated to boiling, when a white crystalline baryum salt separates, and can be collected on a hot funnel and washed with boiling water. The concentrated mother liquors give by repeated treatment like this nearly all the cholanate in a crystalline form ; the last residues of the acid can be obtained by precipitation with hydrochloric acid. Cholanic acid is soluble in water, alcohol and ether. One hundred c.c. of absolute alcohol of 20° dissolve 2'55 grms. of the acid ; hot alcohol dissolves more, and deposits the acid on cooling in very thin prisms free from water of crystallisation. Similar crystals are obtained when the acid crystallises from ether, or from the solution of one of its salts in water, to which acid and some ether have been added. The amorphous flakes at first deposited pass into the crystalline state within a few hours. Without the ether the precipitates do not become crystalline even after weeks of repose. Cholanic acid is little soluble in cold water, but more soluble in boiling water ; from this it is deposited on cooling in fine needles which, after drying on the filter, form a shining felt. One part of acid requires for solution 4000 parts of boiling and 9174ofwater of 20°. The acid crystallised from alcohol may be heated to 250° without change, but at higher temperatures it fuses and is decomposed. The alcoholic solution turns the ray of polarised light to the right. Specific rotation about + 53°. The acid does not give Pettenkofer's reaction. It dissolves in oil of vitriol, the solution fluoresces similar to that of cholic acid ; on addition of water the acid crystallises on cooling, apparently unchanged. 1 grm. of its sodium salt, injected into the jugular vein of dogs (5 to 8 kilos) produces no symptoms. Two grms. intro- duced into the stomach act as a cathartic. Cholanic acid forms with bases two series of salts, CjjHjjOgM', and C^^HsjOj^M's; the latter are probably double salts of the components Cj-H, 0,M, + C,oH,,06M'3. Potassium salt, C^jHjjOuK^ + 6 Hfi, is obtained by boiling the alcoholic solution of the acid with potassic carbonate during at least twelve hours. The alcoholic solution is evaporated to 126 ANNALS OF CHEMICAL MEDICINE. dryness and extracted with absolute alcohol. On addition of ether to this solution a turbidity ensues, and after some time the salt is crystallised. It is soluble in water and very deli- quescent in air. H20=11'7 per cent. K =21-24 „ There are other salts containing from 9-9 to 13'4, and 15"0 per cent. K. They are not yet well defined. Baryum salts; C43Hg,0,2bag + 5 H^O. — This is the salt obtained in the first separation of the acid after removal of the fatty acids, by boiling. Theory: C . . 45-05 H . . 4-78 Ba . . 32-14 H^O. . 8-4 The same salt crystallises from cold water with 7 H^O, or 11-6 per cent., in white crusts of felted fine needles. CjoHgjOgba + HjO. When the solution of the salt with 5 baryum equivalents is treated with carbonic acid to complete saturation with this gas, the salt of the foregoing composition is gradually deposited in fine needles, without any barytic car- bonate being mixed with it. The salt is little soluble in cold water, more in hot, and crystallises from this solution on cooling in needles. It is insoluble in alcohol. Theory : c . . 55-61 H . . 6-25 ba . . 15-87 H,0. . 4-1 The lead salt, C4„H5jOj2pb5, is precipitated from an alcoholic solution of cholanic acid by lead acetate ; white amorphous powder, very little soluble in even hot alcohol. Contains 41-66 per cent, of Pb. The silver salt, C4QHjjOj2Ag5 is obtained by adding to the baryum salt of analogous composition silver nitrate, as a white curdy precipitate, very little soluble in water or alcohol, soon blaclsening on exposure to light. CHEMOLYSIS OF BILE. J 27 Theory : c . . 38-0 H . . 4-0 Aff . . 42-75 Cholanate of ethyle, CjjH^jOg . C^Hj, is formed by passing hydrochloric acid gas into a solution of the acid in alcohol. Is precipitated by water after 24 hours, and washed with dilute soda, recrystallised from alcohol. Bearing with hyd/roohloric and with nitric acid. — Cholanic acid can be boiled with a mixture of hydrochloric acid and water in equal parts without undergoing much change. But a hot mixture of equal volumes of nitric acid and water dissolves cholanic acid rapidly under evolution of nitrous acid. When the action is completed long hair-fine crystals are deposited which are very little soluble in cold, slightly more soluble in hot water. They resemble choloidanic acid, first obtained by Theyer and Schlosser (Ann. Chem. 50, 243) and later by Eedten- bacher (Ann. Chem. 57, 145) CjgH ^0,. Theory: Cig . . 58-53 H,, . . 7-31 Oxydation with permanganate in the cold of a dilute solution of the acid gives oxalic acid, and traces of butyric, and several other products, of which the most abundant is an acid of the formula C24H3gOjg. This acid dried in vacuo, is a glass- like brittle mass, very soluble in water and alcohol, little soluble in ether ; dissolved in absolute alcohol and treated with a stream of hydrochloric acid gas, it forms an ether. Treatment of cholio add with reducing agents at high temperatures (A. Destrem, Compt. rend. 87, 1878, .880). — ^The acid was distilled with zinc-po,wder and gave a hydrocarbon corresponding to the formula Cj^Hg^ ; it begins to distil at 215°, and the temperature rises to 325° ; the last portions which distil over are very viscous ; crystalline needles are deposited in the neck of the retort. Theory of the oxydation of cholio add. — As in this process cholesteric acid is formed before stearic and cholanic acid, it cannot be formed from either of them : moreover neither gives by oxydation cholesteric acid ; further, cholanic acid cannot be 128 ANNALS OF CHEMICAL MEDICINE. formed from stearic. The three bodies can therefore be derived from the molecle of cholic acid as ordinarily written, G^^R^gO^, only on the supposition, that the process of cleavage takes place in different molecles of the same acid according to different modes ; it is even necessary to assume that some molecles take up hydrogen ; neither of these assumptions could claim attention in view of the probability that stearic acid is not the highest fatty acid formed in the process, and that already at the beginning of the oxydation considerable quantities of acetic and carbonic acid are set free. The bodies obtained can be derived from cholic acid on the supposition either that the molecle is in- reality two or three .times as great as is at present assumed, or that during the progress of oxydation fragments of ^24^40^6 combine, and form products of condensation so called. The latter hypothesis has not much in its favour, and the circumstance that nitric acid gives products analogous to those obtained by chromate mixture is particularly against it. The acetic, butyric, caprylic and caprinic acid formed cannot be derived from choloidanic or cholesteric acid, for choloidanic acid is not changed by nitric acid, and cholesteric acid is only changed slowly into pyrocholesteric acid. The oxydation products of cholic acid show that the forma- tion of the biliary acids in the liver has probably some connec- tion with the economy of the liver as regards fats. Tappeiner does not think it probable that the biliary acids are produced in the liver synthetically, but accepts them as derivates of albumen, and claims thus to have produced stearic and laurie acid from albuminous matter. The coincidence of increased formation of bile and accu- mulation of fat in the liver after every meal is probably not accidental but relational. The cohesion of glycogen with these processes Tappeiner does not even allude to. The hypothesis that there may be several cholic acids, all of the formula C24H^„05, and yet differently constituted, isomers, not separable by ordinary means, is one of the few possible ways out of what is at present a very complicated position. CHITIN, AN OKGANOPLASTIC AMYLONIDE. 129 VIII. CHITIN, THE SIMPLEST NITROGENISED ORGANO- PLASTIC AMYLONIDE. {Summary and Additions.) Chitin was first distinguished from horn substance hy Odier in 1823, and supposed to be free from nitrogen (ifem. Sog. d'Hist. Nat. de Paris, 1, 29). Lassaigne recognised that it (named by him entomaderm) contained nitrogen {Gompt. rend. 16, 1087; Journ. Chim. Med. 19, 379). It was next studied by Payen {Gom/pt. rend. 17, 227), and more intimately by K. Schmidt {Ann. Pharm. 54, 298). Schlossberger {Ann. Chem. 98, 105) contributed to its more accurate knowledge by defining the organic matter contained in some shells, and in the byssus of shells which had been confounded with chitin, as conchiolin. Berthelot {Compt. rend. 47, 227 ; and in detail N. Ann. Chim. Phys. 56, 149) found that chitin on chemolysis with acid gave a kind of sugar. Peligot {Gompt. rend. 47, 1034 ; N. Ann. Chim. Phys. 58, 83), in consequence, endea- voured to find the amylumlike body in chitin which gave this sugar. Stadeler {Ann. Pharm. Ill, 21) also studied chitin, and gave a new formula differing from that first given by Schmidt. Gr. Ledderhose {Zeitschr. Physiol. Chem. 2, 1878, 213) found the sugarlike body to contain nitrogen, and termed it glycosamin. He also proposed a new formula for chitin. Fremy's and Eouget's chitin, see below, pp. 138 and 139. Occurrence. — Chitin forms the principal constituent of the dermatic envelope and entire firm skeleton, tendonlike parts, and attachments of muscles of nearly all articulate animals, and of insects not only in the fully developed, but also in the larval, and pupal state. It is also said to be present in the lining membranes of the tracheae of air-breathing articulates, and forms part of the gills of those breathing water ; it is also a constituent of the lining membrane of the iptestinal canal. It K 130 ANNALS OF CHEMICAL MEDICINE. possesses great flexibility, when it forms the external organs, membranes and hairs of animals by itself, combined only with little inorganic salt and organic colouring matter ; but it ac- quires great hardness by an impregnation, in the course of its structural development, with carbonate and phosphate of lime, and then shows as dermatic skeleton analogy with the ossein of the skeleton of the vertebrate animals. As the external envelope of animals of the class of articu- lates is formed gradually, and is changed repeatedly during their growth and life, it is advisable to bear in mind that the chitin obtainable by chemical proceedings, may not be the same in the same species at all times, and may differ in different species. The bones of young vertebrates yield' chondrin, those of adult ones ossein; the growing antlers of the stag yield chondrin only, when just fullgrown they yield chondrin and ossein, and after maturation and scouring ossein only. Modes of preparation. — The materials which yield chitin with the least trouble are perhaps, in places near the seashore the transparent crustaceans, such as shrimps, prawns, and langostinos ; but in inliand countries the wing-cases of coleoptera. If entire insects are taken it is necessary to remove mechanically the internal organs, particularly the intestinal canal, to exclude the possible admixtiu-e with the chitin of any cellulose from leaves or other vegetable structures which have served as food. The shells of larger aqxiatic crustaceans are also a convenient and easily procurable material, although they require prolonged treatment with much acid for the removal of the calcic carbonate and phosphate. The materials selected are exhausted by boiling with caustic soda, prolonged treatment with dilute hydrochloric acid, second boiling with caustic soda, then with water, and long boiling with alcohol in succession. If the chalky shells of large articu- lates are used the following succession of operations may be adopted. The shells are pounded in a metal mortar, until all tubes are crushed, and all stiff parts comminuted. The pasty mass is now boiled with strong caustic alkaline ley, and the latter is repeatedly changed ; when all matter soluble in alkali is removed, the well-washed mass is placed in dilute hydrochloric acid, and treated with this agent until no further effervescence CHITISr, AN OKGANOPLASTIC AMYLONIDE. 131 is perceived on standing, and until all particles are soft and translucent. The residue is now washed, and boiled with spirit until all colouring and other soluble matter is extracted. The chitin thus obtained is dried on the water-bath, and shrinks to a tough, shrivelled, greyish white mass of particles. These may be ground in a cofifee-mill. Chitin thus prepared can be puri- fied, according to Stadeler, by boiling it for 12 hours or longer with a mixture of 1 vol. of oil of vitriol and 4 vols, of water, when a part of it is changed into soluble matters, crustacin (alkaloid), and chitose (sugar), while another part remains as a swelled pasty mass, being almost pure chitin free from ash. This may be washed by decantation as long as it contains free acid, but as soon as all the acid is removed it remains partly sus- pended in the liquid ; the latter must therefore be evaporated to dryness. When Peligot treated the skins of silkworms with caustic ley, alcohol, ether, and acetic acid, he obtained translucent bags, open at both ends, consisting of a chitin which contained 48-13 per cent. C, 6-90 per cent. H, 8*30 per cent. N, and 36*67 per cent. 0. After this had been digested with dilute permanganate of potash, it contained 47-38 per cent. C, 7*02 per cent. H, 6*15 per cent. N, and 39*45 per cent. 0. When this was treated with sulphuric acid containing 6 molecles of water, 44 per cent, of its weight was transformed into soluble products, while the residue still contained 5*8 per cent, of nitrogen. The latter was then treated for several hours with hot, or for some days with cold concentrated watery solution of potassic per- manganate, then with potassic bisulphite, and finally washed with water. The remaining white mass did not evolve ammonia when boiled with potash ley. But when it was made up into a paste with potash hydrate and water, and heated to 100° for eight days it dissolved, evolving ammonia on the one hand, and forming fatty acids on the other, which were set free by acids. Even after two-thirds of the chitin had thus been decomposed, the remainder recovered from the alkali still con- tained 6*2 per cent, of nitrogen, and continued to evolve am- monia on renewed application of potash. When this chitin, after having been softened in water, was treated with oil of vitriol and iodine, it exhibited under the microscope brown k2 132 ANNALS OF CHEMICAL MEDICINE. spots and blue particles of irregular shape. The blue colour became more plainly visible, if the skin was left for some days in the iodine solution, and then moistened with oil of vitriol whereupon entire pieces of the membrane became greenish at first, but changed to indigo blue as the iodine evaporated. From this chitin moreover, watery cuprammonia extracted cellulose, which was precipitated from the solution by acids. The horny substance of the lobster also exhibited the reaction of cellulose with oil of vitriol and iodine. Stadeler declared this chitin to have been impure, as he himself could extract nothing from pure chitin by cuprammonia. But it must be borne in mind that this impurity could only have arisen from a structural admixture of cellulose with chitin, and therefore chitin which gives this reaction is unsuitable for experiments on pure chitin, as at present chitin and cellulose cannot be separated by the means ordinarily applied for the purification of chitin. Properties. — Chitin, when purified by boiling with dilute sulphuric acid, or by solution in concentrated hydrochloric acid and precipitation with water, is a white amorphous mass, some-" times tough and difficult to reduce to a powder. Composition. — Its elementary composition has been esti- mated by many observers with the following results. C H N Children and DanielP . 46-08 5-96 10-29 37-67 K. Schmidt (mean of 16 combustions) 46-66 6-60 6-53 40-21 K. Schmidt's maxima . 46-80 6-77 6-79 — „ minima . 46-48 6-43 6-32 Stadeler 46-32 6-40 6-14 41-14 Lehmann (mean) " 46-734 6-594 6-493 40179 Ledderhose (mean of 12 combustions) . 45-69 6-42 7-00 — Ledderhose (mean of analyses with more C) 46-026 6-256 7-00 ^ K. Schmidt had calculated the empirical formula CjjHjgNjO,, from his analyses, having deducted from 0'6 to 2*0 per cent, of ' Todd's CyohpcBdia of Anat. and Physiol. 2, 282. 2 LeArb. 1853, i. 383. CHITIN, AN OEGANOPLASTIC AMYLONIBE. 133 ash (chitin from Crustacea). Schmidt's formula was CijHi^NOj,, when C=6 ; 0=8; on account of its decomposition by heat and acids, he was of opinion that chitin contained the ele- ments of the primitive fibres of the muscles of arthropodes (CgHjNOj) with a carbohydrate (CgHjOg). But Stadeler cal- culated the formula CgHijNOg, which being more simple and apparently supported by the decomposition of chitin into a glucose and a hypothetical amido-acid was more generally adopted. Its theory agreed with Stadeler's results as follows : Stadeler 9C . . 108 46-35 46-32 15 H . . 15 6-44 6-40 N . 14 6-01 6-14 60 . . 96 41-20 41-14 233 100-00 The nitrogen had been found much higher by Children and Daniell, namely, 10-29 per cent. ; Payen found 8*99 per cent. ; Schlossberger 6"4 per cent. N. Biitschli and Emmerling (Du Bois and 'ReiehevVs Archiv, 1874, 362), estimated the nitrogen in chitin by the two rival methods of analysis. Biitschli found by combustion with soda lime as the mean of three experiments 6-32 per cent., by combustion with cupric oxide in carbonic acid atmosphere as the mean of two experiments 7-385 per cent. N. Emmerling {loc. cit. p. 370), analysed chitin which had been precipitated from hydrochloric acid solution by water and obtained by combustion with soda lime 6*24 per cent., by com- bustion with cupric oxide 7-02 per cent. N. In the last analysis a loss is noted to have been incurred. Ledderhose estimated the nitrogen in chitin twice by combustion with soda lime, observing the precautions otherwise known, but lately again pointed out by Makris {Ann. Chem. 184, 371), and obtained 6-96 per cent, and 7*049 per cent., or mean 7-00 per cent, nitrogen. With the aid of these later data concerning the percentage of nitrogen, Ledderhose came to the formula CjHjgNOg, which requires Mean of all Ms analyses 45-685 45-69 6-598 6-42 7-10 7-00 134 ANNALS OF CHEMICAL MEDICINE. C . . 47-29 H . . 6-40 N . . 6-896 but thinking that the amount of carbon required by it was too high, as compared with that found by analysis, he preferred CisHaeNjjOio, which requires C . H . N . But in the endeavour to support this formula by an equation intended to express the structure of the body, he unfortunately fell into an error, whereby CijH^g in the left half of the equation are supposed to be counterbalanced in the right half by CjgHjg. The error is not a misprint, but is stated in words printed in emphatic type (loc. cit. p. 224 to 225), and the supposed equation is repeated twice over. That chitin was free from sulphur and phosphorus was expressly proved by Schmidt. Decompositions. — When heated in the open chitin does not change in form, but simply chars, without evolving carbonate of ammonia. But when subjected to thermolysis in a closed apparatus it gives out water, acetic acid, ammonic acetate, and a small quantity of an empyreumatic oil, while a charcoal of the original form of the chitin remains. Under the influence of air and water it is only slightly altered even after long periods ; thus Schlossberger kept some chitin under water for a year, and found it only partly dissolved, the other part changed into a slimy mass. Long boiling with water does not alter the purified skins of silkworms, but the water takes up a small quantity of a nitrogenous substance, which when the solution is evaporated to a small bulk, is precipitated by tannic acid, chlorine or alcohol. Owing to this reaction Lassaigne believed that some gelatin had been formed from the skins ; but this has probably no relation to the chitin itself, but was only the effect of the adhesion to the skin of, or penetration of its structure by a collogenetic matter not yet isolated. When Schmidt heated chitin with water in a sealed tube to 280°, he CHITIN, AIT OEGANOPLASTIC AMYLONIDE. 135 observed it to become brown and brittle, but not to alter other- vdse. CMtin dissolves readily in oil of vitriol, forming a thick solution ; this gives a copious precipitate when immediately or after 12 hours thrown into water. The solution in oil of vitriol is colourless at first ; on standing it deposits a few dark flakes ; I have not observed it to become black after 12 hours, but Schmidt saw it become black after 48 hours, and perceived that it had deposited a small quantity of insoluble matter, had a choking smell, and contained acetic acid and an ammonium salt. The colourless solution of chitin in oil of vitriol may, after standing some hours, be diluted and then yields a precipi- tate of unchanged chitin, but if it has been heated and decom- posed it yields, neutralised with barytic carbonate, a soluble baryum salt (acetate), and reduces alkaline copper solution (chitose). Berthelot dissolved chitin in oil of vitriol and dropped the solution into a hundred times its weight of water ; he then boiled for an hour, and neutralised with chalk ; on evaporation he obtained a sugar, which was fermentable (alcohol was isolated), and reduced alkaline copper solution. Stadeler now gave to this experiment a more precise explanation. He boiled chitin with a mixture of one volume of oil of vitriol and four volumes of water for 12 hours or longer, separated what remained insoluble, supersaturated with lime (whereupon ammonia was evolved), and evaporated the filtrate to a syrup. This remained amorphous, and reduced cupro-potassic tartrate. No tyrosin, leucin, or glykokoU could be obtained from the syrup, but it contained a small quantity of an amorphous matter, the nature of which he failed to ascertain. He formu- lated the splitting up of chitin as follows, overlooking entirely the formation of acetic acid first found by Schmidt, C9Hj,NOe + 2 H,0=C«H,,06+C,H,N0^. Sericin is C3HJNO3. Chitin is soluble in cold concentrated hydrochloric acid without change of colour. The skins of insects when immersed in hydrochloric acid containing six molecles of water, become transparent, are disintegrated, and after a few moments dissolved. The shells of crustaceans are never entirely soluble in hydrochloric acid in the cold. After neutralisation of the solution by alkalies, tannic acid throws 136 ANNALS OF CHEMICAL MEDICINE. down a precipitate containing nitrogen (crustacin ?). The solution of chitin in concentrated hydrochloric acid is not changed quickly, but deposits the entire amount of the chitin which it contains by suitable dilution with water. When the hydrochloric solution is mixed with alcohol two bodies free from nitrogen are obtained, the nature of which has not been ascertained by their discoverer Biitschli. One part of purified lobster shell dissolves in twelve parts of hydrochloric acid at 40°, and is immediately chemolysed. A cold hydrochloric acid solution on being boiled for some time becomes blackish-brown, and after an hour's boiling the chitin is completely chemolysed. If it is now evaporated on the water-bath, a compound crystal- lises in large quantities, which Ledderhose has described as hydro(!hlorate of glycosamin, and to which he ascribes the formula C0H.(CH.0H),.0H2.NHj + HCl, or CgH^sNOj -1- HCl, being that of a carbohydrate in which one group OH is substituted by NHj, while a molecle of HCl is in so-called molecular combination. The crystals are mixed with a black humuslike matter, which is completely insoluble in water, and can be separated from the crystals after expulsion of all free hydro- chloric acid, by repeated crystallisation of the salt from water. The quantity of the new salt containing the new base (which as an amide of a previously known body, chitose, we term chitosamin) obtainable from a given weight of chitin was estimated as follows. A weighed portion of chitin dried at 110° was boiled during an hour with concentrated hydrochloric acid, and evaporated to dryness ; the residue was dissolved in water, and digested for some time on the water-bath, and freed from black flakes by filtration ; the Solution was again evaporated, dried at 100° and weighed. The product amounted to 91 per cent, of the chitin employed. This salt lost but little in weight by a repetition of the purifying process. The amount of chitos- amin formed from chitin may therefore be calculated, deducting the hydrochloric acid, and allowing for some impurity, to be from 70 to 75 per cent, of the chitin employed. The solution contains no amnionic chloride, so that it seems as if the entire amount of the nitrogen contained in chitin was transferred inio tHe chitosamin. No oxalic acid is formed, and it seems as if chitosamin was the only fixed product of the chemolysis. CHIXm, AN OKGANOPLASTIC AEYLONIDE. 137 The solution of chitin ia concentrated hydrocliloric acid is subjected to distillation to collect the volatile products. The distillation is repeated after addition of water to the liquid in the retort. The distillates are neutralised with silver oxide, the dissolved silver is removed by hydrothion, and the acid filtrate is neutralised with barytic carbonate and evaporated. A crystalline salt is obtained having the character of acetate. But there is a small amount of a higher fatty acid present, which depresses the baryta a little, as butyroacetic probably, and can be recog- nised by its peculiar smelL By a comparison of the results of Berthelot with his own, Ledderhose comes to the result, that chitin by treatment with concentrated sulphuric acid, is chemolysed in the same manner as by concentrated hydrochloric acid, giving chitosamin and acetic acid ; but when these products are boiled long in very dilute solution, the chitosamin is further split up into ferment- able sugar and ammonia. The decomposition of chitin under the influence of acids and heat might therefore find its simplest expression in the formula C,H,3N05+ 2H,0 = C,Hj3NO, + C,H,0,. The small amount of butyric acid obtained seems to indicate that with the simplest chitin, aceto-chitin, there occurs mixed a butyro-chitin, which contains the radicle butyryl in the place where the simplest chitin contains the radicle acetyl. Chemolysis of chitin with sulphuric acid. — Twenty grms. dry pure chitin were heated in a sealed lead tube with 360 c.c. of sulphuric acid of 10 per cent, strength for 48 hours. The liquid was then found clear and to contain a reducing body and acetic acid, but a large proportion of the. chitin was only softened, of the consistence of cheese, another flaky. The liquid was filtered off and the solid residue treated with oil of vitriol. It dissolved, forming a transparent thick fluid. This with water gave a copious white precipitate of pure chitin. All the portions of liquid and precipitate were boiled for eight hours in platinum still, and three litres of distillate were drawn one after the other, water being added after each litre drawn off. The 138 ANNALS OF CHEMICAL MEDICINE, Uqiiid in the still contained yet much undissolved and appa- rently undecomposed chitin. The sulphuric acid solution gave a precipitate with phoaphotungstic acid. The undecomposed chitin was treated with fresh oil of vitriol and the solution treated with water. Pure white chitin was precipitated, which after washing with water and absolute alcohol, appeared as a white amorphous mass, like starch. The solution contained an alkaloid, crustacin, precipitable by phosphomolybdic acid, and a sugar, chitose. Chitin is coloured orange yellow to dark brown red by iodine water ; addition of oil of vitriol to this test causes the chitin to dissolve, but does not produce either a violet or a blue colouration. Concentrated nitric acid dissolves chitin without discolouration ; one part of chitin dissolves in an equal weight of cold nitric acid containing four molecles of water. By the addition of a large amount of water chitin is again pre- cipitated ; but some acetic acid is always formed, the more the longer the action of the acid has continued. Payen observed that crabs' claws immersed in nitric acid became transparent after a few moments ; when the acid was drained oflF, the mem- brane retained at first its original form, but afterwards deli- quesced to a colourless liquid, beginning at the edges. Alkalies in the dilute or even pretty concentrated state alter or affect chitin so little, that they can be employed for its purification. The alkali may be as strong as one part of potash hydrate in three parts of water, and the chitin may be boiled in this for days, or even heated with it to 210° in sealed tubes without being sensibly attacked. But when it is fused with solid hydrate of potash, it evolves ammonia (Payen) and hydrc^en (Ledderhose) without charring, and leaves a residue soluble in water. If the fusion is not continued until this mass is white, but interrupted, and the fused mass is dissolved in water, mixed with sulphmic acid, and distilled, acetic and a little butyric acid are obtained, and recognised by their baryum salt. Frimy's chitin, free from nitrogen. (Gompt. rend. 39, 1052 ; N. Ann. Chim. Phya. 43, 1855, 93).— The skeletons of Crustacea are treated with cold dilute hydrochloric acid, the undissolved residue is boiled for several hours with potash-ley CHITIN, AN ORGANOPLASTIC AMYIONIDE. 139 and then washed with water, alcohol and ether. A transparent horny substance is thus obtained, containing on the average 43'35 per cent. C, 6'65 H, and 50-00 per cent. 0, but no nitrogen, and therefore isomeric with cellulose (CgHj^Oj). This substance resists the influence of dilute acids and alkalies ; acids in particular do not convert it into sugar, and fuming nitric acid has no action on it whatever. By other concentrated mineral acids it is broken up, dissolved and converted into an acid, which Fremy compares with his metapectic acid ; by boiling nitric acid he obtained oxalic acid. Eouget's chitin and Tfwdified chitin. (Compt. rend. 48, 793.) — The dermatic envelopes of the Crustacea, in their natural state, or dissolved in hydrochloric acid, when treated with mer- curic nitrate give a rose-red colouration ; when treated with nitric acid and ammonia an orange-red colouration, and when mixed with iodo-chloride of zinc a violet reaction. (Zinc chloride solution sp. gr. 1'8 at 15° 100 parts; potassic iodide 6 parts to be dissolved in the syrup, and then as much iodine as the mixture will take up.) The skins, after ebullition with potash ley of 40°, which does not alter their appearance or structure, react more quickly with the iodo-chloride of zinc, producing a more intense bluish violet colour ; but they remain still unchanged by concentrated potash ley, acetic or tartaric acid. Boiled for half an hour with five times their weight of potassic hydrate and such a quantity of water that the mixture would immediately solidify if allowed to cool, a large quantity of ammonia is liberated and half the chitin is dissolved. The residue (modified chitin) shows traces of the microscopic struc- ture of the original tissue, is translucent, gelatinous and easily broken up while moist ; in the dry state it is greyish white, dull of appearance, and light in weight. With tincture of iodine, or with iodine and dilute acetic acid, this residue becomes violet throughout ; iodo-chloride of zinc colours it pure blue. It dissolves almost instantly in acetic or tartaric acid, or in warm water containing 0*5 per cent, of hydrochloric or nitric acid, and is precipitated from these solutions by alcohol or by caustic ley as a somewhat translucent paste, which dries up to a yellowish translucent gum. The acid solution and the precipi- tate are coloured reddish violet by a few drops of a solution of 140 ANNALS OF CHEMICAL MEDICINE. iodine in potassic iodide. It dissolves in oil of vitriol with yellow or brown colour and is precipitated by water from the freshly prepared solution as a white powder. The solution is less completely precipitated by water after having been allowed to stand for 12 to 24 hours, and is then found to contain sugar. After being precipitated by alcohol or alkalies from its solution in acid, it is still soluble in acids, and contains nitrogen. Purified chitin is insoluble in water, aqueous ammonia, acetic acid, alcohol and cuprammonia. The dark amorphous humus-like substances obtained during chemolysis of chitin remain to be investigated. GhitosaTni/n, CgHjjNOj,- is obtained from the sulphate by treatment with barytic hydrate without employment of heat. It crystallises from alcohol in needles. The nitrate and sulphate are obtained by treating the hydrochlorate with nitrate or sulphate of silver ; both salts crystallise in fine needles. A double salt of chitosamin and lead chloride crystallises from water. The hydrochlorate is obtained directly from chitin in the manner above described. It turns the ray of polarised light to the right. The limited, or so-called specific rotation according to Ledderhose is + 70°, according to observation made by me in the Pathological Institute +72° 15'. The rotation is more considerable immediately after solution, and falls gradually during 24 hours, after which it becomes stable at the point related. A hundred parts of satm-ated solution of chitosamiur hydro- chlorate contain 21*83 per cent, of salt dry at 100. All compounds reduce potassio-cupric tartrate, but do not ferment with yeast. Behaviour of chitvn in various species of a/nimals. — As to organs see introduction to this chapter above, also K. Schmidt, Zur vergl. Physiol, der wirbellosen Thiere, Braunschweig, 1845 ; Frey and Leuckart, Wagner's Zootomie, 2, 133 and 167; Grrube, Wiegm. Archiv, 1853, 104; Leydig, MiiUer's Arch. 1855, 376. On other (problematical) statements concerning the occur- Ul rence of tissues containing chitin' in other nonvertebrates compare Schlossberger, Vers. etc. Thierchemie, 1856, 1, 1, 230. Leyer and KoUer {Ann. Chem. 83, 336) and Schlossberger (loc. cit. 228) when chemolysing entire wing-cases of cockchafers obtained leucin and tyrosin. This therefore was due to a constituent other than chitin. Schmidt esamined chitin from Melolontha vulgaris, Ateuchus sacer, Astaous fluviatilis, Astacus marinus, Squilla mantis, and found the elementary composition as stated above. Schlossberger {loc. cit. 227) examined the shell of Palinurus, and found in its chitin 6*4 per cent. N. The shells of Crustacea contain up to 63 per cent, of organic matter ; the mineral ingredients are mainly carbonate and phosphate of calcium. Thorax of crayfish contained : organic matter 33*3 per cent. ; calcic carbonate with little sodic chloride, iron, manganese, and pigment 61*0 per cent. ; calcic phosphate 5'7 per cent. (John, Chem. Schriften, 3, 49.) Crayfish, shells : organic matter 28"0 per cent. ; calcic carbonate 60*0 per cent. ; calcic phosphate 12-0 per cent. (Merat and Gruillot, Ann. Chim. 34, 71.) Crayfish, thorax: chitin 46*73 per cent.; ash 53-27 per cent. In this 86*83 per cent, calcic carbonate, and 13'17 per cent, calcic phosphate. (K. Schmidt, loc. cit.) Shell of crayfish : organic matter 36"5 per cent. ; calcic carbonate 56*8 per cent. ; calcic phosphate 6*7 per cent. (Fremy, loc. cit.) Shell of crab : organic matter and water 28"6 per cent. ; sodium salts 1'6 per cent.; calcic carbonate 62*8 per cent.; calcic phosphate 6*0 per cent.; magnesic phosphate 1-0 per cent. (Groebel, Schweigger's Journ. 39, 440.) The same inquirers found in the claws of crab: organic matter 17-18 per cent.; calcic carbonate 68*36 per cent. ; calcic phosphate 14*06 per cent. ; in the teeth of the claws : organic matter 12*75 per cent. ; calcic carbonate 68*25 per cent. ; calcic phosphate 18*75 per cent.; shell of crab : organic matter 28*6 per cent. ; calcic carbonate 62*80 per cent. ; calcic phosphate 6*0 per cent. (Chevreul, Ann. gen. des. sc. phys. 4, 124^ Schweigg. Journ. 2,495.) 142 ANNALS OF CHEMICAL MEDICINE. Shells of squilla mantis : chitin 62-84 per cent. ; salts 37-17 per cent. (Of these calcic phosphate 47-52 per cent., and calcic carbonate 52-48 per cent. K. Schmidt, loc. cit.) Claws of lobster : chitin 22-94 per cent. In the ash of these claws 87-94 calcic carb. and 12-06 calcic phosphate. (Schmidt, loc. dt.) Claws of lobster : organic matter and water 44-76 per cent. ; sodium salts 1-5 percent.; calcic carbonate 49-26 per cent.; calcic phosphate 3-22 per cent. ; magnesic phosphate 1-26 per cent. (Chevreul, loc. dt.) Claws of lobster : organic matter 28-0 ; calcic carbonate 40 per cent.; calcic phosphate 14-0 per cent. ; (Gruillot, loc. dt.) Shell of lobster : organic matter 44-3 per cent. ; calcic carbonate 49-0 per cent. ; calcic phosphate 6-7 per cent. (Fremy, loc. cit.) Considering that the dermatic envelope and motor parts in connection therewith, as well as respiring and digesting surfaces of three entire classes of invertebrate animals, are to a large extent made up of chitin, the foregoing data, which include all that we have been able to collect from readily accessible sources, must be considered very scanty. It is therefore to be hoped that students of comparative biology will give some attention to this interesting subject. HEMISYMMETKY OF ALBUMIN. 143 IX. ON HEMISYMMETMY IN THE CHEMICAL CONSTITUTION OF ORGANOPLASTIC SUBSTANCES.— HEMIPROTEIN AND BEMIALB UMIN. (Summary and Addition.) The experiments of chemists who operated on the albumi- nous substances with acids were mostly directed to effect a complete splitting up of the molecle into the most simple final products. The intermediate products of the processes were, as a rule, not studied, and with a view to avoiding their formation, or making their existence as transitional as possible, the acids were chosen of such concentration as would effect the desired purpose in the shortest time. From this practice Schiitzenberger deviated in one of his earlier experiments, when he subjected albumin to the influence of boiling dilute sulphuric acid for a short time, and examined the products (BuUet. Soc. Chim. Paris. N.S. 23, 161). He took a quantity of coagulated moist egg-albumin corresponding to 1 kilogramme of dry albumin, distributed it in 6-8 litres of water, and added 200 grms. of oil of vitriol of 66° B. The mixture was then heated during from one and a half to two hom-s by steam. The liqiud was kept on a fixed level to avoid the formation of brown products by drying on the margins. The albumin became disintegrated and formed at last a homogeneous white gruel. To effect a more even influence of the acid the boiling may be interrupted when the albumin has fallen to small pieces, and the mixture may be worked with a brush through a fine-meshed tammy. After this operation the boiling is resumed and continued to the end of the two hours. The mixture is allowed to cool, and filtered through a bag of felt. There remains a precipitate which has great resemblance to freshly precipitated silica or 144 ANNALS OF CHEMICAI. MEDICINE. alumina, and a liquid containing the acid and its pr&ducts in solution passes through. The insoluble precipitate on the filter is termed hemiprotein, the soluble matter remaining in the acid solution hemialbumin. Hemiprotei7b. — It is washed with water until the filtrates cease to show an acid reaction. It is then white, tasteless, and has a very feeble acid reaction ; it dries to a friable mass, which breaks up spontaneously into small, irregular, polyhedric, trans- parent, yellowish fragments, and when ground up in a mortar, yields a nearly white, non-hygroscopic powder, which is insoluble in water, alcohol and ether. The weight of the perfectly dry substance amounts to about one-half of that of the dry coagu- lated albumin employed. The albumin is therefore split up by a short influence of hot dilute sulphuric acid into two nearly equal halves, one soluble, the other insoluble in the acid, being substances which in consequence of the constancy of their properties and their composition may be considered as proximate constituents of albumin. It might be assumed that the boiling of the albumin with dilute sulphuric acid during a limited time effected nothing but the transformation of a part of the originally coagulated substance into soluble compounds, and that the remaining insoluble matter was only unchanged or nearly unchanged albumin. But this explanation is found to be inadmissible on closer examination. For if this were the case, the residue, treated again with hot sulpharic acid during the same period as in the first operation, would have to decrease in quantity, and that in a direct ratio to the duration of the heating. This is however not the case, for the insoluble residue, the hemiprotein, is under these circumstances only very slightly and slowly changed ; its weight diminishes at a ratio which, in comparison with the first diminution of the weight of the coagulated albumin, is hardly perceptible ; further, the products which are obtained by the subsequent influence of acid upon hemiprotein are entirely different from those which are obtained in solution by the first cleavage. Hemiprotein is amorphous. In certain respects it is like the amido acids (leucin etc.) combining with acids and bases without altering its constitution ; it is easily soluble in alkalies, HEMISYMMETRY OF ALBUMIN. 145 and the solution is again precipitated by hydrochloric acid, but the precipitate is soluble in excess of acid. By solution in dilute soda ley, accurate neutralisation with hydrochlor, and washing of the precipitate with distilled water and alcohol, hemiprotein is obtained free from incombustible ingredients. Dried at 100° it gives on quantation the following mean percentage of elements : G 53-45 ; H 7-19 ; N 14-4o. Treatment with hot dilute sulphmic acid seems to raise the carbon in the hemiprotein. It contains sulphur, the amount of which has not been estimated ; it gives the colour reactions of the albuminous substances, becomes yellow with nitric acid, red with mercuric nitrate ; it dissolves in moderately concentrated sulphuric acid, and is precipitated from it by water unchanged. If to such a solution a few drops of a sugar solution are added, and the mixture is heated to 60°, a beautiful violet colouration is produced, which reminds of Pettenkofer's reaction for biliary acids. Ghemolysis of hemiprotem by dilute sulphuric add and hoU/mg. — When hemiprotein is boiled for a longer period with dilute sulphuric acid, it is split up gradually and slowly into new compounds, all soluble ; amongst these are always tyrosin and leucin, and their homologues, but the most peculiar product is an amorphous colourless body of sweetish taste, soluble in water and alcohol, and precipitable by mercuric nitrate. The solubility in alcohol and the bearing with barytic hydrate, to be described lower down, distinguish hemiproteidine, as the new compound is called, from the hemialbumin which is obtained in the first chemolysis of albumin; hemialbumin is soluble in water but insoluble in alcohol. Hemiproteidine gave the following results on quantation of its elements. Dried at 100° Dried at 120° C . . . 45-9 47-73 H . . . 6-65 6-48 N . . . 14-00 14-50 From these figures the formula 0,41142X50,2 + 11^0 may be calculated as an abbreviated expression of the results of analysis. At 100° hemiproteidine is therefore a hydrate. 146 ANKALS OF CHEMICAL MEDICINE. Hemialhum/in and oxyhemialhuim/n. — The sulphuric acid solution which has been separated from the hemiprotein, and which contains about half the amount of albumin originally acted upon in solution, is neutralised by baryta, separated from the sulphate, and from the excess of baryta by carbonic acid, and concentrated. On the addition of an excess of alcohol to this concentrated solution a light yellow, plaster-like mass, consisting of a deliquescent baryum-salt, insoluble in alcohol, is separated, while only small quantities of matter remain in solution. Treatment of the haryum-salt with mercuric nitrate. — The solution in water of the baryum-salt is precipitated with acid mercuric nitrate, while the reaction of the solution is kept neutral by suitable addition of baryta water. The white volu- minous precipitate is washed with hot water, suspended in water, and decomposed by hydration, the filtered solution is evaporated in vacuo, and yields dry, friable, amorphous, feebly hygroscopic matter, which is easily soluble in water, but insoluble in alcohol. It has the properties of the protein tritoxyde of Mulder, obtained by boiling coagulated albumin with water in contact with air, or at 150° under pressure. This body, which constitutes the greater part of the sub- stances dissolved in sulphuric acid, does no longer yield the colour reactions which characterise the albuminous substances. Sulphuric acid does not colour it yellow, mercuric nitrate does not make it red. With sugar and sulphuric acid it gives no reaction. Its principal positive properties are that it is pre- cipitated from its watery solution by acid mercuric nitrate, tannin, and ammoniacal lead acetate ; an excess of the latter reagent redissolves the precipitate. The mercury precipitate contains a mixture of two substances of which one contains more oxygen than the other. They can be separated to some extent by precipitation with basic and neutral ammoniacal lead acetate. The more oxygenated product has a clearly acid reaction, and seems to be more easily pre- cipitated by basic acetate of lead. From a number of elementary quantations it is probable that the hemialbumin, obtained by cleavage with dilute sulphuric acid, consists mainly of an amorphous body of feebly HEMISYMMETEY OF ALBUMIN. 1-17 acid properties, containing about 50 per cent. C, 7 per cent. H, and 15*4 per cent. N. From these numbers the formula C24H4QNgO,o may approximately be calculated, which differs from the soluble derivate obtained from hemiprotein by a minus of 2 H,0 and a plus of H^ ; C^^H^oNgOio + H^ - 2 H^O = C,JI,iN^O^^. Mixed ■with the hemialbumin is found, but only in small quan- tity, an acid preeipitable by basic lead acetate and mercuric nitrate, whose composition can be expressed by the formula Cg^H^pNjOij. This formula differs from that of the main product only by a plus of three oxygen. The mother liquors of the mercuric nitrate precipitate (from the hemialbumin solution) contain a nitrogenised body, which strongly reduces potassio-cupric tartrate, is precipitated by amm,oniaoal plumbic acetate, gives no precipitate with mer- curic nitrate, and is probably an amidated glykose. The sulphuric acid solution moreover contains small quantities of compounds, which are in relation to hypoxanthin, and can be transformed into this body, by precipitating the boiling solu- tion with cupric acetate, dissolving the precipitate in nitric acid, and precipitating the solution with ammoniacal silver nitrate. Hemialbumin can be further spb't up into relatively simple crystalloids by chemolysis with barytic hydrate. 1 2 148 ANNALS OF CHEMICAL MEDICINE. CHEMOLTSIS OF ALBUMIN BT FUSING CAUSTIC ALKALI PRODUCTION OF INBOL, SKATOL, FYRROL, PHENOL AND BUTYRIC ACID. {Summary.) The chemolytic action of . dilute caustic alkaline leys upon albuminous substances as applied by Mulder, showed that the products of these agents were essentially the same as those of the chemolytic actions of acids, and those of the physiolytic action of putrefaction. The chemolytic power of fusing caustic potash was first applied to albumin by Liebig, and led to the discovery of tyrosin. It was subsequently used by Bopp {Ann. Chem. 69, 31) who observed that besides the fixed products already known a volatile body was formed, which had the odour of faeces, and was probably identical with a body of similar general properties obtained during the putrefaction of casein. Similar experiments were lately made by Kiihne (Ber. D. Chem. Ges. 1875, 206). But instead of fusing, as Bopp had done, equal parts of caustic potash and albumin, he heated one part of albumin with ten parts of caustic potash gradually in an iron retort to a dark red heat. He obtained a body which was identical with indol in all its properties except one, namely the fusing point. These experiments were next repeated by Engler and Janecke {Ber. D, Chem. Ges. 1876, 1411); they heated the mixture to a still higher degree, namely to the point at which brown drops of oil were deposited in the upper part of the tube leading from the retort. They then allowed the retort to cool, added some water to its contents and distilled again. This process was repeated as long as crystalline deposits were observed in the condensers. These deposits ceased to appear in each experiment after five days of heating. They obtained in PiEO-CHEMGLYSIS OF ALBUMIN. 149 this way from blood albumin about 0*25 per cent, of crystalline matter ■which had the peculiar smell of indol ; its solution im- parted a red colour to a chip of deal wood moistened with hydrochloric acid, and on addition of fuming nitric acid gave a precipitate of nitrate of nitrosoindol. This product, however, differed from indol in its reactions with heat ; while indol from indigo fuses at 52°, the crystals obtained by these authors began partially to fuse at 50° ; the principal bulk however fused only above 70°. When they were recrystallised repeatedly from hot water the fusing point rose to from 85 to 86° and then became constant at that temperatm-e, or according to Kiihne at 89 to 91°. The recrystallisation did not, however, change either the external appearance or the smell of the crystals. The volume of the vapour of this body was to that of indol under otherwise equal conditions in the proportion of 0-9 : 1. As the crystals on oxydation with ozone did not give indigo, Engler and Janecke considered their product as a matter isomeric with indol, and termed it pseudoindol. Nencki {J. pract. Ghem. 17, 1878, 97) has now furnished the results of new experiments with this process, from which it appears that this pseudoindol is a mixture of indol and of a similar but essentially different body, originally discovered by Secretan {^Archives des sciences de la bibliotkeque universeUe. Greneve, 1876; Fevrier) in a mixture of water and albumin which had been allowed to putrefy during a period of six months, and then also met with in faeces by Brieger {J. pract. Ghem. 17, 1878, 97) and by him termed skatol (from to o-kutos, faeces). Nencki heated the mixture of 10 potash and 1 albumin during from five to six hours, added water and heated again, and repeated this until the distillates ceased to give any red reaction with fuming nitric acid. He extracted the distillates with ether, or, after oversaturating them with hydrochloric acid, precipitated the indol and skatol by picric acid. The red needles of picrate of indol and skatol were distilled with dilute ammonia, whereby both indol and skatol were obtained in the receiver in the crystaUine state. They were separated by recrystallisation from hot water ; the skatol crystallised first and almost completely, while indol remained in the solution, Skatol fused at 93°"5, and its solution in water gave no red precijpitate with fuming nitric acid,, but only 150 ANNALS OF CHEMICAL MEDICINE. a whitish turbidity. Nencki did not obtain a sufficient quantity' of skatol by this process to subject it to elementary analysis. Formation of oily products and of pyrrol. — When the potash and albumin are heated quickly to a high temperature, oily products and pyrrol are formed besides indol and skatolt Small quantities of pyrrol in watery solution are easily recog- nised, if some drops of fuming nitric acid are added to it, or nitrous acid is produced in the solution from nitrite and acid. The mixture becomes at once dark brown, and an amorphous black precipitate is produced, which is soluble in dilute alkalies with a brown colour. Picric acid also gives with pyrrol a crys talline compound, more easily soluble in water than the picrate of indol and skatol. Heating the fusing mixture in an oil or paraffin bath. — Nencki found it practicable to efifect the fusion of 50 grms. albumin, and 500 grms. potash, in a glass flask immersed in an oil bath. Chemical action began at 230°; the mixture became brown, frothed greatly, and evolved great quantities of ammonia. After about an hour the high frothing ceased and the gas escaped in the form of small bubbles. The heating was continued during about live hours at temperatures varying between 260° and 290°, until the watery milky distillate ceased to come over. The flask was allowed to cool ; 15 c.c. of water were added to the fused mass, and the mixture heated anew. This operation was repeated on five days until the distillates ceased to react with hydrochloric and picric acid. The picric acid precipitate produced in the united distillates acidified with hydrochloric acid weighed after drying over oil of vitriol 1'2 grm. By distillation with ammonia it yielded only 0*048 grm. skatol, which when recrystallised fused at 93° to 94° in the narrow tube. Treatment of the potash &c. residue. Eoctr action of phenol. — The residue in the flask was dissolved in water, acidified with dilute sulphuric acid consisting of 500 grms. oil of vitriol mixed with 2 litres of water, and subjected to distillation. The strongly acid distillate had a repugnant faecal odour, a milky appearance, and deposited sulphur on standing. The filtrate from this (2400 c.c.) was neutralised with soda ley and extracted with ether. The ether was removed by distillation, and the PYEO-CHEMOLYSIS OF ALBUMIN. 151 residue of the ether extract placed into a small retort, and distilled with dilute sulphuric acid, until the distillate on addition of bromine water did not any longer become milky. The distillate smelt of phenol, assumed a blue violet colour when mixed with ferric chloride, and with bromine water gave immediately a strong precipitate of tribromophenol, amounting to 0'152 grms. equal to 0-043 grms. as the result of the whole operation. The tribromophenol had to be purified by solution in dilute potash ley and precipitation with hydrochloric acid ; the adhering impurities were perhaps a little kresol and analogous compounds. Extraction of fatty acids. — The distillate, from which ether had extracted the phenol contained the volatile acids united with soda. It was evaporated to a syrupy consistency, and as it did not crystallise, was decomposed by sulphuric acid. The oily fatty acids were dried over calcic chloride to which a trace of caustic baryta had been added, and subjected to frac- tional distillation. The thermometer rose quickly to 150°, remained some time at 160°, and rose to 170°, when the last portion of acid distilled over. The distillate therefore consisted almost entirely of normal butyric acid : this was controlled by the quantation of a silver salt. If all acid is assumed to have been butyric, the 50 grms. of air-dry albumin, containing after deduction of ash and moisture 40-2 ■ grms. pure albumin, yielded 14'36 grms. butyric acid, or 35"7 per cent. Extraction of leuoin. — The residue, from which the fatty acids had been distilled was concentrated, and the crystallising sulphate removed. The acid mother liquor was neutralised with baryta and evaporated to a syrup ; it yielded 1 "4 grms. of crude leucin, but no tyrosin. A peptone-like residue in small quantity still gave the protein-reaction with Millon's reagent. It is long since known that as caustic potash in the dilute state produces at first so-called peptones by hydration, so the fusing potash produces peptones, but quickly splits them up into simpler products, amongst them leucin and tyrosin, then, under evolution of hydrogen volatile acid, particularly valerianic. At the same time, as we now know, indol and skatol appear. "When the fusion is continued long, the amount of the peptone-like matters 152 ANNALS OF CHEMICAL MEDICINE. is diminished, and leucin and valerianic acid are gradually changed to butyric acid. The tyrosin is completely decomposed, and in its stead phenol appears. Nencki believes that the phenol which is obtained by the fusing operation as well as that which is obtained by putrefaction from albumin, originates from the tyrosin. Whenever he obtained by either experiment phenol, he missed the tyrosin in the residues, and vice versa. Fusion with potash, like putrefaction, acts only in the first stage upon albumin by hydration. When hydrogen is developed, products of oxydation as well as reduction appear. Nencki therefore hopes that the study of the mode in which potash decomposes albumin wiU. assist in finding out the manner in which it is decomposed by putrefaction organisms. PHYSIOLYSIS OF ALBUMINOUS MATTERS. 153 XL PJIOCESS AND PRODUCTS OF THE PUTREFACTION OF ELASTIN, MUCIN, GLUTIN, ALBUMIN, HEMOCHROME AND BLOOD. (^Summary.) Glutin is decomposed by physiolysis in a manner similar to that in which it is chemolysed by boiling dilute acids or alkalies ; it yields no tyrosin, no indol or phenol, but of crystalloids prin- cipally leucin and glycin, and of volatile acids principally acetic. The higher albuminous substances yield indol, phenol, tyrosin and leucin, but little or no glycin, and of volatile acids principally butyric, besides some valerianic acid. There are therefore in glutin not only a lesser number of crystalloid insertions, but they are also of a less complex constitution than those contained in the higher albuminous substances. Never- theless they belong mostly to the same homologous series as those contained in albumin. There is another difference which must not be lost sight of, namely the number of times which each radicle may occur in each kind of compound. Thus if in albumin the radicle of tyrosin be taken as one, that of leucin must be present at least six or seven times. On these quantitative pro- portions we have as yet few data, but they will quickly be collected when once our knowledge of the qualities of the products is completed and our methods for their quantitative estimation are perfected. Meanwhile the methods which have yielded so much in- formation on the substances mentioned, have been extended to a number of organoplastic substances which have not as yet been frequently studied, amongst them elastin and mucin. Putrefaction of elastin (Walchli, G-., J. pr. Chem. 17, 1878, 71). — Elastic tissue, principally the ligamentum nuchse of the ox, was purified in the manner practised by W. Miiller {Zeitschr. 154 ANNALS OF CHEMICAL MEDICINE. rat. Med. 10, 1861, 180). It deserves to be noticed that this method is very much like that by which cellulin is purified. The nuchal ligament is carefully dissected out of all visible connective tissue, is then torn into fibres and minced, and washed with cold water; it is then boiled with alcohol and ether to remove fats; and next boiled with water during at least twenty-four hours to dissolve all invisible connective tissue. It is further boiled with acetic acid, then with water, then with dilute caustic potash until it begins to swell, and is then finally purified by boiling with dilute acetic acid, and lastly with water. The tissue thus treated shows the specific fibres under the microscope unchanged. It is free from sulphur, and contains on an average W. Miiller, Hilger, mean of 4 analyses serpents' eggs C . 55*48 per cent. 54*68 per cent. H . 7*41 „ 7*24 „ N . 16*19 „ 16*37 „ As regards the products obtained from elastin by chemolysis with, dilute sulphuric add, the data given by several authors are not very concordant. Zollikofer (ATvn. Chem, 82, 176) found leucin to be the only crystalloid product, while Erlen- meyer and Schoflfer {J. pr. Chem. 80, 1866, 367) foimd besides from 36 to 45 per cent, of leucin, also 0*25 per cent, of tyrosin. W. Miiller also had obtained small quantities of tyrosin besides much leucin. Walchli submitted 100 grms. elastin, purified as above described, to putrefaction with four litres distilled water and 5 grms. of fresh minced pancreas from the ox in a water-bath at a heat from 35° to 40°. The elastin gradually swelled and dissolved, and the reaction of the solution, neutral at first, became alkaline on the sixth day. After fifteen days the elastin all but 7 grms. dry was dissolved. The filtered liquid was treated as follows. Distillation. The putrid-smelling liquid was distilled from a tubulated retort, without any addition, to half its bulk. The distillate, which was slightly ammoniacal, was extracted with ether ; the ether was distilled off, and the residue finally evaporated on a small dish: it weighed 0*0515 grms, and consisted only of a little fat derived from the ox- PHYSIOLYSIS OF ALBUMINOUS MATTERS. 155 pancreas, ■without a trace of either indol or phenol. The residue in the retort was mixed with 100 grms. of caustic baryta and again distilled. Nearly a litre of distillate contained 1*74 grms. of NHg, or 1-433 grms. nitrogen. The residue iiu the retort from, which this ammonia had been expelled, was freed from baryta by sulphuric acid, and again subjected to distillation. It yielded no acetic acid, but priacipally valerianic with little butyric acid. If the whole of the acid obtained were calculated as valerianic, it would be equal to 8* 15 grms. The residue free from ammonia and volatile acids was evaporated on the water- bath to a thick syrup, and then mixed with absolute alcohol until a lasting turbidity was produced ; it deposited crystals, which after twenty-four hours' standing were filtered from the mother liquor, and found to be a mixture of glycin and leucin in nearly equal parts. The entire weight of the crude crystals was 9*4 grms. The substances were separated by fractional crystallisation, and the glycin was identified by the analysis of its copper-compound 2(NH2— CHj— COj) Cu + HjO. The mother liquor filtered from the glycin and leucin gave no further crystals, and after evaporation on the water-bath remained as a viscid, gluelike mass, which on treatment with caustic soda and solution of copper gave the red reaction charac- terising biuret. The 100 grms. elastin, of which 93 grms. had been dissolved during the fourteen days of putrefaction, yielded therefore Matter left undissolved , . 7"00 grms. Ammonia .... 1'74 „ Valerianic acid . . . 8"15 „ Glycin and leucin . . . 9 '40 „ Carbonic acid, saturating some ammonia, syrupy peptone- like matter . . . 73*7 1 „ Total . . 100-00 „ It must be borne in mind that it is not certain whether the 7 grms. left undissolved were elastin in its unchanged state only, or contained other less putrefiable tissue-elements such as characterise the nuclei of cells and their residues. Seeing that 156 ANNALS OF CHEMICAL MEDICINE. nearly three quarters of the elastin employed were transformed only into the syrupy peptone-like matter, ■which was not studied any further, the experiment will have to be continued. But the matters actually obtained, and the absence of aromatic radicles, characterise elastin as related to the gelatinogenous tissues ; but it is again different from these and resembles the higher albumins by yielding in the operation for volatile acids the higher valerianic, while gelatin yields the lower acetic acid. Putrefaction of mucin. — ^Walchli {loc. dt. p. 75) prepared the mucin used in his experiment from the large vineyard snails, Helix poTnatia ; the crushed and minced snails were ground up with glass powder, and the paste on a felt funnel was extracted with boiling water. The yellowish filtrate was mixed with an excess of strong acetic acid, and the precipitated mucia was washed with water by decantation, then collected on a filter, when in a solid state extracted with ether to remove fat, and dried. The mucin thus prepared was a blackish tough mass. 223 grms. of the air-dry substance, corresponding to 163 grms. muciu dried at 120°, were digested with 4 litres of water and 5 grms. of ox-pancreas in a water-bath at 35° to 40°. On the 9th day the mucin, all but 6-8 grms. dry, had dissolved. The fluid, of strongly putrid odour, was subjected to distillation. The distillate gave no reaction for indol with fuming nitric acid. It was saturated with soda ley, and extracted with ether. This solvent left a small amount of an oil, which did not crystallise on long standing, and had the smell of the volatile principle obtained from human excreinents and from those of dogs and from offensive-smelling products of disease (skatol, CjoHjjN). The oil was boiled with water, which dissolved a small portion of it. The hot filtrate became milky on cooling, and after some hours deposited crystals of indol. The filtrate from these crystals was diluted with some water, mixed with a few drops of potash ley, and subjected to distillation from a small retort, until the distillate gave the reaction for indol. The residue in the retort was now oversaturated with dilute sulphuric acid and again distilled. The distillate smelled of phenol, and with bromine water gave white, crystalline tribromophenol. The volatile product therefore contained both indol and phenol, and PHYSIOLYSIS OF AXBUMINOO'S MATTEES. 167 a peculiar-smelling principle in prerailing ataount, which was not suitable for further analysis. The liquid, from which these matters had been expelled, was now mixed with baryta, and again distilled it yielded 3"4 grms. of NHj, equal to 2*87 grms. N. The acidified liquid gave almost pure butyric acid, amounting to 12'3 grms. The residue, now free from ammonia and acids, contained an uncrystallisable, sweet-tasting substance, which reduced potassio-cupric tartrate, decomposed cupric and barytic car- bonate, and formed with these bases amorphous salts precipitable by alcohol. The author believes this body to be the same as the sugar-like body which was first obtained from mucin by Eichwald. He lost his preparation, stored in a dish of • toughened glass,' by the said glass spontaneously bursting up into little splinters and spilling the contents. Changes in the appearance of blood-corpuscles during the decomposition of blood in the presence of Bacillus subtilis and of different gases. — The life of bacteria in gases has been investigated by Grossmann and Mayerhausen (Archiv Physiol. 15, 245). Oxygen caused the bacteria to move strongly and change quickly in form, while ozone had the opposite efiect, and when applied in a concentrated state destroyed the bacteria almost instantaneously. As several oxydations by ozone have the same character as oxydations of the same substances in the animal economy (e.g. that of uric acid, which yields in both cases allantoin and urea) and as it had been stated by A. Schmidt, that the blood-corpuscles ozonise oxygen in contact with them, Kaufmann (J. pr. Chem. 17, 1878, 70) examined the bearing of putrefaction-ferments in oxygen in the presence of red blood-corpuscles. The latter were enclosed in a so-called moist capillary chamber, a double capillary lens, invented by Eecklingshausen and manufactured by Greissler. The experi- ment was in each case made as follows : a few drops of defibri- nated blood were sucked into the chamber, then mixed with about an equal bulk of liquid containing bacteria in suspension, and after complete mixture in the chamber most of the liquid was again removed from it, so that only the capUlary space and its immediate surroundings retained any liquid. Through the chamber thus supplied, oxygen, passed through potash and 158 ANNALS OF CHEMICAL MEDICINE. sulphuric acid, was conducted. The blood, operated upon was in one experiment from the frog, in four other experiments from rabbits. The uniform result was, that while bacilli in putrid liquid without blood, maintained in oxygen, remained moving and unchanged, bacilli of the same breed mixed with blood became after a longer or shorter time perfectly motionless. Therefore, although the oxygen is perhaps not transformed into ozone by the blood-corpuscles, its action in the moist chamber is such as to produce an effect similar to that of ozone upon bacilli. The corpuscles of the blood of the frog remained unaltered in shape in the moist chamber, though in contact with putrid ferment bacilli during five days, oxygen being constantly passed through the chamber ; on the sixth day the nucleus was granu- lated; on the eighth day many corpuscles were completely decolourised and appeared only by their nuclei and stromata. The changes were not followed further. The changes in the blood-corpuscles of rabbits did not appear to differ much, but to be slower than those which are observed when the blood is placed under the microscope on an ordinary slide and covered with thin glass. The author thinks that oxygen in presence of blood-corpuscles, and somehow modified by them, kills, or keeps inert and prevents the development of, the micro-organisms, the germs of which he believes, after Bechamp {Des Microzymas &c. Paris, 1875) and Tiegel (Archiv Pathol. 60, 453), to be present in nearly aU tissues of the healthy body. When the passage of oxygen through the moist chamber was stopped, the bacilli regained their motility and power of multiplication. These bacilli therefore obey conditions of life the opposite of those by which the Bacillus anthracis abides, which thrives well in oxyge- nated blood, but dies in putrefying blood or other liquids. Putrefaction of blood-corpuscles and of blood in the presence of bacilli. — Some experiments on these processes were made by Kaufmann {J. pract. Chem. 17, 1878, 90). In the two first experiments two litres of blood were defibrinated and mixed with their tenfold volume of a mixture of one volume of saturated solution of common salt and nine volumes of water. After 40 hours the corpuscles had settled and the supernatant fluid was PHYSIOLYSIS OF ALBUMINOUS MATTEfiS. 159 decanted. The muddy deposit was diluted with five litres of water, mixed with 5 grms. of minced ox-pancreas, and digested on the water-bath at 40°. After 24 hours the mixture exhaled a putrid odour, and a thin reddish-brown crust floated on its surface. The blood-corpuscles were all destroyed (by the water ?) and the solid matters visible consisted of micro-organisms, particularly heaps of zooglcea. The crusts were examined on the second day and found to consist of hematin. On the fifth day the liquid contained mainly bacilli ; on the tenth day cocci were added. From this time to the sixteenth day the micro- organisms consisted of cocci and bacilli in equal parts. The surface of the fluid exhibited much hematin in crusts, but the liquid still contained hemochrome in solution. The liquid subjected to distillation yielded a little indol, but no phenol. The residue in the retort gave leucin and tyrosin. The mother liquor contained fatty acids, combined with ammonia and peptones. These were unfortunately not examined any further. In a second experiment with the corpuscles from two litres of blood, the pancreas was omitted, and the results did not differ from those of the first experiment, although they were perhaps attained at a greater expenditure of time (29 days). The third experiment was made upon three litres of blood with five grms. of ox-pancreas. The micro-organisms multiplied rapidly ; after five days the blood-corpuscles had all disappeared. After six weeks of putrefaction the liquid was distilled. It yielded much indol and phenol, but no tyrosin, little leucin, fatty volatile acids, ammonia, and peptones. As, before being boiled, the liquid still contained some hemochrome, the author comes to the conclusion that hemochrome possesses great stability and power of resistance against the attacks of micro- organisms. As regards the use of pancreas the author comes to the result that the experiments without it were just as successful as those with it, because, as he thinks, the air of his laboratory was provided with abimdance of bacteria or their germs. 160 ANNALS OF CHEMICAL MEDICINE. XII. ON THE ALKALOIDS OF THE HUMAN URINE. {Cm- solidated Accoimt cf Sesearchei, From the Pathological Institvte.) Alkaloids may be defined as bodies derived from typical ammonia, by substitution of compound radicles for one or more of the atoms of hydrogen ; the radicles may be homogeneous or heterogeneous, the typical ammonia may be one, or may be present several times over, constituting a multiple type, -which gives rise to the possibility of countless substitutions by com- pound radicles. The best known representatives of this class are the so-called amines and ammonium bases, obtained syn- thetically ; but the most important are the alkaloids of vegetable origin and great poisoning or healing power, such as the principles extracted from opium, cinchona bark, and nux vomica. Of these bodies above a hundred are at present known to science, and when we add the bases derived from the animal economy and obtained by other means, we arrive at a number exceeding two hundred. It appears, however, that the defini- tion has hitherto been too narrow, just as the modes of search- ing for alkaloids were far too one-sided and limited. For alkaloids were supposed to be very insoluble, in water at least, and it- is not long since this error was recognised when a few highly soluble alkaloids such as colchicine were discovered. It will now be necessary further to widen the definition of alka- loids, so that the bodies, of which the alkaloid from brain- matter is a representative, may be ranged under it. I have shown with regard to the phosphorised ingredients of the brain that they possess both basic and acid properties at the same time. The same is the fact with regard to the new alkaloids to be described ; they possess most of the combining-powers ALKALOIDS OF UEINE. 161 wMcli are considered to be characteristic of alkaloids, but at the same time they possess combining-powers characteristic of acids. Such alkaloids I have extracted from the muscular and brain tissue in great quantity, from several other tissues of organs, particularly the liver, and from secretions, such as milk and urine. Proceedings for the isolation of the alkaloids of the urine. First modification ; new alkaloid : reduGvn. — Fresh healthy human urine was shaken with a little animal charcoal to collect the mucus and epithelial elements, filtered, strongly acidified with sulphuric acid, and then, precipitated by phosphomolybdic acid. The collected precipitate was washed with water con- taining a little sulphuric acid. It was then decomposed with hot baryta-water in slight excess ; this excess was removed by carbonic acid and boiling, and the filtrate evaporated to a small bulk. On cooling it formed a deposit, which was proved to be jpure VA-ate of haryum. It was specially proved that it con- tained no hypoxanthin. The yellow filtrate contained much urochrome, giving on decomposition with hydrochloric acid and boiling uromelanin, uropittin, and evolving smell of omicholic products. It was therefore treated with neutral lead acetate after this with the basic lead acetate, and ultimately with this and some ammonia. The united precipitates were further treated, but the results were not so satisfactory as those of the copper process to be stated below. The filtrate from the lead precipitates was freed from excess of lead by hydrothion and evaporated to dryness while being stirred on the water- bath. The residue was treated with boiling absolute alcohol and the decoction filtered hot, A voluminous Kaiyum salt remained on the filter, while a yellowish matter dissolved in the alcohol. The former was reduoin-baryum, the latter mainly kreatvnin, Eedudn-baryum. — It was easily soluble in water, and after burning left baryum carbonate. On addition to the solution of some nitric acid and silver nitrate a precipitate was produced which immediately became dark and black in the cold. With mercurous nitrate and nitrite it gave an immediate black pre- cipitate; with mercmric chloride it gave a white precipitate which was not changed by boiling; with cupric acetate and 162 ANNAIiS OF CHEMICAL MEDICINE, boiling, it gave a flaky precipitate which became brown ; with Fehjing's solution and boiling it gave no reduction. Analysis of the baryum compound dried at 100°. 0-4540 grms. gave 0-4520 002=27-092 per cent. C, and 0-1750 grms. H = 4-282 per cent. H. 0-2628 grms, gave 33-3 c,e, gasnormal= 15-850 per cent, N. 0-4490 grms. gave 0-1940 BaSO4 = 25-400 per cent. Ba, Per cent. -5- AtW. -5-Ba.=l c 27-092 2-257 12-2 H 4-282 4-282 23-1 N 15-850 1-132 6-0 Ba . 25-400 0-185 • 1-0 27-376 1-711 9-2 If the substance be considered as a monobasic acid, these figures lead to the formula H„0 Ba. If it be considered as a dibasic acid they lead to CijHj^NgOg as the formula of the free substance. Of this the molecular weight would be 396, while the molecular weight pointed to by the quantity of baryum is 402, The foregoing figures and formulae are given with the reserve imposed by the limited quantity of material ; but small changes in proportions can detract nothing from the essence of the new facts. The isolation of reducin has been effected by the application of a reagent of special action on alkaloids. This is the principal proof of the alkaloidal nature of the new body ; that it combines with bases after the manner of acids need cause no surprise, as there are many substances known to chemistry which have similar properties ; the best known amongst these are the amido-acids, like glycin. Kreatin and kreatvnin. — The alcoholic solution filtered from the redudn just described, was evaporated and the residue extracted with little absolute alcohol to remove kreatinin and leave kreatin ; the residue was dissolved in hot water, and de- colourised by boiling with animal charcoal. It was then allowed to crystallise,, and yielded three successive crops of crystals of ALKALOIDS OF URINE. 163 s kreatin. The air-dry crystals had the appearance of kreatin, and their nature was verified by a determination of the water of crystallisation. 0'4596 grms. dried in vacuo over sulphuric acid, lost 0*5076 grms. or 12-53 per cent. H 0. The theory of C^HgNgO^, H^O requires 12"08 per cent. The crystals were therefore kreatin. It is known that kreatinin, when freed from its combinations and particularly in warm baryta-water, is easily transformed into kreatin, and these bodies are therefore nearly always obtained side by side in certain proportions. The alcoholic extract contained kreatinin and the peculiar alkaloid which gives an insoluble compound with zinc to be described lower down, and a last fourth new alkaloid which remains in the ultimate mother liquor and is precipitated from alcoholic solution by platinic chloride. The compound is de- composed diuring resolution with water, but a stable platinum compound results, which contains an aromatic ingredient. It has not yet been analysed. Second modification of proceedings for the isolation of alkaloids of the urine. Second and third new alkaloid.' — In the researches now to be described the processes adopted were slightly modified. The raw material was filtered only, and not treated with even the smallest quantity of animal charcoal, as it was found that the agent removed considerable quantities of the matters to be studied. It was next strongly acidified with sulphuric acid in the following manner and proportions. Fifty cubic centimetres of oil of vitxiol were poured into 100 c.c. of water, and the resulting 150 c.c. of dilute acid were pom-ed into a litre of filtered urine. This mixture was now fully precipi- tated with a solution of phosphomolybdate of soda acidified with a sufficient excess of sulphuric acid to represent a deep yellow solution of phosphomolybdic'acid, containing 200 grms. of the solid acid in each litre. In each operation the quantities of phosphomolybdic acid solution required were found by prelimi- nary experiment, and the completion of the actual operation on the large scale was ascertained by the mother liquor of tlie precipitate giving no further reaction with either sulphuric or phosphomolybdic acid. The collected buff-coloured precipitate was washed with u 2 164 ANNALS OF CHEMICAL MEDICINE. water containing about five per cent, of sulphuric acid. This Was effected by washing the precipitate into a large stoppered bottle by means of the spray from a continuous pressure wash- bottle (described in another place), then adding a large amount of acidified water, shaking the mixture strongly, allowing the precipitate to settle, and removing the washing water with the syphon. By repetition of this treatment the precipitate could be almost entirely freed from alkalies and chlorine. The precipitate placed in a flask with some water and warmed in a water-bath was now decomposed with hot concentrated solution of caustic baryta in slight excess ; the point at which the excess of the alkali is present is indicated by a change in the colour of the liquid from blue to yellow, and in the colour of the precipitate from greenish buff to pale bluish grey. The excess of caustic baryta was now neutralised by carbonic acid conducted into the flask with the fluid and phosphomolybdate of baryum kept continuously hot in the water-bath. "When a filtered sample was no longer affected by carbonic acid, the solution was separated from the precipitate by filtration with the usual precautions. Volatile bases, — ^In some of the experiments the volatile bases which are always set free on the first addition of the baryta were fixed by being passed through a vessel containing hydro- chloric acid. The solution was evaporated, thechlorides again distilled with caustic soda, and the peculiar and fetid smelling distillate neutralised with dilute sulphuric acid. The dry sulphates gave a portion to absolute alcohol, which contained a compound ammonia, probably trimethylamine, while another portion remained insoluble in absolute alcohol, and was recog- nised as ammonic sulphate. Further proceedings for separation of alkaloids. — The liquid from which all volatile alkalies had been expelled by heat and the carbonic acid treatment was concentrated to a con- venient bulk, allowed to cool, and after some standing filtered from the small deposit of urate of ha/ryum, which was mostly formed. The uric acid is probably present in the phospho- molybdic precipitate as an admixture felled by the sulphuric acid, and not in combination. It is small in quantity and the trace which may possibly remain in solution after deposition of ALKA.LOIDS OF URINE. 165 the baryum salt does not appear in any subsequent operations, and at one stage of the subsequent proceedings is necessarily destroyed. There remains now a solution of mixed alkaloids, coloured yellow, and containing baryta in solution not removable by carbonic acid. I have elsewhere described the application to this solution of lead acetate both basic and neutral, and the further treatment of the lead precipitates, resulting in the isolation of a body having the reaction of xanthin, but differing from it in the composition of its silver salt. It was shown that there were at least two bodies present, imitating the bearing of xanthin with silver nitrate and ammonia, but that the second one differed from xanthin not only in the amount of silver contained in the argentide, but also by the solubility of the argentide in excess of concentrated caustic ammonia. In the experiments here to be related, the united alkaloids were not treated with lead salts, but with cupric acetate, which produced a voluminous greenish, later bufif-coloured precipitate. (On the application of acetate of copper to urine for the pre- cipitation of a base described by Strecker as sarcin, sarkin, hypoxanthin, but also suspected to be guanin, see my Patho- logy of the Urine, first edition, 1858, Appendix.) The change of the precipitate to buff colour was found to be due to a reduction of the cupric to cuprous salt ; this occurred in an acid liquid, and did not occur when the liquid was made alkaline ; the reduction became very copious when the mixture of liquid and copper precipitate was heated ; in that case the precipitate became purely yellow. When the precipitate was quickly filtered from the solution in which it had been formed, and washed with water, so as to be free from mother liquor, it could be treated with hot water without changing its colour ; but it always contained cuprous salt from the beginning. This cuproso-cuprie precipitate was decomposed with hydro- thion, and the filtered liquid boiled and evaporated. At a certain stage of concentration it deposited crusts and pellicles, and then was allowed to cool, when it deposited a pulverulent body. All deposits were separated from the deeply coloured mother liquor, and treated as will be immediately described. The deeply coloured mother liquor contained yet much of tb© 166 ANNALS OF CHEMICAL MEDICINE. pulverulent body, and was separately treated for its separation by evaporation and cooling ; but its main ingredient was urochrome, precipitable by ferric chloride, and giving the same reactions as that precipitated by'the lead process. Second new crystallised alkaloid. — The deposits were washed with cold water, and redissolved in hot, and filtered through Swedish filtering paper, as often as was necessary to free them from all matters which became gradually insoluble in the course of that treatment. The granular matter gradually assumed a crystalline appearance, and after the removal of several mother liquors became perfectly crystallised in rhombic scales of the lustre of mother of pearl. When the crystals were homogeneous under the microscope and p^erfectly white they were analysed. (1) Combustion in ■yacwo.— 0-0222 grm. dried during 48 hours m vacuo over H2SO4, was burned with cupric oxyde in vacuo and gave: Total gas =28-5 c.c. ; after KHO = 7-6 c.c. ; KHO column = 3-7 m.m. Hg. Hg column = 82 m.m. ; B = 764-5 ; T=25°. Therefore total normal gas = 25-45 c.c. ; total CO^ = 1 9-45 c.c. ; N= 6.00 ; Vol. ofN: Vol. ofC = l : 1-62 or =4 : 6-48 or in percentages equal to 46-95 C and 33-81 N. (2) Nitrogen quantation by CuO and COj mixture 0-0988 grm. gave 29-5 c.c. gas. KHO column = 5-2 m.m. Hg; B = 759 m.m.; T = 21°; therefore =26-5 c.c. gas normal= 33-56 per cent. N. (3) 0-3704 grm. recrystallised from alcohol, and dried at 100°, burned with cupric oxyde gave 0-1556 H^O, equal to 4-69 per cent of hydrogen. Found. CjHgN.O, Theory. 7C . . 84 46-67 8H . . 8 4-44 4N . . 56 31-11 20 . . 32 17-78 1. 2. 3. 46-95 — — — — 4-69 33-81 33-5 — 180 100-00 ALKALOIDS OF UBINE. 167 By combustion of pure theobromin from cacao in vacuo a mixture of gases should be obtained in which CO2 : N= 14 : 4 ; the proportion obtained in the analysis of the new base was = 12*96 : 4. The excess of nitrogen may perhaps be due to a slight admixture of hydrogen with the nitrogen gas. For at that period I had not yet ascertained the error introduced into the nitrogen quantation, particularly in the combustion in vacuo, by the hydrogen occluded by the copper reduced in the hydro- gen current. This error was first eliminated by a research communicated to the Chemical Society, and published in its Journal, September 1876, under the title : .' On the Estimation of Hydrogen occluded by Copper, with special reference to Organic Analysis,' by J. L. W. Thudichum and Henry W. Hake. The final decision of this point must be reserved for further research. This body, therefore, has very nearly the composition of theobromin, the alkaloid from cacao. Like this it is sublimable ; fusing just before yielding the vapour which condenses in white clouds; it is tasteless on the tongue at first, but evolves a bitter taste after some time, which is never very intense ; it is easily soluble in absolute alcohol, more in hot than in cold ; and forms salts with acids. But it is not identical with theobromin, as it is much more soluble in water than this base, and with nitrate of silver in dilute nitric acid solution does not yield the crystallised double salt which theobromin from cacao so easily produces. On further comparison with theobromin it appears moreover that this base is not precipitated to any extent by cupric acetate, whereas the new base forms with this reagent a precipitate which is completely insoluble in even boiling water. From xanthin it is distinguished by its easy solubUity in hot water ; solubility in alcohol, in which xanthin is quite in- soluble ; and by its subliming without decomposition ; further by its elementary composition ; for xanthin, CgHjN^Oj, has C . . 39-48 H . . 2-63 N . . 36-84 . . 21-05 100-00 168 Al^NALS OF CHEMICAL MEDICINE. figures which differ so much from those above quoted for the new base as to exclude absolutely any probability of identity. Study of hreat-min obtained by the 'phosphomolybdAc add process, (a) Reaction with ferric chloride. — A faintly yeUow solution of ferric chloride is coloured dark red by a dilute solution of pure kreatinin. The depth of colour is increased by boiling. Chloride of zinc-kreatin produces the same reaction. (6) Govrvpounds of kreatvnin with gold chloride. — A quan- tity of kreatin, obtained by the phosphomolybdic process, was treated with sulphuric acid for the purpose of being transformed into kreatinin, and the product was three times recrystallised from alcohol, the white plates of kreatinin sulphate were dissolved in water, and decomposed accurately with barytic chloride. The neutral kreatinin hydrochlorate was concen- trated and mixed with a concentrated solution of gold chloride. No immediate precipitate ensued. On standing warts of a compound formed, which was indistinctly crystalline. The corapound recrystallised from hot water seemed less soluble than the original mixture, and became turbid at once on slight cooling. On standing some metallic gold was deposited. The reformed compound was apparently very indistinctly crystalline, not crystallised. It was collected, washed, and dried in vacuo. Analyses : (1) 0-0836, after combustion, left 0-0400 metallic gold =47 -84 per cent. Au. (2) 0-1394 gave 12-5 c.c. gas at B=765 m.m. T = 18°; KHO column = 110 m.m. ; equal to 11-39 c.c gas normal = 10-25 per cent. N. (3) 0-994 by fusion with caustic soda and nitre gave 0-0484 Au, equal to 48-69 per cent., and 0-0903 AgCl, equal to 22-46 per cent. CI. A preparation of kreatinin, which had been obtained directly by the phosphomolybdic acid process, and been purified by recrystallisation from absolute alcohol, was dissolved in excess of hydrochloric acid, and mixed with auric chloride solution. Crystals formed, which were separated and pressed. The mother liquor was yet three times treated with gold chloride, and yielded altogether four crystallised deposits, 3-2 grms, in weight. The first and second crystals were recrystallised. ALKALOIDS OF UEINE. 169 Analysis: 0*1043, fused in caustic soda and nitre, gave 0-0449 gold, or 43-04 per cent. Au, and 0-1332 AgCl, or 31-59 per cent. 01. The salt could not be dried at a higher temperature ; heated to near 100° during one hour, it became dark in colour; 0-1662 lost 0-0052 ; 0-1602 burnt left 0-0718, or 44-81 per cent. Au. The same salt by fusion gave 44-41 per cent. Au. The third crystals were not recrystallised but tested separately; 0-1400 grm. dried m vacuo, \eii 0'0602 gold on combustion, equal to 43-00 per cent. Au. A similar preparation was obtained by dissolving some kreatinin zinc chloride in hydrochloric acid, and adding auric chloride; it weighed 1-5 grm. The whole of the products were dissolved in warm water, filtered from some insoluble matters, and the solution placed in a vacuum ■ over sulphuric acid. A crystalline deposit formed, consisting of spherical masses of crystals. On the salt being dried in vacuo over sulphuric acid, it became slightly discoloured, though it was kept in the dark. Analyses : (1) 0-1552 left 0-0726 metallic gold = 46-77 per cent. Au. (2) 0-3734 gave 33-0 c.c. gas at B. 757 m.m. T = 15°; KHO column=113 m.m. equal to 30-16 c.c. gas normal, or 10-46 per cent. N. Consequently by recrystallisation of the dry salts from pure water the percentage of gold increased from 43-0 to 46-77, and the nitrogen rose above the quantity theoretically to be expected. Some further gold chloride compounds were made with kreatinin obtained from zinc chloride compound by silver oxyde : excess of hydrochloric acid and of gold chloride were added. Three crystallisations were obtained in succession : Gold Quantations: No. (1) 0-1936 left 0-0832, or 42-98 per cent. Au. No. (2) 0-1322 left 0-0568, or 43-12 per cent. Au. . , (a. 0-08 10 left 0-0348, or 42-91 per cent. Au. ^°' ^'^^ \b. 0-0614 left 0-0624, or 42-99 per cent. Au. 170 ANNALS OF CHEMICAL MEDICINE. It was thus evident that kreatinin in the presence of excess of hydrochloric acid would yield a hydrochlorate aurochloride in crystals, but that this salt (except in one case, in which only a small amount of crystals was obtained from a larger amount of solution) could not be recrystaUised from water without losing hydrochloric acid and forming a deposit of metallic gold. When neutral kreatinin hydrochlorate was mixed with auric terchloride, the hydrochloric acid was also ejected, and a compound free from hydrochloric acid obtained. A considerable excess of hydro- chloric acid was required to insure a good crystallisation of the double salt. This bearing is shown conspicuously by the follow- ing synopsis of the several theories and results of analyses : Kreatinin Sydrochlorate Awrochloride from. Solution with excess of Acid, Theory. Found. f ..A ^ « Atoms. in 100. 1. 2. 3. 4. 6. "40 48 ■ — _ — _ _ _ 8H8 — _____ 3N 42 9-27 _ — _ _ _ 16 — _____ Au 196-7 43-45 43-00 43-04 42-98 43-12 42-95 4 01 142 31-36 31-59 _ _ _ _ 452-7 Preparation (1) and (2) united and recrystaUised from water gave N . . 10-46 Au . . 46-47 Kreatinin Aurochloride from Neutral Hydrochlorate. Atoms. in 100. Found. ' " ^ , — ■ ' , 4 48 11-53 ~ — IL 7 H 7 — _ _ _ 3N 42 10-09 _ 10-25 — O 16 _ _ _ _ Au 196-7 47-26 47-84 _ 48-69 3 01 106-5 25.58 — _ 22-46 416-2 ALKALOIDS OF URINE. 171 All salts obtained and analysed in the foregoing were now united and recrystaUised with addition to the solution of hydro- chloric acid. The first deposit of crystals was dried m vacuo and analysed. (1) 0-1770 burned left 0-0768 = 43-39 per cent. Au. (2) 0-3426 fused with soda and nitre, &c., gave 0-1507 = 43-98 per cent. Au and 0-4308 AgCl= 31-12 per cent. CI. (3) 0-1662 gave 13-0 c.c. gas, normal, =9*25 per cent. N. (4) 0-2656 gave 0-1058 COj= 10-86 per cent. C, and 0-0468 HjO = l-93 percent. H. ^nojpais of Results. Theory. Found. r ' Atoms. Per cents. 4C 10-60 8H 1-77 3N 9-27 — Au 43-45 4 CI 31-36 4. 10-86 , 1-93 9-25 43-39 43-98 — — — 31-12 — — It is thus evident that kreatinin forms a compound with gold chloride of the formula C4HjN30,AuCl3, which is not very well defined, owing to its instability. This compound is formed when kreatinin .hydrochlorate and gold chloride are mixed together, the molecle of hydrochloric acid being ejected and remaining dissolved in the mother liquor. The same compound is formed as often as it is attempted to recrystallise the salt C^HjNjOjHCljAuClj from water. The most stable and best-defined salt is the kreatinin hydro- chlorate aurochloride of the formula just quoted; this is formed only in the presence of a considerable excess of free hydrochloric acid in the solution from which it deposits ; if the excess of hydrochloric acid over and above the proportion required to form a hydrochlorate is insufficient, a mixture of the aurochloride with the hydrochlorate aurochloride is produced. Third new alJcaZoid givvng i/neoluble yellow compound with zinc (pxyde), (CgHgNgOjZnO). — The precipitate by zinc chloride in the absolute alcohol solution of the alkaloids (from which the new crystallised alkaloid described and 172 ANNALS OF- CHEMICAL MLEDICINE. kreatinin and kreatin had been removed to a great extent by precipitation and crystallisation) was flaky and adhesive; it was allowed to stand in the mother liquor for twelve hours, until the latter was clear, and then separated. The precipitate, which had become hard, dissolved in water only partially, leaving a yellow zinc cornpound insoluble (Preparation I.). The watery mother liquor was evaporated, and yielded several crops of crystallised kreatinin zinc chloride. At last it became thick and syrupy, and contained a deposit of crystals and flakes, which could not be isolated from the syrup. The whole was therefore again poured into a large excess of water, and more water was added as long as the mixture became turbid. The yellow precipitate was filtered off (Preparation II.). The same process was repeated upon the mother liquor and yielded a third product (Preparation III.). All three preparations were dried in vacuo. ■ The precipitates were easily soluble in acids, such as acetic and sulphuric ; in these solutions phosphomolybdic acid gave a copious precipitate ; the substance had therefore preserved its character of alkaloid. It was easily soluble in excess of caustic ammonia. On being heated it evolved a repugnant smell, and left zinc oxyde. It was quite insoluble in water, and could be boiled with it apparently without undergoing any change. From preliminary quantations it appeared that the body gave up a certain amount of water in vacuo, and then gradually further quantities up to a temperature of 170°, at which its weight became stable. 0*5600 grm. taken for experiment : Temperature Time Loss 100°-110° . 20 hours •0519 130° (about) 2 „ •0045 ]35°-140° . 2i „ •0096 150° . 2i „ •0052 150°-170° . 2i „ •0024 170°-175° . 4i „ •0002 Total loss . . 0-0738 or 13'18 per cent, of substance. ALKALOIDS OF URINE. 173 A second experiment upon another quantity of substance gave precisely the same result. The preparations were mixed and • subjected to a careful qualitation. The elements found were carbon, hydrogen, nitro- gen, oxygen, and zinc, with traces of calcium, copper, and iron. The last three elements were evidently non-essential admixtures or impurities, and were eliminated. The quantations led to the following results : Per cent. At. "Weights c 33-34 72 H 3-90 9 N 18-91 42 ZnO 36-84 81 7-01 16 Total 100-00 220 These data lead to the empirical formula C5HgN30,ZnO. It will at once be seen that this is the formula of reducin, minus two atoms of oxygen, plus a molecle of zinc oxyde. Although the compoimd had been precipitated from the alco- holic solution of the mixed alkaloids by zinc chloride, yet it was quite free from chlorine, not a trace being discovered by a quantation. Consequently the zinc chloride compound of this alkaloid undergoes the same change as the kreatinin hydro- chlorate auric chloride, in the presence of water ; it loses the hydrochloric acid completely, and becomes insoluble. The form in which the zinc may be present has in the foregoing been assumed to be that of oxyde ; but this is only hypothesis, and further analysis must show whether or not it may be present as metal replacing hydrogen, and therefore lead to the formula C,H,ZnN30,. The foregoing process was repeated upon 65 litres of material, which had been treated with zinc and hydrochloric acid. The zinc compound was obtained, but like the other alkaloids, in apparently much smaller quantities than from material which had not been reduced by zinc and hydrochloric acid. One hundred parts of the air-dry compoimd, powdered lost in vacuo and over sulphuric acid, and then by heating to 170° 174 ANNALS OF CHEMICAL MEDICINE. 15'73 parts in weight; the loss assumed to be water only; of this loss almost two-thirds occurred in the vacuum, a little more than one-third at between 100° and 170°; the total loss of water almost amounts to two molecles, so that the formula for the air-dry substance might be written C6H3N3O, ZnO-|-2H20. The phosphomolybdic acid process is thus shown to remove from urine at least six fixed alkaloids besides the volatile bases. Of these alkaloids hreatinin is well known ; v/rochrome is now shown for the first time to be an alkaloid, and to be precipi- table by ferric chloride ; reducin is distinguished by a baryum- eompound and a remarkable reaction ; the second new alkaloid is distinguished by its crystaUisability, and by features which resemble those of the theine group ; the third new alkaloid gives an insoluble compound with zinc ; and the fourth new alkaloid gives a compound with platinic chloride, and contains an aromatic nucleus. The physiological quantities of these substances are probably not so small as one might be induced to believe from the small quantities actually isolated even from considerable quantities of material by the above process ; for the phosphomolybdic pre- cipitate is not very insoluble, and consequently some is lost in the voluminous mother liquors ; these had in the first instance to be operated upon without the employment of heat; but evaporation may in future be tried with certain precautions without fear of destroying the substances above described. The phosphomolybdic acid undergoes some reduction in the urine, which becomes deep green from the admixtion of blue oxyde with its naturally yellow colour. It is probable that a portion of the reducin contributes to this efiect, and after its oxydation escapes the further steps of the process. Then the baryta process is undoubtedly unwieldy, and likely to produce loss, particularly of bodies of the xanthin group. On the whole, however, the process yields a considerable amoimt of new and useful informa- tion. SOLUBLE ALBUMIN. 175 XIIL THE PROPERTIES AND METAMORPHOSES OF SOLUBLE ALBUMIN. {Summary and Additions.) Albumin, popularly so-called, is the well-known white of egg. It is obtained by breaking a fresh hen's, or other bird's egg, separating off the yetk and its envelopes (chalazse), keeping the chalazae and all mucous and ropy parts away with particular care, shaking the white in a bottle with some fragments of glass, and filtering the liquid through calico, lastly through paper by means of a pressiire-filter. The substance thus prepared contains, besides albiunin ■ chemically so-called, some albuminate and plasmin (paraglobu- lin), traces of fats, and soaps, grape sugar, and a quantity of salts amounting to about three per cent, of the solid residues- Crystals of an alleged albimainous body were observed during the drying of albumin by A. Boettcher {Arch. Path. Anat. 32, 1865, 525 and 533). They were found in different organs and liquids, and were variously interpreted by different authors, but according to Schreiner {Liebig'e Ann. 194, 1878, 73), they are the phosphate of an organic base, P04H,2(C2H5N") 4 H^O. At 100 it loses 3 H^O. (See Spermatin.) Oonin was separated from white of egg by freezing by Couerbe {J. Pharm. 15, 497, Ann. Chim. Phys. 41, 323). Liebig in his lectures mentioned this experiment and termed the matter a mucous substance ; when it is removed the white of egg is quite liquid and filterable. Some differences between the whites of the eggs of fowls and those of pigeons were discussed by Jahn {N. Br. Arch. 37, 259 ; Berzel. Jahresb. 25, 875). Observations on the whites of wild birds' eggs were made at the Pathological Institute, and will be communicated on a future occasion. 176 ANNALS OF CHEMICAL MEDICINE. Modes of purifying albumin. — Among older modes is that of Wiirtz (Compt. rend. 18, 700) who precipitated diluted and filtered white of egg with basic lead acetate not in excess ; suspended the washed precipitate in water, and passed carbonic acid through the liquid ; filtered, precipitated traces of lead from the filtrate by a few drops of aqueous hydrothion ; heated the clear brown liquid to 60°, whereupon the sulphide of lead was precipitated with the first flocks of albumin. He then filtered again and evaporated the filtrate at 50°. The product still contains salts on its own showing, as pure albumin is not precipitable by boiling ; it also contains acetic acid, and is precipitable by ammonia. Von Wittich's process (iT. pr. Chem. 73, 18) produces an altered albumin, probably syntonin. The only process which is known to produce albumin absolutely free from ash is that of Grraham (Liebig's Ann. 121, 61); see also Aronstein {Archiv Physiol. 8, 1874, 75). The albumin to be purified is freed from membranes by agitation and filtration ; the albumin may be diluted with an equal volume of water, or it may be filtered without dilution, by means of the pressure- filter described in my researches on the brain. The albumin thus prepared may be acidulated with dilute acetic acid, if it is desired to expedite the following processes ; but when it is desired to separate pure plasmin (paraglobulin) it is perhaps advisable to avoid the use of acetic acid, as it might possibly transform some of the albumin into syntonin. It has, however, not been proved that such a transformation does actually take place under these circumstances. The albumin, whether acidulated, neutral, or feebly alkaline is now placed into a cell of a size and shape found by experience to be convenient for dialysis. This cell is made, as far as its active surface is concerned, of parchment paper prepared by the process of Graine. Great care must be taken to procure actual parchment paper of the best quality, and to avoid the counter- feit paper made by means of gelatin and alum. The layer of albumin in the cell should be thin, and the water into which it is placed should be renewed two or three times a day. The apparatus should stand in a room the temperature of which is about 10° to 12°. Four to five days of dialytic action are sutficient to free the albumin from all salts. SOLUBLE ALBUMIN. 177 The albumin to be placed into the dialysing cell should first of all be raeasured, and should again be measured after the dialysing is completed, in order to ascertain the quantity of water which passes into the cell and becomes mixed with the albumin. It is further advisable to keep a measured quantity of the same albumin as that which is placed in the dialyser in an unmixed state, and allow it to stand by the side of the dialysing albumin, in order to be able to compare the reactions of the original impure albumin from time to time with the specimen which is being purified by diffusion. If it is desired to obviate the difference in concentration between the original and the dialysed albumin, the latter may be concentrated to its original volume over sulphuric acid in vacuo, or the former may be diluted with water to the bulk of the dialysed specimen. The amount of the increase in volume by complete dialysis amounts to about one-half the original volume. Should the liquid become undesirably voluminous, and should diffusion proceed but slowly, it is sometimes advisable to remove the albumin from the dialyser, filter it from any plasmin which may be deposited, and evaporate it to a smaller bulk in vacuo over sulphuric acid. The concentrated liquid, on being returned to the dialyser, parts with its salts much quicker. The diffusate or dialyaate. — The diffusates are united and evaporated to the volume of the albumin originally placed in the cell ; during this evaporation small quantities of albumin, which pass into the diffusate, are coagulated, and have to be removed. After this the diffusate is free from aU albuminous matter. It contains sodic chloride, and sulphate, sodie and potassic phosphate and carbonate ; but even when strongly over- saturated with ammonia, it deposits no earthy phosphates. The diffusate contains peculiar nitrogenous matters in solution which have not yet been investigated. On evaporation to dryness it leaves a yellowish-brown residue, which burns with a strong odour- of horn. When the matter is completely calcined, it leaves an ash, of which a part is soluble, another part insoluble in water. The latter is soluble in hydrochloric acid, and consists of phosphates of calcium and magnesium with excess of lime (and magnesia ?). N 178 ANNAI^, OF CHEMICAL MEDICINE. It is therefore clear that the solution of the earthy salts in the albumin (serum or egg) is caused by one or several of the nitrogenous matters which also accompany these salts into the dififusate. When the dialysis is complete the whole of the soluble and insoluble salts have passed into the diffusate, and the albumin in the cell is completely devoid of them ; this proves that the phosphates of earths and the earths cannot be, as is sometimes assumed, in combination with the albumin ; they are however in a soluble combination with nitrogenous matters, which are not albuminous. Deposition of the plasmin {jpa/raglohulin) during dialysis. Soon after the albumin has been put into the dialysing cell it begins to become turbid, and at the same time the presence of alkaline salts in the diffusate can be proved by reagents. If the albumin be filtered every 24 hours, and the water surrounding the cell be renewed, it will be perceived that each time a new turbidity is produced in the albumin, and the diffusate contains salts anew. But the quantities of deposit in the albumin and of salts in the diffusate diminish at a certain rate, so that in equal times more deposit and salts separate from the albumin at the beginning of the experiment than towards the conclusion. At last there comes a period in the experiment, at which the albumin remains clear, and the diffusate contains no trace of salt. The solution in the cell then contains the albumin in a pure state ; the diffusates contain all the soluble salts, and the insoluble salts dissolved by the peculiar nitrogenous matters, and all the plasmin has been collected on filters. The plasmin consequently was dissolved in the original (serum or egg) albumin, by means of the substances which pass into the diffusate. If the plasmin collected on filters, is shaken up with a little water, and if then the diffusate concentrated to the volume of the original albumin solution is added to it, it redissolvies immediately and completely. This plasmin is identical with the plasmin which can be precipitated from serum and egg albumin by dilution with much water, acidification and filtration ; when serum or egg albumin thus treated and again concentrated to its primitive volume, is subjected to dialysis, it does not any longer form the deposits above described. Inversely, when the albumin, freed SOLUBLE ALBUMIN. 179 from all salts by dialysis and filtered from the insoluble deposit which forms during that process, is diluted with much water, and very slightly acidified, no deposit takes place. It is there- fore almost certain that both precipitates are made up of one and the same substance, namely plasmin. It is insoluble in water, easily soluble in dilute acids and alkalies, in carbonates of alkalies, and in neutral salts of alkalies. It also can be utilised for the production of fibrin, a circumstance to which it owes its name. Characters of the alhuTnin freed from salts &o. by dialysis, and from plasmvn by filtration. — ^Evaporated to dryness and burned with all necessary precautions in platinum vessels it leaves not a trace of ash or residue of any kind. Diluted with from 8 to 10 parts of water, and boiled in that state, or if it is alkaline or neutral, previously acidified by a little acetic acid and boiled, it remains perfectly clear. It is not changed in any manner by the addition of strong alcohol, even of absolute alcohol up to twenty volumes. If the albumin is mixed again with the difiiisate, reduced to the concentration which corre- sponds to its share of the bulk of the original albumin, a solution is produced which has all the properties of the original albumin, inasmuch as it curdles on boiling and by the addition of alcohol. The process of diffusion, though extending over several days has therefore not produced any material changes in the properties of the albumin itself; while the pure albumin of serum and eggs is not coagulable by heat or alcohol, the albumin containing salts is so coagulable. It is therefore clear that the coagulability of the natural solution of albumin depends essentially upon the presence of crystalloid bodies. When the part of the ash of the diffusate soluble in water is mixed with pure albumin, it also regains its coagulability by heat and alcohol. The addition of only 0*25 grm. of sodie chloride to 100 c.c. of pure albumin solution restores its coagulability. Potassic chloride, and iodide, sodic iodide magnesic sulphate, and ammonic carbonate have the same effect. The coagulability of natural solutions of albumin therefore depends on the presence in them of soluble salts. Quantities of salts necessary for the production of the coagulable state. — If during the progress of the dialysis small N 2 180 ANNALS OF CHEMICAX MEDICINE. samples of the albumin are taken from the dialyser from time to time, it will be found that the precipitates produced by alcohol or boiling are constantly decreasing in bujk and im- portance ; towards the end of the dialysis only turbidities are observed on the application of the reagents, a little later on opalescence constitutes the result, and ultimately the albumin remains quite clear, indicating the complete removal of the salts. This proves that for the coagulation of a certain amount of albumin a certain minimum amount of salt is required to be present. It is probable that this minimum has as determinants the amount of water in which it is contained, as well as the amount of albumin which is dissolved in it. When to portions of a solution of albumin freed from salts by dialysis very small but increasing quantities of sodic chloride are added, a series of liquids are obtained which, on application of heat or of alcohol, present all degrees of coagulation, from a mere opalescence to the complete precipitation of all albumin. The amount of sodic chloride necessary to effect a complete coagulation of the albumin from either white of egg or blood- serum is much smaller than the amount of salt usually contained in these natural fluids ; or these fluids contain more salt than is required to produce coagulation by heat or alcohol. If to pure albumin very small quantities of salt are added, a portion only of the albumin can be precipitated, and this part stands in a certain relation to the amount of salt present. This relation can be ascertained by adding known quantities of salt solution to known quantities of pure albumin of given concen- tration, and boiling or adding alcohol. The precipitate pro- duced is filtered and weighed, and referred to the sodic chloride employed ; the filtrate is then completely precipitated with the aid of excess of sodic chloride ; the sum of the two precipitates gives the total albumin. The effects of such small quantities of salt as lead to the production of only an opalescence or a turbidity cannot be measured by weighing the precipitates, which would probably run through any filter, but can only be estimated by measuring the amount of most dilute salt solution which was required to engender their production, and calculat- ing from this and the data given by more concentrated solution their probable amount. One hundred c.c. blood-serum, which SOLUBLE ALBUMIN. 181 during dialysis had lost almost the whole of its salts and had gained 21 c.c. of water, were subjected to the following tests by Aronstein. The tests were compared with similar tests made upon a portion of the same blood-serum which had not been subjected to dialysis but had been diluted with water in the proportion of 21 c.c. of the latter to 100 of the serum. The amount of alcohol added in each case was sixteen times the volume of the liquid to be precipitated. The precipitates were collected on filters and dried, and weighed with the usual pre- cautions. The following numbers refer to 100 c.c. of the un- diluted serum. One hundred c.c. of natural serum gave 6'521 grms. coagu- lated albumin. One hundred c.c. of serum freed from salt by dialysis gave on addition of 0-030 grms. NaOl 2-423 grms. coagiilated albumin ; on addition of 0-080 grms. NaCl 3-831 grms. coagulated albu- min; on addition of 0-160 grms. NaCl 4-859 grms. coagulated albumin. The serum free from salt gave a lesser maximum of precipi- tate than natural serum, because the process of dialysis removes small quantities of albumin, which pass into the diffusates, or precipitates matters like plasmin, which are precipitated from serum together with the albumin and weighed with it. Aron- stein believes that extractive matters also might be included in the serum precipitates; of this however there is no direct proof. The data further show that as the amount of salt added to the solution of albumin rises, so the quantity of albumin preci- pitated from it by alcohol rises ; further that the whole of the albumin is precipitated from a solution of the strength of the- one operated upon (which contained nearly five per cent, of albumin) when the amount of salt contained in it amounts to 0-160 per cent. Ordinary blood-serum therefore contains about four times as much soluble salt as is necessary to ensure its coagulation by heat or alcohol, namely about 0*6 per cent. These data prove that the statements of Kiihne and Hoppe- Seyler, according to which albumin free from salt could not be obtained by dialysis are not well founded, and that their pro- ceedings must have been vitiated by either faulty manipulation or bad materials. 182 ANNALS OF CHEMICAL MEDICINE. Belation between the quantity of salt and the strength of the alcohol required for precipitation of albumin. — The amount of salt and the strength of alcohol stand in an inverse propor- tion to each other. Two equal volumes of a pure solution of albumin were mixed the one with 16 vols, of spirit of 45 per cent, strength, the other with 16 vols, of alcohol of 90 per cent, strength. Both liquids remained perfectly clear. To each was now added a very dilute solution of sodic chloride in very small quantities from a very finely divided burette admitting of the reading of a hundredth part of a cubic centimetre, until the albumin began to be precipitated. The mixture with the spirit of 45 per cent, required 0*860 per cent, sodic chloride to pre- cipitate the albumin ; that with the 90 per cent, spirit required only 0*017 per cent, sodic chloride, or only one fiftieth part of that which the more dilute spirit made reqiiisite. The action of the salt in alcoholic solution is therefore not uniform with the per- centage, but increases manifold with the strength of the alcohol. A solution of albumin freed from salts by dialysis which will remain perfectly clear with 1 6 vols, of spirit of 90 per cent, will yet sometimes give a turbidity or opalescence on the addition of 1 6 vols, of absolute alcohol. Such albumin on incineration gives no ash and no reaction for chlorine in the platinum vessel used for the combustion. The opalescence is therefore the only in- dication of the presence of salts, and is a more delicate reaction for chlorine than the silver test. This shows the necessity of working in these experiments with absolutely pure reagents and vessels. The mere presence of a few drops of ordinary well water, or some atmospheric dust containing as it always does . chloride of sodium, is capable of vitiating the experiment, and to produce precipitates where if they had been excluded there would not have been any. Incipient decomposition restores coagulability of solutions of pure albumin. — When a solution of pure albumin, obtained by dialysis, is allowed to stand for some days, it begins to de- compose, and then is again precipitated by heat as well as alcohol. The precipitability begins with a feeble opalescence, and gradually increases so much that all the albumin nearly can be coagulated. This return of the original properties is due to the formation, by decomposition of some part of the albumin, SOLUBLE ALBUMIN. 183 of salts, particularly ammoniacal salts. Aronstein tested the carbonate of ammonium, and found it as active in engendering coagulability as sodic chloride. It is therefore necessary to keep all dialysing fluids at a very low temperature, below 12° at least, and as near zero as circumstances permit. The present experiment however also shows, that an albumin which gives, after dialysis, the reactions of pure albumin, cannot be decomposed. Difference of serum,, and egg-atbumin in their behaviour towwrds ether. — Serum and egg-albumin show the same reactions towards all bodies known to react with them, except ether ; from white of egg the albumin is precipitated by etlier, from serum it is not precipitated by this agent. But when the albumins are free from salts, they show the opposite bearing ; the pure egg-albumin is not precipitated by ether, while the pure serum-albumin is precipitated. If to the pure solutionSj a little salt is added, the properties of the natural solutions are restored. With egg-albumin, the presence of salt is a condi- tion of the coagulability by ether, while as regards serum- albumin, the presence of salts prevents coagulation by heat. From the foregoing it follows that the views put forward by Scherer, Denis {Memoire sur le sang, 187), and Eichwald {Wurzh. Med. Zeitschr. 5, 318), (probably under the influence of Scherer) viz. that egg-albumin and animal albuminous sub- stances generally are in themselves insoluble in water, and be- come soluble only after combination with salts or alkalies, is not tenable as regards the great bulk of the substance termed albumin, but is correct only as regards a small portion of the albuminous matter occurring in animal liquids, namely the plasmin (paraglobulin, or serum-casein), and the fibrinogenous matter so called. Quantation of albumin in animal liquids, with the aid of dialysis, according to Heynsius {Archiv Physiol. 10, 1875, 239). Heynsius objects to the use of nitric acid for the pre- cipitation of albumin ; jBrstly, because precipitates of uric acid might simulate albumin ; secondly, because in liquids containing a small proportion of salts nitric acid redissolves a small quantity of albumin, and forms acid-albumin. He formerly recom- mended the precipitation of albumin by boiling in presence of 184 ANNALS OF CHEMICAL MEDICINE. acetic acid and sodic chloride (1870; he does not claim the method as having been new then), but he now finds the method, though stiU suitable for removing all albumin out of animal liquids, unsuitable for its quantation, as the washing required dissolves much curdled albumin and carries it into the filtrates. In some experiments on partially dialysed white of egg, Heynsius found, that the addition to 50 c.c. of the liquid of 2 c.c. of ordinary acetic acid and quantities of sodic chloride varying from 32 per cent, (satiuration) to 4 per cent., and boiling, all albumin was precipitated, so that the filtrates did not contain any ; from 2 to O'l per cent, of sodic chloride enabled some albumin to remain in solution. Heynsius then criticises the method of Scherer, namely addition of acetic acid to the animal liquid in such quantity that the mixture after boiling shall yet have an acid reaction, a process in which he alleges that acid- albumin is formed ; and says that it gives results which are too low, because a part of the albumin remains dissolved — in two eventualities, either when (and because) the acid was in excess relatively to the salt, or the salt was in excess relatively to the acid. The statement of Heynsius on this point is by no means easily intelligible. Liborius {Deutsch. Archiv Min. Med. 10, 1872, 320) had examined the bearing of albuminous solutions, diu:ing boiling, in two series of experiments; one in which the quantity of acetic acid was stationary, while that of the sodic chloride varied; and another in which the amount of sodic chloride was stiitionary, while that of the acetic acid varied, and had found that in both series the amount of albumin obtained was less than that actually contained in the liquid. Heynsius made a number of similar experiments on white of egg and serum, and came to the same result. A minimum of sodic chloride and of acetic acid gave the largest amount of albumin. But the amount obtained never was the whole amount present, as the washing water always dissolved albumin, which could be identified. He therefore declared this method (of Scherer) unsuitable for the quantation of albumin. The so-called method of Berzelius, consisting in evaporating the albuminous liquid, mixed with a little acetic acid, to dryness, and extracting the residue with alcohol, also gives results which SOLUBLE ALBUMIN. 185 do not express the whole of the albumin present, some of it going into the extracts. Liborius and Heynsius now come to the result, that the albumin is probably most accurately estimated by precipitation with alcohol,- after its solution has been carefully neutralised. But in this case a considerable amount of ash remains in the albumin (from 10 to 20 per cent, of the entire ash), which has to be found by incineration and deducted. To avoid this drawback, Heynsius subjects the albuminous solution in which the albumin is to be estimated, to dialysis, and evaporating the solution, after removal of crystalloids, to dryness, assumes this residue, less 2 per cent, of ash, supposed not to be removable by dialysis, as the amount of albumin in the original fluid. It deserves to be mentioned that Heynsius employed parchment paper, which allowed so little albumin to pass into the diffusate, that it did not affect even the second decimal of the quantities dealt with in his experiments. If albumin had to be determined in urine by this method, salts might be deposited and escape dialysis. In such a case it might be useful to dialyse the liquid successively, first in the alkaline, next in the acid condition. From an inspection of the data adduced by Heynsius in proof of the advantages of his method, it appears that they are literally very small. If it is considered that the amount of ash retained in the dialysed albumin is foimd to vary with every specimen of dialysing paper employed, and that notwithstand- ing this experience, a uniform 2 per cent, of ash is to be deducted, it cannot be admitted that the method can be employed by anybody else besides Heynsius, unless they procured paper which permits no albumin to pass into the diffusate, and unless they determine the average amount of ash (if any) retained by the dialysed albumin. It is more than doubtful whether this amount will be a law of nature so fixed as to enable the observer to dispense with a quantation in each case. And if such be necessary, then it would be more practical to estimate the ash in the albumin precipitated by alcohol (10 to 20 per cent, of the entire ash) than the much smaller quantity contained in the dialysed albumin, and to save the trouble and time expended upon the dialysis. 186 Al^NALS OF CHEMICAL MEUTCINE. D. Haizinga's dialyser {Archiv Physiol. 11, 1875, 392) consists of a frame of vulcanite (hardened caoutchouc) 5 milli- metres thick, 1 centimetre broad, and enclosing an area of about 70 square centimetres. The lower or square part of this is covered on both sides with parchment paper. The papers are cut a little smaller than the external margin of the frame, as they expand when they are moistened. The vulcanite must be made rough with sand or emery on the outer sides, where the paper is in- tended to be fixed. The paper in the wet state is fixed upon the frame by means of chromate glue. To produce the latter 10 grms. of dry gelatin are dissolved in 50 grms. of water, and to this solu- tion 0'5 grm. potassic bi- chromate dissolved in 10 grms. of water is added. This glue must be pre- served in the dark, as it is made insoluble by light. It must also be made fresh from time to time, as re- peated heating has a ten- ' dency to make it lumpy. The vulcanite frame to which two pieces of parchment paper have been pasted one on each side by means of this glue, is exposed to diffuse daylight, whereby the glue becomes in- soluble; the parchment paper must remain a little moist, to prevent its Assuring. The cell thus made is tested with water, and if it is tight, is freed from the excess of chromate by immersion in water. It is now ready for use with albumin or other matters. It is placed in a flat glass cell which is SOLUBLE ALBUMIN. 187 only a little larger than itself, and fiUed with albumin, pre- pared as above described, to about two-thirds of its height. The space between the glass and the dialysing cell is filled with distilled water. This latter is frequently renewed by an auto- matic arrangement (such as is described on p. 127 of my re- searches on the brain, Rep., New Series, Nr. III.), termed a perpetilal syphon, while the water which has performed dialysis and is charged with products is drawn off at intervals and in a larger stream, or constantly and in a smaller current, by a second syphon, delivering into a receptacle for waste, and drawing the waste from the bottom of the glass cell. The principal advantages are that the thin layer of liquid is dialysed on two sides, and therefore in a lesser time than by the tam- bonrine-dialyser ; and further that any deposit which forms in the liquid sinks to the bottom of the frame, and has little chance of obstructing the paper, an accident which not rarely happens with the horizontal paper, particularly in the case of albumin, from which plasmin is always precipitated during dialysis. The albumin to be placed into this cell should be carefully neutralised ; acid albumin dialyses very slowly, as already ob- served by Grraham. 80 c.c. of albumin in the cell should be acted upon by a litre of water per hour for 24 hours ; in that case the albumin does not coagulate any more either by heat or alcohol when diluted with the fourfold volume of water ; after 36 hours the albumin ceases to react with silver nitrate or mercuric chloride. After 48 hours the albumin is no longer changed by the dialytic process. It has increased in bulk but little, is turbid by flakes, but passes through a filter of Swedish paper, freed from ash by treatment with acids etc., quickly and clear. A portion of albumin thus treated is evaporated in a weighed platinum dish on the water-bath, the weight of the residue is determined, and the residue is charred. On extraction of the char with hot water, it is found that it does not contain a trace of soluble salt. The water, though perhaps containing a trace of chlorine, Teaves no residue and no weighable matter in the platinum dish in which it has been evaporated. The charcoal is now burned white, and leaves a small quantity of incombus- tible matter containing calcium, iron, a trace of magnesium, 188 ANNALS OF CHEMICAL MEDICINE. and phosphoric acid. In Huizinga's qaantation the amount of ash thus obtained varied from 0-35 to 0*56 per cent, of the albumin burnt ; and the amount of dry dialysed albumin burnt varied between 0-310 grms. and 1*1376 grms. The dialysed solutions of albumin, if undiluted, give a pre- cipitate with silver nitrate and with mercuric chloride. When the albumin is diluted with four volumes of water, these re- agents no longer produce a precipitate by themselves : but when to the mixture with silver nitrate a drop of nitric acid is added, a considerable flaky precipitate is at once produced. Bewring of dialysed cdbumvn solution with acetic acid. — Schmidt found that the addition of very dilute acetic acid to dialysed albumin restored its coagulability by heat, but a more considerable addition of acetic acid produced again incoagula- bility. When Huizinga added to 5 c.c. of a solution of albumin in 4 water, containing therefore 1 c.c. of original albumin, — 1 c.c. of acetic acid of one-fiftieth per cent, strength (there- fore equal to 0"2 milligrams CjH^Oj), the mixture coagulated strongly on boiling ; but when acetic acid of one-hundredth per cent, strength equal to O'l milligram G^Ufi^ was taken, only an opalescence and no precipitate was produced. It was easy to suppose that the acid in the first case (0*2 milligr. CgH^O^) had been sufficient to decompose an assumed albuminate, while the 0-1 milligr. in the second case might have been insufficient for that purpose. If this explanation was correct 5 c.c. of a solution of albumin diluted to ten times its bulk, therefore containing 0*5 c.c. of original albumin, should have been coagulated by boiling after addition of 1 c.c. of acetic acid of one-hundredth per cent, strength. But this was not the case ; no coagulation took place ; while 1 c.c. of the one- fiftieth per cent, acetic acid produced coagulation. On diluting albumin twentyfold the same result was obtained. 5 c.c. of this albumin mixed with 1 c.c. of one-fiftieth per cent, acetic acid coagulated in flakes on boiling ; while 5 c.c. of the same solution mixed with 1 c.c. of ^-^ per cent, acetic acid only opa- lesced on heating. Only when the dialysed albumin was diluted to forty times its bulk did boiling after the addition of 1 c.c. of yi-g^ per cent, acetic acid to 5 c.c. of the solution produce a precipitate. SOLUBLE ALBUMIN. 189 There is therefore no direct proportionality between the quantity of albumin and the quantity of acetic acid which makes the albumin coagulable, and therefore it is not probable that the acid acts by displacing a base from its combination with albumin. Bearing of dialysed alhumin with increasing quantities of acetic acid. — When to 5 c.c. of a dialysed solution of albumin diluted to its tenfold bulk 1 c.c. of acetic acid of 0*02 per cent, strength was added, and the liquid was boiled, flaky coagulation took place. With the same quantity of albumin and 1 c.c. of acetic acid of 0*04 per cent, strength, the coagulation on boiling was imperfect. Upon the same quantity of albumin, the same volume of acetic acid, increasing in strength by 0'02 with each experiment acted so that only opalescence was produced, which decreased in density as the acid increased in strength ; 5 c.c. of the albumin solution with 1 c.c. of acetic acid of 0-25 per cent, strength remained clear on boiling, and double the amount of acid also left the liquid clear. In order therefore to retain the albumin in solution on boiling (after having destroyed its solu- bility on boiling by the addition of most minute quantities of acetic acid), it is necessary to add so much acetic acid that its amount in the mixture shall be at least -^ per cent, of the mixture. When the dialysed albumin diluted to ten times its bulk has become opalescent by boiling, much larger quantities of acetic acid can be added to it, without any precipitate being produced by boiling. The fluid however which has become opalescent by boiling, gives with silver nitrate a flocculent pre- cipitate. Larger quantities of acetic acid keep the albumin in solution on boiling, so that the fluid remains clear. But the amounts of acid necessary vary with the concentration of the albumin. We have seen before that in order to cause 5 c.c. of the dialysed albumin diluted to its tenfold bulk to remain clear on boiling, the addition of 1 c.c. of acetic acid of ^ per cent, strength is requisite. From this it might be supposed, that in order to produce the same effect upon a solution of albumin of double the concentration, the addition of 1 c.c. of acetic acid of -i per cent, strength would be sufficient. This, however, is not the case ; 5 c.c. of the albumin solution diluted to fiyefold its, bulk 190 ANNALS OF CHEMICAL MEDICINE. requires, in order to remain clear on boiling, the addition of 1 c.c. of acetic acid of 1 per cent, strength. With rising concentrations of the albumin, the quantity of acetic acid necessary to keep it in solution on boiling also increases, but not in an equal proportion. For in order to remain clear on boiling, 5 c.c. of a solution of albumin diluted to fourfold bulk require 1 c.c. of acetic acid of 2 per cent, strength ; 5 c.c. of a solution diluted to twofold bulk, require 1 c.c. of acid of 20 per cent, strength. The undiluted dialysed albumin solution requires upon 5 c.c, 2 c.c. of concentrated acetic acid, to retain all the albumin in solution on boiling. The quantity of acid necessary to retain the albumin in solution, rises much quicker than the amount of albumin to be retained. The albumin solution as it leaves the dialyser of Huizinga, contains from 1|- to 3^ per cent, of albumin, when the albumin on entering the dialyser amounted to about 5 per cent, of the solution. Taste of dialysed albumin. — ^The taste of a dialysed solution of albumin of from 2 to 3 per cent, strength is clearly sweet ; the taste is an aftertaste, and resembles that of liquorice root. When the dialysed albumin has been dried and heated, it is perfectly tasteless. A. Winogradoff (jdrcAw Physiol. 11, 1875, 605) also sub- jected white of egg to dialysis, on the so-called parchment paper used by A. Schmidt; the dialysers were 18 centimetres in diameter. The albamin was always diluted with three times its volume of water, and filtered through paper, but not neutral- ised. When the outer water did not any longer contain chlorides the process was interrupted : the albumin had the properties described by Aronstein, but possessed an alkaline reaction, colouring reddened tincture of litmus blue ; —^ normal acetic acid, that is to say a solution of 60 grms. of crystallisable acetic acid in 940 grms. of water, was further diluted with two volumes of water (so as to contain 60 grms. of acid and 2940 grms. of water), and used for neutralising the albumin ; the acid having been added drop by drop until the albumin had assumed a violet-red colour, the solution was now heated, when all the albumin was precipitated ; the filtrate was so free from albumin that when it was mixed with an equal volume of solution of salt SOLUBLE ALBUMIN. 191 and some acetic acid, it remained perfectly clear, and when evaporated by itself and the residue burned only a slight char- ring was observed. This experiment showed therefore that a solution of albumin which had lost its coagulability by heat and alcohol in consequence of its having imdergone dialysis, re- covered its coagulability by an addition of acetic acid. Winogradoff now made some experiments to determine the amount of ash, if any, which solutions of albumin subjected to dialysis, might retain. Each dialysis was made with 30 c.c. of egg albumin, to which 90 c.c. of water had been added. The dialysis was continued until the diffusate was free from chlorine, the solution of albumin on the dialyser was then measured, treated with acetic acid in the manner described, and coagulated on the water-bath. The albumin was collected on weighed filters, dried and weighed, and then incinerated. The filtrate from the coagulated albumin and the washing water were also evaporated and incinerated, the dififusates were treated in the same manner. The following tables show the results of the experiments as obtained by means of two different kinds of dialysing paper, one sized so-called English, as used by A. Schmidt, another German, thicker than the English, of uncertain quality, as used by Heynsius. I have divided the table of Winogradoff into two tables, so as to keep apart statements referring to absolute quantities operated upon, and figures derived from absolute quantities by calculation referred to percentages. Experimeiit I. Experiment 11. Experiment III. 120 c.c. of di- lute albumin, containing 30 c.c. of white ofegg, rose to Sized •218 C.C. tTncertam 140 c.c. Sized 240 c.c. Uncertain 138 c.c. Sized 230 c.c. TTncertain 154 c.c. It is at once evident that the amount of water which passed into the albumin on the dialyser, and inversely the amount of albumin which passed from the dialyser into the diffusate, were much greater when so-called English sized paper was used, than when German paper of uncertain quality was employed. 192 ANNALS OF CHEMICAL MEDICINE, Winogradoff does not describe the nature of the German paper used by him, but of the so-called English paper he says that it was the same as that used by A. Schmidt, and actually forwarded to him by this author. The latter had in the interval ascer- tained that this so-called English parchment paper, though obtained from London, and bearing the watermark of a London firm, was in reality made in Germany, and was not parchment paper made by the process of Gaine, such as had been used by Graham, but a paper to which hardness and transparency had been given by impregnation with gelatin and alum ; the dialysis was therefore in reality carried on through a septum of gelatin and alum, or rather through two septa of gelatin and alum one on each side of a piece of common paper. The following table wiU. after this explanation be much more instructive than it could have been without it. For it will show that the size and alum paper besides passing much more albumin into the diffusate than the other paper, diffused a lesser quantity of the salts of the albumin into the dialysate, and left more salt in the albumin on the dialyser, so that both the curdled albumin and its mother liquor left more ash than the corre- sponding preparation obtained with the other (German) paper. Neither of the papers gave the results obtained by Graham, possibly because the nondescript German paper also was not Expenment I. Ezpenment n. Experiment m. Sized TJncertain Sized XTncertaiii Sized tTncertain Albumin in 100 1 parts of white of egg . Not known 10-49 10-83 Albumin in solution after dialysis 7-87 9-87 7-26 869 6-34 9-60 Ash in diflFii- sate 0-467 0-548 0-591 0-625 0-614 0-636 Ash in curdled albumin 0-0186 0-0296 0-0226 00240 00233 0-0206 Ash in filtrate from curdled albumin 0-1671 0-1493 0-1446 0-1120 0-1846 0-1463 Sum of ash in dialysed albu- min solution 0-1857 0-1789 0-1672 0-1360 0-2079 0-1669 SOLUBLE ALBUMIN. 193 paper prepared by the process of Graine, but some imperfect imitation, though a better dialyser than the thinner size and alum paper. The foregoing table gives the results of the dialysis in three experiments made each with the two kinds of paper. They are calculated upon 100 parts of the original white of egg, the dilution by water added before dialysis, and by water transferred during dialysis, being deducted. The salts consisted of carbonates and chlorides of alkalies, and of phosphates of earths. The dialysates of the sized paper moreover gave sulphates. The inequality in the action of the two kinds of paper is not only seen in the first table but in almost all the items of the foregoing second table. The second (uncertain) paper removes much less albumin into the diffusate than the sized paper ; it removes, on the contrary, a much larger quantity of salt out of the albumin, a quantity which (considered as work performed by the dialyser) would appear much greater than in the table, if the salts, which must have passed with the albumin to which they were potentially related, are deducted from the ash in the diffusate, not accompanied, so to say, by the albumin previously related to it. It is conceivable that as of two indifferent dialysing papers one removed more, the other less of salts out of the white of egg to be purified, a paper of the quality such as Graham employed made by the process of Graine, and not impregnated with any size or chemical ingredient whatever, may produce the effects described by Graham. Until that point is decided by incontro- vertible experiment, a final view concerning the nature of pure albumin solutions cannot perhaps be formed. But how little the experiments hitherto made are comparable is not only evident from their study, but from even slight experimental epicrisis. While writing these lines I dialysed the white of three eggs through thin good parchment paper, until it was incoagulable by heat and alcohol. At no stage of the dialysis was there any albumim, vjhatever in the diffusate, Winogradoff made three further experiments, but without specifying the paper used as dialyser. and without comparing the 194 ANNALS OF CHEMICAL MEDICINE. two papers with each other. We will term the experiments N"- IV. V, and VI. Exp. IV. — Twenty-five c.c. of albumin were mixed with 25 c.c. of water and dialysed in a bell-shaped dialyser suspended in water for forty-six hours, during which the water was renewed eleven times. At the conclusion of the dialysis the volume of the 50 c.c. of liquid on the dialyser had increased to 224 c.c. It did not curdle on being boiled, but became slightly turbid. Considered as condensed to the original 25 c.c. and multiplied by four, so as to represent one hundred parts of albumin changed only by the loss of salts (and albumin), the fluid gave on analysis the . following data: Albumin 10'98 per cent.; ash (earthy phosphates) in curdled albumin 0-0236 per cent. ; ash in filtrate from coagulum (being chloride, phosphate and sulphate) 0*1224 per cent., or total ash of the dialysed liquid 0'1451 per cent. (Winogradoff says that these data are ' = 1'32 per cent, ash in the albumin, and of this 0*215 per cent, earthy phosphates.' Here albumin means the imaginary dry residue of the dialysate. I have been obHged frequently to surmise the cohesion of data and calculations in his paper, putting on them the best con- structions possible, a trouble which the author might with advantage to himself have spared to his readers.) Exp. V. — The fresh white of egg contained 12-31 per cent, albumin ; its coagulum contained salts 0*0333 per cent, of the white of egg, and the filtrate from this 0-6626 per cent. Thirty c.c. of this white of .egg were diluted with 30 c.c. of water and dialysed during 62 hours in water which was thirteen times renewed. The volume of the albumin had risen to 370 c.c. or more than six times the initial volume. Considered as condensed to the original 30 c.c. and multi- plied so as to represent one hundred parts of white of egg changed only by the loss of salts (and of some albumin), the fluid gave on analysis the following data : albumin 8-96 per cent. ; earthy phosphates in the coagulum 0-0176 per cent.; earthy phosphates in the filtrate 0-0986 per cent.; total ash 0-1162. (Here Winogradoif also says that this was equal to ' 1-29 per cent, ash in the albumin, and of this 0-16 per cent, earthy phosphates ; ' from which we may conclude that by albumin he means here also the imaginary dry residue of the dialysate. SOLUBLE ALBUMIN. 195 Exp. VI. — This dialysis was made with 30 c.c. of white of egg diluted with 30 c.c. of water, in a bell-shaped dialyser suspended in water which was kept constantly fresh by a current of 250 c.c. per hour, increased after 36 hours to half a litre per hour (time during which the latter treatment lasted not stated). At the conclusion of the dialysis the liquid in the cell measured 175 c.c. ; it contained a considerable deposit of paraglobulin, and had a feeble alkaline reaction. Addition of acetic acid and boiling curdled aU the albumin. The analyses of the liquid calculated as in the previous two experiments gave albumin only 2*37 per cent. ; salts in the coagulum 0-0020 per cent. ; salts in the filtrate 0-0173 per cent.; total salts 0*0193 per cent ; or the dried residue of the dialysed liquid con- tained 0*81 per cent, of ash, and of this 0-084 per cent, were earthy phosphates. Although in this liquid the salts liad been more diminished than in any previous one, it coagu- lated on the application of heat alone, without any addition of acid. The enormous loss of albumin shows that the dialyser was very bad in one respect, although it effected a great removal of crystalloids. The coagulability Winogradoff refers to a return of this faculty (which he considers, so it seems from the context, had been lost in the course of the earlier period of the dialysis) but be does not believe it to be due to beginning putrefaction, as Aronstein does, who also assumes the formation of some ammonic carbonate, and ascribes to this the return of coagula- bility by heat. This return of the coagulability by heat and alcohol in albumin freed from this liability by dialysis I have also observed, after filtering it through paper not previously expressly freed from salts by acids and water etc. I have however not yet proved that the renewed coagulability is due to the admixture of salts from this source. It is possible that the albumin, though not smelling in the least of decomposition, may never- theless be altered, as by time it alters in almost all known solutions. Properties of the natural white of egg. — It can be dried, when in thin layers, quickly in a current of air, and then forms o 2 196 ANNALS OF CHEMICAL MEDICINE. a fissured, hard, brittle, white or yellowish translucent mass, which has neither taste nor smell. It is again soluble in water, but leaves a slight insoluble residue. With four parts of cold water it forms a mass resembling fresh albumin, and with more water a slimy solution. This is perhaps referable to the oonin, the mucus-like substance removable by freezing, which swells in contact with water to a large bulk, and can only with difficulty be filtered from the albumin. Polarisation. — The clear solution of egg albumin rotates a ray of polarised light to the left, the specific limited rotation being (^* = —35° 5'; the addition of hydrochloric acid in- creases this rotation to — 37°7' ; strong caustic potash at first increases the rotation to —47°, but reduces it later. Danilewsky (Krit Zeitschr. f. Chem. [2] IV., 12, 41) has stated that in hen's egg albumin there is contained an optically inactive albumin containing 2 per cent, of sulphur, by the splitting up of which albuminous substances are produced having a left-handed rotatory action on polarised light. In these bodies the sulphur is said to be combined in part directly and in part indirectly with oxygen ; but an albumin may be obtained in which the sulphur exists only in direct combination with oxygen. Albumin containing 2 per cent, of sulphur, in being resolved iuto a— , b — , c— albumin, containing 1"3, 1*1, 0'9 per cent, of sulphur, yields at the same time fatty acids, neurine and cholic acid as decomposition products. These last statements make it probable that Danilewsky had a specimen of albumin imder his hands, which contained a phosphorised substance ; the greatest doubt attaches to the alleged formation of cholic acid. When a solution of white of egg free from membrane is allowed to drop into water, each drop becomes covered with a fine insoluble pellicle, and falls to the bottom imdissolved. The gelatinous parts of the chalazse show the same bearing, and remain as roundish swelled transparent particles surrounded by a white thin layer even on shaking with much water. The phenomenon is probably one of diffusion, by which insoluble albumin forms a crust round the drop, and prevents further diffusion of albumin, while favouring the diffusion of salts out of the drops of albumin into the water. SOLUBLE ALBUMIN. 197 Albumm not altered by frost. — When albumin bj itself or mixed with chloride of sodium, contained in a closed tube, is frozen in a mixture of solid carbonic acid and ether, it remains apparently unchanged and retains its fluidity after thawing and on exposure to the air. (Melsens.) Albumin altered by dilution and exposure to air. — When common albumin mixed with an equal volume of water and strained is evaporated in the sun, or exposed in an open vessel for six days, it no longer coagulates on boiling, but at a certain degree of concentration solidifies to a translucent jelly which is soluble in water. The addition of very dilute acetic, formic, tartaric, or citric acid, by which the solution is not precipitated, restores the property of coagulation (Monnier, Par. Chem. Soc. Bull. 1 1 , 470). According to this author a peculiar modification of albumin is thus produced. Coagulation of albumin. — Ordinary natural albumin may be rendered insoluble by motion, and then assumes the appear- ance of fibres or membranes. This formation of membranes takes place also with albumin purified by Wiirtz's method. It occurs when albumin in drops is made to fall from a height, or dashed in spray against a surface ; when dry or moist air, hydrogen, or carbonic acid, is passed through the solution, even under a layer of oil (in the case of precipitation by carbonic acid the product contains plasmin) ; when the solution is shaken for 24 hours in a closed flask, or when it is shaken in a vacuum. On evaporating in vacuo a solution of albumin saturated with sodic chloride, there remains a residue soluble in water, from which the insoluble membranes separate on passing carbonic acid through the solution, or on agitation (Melsens). See Harting's objections (Schmidt's Jahrb. 75, 148; Jahresber. 1852, 691), and Melsens' reply {Instit. 1857, 201 ; Jahresber. 1857, 531). Denis a^so obtained a large quantity of fibrillse, insoluble in water and chloride of sodium, by prolonged agitation of albumin. Another mode of rendering albumin insoluble is by heating its solution containing a sufficiency of a neutral salt, to from 63° to 75°. It is then converted into a white, slightly translu- cent, solid, somewhat elastic mass ; this contains water in a colloid state, and dries up to a yellow horny substance, which 198 ANNALS OF CHEMICAL MEDICINE. when immersed in water assumes the appearance of freshly coagulated albumin, but does not dissolve. Special features of coagulation by heat. — The white of hen's eggs remains clear at 60°, becomes slightly turbid at 63°, begins to soKdify at 65°, and becomes completely solid at 70° to 75° (Dumas and Prevost, Ann. Chim. 23, 52). Perfectly neutral solution of the white of hens' eggs (free from albuminate of soda (egg-casein) and globulin (or plasmin, etc.), becomes turbid at 57°, forms a white pulp at 60°, and deposits large flocks at 62° to 64° (Lehmann, Arch. f. Pathol'. 36, 1 1 5). The white of ducks' eggs begins to curdle at 63° (Chevreul, Ann, Chim. 19, 46 ; Gilb. 70, 379). Behaviour on heating of white of egg diluted with water. — Fresh albumin, mixed with half part of water curdles com- pletely when heated ; with one part of water it remains slightly fluid ; with ten parts no coagulum is produced but a milky turbidity which is perceptible even with 1000 parts of water (Bostock, N. Gehl. 4, 547 ; Schw. 29, 397). In these experi- ments the turbidity which ensues by mere dilution must be taken into account. The experiment of Bostock explains the error of Matthieu and Urbain, who ascribed the non-coagulation of diluted albumin to the abstraction of carbonic acid by the vacuum, to which they had subjected their diluted white of egg. On the behaviour of albumin diluted with water at 38°, see Arnold (Schmidt's Jahrb. 103, 1). A solution of Wiirtz's egg- albumin becomes turbid at 59°5, forms flocks at 63°, and solidi- fies at a somewhat higher temperature. The coagulation by heat takes place also in vessels exhausted of air, without evolution of gas or change of volume. Fresh- laid eggs, when covered at once with oil, do not coagulate completely, but only become milky. The coagulation by heat is accompanied with the formation of hydrothion. Metallic silver is blackened by albumin when hot, but not when cold ; coagulated albumin blackens lead acetate and also lead-paper held over it, after addition of acids (Grobley, N. J. Pharm. 18, 347). The coagulation is accompanied by elimination of alkali [ammonia (Eichwald, Ghem. Centr. 1869, 568)], which com- bines with another portion of albumin to form albuminate (Scherer). Egg-albumin dried at a low temperature is not SOLUBLE ALBaMIN. 199 coagulated, even at 100° [or above 100° (Briicke)] in the dry state (Al. Schmidt, Miiller's Arch. 1862, 447). Commaille distinguishes boiled albumin as pexin. It dissolves with diffi- culty in slightly acidulated water. When its solution in caustic soda is precipitated by hydrochloric acid, and the precipitate is dissolved in water, a solution is formed, from which tetrachloride of platinum throws down a dark orange-yellow precipitate containing 7"80 per cent, of platinum (Commaille). The coagulated albumin employed for the analyses given below was prepared either by boiling filtered white of egg and washing the coagulum with water and alcohol (Mulder, Fogg. 40, 271. — /. pr. Chem. 44, 489), or by precipitating filtered white of egg with alcohol, and washing with alcohol, water and ether. (Lassaigne, Ann. Ghim. Phys. 20, 98 ; Dumas and Cahours, N. Ann. Ghim. Phys. 6, 407 ; Theile, Jenaer Med. Zeitschr. 3, 147 ; Jahresber. 1867, 774). Eiiling (Ann. Pharm. 58, 306) adds a few drops of hydrochloric acid before precipi- tating with alcohol. Elementary composition of coagulated amd coagulable albumin. (Ash deducted in all cases.) Gay-Luesac and Thdnard Mulder. Dried at 125° Bcherer. Dried at 100° Dumas & Caliours. Dried at 140° H N s 52-88 7-54 15-71 53-73 7-02 15-52 1-6 53-78 7-07 15-92 53-37 7-10 15-77 Ash — 2 03 2-0 1-13 EiUing Wurtz TheUe Dried at 100° Dried at 140° a b Dried at 130° H N s 52-81 7-26 1-77 54-33 7-13 52-79 7-13 15-55 52-92 7-15 15-65 53-98 7-51 14-24 22-34 1-93 Ash 1-68 — — — 2-30 200 ANNALS OF CHEMICAL MEDICINE. Thiry SchUtzenberger H N 165 62-80 7-16 16-56 16-7 16-2 — E Ash — — — — — 3-0 1-49 0-7 Wiirtz analysed the albumin prepared by his process in the soluble dried state (a) and in the coagulated state (6). Theile's numbers are calculated from the analysis of albumin dried over calcic chloride, allowance being made for water expelled at 130°. Mulder's albumin contained also 0*4 per cent, of phosphorus. Yexdeil (Ann. Pha/rm. 58, 317)found 2'109 per cent, sulphur. Schwarzenbach {Ann. Pharm. 133, 185) foimd 1*85 to 2-2 per cent, of sulphur. Schiitzenberger's analyses (J-wm. Chim.Phys. 16, 1879, 305) were made upon albumin coagulated by heat, dried at 140° and exhausted with ether, reduced to an impalpable powder, and passed through a silk tammy. The amount of nitrogen was determined as nitrogen gas, and was found much higher than in the previous analyses, those of Thiry excepted {Physiol. Zeitachr. 1863). Older Formulce of albumin : C9oH,39N,,S03„ (Mulder) C^jHijgNjgSOjg (Liebig, as empirical formula) CjjHiijNjgSOjj (Lieberkiihn, for albuminic acid) C,,H.,,N„S0,3 (Theile) In the second article, p. 43, it has been shown that the formula of albumin (according to Schiitzenberger) is at least C240^387-''^65O75®3' correspoudiug to a molecular weight of 5473. Egg-albumin dried over calcic chloride contains 45*85 to 46-58 per cent. C, 7-62 to 7-96 per cent. H, 12-14 per cent. N, 1-63 per cent. S, after deducting 2-3 per cent, of ash. It loses 12-76 per cent, of water at 100° to 130°, turning slightly brown even at 100°. At 165° it suffers no further loss, and at a still higher temperature empyreumatic ammoniacal decomposition- products ai-e formed. SOLUBLE ALBUMIN. 201 The albumins thus far analysed were all mixed with the plasmin, which can be removed by dialysis and filtration, or dilution, neutralisation, and carbonic acid ; and with the nitro- genous body which passes with the salts into the dialysate and holds phosphates and free earths in combined solution. The pure soluble albumin of Grraham has not yet been subjected to elementary analysis. It is therefore premature to speculate on the formulae derived from the analysis of the coagulated impure albumin, although we may hope that the information already gathered represents something like the beginning of a truth. Other properties of soluble and coagulated dihumvn. Effects of heat upon coagulated albumin in the presence of water. — Hard-boiled white of egg heated to 150° with water for two or three hours, dissolves and forms a solution which is pre- cipitated by acids (Wohler, Ann. Pharm. 41, 238). When it is heated to near 200° with water in a copper digester, the walls of the vessel become covered with a film of cupric sulphide, and the albumin dissolves, with exception of a portion which remains unaltered. The solution contains a little free acid, and sub- stances partly soluble, partly insoluble in water. When coagu- lated albumin is boiled for some time in water it swells up strongly, softens, decomposes, and gives up animal matters to the water (Grmelin). Mulder {Ann. Pharm. 47, 314) boiled white of hens' eggs in a Papin's digester for 150 hours, and obtained a solution as well as an undissolved portion, which latter Mulder declared to be unaltered albumin. It contained 54*34 per cent. C, 7"16 per cent. H, and 15"33 per cent. N. The watery solution on evaporation left a residue, which after exhaustion with alcohol, dissolved in water and gave a precipitate with neutral lead acetate and ammonia. Both precipitates were decomposed by hydrothion, and yielded a substance which Mulder termed protein trioxide and found to contain 50-75 to 51"36 per cent. C, 6*69 per cent. H, and 15*01 per cent. N. It was neutral, soluble in cold water, precipitable by dilute mineral acids, chlorine and metallic salts, but not by acetic acid, potassic ferrocyanide, ammonic chloride or barytic chloride. Effect of electrolysis upon undiluted white of hens^ eggs. — Many experiments have been made upon this subject, but with 202 AKNALS OF CHEMICAL MKDICINE. the exception of the one of Lassaigne, never upon pure or purified albumins. Arnim (Gilb. 8, 259) and Marx {Schweig. 54, 209) found albumin alone or diluted with water to be completely coagulated at the negative pole. With a strong current, a rapid deposit takes place at the negative, and a slower deposit at the positive pole ; with a very weak current, there is a deposit at the positive pole, while an alkaline solution of albumin is formed at the negative (Brande, Gilb. 64, 354). With a weak current C. Gr. Grmelin (Gilb. 64, 347) observed coagulated albumin at the positive pole only, never at the negative ; a strong current caused coagulation at both poles, which G-melin attributes to the heat evolved. Lassaigne {Ann. Chim. Phys. 20, 97) precipi- tated albumin with alcohol, washed the precipitate with weak spirit to free it from chlorides, and submitted its aqueous solu- tion to electrolysis, which did not cause coagulation excepting after addition of sodic chloride and then only at the positive pole. According to Morin {Par. Soc. Bull. 1861, 104) this result is due to the low conducting power of the liquid. Ac- cording to Smee {Proceed. Roy. Soc. 12,399 and 505; 13,350) fibrin is formed by the electrolysis of egg or serum albumins ; and Wittich {Journ. pract. Chem. 73, 18) also subjecte'd un- diluted white of hens' eggs to electrolysis and obtained a dense white layer of coagulated albumin at the positive pole, and when the experiment was continued long enough the negative pole became covered with a clear jelly of albuminate of soda, which last did not appear with diluted albumin. Solutions of alkaline albuminates likewise deposited the albumin in films at the positive pole ; acid solutions of albumin deposited it more slowly at the negative pole, in the form of a difiused cloud, while alkali or acid was set free at the opposite pole. When the solution contained free acid, free alkali, or carbonate or bicarbonate of alkali, the deposit of albumiu did not take place, or took place very slowly ; but when the solution contained neutral salts, such as sodic chloride, sodic phosphate, sodic sulphate, potassic sulphate or nitrate, the deposit at the positive pole was much more abundant. Putrefaction of albumin. — Natural albumin putrefies with evolution of hydrogen, nitrogen, carbonic acid, and hydrothion, the quicker the higher the natural temperature to which it is SOLUBLE ALBUMIN. 203 exposed, provided it is protected from being dried up by cur- rents of air and other conditions favouring rapid exsiccation; the presence of added water favours rapid decomposition. Coagulated albumin does not quickly putrefy even under water. The earliest systematic experiments for ascertaining the pro- ducts of putrefaction of albiimin were made by Bopp. Albumin was dissolved in 40 to 50 parts of water and exposed to the air for a few weeks, at the temperature of 25°. The gaseous pro- ducts were apparently not noticed, the other products were separated as follows. a. The liquid was distilled with milk of lime, and yielded ammonia and a body of penetrating odour, part of which passed over in crystalline scales when the amraoniacal liquid was dis- tilled in its turn with phosphoric acid, while another portion was extracted from the distillate by ether. It was very volatile and assumed a rose-red to brown-red colour with dilute sulphuric acid, forming oily drops. h. The residue containing lime was distilled with a slight excess of sulphuric acid, and yielded volatile acids. c. The liquid resulting from the last operation was mixed with neutral lead acetate, by which there was thrown down, besides the sulphate of, lead, a plaster-like precipitate from which hydrothion separated brown acid oily drops. d. When the liquid remaining after the separation of the plaster-like lead salt was freed from lead by hydrothion, it yielded on evaporation a viscid syrup, which was resolved by alcohol into leucin and a soluble portion. The latter was free from sulphur, had an acid reaction, formed with lead oxide a salt soluble in alcohol, and when boiled with sulphuric acid yielded tyrosin and a brown colouring matter. The subject here under consideration will be further treated of in other essays of this volume. The limitation of our space does not allow us to complete this exhaustive survey. This, however, we hope to do in a future edition of the Annals. 204 ANNALS OF CHEMICAL MEDICINE. XIV. ON INFECTION, AND THE VAJRIOUS STATES OF AGGREGATION OF INFECTIOUS MATTER. The consideration of infection is a part of medical aetiology, or that branch of medical science which treats of the causes of diseases. It is therefore by no means, as is not rarely sup- posed, a merely chemical, or as is maintained by others, a microscopical question. For some processes of infection are by no means chemical. Thus a most important lately dis- covered disease, viz. fleshworm disease or trichiniasis ' is pro- pagated by the transfer into the human (or animal) intestine of living wormlets, which there propagate, and their young migrating into the body cause the general effects of the disease. Here the infection is due to a living parasitical animal species. In other diseases no such weU-defined beings can be ascer- tained to exist, and however curious may be the resemblance which such diseases bear to parasitical diseases, the individual agents, by which the infection is carried, maintained, and repro- duced have not yet been discovered. Accordingly medical inquirers are in a state of doubt and perplexity. The doctrine accepted by many during the last thirty years according to which the ordinary infectious, febrile diseases were mere chemical processes, and engendered by the introduction into the body of agents which were likened to ferments, then believed to be unorganised, has been somewhat shaken by the deve- lopment of the views concerning ferments properly so called, their division into two classes, of which one was termed shape- less or non-organised, the other shaped or organised. This development led many setiologists into the paths which their ' For a complete account of this disease, see Re^. Med. Off. Privy Council, 1865. INFECTION AND INFECTIOUS MATTER. 205 science has repeatedly taken on former occasions, namely, to the belief that disease-causes were parasitical beings of a specific kingdom amongst things extant and organised ; that all living things had to be divided into three kingdoms instead of as hitherto into two only, namely into animals, plants, and disease- causes; and that the latter being, like animals and plants, things created, or, speaking less dogmatically, of the same origin as animals and plants, had an independent existence as beings, and infected the individuals of the other kingdoms in the same manner as parasites. In fact pathology became again to some extent, under the impulse of these hypotheses, what it had been repeatedly before^ namely ontology (doctrine of disease-causes, as separate beings, to be distinguished from the doctrine which considered the disease (groups of symptoms produced by the cause) as a parasitic being). The philosopher who ponders over the great number of different diseases to which organised beings are subject, and -who particularly appreciates the specificity of diseases, that is to say their peculiar unchangeable character within certain limits, and their exclusive attachment either to men, or to species of animals, cannot but be forced to consider diseases with the aid of the outological hypothesis. But on the other hand, when he investigates the reasons which speak against the hypothesis, he perceives that there are many and strong reasons which prevent him from accepting the hypothesis as a generally applicable or valid one. As it is impossible for him to draw general conclusions concerning a whole field of morbid phenomena, so also he cannot devise measures for the prevention or cure of diseases as a mass. He is reduced to the necessity of studying each individual disease by itself, and adapting to every single case what measures are possible ; but the measures, equally with his views of the nature of the case, are mere approximations of his mind by means of a reasonable hypothesis. In times when epidemics of infectious diseases prevail, society takes the alarm, and many persons then endeavour to protect themselves from infection by leaving the places where the disease prevails, and going to places supposed to be free from it. In times of less alarm, all society, and in times of epidemics that part of society which cannot migrate, rely upon 206 ANNALS OF CHEMICAL MEDICINE. chemical means either to destroy the infecting matter out of the body (disinfectants), or to stop the infection which has akeady reached and affected the body (specifics, medicines). The use of these agents is mainly the result of chemical hypo- theses concerning the nature of the causes and products of disease. As regards the applicability of these agents it is of no account practically whether infections and products of disease be living agents or whether they be chemical matters possessed only of the powers pertaining to chemical ferments having no organisation or specifically shaped individuality, so long as the chemical means adopted for their destruction are strong enough to destroy either. Infection, as the act or process committed by a thing or specific being, admits of a definition. Such a definition can be correct only within the limits of positive science concerning the subject, and must, if possible, not be liable to any controversy. The strictest logic has to be applied, the use of words has to be restricted to its most legitimate limits, and no latitude or uncertainty of expression can be allowed, which would lead to confusion. Infection is the 'process or act hy which cm animal or a man receives into his body a substance or matter, or agent, which is capable of producing disease, and is actually thereby induced into a diseased state; the disease being admitted, for the purposes of this definition, to be specific and its causing agent to be self -reproducing. — It follows from this that the accession of mere dirt, technically foreign bodies, the collection of mere impurity, the contamination of the air by offensive effluvia, the pollution of water earth and air by the offal of human operations or the excretions of man and animals, do not constitute infection ; and it also follows that the counteracting of these influences, the removal of these pollutions and contami- nations, must not be termed disinfection. For disinfection being the negation or counteraction of infection, can only be defined as an operation which destroys matter capable of pro- ducing infection as above defined ; now as these influences are unable to produce infection, the counteracting or destroying them is not disinfection. Infectious matters are carried by men and animals ; they may be contained in and about places and INFECTION AND INFECTIOUS MATTER. 207 things. Men, animals, places, and things may receive and harbour infectious matters, change them in a manner chemically and physically, or retain them without change. The infectious matters may from these temporary abodes pass again into animals to produce their specific effects, or may be destroyed. Places and things carrying agents of infection are also termed infected, although they are only carriers of an infection imported, and not like men or animals (passive) reproducers of such infection. In practice processes which could truly be termed disin- fections have not been applied to the interior of infected men or animals, but only to their external surfaces, their fur or hair, or to the clothes or uncovered surfaces of men's bodies such as faces and bands or to places and things. Only few carried the doctrine of disinfection farther into practice, and gave strong disinfectants internally, such as chlorine water in enteric fever. This practice is now a thing of the past. If lately a chemist revived a similar practice, and caused to be injected into the blood-vessels of cows affected with the cattle plague watery solution of carbolic acid, it was a proceeding for which he had no medical or scientific authority, and the death of the cows showed the futility of the practice. In a German university some men were disinfected by having their bodies anointed with carbolic acid. The death of the patients was the result of this rash and deplorable practice. Examples of this kind (of which more might be quoted), show that substances termed (by erroneous hypothesis) disinfec- tants, have no specific counteracting effects upon infections ; that they do not destroy infections as quinine destroys inter- mittent fever ; but that they are only agents of general efficiency which they exercise upon living, healthy, diseased or dead chem- ical matter ; and that in a direct proportion to their general power over all these matters stands their special effect upon infectious ones. Now whereas some of these agents employed for the destruction of infectious matters have actually opposite properties, e.g. one preserving organic matter while withdrawing it from the influence of oxygen, the other supplanting or assist- ing oxygen in the destruction of these matters which the first one preserves, we learn, in a manner ex juvantihus et nocentihus. 208 ANNALS OF CHEMICAL MEDICINE. what we can also ascertain otherwise, and in fact have already admitted in the foregoing, that the nature of infectious matters, or of infections, contagions, and miasms is with few exceptions unknown. From this the conclusion may be drawn that it is at least useless to try to affect materially that which does not present itself materially to our senses. But practical necessities cause us to dispense, for the time being and pending inquiry, with data, and to substitute for them hypotheses ; with the aid of these we conjecture the possible and the probable, and to the results we adapt certain agents of known power. On the various modes of infection. — It is useful to consider the simplest cases of infection, in the first instance those in which the direct mechanical process is known and undoubted. Hydrophobia, as an example, is transferred mechanically upon man by the dog. It is transferred from dog to dog most com- nionly by- the same method by which it is transferred to man. But whether amongst dogs transfer (by inoculation) by a bite is the only mode of transfer, or whether there are other modes of transfer (as is probable from the experience of certain kennels in which rabies becomes general and causes the necessity of destroying the entire kennels) or whether a dog may become affected with rabies spontaneously is not at present known. In the spei;ial case of a man who receives a wound from a mad dog, a certain amount of saliva enters the wound, in which an infection is transferred, which works out the death of the man. In such a case we are in the habit of cauterising the wound so as to destroy, as we believe, chemically, the poison adhering to it, and protect the rest of the body from its agency. Or we cut out the piece of flesh in the midst of which the wound is situated, if we have some reason to fear that the poison might in part already be absorbed. We concentrate the whole of our activity upon the isolation of the affected saliva, and upon its destruction, but we do not attempt to disinfect the body after the bitten man has become hydrophobic ; we have at present no means of destroying the infection of hydrophobia within the body of either man or animal. While hydrophobia is distinctly carried by a morbid poison; a product of disease, snake poison, on the other hand, is not a INFECTION AND INFECTIOUS MATTER. 209 product of disease, but a normal secretion of organs specially provided and arranged with purpose and effect.. We range it in our classification of hurtful substances with poisons proper. Snake poison acts almost immediately ; there is no time of incubation ; no long period elapses between the insertion of the poison and the production of its effects. It acts in proportion to the quantity in which it is inserted in the wound, and when that quantity is exhausted its effect ceases. Not so the hydro- phobic poison or infection ; its effect is scarcely ever immediate ; a period however short, but which may be prolonged to months and years, always elapses between the bite and the outbreak of the disease. This is one of the diagnostic features of specific contagions, that they require a time of incubation, a time during which, so to say, the eggs of the disease laid in the body, are being hatched, a time which is mainly used for the multiplication of the contagion, and the termination of which is indicated by the earliest objective symptom of the outbreak of disease, a rise in the temperature of the body from the normal towards the febrile. We have alluded to a natural poison inserted by teeth in order to contrast it with a diseased contagion inserted by teeth ; we have thus shown that in the production of hydrophobia something passes into the body which is being infected ; a germ is transferred, a something which acts as the beginning and centre of a portentous effect : we must hold fast this lesson in the consideration of other diseases, in which the transfer of germs by some means or other becomes probable, has to be assumed, but cannot be proved ; at least not in the same direct manner as in hydrophobia. While we thus clearly understand that contagion consists in the transfer of a small portion of disease-producing matter from one person to another, we in no case, pig typhoid and splenic apoplexy in the ox and antlirax in man excepted, know the nature of that matter by which the transfer is immediately effected. The 70,000 cases of successful infection with typhus and typhoid fever poison which occur annually in this country, and an at least equal number of smallpox, scarlet fever, and other similar infectious diseases, are all assumed to be produced by the transfer of a small particle of disease-producing matter from 210 ANNALS OF CHEMICAL MEDICINE. one individual to another. If we were to assert with the late Dr. Murchison, the author of a treatise on Continued Fevers, that the poison or infective seedling of typhus was ' probably a compound ammonia ' we should not be nearer to a true know- ledge of the body in question. It is necessary to be quite clear on this point, that of the exact nature of the infecting agents of the most important diseases including typhus and cholera we know nothing. And the clearer this is enunciated, the nearer are we to the possibility of finding something about them. For as they are unquestionably tangible matters, unquestionably chemical compounds, whether organised or hemiorganised (Fremy) consisting of three or four elements, perhaps five or six, and as the elements so contained in their composition preserve through all changes of molecular form the character of chemical elements, we comprehend that these matters admit of the same amount' of definition as any other matters of organic or inorganic nature which enter into the researches of biologists or chemists, and that even should these matters belong to an independent range or grade of things standing to the human and animal body in the relation of parasites, even then they would obey laws of organisation of their own, even then their constituent elements would obey the general laws of matter. Infection by definite acts and agencies; inoculation. — We come nearer to the comprehension of the nature of disease by considering the variety of means by which it may be propagated. Knowing that diseases may at times have a more virulent, at other times a more mild character, and knowing on the other hand that persons who had once been affected with a disease of the particular kind would not be liable to be infected by it again as before, some men of particular wisdom and courage (the earliest ones living in remote parts of China and in remote times) have availed themselves of this information, and inflicted upon persons under their control mild infections to escape severer types and obtain immunity. The oldest example of tl^s kind is smallpox. Smallpox is the most infectious of human diseases ; it spreads through families, villages, towns and countries with great rapidity, and in this respect only one disease, cattle-plague, is equal ^r superior to it, HavLjg recognised the refined insidiousne^s of smallpox and that they INFECTION AND INFECTIOUS MATTEE. 211 had no escape from it, and that there were mild types and severe types, men argued that as they mTist have the disease at one time or another, they might as well produce it upon their bodies when it was of a mild type, and thus procure immunity; moreover they could select the season at which experience shows that aU diseases are the most tractable or are suffered with the least domestic inconvenience. The earliest mode of inoculating, or rather intentionally imparting, smallpox, consisted in blowing into the nose of the person to be infected some dried and powdered matter from smallpox pustules. Physicians continued to experimentalise and discovered inoculation. For this great result of reflection and experience, for this most courageous resolution, which contains the germ of the practice we now know as vaccination, mankind is indebted to the Chinese. Amongst the Chinese smallpox was inoculated as early as the year 1000 of our era. The Hindoos learned the practice from the Chinese, and some of their tribes used the marks left by the pustules to inscribe upon the foreheads of their children a mark of the particular caste of which they were members. Through the Hindoos the practice came to the Persians, Armenians and Greeks, and at the beginning of the last century the practice of inoculation became common amongst Greek physicians at Constantinople. There it was noticed by Lady Mary Wortley Montague, and through her letters it was made known in England, and Western Europe. It then became a common practice amongst the rich, and an English physician received a fee of ten thousand pounds for inoculating the children of a Eussian Empress. After the discovery and introduction of vaccination, however, inoculation was made illegal, and vaccina- tion compulsory, and in the present day 95 per cent, of all those born and living during the first year are protected from small- pox by vaccination. Varying states of aggregation of infectious matter. — In order to obtain an approximation to an accurate idea of the nature of infectious matters, particularly the specific poisons of diseases, it is necessary to consider their various states of ag- gregation. The matter which carries smallpox must be capable of assuming an extremely minutely divided state ; but it also exists in an apparently fluid state, inasmuch as fluid (lymph) P 2 212 ANNALS OF CHEMICAL MEDICINE. taken from the pocks themselves is capable of transmitting the ■disease. Such lymph might, however, be supposed to contain the infectious matter in a particular state and not in solution, Chauveau's experiments on vaccine speak in favour of this idea, Bert's experiments on splenic fever blood against it. The in- fectious matter also exists in a dry and solid state in dried lymph. Other specific infectious matters are capable of existing in the same different states as smallpox ; for example, cowpox. The lymph from cowpox can be preserved by being dried upon a lancet, or upon a chip of ivory, or soaked in a thread. Thus fixed it will retain its power of exciting vaccine pustules through the medium of a wound in and around that wound for a considerable time, nay it seems as if such poison in a dry state had an in- creased power of endurance against destructive influences. Dr. Budd of Bristol caused some vaccine lymph dried upon so-called ivory points, to be enclosed in glass tubes, and the latter to be sunk into the centre of a leg of mutton near the bone. The leg of mutton was then roasted in the ordinary fashion in the kitchen. The tubes were afterwards drawn forth and the ivory points used for infection, which proved perfectly successful. It is con- sequently clear that a higher temperature than the ordinary one does not always destroy the activity of lymph in the dry state. The experiment was no doubt made under such condi- tions that it is not necessary or possible for us to assume that the lymph had reached the heat of the coagulating point of albumin (64°). It was almost certainly below the coagulating point of myochrome (about 90°), and then it was under pecu- liar conditions, which admit no direct conclusion as to what would have been its fate if it had been heated only to the co- agulating point of albumin in the moist state. Dry white " of egg can also be heated to a much higher point than moist or dissolved white of egg without undergoing any change such as is expressed by a change of its solubility. The heating experi- ments on dry vaccine are therefore not conclusive as to the effect of heat upon lymph as it comes from the pustule. Infectious matters are not supposed to exist in a gaseous state. When contained in liquids they may, as already stated, be considered as dissolved or suspended ; in the solid state they may be free or combined, retaining their specific energy in the INFECTION AND INFECTIOUS MATTER. 213 combination, as quinine does in the sulphate. What has given rise to the idea of infectious matters assuming the gaseous state is probably their extreme divisibility, by which they assume the form of very fine dust, and are easily carried over great distances. The class of infectious matters which present these characters of apparent volatility, fluidity and solidity, and can pass from one to the other, we will term volatile inocvlable infections. Of such infections smallpox is the type, and there is only one disease of animals at all comparable with it, and that is the cattle-plague or rinderpest. There are a number of human diseases, which although pro- ducing volatile infections, are not proved to be inoculable, such as scarlet fever, whooping cough, (diphtheria ?), croup, typhus fever, and typhoid fever. This class of diseases we range under volatile infections not proved to be inoculable. Distinct from these we have another group of infections which we will call the less volatile, more fixed, not proved to be inoculable ; the type of these is yellow fever. If we go on to consider all other diseases from the same point of standing, we are obliged to construct some more classes ; we thus get the important class of fiooed infections, contagious and inoculable. These cannot assume the volatile condition, and hence can be propagated only by bringing into actual contact with the subject to be in- fected a certain amount of the fluid or suspended infecting matter. Amongst these the representative is syphilis. Then follow the diseases of animals transferable upon man, namely glanders, hydrophobia, pustula maligna, and vaccinia. For however analogous vaccinia may be to human smallpox (so analogous that some believe vaccinia to be only human small- pox modified by transmission through the cow), it difiers from it in this important point, that its poison is not transferable from man to man in the volatile state. The insertion into his skin-tissue of a certain amount of vaccinia is required for man to become infected. In those agricultural districts where vaccinia was first observed it was and is now generally believed that the transfer of the vaccinia upon man takes place by direct contact, and the observations of Jenner mostly referred to cases where, in proof of this assertion, the pustules were observed upon the hands. The hands of the milkers were employed upon the 214 Al^NALS OF CHEMICAL MEDICINE. udders of the cows,,and thus the disease was probably transferred- Whether vaccinia is volatUe, so as to be transferable from cow to cow is not known, but it is very likely. The cow can be in- oculated with human smallpox, but human smallpox poison does not appear to affect cows spontaneously. All the above infections are specific diseases of a type which within certain latitudes and limitations is unalterable. The following disease however is not, perhaps, so specific although more fatal, and may be called a volatile and fixed infection, contagious in one sense, and that is the pysemic infection. It is undoubtedly volatile, inasmuch as it may be contained in overcrowded hospitals and fly from person to person, infecting those who are slightly wounded as well as those, and more particularly those, who are severely so. But it apparently only enters through suppurating wounds, and is as yet not proved to be inoculable ; that is to say, we have no proof that by taking a small portion of pus from a pysemic person and inserting it into a wound made for the purpose of infection on a healthy person the disease can be reproduced. There is however such experience upon animals, altliough the first material used in such experiments is not generally taken from animals suffering from pyemia, but consists, in accordance with certain ideas and facts concerning pyaemia, of simply putrescent matters having no claim to be termed products of disease. The disease-cause of pysemic infection seems analogous to that of cholera in this, that before taking effect upon an individual it has to be fermented or caused to undergo a change, and that change is effected in a wound of which the person to be infected with the pysemic poison must be the bearer at the time of the infection. We come now to consider the last of these infections, viz. cOrpse-poison, or dissection-wound poison. This infection is fixed and inoculable only, and no particular state of the body of the recipient is required for its successful transfer. This infection leads to destruction of parts and death of the individual in many cases, but it lacks the specificity which is peculiar to other diseases. Notwithstanding much that has been written and said about cholera, it is difficult to give it a place in a system like the one INFECTION AND INFECTIOUS MATTER. 215 which we are endeavouring to construct. We are not certain whether it is volatile or not ; whether it may be volatile at one time and fixed at another ; for if it be volatile at all it is only moderately so, and in that respect resembles yellow fever. The experiments of Thiersch led to the conclusion that the cholera poison originated in the excretions of cholera patients. They had no power of infection in their fresh state, but only gained that power after exposure to the atmosphere during a number of days. Two days was the shortest time required for cholera excretions to acquire the power of producing morbid effects upon such small animals as dormice. Intestinal contents from the dead body required sometimes seven days before they exhibited morbid action in dormice ; after sixteen or seventeen days from the beginning of the exposure to the air, the evacuations ceased to exhibit any poisonous effects. It is by this acquired and passing poisonous nature of the excretions, that cholera resembles pyemic infection, as above mentioned. Thpse experi- ments of Thiersch upon dormice were repeated in the Patho- logical Institute, and proved to yield the same results as those obtained by this observer. These experiments we shall have to consider when we come to treat of some chemical features of cholera. At first sight they lead to the thought, that cholera, being capable of affecting dormice, is not a specifically human disease, but participates more of the nature of poisoning. But this is negatived by other data to be discussed. Cholera in dor- mice is similar to the disease as it appears in man, but it is not identical with it in all its symptoms ; the symptoms are modified. The Oriental pestilence or plague is a contagious hut not vnoculable vafection. This has been amply proved by the researches of Clot Bey. The opposite experience of physicians who inoculated themselves with the pestilence, and actually became infected, is not cogent. Deggio, a Eussian doctor in Bucharest, successfully inoculated himself with the Oriental plague, and recovered, while Dr. Whyte, an English physician in Egypt, died from the plague, the result, so it was believed, of his own undoubted inoculation. These cases may be either excep- tions, or cases in which an infection took place by the side of ino- culation ; they are contradicted by a great number of experiments 216 ANNALS OF CHEMICAL MEDICINE. in which inoculation of plague poison into healthy bodies was followed by no results. Views of the chemical school of lAebig. — During the last thirty years infection has been considered as a species of fer- mentation ; in consequence diseases of this class were termed zymotic. Fermentations were considered to be caused by what was termed contact action : a body in a state of change (fer- ment) was said to communicate its state of motion to another body (fermentee) and cause its decomposition. These ferment actions, and so-called catalyses in inorganic nature, were all used to explain infection. When the fermentations were again ascribed to the life-action of living organised ferments (yeast), then the different contagions were also assumed to be referable to organised ferments. Many new ferments were supposed to be discovered, such as produce putrefaction, souring of milk, lactic acid, decomposition of urine, of wines and many other substances; This went so far that the fermentative effects in the living body, such as pancreatic action, were ascribed to the presence 'of living ferments in the gland. Grradually the non- shaped ferments, such as diastas, emulsin, pepsin, pancreatia, were separated from the supposed shaped and organised ones. Some non-shaped ferments, previously believed to be organised, had to be admitted to be products of the organised beings which did not themselves participate in producing fermentation. (Musculus). All these contentions will be more closely con- sidered in other articles. ORIGIN OF INFECTION. 217 XV. CONFLICTING VIEWS OF CONTAOIONISTS AND ANTI- CONTAGIONISTS CONCERNING THE ORIGIN OF INFECTION. DIPHTHERIA IN ANIMALS. {Summary and Additions.) This subject has been well discussed by Dr. Thome in a paper read before the Epidemiological Society, April 8, 1878. He put the question whether any case of acute specific fever could arise independently of an antecedent case. By acute specific fever he mea.nt any febrile disease capable of reproducing its own kind ; the power of the disease of reproducing itself is therefore admitted, the question is its origin de novo. Those who maintain that no case of infectious disease can arise independently of a prior case of the same disease (and these are sometimes termed contagionists), support their con- tentions by arguments of the following kind. They say that infectious diseases do largely owe their spread to communication, immediate or mediate, with prior cases of the same disease. With regard to many of these diseases we know, says Dr. Thome for the supposed contagionists, what are at least the ordinary means by which their producing causes are eliminated from the bodies of those affected by them, and these producing causes we can trace, at times through numerous and intricate channels, until at last we find that in some way or other they are received into the systems of some hitherto healthy persons, in whom forthwith the diseases they give rise to are reproduced with all their well-known signs and symptoms. It may then be urged that this ordinary source of infection is the invariable one, and may be maintained to have been open even in those cases where investigation has led to no result. The contagionists further point to the fact that new channels 218 ANNALS OF CHEMICAL MEDICINE. by which the producing causes of these specific diseases can be conveyed frotn one person to another are periodically being discovered; and that consequently outbreaks, which formerly would have been brought forward as affording indubitable evidence as to spontaneous origin are now known to have been due to the conveyance of the poison by means of some hitherto unknown channel. Thus since it was ascertained that enteric fever and scarlet fever could be transmitted through the agency of milk as a vehicle, numerous outbreaks of these diseases, particularly of enteric fever, have been ascertained to have owed their spread to this means, and thus that which must formerly have remained unexplained on the hypothesis of the contagionist, now tends to confirm his views. The same remark applies, according to Dr. Thorne, to the outbreaks of enteric fever, which have owed their spread to that form of water-pollution which in intermittent water-services leads to the specific poison of the disease being drawn with other filth into the water-mains during periods of intermission, and to its being subsequently distributed to the general body of consumers. (To sucb agency the latest (1878) outbreaks of enteric fever at Croydon are attributed). Therefore, say the contagionists, where aU known methods for the conveyance of these producing causes fail to explain the existence of the diseases produced by them, it is fair to assume that the infection has really been communicated by some hitherto unknown channel. Thus-a woman became ill in the presence of a mass of putrid manure cairried on a railway truck, and attributed her illness to these emanations ; it was however subsequently found that before she had been in the presence of the manure, and well within the incubation period of enteric fever, she had stayed at a house where a fatal case of enteric fever had occurred, and had there been exposed to the very conditions which had led to the outbreak in the house she visited. Dr. Thorne further urges on behalf of the contagionists, that periods of incubation, ordinarily known to be subject to certain variations in length, do in exceptional instances vary much more than is generally recognised, and in consequence, constitute a source of error for which allowance must be made. Dr. Budd in his work on typhoid fever, refers to cases in which the incu- ORIGIN OF INFECTION. 219 bation was probably limited to four and two days, and says that the short period of incubation afiforded evidence of the high degree of concentration of the poison received. On the other hand Dr. Thome relates the case of a female, in which there must have been a period of incubation of enteric fever all but a month in length. In the middle of that period the patient had however for two days suffered from a sharp attack of diarrhCea. Dr. Thome also believes that the specific producing cause can remain in existence out of the body, and retain its vitality for a long period of time. The two alleged states of splenic fever are quoted, as described by Pasteur, and it is maintained that the spores of splenic fever, though reduced to dust, melted and dried repeatedly, and kept in putrefying liquids, yet re- tained their virulence undiminished at the end of four years of such treatment. With regard to scarlet fever Dr. Thome says there are several instances on record which tend to show that its producing cause will remain in existence out of the body for more than a year, and then act with unabated virulence. Instances have come under his notice in which the facts warranted the con- clusion that the producing cause of diphtheria had been retained for months about premises in which cases of this disease had previously occurred. Dr. A. Carpenter, in a paper read before the British Medical Association in 1875, related an experience regarding smallpox, which a practitioner in Oban had related to him as having occurred in Mull. A proprietor of that island ordered the removal of some old houses which had lain in a state of ruin and uninhabited for many years. Eight men were em- ployed in digging the walls, and everyone was attacked with smallpox ; the germs of that disease having (been supposed to have) lain in these houses all those years. The following are further cases illustrating the extremely long vitality of the typhoid fever-producing cause. Dr. W. Ogle, in some of his reports on the sanitary condition of the East Herts district, refers to cases in which attacks of enteric fever halve in successive autumns occurred in isolated dwellings, where so far as could be ascertained by the strictest investiga- tion, no new source of infection could have come into operation. Dr. Budd once knew a labourer's cottage which remained vacant nearly two years, in consequence of nearly every inmate of it 220 ANNALS OP CHEMICAL MEDICINE. having contracted typhoid fever. At the end of that time it was retenanted, and three weeks had scarcely elapsed before several of the new inmates were simultaneously seized with the same fever. The cottage stood alone in a secluded spot, and there was no fever in the neighbourhood at the time of the second attack. Dr. Thome states that he knows a detached house which stands in a country district, and which was occupied by a groom and his family, amongst whom enteric fever prevailed in the autumn of 1872, one case being fatal. This family con- tinued to occupy the house for nearly two years after this occur- rence, but they left it some time in 1874, in consequence of the departure of the owner of the estate on which it stood. From that date the house remained unoccupied until February 1876, when it was tenanted by new inmates ; and exactly within foinrteen days of these latter taking up their abode there, enteric fever broke out amongst them, and a most careful inquiry led both the medical man in attendance and Dr. Thorne to the con- clusion that the disease was not imported. Dr. Thorne also knows an isolated parsonage in which the families of two suc- cessive vicars have been attacked with enteric fever. When I (the Editor) studied the causes of enteric fever in the town of Giessen, I recorded a history of every house concerning which I could obtain data. In several houses successions of tenants were seized with the disease. The most remarkable house was the official residence of the bursar of the university {Rentamtmann) ; in this house three successive families of three successive bursars, were, shortly after having moved into the house, I believe all of them from healthy country towns, attacked with enteric fever ; of each family several members died, others were much invalided, and their happiness destroyed for life. If the infection can be supposed to have resided in this house, it must have lain dor- mant twice during more than twenty years. The cases of the first two families I learnt by personal in- quiry, the last being notorious at the time of my inquiry. The case of the first family I learnt from a son of that family now deceased, when I related the cases of the second and third families to a colleague in the presence of that gentleman ; the latter had been down with enteric fever with other members of the family in the very Rentamt I was speaking of. ORIGIN OP INFECTION. 221 Dr. Grilbert Child has published some evidence regarding the transfer of the infection of enteric fever by means of dirty bedding and clothes. On November 20, 1873, a man arrived at a house in an Oxfordshire village, where his mother and his brother lived, bringing with him several boxes of some very filthy bedding, clothes, etc. On the 29th of the same month the brother was seized with a fatal attack of enteric fever, and on the following day the mother fell sick of the same disease. No source of infection being known, the man referred to was questioned as to his prior movements, and it transpired that in the previous September his wife had died of enteric fever at Toronto, and that he, on subsequently leaving America, had brought with him her dirty bedding and clothing. The only other case of enteric fever which could be heard of anywhere within the district was that of a woman who was attacked on December 5, and she, being also one of the man's relations, had taken to her home some of the dirty things to wash. That contagious diseases are self-propagating may also be urged in view of the long-continued and complete immunity from them which certain isolated countries have enjoyed, and of the virulence which such diseases have exhibited when once introduced into them. America is believed to have been free from smallpox prior to its discovery by Columbus in 1492 ; Iceland and Grreenland are also said to have been free from it for long periods; the Faroe and Fiji Islands enjoyed a long immunity from measles ; but when the disease was imported it spread through them with terrible fatality. The case of the anti-contagionists is stated by Dr. Thorne with some diffidence ; according to him they freely admit that the diseases in question are self-propagating, but they unhesi- tatingly assert that some of them at least do at times arise independently of antecedent cases, that they are in fact of spontaneous origin. Their opinions derive support from the well-known fact that spontaneous generation of the traumatic infections does apparently take place {Sixth Rep. of Med. Off. Privy Council, 1863), and that their respective contagia do arise, settle, grow and thrive in putrefactive wound-products. Thus if the exudation of a simple (deceptive adjective !) peri- tonitis be injected fresh into the peritoneum of another animal. 222 ANNALS OF CHEMICAL MEDICINE. the disease assumes a more intense form in the second than in the first, and if in this way the disease be communicated to several animals at the same time, and the most severe cases be picked out- for the further transmission of their exudation, a still more intense inflammation will result, until at last a virus is obtained of which the virulence resembles that of the specific cases of pernicious peritonitis in the human subject. A simple inflammatory fluid is by artificial selection transformed into an apparently specific poison. The outbreaks of typhus amongst troops when they are ill- fed and badly cared for, are also adduced by the anti-contagion- ists as supporting their view of the possible spontaneous de- velopment of the typhus poison under favourable conditions. But we think this illustration rather unhappy, as with all armies the opportunities of importation of the infection from extraneous foci literally abound. Next the cases of sporadic disease are adduced, in which the most careful inquiry fails to elicit information justifying the assertion that the disease is due to an antecedent case. There are a number of instances in which trained observers carrying on their observations in isolated localities, free from the ordinary sources of error attaching to life in large towns, and making full allowance for all known sources of error, entirely fail to connect outbreaks of some of those diseases, especially of diphtheria and enteric fever, with any antecedent case, whether near or remote ; and hence these observers feel themselves compelled to fall back upon the view that such diseases do, at times, have an independent origin. (See Reports, by Dr. Charles Kelly, on the condition of the Combined Sanitary District of West Sussex). Dr. Thome's failures to elicit any history of previous infection as regards cases of infectious in- flammation of the throat — of diphtheria, which have occurred in isolated houses lying in districts far removed from other human habitations, have forcibly struck him, the more so as in the majority of instances those first attacked have been children of such tender years that all their movements could be ascer- tained with remarkable accuracy. Instances in which a disease such as enteric fever recurs in successive years, or after a lapse of years, in houses where it has ORIGIN OF INFECTION. 223 previously existed, — instances which are assumed by the con- tagionists to afford evidence of the disease-cause being retained as it were in a dormant condition, — such instances may be explained by the anti-contagionists as due to the circumstance that the conditions favourable to the development of the poisons being present, each separate outbreak is but the result of a recurrence of the process by which a new poison is each time brought into existence. Of course this view excludes the hypothesis of contagi/wm vwum, or if not, it includes the hypothesis of spontaneous generation ; in either case it has a most difficult position to maintain. Dr. Thorne adduces yet a more powerful argument on behalf of the anti-contagionists to the following effect. He says that those who believe that the poisons of the acute specific fevers may and do arise sponta- neously have a perfect right to remind their opponents that there must have been first cases of each of the diseases under consideration, and that unless we are prepared to accept the view that the several contagia, which breed truly by a process said to be as regular as that by which dog breeds dog and cat breeds cat, were originally the result of definite acts of creation, it must be assumed that they in some other way did arise independently of antecedent cases ; and what right, they may ask, have we for asserting that a process once possible is now impossible? Dr. Budd says an argument such as this constitutes the last refuge of the partisans of spontaneous origin ; but if it be so, says Dr. Thorne, it is a stronghold in itself, and one which must at least be noticed before it is passed by. Dr. Thome himself believes that there are some grounds for doubting whether certain of these diseases, as at present met with, are exclusively self-propagating, and for raising the question, whether they may not occasionally arise independently of ante- cedent cases. Such personal experience as has inclined him towards this view is mainly limited to investigations into the origin of outbreaks of diphtheria, and one point with which he has been specially struck is the following. In isolated districts, and in houses situated at times miles away from other habita- tions, and in some instances lying in lonely spots among mountain-ranges, where a visit to or a visit from the nearest town or village would be a circumstance too important to be 224 ANNAIS OF CHEMICAL MEDICINE. forgotten, Dr. Thome has met with instances of what appeared to him to be nothing more than a simple inflammation of the throat, at times so trivial that it has passed all but unnoticed, and yet it has led by transmission through other persons to cases of well-marked and severe diphtheria. The first attacks have often happened in children whose former movements could apparently be traced with the strictest accuracy; they have occurred imder circumstances which did not appear to admit of previous infection, and it has been difficult to interpret their occurrence except on the supposition that in some way they have arisen independently of prior cases. With regard also to the well-marked attacks of diphtheria to which they seemed to give rise, all other sources of infection could be excluded with a degree of certainty rarely to be met with. And as to those first affected, whose cases appeared to be earliest in a series which led up to attacks of well-marked diphtheria, it has more than once happened that they were children in whom so-called ' sore throat ' was a common affection, and whose fauces when examined exhibited a loss of tissue indicative of former throat- attacks. Dr. Thorne has further noticed that over an area of some miles around the district in which genuine diphtheria was prevailing, there existed prior to as well as during the diph- theria epidemic numerous instances of sore throat which, so far as examination of the patients was concerned, in every way resembled the early cases above referred to, and yet which gave no indication of being infectious ; sore throat being in fact in the surrounding district a prevailing aihnent. And from this he was inclined to draw the conclusion that conditions very similar to those under which genuine diphtheria was epidemic in a limited district obtained, and had obtained before genuine diphtheria was anywhere seen, over a wide area around the im- mediately infected locality, and that these conditions, leading to a somewhat general predisposition to simple and apparently non-infectious inflammatory sore throat, had further, probably under somewhat modified circumstances, tended at certain points to produce an affection capable of putting on the property of infectiveness, which thus led to the transmission of the disease in a distinctly communicable form to others. ORIGIN OF INFECTION. 225 From such data and explanation Dr. Thome constructs the hypothesis of the progressive development of the property of infectiveness. And if the contagia of the acute specific diseases belong to the vegetable world, he is inclined to admit that organisms capable of producing a minor and an incommunicable disease in particular stages of their growth, become capable, in the course of their subsequent development, of producing a major disease communicable from person to person ; the affair being essentially one of soil. Dr. Thome does not consider this to be a question of the development of a living organism out of matter independently of antecedent life, but merely the production by means of a process of evolution of that which gives to an already existing organism that property by which it becomes infective, a property which it may perhaps lose directly it is deprived of the circumstances which favoured its development, in much the same way as special characteristics may be artificially developed in higher plant life, and be as easily lost again. This hypothesis thus far chimes in with that of Pasteur, according to which the organised cause of splenic fever has two different forms of existence, one in which it is actively infectious, another in which it is quite innocuous ; one can be transformed into the other at will. But these data are not conclusive on account of the data adduced by Bert, which will be considered in the next article. In all these discussions it has been entirely omitted to inquire, whether some of the lower animals could not be per- manent or temporary carriers of some of the infections thus far almost exclusively observed in man. I think it therefore of particular importance that I should be able to direct the attention of the reader to some observations on diphtheria in fowls by Nicati of Marseilles {Gomft. rend. 88, 1879, 297), and to add thereto some notes of observations of the same kind, which I had an opportunity of making in 1875. The attention of Nicati was directed to the subject by M. Gravard, a veterinary surgeon of Marseilles. A disease had during the winter of 1877-8, decimated a fowl-yard at Long- champ. On examination of the victims it was found that thick false membranes of yellow colour covered either the Q 226 ANNALS OF CHEMICAL MEDICINE. mouth and pharynx or the eyes. In one case they penetrated into the bronchi, and the lungs showed yellow hepatisation. A hen died twenty-four hours after the manifestation of the first symptoms, having yet laid the day before. Another of the affected animals lived three days, a third five ; some animals were ill during weeks ; some recovered. Nicati extracted some of the materials from the lower layer of the false membranes nearest to the normal tissue, and inoculated it upon the cornea of rabbits and fowls. He made a great number of pricks into each cornea, and then rubbed into them, by means of the lids, the dSbris of the false membranes. All inoculations succeeded completely. Gribert then showed that during the prevalence of this epizootic disease in the Boulevard de Longchamp, there had been an increased mortality from diphtheria amongst the human inhabitants also. In consequence of this, further inquiries regarding domestic birds were instituted, and the presence of diphtheria in pigeons was discovered. In a pigeon-house attached to a villa named Talabot, in the suburbs of Marseilles, many birds died. Two were examined after death ; in both the mucous membrane of the back of the mouth was covered with a pultaceous layer ; one only exhibited a distinct diph- theritic membrane, which could be easily detached. Another epizootic, attacking both pigeons and fowls, was observed by Dr. Queirel in a fowl-yard in the Eue de I'Academie in the centre of Marseilles. Of the surviving animals three, a fowl and two pigeons, were examined by Nicati. The fowl suffered from a second attack after having apparently recovered from a previous one a few weeks before. The three animals died from increasing weakness, the fowl after having refused food for twenty-four hours. In all three animals the throat was covered with a false membrane which reached up into the nasal fossae. These epizootics also occurred during November and December, there being at the same time an increase of mortality amongst the population from croup, membranous angina, and diphtheria. While these epizootics were under his observation, Nicati observed four cases of diphtheria of the eye in hiunan beings. These facts together with the inoculability upon rabbits brought him to surmise that there might be some relation ORIGIN OF INFECTION. 227 between the diphtheria of fowls and that of man, and that perhaps they might be one and the same disease. In the summer of 1875 a friend and neighbour of mine lost many of the young pigeons which were in course of being reared in his pigeon-house. The birds could take little or no food from the parents, and became emaciated and died. On examination their throats were found occupied, in situations equivalent to the tonsils in man, by thick false membranes of yellow colour and great consistency. The membranes had pro- jections which reached deep into the tissues of the throat, with which they became intermingled ; they could be removed with the aid of suitable instruments and cautious manipulation of the pigeon's throat. In the interior the masses showed no particular cohesion, and appeared like masses of favus. The corroded holes from which they were removed looked raw, but did not bleed. After the removal of the false growth the young pigeons again took food, but few were saved by this in itself difficult operation. The parents, which fed the young pigeons thus affected, did not appear to take the disease. The same disease I observed upon a fowl in my own fowl- yard ; the membrane and prolongations into the tissue of the throat, formed a mass of the size of a haricot bean, and pressed the glottis on one side, so that there was stridulous voice and difficulty of respiration. I had the animal killed and examined. The microscopic appearance was the same as in the pigeon : minute particles, in which with a high power of the microscope no organisation was distinguishable, mixed with debris of destroyed tissue. I was credibly informed that a great breeder of fancy- pigeons at Acton had during the summer of 1875 lost a great number of young, mostly half-grown pigeons from the disease described in the foregoing paragraphs. The question raised by these observations upon animals must be definitely answered before the position assumed by the anti- contagionists can be considered tenable. The observations fur- nish a new means for studying the nature of an infectious disease experimentally, and for approaching the chemical problem of its zymosis on ground which can be extended at will. Q 2 228 ANNALS OF CHEMICAL MEDICINE. XVI. RMCENT DATA ANB ARGUMENTS IN FAVOUR OF AND OPPOSITION TO THE HYPOTHESIS OF THE LIVING CONTAGIUM. {Summary.) Amongst the morbid anatomists the hypothesis of the living contagium has hitherto found few adherents. There are however a few, and Professor Klebs of Prague (^Amtl. Ber. 50 Versamml, Deutach. Naturf. unci Aerzte. Miinchen, 1877, p. 41) par exemple is not only an adherent, but an investigator and ex- pounder of this hypothesis. He believes that the various data already obtained necessitate the assumption that the causes of many and important diseases are to be searched for and found outside of the human body, and that they are, while producing diseases in the body, of a parasitical nature. The parasites are assumed to be 'low organisms' regarding which it is stated more by implication than by direct enunciation that they are plants, of the same category as those which produce processes of fer- mentation properly so-called. The names of Schwann, Helm- holtz and Pasteur are quoted as the scientific expounders of the hypothesis that fermentations are caused by organisms. This hypothesis is considered by Klebs to be something like a proved law of nature, and from it he argues with the aid of analogy in support of the zymotic hypothesis of disease. He first speaks of the close genetic connection which according to him exists between certain forms of gangrene and so-called inflammatory and febrile infective diseases, and argues that this connection made it probable that both diseases had the same mode of origination. The hospital gangrene, and the purulent aflfections following gunshot wounds, as observed in the last great conti- nental wars, are said to prove this already to the naked eye of the observer. But the final proof is afforded by the discovery HYPOTHESIS OF THE XIVING CONTAGIUM. 229 that in, the bodies of persons suffering from pysemia or from hospital gangrene the same varieties of fungi are met with, when the parts are examined under the microscope. The immediate effects of the presence of these parasitic organisms are either mechanical or chemical. (Here a distinc- tion of the infective process from fermentation is admitted, namely the mechanical action of the supposed fungi determined by locality, which does not take place in ordinary fermentations.) The fungi can produce death by the destruction of important parts, particularly the central nervous system (brain and spinal cord), or the heart. Before the fungi produce death by their effects, these effects cause a series of phenomena on the part of the body which they have invaded. These are termed 'reaction,' and this reaction may in some cases lead to a spontaneous cure. Eeaction is not necessary to ensure the healing of wounds, and to have shown this, says Klebs, is the merit of Lister. It is admitted that a division of the phenomena of a disease into those which are due to the infecting organisms, and those which are reactions of the infected body cannot be carried out with the same precision in all infective diseases ; but it is maintained that the proof, that all infective diseases have similar causes can be furnished by three different modes of investiga- tion: 1. By the anatomical investigation of the diseased organs ; 2. By isolation and propagation by intentional rearing (also termed ' cultivation ') of the disease-germs ; and 3. By the re- production in healthy animals of the disease-process through the instrumentality of such intentionally reared fungi or germs. The proof of these propositions begins with the consideration of splenic fever in the ox. The Austrians claim that Pollender, and the Eussians that Brauell discovered, what the French as we think with the greatest justice claim for their Rayer, namely to have made the first observation to the effect, that the blood of animals which have died of splenic fever contains a great number of little rods, which some claimed to be organisms, others alleged to have the properties of crystals. Rayer, and later Davaine, experimented upon this question, and found that the transfer of a very small number of these rods into the veins of a healthy animal was sufficient to cause the splenic fever and rapid death of the inoculated animal. From this it was concluded that 230 ANNALS OF CHEMICAL MEDICINE, these rods were organisms endowed with the faculty of reproduc- tion, and that the reproduced generations represented the true cause of the disease. The usual counterproofs were instituted, such as excision of the inoculated part at a time intermediate between the inoculation and the period at which general symptoms would have appeared, if the part had not been excised ; the excision prevented the appearance of general symptoms, and of any disease whatever, although it was shown that in the excised part a multiplication of rods had already taken place. Davaine termed the rods bacteridia, and claimed for them to be specific low organisms, and the true cause of splenic fever. There remained the objection that the inoculated matter contained not only the bacteridia, but also larger or smaller quantities of liquid adhering to them and coming from the juices of the animal which furiiished the bacteridia. Such liquid might of course be supposed to be able to act as an unorganised or shapeless ferment. To remove these objections Klebs and Tiegel endeavoured to filter the liquid from the bacteridia, by pressing it through septa of porous burnt clay. They were successful, under what they term favourable conditions, in obtaining a liquid free from bacteridia, which on inoculation proved perfectly inactive. Pasteur afterwards, so it is said, applied the same principle and obtained the same results as Klebs and Tiegel. The reservation covered by the term ' favourable conditions ' just mentioned implies perhaps mainly that in some conditions, which are unfavourable, bacteridia pass through the porous clay-septa ; of course such a liquid is unsuited for the experi- ment here in question. But it does not exclude that other Variety of unfavourable conditions, under which a liquid free from bacteridia causes infection. I do not know whether Klebs and Tiegel have observed this amongst their unfavourable experience, but it is certain, and we shall see that other observers have. Indeed Pasteur has thought these contrary experiences so telling that he abandoned the argument derived from the exclusion of the organisms by filtration from the fluid to be tested, and substituted the method of investigation mentioned previously under 2, namely the isolation and propagation by HYPOTHESIS OF THE LIVING CONTAGIUM. 231 intentional rearing out of the body of tlie disease-germs. This might be termed the method of neatralising any possibly adhering unformed ferment by almost infinite dilution ; or, this method of freeing bacteridia from the suspicion' of carrying splenic fever ferment only as an adhering impurity on their surface by subjecting a series of twelve or more successive generations to pure liquids twelve times renewed may be termed a process of washing, which could not be supposed to leave anything unclean in the sense here required amongst their surroundings. Pasteur found that the epigones of these experi- mentally reared bacteridia were as destructive as those taken from the blood of the animal which furnished the first stock of organisms. However, now came this experiment of P. Bert (Compt. rend. 84, 1877, 1130) which he performed in the course of his trials regarding the influence of oxygen under high pressure upon ferments, poisons and infectious matters. Splenic fever blood, which did not lose its virulence by oxygen under high pressure, but killed guinea-pigs as before, was mixed with abso- lute alcohol drop by drop, until to one volume three volumes had been added ; the coagulum was filtered, washed well with alcohol and dried in vacuo. A piece of this cake killed a guinea-pig, and blood from this guinea-pig kiUed several succes- sions of guinea-pigs and a dog ; the blood of these animals contained no bacteridia since the one poisoned with the alcohol precipitate. The precipitate by alcohol yielded to water the virulent principle, and from this it could again be precipitated by alcohol in the shape of light white flakes. It lost in intensity by this treatment, so as to be unable to kill a dog, but it killed yet three successions of experimental guinea-pigs. From this the conclusion was unavoidable that there is contained in the blood of splenic fever a toxic and virulent matter, which with- stands the action of compressed oxygen and of alcohol, and of drying in vacuo, and can be isolated in the same manner as the shapeless ferments called diastases. The fact that oxygen under a pressure of fifty atmospheres does not destroy the virulence of splenic fever blood is a negative which by itself would have no significance. But it acquires a meaning by ranging splenic fever poison with other ferments 232 ANNALS OF CHEMICAL MEDICINE. which also show this resistance. For according to Bert, ferment- ations, which are supposed to be due to the influence (whatever that may exactly mean) of living beings (putrefaction, acetifica- tion of wine, alcoholic fermentation) are arrested by the influence of oxygen under the pressure of fifty atmospheres, whereas fermentations due to (undoubtedly) dissolved matters (such as diastas, pancreatin, mycosin, emulsin), withstand the influence of this agent. If therefore bodies acting in the manner of ferments, but the nature of which is not exactly known, are tested by oxygen under fifty atmospheres, they will exhibit either liability to be changed, i.e. destroyed, or remain un- changed. If they remain unchanged, they show analogy to the shapeless non-organised ferments; if they are destroyed, they show analogy to the ferments at present most frequently admitted to be organised. Now splenic fever blood sides with the shapeless ferments by remaining unaltered ; the same is the case with the poison of the scorpion, and the infection-matters of vaccine and glanders ; by this test these infection poisons appear prima fade as non-organised. These experiments of Bert created the utmost consternation amongst the adherents of the hypothesis of the living contagium. For splenic fever had been the strongest support of that hypo- thesis, no other demonstration approaching that which its materials furnished in certainty of effect and distinctness of form. Moreover it fulfilled a condition upon which alone the hypothesis of an acute vegetable parasitism, developing itself by multiplication of the parasites within .the body, would be ad- mitted by botanists well acquainted with the low forms of vege- tals ; the condition namely that the disease should either end shortly in death or be incapable of spontaneous cessation, that is, be in the individual once seized for the rest of its lifetime, while quite able to destroy life by a chronic course such as obtains in glanders. These conditions of death or incurability were admitted about 1872 by De Bary to be good criteria for vegetable parasitisms. Upon the evidence before us thus far we should be able to explain splenic fever as a zymotic infection, for the perpetra- tion of which, so to say, the supposed shaped ferment of the parasitic bacteridia is not essential. And if this were true, there HYPOTHESIS OF THE LIVING CONTAGIITM. 233 would be an end at once of the better half of the hypothesis. For the other diseases in which parasitic organisms are stated to have been discovered, offer so many difficulties in their nature and course to the identification of their essential causes with these organisms, that it is at present impossible even to construct a plausible hypothesis embodying all the known data ; we are therefore far from a theory of acute febrile diseases of man. The filtration of an infectious poison through porous burnt clay, even if it keep bacteridia and other organisms on the side where the liquid enters the septum, does not necessarily permit the liquid to pass unaltered, and does not necessarily allow any and every liquid to pass, merely because there is some pressure behind it; no amount of pressure which does not burst a membrane (bladder) will drive alcohol through it; and it is quite conceivable that shapeless ferments should have as little affinity for clay-septa, as alcohol has for membranous septa, and colloids have for parchment paper, or sized paper. Indeed it is difficult to see by what manner of collateral argumentation the filtration throiigh clay-cells of infectious liquids can ever be raised to the value of a positive demonstration ; and unless it be so improved it has only a small force in the shape of pointing to a probability ; the objectors may always say, that the porous septum retained both the shaped and shapeless ferment (observe, a ferment isolated otherwise by chemical pro- ceedings, and therefore present by the side of the organisms supposed to be also ferments by the other hypothesis) on the near or entrance side of the porous clay-septum. Klebs and Tiegel claim to have diagnosed and isolated the microscopic organism which causes the septic condition after wounds ; they term it the microsporon septiGum, and remove it out of infectious liquids by the filtration through porous clay already alluded to, and by other means, which are confronted by many opposing considerations. Pyaemia is infectious, no doubt, but the least infectious of the diseases here most to be con- sidered ; it is also the least typical ; but it fulfils a condition already stated above, namely that it is nearly always fatal. Klebs and Soyka further claim to have applied the same process of exclusion to a series of organisms which according to them are originators of inflammation, such as the various 234 ANNALS OP CHEMICAL MEDICINE. forms of pneumonia, nephritis, carditis, and the acute forms of rheumatism or rheumatic fever. To these organisms Klebs has given the genus name of ' monadines,' the species being perhaps left to be signalised by adjectives derived from the diseases to which they are supposed to be specific. Other diseases there are in which other forms of organisms supposed to be specific have been found ; one of them is re- lapsing fever, in which a spirally twisted formation and from its shape termed ' spirilla ''is observed in the blood (Obermeyer). But here the connection of the disease with the spirilla as cause is more problematical than in any other disease where an undoubted peculiar anomalous formation supposed to be an organism has been found. For the spirilla is quiescent or entirely absent at intervals in the disease, and only appears at periods of exacerbation, or as the expression goes, it wells up during the relapses. The virus of the cowpox, so accessible to microscopic studies, has never yet been pronounced to be an organised body or organism, i.e. body consisting of organs. The argument in this case has hitherto been confined to inferences. Chauveau experimented upon the lymph taken from cowpox, and came to the result that it consisted of solid particles, and of a liquid, a result which had always been admitted by micro- scopic observers. He then allowed the lymph to repose in high cylindrio vessels, and testing it by performing inoculation with samples taken from various strata at various times, he ascer- tained that the infecting quality of the lymph decreases as the solid particles become deposited ; that the upper layers of the lymph can lose their infecting qualities entirely, while the in- fecting qualities of the lower layers containing the sediment increase at the same time. This mode of separating the infectious particles is equivalent to filtration ; its result excludes the presence of a dissolved disease-ferment. Klebs terms diphtheria an accidental disease incidental to wounds, as well as a primary affection (i.e. occurring without wounds) ; he deplores the losses which the medical profession annually suffers through this disease, particularly amongst sur- geons, and says that in some countries diphtheria has seriously thinned the ranks of the younger generation even to the extent HYPOTHESIS OP THE LIVING CONTAGION. 235 of threatening the extinction of families. According to him there is in this disease constantly present a well-characterised form of fungus, the specific action of which has been proved by specimens cultivated out of the human body. But what is said in the foregoing lines is almost all that we are vouchsafed to know ; the fungus goes by the name of the microsporon diph- theriticum ; it receives the attention of some disinfecting experiments which seem to show (a) that after it has been for a few hours in contact with a solution of sodic benzoate it loses its energy; (&) that animals which have been impregnated with sodio benzoate (it is not said how) to the extent of one per mille of their body weight are not a suitable soil for the vegetation of these fungi. We may here remind our readers that the benzoate was tried in the cases which occurred in the Grrand-ducal family at Darmstadt, and found to be without any influence on the disease. Of lepra Klebs speaks with much preamble and little essence; there are fungi active in this disease, but they are least so where the cellular growth is most developed. Bidenkap of Christiania, Sweden, excised a nodule from a leper, and in this Klebs was able, without much difficulty, to ascertain the presence of groups of bacteria, which in their forms and arrange- ment differed from the bacteria found in other diseaocs. To syphilis also Klebs attributes a ' bacteric origin,' which he has lately investigated at length by inoculating experiments upon monkeys. In a postscript to his essay Klebs says that he felt it a command (of the intellect, we suppose) to formulate the parasitary theory of the infectious diseases according to the available observed facts, but that he had not claimed uncon- ditional validity for it. Indeed he now comes to term what he before called a theory, an hypothesis, and demands liberty to change his opinions when new facts might make such a change desirable. Such are the opinions taken of the parasitological hjrpothesisby a professor of morbid anatomy brought up in the views of the Berlin school, and advanced to the point which he has reached by his personal experience amongst large oppor- tunities. 236 ANNALS OF CHEMICAL MEDICINE. XVII. ON TRB SPECIFIC ALVINE FLUX OF CHOLERA, WITS SPECIAL REFERENCE TO ITS INORGANIC CONSTI- TUENTS. WITH HINTS CONCERNING THE THEORY OF CHOLERA. (From the Pathological Institute.) Many pathologists admit that the most characteristic sign of cholera is the evacuation of the peculiar liquid discharges, which owing to their similarity as regards external appearance to a decoction of rice, have been termed rice-water discharges. It might therefore be expected that this rice-water discharge of cholera contained the characteristic products of the chemical process in which cholera consists or hy which it is accompanied. This process is, in the beginning of the disease at least, almost exclusively carried on in the intestinal canal, and the symptoms which are exhibited by the rest of the body are all referable to physical effects of this chemical process. It is carried on in its earliest stages upon the layer of mucus and epithelium which covers the ceUs of the intestinal membrane. Whatever kind or quantity of adventitious matter the cholera infection, the very particles which import the disease, may bring, this mass of mucus and epithelium breaks up according to certain conditions inherent in its chemical constitution. As its chemical consti- tution approaches that of the albuminous bodies, the products of its chemical decomposition are also similar to those furnished by the albuminous bodies. And as the products of decompo- sition contained in rice-water are probably not derived from mucin only, but also from the albumin which transudes into the cavity of the intestine in consequence of the destruction of its epithelium, their examination becomes a much more compli- cated problem than is at first sight apparent. In the course of some researches on cholera which I carried ALVINE FLUX OF CHOLEBA. 237 on during the epidemic of 1866 (Ninth Rep. of the Med. Off. Privy Council, 1866, Appendix N""" 10, p. 458), I had subjected rice-water to dialysis, and found that the dialysate from the alkaline rice-water had an acid reaction ; it yielded on further analysis crystallised leucin, an oily substance soluble in water, and giving a peculiar pink reaction with nitric acid, probably indol, butyric acid, and inorganic salts in considerable quantity, but no urea. As the rice-water continued to decompose during dialysis, or during any operaition, even filtration, with evolution of gas, this process was observed as far as opportunities permitted, and it was found that the gas evolved consisted at first almost entirely of nitrogen, with only little carbonic acid ; gradually the latter increased, while the nitrogen evolved decreased, until at last the carbonic acid was almost double the volume of the nitrogen, 400 c.c. of rice-water in a suitable flask and apparatus emitted the following amounts and kinds of gas into a measuring tube over mercury. Mixed gases Nitrogen Carbonic acid First twenty-four hours 75*25 c.c. 99 per cent. 1 per cent. Second „ „ 51-00 „ 52 „ 48 „ Third „ „ 100-00 „ 37-5 „ 62-5 „ On the fourth day some hydrogen appeared in the tube ; on the fifth day the hydrogen continued to be evolved, but carbonic acid still increased relatively to the other gases. On the sixth day the hydrogen had ceased to appear, and the gas consisted almost entirely of carbonic acid, with but very little nitrogen. Soon after this point had been reached the evolution of gas ceased almost entirely, and the liquid .presented a feeble somewhat modified hemochrome spectrum. By other reactions the presence of albumin, and peculiar albuminous bodies, was proved ; and I enumerated the micro- scopical and chemical ingredients of rice-water isolated as follows : Vibriones, cells from the surface of the intestine, granular debris of cells, mucin, modified hemochrome, albumin, albuminous body giving rose-pink reaction, and another one, diffusible, and giving red reaction with nitric acid, probably indol, butyric acid, acetic acid, ammonia, inorganic salts. 238 ANNALS OF CHEMICAL IHELICINE. The inorganic constituents of cholera rice-wat^ were examined some years ago in the Pathological Institute, with the following results, here published for the first time. The liquid with some slimy deposit, was evaporated to dryness. It left a blackish deliquescent mass, which when dry at 100° weighed 82-5 grms. This was ignited in a platinum dish ; it gave out a horrible smell, much vapour, and after incineration left a white fusible ash, weighing 7 -8440 grms. This ash was analysed as follows. 1"6930 grm. were treated with nitric acid, and left 0*0465 grm. or 2-74 per cent, of residue insoluble in the acid. This residue contained silica, lime, and sulphuric acid, but no fluorine. 1 '448 grm. were dissolved in nitric acid, the insoluble residue was filtered off, and the filtrate treated with silver nitrate. There were obtained 1*1420 grm. silver chloride= 19*49 per cent, of ash chlorine. The phosphoric acid in the filtrate was removed by silver oxide etc., and the lime precipitated as oxalate gave 0*1530 grm. calcic carbonate = 4*22 per cent, of ash calcium. 0*714 grm. of ash were dissolved in hydrochloric acid and the insoluble residue was filtered off. The sulphuric acid precipitated by baryum chloride etc. gave 0*1650 grm. baryum sulphate or 7*93 per cent, of ash sulphuric acid. The filtrate, by treatment with ammonia and its carbon- ate, gave (a) a precipitate consisting of baryum carbonate and calcium phosphate etc. ; this was dissolved in hydrochloric acid ; sulphuric acid and alcohol were then added and preci- pitated sulphates of baryum and calcium filtered off; the filtrate was treated with ammonia, ammonic chloride, and sodic phosphate, and gave 0*2850 grm. magnesic pyrophosphate, equal to 8*62 per cent, of ash of magnesium, (/3) The filtrate from the precipitate a obtained by ammonia etc. was evaporated to dryness in a platinum dish, ignited and weighed 0*6208 grm. Of this a small portion remained in- soluble in water, and was removed by the filter. The soluble part was again evaporated to dryness and heated and found to weigh 0*5643 grm. In this the potassium was estimated by platinic chloride ; 0-7675 grm. of the double salt showed the presence in the ash ALVINE FLUX OF CHOIxEEA. 239 of 17'19 per cent, potassium. The solution containing excess of platinum etc. was treated with hydrothion (H^S) and the filtrate evaporated to dryness, ignited and weighed; 0-2850 grm. indicated the presence in the ash of 15"68 per cent, of sodium. Synopsis of results. Total quantity of rice-water discharge = i i litres. Total dry residue =82'5 grms. Total ash= 7-8440 grms. Composition of the ash in 100 parts: — Sulphuric acid (SOg) . . 7-93 Chlorine (CI) 19-49 Phosphoric acid (PjOs) 23-16 Calcium (Ca) 4-22 Magnesium (Mg) . 8-63 Potassium (K) 17-19 Sodium (Na) . 15-68 Iron (Fe) . a trace Silica and trace of gypsum, insoluh le . 2-74 Loss in operations 0-96 G Dotal . 100-00 The prevalence of potassium over sodium in this ash shows conclusively that the liquid in which it is observed is not derived to any large extent from the blood, in other words is not a serous discharge. For in blood-serum the sodium salts are always larger in quantity than the potassium salts. In the juices of muscle, on the other hand, the potassium salts are always larger in quantity than the sodium salts. A liquid, like the rice-water discharge examined in the foregoing, could therefore be derived in part at least, and perhaps to a large extent, from the muscles. At once we are led to think of the symptoms exhibited by the muscles in the cholera process, and find them to be loss of heat, loss of size, cramps and consequent pain. Seeing that there is no obstruction to either respiration or circulation in the cholera patient, we cannot ascribe the great loss of body-heat to a mere diminution of natural oxy- dation, but are constrained to assume that there is an action 240 ANNALS OF CHEMICAL MEDICINE. going on in the cholera-body by which heat becomes latent. This action may reasonably be supposed to be the liquefaction of a part of the colloid constituents of the muscles. For every body, which passes from the solid into the liquid state, by what- ever cause, makes a quantity of heat latent, and takes that heat from its surroundings whatever they may be. These liquefied muscular constituents dififuse into the blood, by necessity, as the blood has been concentrated by diffusion of its serum into the intestinal canal ; and from the blood they are secreted and diffused by pathic exudation into the intestinal canal. It is thus that we find in the rice-water discharge mineral constituents, the proportions of the ingredients of which correspond only with those in which the same mineral ingredients are present in muscular tissue. The potential nature of the rapid sinking of animal heat in the body affected by cholera is so striking that it has never escaped even popular observation ; it is owing to this potential nature of the loss of heat that a cholera-patient either cannot be warmed at all by artificial means, or becomes much worse from the moment that the artificial heat supply takes effect upon him. His comfort is for a time increased by the relaxa- tion of cramps, but he loses more plastic material, and has less chance of recovery. The value of this hypothesis is not diminished by the con- sideration, that some hold cholera to be a heat-making process like other pyrexias not complicated with the state or stage of coUapse. Only two or three cases out of, say, a thousand cases examined ha"ve ever shown increased temperature of the body during the state of collapse, and those only, or principally, in the cavities of the trunk. But it is evident that these higher temperatures occurring in highly exceptional cases can be ex- plained just like the increased temperature of moribund persons, by a local accumulation of heat, in consequence of diminished circulating blood-supply, from contraction of blood-vessels — asphyxia truly so-called. But even if they could not be thus explained, they are exceptions, and are cases in which the state of collapse and of pyrexia of reaction so to say overlap each other, and in which, if they were attended by rice-water dis- charges, the fluidification of colloid matters, and the consequent ALVINE FXUX OF CHOLERA. .241 loss of heat, must have taken place all the same ; of physical necessity, however the loss of heat was masked by a concurrent pyrexia in special protoplasmic regions not in contact with large masses of muscular tissue. This hypothesis of the occurrence of dehydration of colloid tissue in cholera, and consequent potential sinking of tempera- ture, arises out of the results of the examination of the mineral ingredients of rice-water discharges as ahove stated. The evidence is derived from materials being portions of those obtained from about twenty cases, and is therefore represen- tative of more than exceptions. The hypothesis is greatly supported by the symptoms of cholera-collapse, and these in their turn are explicable upon the basis of the hypothesis, and have not yet found any other interpretation that could be said to be in harmony with fundamental physical laws. 242 ANNALS OF CHEMICAL MEDICINE. XVIII. EXPERIMENTS ON ANIMALS EOS ASCERTAINING THE STATE OF GREATEST INFECTIOUSNESS OF FERMENT- ING CHOLERA POISON. (Pi-om the Pathological Institute.-) It had been asserted, and apparently on good grounds, that the specific morbid secretion ejected by cholera patients had no infective properties immediately after emission, but acquired such properties, or the power of giving cholera to fresh subjects, only after a period of decomposition lasting during from- 5 to 1 5 days. This belief was raised to a high degree of probability by the experiments of Thiersch on the infection of white mice with fermenting cholera poison. With a view of obtaining some personal information on this subject, I imitated, in 1866, in the best manner time and opportunities permitted, the ex- periments of Thiersch. As no account of these experiments has ever been published, and as they are an element of consideration in the admeasuring of the significance of experiments to which the surviving animals were subjected at a later period, I here give an abstract of my records. The matter to be tested for its infectiousness was given to the animals in the following manner. Strips of bibulous paper an inch wide were dipped into the liquid, and dried in air. It was ascertained that such paper retained only a veiy small amount of solid matter after drying. Of such papers an inch in length, representing a square inch, was cut off, cut small, and mixed with the food of the animals in such a manner that they had either to eat or to chew some of the paper. In a few cases only, to be specially mentioned, was a minute quantity of liquid diseased matter mixed directly with the food. The white mice employed were 51 in number, distributed in couples of a male and female each in 25 cages, one cage containiDg a single mouse. EXPERIMENTS ON CHOLERA POISON. 243 Particular history of each cage. iV''- 1. Two mice. — On Aug. 10 they were fed with one square inch of paper once dipped into rice-water taken from jejunum of E. W. Aug. 17, feeding repeated with same paper; Aug. 18, ditto; Aug. 19, ditto ; Aug. 21, ditto. On Aug. 27 they were fed with 6 drops of liquid from small intestine of H., who died Aug. 24 ; Aug. 28, ditto ; Aug. 29, ditto. iV' 2. Two mice. — On Aug. 1 1 they were fed with 1 square inch of paper soaked with rice-water contents of ileum of E. W., dipped Aug. 10. Aug. 17, feeding repeated with same paper. Aug. 18, ditto ; Aug. 19, ditto; Aug. 21, ditto. On Aug. 27, 28, and 29, fed as N'- 1. N'^' 3. Two mice, — On Aug. 11 they were fed with 1 square inch of paper dipped Aug. 10 in contents of colon of E. W. Aug. 19, feeding with same paper repeated. Aug. 18, 19, and 21, ditto. iV'" 4. Two m,ice. — Aug. 11,1 square inch of paper dipped Aug. 11 in contents of jejunum of E. W; Eepeated Aug. 17 with same paper. Aug. 18, 19, and 21, ditto. On Aug. 27, 28, and 29, they were fed as N'. 3. iV''- 5. Two mice. — Aug. 11, 1 square inch, dipped Aug. 11, ileum of E. W. Aug. 17, feeding repeated with same paper. Aug. 18, 19, 21, ditto. On 22nd they were apparently well, but one was found dead on morning of Aug. 23. The female mouse had the right aiiricle of the heart very full of clotted blood, as also both cavse. The lungs were congested in patches. The liver contained a large tsenia-like cysticercus fasdolaris ; which had caused the gall-bladder to atrophy. The liver appeared otherwise normal. The stomach was distended with starchy matter, and some cylinder cells. The small intestines were much distended with liquid of a pinkish brown colour, that in the upper part of the bowel being frothy. It consisted of free blood-corpuscles, many pale pus-like cells of various sizes, a moderate number of cylindrical epithelial cells, with a few portions of villi retaining their shape. There was also a very large number of peculiar oval nucleated bodies, but no starch was visible. The large intestine contained formed faeces, presenting no anomaly under the microscope. The he-mouse had the right auricle of the heart distended K 2 244 ANNALS OF CHEMICAL MEDICINE. with fluid reddish blood, the lungs congested throughout, the liver pale in patches, but dark behind and at circumference, the gall-bladder empty. The stomach was distended with starchy matter, which was whitish at the cardiac, slightly brown towards the pyloric end. The duodenum and upper part of small intestine were extremely distended with air; the lower part contained a brownish semi-fluid substance, which under the microscope showed abundance of columnar epithelial cells, several entire villi, and a large quantity of granular epithelium and several blood-corpuscles. The csecum was loaded with solid faecal matter, and the colon considerably distended with air, N''' 6. Two mice. — On Aug. 11 were fed upon one square inch of paper soaked with contents of colon of E. W. Aug. 17, feeding with same paper repeated; 18th, ditto ; 19th and 21stj ditto ; on 22nd both were apparently well, but were dead, on Aug. 23. The post-mortem appearances in both mice were very similar. The intestines were much distended with gas, but there was not much fluid present. Under the microscope the contents showed a large quantity of free epithelium of the intestine. The large intestine contained faeces of normal size and composition, but no epithelium. The stomach was full of farinaceous matter. iV"' 7. Two mice. — One square inch of paper dipped in contents of jejunum of E. W. on Aug. 12 ; was fed the same day. Aug. 19, feeding repeated ; ditto, 21st, 22nd, and 23rd. On Aug. 27, 28, and 29 they were fed as N''* 4. iV''- 8. Two Tnice. — One square inch, dipped Aug. 12, ileum, E. W. Aug. 19, 21, 22, and 23, feeding repeated. On 27th and 28th fed with six drops of fluid from colon of H. (Died Aug, 24.) iV"" 9. Two mice. — They were not quite full-grown animals. Fed on Aug. 12 upon one square inch of paper dipped same day in colon contents of E. W. Aug. 19, 21, 22, and 23, feeding repeated. On 27th and 28th they were fed as N'. 8, and on morning of September 3 were found dead, much con- tracted, with their bellies distended, and the evidence of diarrhcea round the roots of tails. Few shaped faeces in cage. iV"''' 10. Two mice. — They got one square inch of paper, dipped Aug. 13 in contents of jejunum of E. W. In the evening EXPEEIMENTS ON CHOLERA POISON. 245 of Aug. 15 one of the mice was noticed to have considerable difficulty in walking ; the back was bent upwards, and the hind legs spread out on each side, and somewhat dragged after the body in walking. On the cotton wool was a quantity of bloody mucus, and under the tail of the affected mouse was the same substance, leaving no doubt as to the origin of the matter which was about. The temperature of the affected mouse was notably lower than that of the non-affected animal. In the morning of Aug. 16 the Animal was worse. Its skiu was colder than that of the healthy mouse. A large quantity of the bloody evacuation was lying about ; the back was arched, there was more difficulty in progression, the fur was staring. It was put in a separate cage, and died two hours later. The lungs were very pale, but got much redder after exposure to the air. The heart contained much blood, the right side more than the left. The small intestine contained bile-stained matter, especially in its upper part, of slimy consistence. The large intestine was full of slimy blood-stained matter. The contents of the small intestine under the microscope showed remnants of food and coverings of viUi ; those of the large intestine a few scattered blood-corpuscles and a great many cells having somewhat the appearance of pus, but no columnar epithelial cells. On Aug. 19 the feeding of the remaining mouse was re- peated. Ditto on Aug. 21, 22, 23. On Aug. 27 and 28 it was fed as N'- 9. N''' 11. Two mice.— One square inch, ileuin, E. W. Dipped Aug. 13. On Aug. 15 at 8 p.m. one of the mice was found in the inner box apparently dead. It was bent up, stiff, but still warm. There was no bloody matter in the cage or on the mouse. The mouse was placed on the outside of the cage for further observation, but on the 1 6th in the morning it had disappeared. The other mouse appeared healthy. With this animal the feeding was repeated on Aug. 19, 21, 22, and 23. On Aug. 27 and 28 it was fed as N""- 10. N^' 12. Two mice. — One square inch, colon E. W., Aug. 13. Feeding repeated on Aug. 19, 21, 22, 23. On Aug. 27 and 28, fed as N'- 11. N'^- 13. Two mice. — One square inch jejunum, E. W., Aug. 14. Feeding repeated Aug. 19, 21, 22, and 23. On Aug. 28 246 ANNALS OF CHEMICAL MEDICINE. these animals were fed with six drops of rice-water .taken from small intestine of S. forty-eight hours after the death of the patient. The same repeated on Aug. 29. iV'* 14. Two mice. — One square inch, ileum, E. W., Aug. 14. Aug. 19, 21, 22, and 23, feeding repeated. On Aug. 28 and 29, fed as W- 13. . iV"" 15. Two imice. — One square inch, colon, E. W., Aug. 14. Aug. 19, 21, 22, and 23, feeding repeated. On Aug. 28 and 29, fed as N''- 14. !>}''• 16. Two mice. — One square inch, jejunum, E. W., dipped Aug. 15. Feeding repeated Aug. 19, 21, 22, 23. On Aug. 28 and 29, fed as N"^- 15. N''- 17. Two mice. — Ouq square inch, ileum, E. W., dipped Aug. 15. Aug. 19, 21, 22, and 23, feeding repeated. On Aug. 28 and 29, fed as No. 16. These two mice were found dead on the morning of Sept. 3. There were some cake- shaped fseces. The male had fsecal incrustations round anus and root of tail. The female was covered on its belly and side with fluid matter. iV""- 18. Two mice. — One square inch, colon, E. "W., dipped Aug. 15. Feeding repeated Aug. 19, 21, 22, and 23. On 28 and 29, fed with six drops of rice-water stool passed by M. A. T. on Aug. 16. N''' 19. Two mice. — One square inch, jejunum, E. W., dipped Aug. 13, and dried ; fed Aug. 16. This was the same paper as that which had been given to N'* 10 mice. W' 10 mice. On Aug. 19 they were fed with bread on which six drops of mixed rice-water from a cholera patient, passed Aug. 17, had been dropped. This feeding was repeated on Aug. 21, 22, and 23. On Aug. 28 and 29, the mice were fed as N"-- 18. iV''- 20, Two mice. — On Aug. 16 these mice received one square inch of paper, dipped on Aug. 1 3 in contents of ileum of E. W-., being the same paper as that upon which mice No. 1 1 were fed. On Aug, 19 they were fed in the same way as N'* 19, also on Aug. 21, 22, 23, On Aug. 28 and 29, fed like W- 19. iV'* 21. Two mice.^-One square inch, dipped Aug. 16, jejunum, E. "W. Aug. 1 9, fed with one square inch more. On EXPERIMENTS ON CHOLEEA POISON. 247 Aug. 20 they were fed with sopped bread at 1 a.m. They ate some, but about half an hour later they were found both dead, and yet quite warm. There had been^io purging, and their skins had not felt colder than those of healthy mice. They were left in the cage till evening, but did not revive. The white mouse was examined ten hours after death. Its abdomen was much distended, so that on opening it the intestines immediately protruded. The large intestine was moderately filled with solid contents of a greyish colour. From the small intestine, when cut across, nearly half a tablespoonful of pinkish semi-fluid matter escaped. This under the microscope appeared to consist of cells of columnar epithelium which lay sometimes in masses, of which several had the shape of distinct caps of villi. The brown and white mouse was examined thirty hours after death. Its belly was distended and its hair somewhat matted as if from wet. The lungs were completely sunk into the back of their cavity ; they were congested behind, with pale patches on the anterior margins, and on exposure to air became much redder than they had been. The right auricle of the heart was much distended with dark blood. The stomach was distended by food and gas. At the cardiac end was a mass of almost pure sopped bread, in the centre a large mass of gas, and at the pyloric end a smaller quantity of semi-fluid pinkish buff- coloured matter, alniost exactly like that in the duodenum. The line of demarcation between the white bread and the pinkish pyloric portion was very distinct. The pyloric part contained much starch, but the bulk of its contents was composed of cylindric epithelium, so that a retroperistaltic motion must have taken place. In the duodenum and entire small intestine there was nearly the same substance as in the pyloric end of the stomach ; it consisted almost entirely of cylindric epithelium, with a number of fat-globules, but, as was shown by means of iodine, no starch. In order to prevent the admixture of any intestinal epithelium which might be'detached by the mechanism of the examination, the intestine was cut across, and the con- tents allowed to flow out assisted by the very slightest pressure required. Some cells were thus obtained in the form of caps of villi. The large intestine contained semi-fluid faecal matter of greyish green colour in the upper part, and soft faeces in the 248 ANNALS OF CHEMICAL MEDICINE. lower ; in neither was there any notable amount of epithelium, if the intestine had not been forcibly pressed. The liver was very pale, the gall-bladder could not be detected, the urinary bladder, though empty, was more collapsed than contracted. N'- 22. Two mice. — One square inch, dipped Aug. 16, ileum, E. W. Eepeated Aug. 19, 21, 22, 23. On Aug. 28 and 29, fed as N'- 20. iV''" 23. Ttvo tnice. — One square inch, dipped Aug. 16, colon, E. W. Eepeated Aug. 19, 21, 22, 23. On Aug. 28 and 29, fed as N'- 22. N'^' 24. Two mice. — One square iach, dipped Aug. 17, jejunum, E. W. Aug. 19, fed with bread on which six drops of fluid from jejimum of E. W. had been placed. Eepeated Aug. 21 and 22. On Aug. 23 there were several loose motions in the cage of reddish-brown colour and slimy consistence ; they contained a good many blood-corpuscles, and the usual materials of faeces, but no epithelium. There were also some bacteria, and numerous bodies like eggs of entozoa, of unexplained significance. N"' 25. Two mice. — One square inch, dipped Aug. 17, ileum, E. W. Aug. 19, fed with bread upon which six drops of fluid from ileum of E. W. had been placed. Eepeated on Aug, 21, 22, and 23. N"' 26. One mouse. — One square inch, dipped Aug. 17, colon, E. W. Aug. 19, fed with bread upon which six drops of fluid from colon of E. W. had been placed. Eepeated same Aug. 21, 22, and 23. The experiments were terminated towards the end of August, and the remaining forty mice forwarded to Dr. J. B. Sanderson, who then made upon them the series of experiments which he himself has recorded. It will be at once observed how small is the number of recovering animals compared to the number which died. Most remarkable however is the circumstance that out of the eleven deaths ten were those of pairs, of which each was living in a cage together, and that these deaths of the individuals of each couple took place almost at the same time. This points to a very definite cause of disease, a simultaneous introduction of its poison, and a great similarity in the manner in which the EXPEEIMENTS ON CHOLEEA POISOS. 249 animals susceptible of it reacted upon it. The cause of the immunity of other animals apparently subject to the same influences remains unexplained. List op 0a©b3 ajstd AifniAis, showins Cases of Siokitess (*) ajtd Death (f), and Animals not apfecibd. 1. Two mice. 13. Two mice. 2. Two mice. 14. Two mice. 3. Two mice. 15. Two mice. 4. Two mice. 16. Two mice. 6. Two mice ; both died, Aug. 23.tt 17. Two mice 3.tt _ both died, Sept. 6. Two mice ; both died, 18. Two mice. Aug. 23. tt 19. Two mice. 7. Two mice. 20. Two mice. 8. Two mice. 21. Two mice (one brown and 9. Two young mice ; both died, Sept. 3.tt white) ; 20.tt both died, Aug. 10. Two mice ; one died,' Aug. 22. Two mice. 16. 23. Two mice. 11. Two mice; one sick (disap- 24. Two mice ; bloody diarrhoea.* peared).* '25. Two mice. 12. Two mice. Total number of animals 26. One mouse 51 Total number of sick , . 13 Total number of deaths 11 Recovered , , , 2 Not affected , , 38 51 The symptoms exhibited by the infected mice were very uniform. They had diarrhoea, bloody diarrhcea, loss of urinary secretion ; they became cold, their temperature sank ; they became stiff, so as to appear dead while yet feebly living ; they died, and in such positions that it was probable that spasm was one of the ultimate symptoms. Their intestines were filled with whitish matter, and their abdomens always distended. In short, there was a resemblance to human cholera which could not escape consideration. Most of the animals were pure albinos, a circumstance to be remembered, as we know, from experiments concerning the excision of the supra-renal capsules, that albinos have a physiological economy differing in some 250 ANNALS OF CHEMICAL MEDICINE. respects, besides their peculiarities as to the chemistry of pigment, from that of other animals not albinos. In this respect it must be noted that one of the mice which died was grey and white, but as it was bred from albino stock by crossing with a rare variety it forms a singular exception to the fact that all the animals employed were albinos. In the following seven tables the experiments are arranged in such a manner that the effects of the cholera poison taken from different parts of the intestinal canal after death become conspicuous. The seventh table alone relates to experiments with excretions from living cholera- patients. , Table I. — Results op Fibst Seeibs op Expekimenis. Contents of JeQV/mim of E. W.; removed Aug. 10, 1866. Paper dipped Aug. 10, or (Mice N" 1. Fed Aug. 10, also Aug. 17, 18, 19, 1st fermentation day \ 21. No effect. Paper dipped Aug. 11, or [Mice N'- 4. Fed Aug. 11, also 17, 18, 19, 21. 2nd fermentation day \ No effect. Paper dipped Aug. 12, or (Mice N'- 7. Fed Aug. 12, also 19, 20, 21, 22, 3rd fermentation day \ 23. No effect. /Mice N'" 10. Fed Aug. 13. A mouse sick on Aug. 15. t Aug. 16. 2nd mouse fed Aug. 19, 20, 21 ; 2nd mouse well, 22, 23. Mice N'' 19. Fed upon 4tli fermentation dip, on Aug. 13, to test the case of Mice N'- TO, V but no effect. Paper dipped Aug. 14, or (Mice N'- 13. Fed Aug. 14, also 19, 20, 21, 22, Stli fermentation day \ 23. No effect. Paper dipped Aug. 15, or] Mice N'- 16. Fed Aug. 16, also 19, 20, 21, 22, 6tli fermentation day \ 23. No effect. Paper dipped Aug. 16, or (Mice N'- 21. Fed Aug. 16, also Aug. 19, 20. 7th fermentation day \ Both died Aug. 20.tt fMice N'- 24. Fed Aug. 17; on Aug. 19, 20, 21, 22, with 6 drops, with paper of fluid 8th fermentation day. 1st mouse, bloody diarrhoea. 10th, 12th, 13th, 14th fer-N mentation days, 6 drops ■ No effect observed, of, each day Consequently poisonous properties appeared first on the 4th fermentation day, reached maximum on the 7th, and declined again on the 8th, The latest effect was on 4th day after feeding ; the earliest may have been on 1st, probably was on 3rd day. Paper dipped Aug. 13, or 4th ferpientation day Paper dipped Aug. 17, or 8th fermentation day Paper dipped Aug, 13, or 4th fermentation day EXPEEIMENTS ON CHOLERA POISON. 251 Table II. — Ebsums op Secoitd Seeies of Experiments. Contents of Ileum of E. W. ; removed Aug. 10, 1866. Paper dipped Aug. 10, or (Mice N'- 2. Fed Aug, 10, also 17, 18, 19, 21. 1st fermentation day \ No effect. Paper dipped Aug. 11, or fMice N' 6. Fed Aug. 11, also 17, 18, 19, 21. 2nd fermentation day \ Both dead, Aug. 23.tt Paper dipped Aug. 12, or (Mice N" 8. Fed Aug. 12, also 19, 21, 22, 23. 3rd fermentation day \ No effect. (•Mice W- 11. Fed Aug. 13. On 15th one mouse was apparently dead and stiff; disap- peared. The other mouse was fed on Aug. 19, 21, 22, 23. No effect. Mice N'- 20. Fed on Aug. 16, upon 4th ferr ^ mentation day's paper, as mice N"' 11. No effect. Paper dipped Aug. 14, or (Mice N" 14. Fed Aug. 14, also 19, 21, 22, 23. 5th fermentation day \ No effect. „ ^ . ,^ fMice N'- 17. Fed Aug. 15, also 19, 21, 22, 23. Paper dipped Aug. 15, or jf^ ^q^^_ ^.g^^ ^^^^^^^ ggrigg yj.^ rice-water, 6th fermentation day ( Aug. 28 and 29.) Paper dipped Aug. 16, or (Mice. N" 22. Fed Aug. 16, also 19, 21, 22, 23. 7th fermentation day \ No effect. Paper dipped Aug. 17, or J Mice N'- 25. Fed Aug. 17, also with 6 drops of 8th fermentation day \ fluid on Aug. 19, 21, 22, 23. No effect. Also 10th, 12th, 13th, 14th | fermentation days, 6 [• No effect, drops each day ) The contents of the ileum reached their maximum infectiousness on the 2nd fermentation day; this declined on the 4th, and did not afterwards appear at all. Table m. — Showing Rebttlts oi? Third Series as Expekimbnis. Contents of Colon of E. W. ; removed Aug. 10, 1866. Paper dipped Aug. 10, or (Mice N'- 3 Fed Aug. 10, also 17, 18, 19, 21. 1st fermentation day | No effect. Paper dipped Aug. 11, or (Mice N" 6. Fed Aug. 11, also 17, 18, 19, 21. 2nd fermentation day \ Aug. 23, both dead.ft (•Mice N'- 9. Fed Aug. 12, also 19, 21, 22, 23. Paper dipped Aug. 12, or ^^ ^q^^_ ^g^^ j^^^^^^^ g^^^^ ^ ^^^ 3rd fermentation day [ 0.^,+ 3 -> Paper dipped Aug. 13, or (Mice N'- 12. Fed Aug. 13, also 19, 21, 22, 23. 4th fermentation day \ No effect. 252 • ANNALS OF CHEMICAL MEDICINE Paper dipped Aug. 14, or (Mice N'- 16. Fed Aug. 14, also 19, 21, 22, 23, 6th fermentation day | No effect. Paper dipped Aug. 15, or (Mice N'- 18. Fed Aug. 16, also 19, 21, 22, 23. eth fermentation day | No effect. Paper dipped Aug. 16, or (Mice N'- 23. Fed Aug. 16, also .9, 21, 22, 23. . 7th fermentation day \ No effect. Paper dipped Aug. 17, or\ 8th fermentation day; ^.^^ ^„ ^^_ j,^^ ^^^ 17, also with 6 drops on Aug. 19, 21, 22, 23. No effect. also 10th, 12th, 13th, and 14th fermentation days The contents of the colon had infectious power only on the 2nd fermenta- tion day, and were inert from the Srd to the 14th fermentation day. Table IV. — ^Rbstjits of Fourth Series of ExpERrMKNTS. Contents of Small Intestine {S,, died Aug. 24). Day of Month Day of fermentation Mice N" 1 Mice N" 2 August 27 ■ ;; i 3rd 4th 6th Fed upon 6 drops. No effect. Fed upon 6 drops. No effect. Day of month Mice N'- 3 Mice N'- 5 Mice N" 7 August 27 „ 28 „ 29 1 Fed upon \ 6 drops. J No effect. Fed upon 6 drops. No effect. Fed upon 6 drops. No effect. Tabie V, — Ebsuits Of Fifth Series of Expekimbnts. Contemts of Colon (-H.). Day of month Day of fermentation Mice H" 8 Mice N" 9 August 27 „ 28 3rd 4th Fed upon 6 drops. No effect. Fed upon 6 drops. Both died, ft Sept. 3. Day of month Mice N'- 10 Mice N'- 11 Mice N>- 12 August 27 „ 28 Fed upon 6 drops. No effect. Fed upon 6 drops. No effect. Fed upon 6 drops. No effect. EXPERIMENTS ON CHOLERA POISON. 253 TABias VI. — Results op Sixth SEEtEs op ExpEEtmiifTS. Rice-water from Small Intestine {8.). Day of month Day of fermentation Mice N'- 13 Mice N'- 14 August 28 „ 29 2nd 3rd Fed upon 6 drops. No effect. Fed upon 6 drops. No effect. Day of month Mice N" 15 Mice N" 16 Mice S'- 17 August 28 „ 29 [ Fed upon 6 drops. \ No effect. Fed upon 6 drops. No efiect. Fed upon 6 drops. Both died,tt Sept. 3. Tlie date of the death of N'* 17 heing the same as that of N'" 9 mice suggests some cause of infection to which hoth were simultaneously subjected previously. Mice N'- 9 had taken contents of colon, mice N'* 17 of ileum of B. W., but ten days previously. Table VII. — Rbsttlis op Setenth Sebjdes of ExPEKiMEtfTS. Rice-water Stool from M. A. T. Day of month Mice N'- 18 Mice N'- 19 August 28 „ 29 2nd 3rd Fed upon 6 drops. No effect. Fed upon 6 drops. No effect. Day of month Mice M'- 20 Mice N" 22 Mice N" 23 August 28 „ 29 Fed upon 6 drops. No efiect. Fed upon 6 drops. No effect. Fed upon 6 drops. No effect. 254 ANNALS OF CHEMICAL MEDICINE. XIX, NOTE AND EXPERIMENTS' ON TRE ALLEGED EXISTENCE IN THE BRAIN OF A BODY TERMED ' PROTAGON' {From the Pathological Institute.) Some authors have lately revived what could have heen called at one time the hypothesis of ' protagon,' and have reasserted the existence of this matter as a definite chemical entity or im- mediate principle in the brain. The result of some new analyses is an empirical formula, CigjHjjgNjPOgj, which differs from the earlier formula CjigHjiiNjPO^j by a quantity of no less that C54H5,NO,3. In these analyses also much less hydrogen in pro- portion to the carbon was found than in the earlier ones, although they refer to virtually the sanie substance. The processes by which the operators on 'protagon' obtained their matter, are essentially the same as those which were employed by Vauquelin, Couerbe, Fremy, Grobley, Bibra, Thomson and others, for the extraction from brain of a matter which had the same percentic composition as 'protagon' with regard to all elements, phos- phorus alone excepted, as is evident from the following com- parison of the analytical data. Fr6my's cerebric acid Gobley's — cerebrin- Bibra's cerebric acid Thomson's eerebric acid Liebreicb's protagon Gamgee and another's product H N P 66-7 10-6 2-3 0-9 19-5 66-85 10-82 2-29 0'43 19-61 66-78 10-65 2-51 0-52 19-62 67-04 10-85 2-24 0-46 19-41 66-74 11-74 2-80 1-23 17-49 66-39 10-96 2-39 1-06 19-47 ' Protagon ' was therefore by no means a needful discovery, and if this substance were really a definite educt, it would be absolutely the same as Fremy's cerebric acid. This fact was EXPERIMENTS ON « PROTAGON.' 255 somewhat obscured by the circumstance that authors on ' pro- tagon ' ignored the existence of previous researches, and compli- cated the otherwise well-known processes of preparation of the white matter from brain by fanciful additions ; and these ampli- fications were so unmeaning, that by their inventors themselves they were in ultimate proceedings entirely abandoned. Now in spite of this unquestionable priority of Freiny, whose name the authors who treat of ' protagon ' never mention, and whose work they have unquestionably either not studied or not heeded, they state that what they believe or pretend to be their discovery concerned the first definitively ascertained specific constituent of the brain ; they boast that their own investigations had been carried out with all the necessary care and even minute- ness, and that they had ascertained not merely the amount of phosphorus, but also that of all the other elements contained in their production. Fremy had shown that the body to which he gave the name of cerebric acid, was often combined with phosphate of lime, or with soda, and had to be freed from these by treatment with a little sulphuric acid in alcoholic solution. It was this circum- stance which induced him to term the body which he had isolated an acid. In consequence of these observations of Fremy's I ex- amined all educts from the brain which I have described in my researches for mineral ingredients, and found that when they are isolated as far as neutral solvents will isolate them, they all contain considerable quantities of all, or some, or one of the following bodies : ammonium, sodium, potassium^, calciuTn, iron, copper, calcic and magnesia phosphates, (See my ' Eesearches on the Chemical Constitution of the Brain,' Reports of the Medical Officer of the Privy Council and Local Govern- ment Board, New Series, N'- III. 1874, p. 129 ; also 'Further Eesearches on the Chemical Constitution of the Brain.' Re- ports, etc.. New Series, N'- VIII. 1876, p. 131.) I have given complete quantitative analyses of these bodies as contained in the phosphorised principles discovered by me, namely, kephalin and myelin. In one experiment in which the bases were ex- tracted by hydrochloric acid, as much as seven grammes of alkaline chlorides were obtained. These inquiries were already several years ago extended to 256 AHNALS OF CHEMICAL MEDICINE. the product termed ' protagon,' and this too was found to contain inorganic constituents irremovable by recrystallisation from alcohol, however frequently repeated. It was found more particularly that ' protagon ' and the bodies into which it can be separated, according to my researches, always retain consider- able quantities of potassium in combination. As the quantity of inorganic ingredients in ' protagon ' had never been estimated, I prepared a specimen of this matter, and on analysis found it to contain the better part of one per cent, of inorganic incom- bustible matter, phosphoric acid not included, and in this no less than 0*76 per cent, of the 'protagon ' of potassium. This same 'protagon ' contained 1"057 per cent, of phosphorus, and ascribing to it (hypothetically) the mean amount of carbon, hydrogen and nitrogen found by the latest operators, it would be necessary to consider and rename ' protagon ' as the potas- sium salt (mainly) of an acid (in justice to Fremy to be termed cerebric acid), and to give it the forrnula no longer empirical, but compulsory, if ' protagon ' were a definite unitary educt of C284Hj4gNgP2KOgo. If this hypothesis should fail to account for the data, the following conclusion becomes unavoidable, namely, that it is not the fact that any easy process such as that described for the isolation of ' protagon ' yields a product in a state of purity, but that the ' protagon ' thus far obtained, apart from the question whether or not it is a mixture of organic compounds, is yet very impure. The special test experiment {Exp. L) alluded to in the fore- going, gave results of permanent interest in the consideration of the general question, which deserve to be here recorded. Twelve ox-brains were extracted with spirit of 85 per cent, strength at 45°. All the extracts were cooled to 0° and allowed to stand over night. The deposited white matter was collected and exhausted with ether. It was now subjected to the process termed recrystallisa- tion, and thereby split up into the following eight different products. A. Extraction by five litres of spirit at 45° gave a solution, which on standing during 16 hours at 17°, without being cooled to 0°, deposited a white matter, the principal bulk of the ' pro- tagon,' weighing, dry, 30*9 grms. From this large volumes of EXPERIMENTS ON ' PEOTAGON,' 257 ether extracted nearly one grm. of matter (product N''' I. IV containing 0*52 per cent, of phosphorus. The remaining 30 grms. of protagon once recrystallised (product N I. 2) con- tained 1'057 per cent, of phosphorus and 0-76 per cent, of potassium. The spirituous mother liquor, which had deposited the fore^ going white matter yielding products N I. 1, and I. 2, on standing during five days at an average temperature of 17% deposited a voliuninous mass of matter which when dry, weighed 3 grms. and contained 1*13 per cent. P, and 0'22 per cent. K (product N'- 1. 3). The filtrate from this was now placed in water, kept cold by ice during 18 hours, and deposited a matter (product N'- 1. 4) which when dry weighed 5 grms. and contained 2'02 per cent. P and 0*39 per cent. K. The spirituous filtrate from this fourth product gave precipitates with cadmic chloride, platinic chloride, and lead acetate, and after distillation of the spirit and drying left 10 grms. of a yellowish, highly hygroscopic residue containing 2-91 per cent, of P and traces of K (product N'. I. 5). B. The white matter from which five litres of spirit had extracted the mixture of the five several products described in the foregoing was extracted twice more with on the whole about four litres of spirit at 45°. The solutions deposited on cooling and standing 8 grms. dry, of a white matter containing 0*76 per cent. P and 1'44 per cent. K (product N'- 1. 6). C. A considerable quantity of white matter, which had been dissolved in the first spirit at 45°, was left undissolved by spirit at 45° even on very long digestion. It weighed, dry, 13 grms., and contained large rhombic crystals, resembling cholesterin, but insoluble in either ether or alcohol, and therefore not being cholesterin. Ether extracted from these 13 grms. of residue 0*4508 grm. of a matter which contained 0-41 per cent. P (product N'- 1. 7). The remainder of the matter insoluble in alcohol at 45° and in ether, weighing more than 10 grms., is product N^I. 8. The following list shows the quantities of the eight products obtained, and their percentages of phosphorus and potassium. 258 AKNALS OF CHEMICAIi MKDICINE. Numbers of Products Quantity Phosphoms Potassium Grammes Per ceut. Per cent. N'-I. 1 1-0 0-52 p « 2 SO-0 1-057 0-76 „ 3 3-0 1-13 0-22 „ 4 6-0 2-02 0-39 ,, 5 10-0 2-91 traces „ 6 8-0 0-76 1-44 „ 7 0-4608 0-41 „ 8 120 — recrystallised 1 10-0 0-108 traces Here it mnst be pointed out that all writers on ' protagon ' have left these products all but N''- 2 entirely out of consideration, just as they have ignored a large quantity of white matter which is not extracted at all from brain-pulp by spirit at 45°, but requires boiling spirit for solution. The so-called ' protagon ' forms only thirty parts out of nearly seventy parts of what remained of the white matter extracted by spirit at 45° from twelve ox-brains after exhaustion with large volumes of ether to such a degree that two litres of ether digested with the matter during six hours left only one decigramme of residue. Attention may be directed to the fact that in the different products phosphorus and potassium do apparently observe an opposite ratio, potassium sinking when phosphorus rises, and phosphorus sinking when potassium rises in quantity. The further investigation of these relations will no doubt lead to important and interesting results, but they are foreign to our present purpose. D. Separation of ^protagon ' vnto bodies differing in com- position by absolute alcohol at 45°. — The product N"'- I. 2 above described, corresponding to 'protagon once recrystallised' and containing 0-76 per cent of potassium was now treated with l^ litres absolute alcohol during 20. hours at 45°. There remained 7-6 grms. undissolved, and this (product N'- I. 9) contained 0-94 per cent. P and 1-67 per cent. K. The deposit from the solution on cooling during 3 hours at 20°, corresponding to ' protagon twice recrystallised ' weighed 8 grms. (product N""- I. 10) and contained 0-83 per cent. P with 1-41 per cent K. .EXPERIMENTS ON 'PROTAGON.' 259 The solution filtered from this on standing during three days at a mean temperature of 21° made a further deposit which weighed 4"3 grms. (product N"^' I. 11) and contained 0-26 per cent. P and 0"22 per cent. K. The mother liquor on distillation yielded 5"9 grms of matter (product N'- 1. 12) containing 1*77 per cent. P and 0*14 per cent. K. Synopsis of theprod/ucts obtavned from ^protagon' by resolu- tion in absohite alcohol at 45° etc. Numbers of products Quantity Phosphorus Potassium N'- 1. 9 „ 10 " \l Grammes 76 8-0 4-3 6'9 Per cent. 094 0-83 0-26 1-77 Per cent. 1-67 1-41 0-22 0-14 The matter insoluble in alcohol at 45, weighing more than 12 grms. (product W- 1. 8) was found to dissolve entirely in 300 c.c, of boiling spirit, leaving albumin and paper fibre, together 1 grm. undissolved. On cooling a deposit rapidly separated which weighed 10 grms. and contained 0-108 per cent. P and mere traces of K. AH these twelve deposits contained the usual phosphorised and cerebrin bodies, namely myelin, phrenosin, and kerasin ; the mother liquor deposited kerasin like all other mother liquors of the cerebrin bodies (including the boiled ones, as we shall presently show), and gave copious myelin precipitates with cadmic and platinic chloride. In another experiment (Exp. II,), six ox-brains were ex- tracted with large volumes of spirit of 85 per cent, strength at 45°. The solution was allowed to stand at 16° but was not cooled to 0°. The white matter was isolated and exhausted with ether. It weighed dry 25 grms. and contained 0*87 per cent. P. The whole of this was now boiled in spirit of 85 per cent., in which it was completely soluble. It was again de- posited on cooling, and the deposit now contained 0*84 per cent, of P. The P had therefore been diminished by one recrystal- lisation. This matter was now differentiated into four fractions by treatment with spirit of 85 per cent, at 45°. A, Two litres of spirit digested with the 'protagon' gave on cooling a deposit (product W' II. 1), which weighed dry s 2 260 ANNALS OF CHEMICAL MEDICINE. 11 grms. and contained 0*91 per cent, of P and 0*88 per cent, of K. The mother liquor left 2*3 grms. of yellowish white matter (product N""- II. 2), which contained 1*83 per cent, of P and 0*87 per cent, of K. B. One litre of spirit was digested with the matter which spirit in A had left undissolved for 18 hours at 45°, and gave on cooling a deposit, weighing 3*9 grms. (product N"^- II. 3) containing 0*74 per cent. P and 1*04 per cent. K. C. The matter left undissolved by spirit in B was again digested at 45° with 1 litre of spirit for 6 hours. On cooling the solution gave a deposit, weighing 1*6 grm. (product N'- II. 4) containing 0'46 per cent. P and 1'23 per cent. K. D. There were left undissolved by these three extractions with spirit at 45°, 4-4 grms. of matter (product N'^ II. 5), which contained 0*12 per cent. P and 0*54 per cent. K. The mother liquors of B and G contained very little matter. All the fractions summed up, including parts used for analysis, amount to 23 grms. out of 25 grms. originally operated on, so that the amount left in the mother liquors and lost in opera- tions could not amount to more than 2 grms. Synopsis of the fractions ohtavned in Exp. II. by extracting ' protagon ' with spirit at 45°. Numbers of fracUons Quantity Phosphoms Potassium N'-n. 1 " 3 :: 4 „ 5 G-rammes 11-0 2-3 3-9 1-6 4-4 Per cent. 0-91 1-83 0-74 0-46 0-12 Per cent. 0-88 0-87 1-04 123 0-54 Here we observe again, as in Exp. I., the peculiarly shifting proportions between phosphorus and potassium. Not a single fraction is free from potassium, and no two fractions contain the same amount of either phosphorus or potassium. All the fractions are mixtures of the same kind, as the fractions obtained in Exp. I. at the same stages ; in the corresponding fractions of both experiments P and K observe the same relations, and are present in almost the same quantity ; each element at all events observes the same curve in both experiments. The boiling in EXPERIMENTS ON ' PKOTAGON.' 261 spirit had therefore not effected any greater difference than the fractional solution at 45° and precipitations in Exp. I. The appearance of the fractions was the same as that of the corre- sponding fractions from Exp. L, and on further testing and differentiation they yielded the well-known constituents just as the products from Exp. I. In a third experiment {Exp. ITL), a quantity of white matter from the human subject was exhausted with ether, and the product, about 60 grms., digested with 4 litres of spirit at 45° for 18 hours. There was left undissolved 4*5 grms. of matter (product N'- III. 1), which after solution in boiling spirit and filtration from some albumin contained 0-46 per cent. P and 0'26 per cent. K. The solution was cooled rapidly to 21°, and allowed to deposit during four hours ; the deposit collected and dried weighed 32 grms. (product N"'- III. 2), and contained 0-87 per cent. P and 0" 41 per cent. K. The filtrate from this principal precipitate was now put into ice-cold water for 18 hours, and gave a deposit which after isolation was white and waxy, weighed 8*6 grms. (product W- III. Ji), and contained 1*69 per cent. P and 0'17 per cent. K. The mother-liquor on distillation left nearly 1 2 grms. of a yellowish waxy hygroscopic body (product N''- IIL 4), containing 2-85 per cent. P and 0-09 per cent. K. Synopsis of the fractions obtained by extracting human ' protagon ' with spirit at 45°. Numbers of fraotions Quantity Phosphorus Potassram N'lII. 1 : I „ 4 Grammes 4-5 320 8-6 12-0 Percent. 0-46 0-87 1-69 2-85 Per cent. 0-26 0-41 0-17 0-09 The state of aggregation of the particles of ' protagon ' has been described as crystallised, mostly in microscopic rosettes, but it is admitted that they differ somewhat according to the degree of concentration of the solution from which they are deposited. I inspected microscopically the forms obtained by the recrystallisation of the product N''- I. 2, which contained 1*05 per cent, of phosphorus, and 0'76 per cent, of potassium. 262 A25 497 Hcdf-acid CholopJwemate or Sesquir-cholophcevnate of Bcu- ryum. — This salt, prepared by precipitation from a neutral ammoniacal solution by BaCl^, washing with water, treating and boiling with alcohol until the alcohol on filtration and washing passed clear and colourless, was dried in the steam closet. It remained of a brownish-red colour, but after being powdered it became of a darker brown colour on the surface during drying. Dried at 100° until constant, and then at 110° C, it yielded on analysis data which showed that in this BILIAfiy PIGMENTS. 273 cboldpliaemate one atom of baryum is in combination with tltte© molecles of cholophsein. If to the dihydrated neutral cbolo- phseinate of baryum described above we add one molecle' of cholophsein, thus — 1 cholophseinate of baryum C,gH2oBaN205=497 At. w. 1 cholophsBin . , G^IS, N 02=163 „ We obtain . . 'c^^H^^Bs^^fi^^660~^, To the theory of this' compound the above analyses exactly correspond, as is evident from the following comparison : — Symbols Ba Atomic weights Percentages Found mean o„ 3 324 49-09 50-63 29 4-39 4-37 137 20-75 20-66 42 128 660 By the following comparison the differences in the composi- tion of the neutral and the half-acid cholophseinate of baryum will be exhibited in a striking manner. NEUTK AL CHOLOPH^INATE OF HALF-ACID CHOLOPHiEINATE OF BARH UM. BAKTUM. C.,H,„BaN,0, At. W.=497. C,,H,,BaN30, At. W. = 660. c . Theory Found 43-46 44-58 C . Theory Found 49-09 50-63 H . 4-02 3-98 H . 4-39 4-37 Ba . 27-56 27-55 Ba. 20-75 20-66 Neutral Cholophcevndte of Ccdcium.-^The excess of cholo- phsein, which had served to make the neutral-ammoniacal solution used for the production of cholophseinate of silver, was dissolved in a slight excess of ammonia and precipitated by chloride of calcium. The precipitate was red. Dried at 100° it yielded on analysis data which led to the formula of a neutral cholophseinate of calcium CigHj^CaNgOg, a salt in every respect 274 ANNALS OF CHEMICAL MEDICINE. analogous to the neutral baryum compound above des6ribed.i We must assume in it the existence of. two molecles of water, which are not expelled even at the temperature of 100°. The following comparison of theory with the analytical data will make the correctness of these conclusions more conspicuous : — Symbols Atomic weights Percentages -Pound meap - 216 ■ 54-bo 53-86 20 • 5-- 4-9d 40 10- 10-17 ^20 H, Ca 0. 28 96 400 Half-acid Cholophminate or Sesqui-cholophadnaie of Calcium.~-1 had first of all cholophseinateS prep&,red the baryum salt, which was found to be a sesquisalt. The" neutral calcium salt was next obtained, and indicated the dxisteUce of two classes of salts, neutral and half-acid. The correctness of this con- clusion was put to the test by the endeavour to obtain the neutral baryum salt, and the experiment proved successful. It was next necessary to obtain the half-acid, cholophseinate of calcium. A neutral solution of bilirubin was therefore precipi- tated by chloride of calcium washed and. dried. The conipound was found to be analogous to the half-acid baryum compound already described, and has the formula CjjHjgCaNaOg, and the following theory : — Theory of ato^s of percentages ^27 .• . 324 57-54 Hag.- _, 29 5-15 Ca . . 40 7-1 Na'.. . 42 0„ . , 128 563 The following comparison exhibits the differences in the composition of the neutral and the half-acid cholophseinate of calcium. BILIARY PIGMENTS. 275 mvT RAt CHOLOPHjEINATE OF HALF- ACID CHOLOPK^ISATE OF CALCIUM. CALGItTM. C,AoCaN,0, At.W. = 400. . C,,H,,CaN30, At. W. = 563. C Theory . . 54- Found 53-86 C . Theory Found 57-54 — H 5- 4-9 H . 5-15 5-74 Ca^ . '. 10- 10-17 Ca . 7-1 6-91 In his researches on the colouring matter of human gall- Stones, Stadeler produced a combination of bilirubin- with calcium, which on analysis yieldied him 9-1 per cent, of calciuni oxy(]e. Assuming this compound to be the normal neutral salt< he allowed the atomic weight of bilirubin to be thereby deter- mined, and rejecting his former analyses of crystallised cholo- phsein as given in Frerichs' Treatise on Diseases of ttie Liver, he abandoned the empirical formula C,8H5N04, and substituted C32HjjN20g as the formula of bilirubin,' and CjjHjjCaNgOg as that of bilirubate of calcium. (The foregoing three formulae are given in the old notation,) As this formula is supported' by only one, and that a very unsatisfactory calcium determina- tion, and as all the analyses of Stadeler concerning bilirubin or cholophajin can be brought into harmony with my results, I can have no hesitation in considering his formulae, both of bilirubin and bilirubate of calcium, as erroneous. ' The bilirubate analysed by Stadeler was evidently the sesquisalf. Theory of C^H^^CaN.OTe re- quires CaO or Ca 9-94 per cent. 7-1 per cent. Stadeler found CaO or Ca 9-1 per cent. 6'5 per cent. With Stadeler's formula. of bilirubin fall .the formulae of all other substances described by him under the names of bili- verdin, .biliprasin, bilifuscin, and bilihumin. It remains to point out in this place the circumstances under which cholophaein will forni half-acid, neutral, or basic salts. A watery solution of ammonia -which is saturated witl^ T 2 276 ANNAXS OF CHEMICAL MEDICINE. cholophsein by digestion with an insoluble excess yields on the addition of neutral salts of metals the sesquisalt. Possibly the ammonia salt is itself a sesquisalt while dissolved in water. From somewhat alkaline solutions the basic salts of silver and lead are precipitated by the opportune addition of an excess of metallic salt. But neutral salts of earths added to the solutions containing excess of a,mmonia precipitate the neutral compounds with earths. CholophcBinate of ZItw. — A neutral ammoniacal solution of cholophaein was precipitated with sulphate of zinc. The reddish brown precipitate was easily washed, and quite insoluble in Wftter.; Dried at 100° C. it was subjected to analysis. From ,the amount of zinc found it is evident that this compound is constructed upon the plan of the half-acid salts of calcium and baryum. Thus — 1 neutral cholophaeinate of zinc CigS^gZnNfi^ 2 water .... H^ 0^ 1 cholophsein . . . Cg Hg NO^ 1 molecle half-acid eholo- phseinate of zinc . C^^K^^ZqN fi^ Theory of C^HagZnNjOg Percentages 11-05 588 Neutral Cholophodnate of Lead. — Precipitated from a neutral solution by acetate of lead. Dried at 100°. Figures were obtained which keep in proximity of those required by a neutral dihydrated cholophseinate CigHjgPbNjOg. At. 'W'.= 567, Pb= 36-50 per cent. The half-acid cholophseinate of lead G^^ia.^^T})Nfi^=7Z0 requires 28-21 per cent, of Pb. Sums of Atoms Atomic weights c., . 324 H.,. 29 Zn . 65 N3 . 42 Os . 128 BILIARY PIGMENTS. 277 Basic Cholophcdnate of Lead. — The compound was pre- cipitated by acetate of lead in great excess from a neutral ammoniacal solution of cholophsein, wliich although it did not dissolve any more cholophsein, smelled of ammonia, and had an alkaline reaction. Dried at 100° C. This compound may be explained as being a basic cholo- phseinate, or cholophsein, in which two atoms of hydrogen are replaced by one didynamic atom of lead. Atoms At weights Percentages Found mean c, . . 108 — — H, . . 7 — — Pb . 207 56-25 58-1 N . 14 0, . 32 368 This compound corresponds to the basic-cholophseinate of silver, or binargentic cholophsein, described above, CgH,Ag2N02. Neutral Cfholophceinate of Copper. — This salt was precipi* tated by sulphate of copper from a neutral solution. Basic Gholophceinate of Copper. — This salt was obtained by precipitating an ammoniacal solution with excess of acetate of copper. The only probable formula for this compound is that of a cholophseinate containing 2 cholophsein and 3 copper. Atoms Atomic weights Percentages C.s ■ 216 - — H., 12 — CU3 190-5 37-31 N, 28 0. , 64 510-5 Found 38-70 Action of oxygen upon bilirubin in alkaline solution,^ CholocMorin or biliverdin. — When bilirubin is dissolved in caustic potash and exposed to the air, it becomes gradually green. This change can be accelerated by warming the solution and 278 ANNALS OF CHEMICAL KEDICINE. passing a current of air through it. When in thin layers it is purely green, without any admixture of red or yellow," the reaction is complete. On addition of hydrochloric acid a green jnatter is precipitated in flakes, which is first washed by deean- tation, and lastly edulcorated on the filter. This is biliyerdin. ; Chemical Pr&p&rties'. — Biliverdin is easily soluble in alcohol with a splendid green colour, particularly while in the wet state; after drying it is soluble with great difficulty only ; ii js more soluble in hot than in cold alcohol. It is soluble in hydro- chloric acid, forming a green solution, in which platinum tetrachloride and mercury dichloride produce amorphous green precipitates; A quantity of alcoholic solution boiled down with tincture <)f iodine, and the residue shaken with -dilute caustic potash, left a greenish-black resin, which- was "not soluble in sulphuric acid except under evolution of sulphurous acid, indicating decomposition. ' By reducing agents the biliverdin cannot be retransformed into cholophsein. Dissolved in caustic potash and treated with hydrothion it assumes a greenish-browri colour, but the original red is not produced. When metallic 2inc is added to a solution of biliverdin in hydrochloric acid, the green colour disappears and gives way to a brownish-red onej but the" produce is insoluble in chloroform. An alkaline solution treated with sodium-amailga.m changes its colour to a reddishr brown, and a small quantity of a brown matter is deposited in flakes. But the reddish-brown matter on exposure to air does not become ^reen again. Hydrochloric acid precipitates a brown flaky matter from the alkaline solution. When chlorine gas is passed into water containing biliverdin in suspension, dark yellow flakes are obtained, which while in- soluble in water and ether dissolve easilydn alcohol. When a few bubbles of chlorine gas are conducted into an alcoholic solu- tion of biliverdin the dark-green fluid is instantaneously dis- coloured. After evaporation of the alcohol, yellowish white flakes remain, which by a gentle heat fuse to a reddish-yellow mass. They contain chlorine, and are a mixture of at least three com- pounds, which can be separated by chloroform. Biliverdin when repeatedly purified and subjected to varied analyses gave data which led to the formula C„H„NO„. BILIARY PIGMENTS. '279 Theory , ■■'• of atoms in 100 Mean found 8C • • 96 63-57 ■ 62-94 9H • ■ 9 5-96 6-13 N' , , 14 .9-27 9-34 2,0 ■ • 32 21-20 , 22-10 151 100-00 100-00 This shows that biliverdin arises from bilirubin by the addition of 2 oxygen, and the subsequent subtraction of 1 carbonic acid. Bilirubin. Oxygen. Biliverdin. Carbonic Acid. C,HgNO, + 20 = C^HgNO, +• CO,. On boiling an alcoholic solutioii of. biliverdin witjh caustic ammonia and nitrate of silver, a reduction of the dissolved silver oxyde is observed. The solution remains green, but on addition of an acid, such as nitric, hydrochloric, or sulphuric, assumes a splendid purple colour. The new body produced is a product of oxydation and termed bilipurpin. The alcoholic solution of biliverdin is not altered by boiling with red oxyde of mercury. Lead peroxyde immediately changes its colour to brown, and precipitates a portion of the dissolved matter. The same change of colour without any accompany- ing precipitation is produced by peroxyde in the presence of ammonia. A mixture of peroxyde of lead and sulphuric acid changes the colour of the alcoholic solution to a light yellow, and evolves aldehyde and acetic ether jfrom the alcohol. Biliverdin dissolves easily in caustic alkalies, and is not pre- cipitated from their solution by a,iiy metallic salt. A saturated solution in alcobol gives a precipitate with baryta water. When this reagent is added until a sample filtrate is only pale green, and boiled, all biliverdin is in combination. It must be filtered quickly, air being excluded, and washed with strong alcohol. It cannot be washed with water, as the purer it becomes the more soluble it is, and if washing with water were indefinitely continued it would be dissolved entirely. The analyses of this salt led to the conclusion that it contains three molecles of biliverdin and one didynamic atom of baryum, and has the 280 ANNALg OF CHEMICAI/ MEDICINE. formula Cg^Hj^BaNgO,, or C,,H, BaNjOg + H^O. This is half- acid bi^^iverdate with ofle mdlecle of water, according to the following theory : — 1 inol; neutral biliverdate GigH.igBsil!i-fi^ = 4:37 1 mol. Mliverdine . • Cg Hg- N03 = 151 1 mol. water . • . H. ==: 18 mol. of Imlf-acid biliverdate, G^fi^i BaN3O,= 606 Atoms At. weights Percentages Mean found 24 C . . 288 47-52 48-79 27 H . . 27 .4-45 4-38 B^ . , 137 22-60 22-41 . 3 N . . 42 7 . . 112 606 If the one molecle of water be omitted from the calculation, the theory of carbon corresponds better With the experience, but that of the baryum less well. Atoms At. weights Theory of percentages Mean found Cm^ - . 28S ■48-97 48-79 H,a - . 25 4.25 4-38 Ba . • 137 23-29 22-41 K ■ . 42 7-14 o„ . . 96 588 Lime water yjelds a precipitate in an alcoholic solution of biUverdin, which does not, however, appear to be a compound in atomic proportions but a mixture of such a one with excess of free biliverdin. Lead acetate produces a precipitate in the alcoholic solution of biliverdin, which is more soluble in hot than cold alcohol. Basic lead acetate produces a voluminous precipitate, which is so complete that the fluid has the merest green tinge. Acetate of mercury also produces a precipitate liiore soluble in hot than cold alcohol. Acetate of copper produces a . brownish green precipitate, and the filtrate is nearly colourless. The precipi- BILIAEY PI&MENTS. 281 tates of lead, mercury, and copper can be washed ■with. ■Water ■without dissolving. Freshly precipitated silver-oxyde does riot combine ■with biliyerdin ■without change, but produces oxydation. The derivate is yello^w, and soluble in ■water and alcohol. In an experiment instituted to measure the quantity of oxygen necessary for the transformation of a given quantity of bilirubin into biliverdin, by means of the deoxydation of an alkaline copper solution, several copper compounds were pro- duced, of which two contained the copper in such a form that it could not be removed by carbonated soda, but followed the product into the alcoholic solution. One of these products had a peculiar spectrum, and exhibited an absorption band in blue when viewed by the aid of Drummond's light. Action of Bromine upon Biliverdin. — In the following experiments a quantity of biliverdin was used, which had been prepared from bilirubin by Heintz's process, and on analysis had yielded the formula CjHgNOj, 1. Monohrominated biliverdin. — A quantity of finely pow- dered biliverdin, which when quite dry at 100° weighed 0'8064 grm., was treated in a Liebig's drying apparatus with dry bromine vapour mixed ■with dry air. It absorbed the bromine and became perfectly black. When the action of bromine had been allowed to complete itself during many hours, at the ordinary temperature, the excess of bromine was displaced by dry air. The product now weighed 2*3684 grms., or almost threefold the weight of the original biliverdin. The apparatus was now heated to 100° and dry air passed over the product for many hours. A little bromine and much hydrobromic acid escaped, and after the passing of more than two hundred litres of air (measured by the displacement of the water in the aspirator) the apparatus became of constant weight, and the substance lost mere vestiges of HBr. It now weighed I -22236 grm. The equation CjHgNO, + Br, = HBr + CjHsBrNO,, requires that 151 parts biliverdin should become 230 parts of brominated substitution-product, and therefore the 0"8064 biliverdin should have increased to 1'2282, equal to an addition of 0'4218 grm. of bromine. This hypothesis is therefore very nearly satisfied by the experiment. 282 ANNAIiS OF CHEMICAL MEDICINE. The new product is a perfectly black powder, insoluble iit ether, very little soluble in alcohol ; soluble in oil of vitriol, precipitated from this by dilution with water ; the solution has a feeble purplish tint ; soluble in caustic soda, precipitated froni this in brown flakes by acetic acid. From the aspect of the reaction it is probable that the substance cannot be dissolved in either sulphuric acid or soda without change. Analysis. — a. 0*0282 grm. burned with copper oxyde m V€bcuo yielded a mixture of gas which after the necessary corrections amounted to 23-78 c.c. Of this 22-4 c.c. were CO , and 1-38 were N. This is equal to 42-58 per cent. C, and 6-12 per cent. N. The relation of C : N is therefore = 8-1 : 1-0. 6. 0-1920 grm. ignited in glass tube with soda and nitre, (S;c., gave 0-1613 AgBr, equal to 35-72 per cent. Br. c. A combustion - with lead chromate in a very long tube yielded quantities which showed that the quotient of carbon by its atomic weight was to that of hydrogen as'3-22 to 3-179, or, in atoms, very nearly 8 ; 8. These analyses, 'therefore, confirm the presumption derivable from the synthetical genesis of the product, nainely that it is CgHgBrNOj. Theory requires 41-73 ^er cent. C; 3-47 per cent. H; .34-73 per cent. Br; 6-08 per cent. N; leaving for 13-99 per cent. , This reaction, therefore, confirms the formula which in consequence of my researches, first communicated in the Tenth Report of the Medical. Officer of the Privy Council, 1867, p. 240 to 251 ; also Jour. pr. Chem., 104 (1868), 4 ei seq., and Proc. Roy. Soc. xvi. 217, I have attributed to biliverdin, . namely CgH-jNOj. Incidentally the reaction by which bilirubin is trans- formed into biliverdin is also confirmed, and some additional light is thrown on the process ; ■ C,H„NO, + 0,= C,H,NO, + CO,. Bilirubin (as I have shown in Chem. Soc. J., May 1875, p. 389, and more fully in a chapter below) when treated with dry bromine vapour, yields up two atoms of hydrogen, and assumes two atoms of Br in their place : CgHgNO, + Br, = 2 HBr + CgH,Br,NO,. It is therefore clear that bilirubin, when passing into biliver-f BILIARY PIGMENTS. 283 Sin, not only loses an atom of carbon, but also undergoes a change regarding the manner in -which one of its atoms of iydrogen is bound, so that this hydrogen-atom, though capable of being replaced in bilirubin, is no longer replaceable by Br in biliverdin. • ; 2. Hyd/robiliverdin. — A quantity of biliverdin was dissolved in caustic soda and water, and some sodium-amalgam added. The mixture was repeatedly agitated. On the third day the solution, at first greenish, was brownish-red. Hydrochloric acid now gave a brown deposit which was collected on a filter and edulcorated with water. The filtrates were coloured reddish, and seemed to- contain a- side product^ soluble ini dilute HCL The precipitate was treated with alcohol, and dissolved to a great extent, but a portion- remained insoluble in even boiling alcohol, and ultimately formed a black powder. The alcoholic solution contained, however, the bulk of the new product. , Spectral Phenomena of the Alcoholic Solution of Hyd/ro- biliverdin.— The concentrated dark-brown solution, with Drum- mond's light, transmits red only. On greater dilution, all red, yellow, and some green rays pass ; green is shaded. Again more diluted, a separate feeble absorption-band appears at the junc- tion of green'and blue, overlying the line F eqiiilaterally. This spectrum is, therefore, not identical with that of hydrobilirubin (see diagraiQS of both spectra below,- p. 295.), in which the absorption-band fills the space between the lines E and F, overlapping them both, and is of unsymmetrical intensity, its greatest intensity being about one-third nearer to E than F. This experiment thus leads to a presumpticpi that hydrobiliverdin and hydrobilirubin, though similar in external appearance and some properties, are not, as has been alleged, identical. The alcoholic solution of hydrobiliverdin is precipitated with water, Ammoniacal solution of zinc chloride added to this dis- solves all, and the solution fluoresces feebly greenish-bro-wn in sunlight only ; -the fluorescence appears to be homochromatic. ■ Product of Bilirubin dmd fuming Sulphuric Acid. — This substance when examined presented the foliowihg' characters: (a) Slightly soluble in water. (6) Insoluble in ether and benzol, (c) Slightly soluble in alcohol, giving a blue-green solution and: ^ specific spectrum, (d) Soluble in ammonia to a. 284 ANNAIS OF CHEMICAL MEDICINE. dark green almost black solution, remarkable foi' its powerful absorption of light, which when diluted, presented the following spectrum: 1 c.c. thick, not transparent; further diluted, red and blue appear, but very dark indeed; again diluted, transparent green with feeble band. AaBC BiUnMn elwmged ly fvmmg BiCbphwrio Acid, dissolved in Ammonia. Alcoholic solution : All colours were very much obscured. Red nearly out. (a) 0-396 gave 0-7800 of carbonic anhydride equal to 53-718 per cent, of carbon, and 0-186 of water equal to 5-218 per cent, of hydrogen. (h) 0-439 gave 30-5 c.c. gas at 20° C, and 767 m.m. Potash column equal to 16-7 m.m. mercury ; equal to 27-4 c.c. Nitrogen normal, or to 7-80 per cent, of K Per cent. -^At. wts. ^N = l Carbon . . 53-718 4-4765 8-03 Hydrogen . 6-218 5-2180 y-3 Nitrogen . 7-800 -5571 1-0 Oxygen . , 33-264 2-0165 3-6 100-000 = C,H,N03.,orC,sHj,N,0, Sul/phate of Sulpho-cholocyanin. — Cholophsein was treated with fifteen times its weight of sulphuric acid hydrate. After standing six hours it had assumed a reddish-green black colour. Diluted with sulphuric acid it was almost impenetrable to light. More diluted red, then a dark band, then some light, but no green, blue or violet was seen. More diluted there appeared red, then band green and blue. This band in the concentrated acid solution is broader in both directions than the band of sulphate of cholocyanin in dilute watery solution prepared by fuming sulphuric acid. The line near D was difficult to read, and therefore somewhat doubtful. A second reading of adilute BILIAEY PIGMENTS. 285 solution' and athird observation of the spectrum of the concen- trated solution between two glass plates, so that a very thin layer Aa BO Diagram of Speetrum of Sulphate of Sulplw-ClwXoeyamn.. only had to be traversed by the light, yielded data from which the diagram was constructed. GholotKallin. — The concentrated solution just described on dilution with much water deposits a green precipitate, of which a portion is insoluble, another soluble in alcohol. The insoluble portion, cholothallin, has the composition CgHjjNOg, and is therefore hydrated cholophaein, an isomer of tyrosin. The soluble portion has in solution a red colour, and shows two re- markable bands of absorption, resembling ' somewhat the bands of blood, but being situated in different parts of the spectrum, i.e. more towards the red end than those of blood. Diagram oj of Soluble CAoloihalUn. The green ends in faintest bluish-black, and the spectrum altogether at b. Action of bromine and hydrobromic add on bilirvhin. — 1-0115 grm. of dry bilirubin were, suspended in 1 litre of dry chloroform and .to it was added a mixture of bromine and hydro- bromic acid, prepared by treating potassie bromide with sulphuric acid, and made absolutely dry by being passed over pumice and sulphuric acid, until the hydrobromic acid was unabsorbed. The chloroform was distilled off in the water-bath.; 286 ANNALg OF CHEMICAL MEDICINE, at first hydrobrbmic acid and bromine alone, and then bromine iand chloroform were evolved; during^ the distillation the appa- ratus, which was perfectly closed,-becanie a vacuum, all caout- chouc tubes collapsing. When the chloroform: was low and the colour perfectly green, the distillation was stopped and water added ; immediately all colouring matiters were deposited in, nearly black, greenish, violet, blue flakes, which on standing became hard. • i i These flakes while still moistwere treated with ether as long as anything was extracted, giving a perfectly violet solution, the ether was now" distilled off, and a green matter remained, show-, ing that the violet body had been changed. The flakes were nbt wholly soluble in ether, but left a green body which was easily soluble in alcobol and on. evaporation left, a green, very hygroscopic body. A small quantity of matter was insoluble in ether or alcohol and' had a dark green colour., .Therefore the main product was the violet dibromobilirubin, which in the presence of water hydrobromic acid and ethier changed into a green body. The following remarks are to bfe madfe about this prepara- tion: — (1) Pumice and sulphuric acid were used, and as the pumice was afterwards found to contain hydrochloric acid, it must have contaminated the experiment, although but slightly. (2) Much sulphurous anhydride must have been evolved by the action of sulphuric acid on hydrobromic acid. Therefore there must have been three main actions. , ■ (a) That by hydrobromic acid ; (h) that by bromine ; and (c) that by sulphurous anhydride, besides a slight action of the hydrochloric acid. ' Each action will have to be accounted for separately in future experiments. The residues from the extracts when weighed gave: ether extract, 0-8300; alcohol extract, b'6530; sediment, 0-4995," giving a total of 2-083. The bromine was then determined in the residue from' the ether ' extract ; 0-2445 gave 0-0968 of argentic bromide, equal to 16-85 per cent, of bromine, and from a second portion of 0-3070, 0-1213 argentic bromide was obtained, equal to 16-808 per cent, of bromine. UILIAEY PIGMENTS. ' ' 287 'Bilirubin and Tiydrohromic acid.^ — 1'016 grm. of dry bilirubin was suspended in a litre of pure dry chloroform, and pure hydrobromic acid added, and the mixtlire allowed to stand for 48 hours, when a green solution resulted. The chloroform was now distilled off; at first hydrobromic acid was evolved in large quantities, afterwards . chloroform saturated with hydro- bromic acid. The residue was made perfectly dry by passing ,a_ curreiit of dry air at 100° C. oveY it. The product .thus obtained weighed 1'3916 grm., and was green by transmitted light in thin layers, but in thick layers reflected a light that was violet and golden. ' Anah/ses.^Thie substance was first dried in a Liebig's apparatus, and then at 100° C. in the air bath, (a) 0-14,38 grm. gave argentic brdmide 0'0666, equal to 19'68 per cent, of bromine. Theory required 20*56 per cent.; (b) 6"374 grm. gave 25 c.c. N at 14° C, and 769 m.m. B. Potash column, equal l0"2 m.m. Mercury ; equal to 24*3 c.c. normal nitrogen, br 8-12 per cent, Theory, 7*19. (c) 0-3872 gave carbonid b.nhydride 0-7146, equal to 50'33 per cent, of carbon and 0-1830 Water, equal to 5-25 per cent, of hydrogen. Per cent. -j-At. wts. -i-jS[ = 2. -fBr = l. Carbon . . 50-33 4-194 14-4 17' Hydrogen 5-25 5-25 .18-0 21 Nitrogen . . . 8-12 0-580 2-00 2-3 Oxygen . . 16-62 1:038 3-4 4-2 Bromine . .. 19-68 0-246 0-84 1 ' The hydrdbrolnic acid was prepared by the action of phosphoric acid oil potassio bromidfe, and the resulting hydrobromic acid well dried according to , H,PDi + 2KBr = 2HBr + K^HPO, 98 + 238 = 162 + 174 6 + 12 = 8 5 grins, glacial phosphoric acid, 12 grtns. potassic brbmide taken. Protest must be entered here against a common practice of traders of sell- ing in the shape of sticks obtained by fusion, a product, which, while sold at a high price as ' pure glacial phosphoric acid,' aotuaUycontains about one-third, or, at all eyents, large: quantities "of sodic phosphate. This salt is added by the makers to give to their product the deceptive appearance and brittleness. The practice amounts to a fraud in every case where the bu^er is not informed of the admixture of the phosphate. , , ' 288 ANNALS OF CHEMICAI. MEDICINE. The theory of a bromobilirubide-bilirubin, C^HgNOBr + CgHg NO2 jequires : — Carbon . Hydrogen Nitrogen . Oxygen , Bromine . 53-06 4-66 6-87 15-72 19-65 Bilvrubin and hrdmiTie. — 0*5570 grm. of dry bilirubin was saturated at the ordinary temperature with dry bromine vapour. The following phenomena were then observed : (1) 'No greei^ colour at any period, but only brown-red, or chocolate. (2) The powder became warm. (3) No fusion at any time. (4) The bromine was quickly absorbed at the beginning of the experiment, and slowly towards the end. When the whole of the powder, which was from time to time shaken, was of a deep purple colour, and bromine passed unchanged, the, action wag considered complete; but the bromine was allowed to be in contact for 12 hours longer. The product was dark purple, nearly black, and weighed 1-1646 grm. This corresponded to an addition of a little more than four atoms of bromine, 163 : 320=0-557 : 1-0934. The excess of bromine is therefore 0-0712. This was evolved on subsequent heating. More air passed would, perhaps, have removed it. Theory shows that the compound produced is C,H,Br,N0, + 2HBr. -■7' This is not a very stable compound; the combination of the hydrobromic acid being very loose, it is not evolved by passing dry air at an ordinary temperature, but is completely removed by a long-continued current at 100° C. On heating it shrinks, but does not fuse, and on cooling becomes again pulverulent. The product is accounted for thus: — 0-5570 grm. taken; after bromine equal to 1-1646; CgHgN02=163; Br2=160. : 163 : 160=0-557 : 0-5467 [ Theory 1-0934 ; Found 1-1646 Dried at 100° C. 0-5312 bromine was found to have been BILIABY PIGMENTS, ' 289 Tidded, which leads to 165 as being the atomic vreight of bili- rubin. Hypothesis.-^'Eithev the adhesion of the two molecles of hydrobromic acid to the dibromo-bilirubin is a peculiar kind of loose combination produced by a chemism or attraction of the dynamicities corresponding to the hydroxyles contained in the bilirubin : — Or one molecle of hydrobromic acid is combined as hydro- bromate simply, attached to the N pole of the new compound ; and the other is differently attached, viz. by the transparent dynamicities, or the excess of attraction, which the dynamicities occupied by the hydroxyl have yet for the hydrobromic acid* The combination is destroyed at 100° C, and hydrobromic acid goes away. The fact that both molecles of hydrobromic acid go away at 100° C, and apparently without, much change of the residue, except in volume, makes it probable that both of them are bound in an equal manner. Ultvmate Analysis of Dibromobilirubin. — (a) 0*1066 grm. gave 0*1210 of argentic bromide, equal to 48*31 per cent, of bromine. Theory requires 49*84. ~ 0*385 grm. gave carbonic anhydride 0*4750 equal to 33*64 per cent, of carbon ; and 0*1150 of water equal to 3*31 per cent.. of hydrogen ; 0*315 grm. gave 14 c.c. gas at 1 2° C. and 769 m.m. B. Potash column= 15*2 m.m. mercury ; equal to 13*1 normal nitrogen; equal to 5*16 per cent. These data lead to the C,H,Br,NO,. Theory Found Carbon . 33*64 33*64 Hydrogen . . 2*18 3*31 Bromine . 49*84 48*31 Nitrogen . 4*36 5-16 .Oxygen . 9*98 9*15 100*00 100-00 Two further experiments for the quantatipn of bromine in dibromobilirubin by fusion with soda, sodic nitrate, and carbo- nate were made. (1) gave 46*3 per cent., (2) 47*1 per cent. Br. The flux in the last determination was washed out with warm water, and nitrate of silver added before the addition of nitric U ' 290 ANKALS OF CHEMICAL MEDICINE. acid. The tnethbd of fusion iii an open platinum dish seems to produce a loss of bromine. Another preparation of Bihromobiliruhin. — -This prepara- tion gave the same relative proportion as the preceding one, and on analysis the following data; all preparations were dried at 110°. (a) 0"4502, fused with the mixture in a tube, gave argentic bromide 0"5277, equal to 49'87 per cent, of bromine. (6) 0-2314 gave 8*4 c.c. at 8° C. and 759 m-m. Bar.; potash column 13 m.m. mercury ; equal to 9*0 normal nitrogen or 4*8 per cent, (c) 0*3043 grm. gave 0*3760 carbonic anhydride or 33*69 per cent, of carbonic acid ; 0*0978 water, or 3*56 per cent, of hydrogen. Per cent -^ Atomic weights -=-Br.= Carbon . 33*69 2-807 9 Hydrogen . 3*56 3*56 11-4 Bromine . 49*87 0*623 2 Nitrogen . 4*86 0-347 1-1 Oxygen . 8*02 0-501 1-6 100*00 Dibromobilirubin dissolves easily in absolute ether with a violet colour: " The solution on spontaneous evaporation deposits Violet crystals, which when thick are nearly black under the microscope. The solution must be evaporated in vacuo over sulphuric acid and caustic soda. AaBC BrovioHUruMn in Alcolwl and Hyd/i-Qbromia Acid {oolour tf tolution deep AbBC D Bb F G HH' BromohiliruHn in Alcohol and Hydroln-omie Acid, more dilute titan tJie pa-evimn Solution. BILIAEY PIGMENTS. 291 AaBC D E b P a HB Hi SromohiUruhin chmged hp Saryfa Hydrate and Sydrochlwic Add (jsolmw fose-rsd). Action of air at 100° C. on bilirubin (Mbromide. — 5 grms. of bilirubin were brominated in the usual way, and it was then sought to expel the 2HBr by heating to a temperature of 100° C. in a-current of dry air. This was effected in a long tube placed in a water-bath of equal length. The operation was con- tinued for more than a fortnight, when it was found that the substance was losing its violet colour at the end of the tube in which the air entered, and becoming brown, and also that hydrobromic acid was being continuously evolved ; the operation was therefore concluded. After a thorough mixing of the sub- stance had been made the bromine was determined ; 0'3238 grms. gave 0-3444 grms. argentic bromide, equal to 45*25 per cent. of bromine. Therefore dibromobilirubin at 100° C. in a current of dry air is gradually oxydised with loss of bromine as hydro- bromic acid. Action of ether, — From the above preparation 0*1 was taken and extracted with ether. It gave a violet-coloiired solution which, when examined spectroscopically, showed an absorption stretching entirely over the yello^ and nearly over the green. Evaporated in vacuo it left a non-crystalline apparently un- changed violet deposit, still soluble in-ether. Action of sulphuric acid. — A portiou was dissolved in con- centrated sulphuric acid, forming a "purple-coloured solution* This was precipitated by water, and this precipitate extracted with ether; the latter took up a portion which was violet, leaving a green portion which was soluble in alcohol, first cqld and then warm, giving a green solution. The ether sohition was allowed to stand,, and when shaken up with water, gave a colourless filtrate which contained free hydrobromic acid. The alcohol solution examined spectroscopically obscured all the red, and when diluted there was a band over yellow and orange, the green being much shaded. Still further diluted c 2 292 ANNALS- OF CHEMICAL MEDICINE. the band in red only remained, and all yellow and orange were red and a little blue appeared. AbBC D Eb F G H H' SulpliMe of BrdmobiliruMn in Alcohol (coluiir vioUt-ilue), AbBC B ^b F G HH' Sulphate of BromoMUrvMn in Alcoftol, changed hy Hyposulphite and Myd/ro- chUiHc Acid. Action of iodine onbilvrubvn. — About 10 c.c. of a chloro- form solution of bilirubin was treated with an excess of iodine in chloroform. On first mixing a darkening in colour occurred, and the iodine was added until the solution had a deep violet colour. This solution was evaporated in vacuo over sulphuric acid ; no crystals were obtained, but only a coloured deposit in two rings, the first of which had a lighter colour than the second. When the liquid had completely evaporated, the dish was heated in a water-bath to drive oflf any free iodine. The residue was insoluble in ■ ether and in water ; boiling alcohol extracted a little green matter and left a residue, a little of which was dissolved in chloroform witli the colour of bilirubin ; the remainder of tihe. residue, from which again alcohol ex* tracted only a trace of green matter was left insoluble in alcohol, ether, chloroform, and benzole. The chlorofoi-m extract left a deposit on spontaneous eva- poration not strictly Crystalline under the microscope. With nitric acid it gave first a blue and then a red colour. The residue from the alcoholic extract gave a red colour only. Bili/rubin' and nitrous acid. Experiment A. — For this preparation 0*5 grm. of bilirubin were placed in a Liebig's drying apparatus, and nitrous acid (prepared by heating plumbic nitrate in a combustion tube) allowed ' to pass over it ; the Liebig's drying apparatus being further connected with a Gray- Jjussac tube containing baryta water, in order to ascertain if any BILIARY PIGMENTS- ' 293 carbonic acid resulted from the action of nitrous acid in the cold. As soon as the nitrous vapours came into contact with the bilirubin, a blackening of the powder ensued, and the reaction increased in violence every moment, much water was formed, and the whole at last took fire in the tube. There is no doubt that a total decomposition of the greater part of the bilirubin took place, carbon being deposited in the tube. Crystals (pro- bably ammonia nitrate) were deposited on the upper surface of the drying apparatus and carbonic acid passed away. Expervment B. — About 0*3 to 0*4 grm. of bilirubin was placed in a U tube with absolute alcohol and nitrous acid, pre- pared as before; the plumbic nitrate was previously fused to insure absence of carbonic acid. The nitrous acid was allowed to bubble through the liquid. No red fumes passed through the alcohol, which gradually became warmer and warmer until it boiled.' The bilirubin product eventually formed a deposit. Carbonic acid,, aldehyd and nitrous ether were formed during the reaction, besides the bilirubin product. Mxperi/ment C. — ^This was carried on in the same manner as the previous one ; the plumbic nitrate was tested for carbonic acid and found to be free from it. Analyses of the resin obtained by action of nitrous add upon bilirubin vn absolute alcohol. — The product weighed ,0'4136 grm. ; on combustion in vacuo O0484 gave 54 c.c. total gas at 12° C. and 763 m.m. ;' after potash, 5*2 c.c. at 13° C. and 754*^4 B. ; potash column= 3'6 m.m. mercmy, mercury column = 77 m.m. ; total gas normal = 49*49 c.c. Total nitrogen = 4-83 c.c» Total carbonic acid = 44*66 50*02 per cent, of carbon. 11*19 per cent, of nitrogen. Tjherefore C : N = 5*2 to 1. ' Experiment. — Nitrons Add and Alcohol. — This experiment was maxJe with a view of ascertaining how far the reactions observed, in B and C were diie to the action of nitrous acid on alcohol. I The nitrous acid was passed through alcohol in a U tube ; no red fumes passed, and the alcohol became warmer and warmer until it boiled. Aldehyd, carbonic acid, and nitrous ether were among the products of the reaction^ ,^94 ANNAIg. OF CHEMICAL- MEDICINE. The; product has consequently lost much carBdn relatively to the nitrogen, or has gained nitrogen relatively to the carton^ most probably by the entrance into its constitution of a molecle of nitroxyle; perhaps loss of some carbon, aiid entrance of nitroxyle have taken place at the same time, and the product is mainly a nitrited biliverdin. Such a body would contain 48"7 per cent, of carbon and 14- per cent of nitrogen. Thus it is shown that the problem is yet far from its solution. Bilirubin and nitric add, Cholocyani/n,. — An ammo- niacal solution of cholophsein Avas treated with concentrated nitric acid until a blue precipitate was formed. This was quickly isolated by filtration, and after washing with water dis- solved in alcohol. It showed an absorption band in yellow. The red was shortened, the green was somewhat obscured ; the first half of the blue was clear, buj the secojid half and the rest of the spectrum entirely obscured. Aa BC D Eb F 11 Siiigram of ChoUcyanin Specti-nm. A sulphate of cholocyanin could also be obtained by the action of fuming sulphuric acid and subsequently water upon cholophaein together with various other green products insoluble in water. The very dilute watery solution showed spectrum with a feeble band on the side towards red of D, yellow obscured, red dimmed, green lively, blue only one half its ordinary length, rest obscured. Aa BC Diagram, of Speetr^i/m of Sulpltate of Cholocyamin dismhed in water. BILIAKV PIGMENTS. 2f5 This spectrum, of sulphate of cholocyanih by sulphuric acid differs from the one by nitric acid only in this, that the terminal of the band on the red side is more advanced towards red than that of cholocyanin by nitric acid, while the Kne nearest to yellow remains the same in both preparations. Influence of SodmrnHnmalgam upon Biliruhvn, and Bilwerdin. — Hydrogenised bilirubin was made by suspending pure bilirubin in water, and treating it with sodium-amalgam in excess.. .It was found that the action was completed in a few minutes, and did not require from two to four days as some authors state. When I continued the action of the amalgam for some days, no other products- resulted than those obtained after the first half-hour. The alkaline solution was red, and on addition of hydrochloric acid deposited a rust-coloured precipitate; it dissolved almost completely in absolute alcohol, leaving a mere trace of what seemed to be unchanged bilirubin on the filter. The solution was red. It cut off the whole of the spectrum from b ta the violet end. On dilution a band became detached, which had more than double the breadth of the band of hydro- genised. bilivei din. A blue and violet space of the width of the AbBC D Eb F a HH' SilwuKn traituforimd iy Sodmrm-mnalgam, mlution reS). in. Aleoliol (colmt/>' of AbBC D Eb P G HH' Biliverdin champed h/ Sodium-amuilgam, dissolved in Alcohol (colour reddish ironin). band also appeared. When the solution was further diluted, :a narrow band remained, and the blue and violet part of the spectrum was as wide as the red, yellow and green part. Even without the aid afforded by the great differences in ""bearing before the spectroscope, it is clear from the above that hydrogenised bilirubin, and urochrome cannot be identical. ."296 ANNALS OF CHKMICAE MDDICINE. because the former is insoluble in water, and may be Tvashed _with large quantities of it on the filter, while urochrome is easily soluble in water, and is not precipitated from its alkaline solutions by hydrochloric acid. A second experiment was now made with a larger quantity of material, and succeeded like the first. ■ The new body was ■precipitated by acid, and after washing with water, dissolved in alcohol. A reddish^brown solution was formed, which on dilu- tion with alcohol, became red, and ultimately light amber- coloured ; it was, if at all, doubtfully and faintly fluorescent. Its spectrum showed a decided band, even when the solution was much diluted with alcohol, at the junction of green, and blue, sharply defined and of deepest intensity at the green edge, intensity gradually decreasing towards blue. The amount of blue which remained by the side of the band was only about one-third of the width of the band. The band contracted on dilution exactly as the band in the first experiment. The main quantity of the precipitated hydrogenised bilirubin was now dissolved in and boiled with hydrochloric acid. No change wals observed ; the solution retained its properties and remained clear. In particular none of the decomposition- products of urochrome were formed, which, had any urochrome been present, must have been formed immediately. On standing exposed to air, the solution assumed a rose- red colour. Hydrogenised. bilirubin is stated to iave the formula, CjaH^o^i^jj ^^^ declared to be a tribasic acid. This construc- tion is supported by only one silver-compound, which yielded 35'75 per cent. 6f Ag, while other preparations gave 37 "1 per cent. Ag ; and by one zinc-compound, which gave 14"2 per cent. Zn, while other preparations gave up to 37 per cent, of Zn. These data therefore do not afford the means for determining either the atomic weight or the basicity of the product, but seem to show that the sodium-reaction produces a variety of new pro- ducts, which remain partly mixed in the precipitate, partly in the mother liquor from which it falls. , For this liquid remains red, and retains a considerable quantity of a by-product. Similar results concerning hydrogenised bilirubin were lately obtained by L, Disque. In the reduction of bilirubin with sodium-amalgam be employed heat (which had not been em- ployed in the former experiments), and obtained further reduction BILIAEY PIGMENTS. ' 297 of the hydrogenised bilirubin to a colourless matter. This shows no absorption spectrum, but when treated in chloroform, with air, it is again oxydised and transformed back into hydro- genised bilirubin. These experiments of Disque were made upon small quantities in test tubes only, and did not lead to the isolation of definite compounds. i JJist andformulce of definite bilia/ry colourvng Tnatters, their derivatea and com/pounds isolated in these resea/rches. — The biliary colouring matters and their compounds, as evolved by my researches, are now the following : — Crystallised bilirubin . CgllgNOj Neutral silver salt . C^HjAgNOj + H^O Basic silver salt . . CjHjAgjNOj Neutral baryum salt . CigHjgBaNjO^ + 2H2O Half acid „ . C,,H,,BaN,0, + C,H,N0, + 2H,0 Neutral calcium salt . CigHigCaNjO^ + 2H2O Half acid „ . C,8H,gCaN,0, + C,H,NO, + 2H,0 Half acid zinc salt . C,gHi5ZnN204 + C3H,N02 + 2H20 Basic lead salt . . CgHjPbNO^ Dibtomo-bilirubin . CgHjBr^NO^ Hydrobromo-bilirubide CgH^BrNO Hydrobromo-bilirubide bilirubin. . . CgH^BrNO + CgH^NO^ Cholothallin . . CgHjiNOg Bilifuscin . . . CjHuNOj Biliverdin . . . CgHgNO^ Bromobiliverdin . . CgHgBrNO^ Notes on ahnormal biliary pigments and their spectra.-^ The following substances are natural products of diseased processes. Gholonsmatin. — The residue from the alcoholic extract of colouring matter from human gallstones was dissolved in ether. The solution appeared green in reflected light, brown in trans- parent dilute solution. The latter showed a remarkable spec- trum of four bands. Of these two were thread-like (hence the name), thin, like sun-lines ; two broader ones were in' green and blue. Green shaded. The fine lines are clearly visible with the sulphide of carbon prism only, but are fused together when seen 298 ANNALS OF CHEMICAL MEDICINE. in tlie magnifying spectroscope with flint-glass prisms. In- the latter red extends apparently imiformly to a, and is there suddenly cut off. Blue is cut off entirely, even in very dilute solution, the four bands remain to the end, S becoming feeblest. Aa BC D Eb E HH' Diagrami. cf Spectrum, of ClwlonemaMn. Addition of sulphuric acid makes S and a very feeble. The spectrum has some similarity to that of xanthophyll (from chlorophyll of grass). The alcoholic solution of ether extract of bilifascin from human gallstones before precipitation with ether, on comparison showed no bands. Most similar to cholonematin is the spectrum of fluopittin, described in my researches on the decomposition products of the albuminous substances. But in this latter the wider of the two thread-like lines is situated towards the red end, and the thinnest one inside of it, the reverse being the case in cholonematin. Boviprasin, a green colourmg matter from gallstones of ox. — The alcoholic extraction from the gallstones left on Aa BO D Bb F aP y JHagram, of Ixpectnum of Boxiprann. evaporation a resinous residue which dissolved in ether was submitted to- spectral analysis. It showed three bands. Seen in the non-magnifying spectroscope, /8 is divided, a phenomenon not caused by the interference of the sodium line, as this latter is on the band 7. BILIAEX: PIGMENTS. <299 ■ ' Bovifuscopittin. — This resin was extracteafrpm ox gallstones ,by alcohol. It was treated with caustic potash, precipitated by hydrochloric acid, and dissolved in ether. It did iiot crystallise-. Fatty acids were then removed by precipitation with lime, and the resin was dissolved in alcohol. The solution was brownish yellow to red, without any green tinge whatsoever. Its spectrum presented a single band overlying the D line, G EH' DiagrariK cf SiJectrma of BoHfuscopittin. Muscoprasin, — This resin is extracted from ox gallstones by means of alcohol. It has a green colour in the solid state, and retains it on solution in alcohol. In the spectrum it shows bands of great distinctness, one in red being very black. The resin has a particular musk flavour. 7 « j3 5 Diagram qf Spectrnm cf Mmcoprasin. Ethochlorm. — Tlie first ether extraction of ox gallstones yielded a green-coloured substance, which in its second ethereal solution showed the following spectrum : — S e P a 7 Diagram of Speetntm of Mlwclihrin. 300 AOTTAIS OF chemical: HEDICINE. In the magnifying' spectroscope with flint-glass prism the band B is hardly seen. Eed is suddenly cut off, and is then cut off a second time. The margin of the band 8 towards yellow, and that of the band s towards the red end, are apparently washed away. This is the most complicated spectrum met with amongst .biliary matters, and should be compared to that of cruentin. SPEEMATm, AN ANIMAL ALKALOID, 301 XXI. SPEB.MATIN, A NEW ORGANIC BASE IN ANIMAL ORGANISMS. (^Summary.) This substance was discovered by Ph. Scbreiner {LiMg's Ann. 194, 1878, 68), but its phosphate had been known nnder the shape of microscopic crystals of peculiar shape occurring in animal tissues and liquids for twenty-five years. The discovery thus illustrates the great use of microscopic observation as a pioneering power ; peculiar" forms are seen perhaps under anomalous conditions, and noted; they are then again noticed on a field of wider observation, and ultimately searched for* The search then leads to the discovery of materials from which the microscopic objects can be isolated in suflBcient quantity for being subjected to chemical inquiry. Charcot "and Eobin {Gompt. renU. de la Soc. de Biologie, 1853, 49) were the first to observe tbe crystals in the spleen of a leuksemic subject. Charcot in 1856 found them in the sputa from a case of emphyseina with catarrh. From this the ciystals have frequently been termed 'Charcot's crystals.' Forster {Atl. d. microscop. ATiat. 1859, Tab. 33, fig. 4, p. 67 of text) saw them in the bronchial discharge of a man suffering from temporary bronchitis, in a so-called mucinous tissue tunioUr of the, optic nerve, and in the inspissated mucus of an enlarged biliary duct, found them insoluble in ether, and surmised that they consisted of a substance related to mucus. Harting (^Das Microscop. 1859, 458; fig. 182, C.) saw the crystals iii the sputa in chronic bronchitis, observed that they were insoluble in water, alcohol, and ether, but soluble in acetic, hydrochloric; and nitric acid, and interpreted them as calcic phosphate. The crystals were again observed by Charcot and Vulpian (Gaz. hebdom. 7 (I860) 47) in the blood from dififerent parts of the 302 ANNALS OF CHEMICAL. MEDICINE.' body of a female dead from leukaemia. In this case it was observed tbat the crystals only began to make their appearance a" day after the post-mortem examination, and that their number kept on increasing during the following days. These authors found the crystals brittle, insoluble in cold water, alcohol, ether, chloroform, glycerin, -watery and alcoholic solution of iodine, but soluble in warm water, acetic, tartaric, lactic, siilphuric, hydrochloric acid, and in solutions of potash, soda, and ammonia, and: supposed that they consisted of an organic body. The crystals were studied by White {Boston Med. and Surg. Jour. Nov. 28, 1861) and termed by him 'leucosin;' he found them in every case of leukaemia. In 1862 they were noticed by Wiguet {Arch. d. Heilk. 3, 379) in the blood of the portal vein of an ansemic woman who had died shortly after her delivery of a child, suddenly and without any previous illness., In 1864 the same crystals were described and figured by Fried- reich (4tcL /. path. Anat. 30, 382) recognised as identical with those described by Forster, and declared to consist of tyrosin. They were found in fibrinous bronchial sputa of a woman eet. 42. In the same year Huppert {Schmidt's Jahrb. 124, 147) observed that the crystals do not rarely occur in the blood of leukaemic patients. In 1865 Bottcher {Arch./, path. Anat. 32, 525) published a paper ' on colourless crystals of an albuminous body, obtained from human sperma.' He foutid them forming in the plasma of the sperma when it was allowed to dry spontaneously. Sub* sequently he observed them On the surface of old patbological- anatomical preparations, and, was able to obtain them from egg- albumin, and :believed that he had confirmed their albuminous nature. In: 1866 Neiirriann {Ardh. f. inicroscop. Anat. 2, 507) found the crystals in the blood taken from the body of a leuksBmii subject; the formation of- "the crystals began a few hours after the post-mortem examination, and increased during thie following days to such an extent, that every drop of blood contained considerable numbers of them. Iii 1868 Eberth {Arch', f. pathol. Anat. 43, 8) noticed the crystals in connection with leukaemia, and in 1869 Neumann {Archw. d. Heilk. 10, 220) found them in nearly all dead human bodies which he exi^njined, in the normal marrow of the bones. In a great SPEEMATIN, AN ANIMAL ALKALOID. 303 humter of cases of bronchial asthma the crystals were found by Leyden {Arch. f. pathol. Anat. 54, 324 and 346) anddfeclared to be specific products of that disorder. In 1875 crystals were found by Zahn {Arch. f. pathol. Anat. 62, 107 j in the curdled . blood of a frog the subject of experiments upon thrombosis, and found to be identical with those observed by Brondgeest {Nederl, Arch, voor Oenees. en Naturh. 5, 1870, 378) in the blood of frogs which had died from the effects of frost. In 1876 a case of leukaemia gave the opportunity for the observation of these crystals in Ihe bone-marrow and in the mesenteric glands to liaxLenstein {Deutsch. Archiv. f. hlin. Med. 18, 1876, 122). In the same place Zenker gave a summary of the observations concerning these formations, which had frequently come under his eyes in cases of splenic leukaemia and in the sputa of bronchial asthma. Instructive are the various surmises which the observers of these crystals, or their commentators, and particularly the writers of systematic treatises, made concerning their nature. Some declared them to be tyrosin; one author believed this to be an indubitable certainty (Huber, Arch. d. JSeilk. 18, 1877, 485) ; a third declared it to be a substance related to mucus, a fourth to mucin; a fifth again believed in their albuminous nature ; a sixth held them to be related to vitellin ; a seventh likened them to yelk-plates, and crystals of aleurone from plants ; an eighth declared them to be magnesic phosphate ; a ninth calcic phosphate. Schreiner set all these surmises at rest by showing that the crystals were the phosphate of an alkaloid hitherto unknown, and widely distributed in the animal economy. Mode of obtaining the phosphate of spermatvn, (a) from sperma. — Fresh sperma, washed out of linen by warm water, is evaporated to dryness, boiled with alcohol, and allowed to cool and stand for several hours. The mixture is now placed on a filter, and the residue on this is washed and dried. This residue containing the spermatin phosphate is now triturated and ex- tracted with warm water to which a few drops of ammonia have been added ; only traces of the albuminous substances pass into solution and remain so, while the new salt on slow evaporation of the solution crystallises again in its peculiar forms. In a 304 ANNALS. OF CHEMICAL MEDICINE. Pig. 1, mBmP Fig.- 2. (jiiantation the dry residue of sperma was found to yield 5'23? per cent, of these crystals. (b)/rom organs of animals, e.g. calfs liver, calf's heart, bull's testicles, the crystals are obtained by letting the organs stand immersed in alcohol in well-closed vessels. After several months of standing, slender double pyramids, sometimes five millimetres in length, are deposited on the surface of the immersed organs. The crystals are scraped off, the matter thus isolated is extracted with warm water mixed with a few drops of ammonia, and the filtrate on evaporation furnishes the former crystals but of smaller size. Forms of the crystals. — Haushofer described them. as combinations of prismatic with pyramidal forms which mix with each other so as to present spindle-shaped bodies with convex surfaces. The prism fig. 1 is so to say wiped out as regards edges which are diagonal to each other, as is shown in fig. 2, where the lower edges of the anterior con^ tiguous narrow and broad perpendicular planes of the prism co P have been smoothed off, as have the diagonal upper "edges on the hindmost side of the crystals. The crystals combine so as to form crosses and rosettes. : Properties of the crystals. — After repeated crystallisation from hot water to which a few drops of ammonia have been added, the crystals, from whatever source, exhibit the following properties: they are brittle, transparent, colourless, insoluble in atcohol, ether, chloroform, salt-water, watery and alcoholic solution of iodine, almost insoluble in cold^ little soluble in hot water, easily soluble in dilute acids, including chromic and nitric, and in caustic and car^ bonated alkalies, including ammonia. The solution of the •crystals in water acts on sensitive red litmus paper just like common sodic phosphate ; the paper becomes blue, but the blue colour disappears when the paper dries in air. When heated on platinum the crystals at a temperature little above 100° become cohesive and assume a yellow colour ; at 170" they 1-P SPEEMATIN, AN ANIMAL ALKALOID. 305 fuse, and the fused mass sets again on cooling ; on the ap- plication of higher degrees of heat, ammoniacal vapours are emitted, and a black, shining, almost incombustible residue of charcoal is left, which has an intensely acid reaction. On raising the heat the charcoal disappears and a glassy shining spot of phosphorus-platinum is left on the platinum employed. The crystals when burned on a platinum-wire colour the extreme point of the blowpipe flame green. Mixed with the magnesia test for phosphoric acid, a precipitate of the double salt is imme- diately obtained. Water of crystallisation. — The crystals lost in a partial vacuum over sulphuric acid 16*58 per cent, and on subsequent heating to 100° in an air bath they lost again 4"57 per cent., together 21 '15 per cent, of water of hydration and crystal- lisation. The same crystals recrystalKsed from hot water lost again 16*667 per cent, m vacuo, and 4*545 per cent, at 100°, together 21*212 per cent, of water. Phosphoric acid. — A portion of the crystals dried at 100°, dissolved in water with a drop of ammonia, and treated with magnesia mixture, gave pyrophosphate of magnesia equal to 35*399 per cent, of phosphoric acid in the crystals. A second quantation gave 35*126 per cent. PjOg. Nitrogen. — Ignited with soda lime the crystals gave am- monia corresponding to 1 4*03 per cent, of nitrogen in the salt. From these data it follows that the salt at 100° loses three molecles of water of crystallisation, and that it contains two atoms of nitrogen upon one of phosphorus. Isolation and properties of sper matin.— The phosphate is decomposed accurately with baryta water, and the filtrate is evaporated in thin layers on the water-bath. The base then remains as a colourless viscous varnish-like mass, which on cooling crystallises. If the solution is evaporated in larger quantities and in thicker layers, a thick syrup is obtained which crystallises only partially at the margins, even on standing for several days over sulphuric acid. The crystals disappear quickly on ex- posure to the air, and the viscous syrup becomes more fluid absorbing both water and carbonic acid. The base is soluble in absolute alcohol, and crystallises from this solvent easily on z 306 ANNALS OF CHEMICAL MEDICINE. evaporation. It is almost insoluble in ether, and seems to be changed by this solvent, being transformed into a brown mass, which after removal of the ether becomes only gradually soluble in water. On adding to the base in the syrupy or carbonated state some phosphoric acid, or ammonic phosphate, the crystals of the phosphate above described and figured are at once obtained. Spermatin therefore expels ammonia from its compounds, and its phosphate is distinguished by a great tendency to crystalli- sation. It is not changeable when exposed to the air. Heated on platinum it evolves thick white fumes, which have only a weak ammoniacal odour. The watery solution of spermatin, which has a strongly alkaline reaction, shows also the following bearing with re- agents : (1) On being warmed with caustic potash or soda it evolves ammonia ; (2) with zinc chloride it gives a white flaky precipitate which is soluble in hydrochloric acid; (3) with watery solution of tannin it gives a white flaky precipitate; (4) with argentic nitrate it yields a white precipitate soluble in nitric, sulphuric and acetic acid ; (5) corrosive subUmate produces only a turbidity in its solution ; (6) with auric ter- chloride the base yields at once a golden yellow precipitate, which becomes gradually crystalline; (7) with platinic tetra- chloride the base yields no immediate compound, but after some time delicate plates are obtained ; (8) phosphomolybdic acid yields a yellowish precipitate with spermatin ; (9) phos- photungstic acid gives a white flaky precipitate, which is soluble in ammonia, potash, and soda, but insoluble in hydrochloric, nitric, sulphuric, phosphoric and acetic acid. With iodide of potassium and with neutral and basic lead acetate the base gives no reaction. Mode of extracting the base from tissues and organs. — Phosphotungstic acid may be employed for isolating the base from the parts of healthy animals, such as liver, spleen, lungs and blood of cattle, and from the parts of bodies which have died from disease. The parts are minced, boiled with water and a little acetic acid, and the filtered liquid is treated with basic plumbic acetate, an excess of this reagent being avoided. The filtrate from this lead precipitate is freed from lead by SPERMATIN, AN ANIMAL ALKALOID. 307 fiydrothion, boiled, and after suitable concentration treated with phosphotungstic acid. The precipitate is washed with water to which sulphuric acid has been added, and then de- composed by baryta water in the hot state. Any excess of baryta is removed by carbonic acid. The concentrated liquid may contain other alkaloids besides spermatin ; from these the latter is best separated by the addition of phosphoric acid, care being taken to keep the mixture as neutral as possible on account of the easy solubility of the phosphate in acids and alkalies. When bases are present which retain baryta in combi- nation, the isolation of the phosphate of spermatin from the barytic phosphate has to be effected by recrystallisation from hot water. Hydrochlorate of spermatin. — The watery solution of free spermatin neutralised with hydrochloric acid gives on slow evaporation on the water-bath the hydrochlorate as six-sided prisms united in tufts ; the salt is not changed by air, insoluble in ether, almost insoluble in alcohol, soluble in spirit, and very easily soluble in water. Addition of absolute alcohol to the watery solution causes the salt to crystallise in anhydrous small crystals, which on analysis yield numbers leading to the formula C2H5N,HC1. This requires in 100 parts c . . 30-204 H . . 7-551 N . . 17-618 CI . . 44-626 These data show that the phosphate of spermatin dried at, 100° does yet contain a molecle of water of crystallisation, which cannot be expelled without decomposing the salt. Plabmic double salt. — After addition of platinic tetra- chloride to spermatin hydrochlorate a double salt is gradually produced, which crystallises in prisms. Auric double salt, CaHgNjHCljAuClg (Au=51-33 per cent., 01=37-146 per cent.) The hydrochlorate gives with auric terchloride immediately a voluminous precipitate, consisting of shining, golden yellow irregular plates. When freshly pre. cipitated this salt is easily soluble in ether, alcohol, and water ; but when dried and exposed to air it loses its solubility in X 2 308 ANNALS OF CHEMICAL MEDICINE. water in part, and when the redissolved part is allowed to stand, a further precipitate of insoluble matter takes place. When the gold salt dissolved in water is treated with mag- nesium, the odour of fresh human sperma is at once produced. From this it is probable that the odour of human sperma is due to the presence of a derivate of spermatin. The odour of sputa, which not rarely resembles that of sperma, is probably also due to the presence of this derivate. When gold and chlorine are removed from the salt by Scheibler's process {Ber. Deut. Ohem. G. 2, 297), and the filtrate from the silver chloride is freed from silver by hy- drothion, the filtrate from the silver sulphide gives again a precipitate with phosphotungstic acid, which is not identical with the precipitate given by this reagent with the original base ; the body isolated by the baryta process is a colourless alkaline, inodorous syrup, but cannot be brought to crystallise with either phosphoric or hydrochloric acid. When gold and chlorine are removed from the gold salt by hydrothion and silver oxyde, the alkaline solution retains silver, and on evaporation on the water-bath forms a silver mirror on the walls of the dish ; on further evaporation in a new dish a brown syrup, free from silver, is obtained, which does not crystal- lise with either hydrochloric or phosphoric acid. 309 XXII. CHEMICAL SVRGERY, WITH SPECIAL REFERENCE TO THE ANTISEPTIC PHARMACOPCEIA.^ (Reviev!.) In the first part of this ' Guide ' the author gives a description of the condition of affairs in the surgical department of the hospital at Munich before the introduction of the antiseptic treatment of wounds. Pyaemia was permanent, and nearly all cases in which amputation had been performed perished by it. In 1872 hospital gangrene appeared, and became both severe and fre- quent, so that in 1874 80 per cent, of all wounds and ulcers were affected by it ; corroded arteries and mortified bones led to mutilations, and frequently to death. Erysipelas appeared upon almost every patient ; every patient became at least once, frequently twice, the victim of hospital gastricism ; a first re- union, or union of wound by prime intention was never observed before 1875. All these evils are now removed by the introduction of Lister's antiseptic treatment of wounds. Erysipelas, phlebitis, hospital gangrene, and pyaemia have disappeared ; the wounds resulting from amputations and plastic operations heal by first reunion ; the mortality in the clinic has been diminished to one-half its former proportions. This very striking success causes the author to become a strong advocate of the antiseptic method, and he fills the first part of his ' Guide ' with arguments to the effect, that every surgeon is in duty bound to know and to practise the antiseptic method. In the second part of this ' Guide ' Nussbaum gives a concise description of the apparatus and chemical preparations required ' Prof. Dr. J. N. Bitter von Nussbaum, Guide to tlie Antiseptic Treatment of Wounds, pwrticulm-ly to the Method of ZAster. Third edition. Stuttgart, 1879. 310 ANNALS OF CHEMICAL MEDICINE. for the successful application of the method, from which we quote the following practical details. 1. Carbolic acid solution of 5 per cent, strength. 5, Acid, carbolic. crystaUisat. puriss. 50-0. Aq, destillat. 950*0 (grammes). This solution is used for the ablution of the hands of the operator and of the assistants before every operation and before every dressing. The surfaces upon which an operation has to be performed are also washed with it, and the area surrounding every ulceration. The surgical instruments are placed in this solution before and during the operation, canulas and catheters ai"e rinsed with it both in and outside. The apparatus for producing the steam spray is filled with it. The steam in dispersing the solution dilutes it to the extent that the condensed spray represents a solution of carbolic acid in water of about 2^ per cent, strength. This watery solution of carbolic acid has the disadvantage that it causes roughness and even painful excoriation of the skin to some who have to operate much and dress frequently. This drawback is avoided by the use of carbolised vaselin, an ointment consisting of 90 g ms. of vaselin and 10 grms. of carbolic acid ; it can be distributed over the whole of the hands by friction, disinfects perfectly, and does not produce any roughness of the skin of the hands. This roughness is always attended by a certain loss of sensibility in the fingers, and is therefore to be dreaded by the surgeon, who requires the utmost sensibility in his finger-tips in daily practice. Vaselin is a paraffin obtained from petroleum, semi-solid at the ordinary temperature, fusing at about 35°, and boiling at about 200°. It has been used since 1875, for the compoimding of ointments, instead of fat. 2. Ca/rbolised water, or watery solution of carbolic add of 2^ per cent, strength. 1^ Acid, carbolic, crystallisat. puriss. 25*0. Aq. destillat. 975-0. This water is used to moisten dressings, wash wounds ; it is also used in the spray-producer. CHEMICAL SURGERY. 311 3. Oarboliaed oil of 5 per cent, strength. 5o Acid, carbolic, crystallisat. puriss. 5*0. Olei olivar. piir. 95*0. With this oil catheters, specula, fingers, and hands are anointed before use. 4. Garbolised oil of 10 per cent, strength. 5i> Acid, carbolic, crystallisat. puriss. lO'O. Olei olivar. pur. 90"0. This mixture is used to drench lint, which it is intended to place into deep wounds, such as arise after carious bones liave been scraped or cancerous tumours have been scooped out. 5. SaUoylic emulsion. 5) Acid, salicyl. crystallisat. 5"0. Aq. destillat. 95. This preparation is actually salicylic acid suspended in a saturated ■ solution of the acid (5 grms. of the acid require 1500 grms. of water for solution). It is used for moistening dressings, when it is intended to leave them undisturbed for a longer time, and is in such cases preferred to carbolised water. It must be shaken up before use. 6. Solution of si/nc-chloride of 8 per cent, strength. R Zinci chlorid. 8-0. Aq. destillat. 92-0. This solution is used to make wounds and ulcers into which septic ferments have penetrated, again aseptic. 7. A steam spray-producer with spirit or gas lamp. 8. Silk or protective, a thin green oil-silk, which is covered with a mixture of I part dextrin, 2 parts amylon, and 16 parts of a watery solution of carbolic acid of 5 per cent, strength. This protective is used to cover up wounds and ulcers, to which it adapts itself easily, owing to its pliability. The cover- ing of carbolised paste is necessary to insure the desttuction of all ferments which may possibly adhere to the oil-silk. 9. Antiseptic grau^e.— Bleached or unbleached cotton gauze is cut into pieces 6 metres long and 1 metre broad. They are 312 ANNALS OF CHEMICAL MEDICINE. heated in a tin case, surrounded by boiling water, during from 2 to 3 hours. The tissue is now spread out and impregnated with a hot mixture consisting of 1 part crystallised carbolic acid, 5 parts rosin, 7 parts solid paraflBn. The rosin is intended to fix the carbolic acid ; the paraflSn is added to remove the ad- hesiveness of the carbolised rosin. The gauze thus impregnated is again placed into a tin box and pressed for an hour or two, to ensure that the .mixture may penetrate all the fibres. Of this gauze 6 or 8 layers of suitable size are dipped into carbolised water, wrung and laid directly upon the silk cover- ing the wound. This piece of gauze has been termed the lost gauze. Over this are laid 8 layers of dry gauze, which must overlap the wound in all directions. Over this is laid a piece of impermeable caoutchouc tissue or gutta-percha paper. The entire dressing is fastened with rollers of the prepared gauze. 10. Caoutchouc tissue, or mackintosh, made of cotton stuff and caoutchouc, or gutta-percha paper, is used to cover the gauze- dressing, and prevent the secretion of the wound from coming into visible contact with the outer air ; the secretion is thus compelled to soak into the gauze and remains unaffected by ferments. 11. Catgut. — The catgut occurring in trade is made from the small intestine of sheep. It must be prepared by submersion during two or three months in an emulsion consisting of 5 parts of olive oil and 1 part of liquid carbolic acid (the crystallised earboUc acid after addition of 10 per cent, of water becomes permanently liquid). The catgut thus prepared is kept in carbolised oil (3). It is used to tie arteries or veins; the ligatures are cut short and allowed to remain in the wound ; they unite with the tissue of the scar as if they were living tissue. Catgut can also be used for sutures. Such do not require ta be removed, as the portions within the tissue are absorbed, while those outside .fall off. Latterly a kind of catgut prepared with chromic acid has been proposed. 12. Salicylated cotton-wool. — Cotton- wool is freed from fat by boiling with caustic ley, washed and dried. This is now dipped, in thin layers, into a solution, consisting of 1 kilogramme of salicylic acid, one litre of spirit of 0-083 spec, gr., and 60 litres of water of 80° C. temperature. It is allowed to remain CHEMICAL SURGERY. 313 for some hours, taken out, allowed to drain without pressure, and then dried on boards. 13. Salicylated jute. — 2^ kilogrammes of jute (or tow) are steeped in a mixture of 500 grms. glycerin and 4^ kilogrammes of water, containing 75 grms. of salicylic acid in solution. These salicylated materials are used for adapting the dressings very closely to the skin surrounding the wound and dressings. Nussbaum thinks it possible that salicylic acid might destroy ferments which carbolic acid might leave untouched, and vice versa, and that both disinfectants might give the best results when employed at the same time. 14. Sponges prepared as usual, are kept ready for use in a watery carbolic acid solution of 5 per cent, strength. They are frequently used to stanch bleeding by gentle compression. The bloody sponges are washed with soap-water two or three times, then with tepid water ten times, then dipped in dilute carbolic or hydrochloric acid, washed again in water, and preserved again in carbolic acid water. 15. Drainage tubes of caoutchouc, or small bundles of horse- hair are placed into the lowest angles of wounds to facilitate the discharge of the wound secretion. These articles are washed with soap-water and disinfected with solution of carbolic acid. 16. Antiseptic silk. — Silk thread of suitable size is steeped in a hot mixture of 1 part of carbolic acid, and 10 parts of bees'- wax. The excess of the wax is removed by drawing the threads through a cloth. This antiseptic silk is useful for sutures, and for ligatures ; it may be left in the wound, which heals over it without difficulty, although the silk, unlike the catgut, is said not to be absorbed. It rivals silver wire and carbolised horsehair for some kinds of sutures. 17. Salicylated water. — A solution of 3 parts of salicylic acid in 900 parts of water. This water is used to wash wounds in cases where the solution and preparations of carbolic acid produce a kind of blood-poisoning, which is indicated by in- disposition of the patient and dark green colour of his urine. It is also placed in the spray-producer, to feed the spray during operations on children. For the young are easily afifected by carbolic acid, and sometimes severely and dangerously so ; the 314 ANNALS OF CHEMICAL MEDICINE. carbolic preparations have therefore to be used in their case with great caution. 18. Boracic acid water. — 35 grms. of boracic acid are dissolved in 96h grms. of water. This solution has a long- lasting antiseptic efifect, and is free from the objections which arise in the case of children to carbolic acid preparations. The solution is used to moisten lint with it, and wash wounds dressed with such Unt. The solution is also useful for injections in gonorrhoea, in blenorrhoea of the bladder, and for other piirposes. 1 9. Boracic lint. — Lint impregnated with boracic acid is a good dressing for ulcers, scalds, and burns ; it is covered over with caoutchouc tissue, or gutta-percha paper. 20. Boracic ointment. ^ Acid, boric, pulverisat. Cerse albse aa 10. Olei amygdalar. dulc. Paraffin aa 20. Ft. unguent. This ointment is spread on disinfected calico in thin layers, and this is placed over wounds of the skin united by sutures ; it is then fixed by gauze dipped in coUodium. Such a dressing is very useful in cases of operation for harelip. In the third part of his ' Guide ' Nussbaum describes in detail and exemplifies by cases the methods of applying these pre- parations. In the fourth part he advocates the earliest possible use of the method in wounds occurring to soldiers in the course of warfare, and recites instructive examples of its success in the hands of Eussian surgeons during the Eusso-Turkish war. Although in some places the case of the antiseptic method is overstated, the examples amply justify the elated tone of the author. Some details no doubt exemplify conditions specially Bavarian, and would perhaps not be met with in any other part of Grermany, the Wendish districts excepted. On the whole however the ' Gruide ' of Prof, von Nussbaum is a valuable, interesting, and concise exposition of the antiseptic method as applicable to most human conditions, and Professor Lister may be congratulated on having found so ardent and so able an ex- positor of his discoveries and views. 315 XXIII. EISrORICAL RETROSPECT ON EARLIER, AND CRITICAL CONSIDERATION OF CONTEMPORANEOUS, RE- SEARCHES ON -BILIARY PIGMENTS. The colouring matter of bile has repeatedly attracted the attention of chemists and physicians. The first attempt to extract it from bile was made by Berzelius. He obtained a yellow substance which was soluble in ether, contained soda, and was named bilifulvin. He also endeavoured to extract the green colouring matter, by combining it with baryta. When the green precipitate was decomposed with hydrochloric acid, biliverdin was left. At a later period Scherer attempted {^Ann. Chem. und Pharm. 53, 377) to isolate the yellow colouring matter of bile contained in the urine of jaundiced persons, and performed the elementary analysis of a substance obtained from such urine, as also the analysis of a colouring matter which by a different proceeding had been obtained from gallstones. The colouring matter from gallstones was also subjected to elemen- tary analysis by Hein and Marchand {Physiol. Chem. 25). As the substance analysed by these chemists was yet very impure, it yielded no satisfactory results. Some addition to out know- ledge regarding the nature of this substance was made by the researches of Heintz (Poggend. Annal, 84, 106). He purified the colouring matter from gallstones by solution in carbonate of soda, and separated by means of alcohol the brown from the green modification. He further investigated the metamorphosis of the brown into the green colouring matter under the influence of alkalies and air, and upon the basis of several elementary analyses he conceived this metamorphosis to consist in an oxydation. One equivalent of biliphsein absorbed, according to the theoiy of Heintz, one equivalent of oxygen, and then 316 ANNALS OF CHEMICAL MEDICINE. split up into two equivalents of biliverdin (CgjHjgNjjO, + = 2 (CijHgNOgV After Valentiner {Giinaburg'a Zdtsehrift N. F. 1, 46, 1859) bad extracted a crystallised matter from gallstones by means of chloroform, which matter he announced to be hema- toidin, Stadeler (Frerichs, Klin. d. Leberhr.) applied the same solvent upon gallstones and obtained crystallised biliphsein, to which in accordance with his analysis, he attributed the formula CijHgNO^. At a later period, however, Stadeler published {Mittheilungen aus dem chem. Lahorator. im, Zurich, 1864) a more extensive paper on the colouring matters in human gallstones, in which, based upon new elementary analysis and one atomic weight determination, he attributed the formula of CgjHjgNjOg to the red colouring matter of bile, and termed it bilirubin. He observed its transformation into biliverdin, as described by Heintz, and formularised the process, without having performed any additional analysis, thus, that one equiva- lent of bilirubin by the absorption of two equivalents of water and two equivalents of oxygen passed into biliverdin, to which latter he attributed the hypothetical formula CjjHjoNjOio. He further distinguished bilifuscin, which was obtained directly from gallstones, and not produced from bilirubin, but placed in relation to this substance. Its empirical formula was given as CgjHjoNjOg, thus being distinguished from bilirubin by exceeding its formula to the amount of 2 HO. But by solution in alkali it yielded no biliverdin. Stadeler further distinguished bUiprasin, which was also extracted ready formed, not produced artificially, and on analysis yielded figures from which it was possible to calculate the formula C^^H^i^fii^, distinguished from the hjrpothetical biliverdin by an excess of 2 HO, and a colour test of its spirituous solution with caustic ammonia. Biliprasin was not obtained from biliverdin ; no processes were indicated by which both substances could be separated. (In the foregoing formulae 0=6, = 8.) Already, in the year 1861, I communicated to the British Association for the Advancement of Science some researches on the meta- morphosis of the colouring matter of bile by oxygen and nitrous acid, which afterwards were embodied with my work on Grallstones (London, 1862). I then published a new theory of bilirubin and its salts, based upon the comparison of many BILIARY PIGMENTS. 317 preparations, and nearly fifty elementary analyses of these pre- parations, and of eight different combinations with metals, in the Tenth Report of the Medical Officer of the Privy Council, 1867, pp. 240 to 251 and Journ. f. prcict. Chemie, 104 (1868), 4. This was made the subject of some hypothetical considerations by Stadeler. They are to be found in the 18th volume of Omelin's Handbook by Kraut, English edition, pp. 75 and 76, and are stated by the editor to be taken from an epistolary communication to him by Stadeler. It is first to be remarked that the analyses of crystallised bilirubin formerly given by Stadeler, and published in Frerichs' Handbuch der Leberkrankheiten, are not given in Kraut's summary, nor is there any reason given either by Stadeler or Kraut for their rejection or omission. They fully coincide with my own analyses and theory, and I,- therefore, claim them in my support, and against Stadeler's theory. In his epistolary communication Stadeler does not adduce a single new fact or analysis, nor does he controvert any of my facts and analyses, but he puts upon the whole of them a new construction, by means of which he endeavours to bring them into some kind of harmony with his theory of bilirubin. He doubles his formula of bilirubin, assuming it to be CgjHgoN^Og (here, and in the rest of the formulae of this article C = 12, = 16), and to contain six atoms of hydrogen replaceable by metals. He gives formulae for all my salts according to that hypothesis ; I quote, as examples, the basic silver salt, CgHjAg^NOj, which in its new dress appears as C32H3(,AggN40g, and the neutral silver salt CgHgAgNOj + HjO, which in its new attire becomes C HgjAgjN^Og. In the half-acid baryum, calcium, and zinc salts two atoms of hydrogen are supposed to be replaced by one atom of metal, thus : CgjHjiBaN^Og. This hypothesis has no foundation in fact. Not a single formula of Stadeler's, and not a single element of any formula, can be derived from my analyses. The quantities of metals, as calculated by Stadeler for his formulae, are all from one to six per cent, below the quantities found by me. To this I must add that the facts regarding Stadeler's lime-salt (the only salt he ever investigated, and that only by one calcium estimation) are misstated in the translation. Stadeler found in his lime-salt 9-10 per cent, of 318 ANNALS OF CHEMICAL MEDICINE. lime, i.e. calcium oxyde, and not as there stated 9*10 per cent, of Ca, i.e. calcium metal. It was this only lime determination which led Stadeler to his second theory of bilirubin, and as he did not know of two classes of salts, which were first discovered by me, this atomic weight determination led him, according to my explanation of it, into error. It must be pointed out that all the figures given on p. 76 of the translation marked ' accord- ing to Stadeler' are not results of analyses, but merely of calculations on paper by the light of the hexabasic hypothesis. I hold the hypothesis of Stadeler to be not merely not proved by facts, but to be directly disproved by all my analyses, without exception, and more particularly by the brominated compound of bilirubin. A hexabrominated bilirubin, according to Stadeler's hypothesis, would require 45 "8 per cent. Br, whereas the dibro- minated bilirubin of my formula requires and actually contains 49 '8 per cent. Br. A number of chemists have operated upon bilirubin with bromine and iodine, and have arrived at the conclusion that the resulting substance was biliverdin. ' Thus Maly (see Gmelin's Chemistry, Cavendish Soc. edit., vol. 18, p. 74) stated, that the conversion of bilirubin into biliverdin may be effected by bromine or iodine, by acids with access of air, by alkalies with access of air, more quickly by peroxyde of lead. In order to appreciate these statements, it must be borne in mind that with the excep- tion of the one referring to the influence of alkalies and air, none were supported by any analytical qualitative or quantitative evidence. Further, that the statements of Maly regarding the nature and relations of bilirubin and biliverdin have from time to time undergone important modifications at his own hands. Thus he considered the change which bilirubin undergoes with glacial acetic acid, as an elimination of ammonia, and believed the green substance formed to be non-nitrogenous and yet identical with biliverdin. By adding ammonia to his supposed biliverdin he again obtained bilirubin. He has himself corrected, but not explained this error. He merely dissolved his bilirubin in the acetic acid, and by heating probably acetylised a small portion, which dyed the bilirubin green. In a similar manner he has mistaken for biliverdin a number of green derivatives of bilirubin, which all differ from each other, and simply have this BILIARY PIGMENTS. 319 in common, that they possess a green colour. With regard to the alleged formation of biliverdin from bilirubin by bromine, Maly said : ' In contact with bromine vapour and moist air, bilirubin quickly turns green. When a dilute alcoholic solution of bromine is dropped into a solution of bilirubin in chloroform, the colour changes, according to the quantity of bromine, to green, dark blue, which remains unaltered for weeks, then dirty violet, dark wine-red, and ultimately light wine-red — the same changes of colour, therefore, as those produced by nitric acid containing nitrous acid.' The change was explained as oxyda- tion. In contact with bromine vapour and moist air, bilirubin perhaps turns green for an instant, namely as long as the orange powder is able to send yellow rays through the blue compound, which quickly covers its surface. But often as I have repeated the experiment, it has had the same result in moist as well as diy air ; never has there been formed a matter or a colour similar to biliverdin, but always the brominated products de- scribed. Further, if the green colour produced in the chloroform solution of biliverdin by bromine had been due to biliverdin, the latter must have been precipitated, as it is insoluble in chloroform. The green colour, according to my explanation, was simply a mixture of the yellow of the original solution, with the blue of the brominated product. The dark blue when once obtained remains unaltered for weeks, a good proof of the difference of this reaction from that of Gmelin, in which the blue produced by nitrous acid is of the most transient nature. The spectroscope easily shows that the two blues are due to entirely different chemical entities. Even the blues produced by nitrous- nitric acid in different bile-colouring matters are different. Their different spectra were originally observed and described by me in 1866 and 1867, in the Ninth and Tenth Reports of the Medical Officer of the Privy Council. See the latter volume, pp. 251 to 260. Cholocyanin; its sulphate; sulphate of sulpho-cholocyanin given above; and hyoccerulin. Therefore in reactions with bile-colouring matters a blue colour is no more a proof of identity than a green. A most elaborate account of the alleged oxydation-products 320 ANNALS OF CHEMICAL MEDICINE. of bile-pigments and their absorption bands was published by A. Heynsiiis and J. F. F. Campbell, in Pfluger'a Arch. f. Physiol. iv. 497-547, extending over fifty pages, A blue substance, hilicyanin, was obtained by what is termed the oxydation of bilirubin by bromine-water. The spectra obtained varied, as also did the solubilities of the products. Not a single product was isolated, and none was analysed. It is easy to see that these products were principally mixtures of the mono- and dibrominated bilirubin. Of oxydation there is no evidence whatever. The same remarks apply to a greenish product, obtained formerly by Stockvis, and termed choleverdin, which after perusal of the paper just quoted, he declared to be identical with and thenceforth termed bilicyanin {Neues Repert. f. d. Pha/rm. 21, 732-737). These discursive papers relate merely to experiments made with dilute impure solutions in test-tubes, and do not start with any pure substance, or arrive at any stoichiometrical conclusion. My researches on this subject had proceeded thus far, when a paper was presented to and printed by the Imperial Academy of Sciences of Vienna, which, based as it was upon a plagiarism committed upon a private letter, compelled me to address to the Academy a remonstrance in the shape of an open letter, of which the following is an abstract. . This letter was published in Pfluger's Arohiv, Liehig'a Annalen, and in the Chemical Ifews, under the title of ' Open Letter to the Imperial Academy of Sciences at Yienna, containing an Examination of the Eesearches on the Colouring Matter of Bile, by Eichard Maly, of Graz.' The 72nd volume of the Proceedings of the Imperial Academy of Sciences (Part 3, October 1875) contains a paper by Eichard Maly, of Grraz, entitled ' On the Action of Bromine upon Bilirubin,' which compels me to communicate to the Imperial Academy the following statement : — The paper by Prof. Maly alluded to is described as the fifth of a series, the first and second members of which are printed in the 57th and 59th volumes of the Proceedings of the Academy : the third paper is contained in Liebig's Annalen (vol. clxiii.); the fourth, again, in the Proceedings of the Academy (vol. Ixx.). I am compelled to refer to these four BILIARY PIGMENTS. 321 previous papers by the contents of the fifth in the. 72nd volume of the Proceedvngs of the Academy. In the introduction to the fifth paper Prof. Maly states that, through some experiments, undertaken with the view of learning something about the blue products of oxydation which nitric acid produces with bilirubin, he ' has been led to the study and discovery of a body which was totally different from the one he had proposed to himself.' Incident- ally he remarks that his experiments are the fruit of a year's labour. Prof. Maly next recants the opinion which he had main- tained in all his former papers, and which in the second paper he had endeavoured to prove by many and laborious volumetric experiments, according to which the products of the action of bromine upon bilirubin owe their origin to a process of oxyda- tion ; and he then describes, after many remarks upon the alleged striking similarities between the action of nitric acid and that of bromine upon bilirubin, his great surprise on find- ing that the products of the action of bromine upon bilirubin are by no means oxydised, but contain bromine in substitution for hydrogen. In a footnote Prof. Maly then states : — ' After I had con- cluded the following research, and drafted the manuscript, I became acquainted with a research by L. W. Thudichum, " Further Eesearches on Bilirubin and its Compounds," which is contained in the lately-published (May) nimiber of the Journal of the Chemical Society, I have learned from this that Thudichum also has recognised that the action of bromine upon bilirubin produces bromo-proclnicts ; therefore this ob- servation, which has also been made by Thudichum, is in any case, on account of prior publication, to be considered as his property.' I interrupt the quotation of the footnote in order to point out that Prof. Maly represents to the Academy that he had dis- covered the substitution by bromine, of hydrogen in bilirubin, by means of his own experiments ; that he had himself inde- pendently found out and corrected his former errors, which he had maintained for years, and which have been repeated in many chemical publications ; and particularly that he had only 322 ANNALS OF CHEMICAL MEDICINE. obtained knowledge of my researches after he had completed his research and written out the draft of his paper. However, on June 11, 1874, I had directed a letter to Prof. Maly, in the course of which I had made to him the following communication, given in the words of the copy which I preserved : — ' I was pleased that as regards biliverdin you now agree with me. You double the formula, but without giving any reason for it. However, in the course of time our agreement will, no doubt, become greater yet. You still believe, 'par exemple, that the products of the action of bromine upon bili- rubin are products of oxydation, whereas I have shown already two years ago, that they are products of substitution. Simple addition of bromine vapour to bilirubin gives CgHjEr^NOj, and determines the atomic weight of bilirubin accurately at 163, the same number which results from all my other researches.' That Prof. Maly had received the letter containing the foregoing passages is proved by the reply which he addressed to me, dated from Innsbruck, June 14 (1874), now before me. Prof, Maly, therefore, before he began the experiments which are so exhaustively described in the fifth paper, was not only informed of his error, but actually in possession of the key to his alleged discovery, and it was therefore impossible that he should have been led to this discovery by his experiments. It is further quite clear, from the connection of the state- ment of Prof. Maly, that if he had not obtained information of my paper in the Journal of the Chemical Society, he would in that case also have ignored my letter to him, and would have claimed the priority of the discovery of the brominated product of bilirubin. For although, in the beginning of his fifth paper, he declares, as he expresses himself, ' the characterisation of the hitherto existing knowledge concerning the blue product of bilirubin ' to be a ' usual duty,' yet in enumerating previous data he neither mentions my letter nor the fact that I had published the existence of the bromo substitution-product of bilirubin already in the year ,1872, in my Manual of Chemical Physiology (London, 1872, p. 72), and had communicated this observation to the Chemical Section of. the Grerman Association for the Advancement of Science, at the meeting at Wiesbaden. BILIAEY PIGMENTS. 323 He is perfectly silent regarding the numerous blue and variously coloured derivatives of the colouring matters of gallstones, which I was the first to characterise from my own researches, chemi- cally as well as spectroscopically, in my Report in the Ninth and Tenth Reports of the Medical Officer of the Privy Council (1866 and 1867, pp. 251 to 260), and in the Manual alluded to. That these researches and publications should have remained unknown to the editor of an annual report on the progress of animal chemistry is not impossible, but that he excluded the contents of my letter from the circumference of the ' usual duty' admits of only one explanation, but not of justifi- cation. In the note alluded to Prof. Maly says — ' Thudichum has not analysed Ms body, but has only drawn a conclusion con- cerning its composition from the increase in weight which bilirubin undergoes when bromine vapour is passed over it. When bromine was passed for a short time Thudichum obtained a body which was soluble in alcohol with almost monochromatic blue colour, and contained 35"30 per cent. Br; and this, in accordance with Thudichum's formula for bilirubin, is said to be monobromo-bilirubin, for which, however, calculation requires 33'0 per cent. Br. This substance, from its properties, may correspond to the body which I describe in this paper^ but without being approximately pure.' These statements are exclusively taken from the prelimi- naries of my paper, which I relate only because they lead to and necessitate the performance of my cardinal experiment. The product containing 35*30 per cent. Br I have never declared to be monobromo-bilirubin; on the contrary, I have stated that by solution in concentrated sulphuric acid, and precipitation with water, to which it had been subjected, it had acquired different properties — among others, a green colour. I say expressly that the produfct before treatment with sulphuric acid appeared to have been a mixture of mono- and dibromo-bilirubin. I then assert that these bodies cannot easily be separated from each other by ordinary solvents. Prof. Maly says further in the note — 'When bromine is passed for a long time (over bilirubin), then, according to Thudichum, a body is produced which is yet richer in bromine, T 2 32-t ANNALS OF CHEMICAL MEDICINE. and this he claims to be bromo-bilirubin.' And further in the text — ' It is therefore ' (namely because according to Maly the proper point of bromination can only be discovered with the aid of solvents) ' impossible to draw any conclusion regarding the composition of the products of the two experiments of Thudichum, in which bilirubin was exposed for some time to bromine vapour, and then weighed, for in this form the body is always fused like a resin.' This statement of Prof. Maly is not only contrary to my actual description, but is directly opposed to the stated motives which led to my experiment. For I say that it was evident, from the preliminary experiments, that bilirubin in the presence of hydrobromic acid and moisture could not be completely brominated, just because it deliquesced, and that for this reason I had instituted an experiment in which this action was com- pletely avoided. I then describe the experiments with bromine given in the substance of my researches and which need not here be repeated. I further describe the properties of the new compound, and particularly that it becomes quickly changed in several solvents. It would have been more to the interest of Prof. Maly if he had studied my statements relating to these changes, and had re- peated the experiments, — if he had heeded my warning, and excluded moisture and hydrobromic acid from his experiments ; it would . have saved him much disappointment had he com- prehended that I saturated bilirubin with bromine by offering it an excess, and did not only, as Maly reports, pass it over for some time. All these necessary precautions Prof. Maly has neglected, and in consequence has arrived at conclusions which have no foundation. Prof. Maly further endeavours to influence the judgment of the Academy by raising doubts in general regarding my experiments ; first, on the ground that I had performed each experiment only once ; secondly, because I had not analysed the final product. In order to meet this objection, I have repeated the experiment described yet two several times, and have analysed the products by determining quantitatively the amounts of carbon, hydrogen, nitrogen, and bromine ; during the ele- mentary combustion none of the hypothetical bromide of carbon BILIAEY PIGMENTS. 325 was observed, with the alleged escape of which Prof. Maly en- deavours to explain his discordant carbon-numbers. In two paragraphs the experiments and analyses here alluded to are described in detail ; as they are given in the text of my researches, they have not here been repeated. In his remarks on the formula assumed for his bromine pro- duct. Prof. Maly says — ' The most simple expression of the composition of bilirubin is, according to the analyses of Prof. Stadeler and myself, CigHigNjOg.' In making this statement Prof. Maly loses sight of ' the usual duty of characterising pre- vious knowledge,' or other knowledge. I therefore felt myself called upon to search for the analyses of Prof. Maly, of which he claims that they prove the above formula, but with the ex- ception of one carbon and two hydrogen determinations, in the Reports of the Vienna Academy (1868, vol. Ivii. p. 97), I could not find any proofs of this assertion. In particular, Prof. Maly has not made any determinations of the nitrogen, and, above all, no determination of the atomic weight, and has not even sub- jected his results to control on different preparations. For these reasons I consider his formula as of little significance opposite my theory, which is based upon numerous preparations and well-defined compounds, and now more than fifty elementary analyses. Prof. Maly, in the paper just quoted, supports his formula for bilirubin by the authority of Stadeler, but on p. 102, note 2, he censures the proceedings of Stadeler, who had produced a formula for biliverdin by recalculating an old analysis of Heintz, which had been executed upon material declared by Stadeler himself to have been a mixture of pigments. Prof. Maly him- self points out that this proceeding was characteristic of the work of Stadeler. I dare not trouble the Academy with a criti- cism of the researches of Stadeler, particularly as I have given one in my paper contained in the Journal of the Chemical Society, 1875. I think it, however, necessary to direct the attention of the Academy to the circumstances that the doubling of the formula CigHigNjOj into Cg^HgeN^Og— which Prof. Maly introduces in the fifth paper, in 1875, as a necessary consequence of his discovery of a brominated compound, and then further particularly as a novelty — had already been adopted by Stadeler, 326 ANNALS OF CHEMICAL MEDICINE. before 1870, in a letter to the editor of Gmelin's Handbuch, Prof. Karl Kraut, and been published by the latter in an Appendix to the last volume of the Handbuch. In the letter alluded to, Stadeler, in view of my researches, abandons all his former formulae, and coerces my results by an utterly unjusti- fiable process of recalculation, in which no single analytical result harmonises with the new hypothesis into some sort of support for his doubled formula and hexabasic acid hypothesis without having produced a single compound or made a single new analysis. Prof. Maly causes to himself many diflBculties by his pre- conceived opinions and uncontrolled imagination, as I am obliged to prove now more in particular. At first he believed every green product of the colouring matter of gallstones to be ' biliverdin ; ' thus the product by chloroform and glacial acetic acid, which seduced him into the belief that he had transformed bilirubin, an alleged amide, into ammonia, on the one hand, and an acid free from nitrogen, viz. biliverdin, on the other ; and, what was still more surprising, that he had retransformed this acid into the original amide, bilirubin, by simply mixing it with ammonia (see 'Preliminary Communication on the Colouring Matter of Bile ' — Reports of the Meetings of the Vienna Academy, vol. xlix.). A part of this conclusion Prof. Maly has withdrawn {Reports of the Vienna Acad., 1868, p. 98), namely the one concerning the decomposition of bilirubin into ammonia and ' biliverdin.' The ammonia which he had before found he then explained to have been an impurity of his prepa- ration. But in 1868 he still insisted upon the erroneous pro- position that the product of heating bilirubin, glacial acetic acid, and chloroform, in sealed glass tubes, was ' biliverdin.' For a long time he also believed his brominated product to be 'biliverdin' {Vienna Acad. Reports, 1868, p. 104). I have shown that in its first form it was a mixture of blue brominated bilirubin with orange bilirubin : this Prof. Maly now admits himself, in his fifth paper, as his original discovery. In the fifth paper Prof. Maly again produces 'biliverdin ' by treating the brominated product with alkalies. But it contains always yet a small trace of bromine, and a little ash, which is deducted. In the analyses 3 and 4 of his former 'biliverdin' BILIARY PIGMENTS. 327 (^Vienna Acad. Reports, vol. Ivii. p. 105) lie was even obliged to deduct as much as ' circa 2 per cent.' of ash. How can an author who works with such preparations call others to account for the alleged impurity of their preparations ! In short, Prof. Maly is unacquainted with the fact that there is a great number of derivatives of the colouring matter of gall- stones, which all have this in common, that they are green, but are not for that reason alone biliverdin (CgHgNOj). Thus the green cholothallin obtained by the action of oil of vitriol upon bilirubin, and subsequent treatment with water, is bilirubin to which the elements of water have been added. In two paragraphs I give a summary of the data concerning the action of bromine and hydrobromic acid upon bilirubin, contained in the text of my researches. Prof. Maly operated with bromine in moist chloroform. Every molecle of bromine yielded him a molecle of hydrobromic acid, which now in its turn attacked the bilirubin. The mois- ture precipitated the mixture. The precipitate consisted proxi- mately of bromo-bilirubin, with some hydrobromic acid, of which it yet lost some on drying, and of perhaps a little hydro- bromo-bilirubide, for the action of HBr requires more time than that of Br. But now the product was put in alcohol, which immediately began its reducing action. Consequently the powder was ' dark blue-green,' while the probable monobromo- biUrubin is monochromatic blue, as I state in my essay (and not 'nearly' monochromatic blue, as Prof. Maly reports my description). Again, the ethereal solution which Prof. Maly obtained when treating bilirubin in ether with bromine was ' dark greenish- blue,' and became blue only when the monobromo-bilirubin prevailed, by means of its greater absorptive power for green . The residue of this ether process was ' always in thin layers, green.' This change from blue to green was evidently the reason which caused Prof. Maly to abstain from further at- tempts at purifying his products. The incessant loss of bromine which the products of substitution, as well as of reduction, experience in alcohol or ether, is so great and marked that in some of my experiments the amount of bromine fell from 49*8 to 16 per cent., and the violet product, which had been spluble 328 ANNALS OF CHEMICAL MEDICINE. in ether with a violet colour, became green, quite insoluble in ether, but soluble in alcohol with a green colour. After the foregoing I hardly know whether there is any part of Prof. Maly's research left which requires refutation or explanation, excepting perhaps the remarkable results of his carbon determinations in which the carbon found varied between 35'51 and 47"83 per cent. However, this is by no means in- cumbent upon me, but upon Prof. Maly. The assumption of ' a very volatile bromide of carbon ' cannot satisfy the demand for explanation, — much less can the proposition that an accurate determination of the quantity of carbon was not essential to the ascertaining of the composition of the bromine product. On the contrary, it must be maintained that such results and corollaries are directly opposed to the principles of chemical science, and slap the endeavour for final accuracy rudely upon the face. We have seen above how Prof. Maly believed every green product of the metamorphosis of the colouring matter of gall- stones to be 'biliverdin.' In the same manner he assumed every blue product to be a result of oxydation, and identical with the blue product which forms transiently during the reaction of bilirubin with nitric containing nitrous acid. But the blue product which is obtained with bromine is now transmuted into a product of substitution, and there is no reason to believe that the blue product obtained by means of nitrous acid is a product of oxydation. Indeed it is impossible to predict what it is. Pure nitrous, acid forms no blue product with bilirubin. It seems that the presence of alcohol is necessary for the production of a blue body. From my experiments, it follows, first, that the formula which Prof. Maly has given for his ' choloteline 'is improbable, and that the diminution of the carbon in this re- action which Prof. Maly observed — but left out of consideration or explained away — is much more probable than the alleged oxydation without any loss of carbon, particularly as the nitrogen in the product is the same quantity as that which was present in the bilirubin employed. The three silver compounds of choloteline, in which the silver rose in quantity from the first to the third, of which, however, only the second one furnished an acceptable theory (Vienna Acad, Hep. vol. lix. p. 605), are BILIAEY PIGMENTS. 329 analogous to the zinc compounds of hydro-bilirabin, in which the zinc rose from 14-6 to above 37 per cent. {Liehig's Ann. clxiii. 86). The observation of the influence of sodium- amalgam upon bilirubin, which led Prof. Maly to the discovery of the so-called hydro-bilirubin, would have been an interesting progress in our knowledge concerning bilirubin. But as the author starts from erroneous views regarding the composition and molecular weight of bilirubin, his conclusions regarding his product and its com- position, and regarding the formula of the change, are necessarily erroneous. The announcement at the head of this article on hydro- biUrubin, of the transformation of bilirubin into the colouring matter of urine caused me to read it with expectations which were speedily disappointed when the absolute ' urine-colouring matter ' became limited to Jafife's urobilin. I thereupon made many experiments which I have d,escribed in my paper (in the Journ. Chem. Soc. May 1875), and which, without exception, negative the alleged metamorphosis. On this point I wrote to Prof. Maly :^' I have now compared the products (of the reduc- tion of bilirubin) with all urinary colouring matters with which I am acquainted, and with Jaffe's product, which I have pro- duced for the purpose, but have not discovered any identity. Urochrome and uroerythrin are quite different as regards solu- bility and chemical properties ; uroxanthin, which it is now the fashion to call " indican," also. The products of the cleavage of urochrome — namely uromelanin, uropittin, and omicholin also. These products can be easily obtained from urochrome by acids, but your hydro-bilirubin yields nothing of the kind, and is not much changed by boiling with hydrochloric acid. Jaffe's uro- bilin has never been isolated, never been analysed. According to my comparison the mass is a mixture of urochrome, uroery- thrin, with a little omicholin already separated. The process (of Jaffe) is not inviting, and the result in any case, a compli- cated tincture. I am therefore unable to adopt your conception of the metamorphosis of the colouring matter of bile, into the or a colouring matter of the urine in even a single particular.' In his fourth paper (^Vienna Acad. Rep, vol, Ixx. 1874) on biliverdin, Prof. Maly relates how he ' already earlier and inde- 330 ANNALS OF CHEMICAL MEDICINE. pendently of that of Thudichum,' had arrived at the formula CjgHjgN204. However I had only arrived at half that formula, namely CgHgNOj; but this difference is unimportant; it is quite true that I arrived at this formula independently of Prof. Maly, but I have also a priority with reference to it which does not appear quite clearly in Prof. Maly's representation ; for my research on biliverdin was communicated to the Eoyal Society already on November 14, 1867, and published in abstract in the Proceedings of that Society, vol. xvi. p. 217. The relative research of Prof. Maly on the other hand was communicated to the Academy of Vienna only on February 6, 1868. In the paper No. IV. just alluded to, concerning biliverdin, Prof. Maly communicates two further elementary analyses of biliverdin, and, as now his results exactly correspond with mine from the year 1867, he is satisfied that the composition of this body can now be considered as definitely established. This time the biliverdin did not contain any ash, and was made according to the orthodox method (of Heintz), with soda and air. There still figures the biliverdin made by means of chloro- form and glacial acetic acid, but it cannot easily be precipitated by water. A new green matter obtained by monochloracetic acid from bilirubin, also makes its first appearance. The meta- morphosis each time yields less ' biliverdin ' than the weight of the employed bilirubin, but was obliged in each case to support the theory without analysis. But now comes the buttress of Prof. Maly's theory, according to which biliverdin is bilirubin p^us oxygen only, CigHigN^Oj 4- = CjgHjjNgO^. Bilirubin is transformed into biliverdin, according to the orthodox method, with soda and air, and the product weighed ; 0'4558 grm. bilirubin yields actually 0*4458 grm. biliverdin, both dried at 100°. A loss was therefore sus- tained. There was no guarantee that the biliverdin did not yet contain bilirubin. But the hypothesis demanded a greater weight of biliverdin than was that of the bilirubin employed. In consequence the filtrates were now evaporated, and what they lost by ignition was scored as biliverdin. Even the washing water is put into requisition and compelled by means of a process termed ' ocular measurement ' {Augenmessung) to supply more than half of the desired increase. Thus by hook BILIARY PIGMENTS. 331 or by crook Prof. Maly succeeds in calculating an increase of 4-3 parts upon 100 of bilirubin, whereas his hypothesis demands an increase of 5'6 parts. ' This,' says Prof. Maly, ' agrees as accurately as can be demanded under such circumstances.' As often as the experiments agree very badly with the hypotheses of Prof. Mafy, and prove rather the contrary than the hypothesis, he consoles himself with the expression that the result was as good as could be demanded under the circumstances. Thus again, in his first synthetical bromination experiment, paper V., he finds an addition of only 2*74 atoms of bromine to his new bilirubin, CjjHggN^Og, but observes — 'This accorded so far with the increase by 3 atoms of bromine which were de- manded by calculation and supported by experiment, as could be demanded in determinations of this kind. But the very next synthesis yields 3"1 atoms of Br ; the ex- periment could therefore be made in a more accurate manner than could previously be demanded. But this experiment also has no value at all. Bilirubin, treated with bromine in excess, yields, as I have proved, always dibromo-bilirubin, CgH^BrgNOj, in which N : Br=l : 2. In Maly's supposed molecle N : Br= 4 : 3. Consequently if my product were quadrupled, in order to let it contain 4 N, it would contain 8 Br, — say 8 atoms of bromine. In any case, therefore, Maly would only have intrO' duced 3 atoms of bromine out of 8 which can be introduced. But this high formula is so entirely negatived by my compounds of bilirubin with silver, calcium, baryum, lead, zinc, and others, that it cannot come into consideration. Ekeoneous Statements of Hoppb-Setlee conceemng the CoLOtJEiHG Matters of Bile. After my research had been published {Journ. f. pract. Chem. 104, 1868, 1) Hoppe-Seyler wrote a notice of it in the Medical Jahresbericht, which had more the character of an aspersion than that of a report, or even of a criticism. He himself had no personal knowledge whatever of these substances, and has up to the present time not published any researches 332 ANNALS OF CHEMICAL MEDICINE. upon them. He had therefore not the slightest qualification to as much as doubt my researches, and the challenge which I addressed to him as well as to the editor-in-chief of the Jdhres- bericht. Prof. Virchow, that he, Hoppe-Seyler, should refute my researches by experiments and analyses, has to this day remained unanswered. On the other hand Hoppe-Seyler has, in his Handbook of Physiol, and Pathol. Analysis, given a representation of the biliary pigments, of which the statements which Stadeler made in 1863, form the sandy foundation, in perfect disregard of the fact, that Stadeler himself had abandoned these, his second variety of formulae, already before 1870. Now as Hoppe-Seyler produces no reasons why this resignation of Stadeler's should not be adopted by others, and brings no new grounds for re- taining Stadeler's formulae, his proceeding does not only mani- fest a want of acquaintance with the subject, but also, and particularly, a want of acquaintance with its literature. Of my results Hoppe-Seyler now says, that ' it would lead too far ' to describe them. This indefinite utterance is a feeble excuse for the omission of a well-recognised literary duty, and it seems to be felt when further on Hoppe-Seyler endeavours to justify his reticence by a surmise (p. 211) to this effect, that the bilirubin examined by me had been a different body from that examined by Stadeler and Maly. This view was not for an instant conceived by Stadeler himself, and has never been put forth by Maly. Stadeler accepted my bodies and my facts, but used them, by means of an arithmetical operation, which was more desperate than reasonable, for the production of an hypothesis, intended to save his double formulee. The assump- tion of ' different kinds of bilirubin ' has therefore no better foundation than the imagination of Hoppe-Seyler. Towards the end of his description of the matters in question, the confusion of Hoppe-Seyler rises to a wild conflict of errors. A colouring matter, said to forin in ox-bile on standing, and formed in other ways not described, also contained in sheep's- bile, is said to be identical with the product of oxydation of bilirubin, biliverdin, and bilifuscin, by nitric acid or bromine water. It is confounded with the choleverdin of Stockvis, and the bilicyanin of Heynsius and Campbell, and it is claimed that BILIARY PIGMENTS. 333 he, Hoppe-Seyler, had described 'the spectral phenomena' already in the earlier edition of his handbook, and that Jaffe and Bogomoloff had described them after him. In short, the entire representation of this subject given by Hoppe-Seyler is unworthy of literature and of science. The other protests referred to in the Preface are the fol- lowing : — ' On Acetic Acid, Formic Acid, and supposed Sulphurous Acid and Nitrous Acid from Human Urine.' Archiv Physiol. 15, 1877, 12. ' Eepetition of the Experiment of Grscheidlen for the Ex- traction of Sulphocyanio Acid from Human Urine.' Archiv Physiol. 15, 1877, 51. ' On Indican, and the relations of Prof. Max Jaffe to the definition of Chemical Purity.' Archiv Physiol. 15, 1877, 343. A more general view of these objectionable practices has been taken by C. T. Kingzett and H. W. Hake, in their impor- tant article entitled 'Physiology and its Chemistry at home and abroad' in the Quarterly Journal of Science, 1877. SOME DEFINITIONS OP NAMES AND SYNONYMS. Amylonicle: «■ body siimilar to amylon, or containing an amylon-like radicle in its constitution ; this radicle appears as a kind of sugar after de- composition of the body. Celhdin : the substance forming the cells of plants, wood, etc. The end- ing in has been substituted for the former terminal ose, as it seems convenient to reserve this latter for sugars only. Cliemolysis : decomposition of organic compounds into more simple sub- stances by merely chemical agents, such as sulphuric acid or baryta. CoXUAd : a body like gelatin, which does not dialyse through parchment paper. Crystalloid : a body which behaves like a crystallising substance in this, that it dialyses through parchment paper. Fell, to : verb ; Saxon equivalent of the Latin to precipitate. Gh/cin : the same as glycosm or glyliocollf amido-acetic acid ; it is proposed to retain glycin only, and discontinue the second and third name. Hydrocklor : abbreviation of hydrochloric acid. HydrotMon : abbreviation of hydrosnlphurio acid, or sulphuretted hydro- gen ; used by Bergmann and Berzelius, and by Gtmelin throughout his great treatise on Chemistry. Molecle: English form of the Latin 'molecula,' ordinarily spelt in the French form 'molecule.' Orgamoplastic : adjective used in physiology to describe the chemical compounds which constitute tissues or organs ; tissue-forming or histogenetic would be the same, but less euphonious. Patholysis : decomposition of organic substances in the living body under the influence of disease-ferments, or disease-causes in general. 336 SOME DEFINITIONS OF NAMES AND S'SNONYMS. Phyiiolysis : decomposition of organic substances under the influence of water, air, and putrefaction-ferments ; therefore equivalent to putrefaction, but wider in its significance. Pyrolym : decomposition of organic substances under the influence of a raised temperature only ; dry distillation ; a process compared to the burning of a body in the presence of that oxygen which is contained in it. QuaUtation : process of finding out the quality, therefore abbreviation for qualitative analysis. Quantation : process of ascertaining the quantity, therefore abbreviation for quantitative analysis or estimation, or determination. RadAcle : English form of the French 'radical,' analogous to 'molecle.' Sarcolactio acid: lactic acid from flesh, formerly termed paralactic acid. Zymolactio acid : lactic acid obtained by fermentation, e.g. of milk, or sugar with cheese. ^molym: decomposition of organic substances by ferments, organised or INDEX. ACI Acid, acetic, from albumins, etc., 29, 44 — amido-acetlo, 44 — amido-benzoio, 44 — amido-butyric, 44 — amido-caproic, 44 — amido-oenanthic, 44 — amido-oxypropionic, 44 — amido-propionio, 44 — asparaginic, series, 33, 44 — butyric, 148, 157 — carbomc, 43 — cerebric, 254 — - cerebrinio, 262 — cholesteric, 120 — cholic, 118 oxydation of, 120 — formic, in brain, 104, 115 — glutamic, 44 — glutimic, 44 — glykooholic, 117 — hydracrylic, 112 T- kryptophanic, 42 — lactic, 104 — lanophanio, 42 — oxalic, 43 — paraphanic, 42 — strong, in amido-mixture, 28 — succinic, 44 in brain, 104, 115 — sulphurous, 43 — taurooholic, 118 — valerianic, from albumin, etc., 155 Acrylic series, of amido-acids, 33 Albumin, 14, 17 — bearing with acetic acid, etc., 189 — of blood, 14 — ohemolysis of by baryta, 20 caustic potash, 148 — coagulation of, 197 — electrolysis of, 201 Albumin, formulse of, 200 — soluble properties and metamor- phoses of, 175 — quantation of, 183 Albumino-dextrose, 17 Albuminol, 21, 34 Albuminose, 17 Alkaloids of urine, 160 — second new, 166 — third new, 171 — fourth new, 174 Alpaca, hair of, 41 Amidocaproio acid, 34 Amido mixture from albumins, 22 elements in, 30 Amidovalerianio acid, 34 Ammonia, 23, 43 — from albumins, 21, 23 — in brain-educts, 255 Amylon, 2 — hepatic, 17 Amylonides, organoplastio, 1 Anti-contagionists, 217 Antiseptic surgery, 309 AscidiEe, 5 Australian wool, 41 Bacillus subtilis in blood-putrefac- tion, 157 Bases, volatile, from urine, 164 Bile, ohemolysis of, 117 — colouring matters of, 264 Bilifusoin, 297 Bilirubin, 264 — action of bromine on, etc., 285, 288 — action of hydro-bromic acid on, 287 — hydrogenised by sodium, 295 — and iodine, 292 — and nitric acid, 292 — and nitrotis acid, 292 338 INDEX. BIL Bilirubin and sulphuric acid, 283 Biliphsein, 268 Blliverdin, 277 — hydrogenlsed, 283 — monobrominated, 281 Birds, eggshells of, 93 — ■ — pigments of, 99 Blood, putrefaction of, 163 Boracicacid in antiseptic surgery, 314 Bovifuscopittin, 299 Boviprasin, 298 Brain, alkaloids and amido-acids in, 105 — organic acids in, 104 Butalanin, 36 Butyric acid, 181 Butyro-leuoein, 44 Byssan, 8 CALCltTM in braiu-educts, 255 Cane-sugar, 52 Carbolic acid in antiseptic surgery, 310 Carbonate from albumin, 22 Cartilage, 10 Casein, 14 Caprono-leucein, 44 Cattle-plague, 213 Cellulin, 1, 17 Cellulin-amide, 44 Cerebrin, 17 Cerebrinic acid, 262 Cerebrose, 17, 18 Chalazse, 175 Chitin, 8, 129 — Frfemy's, 138 — Rouget's, 139 Chitosamin, 140 Chitose, 17, 135 Cholamide, 120 Chelate of ethyle, 119 Cholera, alvine flux in, 23(> — experiments on, 242 Cholin from bile, 118 Choloohlorin, 277 Cholooyauin, 294 Cholonematin, 297 Cholophsein, 270 Cholophseiriates, 270 Oholothallin, 285 Chondrin, 17, 39 Chondrinogen, 10 Ohondrose, 17 Collidin, 37 Colouring matter of bile 264 -of birds' eggs, 99 Conchiolin, 8 Conchiolose, 17 GLT Contact action, 216 Contagionists, 217 Contaginm vivum, 228 Copper in brain-educts, 255 Corpse-poison, 214 Cruentin, 95 — varieties and spectra, 96, 98 Dbhtdkation of muscle in cholera, 240 Dextrin, 3, 44, 55 Dextro-glucose, 3 Dextrose, 17 — potash, 64 Dialysate of albumin, 177 Dialyser, Huizinga's, 1 86 Dialysis of albumin, 176 Diastas, 45 Diphtheria in animals, 217 — origin of, 223 — in pigeons and fowls, 226 Disinfection, 206 Elastic tissue, 11 Elastin, 153 Epipyrrol, 44 Brythrodextrin, 61 Ethoohlorln, 299 Ethyle, chelate of, 119 Fermentations, under pressure, 232 Ferment, pancreatic, 62 Ferments, in plants, 49 — starch-transforming, 45 Fever, enteric by water-pollution, 218 — splenic, 229 — typhoid, 210 Fibrin, 14 Fibroin, 8, 17, 40 Fibrose, 17 Fluopittin, 14 Fluorescentin, 14 Gelatin, 11, 12, 17, 32, 39 Gelatose, 17 Globulin, 14 Glucosides, 8 Glutamic acid, 44 Glutimic acid, 44 Glutin, putrefaction of, 153 Glycin, 40 Glycogen, 17, 63 GlycokoU, 40 Glycosamin, 129 Glycosin, 40 INDEX. 339 GLT Glykocholic acid, 117 Grape sugar, 52 Gum, 3 Haie, human, 41 — of alpaca, 41 Heat, mechanical theory of, 65 Hemialbumin, 143 Hemiprotein, 143 Hemisymmetry of albumins, 143 Hemochrome, putrefaction of, 153 Hepatic amylon, 17 Horn-substance, 12 Hyalin, 7, 8, 17 Hyalose, 17 Hydrophobia, 208 Hypoxanthin, 44 — from albumin, 147 ICHTHTOCOLLIN, 17 Ichthyocollose, 17 Indol, 44, 148 Infection, 204 — origin of, 217 Inoculation, 210 Inosite, 3 Iron in brain-eduots, 255 Isinglass, 39 KbeASIN, 17, 262 Keratin, 12, 17 Keratose, 17 Kephalin, 255 Kreatin, 162 Kreatinin, 162 — study of, and its gold chloride salts, 168 Lactates of calcium, 110 — of zinc from human and ox brains, 107 Lactic acid, 44 — in brain, 104 — constitution of isomers. 111 — polarisation of, 108 Lactin, 17 Lactose, 3 Lanophanio acid, 42, 44 Lepra, 235 Leucin, 44 Leuceins, series of, 33 Leucin in brain, 105 — quantation of in amido-mixture, 31 Leucins, series of, 33 PEO Maltose, 3, 52, 53 Marsh-gas, 43 Mice afEected by cholera, 242 Microsporon diphtheriticum, 235 — septicum, 233 Microzymes, 168 Milk-sugar, 3 Mucilage, vegetal, 3 Mucin, 9, 17 — putrefaction of, 153 Mucinose, 17 Mucus, 9 Muscle in cholera, 240 Muscoprasin, 299 Myelin, 255 Myosin, 14 OONIN, 175 Ossein, 11, 32, 39 Ovocruentin, 99 Ovoviridin, 101 Oxalate from albumin, 22 Oxalic acid from albumin, 24 — organoplastic substances, 24, 28 Oxyhemialbumin, 146 Pancbbatin, 45, 62 Paraglobulin, 14 Paralbumin, 16, 17 Paralbuminose, 17 Parchment paper, different kinds of, 191 — of Gaine, 192 Phenol, 44, 148 Phosphates in educts from brain, 255 — of calcium and magnesium in brain, 255 Phosphorus in ' protagon,' 258 Phreuosin, 17, 262 Pigments of birds' eggs, 99 — placenta, 103 Pine sugar, 3 Finite, 3 Placenta, pigments in, 103 Plague, oriental, 215 Plasmin from white of eggs, 178 Polarisation of albumin, 196 — lactic acid, 108 Potassium in brain-educts, 255 — salt, peculiar, in brain, 106 Protagon, alleged existence of, in brain, 254 — experiments on, 254 Protein, theory of Mulder, 32 — tritoxyde, 146 Protocouia, 37 340 INDEX. Ptyalin, 45 Ptyalose, 62 Putrefaction of albuminous sub- stances, 153 Pysemia, 214, 309 Pyrrol, 21, 44, 148 Pyrrol-red, 84 BbdttciN, 161 Beduoin-barium, 161 Salicylic acid preparations in an- tiseptic surgery, 312 Saliva, influence of on starch, 62 Salts, inorganic in cholera flux, 236 Sarkolactic acid, 109 Soatol, 44, 148 Scyllite, 3 Serioin, 19, 135 Serin, 14, 17 Serinose, 17 • Serosamide, 17 Serpent's skin, 14 — eggs, 154 Serum-albumin, 14, 183 Sheep's wool, 41 Silk-gelatin, 9 Skatol, 44, 148 Small-pox, 210 Sodium in brain-educts, 255 Spermatin, 301 — compounds, 303, 307 ZYM Splenic fever, 229 Spongin, 8, 17 Spongose, 17 Starch, soluble, 61 — transformations of, 45 Succinic acid, 44 — in brain, 115 Sulphite from albumin, 22 Sulpho-cholocyanin, 284 Sulphur-compound from albuiuin, 21 Sulphurous acid, 43 Syntbnin, 14 Tatthocholic acid, 118 Trichiniasis, 204 Trimethylamin, 164 Tunicata, 4 Tunicin, 17 Tunicose, 17 Typhoid fever, 210 Typhus, 209 Tyroleucin, 36, 44 Tyrosin in brain, 105 — quantation of in amtdo-mixture 31, 34, 44 Ueochbome, an alkaloid, 174 VALEEiANO-leucein, 44 Zymolactic acid, 11 LIST OF AUTHORS. Aenim, 202 Arnold, 198 Aronstein, 176 Bakanbtzkt, J., 49, 51 Barfoed, 55 Barreswill, 47 BSchamp, 158 Bernard, 16, 47, 48, 50 Bert, P., 231 Berthelot, 6, 7, 129 Bibra, 254 Biot, 52 Bondonneau, 59 Bopp, 14, 148 Bostock, 198. Bottoher, 175, 302 Bouchardat, 47, 48 Boussingault, 6 Brande, 202 Brieger, 149 Brondgeest, 303 Briicke, 61 Biitschli, 133 Cabpbntbe, a., 219' Charcot and Eobin, 301 — and Vulpian, 301 Chauveau, 234 Chevreul, 141, 198 Child, Gilbert, 221 Children and Daniell, 132 Commaille, 199 Couerbe, 175, 254 Cruikshank, 45 DANILEWSKT, 196 Davaine, 229 De Lnca, 13 HUF Denis, 183 Dubrunfaut, 49, 52 Dumas, 6 — and Cahours, 199 — Prevost, 198 BiCHWALD, 9, 183 EmmerUng, 133 Bngler and Janeoke, 148 Epstein and MuUer, 48 Erlenmeyer, 112, 113 — and SchofEer, 154 EORSTBE, 301 Er^my, 8, 11, 188, 254 Friedreich, 302 G-AMGEE and another, 254 Gavard, 225 Gerhardt, 12 Gibert, 226 Gmelin, 201 Gobley, 198, 254 Goebel, 141 Gorup-Besanez, 48 Graham, 176 Grossmann and Mayerhaussen, 157 Gu6rin-Varry, 51 Guillot, 142 Gundelach and Strecker, 120 Habtiug, 197, 301 Heintz, 112 Hensen, 47 Heynsius, 183 Hilger, 154 Hnber, 303 Hufner, 117 342 LIST OF AUTHORS. HDN Hunt, 11 Huppert, 302 Jahn, 175 John, 141 Kaufmann, 157-158 Kelly, C, 222 Kirohhof, 45 Klebs, 228 — and Soyka, 233 Tiegel, 230, Kllmenko, 113 Kuhne, 148 Lamaek, 4 Lassaigne, 47, 129, 199 Lauenstein, 303 Ledderhose, G., 129 Lehmann, 132, 198 Lepme, 48 Leuchs, 46, 62 Leuokart, 8 Leyden, 303 Leyer and KoUer, 141 Liborius, 184 Liebermann, C, 94 Liebig, 148 Liebreich, 254 Lister, 314 Lowig and EoUiker, 4, 6 Lowitz, 52 Liioke, 7 Magbndib, 47 Makris, 133 Marx, 202 Mayer, E. J., 65 Melsens, 197 M&at and Guillot, 141 Meyer, Hans, 54 Mialhe, 47 Milne-Edwards, 5, 6 Monnier, 197 Morin, 202 Mulder, 47, 51, 199 Muller, W., 153 Musculus, 52 — and Gruber, 53 ' von Mering, 55 NASBB, 0., 20, 62 Nenokl, 149 THU Neumann, 302 Nioati, 225 Nussbaum, J, N. von, 809 Obolknski, 10 Odier, 129 Ogle, W., 219 O'Sullivan, 52, 57 PASTBUE, 225 Payen, 6, 52, 129 — and Persoz, 46 Peligot, 129 Proust, 52 QUBIBBL, 226 Eatbb, 229 Eedtenbacher, 120 Riiling, 199 Eumelin, G., 65, 66 Sandeas and Bouchardat, 47 Saussure, T. de, 46 Scheele, 12 Scherer, 16, 183 SchifE, 47 SoMieper, 120 Schlossberger, 8, 51, 129 Schmidt, C, 4, 7, 129 Schonbein, 48 Schreiuer, 175, 301 SchtsoherbakofE, 64 Sohiitzenberger, 14, 20 — and Bourgois, 34, 38 Schwann, 46 Schwartzer, 52, 57 Schwarzenbach, 200 Secretan, 149 Seegen, 62, 63 — and Kratschmer, 48 Siebold, von, 4, 6 Smee, 202 Sorby, 93 Stadeler, 7, 129 Tappeinbr, 118, 120 Theile, 199 TMnard and Dupuytren, 52 Thomson, R. 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