Monographs on Biochemistry UC-NRLF NATURAL BASES to Mo.\W Lib* U- 2^ ^ feO MONOGRAPHS ON BIOCHEMISTRY EDITED BY R. H. A. PLIMMER, D.Sc. AND F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S. GENERAL PREFACE. THE subject of Physiological Chemistry, or Biochemistry, is enlarging its borders to such an extent at the present time, that no single text-book upon the subject, without being cumbrous, can adequately deal with it as a whole, so as to give both a general and a detailed account of its present position. It is, moreover, difficult, in the case of the larger text-books, to keep abreast of so rapidly growing a science by means of new editions, and such volumes are therefore issued when much of their contents has become obsolete. For this reason, an attempt is being made to place this branch of science in a more accessible position by issuing a series of monographs upon the various chapters of the subject, each independent of and yet dependent upon the others, so that from time to time, as new material and the demand therefor necessitate, a new edition of each mono- graph can be issued without re-issuing the whole series. In this way, both the expenses of publication and the expense to the purchaser will be diminished, and by a moderate outlay it will be possible to obtain a full account of any particular subject as nearly current as possible. The editors of these monographs have kept two objects in view : firstly, that each author should be himself working at the subject with which he deals ; and, secondly, that a Bibliography, as complete as possible, should be included, in order to avoid cross references, which are apt to be wrongly cited, and in order that each monograph may yield full and independent information of the work which has been done upon the subject. It has been decided as a general scheme that the volumes first issued shall deal with the pure chemistry of physiological products and with certain general aspects of the subject. Subsequent monographs will be devoted to such questions as the chemistry of special tissues and particular aspects of metabolism. So the series, if continued, will proceed from physiological chemistry to what may be now more properly termed chemical physiology. This will depend upon the success which the first series achieves, and upon the divisions of the subject which may be of interest at the time. R. H. A. P. F. G. H. MONOGRAPHS ON BIOCHEMISTRY EDITED BY R. H. A. PLIMMER, D.Sc. AND F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S. ROYAL 8vo. THE NATURE OF ENZYME ACTION. By W. M. BAYLISS, D.Sc., F.R.S. Third Edition. 55. net. THE CHEMICAL CONSTITUTION OF THE PROTEINS. By R. H. A. PLIMMER, D.Sc. (Two Parts.) Part I. Analysis. Second Edition, Revised and Enlarged. 55. 6d. net. Part II. Synthesis, etc. Second Edition, Revised and Enlarged. 35. 6d. net. THE GENERAL CHARACTERS OF THE PRO- TEINS. By S. B. SCHRYVER, Ph.D., D.Sc. 2s. 6d. net. THE VEGETABLE PROTEINS. By THOMAS B. OSBORNE, Ph.D. 35. 6d. net. THE SIMPLE CARBOHYDRATES AND THE GLUCOSIDES. By E. FRANKLAND ARMSTRONG, D.Sc., Ph.D. Second Edition, Revised and Enlarged. 55. net. THE FATS. By J. B. LEATHES, F.R.S., M.A., M.B., F.R.C.S. 45. net. ALCOHOLIC FERMENTATION. By A. HARDEN, Ph.D., D.Sc., F.R.S. 4 s. net. THE PHYSIOLOGY OF PROTEIN META- BOLISM. By E. P. CATHCART, M.D., D.Sc. 45. 6d. net. SOIL CONDITIONS AND PLANT GROWTH. By E. J. RUSSELL, D.Sc. 55. net. OXIDATIONS AND REDUCTIONS IN THE ANIMAL BODY. By H. D. DAKIN, D.Sc., F.I.C. 48. net. THE SIMPLER NATURAL BASES. By G. BARGER, M.A., D.Sc. 6s. net. THE DEVELOPMENT AND PRESENT POSI- TION OF BIOLOGICAL CHEMISTRY. By F. GOWLAND HOPKINS, M.A., M.B., D.Sc., F.R.S. THE POLYSACCHARIDES. By ARTHUR R. LING, F.I.C. COLLOIDS. By W. B. HARDY, M.A., F.R.S. RESPIRATORY EXCHANGE IN ANIMALS. By A. KROGH, Ph.D. NUCLEIC ACIDS. THEIR CHEMICAL PRO- PERTIES AND PHYSIOLOGICAL CON- DUCT. By WALTER JONES, Ph.D. PROTAMINES AND HISTONES. By A. KOSSEL, Ph.D. LECITHIN AND ALLIED SUBSTANCES. By H. MACLEAN, M.D., D.Sc. ORGANIC COMPOUNDS OF ARSENIC AND ANTI- MONY. By GILBERT T. MORGAN, D.Sc., F.I.C. LONGMANS, GREEN AND CO., LONDON, NEW YORK, BOMBAY AND CALCUTTA. THE SIMPLER NATURAL BASES BV GEORGE BARGER, M.A., D.Sc. FORMERLY FELLOW OF KING'S COLLEGE, CAMBRIDGE PROFESSOR OF CHEMISTRY IN THE ROYAL HOLLOWAY COLLEGE, UNIVERSITY OF LONDON LONGMANS, GREEN AND CO. 39 PATERNOSTER ROW, LONDON NEW YORK, BOMBAY AND CALCUTTA 1914 0? CALIFORNIA Y TO H. H. D. AGR1C, DEPT, PREFACE. IN the following pages I have endeavoured to give an account of those basic substances of animals and plants which are of general biological interest, either because of their wide distri- bution, or on account of their close relationship to the proteins and phosphatides. In contradistinction to the typical vegetable alkaloids, these bases have a simple chemical constitution. By a more or less arbitrary delimitation of the subject matter, involving for instance the total exclusion of purine bases, I have aimed at giving, in the space at my disposal, a somewhat detailed account of the chemistry of the bases dealt with, and of their derivatives. Some, like the amines and adrenaline, are remarkable on account of their physiological action, and in each case, therefore, a brief description of this action has been added. In this way I have endeavoured to make the mono- graph also of interest to those who are concerned with the biological rather than with the chemical aspect of the subject. A brief chapter on the practical methods used in the isolation of the simple bases has been added, and special attention has been given to the bibliography which extends to the autumn of 1913. It is a pleasant duty to express my great indebtedness to Dr. H. H. Dale, without whose advice and criticism much of the pharmacological sections would have remained unwritten. G. B. ENQLEFIELD GREEN, SURREY, November, 1913. CONTENTS. CHAPTER III. BETAINES PAGE INTRODUCTION AND SCOPE - i CHAPTER I. AMINES DERIVED FROM PROTEIN ----- .7 SECTION 1. The Putrefactive Decomposition of Amino-acids - 7 2. Methylamine, Ethylamine, Dimethylamine - n 3. Trimethylamine - n 4. Isobutylamine - - - - - - - -12 5. Isoamylamine - - - - 13 6. Pyrrolidine - - - 13 7. Amino-ethyl Disulphide - 13 8. Putrescine and Cadaverine - - 14 9. Agmatine ... - - - 16 10. Phenyl-ethylamine - 16 11. p-Hydroxy-phenyl-ethylamine - 18 12. Hordenine - ... 20 13. Indolethylamine (3-/3-Amino-ethylindole) - - 21 14. y8-Iminazolyl-ethylamine - 22 15. Physiological Properties of the Amines derived from Amino- acids - ... 25 CHAPTER II. u>- AMINO-ACIDS AND OTHER BASES CONTAINING A CARBOXYL GROUP - 33 SECTION 1. /?-Alanine (/3-Amino-propionic Acid) - 34 2. y-Amino-n-butyric Acid - - 34 3. 8-Amino-n-valeric Acid - .35 4. e-Amino-caproic Acid - - 35 5. /3-Iminazolyl-propionic Acid - - 35 6. Carnosine (Ignotine) - 36 7. Urocanic Acid (Iminazolyl-acrylic Acid) - - 36 8. Kynurenic Acid - 37 39 SECTION 1. Betaine (Trimethyl-glycine) - - 40 2. Physiological Properties and Importance of Betaine 42 3. Stachydrine (Dimethyl-proline) - 43 4. Betonicine and Turicine (Dimethyl-oxyproline) 44 CONTENTS vii SECTION 5. Trimethyl-histidine . . 45 6. Ergothioneine (Thiolhistidine Betaine) - - - - 46 7. Hypaphorine (Trimethyl-tryptophane) - - - 47 8. Trigonelline (Methylnicotinic Acid) - . . 47 9. Other Pyridine Bases - - - - 48 10. y-n-Butyrobetaine - 49 11. Carnitine (Novaine, a-Hydroxy-y-butyrobetaine) - - - 50 12. Myokynine - 52 CHAPTER IV. CHOLINE AND ALLIED SUBSTANCES - - - - - 53 SECTION 1. Choline- . 54 2. Ammo-ethyl Alcohol (Colamine) and the Origin of Choline ; the possible Presence of other Bases in Phosphatides 58 3. Neurine- 60 4. Physiological Action of Choline and of Neurine - - 61 5. Natural and Synthetic Muscarines and their Physiological Action 64 6. Trimethylamine Oxide - -67 7. Neosine- ......... 55 CHAPTER V. CREATINE, CREATININE, GLYCOCYAMINE AND GUANIDINES - - 69 SECTION 1. Creatine and Creatinine - - 69 2. Physiology of Creatine and Creatinine - 71 a. Distribution - 71 b. Metabolism - - - 73 c. Possible Precursors of Creatine - .77 3. Glycocyamine and Glycocyamidine - - 78 4. Guanidine - - 79 5. Methylguanidine - - 79 6. as-Dimethylguanidine - 80 CHAPTER VI. ADRENALINE - 81 SECTION 1. Historical - 81 2. Nomenclature and Synonyms - 83 3. Preparation and Purification of Natural Adrenaline - 84 4. Syntheses of Adrenaline - 85 5. Adrenaline Substitutes - 87 6. Physical and Chemical Properties of Adrenaline. Salts and Derivatives. Constitution - 87 7. Colour Reactions of Adrenaline and Colorimetric Estimation 89 8. Amount of Adrenaline in the Suprarenal Gland ; Yield ; Distribution in other Organs ; Origin - 92 viii CONTENTS SECTION PAGE 9. Physiological Action of Adrenaline - - 96 a. Action on the Circulatory System - - 96 b. Action on other Organs containing Involuntary Muscle, and on Glands - - 97 c. Action on Carbohydrate Metabolism - - 99 d. Toxic Action - - 100 10. The Physiological Action of Dextro- and of Racemic Adrena- line - - 100 11. Physiological Methods of Estimating Adrenaline - 101 CHAPTER VII. BASES OF UNKNOWN CONSTITUTION - 106 SECTION 1. Spermine - 106 2. Bases from Muscle - 107 3. Bases from Urine - 107 4. Putrefaction Bases - 108 5. The Active Principle of the Pituitary Body - 108 6. Vitamine, Oryzanin, Torulin - 1 1 1 7. Sepsine - - 113 8. Secretine - 114 CHAPTER VIII. (APPENDIX.) PRACTICAL CHEMICAL METHODS AND DETAILS - 116 A. General Methods for the Separation and Isolation of Bases - 1 16 B. Special Methods. Properties of Individual Bases and of their Salts - - 124 Bases of Chapter I. - 124 Bases of Chapter II. - 135 Bases of Chapter III. - 141 Bases of Chapter IV. - - 150 Bases of Chapter V. - 1 5 7 BIBLIOGRAPHY - 167 INDEX -213 INTRODUCTION AND SCOPE. THE substances described in this monograph do not constitute a homogeneous group, like the proteins or carbohydrates, and the choice of a title was therefore difficult. Many are derived in various ways from the amino-acids of protein, a few are constituents of phosphatides ; some are of bio-chemical interest on account of their wide distribution in animals and in plants, others are important because of their phy- siological action. It is common to nearly all the simpler natural bases, however, that they are insoluble in ether and chloroform and readily soluble in water, so that their isolation is generally more difficult than that of the complex vegetable alkaloids, which can be extracted by making the aqueous solutions of their salts alkaline and then shaking with a solvent immiscible with water. The separation of the simpler bases from each other and from non-basic substances like peptones must be carried out by means of suitable precipitants and crystalline derivatives. The special technique required for this purpose constitutes the chief bond between the bases with which we are here concerned. This technique was first elaborated in a systematic manner by Brieger, who employed mercuric chloride in the isolation of putrefaction bases. The introduction of phosphotungstic acid, by Drechsel, as a general precipitant for basic substances and its use for preparative purposes marked a great advance ; later Kossel added the silver method for the separation of imino-bases, such as arginine and histidine. Since then the details of technique have been chiefly elaborated in three centres. Schulze at Zurich, in a long series of researches on plant bases, discovered phenylalanine and arginine and more lately extended our knowledge of betaines. Kutscher and his pupils, in Germany, have isolated bases from a variety of sources, and Gulewitsch, at Moscow, has studied exhaustively the bases in meat-extract. The history of the simpler natural bases has been greatly influenced by the need of special methods for their isolation. Another influence, adverse to their study, was the presence of alkaloids in drugs and stimulants, which directed attention to these complex bases having obvious physiological actions rather than to simpler bases of more I 2 THE SIMPLER NATURAL BASES general biological importance. Thus the basic nature of morphine was recognised as long ago as 1806, and in 1820 quite half a dozen of the most important vegetable alkaloids were known, but our knowledge of animal bases is of a much later date. Pettenkofer prepared creatinine from urine in 1844 and Strecker first obtained choline from pig's bile in 1849, but for a long time hardly any other animal bases were known, and betaine, which is now known to occur in many plants and some animals, was not discovered until 1863. The more volatile amines, trimethylamine and amylamine, were obtained as putrefaction pro- ducts in 1855 and 1857 respectively, and about the year 1866 it became generally recognised that bases are formed in putrefaction, but for a long time these bases were regarded as similar to the vegetable alkaloids, and their isolation was attempted by similar methods. For this there were two reasons. In the first place the poisonous properties of putrid material were considered analogous to those of plant alkaloids, and secondly the medico-legal examination of corpses in murder trials revealed the presence of bases (called ptomaines by Selmi) which gave reactions like those of coniine, nicotine, atropine, etc. In no single instance did these early investigations result in the preparation of a pure substance, so that they do not concern us further. The chemistry of putrefaction bases may be said to begin in 1876 when Nencki correctly analysed a base C 8 H n N, obtained from putrid gelatin ; he afterwards identified it as phenylethylamine. It seems highly probable that this amine, perhaps mixed with diamines, was the " animal coniine " of earlier investigators. The next great advance was due to Brieger who, breaking away from the methods used for plant alkaloids, and relying chiefly on mercuric chloride, platinic chloride and similar reagents, discovered putrescine, cadaverine, and many putrefaction bases which had been overlooked by his predecessors. Gradually it became evident that pto- maines, or putrefaction bases, are the products of bacterial action on protein and phosphatides, and since then our knowledge of these bases has become more and more intimately associated with what we know of the amino-acids from which protein is built up. Two examples of this association may be given. The discovery of phenylalanine by Schulze and Barbieri in 1881 enabled Nencki to surmise the constitu- tion of his base C 8 H n N referred to above ; it is derived from the amino- acid by loss of carbon dioxide. Later Ellinger proved that Brieger's diamines were similarly derived from the amino-acids ornithine and lysine. Since then the amines corresponding to nearly all the known INTRODUCTION AND SCOPE 3 amino-acids have been found to occur as putrefaction products. These amines are described in Chapter I and include substances with inter- esting physiological actions. Another group of bases, likewise derived from protein, is described in Chapter II. The members of this group still retain a carboxyl group of the amino-acid, so that they are but feebly basic, and without marked physiological action. They include the - Ammo-acids are also formed by the deaminization of diamino-acids ; the deaminization of monamino-acid yields non-nitrogenous acids such as isocaproic (from leucine) and succinic (from aspartic acid). Deaminization is accom- panied by reduction, since hydroxy-acids and un saturated acids ap- parently do not occur in putrefaction : R R CHNH 2 + 2H = CH 2 + NH 3 . COOH COOH By a combination of the two processes of decarboxylation and deaminization, methane may be formed from glycine and -butyric acid from glutamic acid (Neuberg and Rosenberg [1907]). A putre- factive process involving only reduction is the conversion of proline into 8-aminovaleric acid. The importance of reduction in the above bacterial actions is ex- pressed by the fact that they chiefly take place under anaerobic con- ditions. Bienstock [1899, 1901], one of the chief workers in this field on the bacteriological side, concludes that putrefaction, in the ordinary sense, cannot take place without an obligate anaerobe, such as Bacillus putrificus. B. coli hinders the action of B. putrificus and B. tetani has no action on fibrin. Rettger [1906, 1907 ; Rettger and Newell, 1912] shares the view that putrefaction is the work of strict anaerobes. The access of oxygen induces further changes ; p-hydroxy-phenyl- propionic acid (formed by the deaminization of tyrosine) is oxidised, according to Baumann and Nencki, to p-hydroxy-phenyl-acetic acid, which is successively converted into p-cresol and phenol, and simi- larly indole-propionic acid (from tryptophane) yields indole-acetic acid, skatole, and indole. Oxidation also accounts for the shortening of AMINES DERIVED FROM PROTEIN 9 the carbon chain in the production of succinic acid from glutamic acid by putrefaction. Some putrefaction bases are formed from substances other than proteins ; thus lecithin is broken down to choline, neurine, trimethyl- amine, monomethylamine, and ammonia; creatine yields monomethyl- guanidine and perhaps also dimethylguanidine ; the trimethylamine of stale urine is derived from more complex betaines ; purine and pyrimid- ine bases probably also contribute to the formation of putrefaction bases. When an entire tissue or organ, and to a less extent when a single protein is putrefied, as in the experiments of Nencki, Gautier, Brieger, Salkowski, Emmerling, Barger and Walpole, and the earlier experi- ments of Ackermann, a complex mixture of bases is obtained from various parent substances. A better insight into the chemistry of putrefaction is possible when a simple substance, such as a single amino-acid, is subjected to bacterial action. This method depends on a knowledge of the constituents of protein, and was first applied to the study of bases by Ellinger, who showed that putrescine and cadaverine are derived from ornithine and lysine respectively. Further work in this direction has been carried out principally by Ackermann and by Neuberg. (The products of the action of bacteria on indole-propionic acid (Nencki) and of yeast on proteins (F. Ehrlich) are not bases, and they are therefore not included in this monograph.) It is generally much more difficult to grow bacteria in a solution of a pure amino-acid than on protein, and Ackermann therefore adds 0^25 per cent. Witte pep- tone to the solution, together with 0*5 percent, glucose and a few drops of sodium phosphate and magnesium sulphate ; calcium carbonate is sometimes added to prevent the solution becoming acid, but a faint alkaline reaction is secured more certainly by adding sodium carbonate from time to time. Although Neuberg [1911, l] has pointed out the theoretical objections to the addition of peptone he yet agrees with Ackermann that in many cases this addition is desirable. For the decomposition of histidine Mellanby and Twort [1912] used a culture medium containing only ammonium tartrate and inorganic salts (see p. 133). A similar medium was used by Berthelot and Bertrand [1912, I]. Of late years nearly all the putrefaction products, which might be expected to result from the known amino-acids, have been obtained by bacterial action. Exceptions are e-amino-caproic acid which might be formed from lysine, guanidino-valeric acid (from arginine), pyrroli- dine (from proline), oxypyrrolidine (from oxyproline) and the amines from cystine and serine. 10 THE SIMPLER NATURAL BASES The decarboxylation of amino-acids is not necessarily accompanied by any obvious sign of bacterial action such as putrefactive odour ; some of these amines occur in cheese and they have repeatedly been obtained in fermentation experiments supposed to be sterile (Langstein, Emerson, Lawrow ; see the section on putrescine and cadaverine). The difficulties of ensuring sterility, particularly in autolysis, have often been underestimated and have been emphasised by Schumm [1905-6], Rothmann [1908], Kikkoji [1909], Salkowski [1909], Ohta [1910], Harden and Maclean [1911], Beker [1913]. Chloroform should not be used in conjunction with toluene, which dissolves the chloroform from the aqueous layer. It is best, according to Schumm and Kikkoji, to use water saturated with chloroform, or chloroform in excess and to ensure continued saturation by means of stoppered bottles and frequent shaking. Sterility tests should be made by smear. In the absence of bacteria, decarboxylation of amino-acids does not occur; at least the corresponding primary amines are not found. (Kutscher and Lohmann [1905], Schumm [1905-6], Bissegger and Stegmann [1908], Schulze [1906], Kiesel [1911].) The occur- rence of methylated bases such as tetramethyl putrescine and hordenine in the higher plants perhaps implies the intermediate formation of primary amines. Apart from putrefaction, putrescine and cadaverine occur in cystinuric urine, agmatine in herring spawn and p-hydroxy- phenyl-ethylamine in the salivary gland of Cephalopoda. It has further been established that fresh fungi may contain amines resulting from the decarboxylation of amino-acids or at any rate these amines are formed by autolysis independently of bacterial action. The close relationship between the fungi proper and bacteria makes this less surprising. Ergot, which has been examined more thoroughly than any other fungus, contains p-hydroxy-phenyl-ethylamine, /3-iminazolyl-ethyl- amine, putrescine, cadaverine, agmatine, and probably isoamylamine, and owes much of its physiological action to the first two of these bases. It is almost certain that they are to some extent present in fresh ergot, but the amount is increased after death, probably by autolysis. Reuter [1912] recently found putrescine in fresh specimens of Boletus edulis and when this fungus was autolysed under sterile conditions, isoamyl- amine, phenyl-ethylamine, probably p-hydroxy-phenyl-ethylamine and possibly iminazolyl-ethylamine were formed in addition. Schenck [1905, I] had previously obtained putrescine from autolysed yeast. Reuter's experiments are of particular interest ; sterility tests showed that bacteria were absent, and he concludes that fungi possess ferments capable of decarboxylating amino-acids. AMINES DERIVED FROM PROTEIN it Methylamine, Ethylamine, Dimethylamine. Methylamine occurs according to Trier [1912, 3; p. 8] in species of Mercurialis and the root of Acorus Calamus and has been frequently met with as a product of bacterial action (see P. Rona, Biochemisches Handlexicon, Band IV, p. 801). It is perhaps formed from glycine, by decarboxylation, but so far it has not been possible to demonstrate this experimentally. The source of methylamine is in most cases more probably trimethylamine (from choline). Thus Hasebroek [1887] obtained this amine along with ammonia by the anaerobic putrefaction of choline, and Morner [1896] found amines present in a peculiar Swedish food (" surfisk "). This fish is pickled with a little salt and allowed to ferment anaerobically ; it probably contains monomethyl- amine, and certainly dimethylamine and choline, but not putrescine or cadaverine. Ackermann and Schiitze [1910, 1911] also found that a little methylamine, together with trimethylamine, is formed by the action of Bacterium prodigiosum on choline. Emmerling [1897] obtained mono- and trimethylamine by the action of Streptococci on fibrin, but here also the amines appear to be derived from admixed lecithin. Ethylamine was said more than fifty years ago to be produced in the putrefaction of yeast and of wheat flour, but these observations re- quire confirmation. It might result by the decarboxylation of alanine, from which it is indeed formed on destructive distillation. Dimethylamine was stated by Bocklisch [1885] and by Morner [1896] to occur in putrid fish, and by Ehrenberg [1887] in cultures from a bacillus isolated from poisonous sausages. In the latter case at least a confusion with putrescine was not unlikely, since the platini- chlorides of the two bases have nearly the same composition. If dimethylamine is formed at all it would be most probably derived from choline and trimethylamine, although it could also result from the decarboxylation of sarcosine (from creatine). Trimethylamine, N(CH 3 ) 3 . Trimethylamine occurs in the leaves of Chenopodium Vulvaria (the Stinking Goosefoot) where it is readily detected by the odour on bruis- ing the leaves ; it is also present in hawthorn flowers (Cratagus Oxyacantha] and in ergot. Unlike the other amines dealt with in this chapter, trimethylamine is not formed from an amino-acid, but is a decomposition product of choline and allied quaternary bases ; it is therefore of common occurrence in putrefaction. Thus it is present in herring brine, the first natural source to be discovered by Winckler in 12 THE SIMPLER NATURAL BASES 1855. On an industrial scale it is formed by the destructive distilla- tion of beet sugar molasses ; here the parent substance is betaine. Examples of the production of trimethylamine by pure cultures are the action of Proteus vulgaris on wheat gluten and on meat, of Bacillus liquefaciens on commercial gelatin and of Bacteriumprodigiosum on choline and on lecithin. Ackermann and Schutze [1910, 1911] found that the last-named organism does not produce trimethylamine from betaine, and that B. vulgatus does not decompose choline. The alleged occurrence of trimethylamine in urine has been the subject of several investigations. Long ago Dessaignes [1856] ob- tained it by distillation of urine with caustic soda (37 grm. of the free base from 65 litres of human urine). He, however, left open the question whether trimethylamine is present as such or is formed by the decomposition of some other compound by the alkali. This question was likewise left unanswered by de Filippi [1906] who worked out a process for the estimation of urinary trimethylamine (see appendix). Takeda [1909] used magnesium oxide instead of caustic soda, and distilled under reduced pressure ; he found no trimethylamine in the urine of horses and of dogs and only doubtful traces in human urine ; it is however formed in putrefaction. Kinoshita [1910, l], using Herzig and Meyer's method for the estimation of N-methyl groups, found only traces, and Erdmann [1910] has also arrived at the con- clusion that " fresh normal urine does not contain trimethylamine ". According to Kutscher the trimethylamine in urine is formed from such bases as novaine and reducto-novaine. Isobutylamine, 3 cH . CH 2 . NH This base was obtained by the putrefaction of racemic a-amino- isovaleric acid (d.l. valine) by Neuberg and Karczag [1909]. A solution of 10 grams of the amino-acid in 450 c.c. of water, with a little KC1, Na 2 HPO 4 and MgSO 4 was rendered alkaline with sodium carbonate and yielded after inoculation and four weeks' incubation at 37 0-424 grm. of a platinichloride (C 4 H n N) 2 H 2 PtCl 6 , mp. 226-227, in all probability that of isobutylamine. A butylamine has also been obtained by Gautier from cod liver oil prepared by the old putrefactive process. In Fagara xanthoxyloides isobutylamine occurs in combination with piperonylacrylic acid as an amide, fagaramide (Thorns and Thumen, Isobutylamine is the lowest amine causing any appreciable rise of blood pressure when injected intravenously. AMINES DERIVED FROM PROTEIN 13 Isoamylamine, > CH . CH 2 . CH 2 . NH 2 . CH/ An amylamine has been obtained from putrid yeast (Muller [1857]), from cod liver oil (Gautier and Mourgues [1888]), from putrid horse meat (Barger and Walpole [1909, i]), putrid placenta (Rosenheim [1909]), from Boletus edulis on sterile autolysis (Reuter [1912]), and probably from fresh ergot (Barger and Dale [1909]). In all these cases isoamylamine (derived from leucine) was pro- bably mixed with the isomeride 2-methylamino-butane (derived from isoleucine), and possibly with normal amylamine. from norleucine. Iso- amylamine is further formed from leucine on rapid heating, and in the dry distillation of bones and horn. Ciamician and Ravenna (quoted by Trier [1912, 3]) found isoamylamine in tobacco. The oxalate of isoamylamine was obtained in an impure form from putrid meat by Abelous, Ribaut, Soulie and Toujan [1906, I, 2]; Abelous and Ribaut [1908] deduced the erroneous formula C G H U ON for the base, and were the first to observe its power of raising the blood pressure when injected intravenously. Extracts of putrid meat were shown by Barger and Walpole to owe their pressor action principally to isoamylamine and to p-hydroxy-phenyl-ethylamine. Pyrrolidine, C 4 H 9 N. This base should result from the amino-acid proline by decarboxy- lation, but has never been isolated as a putrefaction product, probably because putrefactive bacteria rupture the pyrrolidine ring by reduction (see Chapter V). Pyrrolidine has, however, been isolated in minute quantity from carrot leaves (Daucus Carota] by Pictet and Court [1907]- They also found pyrrolidine and N-methylpyrroline in minute quantities in tobacco, and have termed these bases proto-alkaloids. Amino-ethyl Disulphide, S 2 (CH 2 . CH 2 . NH 2 ) 2 . Neuberg and Ascher [1907] obtained this amine in small quantity by the dry distillation of cystine, from which it is derived by loss of carbon dioxide. 'Ite pi crate melts at 197. The amine has no pro- nounced physiological activity, and has so far not been obtained by bacterial action. I 4 THE SIMPLER NATURAL BASES Putrescine and Cadaverine, C 4 H 12 N 2 and C 5 H U N 2 . These two homologous diamines have similar properties and generally accompany each other, so that they may be most conveniently considered together. They were discovered by Brieger [1885, 1, 2] by his new method of investigating putrefaction bases ; cadaverine was soon afterwards shown by Ladenburg [1886] to be identical with the pentamethylene-diamine previously obtained by reduction of tri- methylene dicyanide, and later Udranszky and Baumann [1888, 2] proved the identity of putrescine with tetramethylene-diamine. Putrescine and cadaverine are among the commonest of all putre- faction bases. They probably escaped the notice of earlier investigators on account of their sparing solubility in ether and in chloroform, but Brieger obtained them repeatedly from various sources and they have been isolated many times since. The possibility of the formation of cadaverine from lysine by loss of CO 2 was already considered by Udranszky and Baumann and the origin of both amines was definitely established by Ellinger [1900] who obtained putrescine by the action of putrefactive bacteria on ornithine : NH 2 . CH 2 . CH 2 . CH 2 . CH(NH 2 ) . COOH = NH 2 . CH 2 . CH 2 . CH 2 . CH 2 . NH 2 + CO 2 ; and similarly cadaverine from lysine : NH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH(NH 2 ) . COOH = NH 2 . CH 2 . CH 2 . CH 2 . CH 2 . CH 2 . NH 2 + CO 2 . These important results furnished the first examples of the bacterial decarboxylation of amino-acids. With access of air Ellinger ob- tained a 1 2 per cent, yield of putrescine and under anaerobic conditions a 50-60 per cent, yield (three days at 37) ; with cadaverine the yield was 36 per cent. Ackermann [1909, I], who more recently repeated Ellinger's experiments, was at first unable to obtain putrescine and cadaverine from the pure amino-acids but succeeded in the case of the products of the hydrolysis of caseinogen by acids. He showed that putrescine but not cadaverine is formed in the putrefaction of gliadin [1909, 2], which does not contain lysine, and ultimately he [1910, 3] found that the addition of 0*25 per cent. Witte peptone and 0-5 per cent, glucose to the culture medium greatly facilitated decar- boxylation. In the earlier experiments only traces of inorganic salts had been added. When once formed, cadaverine and putrescine are apparently very resistant to the action of micro-organisms, for Brieger and others isolated the bases in considerable quantity after putrefac- tion had been going on for months. Apart from such bacterial formation of putrescine and cadaverine, AMINES DERIVED FROM PROTEIN 15 both bases have been isolated from ergot by Rielander [1908] and putrescine has been found in autolysed yeast by Schenck [1905, i], in fresh specimens of Boletus edulis by Reuter [1912] and in Datura (a Phanerogam) by Ciamician and Ravenna (Trier [1912, 3]). The diamines further occur in some cases of cystinuria (Udranszky and Baumann [1889], Cammidge and Garrod [1900], Loewy and Neuberg [1904], Garrod and Hurtley [1906]; the last-named paper should be consulted for the literature of other cases). In some cases of cystinuria the diamines are only excreted occasionally, or not at all, in Loewy and Neuberg's case only when arginine and lysine were given by the mouth. On the other hand the diamines do not pass into the urine when given by the mouth to a normal animal (Udranszky and Baumann [1890]). Garrod's impression [1909] is "that the likelihood that diamines will be detected in any given specimen of cystin urine is comparatively small, but that if in any case the examination be continued over sufficiently long periods they are likely to be found eventually". Lately Ackermann and Kutscher [1911] have found a minute quantity of lysine in cystinuric urine. The excretion of diamines in the urine indicates a peculiarity of meta- bolism, probably not intimately connected with the excretion of cystine. Cadaverine was also found by Roos [1892] in the urine in two cases of malaria, but this may have been the result of bacterial action. Other cases of the alleged fermentative formation of the two diamines may safely be ascribed to this cause. Thus Lawrow [1901] ob- tained both bases in the autolysis of pig's stomach, Langstein [1901, 1902] isolated cadaverine after digesting egg white with pepsin for more than a year, Steyrer (referred to by Emerson [1901]) ob- tained the same base from a pancreatic digest and Werigo [1892] from pancreas macerated with chloroform water. In some of Werigo's experiments incipient putrefaction was indeed noticed, and we may well attach more weight to the experiments of Kutscher and Lohmann [1905] and of Schumm [1905-6], who could not isolate either putrescine or cadaverine when pancreas was autolysed under sterile conditions, and to those of Bissegger and Stegmann [1908] who likewise could not obtain the diamines by the tryptic or peptic diges- tion of caseinogen. Schulze showed [1906] that putrescine and cadaverine, unlike their parent substances, are absent from germinating seedlings. Among the cases where putrescine and cadaverine are formed by bacterial action we may further mention that both bases have been obtained from putrid Soy beans (Yoshimura [1910]) and from 16 THE SIMPLER NATURAL BASES Emmenthaler cheese (Winterstein and Thony [1902]). Van Slyke and Hart [1903] found a little putrescine in ordinary Cheddar cheese, but none in a sterile chloroform cheese. According to Garcia [1892-3, 2, 3] the -%\, Tof the diamines from putrid horse meat and from pancreas is dimimsheol by the addition of carbohydrates (compare p. 25); four-fifths is already formed in the first twenty-four hours of incubation and the maximum is reached after three days. Once formed, putrescine and cadaverine appear to be very resistant to bacterial action. Gulewitsch [ 1 894] obtained cadaverine from horse meat kept four months at 15. Hyoscyamus muticus contains tetramethyl-putrescine (see appendix). Agmatine, C 5 H U N 4 . Agmatine, or guanidino-butylamine, was obtained by Kossel [1910, i] from herring spawn after heating with dilute sulphuric acid (5 per cent, by volume) in an autoclave at 4 atmospheres pressure. The base differs from arginine by CO. 2 , the chief amino-acid in herring spawn, so that it may be considered as being derived from arginine by decarboxylation : NH 2 . C( : NH) . NH . CH 2 . CH 2 . CH 2 . CH 2 . NH 3 agmatine. NH 3 . C( : NH) . NH . CH 3 . CH 2 . CH 2 . CH(NH 3 ) . COOH arginine. Agmatine has also been isolated from ergot by Engeland and Kutscher [1910, I, 2] who obtained from their base on oxidation guanidine and guanidino-butyric acid, NH 2 . C( : NH) . NH . CH 2 . CH 2 . CH a . COOH. Kossel [1910, 2] synthesised agmatine from cyanamide and tetra- methylene diamine, NH 2 . CN + NH 2 (CH 2 ) 4 NH 3 =NH 2 . C( : NH) . NH . (CH Q ) 4 . NH 2 . Phenyl-ethylamine, C 6 H 5 . CH 2 . CH 2 . NH 2 . /3-Phenyl-ethylamine is of some interest, since it was the first putre- faction base of which the composition was determined. Nencki [1876] obtained the base from a mixture of 200 grams of ox pancreas and 600 grams of gelatin dissolved in 10 litres of water, which was putrefied at 40 for five days. Nencki, like Selmi and other early investigators of putrefaction bases, was most impressed by their analogy to vegetable alkaloids such as coniine and nicotine, and he at first considered his base to be a pyri- dine homologue, dimethylpyridine or collidine. Finding later that his hydrochloride, unlike that of collidine, yielded on destructive distilla- AMINES DERIVED FROM PROTEIN 17 tion a substance resembling xylene in odour and other properties, he concluded [1882] that the base obtained from gelatin was an aro- matic amine, probably a-phenyl-ethylamine, C 6 H 5 . CH(NH 2 ). CH 3 . Still later he regarded enylalanine, which Schulze and Barbieri had discovered in etiolated lupin seedlings, as the parent substance of his putrefaction base, which he [1889] therefore considered to be /3- phenyl-ethylamine, formed according to the equation : C 6 H 5 . CH a . CH(NH 2 ) . COOH = C 6 H 6 . CH a . CH 2 . NH 2 + CO 2 . Nencki was thus also the first to invoke the decarboxylation of an amino-acid in explanation of the origin of a putrefaction base. Nencki's "collidine" was further obtained from putrefied egg white by his 1 pupil Jeanneret [1877]. The identity of the base from putrid gelatin with /3-phenyl-ethylamine was first rendered absolutely certain by Spiro [1901]. Putrefaction bases of the formula C 8 H U N or of a similar formula, with properties somewhat resembling those of phenyl-ethylamine, have at various times been obtained by other investigators and one is tempted to regard all these bases as identical with that first isolated by Nencki. In some cases this is indeed almost or quite certain. Thus by the action of a Strepto- coccus on fibrin, Emmerling [1897] obtained a base of the formula C 8 H n N of which the picrate melted at the same temperature as that of synthetic /3-phenyl-ethylamine ; the only discrepancy is that the platinichloride is described as readily soluble in water. Similarly a base obtained from putrid horse meat by Barger and Walpole [1909, 1], and having the boiling point and physiological properties of $-phenyl-ethylamine, was doubtless identical with this amine. It is much more difficult to draw the same conclusion with regard to certain bases described as pyridine derivatives and isolated by Gautier and Etard [1882, 1883] and by Oechsnerde Coninck,[ 1886-91]. The former investigators obtained from putrid mackerel a base, boiling at 210, d = 1*0296, which was analysed as platinichloride. The formula deduced was C 8 H 13 N and the base was named dihydrocollidine, but the analyses are in better, although not good, agreement with the formula C 8 H U N. No evidence of its being a pyridine derivative was adduced and Nencki [1882] at first regarded Gautier and Etard's hydrocollidine as identical with phenyl-ethylamine, but subsequently [1889], after a visit to Gautier, he gave up this view. Oechsner de Coninck obtained a base of the formula C 8 H n N from putrid cuttle- fish ; on oxidation it yielded nicotinic acid ; it was examined much more closely than Gautier and Etard's " hydrocollidine " and in this 2 1 8 THE SIMPLER NATURAL BASES case at least, a confusion with phenyl-ethylamine seems completely ex- cluded. Compare further the section on p. 48. Phenyl-ethylamine does not accompany phenyl-alanine in seedlings (Schulze [1906]), but with regard to the higher plants it should be mentioned that Le Prince [1907] has isolated a volatile base C 8 H n N from the European mistletoe (Viscum album} and that Crawford [1911] attributes the pressor action of the U.S.P. extract of the American mistletoe (Phoradendron flavescens) to the presence of abase, C 7 H U N or C 8 H U N, which he thinks is perhaps identical with phenyl- ethylamine. This base requires further investigation ; the presence of phenyl-ethylamine may possibly depend on the fact that the mistletoe is a semi-parasite. Although phenyl-ethylamine has not been found in any fresh fungus, Reuter [1912] obtained it from Boletus edulis by aseptic autolysis. Derivatives of phenyl-ethylamine have been found in various essential oils ; thus phenyl-ethyl-alcohol C 6 H 5 .CH 2 .CH 2 OH occurs in rose oil and is also produced from phenyl-ethylamine by yeast (Ehrlich); phenyl-acetonitrile, C 6 H 5 . CH 2 . CN, was found by Hof- mann [1874] in the essential oil of Nasturtium officinale^ and phenyl- ethyl-z'jtf-thiocyanate is present in the oil from the root of Reseda according to Bertram and Walbaum [1894], and yields phenyl-ethyl- amine on hydrolysis. Possibly phenyl-ethylamine is an intermediate stage in the formation of all three substances from phenyl-alanine. p-Hydroxy-phenyl-ethylamine, OH . C 6 H, . CH 2 . CH 2 . NH 2 . This amine was first obtained by Schmitt and Nasse [1865] by heating tyrosine, when the following change occurs : HO/ \CH 2 . CH (NH 2 ) COOH = HO/'" ~\CH 2 . CH 2 . NH 2 + CO 2 . p-Hydroxy-phenyl-ethylamine was subsequently isolated from auto- lysed pancreas by Emerson [1901] and from a prolonged peptic digestion of egg-albumin by Langstein [1901, 1902]. It seems pretty certain that in these experiments bacterial action was not completely excluded (see p. 10). Gautier and Mourgues [1888] isolated the base from the mother liquors obtained in the putrefaction of cod-livers (in the old process of making cod-liver oil). Gautier also obtained in small quantity a lower homologue C 7 H 7 NO and a higher one C 9 H U NO and named the three bases " tyrosamines ". The last two do not, however, appear to have been sufficiently well characterised. p-Hydroxy-phenyl-ethylamine is fairly abundant in various kinds of cheese. It was found by Van Slyke and Hart [1903] in Cheddar AMINES DERIVED FROM PROTEIN 19 cheese prepared in the usual manner, but not in a cheese prepared with chloroform milk, so as to ensure sterility. The normal cheese was found to give off considerable quantities of carbon dioxide during ripening and Van Slyke and Hart consider that the carbon dioxide arose from the decarboxylation of amino-acids. The chloroformed cheese produced only traces of carbon dioxide and when finally analysed yielded a considerable quantity of arginine, while the normal cheese contained only traces of arginine, but instead of it guanidine and putrescine were present. The cavities in Emmenthaler ("Gruyere") cheese are mostly filled with carbon dioxide, and p-hydroxy-phenyl-ethylamine was isolated from this kind of cheese by Winterstein and Kiing [1909]. It is further almost certain that one of Brieger's ptomaines, my dine [1886, i, p. 26], was identical with p-hydroxy-phenyl-ethylamine. The base had the composition C 8 H n NO, yielded a soluble platinichloride, and a picrate crystallising in broad prisms melting at 190. It was ob- tained from putrid human viscera, and was non-poisonous ; ferric and gold salts were reduced by it. (The picrate of the synthetic amine crystallises in " short prisms " melting at 200 ; the other properties are identical with those described for mydine by Brieger.) The physiological action of p-hydroxy-phenyl-ethylamine was first brought to light by its identification, by Barger and Walpole [1909, i], as the chief pressor constituent in extracts of putrid meat. The blood pressure raising property of such extracts had already been observed by Abelous, Ribaut, Soulie, and Toujan [1906, I, 2]. Dixon and Taylor [1907] had also noticed that extracts of human placenta raised the blood pressure on intravenous injection and caused, in addition, contraction of the pregnant uterus. Rosenheim [1909] showed that this effect was mot produced by extracts of perfectly fresh placenta, and after Barger and Walpole' s identification of the pressor con- stituent of putrid meat, he was further able to show that the active constituent in Dixon and Taylor's placental extracts was also p- hydroxy-phenyl-ethylamine. Finally this amine is the chief pressor constituent of certain extracts of ergot, as shown by Barger and Dale [1909]. A certain quantity is apparently present in perfectly fresh ergot, where it has also been found by Engeland and Kutscher [1910, 2] and by Burmann [1912]. p-Hydroxy-phenyl-ethylamine is pro- bably also present in autolysed Boletus edulis (Reuter [1912]). That tyrosine is indeed the parent substance of p-hydroxy-phenyl-ethylamine was shown by Barger and Walpole [1909, i]; the yield in putrefaction was minute (less than I per cent, of the tyrosine present). Ackermann 20 THE SIMPLER NATURAL BASES [1909, I] also isolated the base after putrefying the mixture of ammo- acids obtained by boiling caseinogen with sulphuric acid. Henze [1913] has made the most interesting observation that p-hydroxyphenyl-ethylamine occurs in the salivary gland of Cephalo- poda and has a paralysing action on crabs, which are the chief food of these Molluscs. Syntheses. Larger quantities of p-hydroxy-phenyl-ethylamine are obtained by synthesis, most conveniently by the reduction of p-hydroxy-phenyl- acetonitrile with sodium and alcohol (Barger [1909, i]), according to the equation : OH . C 6 H 4 . CH 2 . CN + 4 H = OH . C 6 H 4 . CH 2 . CH 2 . NH 2 . Two other syntheses of this amine were described by Barger and Walpole [1909, 2]; according to one of these benzoyl-phenyl-ethyl- amine is nitrated and the p-nitro-derivative is reduced, diazotised, and hydrolysed : C 6 H 5 . CH 2 . CH 2 . NH . CO . C 6 H 5 ->NO 2 . C 6 H 4 . CH 2 . CH 2 . NH . CO . C 6 H 5 -NH 2 . C 6 H 4 . CH 2 . CH 2 . NH . CO . C 6 H 5 -OH . C 6 H 4 . CH 2 . CH 2 . NH . CO . C 6 H 6 -OH - . C 6 H 4 . CH 2 . CH 2 . NH 2 . The other synthesis starts from anisaldehyde which is successively converted into p-methoxy-phenyl-acrylic acid, p-methoxy-phenyl- propionic acid, and its amide, p-methoxy-phenyl-ethylamine and p- hydroxy-phenyl-ethylamine : CH 3 O . C 6 H 4 . CHO->CH S O . C 6 H 4 . CH : CH . COOH->CH 3 O . C 6 H 4 . CH 3 . CH 2 . COOH _CH 3 O . C 6 H 4 . CH 2 . CH 2 . CO . NH 2 -CH 3 O . C 6 H 4 . CH 2 . CH 2 . NH 2 -5.0H . C 6 H 4 . CH 2 . CH 2 . NH 3 . The yield by the last synthesis is poor ; the p-methoxy-phenyl- ethylamine is better prepared by Rosenmund's method [1909], by the reduction of the condensation product of anisaldehyde with nitro- methane : CH 3 . C 8 H 4 . CHO + CH 3 . NO 2 = CH 3 O . C 6 H 4 . CH : CH . NO 2 -CH 8 . C 6 H 4 . CH 2 . CH : NOH-CH 3 O . C 6 H 4 . CH 2 . CH 2 . NH 2 . Rosenmund then boils the latter compound with colourless hydriodic acid and obtains p-hydroxy-phenyl-ethylamirie. Hordenine, OH . C 6 H, . CH 2 . CH 2 . N(CH 3 ) 2 . An infusion of barley germs, a by-product obtained in the pre- paration of malt, had been employed in the South of France against dysentery. This led to the isolation by Leger [1906, l] of an "alkaloid" from barley germs, which he named hordenine. The base was found by Leger [1906, 2,3, I9O7] and independently also by Gaebel [1906] to be p-hydroxy-phenyl-ethyl-dimethylamine ~ 2 . CH a . N (CH 3 ) 2 AMINES DERIVED FROM PROTEIN 21 The constitution of hordenine was deduced by Leger from the oxi- dation of acetyl-hordenine to acetyl-p-hydroxy-benzoic acid and the distillation of the ammonium base from hordenine methiodide methyl-ether, which yielded trimethylamine and p-vinylanisole, CH 3 O.C 6 H 4 .CH:CH 2 . Gaebel, on methylating and oxidising, obtained anisic acid from hordenine. The synthesis of hordenine was first carried out by Barger [ 1 909, 2] from phenyl-ethyl -alcohol, a commercial product, as follows : C 6 H 5 . CH 2 . CH 2 . OH-C 6 H 5 . CH 2 . CH 2 . C1-C 6 H 6 . CH 2 . CH 3 . N(CH 3 ) 2 I HO.C 6 H 4 .CH 2 .CH 3 .N(CH 3 ) 2 <-NH 2 .C 6 H 4 .CH 3 .CH 2 .N(CH 3 ) 2 -N0 2 .C 6 H 4 .CH 3 .CH 3 .N(CH 3 ) 2 Closely related to this is the synthesis from tyrosol, by Ehrlich [1912]:- OH . C 6 H 4 . CH 3 . CH 2 OH->OH . C 6 H 4 . CH 2 . CH 2 C1->OH . C 6 H 4 . CH 2 . CH 2 . N(CH 3 ) 2 The attempted conversion of p-hydroxy-phenyl-ethylamine into horde- nine by methyl-iodide resulted only in the formation of the quaternary iodide, but Rosenmund [1910] has succeeded in methylating p-methoxy- phenyl-ethylamine to the tertiary base, hordenine methyl-ether, from which hordenine was obtained by boiling with hydriodic acid. Other syntheses are by reduction of p-hydroxy-phenyl-dimethyl-amino-methyl- ketone HO . C 6 H 4 . CO . CH 2 . N(CH 3 ), (Voswinckel [1912]) and by distillation in a vacuum of the quaternary hordenine methiodide (prepared from p-hydroxy-phenyl-ethylamine) according to D.R.P. 233069 of Farbenfabriken vorm. F. Bayer & Co.: OH . C 6 H 4 . CR, . CH 2 . N(CH 3 ) 3 I = OH . C 6 H 4 . CH a . CH a . N(CH 3 ) 2 + CH 3 I . Hordenine has only a transitory existence during the germination of barley. According to Torquato Torquati [1910] it is not present in the ungerminated seed and is most abundant after four days, when the rootlets contain 0-4 - 0*45 per cent. It then gradually diminishes and has disappeared after twenty-five days. It is absent in germinating wheat, peas and lupins. Indolethylamine (3-/3-Amino-ethylindole), C 10 H 12 N 2 . 3-/3-Amino-ethylindole is the amine derived from tryptophane by decarboxylation. It was obtained by Ewins and Laidlaw [1910, 2] both synthetically and by the action of putrefactive bacteria on the amino-acid. The synthesis, subsequently described by Ewins [1911], is the 22 THE SIMPLER NATURAL BASES most convenient method for obtaining the base in quantity ; ry-amino- butyrylacetal is heated with phenyl-hydrazine and zinc chloride. CH 2 . CH 2 . CH 2 . NH 3 ,'NH . NH 2 CH (OC 2 H 5 ) 2 ( C.CH 2 .CH 2 .NH 2 CH + NH 3 + 2 C 2 H 6 OH NH From the concentrated solution of the crude hydrochloride (obtained by washing the reaction mixture with ether and removing the zinc as sulphide) the free base is precipitated by sodium hydroxide as an oil, which on keeping crystallises to a mass of fine needles. Laidlaw [1911] dissolved 0*5 grm. tryptophane in 250 c.c. of tap water, together with 0*5 grm. peptone, 2 grm. glucose, traces of sodium phosphate and magnesium sulphate and added 5 grm. of calcium car- bonate ; this is the culture medium employed by Ackermann in the decarboxylation of histidine (p. 132). After infection with a subculture from putrid pancreas and incubation for a fortnight the mixture was boiled with charcoal and concentrated. Picric acid then precipitated the deep orange red picrate of indolethylamine. Yield after purifica- tion = 0-14 grm. = 14 per cent, of the theoretical. The decarboxylation of tryptophane cannot be effected by heat. The author's experiments in this direction were carried out under a pressure of I mm. ; the only substance which could be isolated from the sublimate was a small quantity of unchanged tryptophane. /5-Iminazolyl-ethylamine, C 5 H 9 N 3 . /2-Iminazolyl-ethylamine (4-/3-amino-ethyl-glyoxaline) is the amine derived from histidine by decarboxylation ; it is of considerable in- terest on account of its great physiological activity. The base was first obtained by Windaus and Vogt [1907] who prepared it by Curtius's method from iminazolyl-propionic acid, which can be made by synthesis as well as from histidine. A few years later Ackermann [1910, I] submitted pure histidine hydrochloride to the action of putrefactive bacteria and obtained a relatively large yield of iminazolyl-ethylamine (together with a small quantity of iminazolyl- propionic acid). The physiological activity of the amine, however, remained unknown until the latter was identified as one of the active principles of ergot by Barger and Dale [1910, 2-4]. The same active principle was simultaneously isolated from ergot by Kutscher [1910, l] who at first regarded it as closely related to iminazolyl-ethylamine, but AMINES DERIVED FROM PROTEIN 23 not identical with it, on account of a supposed difference in the physio- logical action of the two bases. Iminazolyl-ethylamine has also been obtained from the intestinal mucosa by Barger and Dale [1911]; it is therefore present in crude solutions of secretine, to which it gives a depressent action. Its formation in the intestinal wall is probably due to bacilli, isolated by Mellanby and Twort [1912] and by Berthelot and Bertrand [1912, I, 2]. The base has further been isolated from putrid Soy beans by Yoshimura 1 [1910]; it probably also occurs in commercial extracts of meat, of yeast, etc. The yield from almost all the above sources is very small ; larger quantities may be prepared from histidine, as well as by direct syn- thesis. The decarboxylation of histidine has been carried out indi- rectly by Windaus and Vogt [1907] as mentioned above. The reactions involved are the transformation of histidine (I) CH-NH^ CH-NH, CH-NH. CH 3 . CH (NH 2 ) . COOH CH 2 . CHC1 . COOH CH a . CH a . COOH I II III CHNH. CH NHv CH <- C -- N 4- C __ CH 2 . CH 2 . NH 3 CH 2 . CH 2 . CONH . NH,, CH 2 . CH. . COOC a H 5 VI V IV into a-chloro-/2-iminazolyl-propionic acid (II) (by sodium nitrite and hydrochloric acid) ; the reduction of this substance to /3-iminazolyl- propionic acid (III), which can also be synthesised from glyoxyl-propi- onic acid ; the successive 'conversion of this acid into the ester (IV) and the hydrazide (V) ; finally the conversion of the latter into the azide and urethane (in alcoholic solution by amylnitrite and hydrogen chloride) and the hydrolysis of the urethane by concentrated hydro- chloric acid, which gives the hydrochloride of the desired amine (VI). The direct decarboxylation of histidine can be carried out more conveniently by bacterial action and is applied industrially, according to patents by Hoffmann, La Roche & Co. [1912], and by Farben- fabriken vorm. F. Bayer & Co. (D.R.P. 250110). Details of the method are given in the appendix. An attempt to decarboxylate histidine by heat alone results only 1 Yoshimura [1909] probably obtained iminazolylethylamine by putrefaction before Ackermann, but he did not identify it. He found that the Japanese beverage Tamari- Shoyu, prepared from Soy beans, contains per litre o'7 grm. of a base C 6 H 9 N 3 , which he surmised was derived from histidine. 24 THE SIMPLER NATURAL BASES in the formation of traces of the amine, and Ackermann, by heating histidine with lime, could only obtain glyoxaline. Ewins and Pyman [1911], however, obtained a 10-20 per cent, yield by heating benzoyl histidine in a vacuum to 240 and subsequent hydrolysis, and a 24 per cent, yield by heating histidine hydrochloride with 20 per cent, sulphuric acid to 265-270. The most convenient method of prepar- ing iminazolyl-ethylamine is, however, by the synthetical method of Pyman [1911]. Diaminoacetone dihydrochloride (I) (obtained from citric acid) is heated with one molecular proportion of potassium sulphocyanide ; the thiolglyoxaline (II), thus formed by CH a .NH 3 .HCl CH.NH, CH . NH X * i * f-^ CSH -> f- CH 2 .NH a .HCl JH..NH, CH 2 OH I II III I CH.NHv CH.NH, CH . NH 11 X ~H II >XH II C N ^ C N< +_ C CH 2 . CH 2 . NH 2 CH 2 . CN CH 2 C1 VI V IV Gabriel's general method, is oxidised with nitric acid ; the nitrous acid formed in the reaction further attacks the amino-group so that a glyoxaline alcohol (III) results. This is successively converted into the chloro-compound (IV) and the cyano-compound (V) ; the latter yields on reduction the desired amine (VI). The lower homologue, iminazolylmethylamine, has been prepared by Windaus and Opitz [191 1]. PHYSIOLOGICAL PROPERTIES OF THE AMINES DERIVED FROM AMINO-ACIDS. The chief interest attached to the amines described in this chapter is due to their physiological action and to the possibility of their forma- tion in the organism, wherever proteins or amino-acids are exposed to bacterial action as, for instance, in the intestine. By far the most active amines are those containing a ring, namely those derived from phenyl-alanine, tyrosine, tryptophane, and histidine. Their formation does not take place in acid solution, and would, therefore, appear to be prevented or lessened by the sour-milk treatment recommended by MetchnikofT. Berthelot and Bertrand [1913, l] find, however, that their Bacillus aminophihis even produces /3-iminazolylethylamine in O'3 per cent, lactic acid, unless much glucose is present, when the sugar alone is attacked. The same investigators [1913, 2] find that rats, fed on a milk diet, are not affected by either Proteus vulgaris or B. amino- philus intestinalis when given separately, but that if the two organisms are given simultaneously, the rats may develop a fatal diarrhoea in from 4-8 days. Normally these putrefactive amines appear to be de- stroyed in the liver; Ewins and Laidlaw [1910, 3; 1913] have shown that p-hydroxy-phenyl-ethylamine and indole-ethylamine are trans- formed by perfusion through a surviving liver into p-hydroxy-phenyl- acetic acid and indole-acetic acid respectively. Oehme [1913] states that 0'6 mg. may kill a rabbit when given intravenously, but that the lethal dose is much higher when injected into the portal circulation. Rabbits will even stand 0*5 grm. by the mouth. Nevertheless the amines may perhaps play a part in certain diseases ; thus p-hydroxy- phenyl-ethylamine may be connected with a persistent high blood pressure, and Mellanby [1911] has attempted to connect /3-iminazolyl- ethyl-amine with cyclic vomiting. Pharmacologically these bases are important on account of their presence in ergot. Ehrlich and Pistschimuka [1912] have shown that they are transformed by yeast into the corresponding alcohols, and according to Czapek [1903] the amines with 3-7 carbon atoms are a good source of nitrogen for Aspergillus. The action of many synthetic amines has been examined ; it seems 25 26 THE SIMPLER NATURAL BASES that the most active are cyclic ones with a side chain of two carbon atoms like the last four naturally occurring ones described in this chapter. This conclusion with regard to the side chain was deduced for aromatic amines by Barger and Dale [1910, I] ; it is further sup- ported by toxicity determinations of several iminazole derivatives by Friedberger and Moreschi [1912]. Von Braun and Deutsch [1912] have found, however, that when the side chain of hordenine is lengthened the pressor action is diminished and the toxicity is in- creased. With four and five carbon atoms in the side chain the toxicity is ten times as great as with three carbon atoms. The natural amines described in this chapter may be arranged in two groups, of monamines and of diamines, and physiological action is more or less of the same type within each group. The monamines (see p. 29) produce effects similar to those caused by stimulation of the sympathetic nervous system. They may be termed sympatho- mimetic (see p. 98). The most powerful sympathomimetic base is adrenaline (see Chapter VI). Of the bases already described the most powerful is p-hydroxy-phenyl-ethylamine : the others in descending order of activity are phenyl-ethylamine, isoamylamine, isobutylamine. One of the most marked of sympathomimetic actions is the raising of the blood pressure on intravenous injection and isobutylamine is the lowest amine which has any marked pressor action. 10-20 mg. of isoamylamine, injected intravenously as the hydrochloride, produce a marked rise of blood pressure in the cat (Dale and Dixon [1909]). The effect of other aliphatic monamines is very similar. Normal amylamine has a slightly greater activity than its isomeride, and hexyl- amine is still more active, but in ascending the series beyond this point the activity again declines, heptylamine being less active than hexyl- amine and octylamine much less so (Barger and Dale [1910, l]). The introduction of a benzene ring in phenyl-ethylamine greatly increases the activity and this base is at least five times as active as any aliphatic amine. Thus 2 mg. of the base may raise the blood pressure of a cat from 30 to 180 mm. Phenyl-ethylamine has the same carbon skeleton as adrenaline. p-Hydroxy~phenyl-ethylamine has an activity something like ^ of that of adrenaline, and has been studied by Dale and Dixon [1909]. Doses of 1-2 mg., injected intravenously, cause a sudden and pronounced rise of arterial blood pressure, which is somewhat less transitory than that caused by adrenaline. As with the latter sub- AMINES DERIVED FROM PROTEIN 27 stance, the output of the heart is increased, the non-pregnant cat's uterus relaxes, the pregnant cat's uterus contracts, the salivary gland is stimulated to secretion. p-Hydroxy-phenyl-ethylamine differs from adrenaline in causing little vase-constriction when applied locally to a mucous surface, and in being hardly toxic. Thus 100 mg. given hypodermically to a cat, produced all the symptoms of intense stimulation of sympathetic nerves, but no after-effects and no glycosuria. Since p-hydroxy-phenyl-ethylamine is formed from tyrosine by the action of faecal bacteria, it doubtless occurs in the alimentary canal and might therefore perhaps play a part in certain pathological states in which a high blood-pressure is the most prominent symptom. A pressor substance has been found in the urine by Abelous and termed urohypertensine (perhaps identical with isoamylamine) and Bain [1909, 1910] obtained from normal urine a pressor base, giving Millon's reaction ; the latter base was not isolated in a state of purity and its identity with p-hydroxyphenylethylamine, suggested by Bain, is very doubtful. Bain found that the amount of this base was diminished in the urine from gouty patients and particularly in that from patients with a high blood pressure ; on the other hand it did not disappear from normal urine during a milk diet or when medicinal doses of antiseptics were administered. On account of the possible clinical significance of p-hydroxy-phenyl- ethylamine, as indicated above, Ewins and Laidlaw [1910, 3] have in- vestigated the fate of this amine in the organism. They found that when given by the mouth to dogs, something like one-half the amount is excreted in the urine as p-hydroxy-phenylacetic acid ; the other half remains unaccounted for. The conversion of the amine into the acid readily takes place in the perfused rabbit's liver, and also to some extent in the perfused isolated uterus, but in the isolated heart the amine, when perfused, was completely destroyed and no p-hydroxy- phenylacetic acid could be isolated. Other papers of clinical interest are those by Harvey [1911], who induced renal disease and vascular sclerosis in rabbits by pro- longed intravenous and oral administration of p-hydroxy-phenyl- ethylamine, by Clark [1910] and by Findlay [1911] who examined the effect of this amine on man. Clark found that large doses (30- 200 mg.) given by the mouth generally gave a slight rise of blood pressure lasting for several hours, and that 20-60 mg., given sub- cutaneously, produced in the healthy subject a considerable rise of blood pressure, lasting for about twenty minutes. The suggestion by 28 THE SIMPLER NATURAL BASES Burmann [1912] and Heimann [1912] that p-hydroxy-phenyl-ethyl- amine can replace ergot, or even that it is the most important con- stituent of this drug, is erroneous (see especially a paper by Guggenheim [1912]). The action of the base has also been studied lately by Frohlich and Pick [1912], by Handovsky and Pick [1913] and by Bickel and Pawlow [1912]. According to Engel [1912] p-hydroxy-phenyl-ethylamine has no necrotising effect on tumours, although this effect is produced by phenyl-ethylamine, which has only one-fifth of the pressor activity of the first-named base. The effect is also shown by hordenine and by adrenaline. p-Hydroxyphenyl-ethylamine has a paralytic action on Crustacea and occurs in the salivary gland of Cephalopoda which feed on crabs [Henze, 1913]. Hordenine, which is the N-dimethyl-derivative of the last-named base, has a much weaker action, and has been studied by Camus [1906]. The minimal lethal dose of the sulphate is 0*3 grm. per kilo, for dogs, injected intravenously, and 2 grm. per kilo, for guinea- pigs injected subcutaneously, so that the toxicity is very slight. The base has a feeble pressor action. Its methiodide, however, causes a very rapid and evanescent rise of blood pressure in cats, when in- jected intravenously in doses of I mg. The effect superficially resembles that of adrenaline but is in reality of the nicotine type (Barger and Dale [1910, I]). Von Braun and Deutsch [1912] have prepared homologues of hordenine, having the formula OH.C (i H 4 .(CH 2 ) n .N(CH 3 ). 2 with ti = 3, 4 and 5. In these the pressor action of hordenine is diminished. The lethal dose for rabbits is respectively cri grm., croi grm.,o*O2 grm., as compared with 0-3 grm. for hordenine. Comp. von Braun, Ber. deutsch. chem. Ges., 1914,47, 492. The physiological action of indolethylamine has been studied by Laidlaw [1911]. Doses of 10-20 mg. of the hydrochloride given intravenously to rabbits and cats, produce a transient stimulant effect upon the central nervous system, causing clonic and tonic convulsions, tremors of limbs, and vaso-constriction. In the spinal cat 2 mg. causes a large rise of blood pressure due to vaso-constriction and in- creased cardiac activity. In this respect the amine resembles p-hydroxy- phenyl-ethylamine. Indolethylamine has further a direct stimulant action on plain muscle, which is most marked in the arterioles, the iris, and the uterus. This action of the amine from tryptophane is on the whole much less than that of the amine from histidine. Speaking AMINES DERIVED FROM PROTEIN 29 very broadly, indolethylamine (with two nitrogen atoms of which only one is basic) has a physiological action intermediate between that of the sympathomimetic monamines such as p-hydroxy-phenyl-ethylamine, and the diamines, like iminazolyl-ethylamine. Ewins and Laidlaw [1913] have more recently studied the fate of indolethylamine in the organism ; in the perfused liver the base is converted into indole-acetic acid, a change quite comparable to the transformation of p-hydroxy-phenyl-ethylamine into p-hydroxy- phenyl-acetic acid (see p. 27). In dogs the indole-acetic acid is however excreted in the urine in combination with glycine as indole- aceturic acid C 8 H 6 N . CH 2 . CO . NH . CH 2 . COOH, mp. 94, forming an orange red picrate which melts at 145. Among diamines /3-iminazolyl-ethylamine is the only one having a cyclic structure, and it is by far the most active. Putrescine and cadaverine have at most a very slight toxicity ; on intravenous injection in the cat they lower the blood pressure. Agmatine has according to Engeland and Kutscher [1910, l] a powerful action on the isolated uterus, causing contraction, but Dale and Laidlaw [1911, p. 194] state that agmatine does not make any significant contribution to the activity of ergot and is only feebly active as compared with /3-iminazolyl-ethylamine, also present in ergot. Thus 5 mgs. of agmatine produced a much smaller effect on the cat's uterus than O'l mg. of the latter base. The physiological action of ^-iminazolyl-ethylamine has been investigated by Ackermann and Kutscher [1910, I] and more fully by Dale and Laidlaw [1910, 191 1]. 1 According to the latter authors the fundamental and characteristic feature of the action is a direct stimulant effect on plain muscle, producing exaggerated rhythm or tonic con- traction, according to the dose. The most sensitive plain muscle is the non-pregnant uterus of some species and it is this reaction which led to the identification of the base in ergot. A marked contraction of the isolated uterus is produced by adding to the bath of Ringer's solution sufficient of the base to give a concentration of I : 25,000,000 and the effect of I : 250,000,000 is often quite definite (compare also Frohlich and Pick [1912] and Sugimoto [1913]). The muscular coats of the bronchioles are also highly sensitive to the action of /3-iminazolyl-ethylamine, especially in rodents, but not in the ox (Trendelenburg [1912]). Baehr and Pick [1913, I] have studied the effect on the musculature of the surviving guinea-pig's lung. Here 1 Many scattered observations on its action occur in the pharmacological literature of the last few years. 30 THE SIMPLER NATURAL BASES the contraction due to /3-iminazolylethylamine is permanently abolished by adrenaline, which is not so in the intact animal. Large guinea- pigs are killed in a few minutes by an intravenous injection of O'5 mg., owing to asphyxia resulting from the constriction of the bronchioles ; post-mortem the lungs are found to be permanently distended. This corresponds closely to the effects of poisoning by Witte's peptone and the toxic effects of serum or other protein in the sensitised guinea-pig, known as anaphylactic shock. Unlike peptone, iminazolyl-ethylamine does not, however, possess in any marked degree the power of rendering the blood incoagulable. Ac- cording to Popielski the physiological effect of peptone is produced by a hypothetical substance " vasodilatin," and he [1910, 2] has suggested that iminazolyl-ethylamine acts by liberation of vasodilatin, when injected intravenously, a supposition rejected by Dale and Laidlaw [1911]. Attention may also be drawn to a possible connection between iminazolyl-ethylamine and the " depressor substances " of various observers, such as the urohypotensine of Abelous and Bardier [1909]; the depressent action of Bayliss and Starling's secretine is indeed explained by the isolation from it of iminazolyl-ethylamine by Barger and Dale [1911]. The resemblance of the symptoms of poisoning with iminazolyl-ethyl- amine to those of anaphylactic shock is indeed very striking (Dale and Laidlaw [1910, 1911], Pfeiffer [1911], Biedl and Kraus [1912], Schittenhelm and Weichardt [1912], Aronson [1912], Friedberger and Moreschi [1912]); not only does it extend to the bronchial constriction in guinea-pigs, mentioned above, but also to a fall of body temperature, which is one of the characteristics of the milder degree of the " shock ". Thus the intraperitoneal injection of 3 mgs. of iminazolyl-ethylamine was found by Dale and Laidlaw to lower the rectal temperature of a guinea-pig gradually from 38*5 to 28*5 in the course of two hours ; next day it was again 38. Extremely minute doses of serum may, on the other hand, cause a rise of body temperature in an anaphylactic animal, and the same applies to iminazolyl-ethylamine when given in sufficiently small doses to a (normal) guinea-pig, as has been shown by Pfeiffer [191 1]. The correspondence is also illus- trated by the relatively great resistance of dogs, both to anaphylactic shock and to the amine. In this connection we may refer to a paper by Engeland [1908, 3] in which evidence is adduced that histidine derivatives are more readily broken down by carnivora than by herbivora. No data are available to fix the lethal dose of /3-iminazolyl- ethylamine in man, but a Macacus monkey of 1*25 kilo, was killed by AMINES DERIVED FROM PROTEIN an intravenous injection of 0*065 grm. of the hydrochloride [Berthelot and Bertrand, 1912, 3], Lately the close similarity between the symptoms of poisoning by /9-iminazolyl-ethylamine and those of anaphylactic shock have been emphasised anew by Oehme [1913]. He and Loewit [1913 ; Ch. V, methyl guanidine] both criticise the conclusion of Heyde [1912 ; Ch. V, methylguanidine] that methylguanidine rather than iminazolyl- ethylamine is of importance in this respect. The supposed connection between /3-iminazolyl-ethylamine and ana- phylactic shock has even led to the statement (by Aronson [1912]) that the amine is formed by incubating histidine with normal guinea-pigs' serum, but this has been disproved by Friedberger and Moreschi [1912] and Modrakowski [1912] denies that the amine is the cause of anaphylactic shock since it does not render the blood incoagulable. In recording the fact, "as a point of interest and possible signifi- cance," that the immediate symptoms with which an animal responds to an injection of a normally inert protein, to which it has been previously sensitised, are to a large extent those of poisoning by /3-iminazolyl-ethylamine, Dale and Laidlaw consider that " the corre- spondence cannot yet be regarded as sufficient basis for theoretical speculation ". Pfeiffer thinks that /3-iminazolyl-ethylamine will cer- tainly be of significance for the solution of the problem of anaphylaxis. The effect of iminazolyl-ethylamine on the vascular system is complex and varies in different species, as well as in the same species under different conditions. In rodents a rise of blood-pressure occurs, owing to constriction of the arterioles, but may be masked by embar- rassed respiration. It was the different behaviour of rabbits to the base from histidine and that from ergot, which led Kutscher [1910, I] to regard the two bases as different. Barger and Dale [1910, 3] have however shown that both kinds of physiological effect are obtainable with the base from either source, so that the identity cannot be doubted. In carnivora, in the fowl, in the monkey (and probably therefore in man) iminazolyl-ethylamine causes vasodilatation and a fall of systemic blood pressure. The following table (Barbour [1913]) gives the effects of the amine, compared with those of adrenaline and p-hydroxy-phenyl- ethylamine : Blood Pressure. Peripheral Vessels. Coronary Vessels (Ox). Non-pregnant Uterus. Epinephrin (adrenaline) .... + + _ _ Tyramin (p-hydroxy-phenyl-ethylamine) Histamin (/3-iminazolyl-ethylamine) + + + + + -r- + means rise of blood pressure or constriction, - the opposite ; the last-named amine may have a pressor effect in some animals. 32 THE SIMPLER NATURAL BASES The pulmonary arterioles, however, are constricted and the pulmon- ary blood pressure is raised. This combination of a vasodilator fall of systemic blood pressure with a vasoconstrictor rise of pulmonary pres- sure has been described as characteristic of the action of ergot (Bradford and Dean [1894]), and is doubtless due to the iminazolyl-ethylamine present in the drug. For the effect of the base on the pulmonary vessels consult Baehr and Pick [1913, 2], and on the frog's blood vessels, Handovsky and Pick [1913]. Finally it should be mentioned that iminazolyl-ethylamine has a weak stimulant action on the salivary glands and on the pancreas, qualitatively resembling that of pilocarpine, which alkaloid also contains a glyoxaline ring. The action on the pancreas is not at all like that of secretine, being abolished by a small dose of atropine. CHAPTER II. o> AMINO-ACIDS AND OTHER BASES DERIVED FROM PROTEIN CONTAINING A CARBOXYL-GROUP (UROCANIC AND KYNURENIC ACIDS). IN the monamino-acids, formed by the hydrolysis of proteins, the acidic properties of the carboxyl-group are neutralised more or less completely by an adjoining amino-group in the a-position, and only the diamino-acids histidine, lysine, and arginine are bases. When the amino-group is not in the a-position the basic character is more pro- nounced, and the so-called w-amino-acids are feeble bases, being pre- cipitated by phosphotungstic acid ; several of them are formed from protein fission products by putrefaction, and these are described in this chapter. The influence of the position of the amino-group on the acid dis- sociation constant K a and on the basic dissociation constant K 6 is evident from the following table (Ley [1909, p. 358]) : Ka K& Glycine o-amino-propionic acid j8-amino-propionic acid 7-amino-butyric acid . 180 x 10 - 12 230 x 10 - ia 71 x io~ 12 37 x 10 _ 12 2*7 X IQ- 12 3-1 x to - 12 51 x io~ 12 170 x 10 ~ 12 An ammo-acid may also be rendered basic by complete methylation of the nitrogen atom, as in the betaines described in Chapter III. o>-Amino-acids are produced by putrefaction in three ways ; 1. By partial deaminization of a diamino-acid, as in the formation of S-amino-valeric acid from ornithine : NH 2 . CH 2 CH 2 CH a CH(NH 2 )COOH + 2H = NH 2 . CH 2 CH 2 CH 2 CH 2 COOH + NH 3 . 2. By the partial decarboxylation of a dibasic amino-acid, e.g. the production of 7-amino-butyric from glutamic acid : COOH . CH(NH 2 ) . CH 2 . CH 2 . COOH = NH 3 . CH 2 . CH a . CH 2 . COOH + CO 2 . 3. By the reduction of a cyclic amino-acid. Ackermann [191 1, 2] and Neuberg [191 1, l] have recently shown that a-pyrrolidine car- boxylic acid (proline) yields S-amino-valeric acid in putrefaction : 33 3 34 THE SIMPLER NATURAL BASES CH 2 CH 2 CH 2 CH . COOH + 2 H = NH 2 . CH 2 . CH 2 . CH 2 . CH 2 . COOH. NH The (o-amino-acids differ from a-amino-acids in being precipitated by phosphotungstic acid, even in dilute solutions ; they yield platini- chlorides soluble in alcohol (Ackermann). The 7-, 8-, and e-amino- acids are so weakly acidic that they do not form blue copper salts on boiling with cupric oxide, or on addition of cupric acetate, this pro- perty belonging only to a- and /3-amino-acids (Fischer and Zemplen [1909, p. 4883]). On heating 7-amino-butyric and S-amino-valeric acids are transformed into their anhydrides, pyrrolidone and piperidone. /9-Alanine, /3-amino-propionic Acid, NH 2 . CH 2 . CH 2 . COOH. This substance, long known synthetically, was first isolated from Liebig's extract of meat by Engeland [1908, I] ; Micko [1905] had previously obtained an alanine from the same source and assumed that it was the a-amino-acid. /3-Alanine is formed from the meat base carnosine by hydrolysis (see next section), and since Engeland's process of isolation involved evaporation in hydrochloric acid solution, Gulewitsch [1911; see under carnosine] questions whether /8-alanine is present as such in muscle. It was to be expected that /?-alanine could also be formed from aspartic acid by putrefaction, according to the second general method given in the preceding section, and after some failures Ackermann [1911, I] has succeeded in demonstrating this. One hundred grm. of aspartic acid in a culture medium similar to that used for preparing ^-iminazolyl-ethylamine yielded 2 grm. of yQ-alanine hydrochloride. ft- Alanine is broken down to urea in the dog (Abderhalden and Schittenhelm [1907]). 7-Amino-n-butyric Acid, NH 2 . CH 2 . CH 2 . CH 2 . COOH. This acid is formed in putrefaction from glutamic acid by the second general process (p. 33). Ackermann [1910, 3] obtained 2'i grm. of 7-amino-butyric acid aurichloride from 50 grm. of glutamic acid. Abderhalden and Kautzsch [1912] lately failed to repeat Ackermann's experiment, but afterwards Abderhalden, Fromme and Hirsch [1913] obtained 0*3 grm, of the platinichloride of 7-amino-butyric acid from 25 grm. of glutamic acid. o>-AMINO-ACIDS 35 S-Amino-n-valeric Acid, NH 2 . CH 2 . CH 2 . CH 2 . CH 2 . COOH. This, the first known example of a natural w-amino-acid, was ob- tained by E. and H. Salkowski [1883] from putrefied fibrin and muscle, and later by H. Salkowski [1898] from putrefied gelatin. Ackermann [1907, 2] isolated it from putrid pancreas (and at first called it putridine, because he failed to identify it). The substance was pre- pared synthetically by Schotten [1884] by the oxidation of benzoyl- piperidine with potassium permanganate. S-Amino-valeric acid is derived in putrefaction from both arginine (ornithine) and proline. Ackermann [1910, 3] submitted 56 grm. of arginine carbonate to putrefaction in the same way as aspartic acid and glutamic acid (preceding sections), and obtained putrescine, ornithine, S-amino-valeric acid (about 1 5 grm. of the aurichloride) but not agmatine. The arginine is no doubt first broken down to ornithine, and the latter by the first general process (p. 33) yields S-amino-valeric acid. The putrefactive formation of S-amino-valeric acid from proline (a-pyrrolidine carboxylic acid) has been observed more recently by both Ackermann and Neuberg ; two hydrogen atoms are added and the ring is opened. e-Amino-caproic Acid, NH 2 . (CH 2 ) 5 . COOH. This substance should be obtainable from lysine by putrefactive deaminization ; an attempt to prove this was made by Ackermann [1910, 3] with 98 grm. of lysine chloride. He obtained a large quantity of cadaverine and a small quantity of a platinichloride fairly readily soluble in alcohol and in water ; the analysis of this salt did not agree with the composition required for the platinichloride of the desired amino-caproic acid. /Mminazolyl-propionic Acid, CH = C CH 2 .CH 2 .COOH N NH V CH This acid was first obtained from histidine by chemical means and was also prepared synthetically by Knoop and Windaus [1906] (see Plimmer's " Chemical Constitution of the Proteins," Part I, p. 1 26). Ac- kermann [1910, i] then showed that it is also formed by putrefaction from pure histidine hydrochloride ; the principal product was imin- 3* 36 THE SIMPLER NATURAL BASES azolyl-ethylamine (described in Chapter I, p. 22), but in addition a small quantity of iminazolyl-propionic acid was obtained. Carnosine (Ignotine), C 9 H 14 O 3 N 4 . This substance is described in this chapter as it is a derivative of y8-alanine. Carnosine is, after creatine, the most abundant base in meat extract. It was discovered by Gulewitsch and Amiradz'ibi [1900, 1,2]; Krim berg [1906, I] obtained 0*13 per cent, from fresh ox meat. Ignotine, subsequently isolated by Kutscher [1905] from meat extract and regarded by him as an isomeride, was shown by Gulewitsch [1906], by direct comparison, to be identical with carnosine, and the identity has been admitted by Kutscher after pro- longed controversy. Carnosine has also been obtained from horse meat, to the extent of 1-82 grm. per kilo. (Smorodinzew [1913]) and from fish, crabs, oysters and wild rabbits. On heating with baryta to 140, carnosine is hydro lysed to histi- dine and /3-alanine in equimolecular proportions (Gulewitsch [1907, 191 1 ]) according to the equation : C 9 H 14 O 3 N 4 + H a O = C 6 H 9 O 2 N 3 + C 3 H 7 O 2 N. It is, therefore, similar to a dipeptide and must be either histidyl-/3- alanine or /3-alanyl-histidine ; it gives the red coloration with sodium p-diazobenzene sulphonate, characteristic of histidine, and yields on boil- ing with cupric carbonate a copper salt similar to that of /3-alanine. Perhaps, therefore, histidyl-yS-alanine is the more likely constitution : CH = C CH 2 . CH . CO . NH . CH a . CH 2 . COOH II 'I N NH NH 2 Urocanic Acid, Iminazolyl-acrylic Acid, CH = C CH = CH . COOH N NH v CH This acid contains two hydrogen atoms less than iminazolyl-pro- pionic acid described above and may be considered to be derived from histidine by loss of ammonia, without reduction. It was discovered by Jarfe" [1874, 1875] in the urine of a dog; after a few days the dog ran away, and, to Jaffe's great disappointment, it was never recaptured. The substance, was not observed again until Siegfried a>-AMINO-AClDS 37 [1898] found it once more in dog's urine. In both cases the substance was constantly present ; no other case of its occurrence in urine has been observed and it would appear that the two dogs presented a rare anomaly of metabolism. Recently Hunter [1912], although unable to find a dog secreting urocanic acid, obtained the same substance by prolonged tryptic digestion of caseinogen and was able to identify it by comparison with a specimen of iminazolylacrylic acid which Barger and Ewins [1911] had obtained as a degradation product of ergo- thioneine and had also synthesised. Among closely related substances from human urine we may men- tion histidine itself, a base yielding a picrolonate C 5 H 7 O 2 N 3 , C 10 H 8 O 5 N 4 melting at 244, and a base giving an aurichloride C 15 H 36 O 13 N 8 , HAuCl 4 very soluble in water and blackening at 100. These bases were ob- tained by Engeland [1908, 3] who regards the second as amino-imin- azolylacetic acid, a lower homologue of histidine, and the third as probably & polypeptide of histidine. According to Engeland histidine is broken down more readily by carnivora than by herbivora ; the urine of rabbits and horses gives a stronger reaction with p-diazobenzene sulphonic acid than that of the cat or dog. OH Kynurenic Acid, v N Long ago Liebig [1853] discovered an acid which occasionally separated from dog's urine in minute quantity. The substance was further investigated by Schmiedeberg and Schultzen [1872] and by Kretschy [1881-84] who showed that the product formed by heating the acid above its melting point, the so-called kynurine, C 9 H 7 ON, was an oxyquinoline, and that kynurenic acid was therefore an oxyquinoline carboxylic acid. Heated with zinc dust kynurine was reduced to quinoline, and on oxidation of kynurenic Kretschy obtained oxalyl- anthranilic acid, COOH \NH . CO . COOH. Hence, when Wenzel [1894] had shown by synthesis that kynurine is 4-hydroxy-quinoline, kynurenic acid was found to be either 4-hydroxy- 3 -quinoline carboxylic acid, or 4-hydroxy-2-quinoline carboxylic acid. OH OH ^\/\ COOH or ^\/\ I II I I I! I I /"*/-\/"\TT >x /\ A L/UUri. N N 38 THE SIMPLER NATURAL BASES Camps [1901, I, 2] prepared both acids and wrongly concluded that the former was identical with the acid from dog's urine, but Miss Homer [1913] has shown, by the mixed melting point, that kynurenic acid has the latter constitution. Liebig [1853], Kretschy [1881] and others had already found that kynurenic acid only makes its appearance, or is most abundant, in the urine of dogs fed on large quantities of meat. Many fruitless investigations were undertaken to find the precursor of the acid, until finally its formation was shown to depend on a product of tryptic digestion of protein (Glaessner and Langstein [1902]). This Ellinger [1904, I, 2] identified as tryptophane (see Plimmer's " Chemical Constitution of the Proteins," Part I, p. 137). Abderhalden, London, and Pincussohn [1909] have shown that the transformation of trypto- phane into kynurenic acid does not take place in the liver. Kynurenic acid, taken by the mouth, is not excreted in the urine in man and in the rabbit (Hauser [1895], Solonin [1897]); the reason is probably that the acid is an intermediate product of metabol- ism which is not destroyed so rapidly in the dog as in man. CHAPTER III. BETAINES. THE betaines are amino-acids in which the nitrogen atom is completely methylated. In addition to trimethyl-glycine, which has been known for a long time and occurs both in plants and in animals, fully methyl- ated derivatives of proline, oxyproline, histidine, and tryptophane have so far been obtained from plants, and corresponding derivatives of y- amino-butyric and of y-amino-hydroxy-butyric acid from animals. Except in the case of trigonelline, which occurs in many plants but is not related to any known decomposition product of protein, the betaine grouping does not occur in the typical vegetable alkaloids ; the two cases of its alleged occurrence, in damascenine and in chrysanthemine, have lately been disproved (respectively by Ewins [1912] and Yoshimura and Trier [1912, section on stachydrine]). The betaines therefore form a fairly natural group comprising feeble bases of simple constitution ; the a-betaines are devoid of marked physiological activity, but the two y-betaines (being presumably stronger bases) have a distinct action. A comprehensive study of the chemical behaviour of betaines has been made by Willstatter [1902, l] whose nomenclature is here employed. He points out that a-betaines and the isomeric esters of dimethyl-amino-acids are interconvertible : /CH 3 /CH, COOCH 3 COO CH 3 In the case of the betaines of $-and-y-amino-acids the above change only proceeds from left to right, but not in the reverse direction. From the methyl ester of /3-dimethyl-amino-propionic acid -propio-betaine is thus obtainable ; when y-dimethyl-amino-butyrate is heated, the y- butyro-betaine which no doubt first results, is unstable and yields trimethylamine and y-butyro-lactone. Further details concerning the interconversion in the case of trimethyl-glycine are given in the next section. The a-betaines differ greatly in the ease with which they split off 39 40 THE SIMPLER NATURAL BASES trimethylamine. Some are so unstable that they cannot be formed by the ordinary process of methylation. Thus aspartic acid, when treated with methyl iodide and alkali, breaks up into trimethylamine and fumaric acid. The same applies to tyrosine and it is noteworthy that the betaines of tyrosine and of phenylalanine have never been found in nature, whereas the corresponding unsaturated acids (p-cumaric and cinnamic acids) are often met with in plants. The betaine of tryptophane is somewhat more stable, and ergothioneine requires heating with concentrated alkali to decompose it into trimethylamine and the un- saturated acid. The free betaines when dried above 1 00 have a composition corresponding to a cyclic anhydride (the second of the above formulae). Salts are formed by direct addition of an acid, when the ring is broken down. Most betaines crystallise with one molecule of water and in this condition their constitution is probably illustrated by the for- mula : /OH (CH 3 ) 3 : N/ \CH 2 .COOH. The main physiological interest of betaines is derived from the question whether they may re-enter the metabolism of plants or whether they are merely waste products ; this question is further discussed in the next section. Pharmacologically the a-betaines are inert, but y-butyro- betaine is toxic to higher animals. Betaine, Trimethylglycine, (CH ) 3 : N/ X CH While searching for alkaloids in Solanacece^ Husemann and Marme [1863, 1864] isolated a base from Lycium barbarum, which was found to have the composition C 5 H n O 2 N and was named by them lycine. Three years later Scheibler [1866] obtained from the sap of the sugar beet (Beta vulgaris) and from beet molasses a " soluble alkaloid" which he described in detail later [1869] and called betaine. Soon afterwards Scheibler [1870] and Liebreich [1870] showed the identity of betaine with oxyneurine, a base prepared by Liebreich [1869, 2] by the oxidation of " bilineurine " ( = choline) and also synthetically by the action of trimethylamine on mono-chloracetic acid. Griess [1875] prepared betaine according to his general method, by methylating glycine and Husemann [1875] proved the identity of lycine with betaine ; the second (and later) name for this base has, however, passed into general use. BETAINES 41 Betaine is of rather widespread occurrence in plants and has also been found repeatedly in animals, but it is by no means so common as choline. Stanek and Domin [1910] have given a list of plants con- taining betaine ; it was found in all species of Chenopodiacece examined ; this natural order includes the sugar beet and also Chenopodium Vulvaria which gives off trimethylamine during life. In the closely related order of Amarantacece betaine was found by Stanek and Domin in some genera only ; in other orders it only occurs sporadically and in small amount. The dry leaves of A triplex canescens (N.O. Cheno- podiaceae) contain as much as 378 per cent, of betaine, but in rye the amount is only 0*3 per cent, of the dry weight. Young sugar beets contain 2-5 per cent, old ones I per cent, of betaine (Scheibler). 1 Various authors have at different times expressed the view that betaine may replace choline in lecithin. According to Trier [1912, 3, p. 83 ; Ch. IV, choline] they were misled on account of the difficulty of purifying the phosphatide. In the manufacture of beet sugar most of the betaine remains in the molasses, but crude beet sugar may contain 0375 per cent, of betaine (Waller and Plimmer [1903]). When the molasses are desaccharified by means of strontium, the final liquor (" Schlempe ") is very rich in betaine (i 15 grm. per kilo., Andrlik [1903-4]). Syntheses of betaine by Liebreich [1869, 2] and by Griess [1875] have been referred to above ; it is also formed by isomeric change from the methyl ester of dimethylamino-acetic acid in sealed tubes at 200 (see below). The estimation of betaine and its separation from choline by Schulze's method [1909 ; Ch. IV, choline] and by Stanek's method [1906, I, 2; Ch. IV, choline] are described on pp. 150-152. 1 Other sources of betaine are : Lycium barbarum (Husemann and Marme [1863]), the press cake of cotton seeds (Ritthausen and Weger [1884]), malt and wheat germs (Schulzeand Frankfurt [1893 ; Ch. IV, choline]) ; (Yoshimura [1910, Ch. IV, choline] recently found 0-06 per cent, of betaine in air dry malt germs) ; sunflower seeds (Schulze and Castoro [1904]), tubers of Helianthus tuberosus (Schulze [1910]), seeds of Avena sativa (Schulze and Pfenninger [1911; Ch. IV, choline]), Kola nuts (Polstorff [1909, 2; Ch. IV, choline]), bamboo shoots (Totani [1910,2; Ch. IV, choline]), green tobacco leaves (Deleano and Trier [1912]), ergot (Kraft [1906, Ch. IV, choline], Rielander [1908, Ch. I]) and com- mercial mushroom extract (Kutscher [1910, 4 ; Ch. IV, choline]). For a long time the only recorded instance of the occurrence of betaine in animals was Brieger's discovery of the base in mussels (Mytilus edulis ; [1886, 1, pp. 77-79; Ch. I)]. Later a number of other animal sources have become known : in commercial shrimp extract (Ackermann and Kutscher [1907, 3]), in the muscles of Acanthias vulgaris, 2 per cent, in embryos, 0*07 per cent, in adults (Suwa [1909, i], Kutscher [1910, 3]), in the crayfish, Astacus fluviatilis (Kutscher [1910, 2]), in a cuttle-fish (Octopus) (Henze [1910]). A sub- stance from the Japanese cuttle-fish Ommastrephcs identified by Suzuki and Yoshimura [1909] as 5-amino-valeric acid is, according to Kutscher [1909], betaine. Betaine is also present in mammalia; Bebeschin [1911] isolated 0*05 per cent, of betaine from ox-kidneys. 42 THE SIMPLER NATURAL BASES Physiological Properties and Importance of Betaine. The question as to whether betaine can be utilised by the animal organism as a source of nitrogen is of some interest on account of the increasing use of molasses as a cattle food. In the dog after intra- venous injection nearly the whole of the betaine is rapidly excreted in the urine, but when given by the mouth only about one quarter is so excreted (Andrlik, Velich and Stanek [1902-3], Voltz [1907]). Ruminants are more able to decompose betaine ; a cow accustomed to molasses excreted no betaine in its urine, and a sheep only during the first few days of feeding on molasses. Nevertheless, according to Voltz, the whole of the betaine nitrogen is excreted in sheep even when there is a deficiency of nitrogen in the food, and the organism only retains the non-nitrogenous part of the betaine. Although betaine is therefore not a food, it appears to be quite harmless. Andrlik, Velich and Stanek for instance gave a rat intra- venously betaine representing 0*24 per cent, of its body weight without any appreciable effect. Riesser [1913 ; Ch. V, creatine] injected betaine into rabbits and thereby increased their muscular creatine content by 6-3-11-3 per cent. He thinks that betaine may condense with an equimolecular proportion of urea to form creatine and methyl alcohol. When betaine chloride is melted with an excess of urea, methyl alcohol is given off. See also pp. 77-78. Waller and Sowton [1903 ; Ch. IV, choline] have described a toxic action of betaine in the excised frog's heart and on isolated nerves, and Waller and Plimmer [1903] on intravenous injection. According to Velich [1904-5] the effects observed were due to hydrochloric acid, owing to insufficient neutralisation of the betaine chloride injected. Further experiments (unpublished) by Waller and Plimmer showed that the injection of the betaine produced a slight lowering of the blood pressure, which allowed some of the magnesium sulphate solution, contained in the cannulae, to enter the circulation and exert a toxic action. A slight effect on the frog's heart has also been noted by Kohlrausch [1909, 1911]. With regard to the physiological importance of betaine in plants, Stanek [1911, I] has recently attempted to prove that the base is not a waste product. He has shown that more betaine is present in the leaves than in the seeds from which the plant has been grown ; the sugar beet may contain as much as I -2 per cent, of its dry weight as betaine. Schulze and Trier [1912, I] have similarly found that betaine BETAINES 43 is formed during germination in Vicia sativa and trigonelline in Pisum sativum. In a later paper Stanek [1911, 2] has concluded that there is more betaine in the dry substance of the young leaves than in that of the old, that betaine is formed during the germination of the seeds and that it travels from the roots to the leaves during the sprouting ; the base collects in the etiolated leaves and on ripening of the organs it disappears, probably because it travels back into the root. This latter conclusion is not shared by Schulze and Trier [1910, I] who consider betaine to be a waste product which no longer takes part in metabolism (see also Trier [1912, 3, pp. 83-7 ; Ch. IV, choline]). These authors point out that yeast cannot utilise betaine as a source of nitrogen (Stanek and Miskovsky [1907]) and that betaines pass unchanged through the animal organism. Some other fungi do utilise betaine, however. Ehrlich and Lange [1913] have shown that, in contra- distinction to ordinary cultivated yeasts, some wild yeasts like Willia anomala transform betaine to glycollic acid : (CH 3 ) 3 N . CH 2 . COO + H 2 O = CH 2 (OH) . COOH + N(CH 3 ) 3 This is analogous to the change of primary amines, described on page 25. In any case it seems justifiable to draw the conclusion from Stanek's experiments that betaine occurs most abundantly in those parts of the plant where the vegetative processes are most active, and Schulze and Trier consider that betaines collect in young leaves be- cause they are formed there. Young orange leaves also contain a greater proportion of stachydrine than the old ones. Stachydrine, C 7 H 13 O 2 N. Von Planta [1890] discovered a base in the edible tubers of Stachys tuberifera. The base closely resembled betaine but yielded an aurichloride with a smaller gold content ; it was further investigated by von Planta and Schulze [1893, i, 2] who found it had the compo- sition C 7 H 13 O 2 N, and Jahns [1896] isolated the same base from the leaves of the orange tree (Citrus vulgaris] and proved the presence of a carboxyl-group. Stachydrine is also present in the flowers of Chry- santhemum cinerariczfolium and in Galeopsis ochroleuca (Yoshimura and Trier [1912]) and (with betonicine) in Betonica officinalis (Schulze and Trier [1912, I, section on betaine]). Stachydrine gives off dimethyl- amine on heating with potassium hydroxide, and since it contains two hydrogen atoms less than is required for a homologue of betaine, Jahns considered it to be dimethylamino-angelic acid. The base is, however, stable to potassium permanganate, and the deficiency of two 44 THE SIMPLER NATURAL BASES hydrogen atoms is not due to unsaturation but to ring formation ; on heating, vapours are formed which give the pyrrole reaction with pine wood, and these facts led Schulze and Trier [1909, 2; 1910, 2] to re- gard the base as a derivative of a-pyrrolidine carboxylic acid (proline) which had meanwhile been recognised as a common fission product of proteins. They suggested for stachydrine the formula I, which was I t H 2 C CH, H 2 C CH 3 H 2 C I / '' - H 2 C C C : \X 1 TJ /" p X"^H TJ f > \x OCHs -^ / \y N N WT CH 3 CH 3 J X\ CH 3 CH, C I II III CH The same base was obtained more recently by Reuter [1912, Ch. I] from the arginine fraction of Boletus edulis (8 grm. of the monopicrate from 2| kilos, of the dried fungus), and Barger and Ewins [1913] have shown that it is also formed by the oxidation of ergothioneine (see next section). The direct methylation of histidine with dimethyl- sulphate leads to the formation of a pentamethyl derivative, since the imino-group of the glyoxaline ring is also attacked (Engeland and Kutscher [1912, 2]). 46 THE SIMPLER NATURAL BASES Ergothioneine, Thiolhistidine-betaine, C 9 H 15 O 2 N 3 S. Tanret [1909] isolated from ergot a base of the composition C 9 H 15 O 2 N 3 S and named it ergothioneine. Barger and Ewins [1911] have shown it to be the betaine of thiolhistidine, as follows : I II III CH NH IV V On heating ergothioneine (I) with concentrated potassium hydroxide solution, trimethylamine was given off almost quantitatively and a yellow unsaturated acid (II) resulted, which still contained sulphur and was almost insoluble m water. On boiling this acid with dilute nitric acid, the sulphur was removed and iminazolylacrylic acid (III) was formed and identified by comparison with a synthetic specimen. This substance was subsequently shown by Hunter [1912; Ch. II, urocanic acid] to be identical with urocanic acid from dog's urine (see p. 36). On boiling ergothioneine with ferric chloride the betaine of histidine itself is formed (IV) (see previous section). On adding iodine in alcoholic solution two molecules combine to form the quaternary iodide (V) which is much less soluble than the salts of ergo- thioneine, to which it bears the same relationship as cystine does to cystei'ne. By reduction with hydrogen sulphide this iodide is recon- verted into ergothioneine. The crystals of the dimeric iodide have the remarkable property of taking up excess of iodine from an aqueous solution and becoming steel grey or blue, like narceine and other substances. The biochemical (interest of ergothioneine is chiefly due to the sulphur atom contained in the glyoxaline ring. Oddly enough the BETAINES 47 thiolglyoxalines are intermediate products in the chief method for synthesising glyoxalines, due to Gabriel. The sulphur of ergothioneine behaves very differently from that in cystine ; it is not removed by alkalies and ergothioneine, therefore does not blacken lead hydroxide solution on boiling. On the other hand the sulphur atom is much more readily attacked by weak oxidising agents such as ferric chloride. Bearing this in mind we may perhaps hope to isolate ergothioneine or similar sulphur compounds from sources other than ergot. The physiological activity of ergothioneine is slight and it does not make any significant contribution to the action of ergot. Hypaphorine, Trimethyltryptophane, C 14 H 18 O 2 N. 2 . Hypaphorine is the betaine of tryptophane and has the constitution CH CH - r.C-CH,. CH . CO rr I ll IL It was discovered by Greshoff [1898] in the seeds of Erythrina Hypaphorus, Boerl., a tree grown for the sake of its shade in the coffee plantations of Eastern Java, and known locally as " dadap minjak ". The constitution of hypaphorine has been investigated by Van Rom- burgh [1911]. On heating with concentrated aqueous potassium hydroxide indole and trimethylamine result. The constitution was, however, determined by the synthesis from tryptophane (Van Rom- burgh and Barger [1911]). On heating tryptophane in methyl alcoholic solution with sodium hydroxide and methyl iodide the quater- nary iodide of methyl-a-trimethylamino-/3-indolepropionate is formed CH CH ^ C CH 2 . CH . COOCH 3 CH \/l\/ CH i(CH 3 KI CH NH and this, on warming with dilute alkali, yields a substance identical with the naturally occurring hypaphorine. Physiological Action of Hypaphorine. The substance has hardly any action on rodents and pigeons; thus intravenous doses of 0-5-1 grm. do not affect rabbits and the unchanged substance is rapidly secreted in the urine. In frogs, however, doses of 12-1 5 mg. produce increased reflex irritability and tetanus, lasting for days in non-fatal cases. Trigonelline, C 7 H 7 O 2 N. This substance, the betaine of nicotinic acid, is not derived from a protein fission product ; it contains a pyridine nucleus and is there- 48 THE SIMPLER NATURAL BASES fore to some extent more akin to the alkaloids. As it is however very similar to stachydrine, and as it has moreover been found in a number of species belonging to widely different natural orders, its inclusion here may be justified. Trigonelline was discovered by Jahns [1885] in the seeds of Trigonella foenum graecum (the Fenu greek). It has also been obtained from the seeds and seedlings of Pisum sativum (Schulze and Winterstein [1910]) ; from the seeds of Phaseolus vulgaris, Cannabis sativa, Avena sativa (Schulze [1896 ; Ch. IV, choline], Stro- phanthus hispidus, and 5. Kombe, Thorns [1898, I, 2 ; Ch. IV, choline] and Coffea arabica, Polstorff [1909, 2; Ch. IV, choline]); from the tubers of Stachys tuberifera and from potatoes (Schulze [1904 ; Ch. IV, choline]); from the roots of Scorzonera hispanica and the tubers of Dahlia (Schulze and Trier [1912, I ; section on betaine]). It is generally present in very small quantity and will doubtless be found to occur in many more species. Nicotinic acid, from which trigonelline is formed by methylation, occurs in rice polishings (Suzuki, Shimamura and Odake [1912], Funk [1913] ; both references in Ch. VII, vitamine, oryzanin). When this acid is given to dogs, trigonelline appears in the urine (Acker- mann [1912, l]). The constitution of trigonelline was established by Jahns [1887]. CH CH CH/^j C . CO CH/^C . COOH CH" ^'CH * CH^JCH N O N ^ 3 On heating with concentrated hydrochloric acid to 270 nicotinic (/?- pyridine-carboxylic) acid was formed and trigonelline was shown to be identical with the " methylbetain " of nicotinic acid, previously synthe- sised by Hantzsch [1886]. Trigonelline is physiologically inert; given subcutaneously, O'I2 grm. had no effect on frogs, nor O'5 grm. on rabbits (Jahns [1887]; compare also Kohlrausch [1909, 1911; section on betaine]). The methylation of nicotinic acid to trigonelline in the dog, discovered by Ackermann [1912, i], is similar to the methylation of pyridine to methylpyridinium hydroxide (see the next section). Other Pyridine Bases. Although they are not betaines, other derivatives of pyridine may be referred to here. For pyridine derivatives formed in putrefaction see Chapter I, p. 17. His [1887] showed that when pyridine acetate is given by the mouth to dogs, about one quarter may be recovered from the urine as the quaternary base, methyl -pyridinium hydroxide. BETAINES 49 II I A CH 3 OH The isolation was carried out by means of potassium mercuric iodide, and conversion into the gold and platinum salts. Kutscher and Lohmann [1906, 4 ; section on butyro-betaine] obtained the same base from normal human urine (at first [1906, 3] they mistook it for neurine). They [1907] consider that it is derived from the pyridine of tobacco smoke and of roasted coffee; 10 litres of men's urine yielded 0*17 grm. of the aurichloride, and 100 litres of women's urine 2'6 grm. ; the greater content of women's urine they ascribe to the " bekannte Vorliebe der Frauen fiir pyridinhaltigen Kaffee ". Roasted coffee beans con- tain 0*02 per cent, of pyridine [Bertrand and Weisweiller, 1913]. Methyl-pyridinium chloride has also been obtained from a commercial shrimp extract (Ackermann and Kutscher [1907, 4 ; under betaine]). The physiological action was investigated by Kohlrausch [1909, 1911 ; under betaine]. The platinichloride (C 6 H 8 N) 2 PtCl 6 forms large orange coloured plates, mp. 205-207, little soluble in cold water, readily in hot, and the aurichloride C 6 H 8 NAuCl 4 yellow needles, mp. 252-253, very little soluble in cold water. Achelis and Kutscher [1907] obtained 0-7 grm. of 7-picoline aurichloride mp. 201 from 10 litres of horse urine. This salt has the same composition as the preceding and is said to be derived from pyridine derivatives of the fodder. 4. u O CO 7-n-Butyro-betaine, (CH 3 ) 3 NSj >CH 2 \CH 2 CH/ Among the ptomaines isolated by Brieger [1886, I, p. 27 ; Ch. I] from horse meat which had putrefied for four months, was a base C 7 H 17 O 2 N. The chemical and physiological properties, as described by Brieger, correspond very closely with those of a betaine C 7 H 15 O 2 N obtained a few years ago by Takeda [1910] from the urine of dogs poisoned with phosphorus ; Engeland and Kutscher [1910, 3] obtained Takeda's base by methylating 7-amino-butyric acid, so that there is no doubt as to its constitution ; the identity with Brieger's base is almost equally certain, in which case his formula should contain two hydrogen atoms less. 1 7-Butyro-betaine was first synthesised by Willstatter [1902, i ; under betaine] and was also obtained by Krimberg [1907, 2] by the reduction of carnitine (see next section). Brieger isolated it from that part of the precipitate with mercuric chloride, which was the more soluble in water. After removal of the mercury, the base was precipitated as aurichloride. The physiological action was studied in some detail by Brieger. On frogs it has a curare action, in accordance with the fact that it is a quaternary base and a 7-betaine. In the a-betaines so far described the 1 Brieger's ptomaine and 7-butyro-betaine have a very similar composition, a gold salt of identical melting point, a soluble picrate and similar reactions to alkaloidal reagents : both arrest the frog's heart in diastole. 4 50 THE SIMPLER NATURAL BASES basic properties are more completely neutralised by the carboxyl-group, which is probably the reason for their physiological inertness (com- pare also the section on w-amino-acids, p. 33). Brieger found that 10 mg. of his hydrochloride arrested the heart of a frog in diastole. In rabbits 0-05-0-3 grm. produced mydriasis, salivation, clonic con- vulsions, often violent lowering of body temperature, dyspnoea, paralysis and ultimately (after several hours) death with the heart in diastole (Brieger [1886, I, pp. 29-31 ; Ch. I]). Brieger obtained two other bases of the composition C 7 H 17 O 2 N. One of these is gadinine, obtained from putrid cod fish (Bocklisch [1885, Ch. I], Brieger [1885, I, p. 49 ; Ch. IJ) and isolated as platinichloride. It " appeared " to be physiologically inert and the solution of the hydrochloride yielded a precipitate with picric acid, but not with gold chloride. Against these differences we may set the fact that the hydrochloride, like that of y-butyro- betaine and of betaine itself, was insoluble in absolute alcohol. The other base C 7 H 17 O 2 N is typhotoxine, obtained from cultures of typhoid bacilli (Brieger [1886, i, p. 86 ; Ch. I]). The melting point of the aurichloride was identical with that of the ptomaine from putrid horse meat (176). Typhotoxine, however, yielded a spar- ingly soluble picrate, a yellow coloration with diazobenzene sulphonic acid, and amorphous precipitates with potassium tri-iodide, potassium mercuric iodide and potassium cadmium iodide. The physiological action of typhotoxine was also somewhat different from that of the ptomaine from putrid horse meat. It does not seem wholly impossible, however, that all three bases were identical with y-butyro-betaine. Carnitine (Novaine, a-Hydroxy-7-butyro-betaine), O --- CO, CHOH - Carnitine, C 7 H 15 O 3 N, is a hydroxy-derivative of the base described in the previous section arid was discovered in extract of muscle by Gulewitsch and Krimberg [1905]. A few months later Kutscher [1905] obtained from Liebig's extract of meat a base " novain " which Krimberg [1908, i] proved to be identical with carnitine ; the identity has been admitted by Kutscher' s pupils, if not explicitly by Kutscher himself. According to Kutscher a base C 7 H 16 O 2 N, isolated by Dombrowski [1902] from normal human urine, was identical with novaine ; Kutscher thinks that in most cases (except in the dog) novaine passes into the urine as its reduction product reducto-novaine. Both carnitine and novaine were found by their discoverers to yield trimethylamine and crotonic acid (or an isomeride) on heating with baryta. By boiling with phosphorus and hydriodic acid Krimberg [1907, 2] reduced carnitine to 7-butyro-betaine. The only doubt now remaining was with regard to the position of the hydroxyl group in carnitine. Krimberg at first favoured the /3-position, but /3-hydroxy-7-butyro-betaine BETAINES 51 /o co\ (CH 3 ) 3 N/ J>CH 2 CH 2 CHOH has been synthesised by Rollett [1910] and by Engeland [1910 2] and was found to differ from carnitine, which is therefore most likely a-hydroxy-7-butyrobetaine i N CHOH. \CH 2 .CH 2 / The a-position of the hydroxyl group seems also to result from the oxidation of carnitine by calcium permanganate (Engeland [1909, I]) to /?-homobetaine o co (CH 3 ) 3 : N/ \CH 2 CH 2 Racemic carnitine has probably been obtained by Fischer and Goddertz [1910] from 7-phthalimido-a-bromobutyric acid; the melting point of the platinichloride agrees with that of natural carnitine, but the aurichloride has a much higher melting point. Carnitine may be pre- pared from meat extract by Gulewitsch and Krimberg's method, or by that of Kutscher ; the former method, in which the filtrate from carno- sine is precipitated with potassium bismuth iodide, gives apparently the better yield (1-3 per cent, of the Liebig's extract employed). Smorodinzew [1913; Ch. II, carnosine] obtained O'O2 per cent, of carnitine from fresh horse meat. Carnitine probably passes unchanged into the urine, for Kutscher and Lohmann [1906, 2] could isolate novaine ( = carnitine) from the urine of a dog fed on meat extract but not from normal dog's urine. In the rabbit carnitine is, perhaps, re- duced to butyrobetaine, according to Engeland [1908, I]. The physi- ological action of novaine ( = carnitine) has been studied by Kutscher and Lohmann [1906, I]. One gram, given hypodermically to a cat, produced serious disturbance of the digestive tract ; given intravenously novaine has a slight depressor action. Oblitine, a base obtained by Kutscher from meat extract, is according to Krimberg merely carnitine ethyl ester formed from carnitine during Kutscher's process of extrac- tion (see appendix). Reductonovaine C 7 H 15 ON was isolated as the aurichloride C 7 H 16 ONC1, AuCl 3 , mp. 155-180, from women's urine by Kutscher [1907, 2] who regards it as formed by loss of water from novaine to which it stands in the same relation as neurine to choline. 4* 52 THE SIMPLER NATURAL BASES Myokynine (1-Hexamethylornithine ?), C n H 28 O 4 N 2 . Working with Kutscher's method, Ackermann [1912, 2] has isolated from the lysine fraction of an extract of dog's muscle a platini- chloride C n H 30 O 4 N 2 PtCl 6 , insoluble in ethyl alcohol, mp. 233-234. The corresponding base was laevo-rotatory and gave off two molecular proportions of trimethylamine on heating with baryta. The composi- tion of the platinichloride agrees with that of a platinum salt of hexa- methylornithine with 2H 2 O. Hexamethylornithine was, therefore, prepared by methylating ornithine, and was found to be dextro-rotatory and to yield a platinichloride with iH 2 O melting at 232-233. It is not unlikely, therefore, that myokynine is the enantiomorph of the synthetic base, having the constitution : /OH HO X (CH 3 ) 3 : N<^ ^>N : (CH 3 ) 3 CH 2 . CH 2 . CH 2 . CH . COOH Later Ackermann [1913, I] obtained 3 grm. of the same platini- chloride from 30 kilos, of fresh horse meat. The base contains one carboxyl group. Unlike the natural base, synthetic hexamethylorni- thine gives a pyrrole reaction when heated with zinc dust. Ackermann points out that ornithine to some extent resembles glycine (compare the formation of ornithuric and hippuric acids) ; trimethyl-glycine or betaine has already been isolated from the muscles of a number of animals. CHAPTER IV. CHOLINE AND ALLIED SUBSTANCES. THE previous chapters have dealt with basic substances derived from the amino-acid units of proteins by various modifications. We must next consider two bases which enter into the composition of the phos- phatides ; they are units or " Bausteine " of these compounds, and are analogous to the amino-acids (described in Plimmer's " Chemical Con- stitution of the Proteins "). One of these units, choline, is apparently present (in a combined form) in every living cell ; the other, amino- ethyl alcohol, is probably the precursor of choline. Allied to choline there are two bases, neurine and muscarine, which are derived from choline by dehydration and probably by esterifkation respectively. These bases do not enter into the composition of phos- phatides ; their physiological behaviour is different from that of choline ; they are modified units and are therefore comparable to the modified amino-acids with which we have been concerned so far. In this chapter are also included two other bases with pentavalent nitrogen and without a carboxyl-group ; they are trimethylamine oxide and neosine ; the latter is perhaps a homologue of choline. Betaine is generally grouped with choline on account of a more or less accidental chemical connection, for it can be obtained in the laboratory by oxidising choline. There is, however, a considerable physiological difference between the two substances, for choline is a structural unit of phosphatides, but betaine plays no such part either in the phosphatide or in the protein molecule. Nor is a genetic relationship between the two substances apparent in the organism. It has been suggested that betaine is formed by the oxidation of choline, but recent work has made the conclusion almost inevitable that betaine is not formed in this way, but by the methylation of glycine (glycocoll), like the other betaines described in Chapter III. Choline and the substances derived from it further differ from the betaines in being strong bases, having a marked physiological action. To em- phasise all these points of difference the two groups of substances are described in separate chapters. 53 54 THE SIMPLER NATURAL BASES Choline, Trimethyl-/3-hydroxy-ethyl-ammonium Hydroxide, /PIT N XT/OH NCH 2 .CH 8 OH. Strecker [1849] obtained from pig's bile the platinichloride of a base, of which he later [1862] published the formula and a further description, and which he then named choline. Meanwhile von Babo and Hirschbrunn [1852], by hydrolysis of the alkaloid sinapin from white mustard seeds, had prepared a strong base which was well characterised by its platinichloride and was named sinkatin (from Sinapis and alkali). The identity of the base from mustard with that from bile was established by Claus and Keese [1867], but never- theless Strecker's (later) name has passed into general use. Con- fusion was introduced when Liebreich [1865] obtained a base by the hydrolysis of the brain substance protagon, and termed it neurin. The analysis of an impure platinichloride led Liebreich to the erroneous formula C 5 H 12 ON, corresponding to vinyl-trimethyl- ammonium hydroxide, and to this substance the name neurine has become definitely attached. The identity of Liebreich's protagon base with choline was established by Dybkowsky [1867] and for some years neurine was used as a synonym for choline, to which the name bilineurine was at one time also applied. The true formula of Liebreich's "neurin" was determined by Baeyer [1866, under neurine] who also converted it into the vinyl base [1869, under neurine], and " nevrine " (= choline) was first synthesised by Wurtz Since choline is a constituent of lecithin, it occurs probably in all living cells. It has been isolated by Schulze and his collaborators from every plant extract examined by them for its presence [Schulze and Trier, 1912, 3], Choline has been found in the following tissues : In the brain: as phosphatide, Liebreich [1865], Gulewitsch [1908, i], Vincent and Cramer [1904], Cramer [1904], Coriat [1904], Thudichum [1884, 1901 ; under amino-ethyl- alcohol] ; it is not present in the free state, Kauffmann [1911]. In the cerebro-spinal fluid in disease (Mott and Halliburton [1899] ; see below for an account of the controversy on this point). In many viscera (Kinoshita [1910, 2]), in the adrenal gland (Hunt [1899-1900], Lohmann [1907, 1911]), in the thymus, thyroid and lymphatic glands, and in the spleen (Schwarz and Lederer [1908]), in blood and in serum (Letsche [1907; Ch. IV, creatine], Gautrelet and Thomas [1909]), in ox testes (Totani [1910, i]), in semen (Florence [1897]), in egg-yolk, the most convenient natural source (Diakonow [1868]), in autolysed pancreas (Kutscher and Lohmann [1903]), in meat extract (Kutscher [1906, i ; Ch. V, creatine]), in putrid horse meat (Gulewitsch [1884, Ch. I]), in human corpses (Brieger [1885, 2, p. 17; Ch. I]), in bile (Strecker [1849]), in secretine (von Fiirth and Schwarz [1908]), in cheese (Winterstein [1904]), in herring brine (Bocklisch [1885, Ch. I]), in salted fish (Morner CHOLINE AND ALLIED SUBSTANCES 55 [1896, Ch. I]), in carnaubon, a glycerine free monophosphatide from ox kidney (Dunham and Jacobson [1910]), in sahidin (Frankel and Linnert [1910]), from sinapin by hydrolysis (von Babo and Hirschbrunn [1852]), in seeds of Vicia sativa and Pisum sativum (Schulze [1890]), of Strophanthus (Thorns [1898, I, 2]), of Avena sativa (Schulze and Pfenninger [1911]), in cotton seeds and beechnuts (Boehm [1885, 2]), in seeds of Trigonella feenum graecum and of Cannabis sativa (Jahns [1885]), in seeds of Artemisia cina (Jahns [1893]), in etiolated seedlings of lupins and of Cucurbita (Schulze [1887]), in seedlings of Soya hispida (Schulze [1888]), in malt and wheat germs (Schulze and Frankfurt [1893]), in rice polishings (Funk [1911]), in potatoes and Dahlia tubers (Schulze [1904]), in tubers of Stachys tuberifera and in orange leaves (Schulze and Trier [1910, 2; Ch. Ill, stachy- drine]), in beet molasses (von Lippmann [1887]), in roots of Atropa Belladona, Hyoscyamus and Ipecacuanha (Kunz [1885, 1887]), in bamboo shoots (Totani [1910, 2]), in the flowers of Chrysanthemum cineraria folium (Yoshimura and Trier [1912; Ch. Ill, stachydrine]), in Areca nuts, in pignuts (Arachis hypogcea] and in lentils (Jahns [1890]), in kola nuts (Ilex Paraguay ensis), Indian tea, and cocoa beans (Polstorff [1909, 2]), in hops and therefore in beer (Griess and Harrow [1885]), in grape juice and wine (Struve [1902]), in Sesame, Cocos, and palm seed press cake (Schulze [1896]), in the subterranean parts of Brassica Napns, Helianthus tubcrosus, Scorzonera hispanica, Cichorium Intybus, Apium graveolens, Daucus carota and in the aerial parts of Salvia pratensis and Betonica officinalis (Schulze and Trier [1912, 3]), in ergot (Brieger [1886, 2; Ch. I], Kraft [1906], Rielander [1908, Ch. I]), in Amanita muscaria (Harnack [1875 '> under muscarine]), in Boletus luridus, Amanita pantherina and Helvetia esculenta (Boehm [1885, I under muscarine]), in Can- tharellus cibarius, Agaricus campestris, and Boletus edulis (o'oi5-o'oo5 per cent. ; Polstorff, [1909, i]), in commercial mushroom extract (Kutscher [1910, 4]), in Russula emetica (Robert [1892]) and in Boletus satanas (Utz [1905]). The amount of choline obtainable from most sources is very small (in animal viscera and in seeds often of the order of 0*02 per cent). Schulze considered that in seeds at least some of the choline is in the free state ; he showed [1892, I] that in Vicia sativa the choline content increases during germination from 0*017 per cent, in the seeds to 0*06 per cent, in the seedlings. The additional choline in the latter is derived from lecithin, of which the seeds contain 0*74 per cent., but four weeks' old seedlings only 0-19 percent. We thus see that choline behaves in the same way as the amino-acids of protein, which are also formed by hydrolysis during germination. Betaine, which is also present in the seeds, on the other hand does not change in amount during germina- tion, for it is not a unit or <( Baustein ". The choline of the brain does not occur even partially in the free state. Liebreich [1865] obtained it by the hydrolysis of protagon ; Gulewitsch [1899] found that at most one-fifteenth of the total amount is free choline, and KaufTmann [1911] has shown that if perfectly fresh ox brain is worked up rapidly, no free choline is obtain- able. According to Coriat [1904] lecithin is not affected by try ps in or pepsin, but in autolysis choline is slowly split off by a ferment, which could not be isolated ; during putrefaction choline is liberated more rapidly. Mott and Halliburton [1899] found choline in the cerebro- 56 THE SIMPLER NATURAL BASES spinal fluid in certain degenerative nervous diseases, such as general paralysis of the insane, and they regard it as a break-down product of nerve substance. They used platinic chloride for the isolation, but since the amount of choline to be detected is at most very small, and since potassium and ammonium salts are also present, a good deal of controversy has taken place as to the identity of the platinichloride obtained. Probably Mott and Halliburton's salt was contaminated with potassium, since even anhydrous alcohol, as employed by Donath [1905-1906], dissolves ammonium chloride. Donath has attempted to utilise the double refraction and chromatic polarisation of choline platinichloride which is not given by the isotropic crystals of the potassium and ammonium salts. The conclusions of Mott and Halliburton and of Donath have been criticised by Vincent and Cramer [1904], by Allen and French [1903] and by Mansfeld [1904] ; Rosenheim [1905-6, 1907] and Allen [1904] have therefore attempted to find a more characteristic test in Florence's periodide reaction (see below) which may be applied to the platinichloride, or directly to the crude choline chloride. 1 According to Rosenheim and to Allen choline is indeed present in the cerebro-spinal fluid in certain diseases, but Donath's suggestion that choline is present in epilepsy and is the cause of the convulsions cannot be upheld (Allen [1904], Kajura [1908], and especially Handelsman [1908]). At most traces are present, wholly inadequate to account for the convulsions. Other authors, however, do not admit that choline has been demonstrated in the cerebro-spinal fluid even in diseases where there is a break-down of nervous tissue. Webster [1909] considers that no choline test hitherto employed is satisfactory. Kauffmann [1908, 1910] thinks that if traces of choline are present they are too small to be recognised with certainty. Kauffmann and Vorlander [1910] consider that the dimorphism of choline platinichloride (and conversion of the regular crystals into those of the monoclinic system, see below) affords a most char- acteristic test, and Kauffmann has concluded that an organic base is present in the cerebro- spinal fluid, which is not identical with choline. Stanford [1913] has recently arrived at the same conclusion, that the base present in disease gives alkaloidal reactions, but no tri- methylamine. Handelsman [1908] has emphasised the fact that on igniting the platini- chloride the odour of trimethylamine is never observed. It would appear that this con- troversy can only be ended by a satisfactory analysis of the platinum salt ; the only published analysis (by Mott and Halliburton) is of little value (Ft found 34-8 per cent. ; calculated 31*6 per cent.). According to Mott and Halliburton the choline set free in nervous lesions passes into the blood, a conclusion shared by Allen [1904], criticised by Vincent and Cramer [1904] and particularly by Vincent's pupil Webster [1909], and maintained by Halliburton [1905], Choline has been synthesised by several methods : 1. By the action of trimethylamine on ethylene oxide in concen- trated aqueous solution (Wurtz [1867]). /O\ /OH (CH 3 ) 3 : N + / \ + H 2 = (CH 3 ) 3 : N/ \CH l .CH Jt QH. 2. Trimethylamine combines with dry ethylene dibromide at 1 1 0-112 to yield trimethylamino-bromethylium bromide (Hofmann [1858, under neurine]). 1 Possibly the very slight solubility of choline nitric acid ester perchlorate might be utilised with advantage. CHOLINE AND ALLIED SUBSTANCES 57 /Br (CH 3 ) 3 : N + Br . CH 2 . CH 3 . Br = (CH 3 ) 3 : N( \CH 2 .CH 2 Br. By acting on the latter substance with silver oxide, Hofmann obtained the vinyl base instead of choline. Choline is however obtainable from it in two ways ; (a) by boiling for eight days with silver nitrate (Bode [1892]) /Br /Br (CH 3 ) 3 : N( + AgN0 8 + H,O = (CH 3 ) 3 1 N/ + AgBr + HNO 3 \CH a . CH 2 . Br \CH 2 . CH 2 OH (fi) by heating with twenty-five parts of water to 160 for a few hours (Kriiger and Bergell [1903]) /Br /Br (CH 3 ) 3 : N/ + H 2 = (CH 3 ) 3 j N< + HBr. \CH 2 . CH 2 Br X CH 2 . CH 2 OH 3. Rather more than one equivalent of trimethylamine gas is passed into ethylene chlorohydrin cooled to - 12 to - 20 in a tube which is subsequently warmed to 80-90 ; the yield is almost quantitative (Renshaw [1910]). /Cl (CH 3 ) 3 i N + Cl . CH 2 . CHoOH = (CH 3 ) 3 N/ \CH 2 . CH 2 OH. 4. By the methylation of amino-ethyl alcohol (Trier [1912, 2; under amino-ethyl-alcohol]) /I 3(CH 3 )I + 2NaOH + NH,CH 2 . CH a OH = (CH 3 ) 3 1 N/ + 2NaI + 2H 2 O. \CH 2 . CH 2 OH The methods of Kriiger and Bergell and of Renshaw appear to be the most convenient. A method for the estimation of choline in animal tissues has been described by Kinoshita [1910, 2]. For the isolation of choline from plant extracts, Jahns [1885] has employed potassium bismuth iodide (Kraut's reagent), Schulze has used phosphotungstic acid and mercuric chloride and Stanek utilises potassium tri-iodide. The two last named methods are more or less quantitative. Stanek 's method [1905, 1906, I, 2] is the most convenient for the quantitative estimation of choline in the presence of betaine when other bases yielding periodides are ab- sent (compare Kiesel [1907]). For a description of Stanek's and Schulze's methods see the appendix (Chapter VIII). The tests for, and chemical properties and salts of, choline are also described in the appendix (Chapter VIII). NH 3 + | = I CH 9 58 THE SIMPLER NATURAL BASES Amino-ethyl Alcohol (Colamine) and the Origin of Choline; the Possible Presence of other Bases in Phosphatides. By the hydrolysis of kephalin (a phosphatide from the brain) by means of baryta, Thudichum [1884, 1901] obtained long ago, in ad- dition to choline, a base having the composition of "oxethylamin," NH 2 .CH 2 .CH 3 OH. During the last few years Trier has isolated a base of the same com- position from lecithin of various sources and has definitely identified it as hydroxy-ethylamine or amino-ethyl alcohol. By hydrolysis of the phosphatide from beans (Phaseolus vulgaris) Trier [1911] obtained a fraction, representing one-seventh of the nitrogen content of the phosphatide, which yielded an aurichloride C 2 H 5 ON . HAuCl 4 , identical with that of a base previously synthesised by Knorr from ammonia and ethylene oxide : CH 2 \ CH 2 OH l>0=|' CH 2 / CH 2 NH 2 . The same base was subsequently obtained from the lecithin of peas and oats and also from commercial ovolecithin of Merck (Trier [1912, i]). The amino-ethyl alcohol can be estimated in phosphatides by means of Van Slyke's method (see Plimmer's " Chemical Constitution of the Proteins," Part I, p. 69). Trier [1913, 2] concludes from this that the base is joined to the rest of the phosphatide molecule by means of its hydroxyl group. In one specimen of ovolecithin the amino-nitrogen was nearly half the total. Baumann [1913] and Renall [1913] also used Van Slyke's method and showed that kephalin from human brain and from that of the sheep and ox contains as only base amino-ethyl alcohol and that here too the primary amino-group is free. They could not find choline and another base, which Thudichum believed to accompany the amino- ethyl alcohol. Trier considers that choline is formed from amino-ethyl alcohol by the biologically common process of methylation, in the same way that the betaines are derived from amino-acids. Thus there would be no genetic relationship between choline and betaine. The question is then : How is amino-ethyl alcohol itself formed ? Winterstein and Trier [1909, p. 31 1] 1 put forward the hypothesis that formaldehyde is condensed to glycollic aldehyde and that the latter is converted by ammonia into amino-acetaldehyde. By simul- 1 This and the subsequent references in this section will be found in the bibliography under choline. CHOLINE AND ALLIED SUBSTANCES 59 taneous oxidation and reduction (Cannizzaro's reaction) amino-ethyl alcohol and amino-acetic acid (glycine) are then supposed to be formed from the aldehyde. CH 3 OH + NH, CH 2 . NH 2 2. CH 2 -> | i | CHO CHO formaldehyde glycollic aldehyde amino-acetaldehyde CH 2 . NH 2 CH 3 . NH 2 CH 2 . NH 2 CHO CH 2 OH COOH + H 2 CHO amino-acetaldehyde amino-ethyl alcohol glycine In his recent book on the simple plant bases Trier [1912, 3, p. 33] has modified the above hypothesis and imagines that glycollic aldehyde first undergoes Cannizzaro's reaction and that the two products of this reaction (glycol and glycollic acid) then condense with ammonia CH 2 OH CH 2 OH CHoOH 2 I + H 3 +1 CHO CH 2 OH COOH glycollic aldehyde glycol glycollic acid CH 2 OH CH,.NH 2 + H O | + NH* = I CH 2 OH CH,,OH glycol amino-ethyl alcohol CH 2 OH CH 2 . NH 2 I + NH 3 = | +. H 2 COOH COOH glycollic acid glycine Amino-ethyl alcohol and glycine are the simplest units for the forma- tion of proteins and phosphatides respectively, and hence it becomes intelligible why, as Stoklasa has pointed out, protein and lecithin formation are two parallel processes. An argument for the biological significance of Cannizzaro's reaction is the occurrence of a number of alcohols as esters of the corresponding acid (e.g. benzyl benzoate and cinnamyl cinnamate in balsams ; cetyl-palmitate C 16 H 31 O 2 . C 16 H 33 occurs in spermaceti and ceryl cerotinate C. 27 H 53 O 2 . C 27 H 55 in Chinese wax). A ferment causing Cannizzaro's reaction (" aldehyde mutase") has been recently found in liver extracts by Parnas and by Batelli and Stern (see Dakin's " Oxidations and Reductions in the Animal Body," pp. 105, 1 06, in this series of monographs). In addition to Thudichum, Trier, and Baumann, who isolated amino-ethyl alcohol, other investigators have suggested that phos- phatides may contain bases similar to choline but containing fewer alkyl groups. These investigations however require careful scrutiny in the light of recent knowledge. Koch [1902] applied Herzig and Meyer's method for the estimation of N-methyl groups to kephalin and cerebrin and concluded that one N-methyl group is present in kephalin none in cerebrin, and three in lecithin. Frankel and Neubauer, like 60 THE SIMPLER NATURAL BASES Koch, failed to isolate Thudichum's non- methylated " ox-ethylamin " from kephalin, and agreed with Koch that one N-methyl group is present. Frankel and Linnert [1910] state that sahidin, from human brain, also contains a base with fewer methyl groups than choline. On the other hand Cousin [1907] could only obtain choline from kephalin. Koch, and Frankel and Neubauer did not isolate their supposed monomethylated base and their results have been criticised by Baumann [1913]; he and Trier [1913, 5] find that amino-ethyl alcohol, when heated with hydriodic acid, gives off some ethyl iodide, thus simulating the presence of an N-methyl group. It should further be remembered that the accuracy of Herzig and Meyer's method for determining N-alkyl groups is not sufficiently great for the certain determination of their number in a molecule of the size of lecithin, and that its application becomes wholly illusory if more than one base is present. Further mention of the presence in phosphatides of bases other than choline is to be found in papers by Erlandsen [1907] (on cuorin from ox hearts), by Baskoff [1908] (on the phosphatides of horse liver), by MacLean [1909], by Njegovan [1911], and in Trier's book on plant bases [1912, 3, pp. 96-101]. According to Trier, Njegovan's base "vidine" was merely choline containing a little ammonia as impurity. Neurine, Vinyltrimethyl-ammonium Hydroxide, /OH (CH 3 ), : N( \CH:CH 2 . Neurine was the name applied by Liebreich to a base obtained in the hydrolysis of protagon. Baeyer [1866] found that Liebreich's neurine yielded a mixture of platinichlorides, difficult to separate, but by means of the aurichlorides he subsequently [1869] showed that the principal base was identical with Strecker's choline. For the other base, which Baeyer obtained pure by the elimination of water from choline by chemical means, he reserved the name neurine, and Brieger [1885, I, p. 32] sharply differentiated the two bases; for a time much confusion was introduced by the continued use, by some authors, of neurine as a synonym for choline, but eventually the term neurine was restricted to the unsaturated base. According to Gulewitsch [1899, under choline] protagon does not yield neurine at all, but only choline. It is very doubtful whether neurine occurs in the body or body fluids, and apart from the old con- fusion of nomenclature, statements concerning its presence should be CHOLINE AND ALLIED SUBSTANCES 61 carefully scrutinised. 1 Neurine occurs as a product of putrefaction and was isolated by Brieger [1885, i, pp. 25-39] from putrid meat (horse, ox, human corpses). Brieger studied the physiological action of neurine in some detail and naturally assumed that the base was formed from choline by bacterial action. This assumption has never been proved rigidly, but the possibility should be taken into account with reference to Kutscher's alleged discovery [1905 ; Ch. V, creatine] of neurine in commercial meat extract. Krimberg [1906, I ; Ch. V, methylguan- idine] could not find neurine in an extract of perfectly fresh meat and concludes [1908,2; Ch. Ill, carnitine] that it is not present in muscle. Lohmann [1909] obtained neurine from the supra- renal gland, but here again it is not clear to what extent sterility was ensured. Brieger [1885, I, p. 61] obtained neurine from fresh human brain by hydrolysis with hydrochloric acid. Neurine is most readily obtained synthetically and was first pre- pared by Hofmann [1858] nine years before the synthesis of choline by Wurtz. Hofmann treated the condensation product of trimethyl- amine and ethylene dibromide with moist silver oxide, which removes hydrobromic acid, and forms neurine bromide : /Br /Br (CH 3 ) 3 I N/ + AgOH = (CH 3 ) 3 i N/ + AgBr + H 2 O. \CH 2 . CH. 2 Br \CH : CH, Baeyer [1869] prepared neurine from choline by heating the latter with concentrated hydriodic acid and then treating the resulting iodo-compound with silver oxide as in Hofmann's synthesis. Neurine is perhaps also formed from choline by boiling with concentrated baryta and this may have caused it to accompany choline in Liebreich's hydrolysis of protagon. According to Brieger [1885, i, pp. 33, 34] neurine appears to be formed from choline by long standing in aqueous solution. Physiological Action of Choline and of Neurine. When given subcutaneously or by the mouth to rabbits in doses of i grm., choline produces no severe symptoms and is not excreted in the urine (von Hoesslin [1906]). Riesser [1913; Ch. V, creatine] found that rabbits often withstood a daily injection of 0*5-1 grm. choline. Similarly the urine of rabbits, fed on lecithin, does not con- tain choline, but only a little glycero-phosphoric and formic acids 1 Thus Kutscher and Lohmann's statement [1906, 2, under choline] that neurine occurs in human urine has passed into the literature (" Biochemisches Handlexicon "), although these authors subsequently [1906, 4 ; Ch. V, methylguanidine] stated that their supposed gold salt of neurine was in reality methylpyridyl ammonium aurichloride. 62 THE SIMPLER NATURAL BASES (Franchini [1908]). Muscarine, neurine and betaine, on the other hand, are at least partially eliminated in the urine, and in this respect choline behaves like an amino-acid unit of protein. Whether choline is oxidised or whether it is synthesised into phosphatides is not known, but the latter alternative is in agreement with the conception of choline as a unit (Baustein) of phosphatides. The formation of choline in seed- lings has been referred to above and its behaviour towards micro- organisms is mentioned in the appendix. Riesser [1913 ; Ch. V, creatine] has recently carried out some ex- periments which suggest that choline, when injected subcutaneously, may be partially converted into creatine. In some rabbits he increased the muscular creatine content 10-15 per cent, by this means. Riesser supposes that choline condenses with urea according to the following equation : CH 2 OH / 2 CH 2 OH NH 8 + CO = I S CH 2 . N(CH 3 ) 3 OH v CH 2 . N(CH 3 ) C +2 CH 3 OH NH, XH and that the alcoholic group of the condensation product is then oxidised to a carboxyl group, yielding creatine. The choline must therefore lose some of its methyl groups, and in support of this theory Riesser quotes an experiment in which choline chloride is carefully heated with sodium tellurite and sodium formate (the latter salt acting as a reducing agent) ; the garlick-like smell of methyl telluride is pro- duced ; see also p. 77. The physiological action of choline has been studied by Gaehtgens, and by Boehm [1885, 2] who observed salivation, myosis, and diastolic arrest of the heart ; in frogs Boehm obtained general paralysis with 0*025-0-1 grm. ; in mammals O'Oi-O'O2 grm. injected intravenously gave a rise of blood pressure. The action is somewhat analogous to that of pseudo-muscarine (synthetic " muscarine "). Brieger [1885, I, p. 38] found that the toxic action of choline is inhibited by atropine (" in pracisester Weise "). A detailed study of the action was made by Mott and Halliburton [1899], who found that small doses of choline injected intravenously cause a fall of blood pressure, but after a preliminary dose of atropine a rise occurs. The antagonism between choline and atropine has been confirmed by all subsequent investigators, but a good deal of confusion and con- troversy has resulted from a statement by Modrakowski [1908] that pure choline always produces a rise of blood pressure and that the CHOLINE AND ALLIED SUBSTANCES 63 depressant action observed by others was the result of an impurity. Popielski [1910, i], in whose laboratory Modrakowski carried out his experiments, shares the latter's views, but Mott and Halliburton's statement that choline has primarily a depressent action has been confirmed by Busquet and Pachon [1909], Abderhalden and Muller [1910, 1911], Mendel and Underbill [1910], Pal [1910, 1911], Muller [1910], Lohmann [1907, 1908], and most recently by Mendel, Under- bill and Renshaw [1912]. The general conclusion is that Modrakowski's and Popielski's aber- rant results are not to be explained by impurities in the choline em- ployed by others, but rather to differences in anaesthesia and dosage. With small doses up to I mg. per kilo, in dogs and cats under ether or urethane, a fall of blood pressure always results, which with some- what large doses may be followed by a slight rise. Larger doses, es- pecially when repeated, may at once exert a pressor action. With slight anaesthesia, or with the medulla oblongata cut, small doses may also produce a rise of blood pressure. The depressent action is partly due to an effect on the heart and partly to vaso-dilatation in the limbs and splanchnic area. After atropine, perfusion of an isolated organ produces only vase-constriction. According to Muller this vaso-motor reversal depends on a paralysis by atropine of the dilator elements of the vascular walls, and resembles the adrenaline vaso-motor reversal by ergotoxine (Dale [1906, Ch. VI]). Choline has a stimulant effect on the isolated muscle of the in- testine, uterus and iris, resembling in this respect physostigmine some- what closely. It further stimulates the secretion of the lachrymal, salivary, and sweat glands. Salivation is one of the first symptoms of choline poisoning in an intact animal (Brieger). The physiological activity of choline is, however, slight, only about T V^j of that of neurine. The minimal lethal dose for rabbits of I kilo, is 0*5 grm. according to Brieger, but Mott and Halliburton were unable to kill an animal by choline injections. Compare also Riesser [1913; Ch. V, creatine]. The action of choline on isolated nerves and the excised heart of the frog has been studied by Waller and Sowton [1903]. Hunt and Taveau [1911] have studied the action of a large number of synthetic choline-like substances and their derivatives. In particular acetyl-choline (CH 3 ) 3 N(OH) . CH 3 . CH 3 . O . OC . CH, is remarkable in being 100,000 times as depressent as choline itself. According to Mr. A. J. Ewins [Bio-Chem. J., 1914, 8, 44] acetyl choline is present in small quantity in some ergot extracts. The lower homologue formocholine (CH 8 ) 8 N(OH) . CH a OH is also more active than choline. The nitrous acid ester of choline is identical with Schmiedeberg and Harnack's /sfo-muscarine (see p. 68). 64 THE SIMPLER NATURAL BASES Other synthetic substances allied to choline have been described by Schmidt [1891, 1904, I, 2], Malengreau and Lebailly [1910, under homocholine], Mengefign], and Berlin [1910, I, 1911, under homocholine] who gives further literature. The action Q{ neurine shows a general resemblance to that of choline and muscarine, and like these, it is antagonised by atropine. To rabbits it is 10-20 times as toxic as choline (Brieger [1885, i, p. 39]) ; on subcutaneous injection the lethal dose is about 40 mg. per kilo. Cats are more susceptible and react violently to doses of a few milli- grams. The effects are profuse salivation, dyspnoea, an initial accel- eration and then a retardation of the heart beat and death in diastole ; the intestine is stimulated to violent peristalsis ; there is often myosis in rabbits and always in cats. Atropine is a powerful antidote. In frogs there is a curare-like paralysis and diastolic arrest of the heart's action, after injection of 1-2 mg. into the dorsal lymph sac. Waller and Sowton [1903] studied the effect of neurine and other bases on isolated nerves and on the excised heart of the frog ; neurine was the most toxic, rather more than muscarine, and very much more so than choline. Lohmann [1911] finds that neurine in doses of 10 mg. first lowers the blood pressure of rabbits and then raises it. The general effect of neurine on the blood pressure is to produce a rise after a preliminary fall (Mott and Halliburton [1899]; Pal [1911]). Minute doses, of TTJW m g-> mav k either pressor or depressor. The rise of blood pressure is due to constriction of the peripheral vessels (compare Samelson [1911] who found, by the Laewen-Trendelenburg method, that neurine acts on the frog's limb in a dilution of I : 800,000). The physiological action of synthetic bases allied to neurine has been described by Schmidt [1891, 1904, i]. Natural and Synthetic Muscarines and their Physiological Action. 1 Muscarine is the name given by Schmiedeberg and Koppe [1869] to an extremely poisonous base which they obtained from Amanita muscaria (the Fly Agaric). Very small amounts arrest the frog's heart in diastole and the action is antagonised by atropine. Other bases of somewhat similar composition and similar physio- logical action have been obtained synthetically, and one of these was at one time considered to be identical with natural muscarine. It seems certain, however, that this is not so. 1 Compare the important addendum on p. 68. CHOLINE AND ALLIED SUBSTANCES 65 Schmiedeberg's base was isolated as the gold salt which Harnack [1875] found to be contaminated with choline ("amanitine ") auri- chloride ; a separation was effected by crystallisation from hot water, the muscarine salt being the more soluble. Harnack found muscarine aurichloride to have the composition C 5 H 14 O 2 N . AuCl 4 ; the base therefore differs from choline in having an additional oxygen atom. Soon afterwards Schmiedeberg and Harnack [1877] obtained a base of this composition by heating dried choline chloride with con- centrated nitric acid on the water bath ; the new base was isolated as the platinichloride ; the chloride, when left in a desiccator, sets to a crystalline mass and the base has according to Schmiedeberg and Harnack the constitution (CH 3 ) 3 : NCI . CH 2 . CH(OH) 2 , being therefore a hydrated aldehyde like chloral hydrate (but compare addendum, p. 68). This synthetic, artificial, or flseudo-muscarinQ is chemically very similar to the natural substance, and the physiological resemblance is sufficiently close to have induced Schmiedeberg and Harnack to believe in the identity of the two bases. Boehm [1885, 2] was the first to point out the differences in the physiological action. He found that ^ mg. of flseudo-muscarine (from choline) was required to stop the frog's heart in diastole, whereas the corresponding dose of natural muscarine is only ^VsV m g-> according to Schmiedeberg and Harnack. Recently this large difference in the activities of the two bases has been confirmed in Schmiedeberg's laboratory by Honda [1911], who again prepared natural muscarine and found it active on the frog's heart in doses of ^VrV mg., according to the season of the year, whereas the same effect was only produced by -J-i-J mg. of ^seudo-muscarme from choline. Boehm further found that in larger doses (10 mg.) pseudo- muscarine produces a curare-effect in mammals, which is not given even by large doses of the natural base ; moreover there is no com- plete antagonism between pseudo-muscanne and atropine : cats which have been poisoned by pseudo-muscanne cannot be kept alive by a subsequent dose of atropine. The curare-like action of pseudo- muscarine on frogs is according to Boehm fifty times as great as that of choline from which it is derived (the minimal paralytic doses being O'l and 50 mg.), and according to Honda [191 1] flseudo-muscarine has one-fifth of the activity of pure curarine in this respect. According to H. Meyer (see below) pseudo-m\\scarmz causes contraction of the pupil in birds, natural muscarine does not. Another synthetic substance, much more distantly related to muscarine than the oxidation product of choline, is trimethylamino- 5 66 THE SIMPLER NATURAL BASES acetaldehyde, (CH 3 ) 3 : N(OH) . CH 2 . CHO, which was first prepared by Berlinerblau [1884] by the action of trimethylamine on mono- chloracetal and subsequent hydrolysis, and later by Fischer [1893] by the methylation of acetalamine. The platinichloride has the com- position [(CH 3 ) 3 N . CH 2 . COH] 2 PtCl 6 . 2H 2 O ; the water of crystallisa- tion is given off at 105. The constitution of this base is quite certain, for Fischer [1894] oxidised it to betaine and accordingly suggested for it the name betaine aldehyde. In an abstract of a dissertation by Nothnagel [1893], E. Schmidt [1904, I, p. 47, under choline] quotes a report by Hans Meyer, who found that the anhydro-muscarine of Berlinerblau (= betaine aldehyde of Fischer) does not arrest the action of the frog's heart in doses of 10 mg., nor does it produce vagus inhibition in the mammalian heart in doses of several centigrams. It causes salivation and sweating, however, and kills by respiratory par- alysis. Betaine aldehyde differs also chemically from muscarine, but on the other hand natural muscarine and Schmiedeberg and Harnack's flseudo-muscarine are chemically very similar, according to Schmidt and Nothnagel. The platinichlorides of both bases have the composition [(CH 3 ) 3 N . CH 2 . CH(OH) 9 ] 2 PtCl 6 . 2H 2 O and do not lose water at 1 00. The physiological differences observed by Boehm.were however also found by Hans Meyer ; pseudo-musc^rmo. in doses of O' 1-0*05 m g- paralyses the intra-muscular nerve-endings of a frog ; natural muscarine does not. The cardiac effect of the natural base, even in doses of 6 mg., is counteracted by atropine, but this is not so with pseudo-muscar'me. Natural muscarine does not affect the pupil of birds, but maximal myosis is produced by a I per cent, solu- tion of;to*dfo-muscarine. Schmidt has suggested that the physiological differences may be due to stereo-isomerism, but in this case the relationship cannot be that between an optically active and a racemic modification, for then the one variety could not be 10-15 times as active as the other. Further investigation of the chemical properties of natural muscarine is very desirable, but the base is unfortunately difficult to obtain in sufficient quantity. Schmiedeberg's process of isolation was a compli- cated one, and Harmsen [1903] calculates from physiological data that Schmiedeberg only isolated about 6 per cent, of the muscarine present in the fungus. According to Harmsen 100 grm. of fresh fungus (=5 grm. of dried material) contain about 16 mg. of mus- carine. The amount seems, however, to be very variable, as does also the amount of choline which accompanies the muscarine. The chief difficulty in isolating natural muscarine is the separation from choline. CHOLINE AND ALLIED SUBSTANCES 67 Honda [1911] first separates a good deal of the latter base by means of its acid tartrate, which is less soluble than the muscarine salt. The discovery of a muscarine salt which is less soluble than the corres- ponding choline salt would greatly facilitate the preparation of pure muscarine. The fate of pseudo-musczrme. (from choline) in the animal organism has been investigated by Fiihner [1908, I ; 1909]. The lethal dose for rabbits of 1-5 kilo, is O'3-O'5 grm. by the mouth and 0^04 -0*05 grm. subcutaneousty ; the drug is partly secreted in the urine unchanged (in the toad the whole is so excreted). In this respect ^seudo-muscarine resembles betaine and differs from choline ; it is not a " Baustein ". Harmsen has concluded that the muscarine content of Amanita muscaria is quite insufficient to account for the poisonous effects of eating this fungus and considers that the effect is mainly due to a complex toxin insoluble in alcohol and not counteracted by atropine. From an allied species Amanita phalloides, Abel and Ford [1906] have prepared a haemolysin which they regard as a nitrogenous glucoside. Muscarine occurs in small quantity in Amanita pantherina and in Bol- etus luridus (Boehm [1885, I, under choline]). Brieger [1885, i, p. 48, Ch. I] isolated from putrid codfish a platinichloride (C 5 H U O 2 N) 2 PtCl 6 ; the physiological action of the base was that of muscarine. The physi- ological action of synthetic bases allied to muscarine has been described by Schmidt [1891, 1904, I, under choline]; Brabant [1913] has re- cently synthesised /3-homo-muscarine (CH 3 ) 3 N(OH)CH 2 . CH 2 . CHO. Trimethylamine Oxide, (CH 3 ) 3 NO. This base, the only member of its class known to occur naturally, was isolated by Suwa [1909, I, 2] from the muscles of Acanthias vul- garis. One dozen of this fish, yielding 23 kilos, of muscle, gave 20 grm. of the hydrochloride of trimethylamine oxide, together with a quantity of betaine, but hardly any creatine, or creatinine. The hydrochloride melts at 205-210, the pier ate forms thin needles, mp. 197, sparingly soluble in ethyl alcohol and cold water ; the platinichloride forms rhombic leaflets, mp. 214 ; the aurichloride C 3 H 9 ON . HAuCl 4 , mp. 250, is sparingly soluble in hot water. In concentrated aqueous solutions of the hydrochloride alcoholic solutions of mercury and cadmium chlorides precipitate C 3 H 10 ONC1 . 4HgCl 2 . H 2 O and C ; ,H 10 ONC1 . CdCL respectively. By putrefaction and also (at least in part) in the organism of the rabbit, trimethylamine oxide is reduced to trimethylamine from which it can be produced by oxidation with hydrogen peroxide. 5* 68 THE SIMPLER NATURAL BASES Neosine, C 6 H 17 O 2 N. There is still a good deal of doubt concerning the nature of this base, one of those obtained by Kutscher [1905, Ch. V, creatine] from extract of meat Krimberg [ 1 906, 1, Ch. V, methylguanidine] could not find neosine in fresh meat and doubted whether it is present in faultless meat extract. Ackermann and Kutscher [1907, 4, Ch. Ill, betaine] afterwards isolated the base from a commercial extract of shrimps which is the most abundant source. They [1908] found that tri- methylamine is given off on heating and accordingly surmised that neosine is a homologue of choline, but various attempts to identify it with synthetic choline homologues have failed, including the most re- cent and thorough attempt of Berlin [191 1] who found that Kutscher's neosine was contaminated with choline. The uncertainty with regard to this base is shown by the various melting points ascribed to the aurichloride. Kutscher found 202-205 '> Kutscher and Ackermann 205 ; Engeland [1908, i] for the base from meat extract 150-152 ; Berlin, after freeing the crude neosine from choline, obtained a few grams of a gold salt melting at 244-245 from 6 kilos, of Liebig's extract of meat. Berlin has also reinvestigated the synthetic homocholines of previous authors and con- cludes that Morley, Weiss, Partheil and more recently Malengreau and Lebailly [1910] obtained -homocholine (CH 3 ) 3 N(OH) . CH 2 . CHOH . CH 3 of which the aurichloride melts at 163-164. By the action of trimethylamine on trimethylene chlorohydrin CH 2 C1 . CH a . CH 2 OH and (less readily) by the methylation of 7-amino-propylalcohol Berlin [1910, 2, 1911] prepared 7-homocholine (CH 3 ) 3 N(OH) . CH 2 . CH 2 . CH 2 OH which yields an aurichloride crystallising in leaflets and melting at 193, a mercurichloride C 6 H 16 ONC1 . 6HgCl 2 , mp. 208, and a picrate exploding at 255. The constitution of this base follows from its oxidation to homo-betaine (CH 3 ) 3 N(OH) . CH 2 . CH 2 . COOH, and since it does not contain an asymmetric carbon atom, neosine, which is optically inactive, was at first regarded as identical with it. But the melting points of neosine aurichloride (244-245) and of neosine mercuric chloride C 6 H 16 ONC1 . 6HgCl 2 (252) render this hypothesis untenable. The physiological action of 7-homocholine is similar to that of choline but slightly more intense (Berlin [1910, I, 1911]). Addendum to Muscarine. While this book was in the press Dr. H. H. Dale and Mr. A. J. Ewins have, according to a private communication, established that the />s7/d0-muscarine of Schmiedeberg and Harnack and of Schmidt and Nothnagel is not an aldehyde at all, but the nitrous acid ester of choline. The platinichloride has the formula [(CH 3 ) 3 N . CH 2 . CH 2 ONO] 2 PtCl 6 , instead of [(CH 3 ) 3 N . CH 2 . CH(OH) 2 ] 2 PtCl 6 . aH 2 O. This explains why no water of crystallisa- tion is given off at 100; the loss of weight at 130 is due to decomposition. The percent- age composition required by the two formulae is very similar, except as regards nitrogen, the estimation of which presents difficulties here. This discovery further disposes of the inherent improbability that two hydroxyls should be attached to the same carbon atom ; such an arrangement has so far only been observed in compounds in which the carbon atom is attached to negative groups, as in chloral hydrate, mesoxalic acid and triketohydrindene hydrate. An analogy for the great modification of the physiological action of choline by esterification is to be found in the case of acetyl choline, p. 63, and of the nitric acid ester, p. 153. In its action the latter, according to Dale and Ewins, resembles natural muscarine even more closely than does the nitrous acid ester, (Comp. PrpQ, Physiol. Soc., March 14, 1914.) CHAPTER V. CREATINE AND CREATININE, GLYCOCYAMINE AND GUANIDINES. A. Creatine and Creatinine. Creatine was described and named as long ago as 1835, by Chevreul [1835], in a report to the French Academy of Sciences on commercial meat extracts. Chevreul did not analyse the substance, but noticed its resemblance to asparagine. Berzelius later failed to prepare creatine, but Wohler succeeded, and when Schlossberger [1844] ob- tained the same substance from the muscles of an alligator, its im- portance as a general constituent of muscle was recognised. Our detailed knowledge of creatine dates from Liebig's classical in- vestigation of the constituents of muscle juice [1847]. Liebig pre- pared creatine from the flesh of various animals, analysed it and con- verted it into its anhydride which he named creatinine and found to be identical with a substance isolated three years previously from urine by Pettenkofer [1844]. By boiling creatine with baryta, Liebig further obtained a new substance, sarcosine. Dessaignes [1854, 1855] showed that creatine is oxidised by mercuric oxide to methyl- guanidine (" methyl-uramine"). Sarcosine was synthesised by Vol- hard, who obtained creatine from it [1868]. Our physiological knowledge of creatine and creatinine did not advance so rapidly as the chemical, largely perhaps owing to the want of a convenient and accurate method of estimation. Such a method was, however, supplied by Folin in 1904, and this, together with his theory of metabolism, has led during recent years to many investiga- tions on the physiology of creatine and creatinine. Creatine was synthesised by Volhard [1868] by the action of cyanamide on sarcosine in alcoholic solution at 100. /CH 3 CH 2 .NH.CH 3 + CN.NH 2 CH-j.N/ OH and II CHOH CH . NH . CH S CHg.NH.CHg CH 2 OH ADRENALINE (EPINEPHRIN, ADRENINE) 83 Jowett [1904] arrived at results similar to those of Pauly ; on complete methylation and subsequent oxidation he obtained veratric acid and trimethylamine ; of the above two formulae he favoured the first, subsequently shown to be the correct one. Further investigations were carried out by Abderhalden and Bergell [1904] and by Ber- trand [1904, I, 2]. In the meantime the problem was being attacked in a different way by Stolz whose results, although not published until 1904, had already led in August 1903 to a patent application of the Farbw. vorm. Meister, Lucius und Briining [1904] describing the synthesis of a substance of the constitution I (above) which could not at first be obtained crystalline but seemed to be physi- ologically identical with adrenaline. Similar synthetic experi- ments were published somewhat later by Dakin [1905, 1-3], but al- though the identity of the synthetic substance with adrenaline was rendered extremely probable, this identity could not be proved rigor- ously, until the former substance had been crystallised and finally resolved into its optically active components, one of which was found to be completely identical with natural adrenaline (Flacher [1908]). Before this, an independent proof of the constitution of adrenaline had been furnished by Friedmann [1904, 1906] who showed that von Fiirth's tribenzenesulphonyl adrenaline, which is optically active, lost its activity on oxidation to the corresponding keto-derivative, which was crystallised. This proved that adrenaline is a secondary alcohol (for- mula I) and its constitution was further established by a comparison of the above-mentioned ketone with a synthetic specimen obtained from the amino-aceto-catechol of Stolz. Nomenclature and Synonyms. It is clear from the above that the active principle of the supra-renal gland has re- ceived different names from various investigators. The three principal ones are "epinephrin" (Abel), " suprarenin " (von Fiirth) and "adrenalin" (Takamine), and these are the only ones in scientific use, together with "adrenine" which has lately been em- ployed in the " Journal of Physiology ". On grounds of scientific priority the name should be adopted, which was suggested by the chemist who first isolated the substance in a pure state ; this was Takamine and we therefore use the name adrenalin(e) in the present mono- graph ; this name also happens to be the one at present in most general use. The objection to adrenalin is that it is a proprietary trade-name. For this reason the English Chemical Society used for some time the name epinephrin, which has also been adopted more recently by the American Medical Association. Apart from the fact that Abel first applied this name to an amorphous and probably impure substance there is the additional confusion, that for a long time he designated by it a supposed artificial alkaloidal anhydride of the active principle, which latter he called epinephrin hydrate ( = adrenalin) and some of his papers speak of epinephrin and adrenalin as two distinct substances. Later, when 6* 84 THE SIMPLER NATURAL BASES the hydrate theory proved to be untenable, epinephrin was made synonymous with adrenalin. 1 Preparation and Purification of Natural Adrenaline. The various processes depend on the fact that the active principle is extracted from the glands by water, neutral or acidulated, that it is not precipitated from its concentrated aqueous solution by alcohol, nor by neutral lead acetate, and that it separates in a crystalline form from suitably purified and concentrated aqueous solutions on the addi- tion of concentrated ammonia. On account of the readiness with which adrenaline undergoes oxidation various precautions have been sug- gested, such as preventing the access of air by means of a current of hydrogen or of carbon dioxide, and carrying out the final precipitation under a layer of petrol. For the same reason it is very convenient to extract with water containing sulphur dioxide. Takamine [1901, 2] extracted the minced gland at 50-80 for five hours with water acidulated with acetic or hydrochloric acid, shaking at intervals. The extract was then raised to 90-95 for one hour to coagulate the proteins, using a layer of fat or current of carbon dioxide to avoid oxidation. The glands were extracted a second time and the mixed extracts were concentrated in vacuo, and then precipitated with 2-3 volumes of alcohol. After filtration, the filtrate was again evaporated to a small bulk and was then precipitated with excess of concentrated ammonia which caused the crude adrenaline to separate in sphaero-crystals. Aldrich [1901] proceeded like Takamine, but before precipitating the concentrated solution with alcohol he added neutral lead acetate, centrifuged and removed the excess of lead from the solution by means of hydrogen sulphide. Then, after concentration, he added four to five volumes of 94 per cent, alcohol, evaporated the alcoholic filtrate to a very small bulk and added ammonia ; after filtration the crude adrenaline is washed with very dilute ammonia. Abel [1903, i] recommends a process illustrated as follows : 11*13 kilos, of minced glands were divided over a number of flasks and to each portion an equal quantity of a solution of 175 grm. trichloracetic acid in 5 litres of absolute alcohol was added, in small quantities at a time, with vigorous shaking. Next day 5-6 litres of filtrate were collected at the pump and evaporated to 380 c.c. After filtering off a flocculent precipitate, ammonia (d = 0-94) was gradually added to the clear filtrate with stirring until the smell of ammonia was per- manent. The adrenaline, which separated at once, was filtered off and washed with water, alcohol and ether ; yield 23-79 grm. = 0-2 per cent. The product, although nearly white, 1 Those interested in this question of nomenclature may refer to a letter by T. Maben in the Pharmaceutical Journal (1907, 78, 388-90 ; "Adrenalin : the Active Principle of the Suprarenal Gland ") and to a reply by W. Martin in the same journal (1907, 78, 447 and 514 ; " Epinephrin or Adrenalin ? "), and particularly to a correspondence entitled " Pro- prietary versus Unprotected Names " between the Council on Pharmacy and Chemistry of the American Medical Association and Messrs. Parke, Davis & Co. (Journ. Amer. Med. Assoc., 1911, 56, 910-5). It is said that 30-40 different trade names for the active principle of the supra-renal gland have been in use. Of these adnephrin, adrenalin, adrin, caprenalin, supra-capsulin and supra-renalin are of American origin ; the following are European : atra- bilin, chelafrinum, epirenan, haemostasin, hemisine, ischemin, paraganglin, paranephrin, renoform, supra-nephran, supra-renaden, tonogen, and vaso-constrictin. Suprarenin is used by the Ho'chst works for their synthetic product. ADRENALINE (EPINEPHRIN, ADRENINE) 85 contained 10-12 per cent, of ash. A second and a third extract, made from the mass of glands with 30-40 grm. of trichloracetic acid in 5-6 litres of 60-70 percent, alcohol, yielded respectively 8*57 and 3 grm. of base ; total = 35*36 grm. or 0*3 per cent, of crude product. Bertrand [1904, i] extracted 600 grm. of the minced glands (of horses) with 2 litres of 95 per cent, alcohol, containing 5 grm. of oxalic acid. On evaporation the extract was shaken with petrol to remove lecithin, etc., and the aqueous layer was exactly precipitated with neutral lead acetate and centrifuged. After removal of the excess of lead and evapora- tion to 100 c.c. a slight excess of ammonia was added. 118 kilos, of fresh minced gland from 3900 horses yielded 125 grm. of adrenaline. This yield is hardly more than one- third of that obtained by Abel (from bullock's glands). The purification of the crude adrenaline may be carried out by dissolving in acid and reprecipitating, but better by Abel's method depending on the solubility of adrenaline oxalate in alcohol. Pauly [1903] used it as follows: 12 grm. of crude adrenaline were ground up with 50 c.c. of 85-90 per cent, alcohol, containing 7 grm. of oxalic acid ; the inorganic impurities remain behind. After filtration and dilution with 100 c.c. of water, ammonia precipitated the base in a crystalline condition ; the base was freed from ammonium oxalate by thoroughly washing. This process was repeated several times and finally the base was washed with alcohol and ether. A more compli- cated process which yielded a substance absolutely free from ash, is also described by Pauly [1904]. Syntheses of Adrenaline. Adrenaline has been synthesised by several methods : (i) By means of phosphorus oxychloride, catechol is condensed with monochloracetic acid and the resulting chloracetocatechol (I), thus first prepared by Dzierzgowski, is suspended in alcohol (50 c.c. for 100 grm. of the ketone). I II III CHOH I CH 2 .NH.CH 8 A 40 per cent, aqueous methylamine solution (200 c.c.) is then added and on standing methylamino-acetocatechol separates out ; the product is washed with water, alcohol and ether. The methylamino- acetocatechol (II) so obtained is reduced to racemic adrenaline (III) by means of aluminium amalgam, or electrolytically. The above process is protected by the German patents Nos. 152814 and I 57300 of the Farb- werke vorm. Meister, Lucius und Briining [1904] and appears to 86 THE SIMPLER NATURAL BASES be the only one which is commercially suitable. The resolution of the racemic adrenaline is effected according to Flacher [1908] by ex- tracting the bitartrate with methyl alcohol ; d-adrenaline d-tartrate dis- solves and 1-adrenaline d-tartrate remains behind. The latter yields commercial synthetic suprarenin. An attempt to synthesise adrenaline by another method was originated by Barger and Jowett [1905] and continued by Pauly and Neukam [1908], Barger [1908], Bottcher [1909] and Mannich [1910], but has not yielded results of practical value (cf. German patents Nos. 209609, 209610, and 212206). Starting from piperonal (I), Barger and Jowett pre- pared the bromohydrin (II) which was converted into adrenalin methylene ether (III) I II III O CH 2 CHOH CHOH CH 2 Br CH 2 . NH . CH 3 . Adrenaline dimethyl ether was prepared from methyl vanillin by a similar method, but neither ether is convertible into adrenaline. Mannich showed that on the addition of methylamine to the bromohydrin, ethers of isoadrenaline^(OH) 3 C 6 H 3 . CH(NHCH 3 ) . CH 2 OH are also formed. The indirect removal of the methylene group by conversion into an un- stable cyclic carbonate e.g. OCO 2 : C 6 H 3 . CH(OH) . CH 2 C1, has also proved impossible. 1 Another synthesis of adrenaline which is theoretically possible and has been referred to in the patent literature, consists in methylating the primary base 3 : 4-dihydroxy-phenylethanol- amine (OH) 2 . C 6 H 3 . CH(OH) . CH 2 . NH 2 . This base, which is about as active as adren- aline itself and is known commercially as " arterenol," may be prepared by the reduction of amino-acetocatechol> (D.R.P. 155632). (OH) 2 C 6 H 3 . CO . CH 2 . NH 2 + 2H = (OH) 2 C 6 H 3 . CH(OH) . CH 2 . NH 2 and also by the reduction of the cyanhydrin of protocatechuic aldehyde with sodium amalgam (D.R.P. 193634). (OH) 2 C 6 H 3 . CH(OH) . CN + 4 H = (OH) 2 C 6 H 3 . CH(OH) . CH 2 . NH 2 . Amino-acetocatechol is obtainable in several ways : 1. From chloro-aceto-catechol and ammonia (the chief method) : (OH) 2 C 6 H 3 . CO.CH 2 C1 + 2NH 3 = (OH) 2 C 6 H 3 . CO . CH 2 . NH 2 + NH 4 C1. 2. By reduction of w-nitroacetocatechol : (OH) 2 C 6 H 3 . CO.CH 2 . NO 2 + 6H = (OH) 2 C 6 H 3 . CO.CH, . NH 2 + 2H 2 O. The w-nitroacetocatechol is obtained by hydrolysis of the corresponding methylene- or dimethylether with aluminium chloride in benzene solution. These ethers, co-nitroaceto- piperone and co-nitroacetoveratrone, may be prepared from piperonal and methylvanillin re- spectively, by successive treatment with nitromethane, bromine, methylalcoholic potash and acids (D.R.P. 195814). 3. By hydrolysis with hydrochloric acid of the condensation product obtained from veratrole and hippurylchloride by means of aluminium chloride (D.R.P. 185598 and 189483) (CH 3 O) 2 C 6 H 4 + C1CO.CH 3 .NH.CO.C 6 H B =(CH 3 0) 2 C 6 H 3 .CO.CH 2 .NH.CO.C 6 H 5 + HC1. (CH 3 O)^C 6 H 3 . CO . CH 2 . NH . CO . C 6 H 5 + 3HC1 + H 2 O- (OH) 2 C 6 H 3 . CO . CH 2 . NH a . A better yield is obtained by the hydrolysis of the similarly constituted phthalimido- acetoveratrole (D.R.P. 209962 and 216640). 1 Compare Pauly's repudiation [1909] of Bottcher's claim [1909] to have synthesised adrenaline by this method and D.R.P. 209609, 209610, 212206. ADRENALINE (EPINEPHRIN, ADRENINE) 87 In order to utilise the d-adrenaline, obtained as a by-product in the resolution of the racemic base (according to Flacher [1908] and D.R.P. 222451), the dextro-variety may be racemised by means of acids (according to D.R.P. 220355). For example, 1*5 grm. d-adrenaline is dissolved in 13-5 c.c. normal hydrochloric acid (= 1*65 mol.) and after adding I 5 c.c. of water the solution is heated to 80-90 for two to three hours, after which the solution is optically inactive and the crystalline hydrochloride of the racemic base can be isolated by means of alcoholic hydrogen chloride. When the natural base was kept for six weeks at 20-30 with the same concentration of hydro- chloric acid, 75 per cent, had been racemised. By repeated resolution and racemisation of the d-base, the whole of the synthetic adrenaline is finally obtained in the 1-form. For an account of the patents relating to the synthesis of adrena- line reference may be made to Friedlander's " Fortschritte der Teerfar- benfabrikation," 1905-7, VIII, 1181-90, and 1907-10, IX, 1024-33; or to the " Chemisches Zentralblatt ". Adrenaline Substitutes. Numerous bases, more or less closely related to adrenaline, have been synthesised and some of these also resemble adrenaline in physiological action. Only three of them, however, have been recom- mended as substitutes for the natural active principle, namely 3 : 4 dihydroxy-phenylethanolamine (OH) 2 C 6 H 3 . CH(OH) . CH 2 . NH 2 ("arterenol ") w-ethylamino-3 : 4-dihydroxy-acetophenone (OH) 2 . C 6 H 3 . CO . CH 2 . NH . C 2 H 5 (" homorenon ") 3 : 4-dihydroxy-phenylethyl-methylamine (OH 2 ) C 6 H 3 . CH a . CH 2 . NH . CH 3 (" epinine ") Of these, arterenol is according to Schultz [1909, I] about as active on the blood pressure as natural 1-adrenaline (and therefore more active than the racemic base). Homorenon and epinine are much less active, the former base having according to Schultz only about one-eightieth of the pressor action of 1-adrenaline. Physical and Chemical Properties of Adrenaline. Salts and Derivatives. Constitution. Adrenaline, when pure, crystallises in colourless sphaerocrystals consisting of super- posed lamellae ; crystals suitable for crystallographic measurement have not been obtained. It melts at 211-212 (uncorr.) with decomposition. According to Bertrand the solubility in water at 20 is 0-0268 per cent. The base is somewhat more soluble in boiling water, but less in alcohol ; it is practically insoluble in most organic solvents but dissolves in glacial acetic acid, in warm ethyl oxalate (Abel) and in benzaldehyde. In the latter solvent Barger and Ewins [1906] found at 90 the molecular weight 170. Adrenaline is lasvo-rotatory. The'more trustworthy determinations in solution in dilute mineral acids are tabulated below : 88 THE SIMPLER NATURAL BASES Author. Source. Temperature. Wo Bertrand [1904, 2] .... horse ; in N/io H 2 SO 4 - 53'3 Abderhalden and Guggenheim [1908] . Flacher (with Korndbrfer) [1908] bullock bullock 20 I9'8 - 5072 - 51-40 Schultz (with Taveau) [1909, i] . bullock 26-4 - 53 '4 Abel and Macht [1912] parotid gland of B ufo Agna 20 - 5i'30 Weidlein [1912] .... whale 25 - 52-00 Flacher [1908] synthetic 1-adrenahne - 51*40 " d- + 51-88 d- Adrenaline has the same physical and chemical properties as 1-adrenaline and melts also at 211-212, but is much less active physiologically. Adrenaline is a fairly strong base and can be dissolved in the theoretical quantity of a mineral acid, or even in somewhat less than one equivalent (Gunn and Harrison [1908]). Being a phenol, it is also soluble in caustic alkalies, but not in ammonia or sodium carbonate. The chief chemical characteristic of adrenaline is the readiness with which it undergoes oxidation, on account of the presence of a catechol nucleus. A large number of mild oxidis- ing agents colour adrenaline solutions pink, rose red, and brown, and the same change takes place on exposure to air, slowly in acid, rapidly in alkaline solution. Adrenaline is most stable in solutions containing a slight excess of acid, for instance one and a half equivalents of acid to one equivalent of the base. The coloration takes place much more rapidly when minute traces of iron are present (Gunn and Harrison [1908]). A number of colour reac- tions, depending on this oxidative change, are described below (pp. 89-91). According to Abel [1902, 3] extracts of the supra-renal gland are more stable to Fehling's solution than solutions of the pure active principle. Adrenaline solutions do not give precipitates with the common alkaloidal reagents, but on heating with dilute acids, or by the action of concentrated hydro- chloric acid in the cold, adrenaline is transformed into a substance yielding alkaloidal re- actions (Abel's epinephrine). The salts of the optically active adrenalines are mostly amorphous and deliquescent ; the bar ate prepared by evaporating 1-83 gr. of the base and 0*93 gr. of boric acid in 5 c.c. of water is said to be more stable (D.R.P. 167317). The chief crystalline salt of adrenaline is the bitartrate, employed in the resolution of the synthetic product, Pauly [1904] prepared a crystalline urate. The racemic base yields, in addition, a crystalline hydrochloride, mp. 157 (D.R.P. 202169), and a crystalline oxalate, but the corresponding salts of both d-and 1-adrenaline are amorphous (Flacher [1908]). No crystalline derivatives of adrenaline are known. Abel and Pauly prepared benzoyl derivatives of somewhat uncertain composition. Von Fiirth obtained a tri-benzenesulphonyl derivative which contains the alcoholic hydroxyl of the side chain intact, for Friedmann [1904, 1906] converted it into m-nitrobenzoyl-tribenzenesulphonyl-adrenaline and oxidised it to tribenzenesulphonyl-adrenalone. Stolz obtained a tri-p-chlorbenzoyl derivative. The constitution of adrenaline was ascertained from the following reactions ; On fusion with potash catechol and protocatechuic acid are formed ; on heating with acids or caustic soda methylamine is eliminated. On methylation and subsequent oxidation with permanganate veratric acid, vanillin and trimethylamine were obtained. The constitution is further proved by Friedmann's work (see above, p. 83) and finally of course by synthesis and resolution. The alleged production of skatole on potash fusion is probably due either to the presence of protein impurities, or to that of a benzoyl ADRENALINE (EPINEPHRIN, ADRENINE) 89 nucleus (in Abel's epinephrine). The constitution of the " alkaloidal " substance formed by the action of acids on adrenaline has not been elucidated, nor of the base C 3 H 4 ON 2 obtained by Abel [1904] on oxidising adrenaline with nitric acid. Adrenaline is readily attacked by various oxidases [Neuberg, 1908; Abderhalden and Guggenheim, 1908]. Colour Reactions of Adrenaline. The principal colour reactions were already observed by Vulpian and have more recently been used for the estimation of adrenaline. A general review of the various quantitative colorimetric methods has lately been furnished by Borberg [1912]. The reactions are as follows : I. Ferric chloride produces in neutral or slightly acid solution a grass green coloration, changing to violet, reddish violet, and red on the careful addition of dilute alkali. This is a reaction characteristic of catechol derivatives. The green coloration is the more fugitive and the less strongly marked, the more acidic the solution is. The limit of sensitiveness is about I : 30000, but the addition of sulphanilic acid increases the sensitiveness tenfold and changes the green colour to reddish brown or brown yellow (Bayer [1909]). Falta and Ivcovic [1909] describe another sensitive modification of the ferric chloride reaction. For the detection of adrenaline in urine Borberg [1912] gives the limit for the green ferric chloride reaction as I : loopoo. On standing a red coloration is produced up to I : 300,000. II. A pink or rose red coloration (" tout a fait remarquable," Vulpian) is produced in adrenaline solutions on prolonged exposure to air and, almost immediately, by various oxidising agents. The change of colour is less rapid in faintly acid solution than in neutral solution, and more rapid in alkaline solution. It is also brought about by oxidases ; from the behaviour of adrenaline to tyrosinase, Gessard [1904] first deduced a relationship to tyrosine. Neuberg [1908] found that an enzyme from the ink-bag of Sepia officinalis produces a black pigment from adrenaline, and Abderhalden and Guggenheim [1908] observed that adrenaline solutions are coloured red by a tyrosinase from the fungus Russula delica ; the laevo- , the dextro- , and the racemic forms are all coloured at the same rate. The formation of pigments from adrenaline has been considered by some to be connected with the pigmentation of the skin in Addison's disease. The oxidising agents employed for the red colour reaction for adrenaline are : 90 THE SIMPLER NATURAL BASES A. Iodine or iodic acid. The excess of iodine may be removed by shaking with ether and the sensitiveness is then according to Schur [1909] I : 1,500,000. Abelous, Soulie" and Toujan [1905] removed the excess of iodine by means of sodium thiosulphate, but according to Bayer [1909] the reaction, when carried out in this way, is not very delicate and the red colour is not permanent. Another modification of the iodine reaction was suggested by L. Krauss [1909] who used iodic acid. Subsequently Frankel and Allers [1909], independently of Krauss, employed an equal volume of O'OOi N-potassium bi-iodate and added a few drops of phosphoric acid ; by heating the mixture nearly to the boiling point, the reaction is said to be obtainable at a dilution of I : 300,000. Hale and Seidell [1911] recommend this test, but do not add phosphoric acid. Frankel and Allers consider their test to be quite distinct from that of Vulpian ; they state that at no stage of the reaction is iodine set free, but both Krauss and Ewins [1910] deny this. Bayer [1909] claims to have greatly increased the sensitiveness of the Frankel-Allers reaction by adding sulphanilic acid, which, however, changes the red colora- tion to an orange or yellow one, which is less specific ; Bayer gives I : 5,000,000 as the limiting dilution. B. Another oxidising agent, which colours adrenaline solutions red, is mercuric chloride, recommended by Comessatti [1909]. Boas [1909] and Frankel and Allers [1909] could not obtain the reac- tion at all readily, but Ewins [1910] has pointed out that Comessatti used solutions of mercuric chloride in tap water, and that the calcium bicarbonate present in the latter acts as a catalyst ; it may be replaced by solutions of other salts of weak acids. This observation is of con- siderable interest in connection with the discovery of Euler and Bolin that the oxidase from Medicago consists of calcium salts of organic hydroxy-acids. It was moreover already noticed by Vulpian, that the spontaneous coloration of the adrenal chromogen by exposure to air takes place slowly in distilled water, but much more rapidly in tap water. Ewins suggests the following conditions for carrying out Comes- satti's reaction. To I c.c. of adrenaline (i : 100,000) an equal volume of a I per cent, sodium acetate solution is added and then four to five drops of a o-i per cent, solution of mercuric chloride in distilled water. A pale rose tint is produced at room temperature in 4 to 5 minutes. Here the sodium acetate solution replaces tap water, in order to secure uniformity. C. The most sensitive oxidising agent is probably a persul- ADRENALINE (EPINEPHRIN, ADRENINE) 91 phate. Pancrazio [1909, 1910] has used the sodium salt and Ewins [1910] the potassium salt. Ewins adds potassium persulphate solution to the adrenaline solution until the concentration of the per- sulphate is about o-i per cent, and then immerses the test tube for a short time in a boiling water bath. Under these conditions a distinct reaction is still obtained at a dilution of I : 5,000,000. The persul- phate reaction for adrenaline seems therefore to be more delicate than any other, with the possible exception of Bayer's modification of the Frankel-Allers reaction (see above) for which an equal degree of delicacy is claimed. According to Ewins potassium persulphate has an additional advantage in the estimation of adrenaline in extracts of the gland, since it discharges the colour of these extracts to a consider- able extent, the colour interfering with the Bayer-Frankel-Allers test. With persulphate a clean and distinct red tint results, which is per- manent for a considerable time. D, Other oxidising agents which colour adrenaline solutions red, are potassium ferri cyanide (Cevidalli [1908]), brown oxides of man- ganese (Zanfrognini [1909]), sodium nitro-prusside and ammonia, bleaching powder, chlorine, bromine, ammoniacal silver solutions, and osmic acid (Mulon [1905]). According to Borberg [1912] all the " red " colour reactions for adrenaline are similar and depend on the formation of the same oxidation product. Borberg gives the limit as I : 300,000, thus perhaps underestimating the sensitiveness of some of the reactions. Ewins [1910] examined the effect of iodine and persulphate and of the Comessatti, Frankel and Allers, and Bayer reagents on a number of synthetic bases, closely related to adrenaline. He found that aminoethanol-catechol (arterenol), as well as dihydroxy-phenyl- ethylamine and its N-alkyl derivatives (including epinine) give the various reactions with about the same degree of sensitiveness as adrenaline, but none of these reactions are given by ketone bases, such as amino-aceto-catechol and its derivatives (including homorenon). Among these synthetic bases there is therefore no close parallelism between chemical reactivity and physiological action. E. Folin, Cannon and Denis [1912] have recently described a new and very sensitive colour reaction for uric acid, which is also given by adrenaline with three times as great a sensitiveness (i : 3,000,000). One hundred grm. of sodium tungstate is dissolved in 750 c.c. of water, and after adding 80 c.c. of 85 percent, phosphoric acid, the solution is boiled gently for one and a half to two hours and then made up to I litre ; -^-^ mg. adrenaline can be detected. 92 THE SIMPLER NATURAL BASES Colorimetric Estimation of Adrenaline. The green coloration with ferric chloride has been employed by Batelli [1902] who found by this means 0*174 per cent, in fresh bullock's glands. Von Fiirth [1901] has used the carmin red coloration produced by ferric chloride in the presence of sodium carbonate and sodium potassium tartrate. The ferric chloride reaction is, however, not very suitable for quantitative work (cf. Cameron [1906]) and the same applies, according to the author's experience, to the iodine-thiosulphate method of Abelous, Soulie and Toujan [1905]. Comessatti [1909] has employed the mercuric chloride reaction a good deal for quantitative purposes, and Cevidalli [1908] and Zanfrognini [1909] have used their re- actions in the same way ; their methods have been adversely criticised by Borberg [1912]. Ewins [1910] found a distinct parallelism between the depth of colour produced by potassium persulphate and the pressor activity of supra-renal extracts. This physiological control has not been applied sufficiently to most other colon' metric methods. A notable exception is found in a recent paper by Folin, Cannon, and Denis [1913] and the colorimetric method of these authors based on the reaction described above (under E) appears to be almost or quite as accurate as the blood pressure method with which its results agree within a few per cent, of the total adrenaline present. The method is even sufficiently sensitive to demonstrate the increase of adrenaline in the supra-renal vein by stimulation of the splanchnic nerve (cf. p. 95). It is not necessary to have pure adrenaline as a standard, for uric acid gives an identical coloration with one-third of the intensity. Amount of Adrenaline in the Supra-renal Gland ; Yield ; Distribution in other Organs ; Origin. By the physiological blood pressure method, which is probably the most accurate, Elliott finds that the adult human gland in health con- tains about O'l per cent, (unpublished observation, referred to below). By the same method Elliott [1912] has found that the normal cafs supra-renal, weighing 0*2 grm., contains on the average 0*22 mg. of adrenaline, or cm per cent. Folin, Cannon and Denis [1913] found in the gland of young cats cri 22-0*1 52, of the dog and monkey 0*2-0-25, of the calf 0-25-0-35, of sheep, cattle, rabbits, 0-3 per cent. Houghton [1902] found Takamine's original adrenaline to be 600 to 800 times as active as fresh bullock's gland; according to Takamine [1901, 4] the specimen contained mineral impurities and pure adrenaline is probably 1000 times as active r dogs and -^nru m g- f r cats (which are more resistant than dogs). Other blood-pressure methods, such as the de- termination of the dose required to compensate for the vaso-dilator action of a given quantity of nitroglycerine (Cameron [1906]) and the determination of the minimal dose necessary to give a perceptible pressor effect, are much less accurate. A second method employing the circulatory system but depending on vaso-constriction instead of on blood pressure is due to Lawen [1903-4] and has been improved by Trendelenburg [1910]. The rate is measured at which, under a constant hydrostatic pressure, blood flows through the vessels of a frog, of which the brain and spinal cord have been destroyed ; the adrenaline to be estimated is added to the blood. This method appears to yield moderately accurate results, but is la- borious when many estimations have to be performed. The significance of determinations by this method of adrenaline in serum has recently been questioned by O'Connor [191 1 , 1912, i] who finds that serum itself causes vaso-constriction, quite apart from the addition of adrenaline (see also Handovsky and Pick [1913, Ch. I]). Stewart [1912], and Dale and Laidlaw [1912, 2] agree with O'Connor's objections to the ADRENALINE (EPINEPHRIN, ADRENINE) 103 use of serum. According to Stewart it is possible to prove the pres- ence of adrenaline only in the blood from the supra-renal vein. Besides those on the circulatory system, the other effects of adrena- line on plain muscle, described in a previous section, are to some extent available for the quantitative estimation of the drug ; the methods which have been suggested, based on these effects, are much less accurate than the blood-pressure method, but, on the other hand, some of them are more suitable for the very rough estimation of extremely minute quantities of adrenaline, such as may occur in the blood or in tissue extracts. In such cases it is, however, necessary to avoid confusion with other ill-defined substances (such as vaso-dilatin, p. 30) which may produce similar effects in plain muscle (cf. Hoskins [1911] and O'Connor [1912, i]). O. B. Meyer [1906] has employed isolated rings of the sub- clavian or carotid artery of the ox, which contract in solutions of adrenaline up to I : 1,000,000,000 (0-000015 mg. in 15 c.c. Ringer's solution). Cow [1911] has investigated other arteries by this method and finds that the only arteries not constricted by adrenaline are the intravisceral portion of the pulmonary, the coronary and the cerebral arteries. Argyll Campbell [1911] also finds by this method that adrenaline causes marked constriction of the vessels of all organs, except those of the heart and lungs. A slight constriction occurs occasionally in the heart and more frequently in the lung vessels. A. Frankel [1909] used the isolated uterus of the rabbit, which still reacts to adrenaline at a dilution of I : 20,000,000, but Hoskins [1911] states that this reaction is not specific and that contractions are caused by a large number of glandular and tissue extracts ; the use of the rabbit's uterus for testing serum has also been criticised by Stewart [1912]. Cannon and de la Paz [1911] employed longitudinal strips of muscle from the rabbit's intestine and Hoskins [191 1] a short length of small intestine from the same animal. These two methods depend on the inhibition, by adrenaline, of the spontaneous contractions. In Hoskins's experiments this inhibition occurred regularly at I : 100,000,000 and sometimes even at I : 500,000,000. Hoskins considers his method and that of O. B. Meyer (above) to be the most sensitive methods known. According to O'Connor [1912, I] substances are formed during the coagulation of blood with actions simulating this and other effects of adrenaline, but by using the plasma, instead of the serum, and rabbit's intestine as test object, he finds that the blood from the 104 THE SIMPLER NATURAL BASES supra-renal vein contains one part of adrenaline in I to 5 millions ; he could not demonstrate adrenaline with certainty in the peripheral blood. Stewart, who employed this method and that depending on the contraction of the rabbit's uterus, also concludes that adrenaline is not detectable in the general circulation, or indeed in blood from the supra- renal vein, except during massage of the gland or stimulation of the splanchnics, when there was respectively I : 500,000 and I : 1,000,000. Dale and Laidlaw [1912, 2] have used as a test object another organ which is inhibited by adrenaline, viz. the non -pregnant uterus of the cat. In a cat under chloroform and ether they find that the blood from the supra-renal vein contains one part of adrenaline in from I to 2 millions. After injection of pilocarpine this amount was increased tenfold. The method which has been most widely used for the detection of small quantities of adrenaline is based on mydriatic action, particu- larly as applied to the excised eye of the frog. This test object was first employed by S. J. and C. Meltzer [1904, i, 2] ; later Ehrmann [1905] brought it into prominence by his experiments on body fluids and by his claim that the excised eye, being much more sensitive than the intact eye, can reveal adrenaline in a concentration of I : 10,000,000. According to Borberg [1912] the sensitiveness is only one-tenth of this. Schultz [1909, i] has elaborated the technique of this method by measuring the pupil under the microscope. Hoskins [191 1] dis- sected the eye, removed the lens and applied the fluid under examina- tion directly to the iris ; in this way results were obtainable at a dilu- tion of i : 5,000,000 and sometimes a positive result was noted at I : 100,000,000, but a mydriatic effect is also shown by pituitary ex- tract, iodothyrin, etc., which renders the method very uncertain when applied to the detection of adrenaline in the blood. Schultz [1909, 2] considers that Ehrmann overstated the sensitiveness of the method. He writes : " At its very best the excised frog's eye as a pharmaco- logical assay for adrenaline is inferior to the blood-pressure method. As a qualitative test it is perhaps one of the most sensitive test-objects known, but it is not a characteristic test (Comessatti, Meltzer) and observations convince me that too much weight ought not to be at- tached to results with it in clinical diagnosis ". This adverse opinion is shared by Cameron [1906] and by Borberg [1912], but the method at least has the advantage that it is applicable to very dilute solutions and that it can be used by the chemist who cannot undertake more elaborate animal experiments. According to Schultz the dilata- tion time is a better index than the degree of mydriasis and one should ADRENALINE (EPINEPHRIN, ADRENINE) 105 aim at making this time equal for both of a pair of eyes. In a recent article on the estimation of adrenaline in the blood, Gottlieb and O'Connor [1912] place the blood-pressure method first in point of accuracy, provided the adrenaline solution is sufficiently concentrated. Next comes the perfusion of the frog's blood vessels, which may be used quantitatively and is more sensitive (up to I : 30,000,000). For the qualitative recognition of the minutest quantities the inhibition of the cat's small intestine is very specific and, in particular, it is not pro- duced by serum (limit I : 400,000,000). CHAPTER VII. BASES OF UNKNOWN CONSTITUTION. THE constitution of nearly all the bases dealt with in the preceding chapters is known with certainty. In addition a large number of bases of unknown constitution have been described at various times. In many cases even their composition has not been fully established. Nevertheless some of the latter class will be included here on account of their great physiological interest. It is of course impossible to say whether they have a " simple constitution," but in any case the methods by which their isolation may be attempted are similar to those used for the other bases of this monograph. Spermine. The phosphate of this base crystallises out when semen dries, and constitutes over 5 per cent, of the solids. It has been most fully in- vestigated by Schreiner [1878] who prepared it in a pure condition by boiling fresh human semen with alcohol, filtering off and drying the precipitate so formed, extracting the latter with very dilute warm aqueous ammonia and then concentrating. The phosphate is hardly soluble in cold, and only a little in hot water, but soluble in dilute acids and alkalies. The salt contains two atoms of nitrogen to one of phos- phorus, and at 100 3H 2 O are given off; it melts at 170. Schreiner found that the crystals on the surface of old anatomical preparations (Bottcher's crystals) are identical with spermine phosphate ; he obtained them by scraping them off the surface of calves' livers and hearts and bulls' testes, kept in alcohol for three months. It has further been suggested that the crystals discovered by Charcot in the spleen, liver, and blood in cases of leucocythaemia, and also found in the sputum in cases of bronchial asthma, are identical with spermine phosphate, but this does not appear to be the case. Schreiner assigned to spermine the formula C 2 H 5 N ; Ladenburg and Abel [1888] considered it to be most probably identical with piperazine C 4 H 10 N 2 , which has the constitution : x NH( \CH 2 .CH/ 106 BASES OF UNKNOWN CONSTITUTION 107 By direct comparison with a specimen of Schreiner's preparation they found a great similarity to piperazine but also some differences. Schreiner's specimen was found to be slightly impure and to contain calcium. Ladenburg and Abel considered that Schreiner's phosphate might conceivably be (C 4 H 10 N 2 ) 2 CaP 2 O 8 which agrees better with his analyses. Poehl [1891] arrived at the formula C 10 H., 6 N 4 for spermine after analysing the platinichloride and the aurichloride, but the formula C 5 H 12 N 2 would also fit his results. Bases from Muscle. In addition to creatine, methylguanidine, carnosine, carnitine, neosine, betaine, myokynine, and trimethylamine-oxide, all described previously, the following may be mentioned : Vitiatine, C 5 H 14 N 6 , has been obtained by Kutscher [1907] from meat extract and is regarded by him as a guanidine derivative of the possible constitution : / : C HN : C C : NH \N(CH 3 ) . CH 2 . CH 2 . NH/ Crangitine, C 13 H 2o O 4 N 2 , and crangonine, C 13 H 26 O 3 N 2 , have been ob- tained by Ackermann and Kutscher [1907, 4, Ch. Ill, betaine] from shrimps. Creatosine has been obtained from commercial meat extract by Krimberg and Izra'ilsky [1913] and yields an aurichloride C u H 28 4 N 3 Au 2 Cl 8 . Bases from Urine. The following bases, already described, have been isolated as normal or occasional constituents of human or animal urine : trime- thylamine, isoamylamine, putrescine, cadaverine, iminazolylacetic acid, urocanic acid, kynurenic acid, methylpyridinium hydroxide, ^-picoline, butyrobetaine, carnitine (= novaine), reductonovaine, creatine, creati- nine, methylguanidine, dimethylguanidine, vitiatine. In addition the following may be mentioned : Mingine, C 13 H 18 O 2 N 2 . Kutscher [1907, Ch. Ill, butyrobetaine] obtained 0-45 grm. of the di-aurichloride from 100 litres of women's urine. Gynesine, C 19 H 23 O 3 N 3 . Kutscher and Lohmann [1906, 4, Ch. Ill, butyrobetaine] obtained 1*5 grm. of the aurichloride C 19 H 2S O 3 N 3 , 2HAuCl 4 , from 100 litres of women's urine. Kynosine, C l3 H., 6 O 4 N 4 , was isolated from normal dog's urine as the aurichloride C 13 H 26 O 4 N 4 , 2HAuCl 4 by Kutscher [1906]. xoB THE SIMPLER NATURAL BASES Putrefaction Bases. In addition to the amines of Chapter I and some other bases mentioned in the previous chapters a large number of less well char- acterised putrefaction bases have been described. A few of these may be mentioned here : Viridine^ C 8 H 12 O a N 2 , was obtained by Ackermann [1908, 2] from putrid pancreas. The hydrochloride has an intense green colour ; on heating the odour of quinone is perceptible. The aurichloride is blackish green to yellow and melts at 176 ; the platinichloride is intense yellow and melts at 212-216. Marcitine, C 8 H 19 N 3 , also obtained by Ackermann [1907, 2] from putrid pancreas, gives an aurichloride C 8 H 19 N 3 , 2HAuCl 4 melting at 175-178. It is perhaps a guanidine derivative. Putrine, C n H 26 O 3 N 2 , likewise isolated by Ackermann [1907, 2] from putrid pancreas, gives a dark orange aurichloride melting at 109-1 10. The formula of this base contains one carbon atom and two oxygen atoms less than the so-called diamino-trihydroxy-dodecanic acid C 12 H 26 O 5 N 2 of Fischer and Abderhalden from which it is perhaps de- rived by decarboxylation. Skatosine, C 10 H 16 O 2 N 2 , has been described by Baum [1903] and Swain [1903] as a product of pancreatic autolysis. It is stated to give a benzoyl derivative melting at 169 and a hydrochloride forming leaflets melting at 345. To the latter the improbable formula C 10 H 16 O 2 N 2 , 3HC1 was given. Mr. A. J. Ewins (private communica- tion) has lately failed to obtain this base by Baum's process. The Active Principle of the Pituitary Body. Soon after their discovery of the pressor action of supra-renal ex- tracts Oliver and Schafer [1895, 3] found that an extract of the pituitary body or hypophysis cerebri (a small appendage at the base of the brain) has the power of raising the blood pressure, when injected intravenously. The active principle is only contained in the infundibular or posterior lobe of this organ. At first stress was laid in the literature on the similarity of the action to that of adrenaline, and some authors even imagined that the two active principles must have a similar chemical constitution. During the' last few years pituitary extracts have come more and more into therapeutic use on account of their great power of producing contractions of the uterus, and the isolation of the active principle has been attempted. Although these attempts have perhaps not been wholly successful as yet, they seem to prove that the active substance is a base ; little else is definitely known about its chemical BASES OF UNKNOWN CONSTITUTION 109 constitution. Its physiological action has, however, been studied in some detail and such correspondence as exists between the action of the pituitary body and of adrenaline has been found to be " superficial and illusory". The chemical investigation of the pituitary active principle is greatly hampered by its instability and by the difficulty of procuring enough material. The infundibular portions, dissected clean from fresh glands, are ground up with sand and boiled with water acidulated with acetic acid. After filtration a clear colourless extract is obtained, which contains a little protein and some phosphates. By the addition of uranyl acetate the phosphates may be precipitated and most of the protein is carried down with the precipitate, but the solution remains physiologically active. Almost the only precipitant for the active principle itself is phosphotungstic acid, as has for instance been found by Engeland and Kutscher [1911] and by Meister, Lucius and Briining (see Fiihner [1913]). The chemists of the Hoechst firm, on decomposing the phosphotungstate with baryta, and removing the excess of baryta with sulphuric acid, obtained on concentration in vacua a pale yellow crystalline sulphate, which was physiologically active and apparently homogeneous, but was afterwards separated by fractional crystallisation into four different substances, all crystalline, and all having some physiological activity. Two of these were more active than the others ; the more abundant of the two is a colourless sulphate, readily soluble in water, but only slightly so in alcohol, acetone, or ethyl acetate. It gives Pauly's histidine reaction with p-diazobenzene- sulphonic acid and also the biuret reaction. Its picrate is readily soluble in water. In contact with alkali a volatile amine is at once given off. According to Fiihner, who has examined physiologically the various substances from the phosphotungstate, they all contribute to the activity of the gland ; thus there would be four active principles. The facts at present available do not, however, absolutely exclude the possibility that these four substances all owe their activity to con- tamination, in various degrees, with one and the same highly active substance which has so far escaped isolation. The further chemical examination of the most active of the four substances should prove of great interest. That this substance gives the biuret reaction may be considered in conjunction with an observation by Dale [1909] that the activity of pituitary extracts is rapidly destroyed by trypsin and much less rapidly by pepsin. This would point to a polypeptide struc- ture. The activity is also fairly rapidly lost when an aqueous solution is evaporated to dryness ; perhaps this is owing to hydrolysis. i io THE SIMPLER NATURAL BASES The fact that the bases from a pituitary extract give the Pauly re- action suggests a connection with histidine, and moreover /3-imina- zolylethylamine, which is obtained from histidine by decarboxylation, also causes powerful contractions of the uterus. Possibly, therefore, the pituitary active principle is a polypeptide-like derivative of histidine. Guggenheim [1913] has lately synthesised a number of bases by combining amines with chloracetylchloride and treating the pro- duct with ammonia. In this way, for example, glycyl-/3-iminazolyl- ethylamine NH . CH^ ^C . CH 2 . CH 2 . NH . CO . CH 2 . NH 2 CH = NX was prepared. The bases of this type, for which the name pep famine is suggested, are therefore decarboxylated polypeptides ; their physio- logical action is of the same kind as the amine from which they are derived, but much weaker. The physiological action of pituitary extracts has been in- vestigated chiefly by Schafer, in conjunction with Oliver [1895, 3], Magnus [1901], Herring* [1906] and Mackenzie [1911], and further by Dale [1909], von Frankl-Hochwart and Frohlich [1910], Pankow [1912] and others. Pituitary extract produces a direct stimula- tion of involuntary muscle, without any relation to innervation. Here there is, therefore, an important difference from adrenaline which stimulates sympathetic nerve endings (see p. 98). The action of pituitary is most nearly allied to that of the digitalis series, but the effect on the heart is slight, that on plain muscle intense. The rise of blood pressure caused by pituitary is thus due to the stimulation of the plain muscle of the arterioles. The rise is much smaller than in the case of adrenaline and lasts much longer. A further difference is, that when the blood pressure has returned to the normal, the rise caused by adrenaline can at once be reproduced by a second dose, but in the case of pituitary the effect of a second dose is much smaller, un- less it is administered after a considerable interval of time. In the birds pituitary extract causes a fall of blood pressure, which is anta- gonised by adrenaline and by barium (Paton and Watson [1912]). The powerful stimulation of uterine plain muscle was first pointed out by Dale [1909] and also studied by von Frankl-Hochwart and Frohlich [1910] and was first applied clinically by Bell [1909] in England and soon afterwards by Foges and Hofstatter in Germany. The supposed pure substances have been used clinically by Herzberg [I9I3J BASES OF UNKNOWN CONSTITUTION in Pituitary extracts bring about contraction of the uterus in the cat, dog, guinea-pig, rat, and rabbit, in all functional conditions. Adrena- line, on the other hand, in some of these species has a motor effect on the pregnant uterus only and inhibits the non-pregnant organ. The effect of pituitary extracts on the uterus can be shown both by intravenous injection into the anaesthetised animal and by means of the surviving uterus in a bath of oxygenated Ringer's solution. The latter method, applied to the uterus of the young virgin guinea-pig, has been worked out by Dale and Laidlaw [1912, i] to a process for standardising pituitary extracts and has also been used more recently by Fiihner [1913]. It has the great advantage over blood pressure experiments that tolerance is practically absent. Dale and Laidlaw find that o-J^- c.c. of an extract obtained by boiling infundibula with five parts of water will produce almost maximal tonus of the uterus in a bath of 250 c.c. Ringer solution. Since such an extract only contains about O'6 per cent, of solids, this represents a concentration of little more than cri mg. of solid matter per litre, most of it being inert material. The pituitary active principle is therefore a very powerful uterine stimulant, the activity being probably at least of the same order as that of y@-iminazolyl-ethylamine. In addition to the above effects on plain muscle, pituitary extracts bring about a profuse flow of urine and also greatly increased secretion of milk. The diuretic action was discovered by Schafer in conjunc- tion with Magnus and with Herring and was at first attributed to a different substance from that causing the rise of blood pressure ; later observers, however, consider that the active principle is the same in both these cases. According to Houghton and Merrill [1908] diuresis is merely a secondary effect of the rise in blood pressure and is also brought about by injecting adrenaline. The galactagogue action was first observed by Ott and Scott [1911] and has subse- quently been described by Schafer and Mackenzie [1911], and Ham- mond [1913]. For the effect on the mammary gland in the human subject see Schafer [1913]. Vitamine, Oryzanine, Toruline. A polyneuritis, resembling the tropical disease beri-beri, can, as Eykman discovered, be induced artifically in fowls by feeding them on an exclusive diet of polished rice. The condition is due to the lack of a substance present in the outer coating of the rice and removed in the process of polishing. During the last year or two several attempts have been made to isolate this curative substance from various sources. ii2 THE SIMPLER NATURAL BASES Funk [1911] in England, and Suzuki with Shimamura and Odake [1912] in Japan, showed independently and about the same time that the substance is a base, is present in very small amount, and has great curative action. To Funk belongs the further credit of having been the first to analyse the substance and to isolate the same or a similar body from yeast. Chemical work in this direction has also been done by Schaumann [1912, I], Moore and his collaborators [1912], Cooper [1913] and others. In spite of the discrepancies which exist between the statements of various authors, it seems fairly well established that the curative sub- stance in rice polishings, for which Funk has suggested the name vitamine and which Suzuki and his collaborators call oryzanine, is a base which can be extracted by water and by alcohol, but not by acetone or ether. It is precipitated by phosphotungstic acid, by tannin, by mercuric chloride in alcoholic solution and by silver nitrate and baryta. The latter property indicates the presence of an imino- group. The mercurichloride is soluble in boiling water. Suzuki, Shimamura and Odake describe a crystalline picrate of their substance, which they did not however analyse. Funk, by utilising the properties indicated above, obtained from rice polishings a minute yield of a crystalline substance, to which he assigned the formula C 17 H 20 O 7 N 2 , but more recently [1913], by fractional crystal- lisation, he separated it into two substances ; one of these was found to give the following average analytical results: C = 58*85 per cent, H = 3-9 per cent, N = 10*6 per cent ; it melted at 233. The other gave on the average C = 58*4 per cent, H = 4*0 per cent, N = 11*05 per cent, and melted at 234. The latter was identified as nicotinic acid, C 6 H 5 O 2 N, which, in the pure state, is inactive and had already been obtained from rice by Suzuki. To the former substance Funk gave the formula C 26 H 20 O 9 N 4 and he stated that it is a tetrabasic acid. It is considered by Funk to be the chief curative substance in rice polishings. Funk separated the " vitamine " fraction of yeast, which he at first considered to be identical with that of rice, into nicotinic acid and an active principle melting at 229 (corr.) which when dried in vacua at room temperature has the formula C 26 H 21 O 9 N 5 , but dried at 1 00 changes to C 24 H 19 O 9 N 5 , implying the somewhat unusual loss of two carbon and two hydrogen atoms. It will be seen that the substance C 26 H 20 O 9 N 4 obtained from rice has a very close resemblance to nicotinic acid, both as regards melting point and chemical composition, and at present the possibility does not seem completely excluded, that this body is merely nicotinic acid con- BASES OF UNKNOWN CONSTITUTION 113 taminated with a small quantity of a highly active substance richer in carbon. Further work will therefore be of the greatest interest. Funk and also Schaumann consider that there are a number of substances capable of preventing and curing polyneuritis. The former [1912, 2] has found that certain purine and pyrimidine derivatives have a weak activity in this direction. The crystalline and apparently homogeneous vitamine fraction from rice and from yeast is active in doses of a few centigrams, and when injected subcutaneously such doses will restore a severely paralysed pigeon within a few hours. A substance curing polyneuritis is also present in ox brain, in milk (Funk [1912, i]), and in muscle (Eykman [1897], Cooper [1913]). Edie, Evans, Moore, Simpson, and Webster [1912] have given the name toruline to an antineuritic base from yeast having the formula C 7 H 17 O 5 N 2 . A concomitant effect of a diet of polished rice is a loss of body weight which has been taken into account more particularly in the experiments of Suzuki and his colleagues. In this connection attention may be drawn to the work of Hopkins [1912] which shows that growth is greatly influenced by some as yet undetermined con- stituents of food. Sepsine. The name sepsine was given more than forty years ago by Schmiedeberg to a poisonous putrefaction product which was more recently isolated by Faust [1903-4] as a crystalline sulphate. Faust used putrid yeast and obtained under the most favourable conditions only 0-03 grm. of sepsine sulphate from 5 kilos, of yeast. The pro- cess of isolation is a complicated one, one of its chief features being that the sepsine is precipitated by mercuric chloride from an aqueous solution rendered strongly alkaline by means of sodium carbonate. Later the sulphate separates out in a crystalline condition by fractional precipitation of the alcoholic solution of the base by means of sulphuric acid dissolved in alcohol. The sulphate can be recrystal- lised and then forms well-developed crystals having according to Faust the composition C 5 H 14 O 2 N 2 , H 2 SO 4 ; his analyses, however, fit equally well or slightly better the formula C 5 H 12 O 2 N 2 , H 2 SO 4 . The free base is a syrup readily soluble in water. Sepsine is very unstable ; on repeated evaporation of the aqueous solution of the sulphate on the water bath this salt is transformed according to Faust into cadaverine sulphate, and the substance loses its physiological activity. This transformation, which involves the loss of two oxygen atoms, is without any analogy and very difficult to 8 ii4 THE 'SIMPLER NATURAL BASES understand. Perhaps the identification of the inactive substance as cadaverine is erroneous, as it is apparently only based on the platinum content of a platinichloride. Perhaps the analyses of sepsine sulphate have been wrongly interpreted. However this may be, it seems clear that a crystalline substance of remarkable physiological properties was obtained, corresponding to those originally possessed by the putrid yeast and described by Schmiedeberg. Twenty mg. of sepsine sulphate injected into a dog of 7-8 kilos, weight very soon cause vomiting and defecation ; finally almost pure blood is passed and the poisoning ends fatally ; sepsine is a capillary poison. Fornet and Heubner [1908] have isolated organisms which they imagined produce sepsine and the chief of these they named Bacterium sepsinogenes, but in a later paper [1911] they greatly modified their original conclusions. The organism referred to was found not to produce sepsine but a colloidal poison having a similar action and being in some respects comparable to the toxin formed in anaphylaxis. A further chemical investigation of Faust's sepsine appears to be very desirable, particularly if it could reveal the constitution of this interesting substance. Secretine. This substance, which causes secretion of pancreatic juice when injected intravenously, appears to be a base, judging from a method of purification described by Dale and Laidlaw [1912, 3]. This is founded on the solubility of the mercury compound in moderately dilute acid and its insolubility in neutral or weakly acid solution. Dale and Laidlaw's method may be given as an additional example of the tech- nique of using mercuric chloride for the separation of bases (cf. p. 1 19). The mucous membrane of the intestine of dogs is scraped off weighed and ground up with one-fifth of its weight of solid mercuric chloride to a smooth paste ; then two parts of water are added for every part of the mucous membrane taken. This mixture can be accumu- lated and kept indefinitely ; the mercuric chloride coagulates the pro- tein and acts as an antiseptic. To work up the mixture it is boiled, filtered through paper or muslin, and pressed dry. The press cake is suspended in an aqueous I per cent, mercuric chloride solution containing acetic acid ; 4 c.c. of this are used for every gram of moist mucous membrane taken. The mixture is boiled and filtered, and the filtrate should be nearly clear. Ten per cent sodium hydroxide is added BASES OF UNKNOWN CONSTITUTION 115 until the filtrate is nearly neutral, i.e. until the yellow mercuric oxide just fails to be permanent. The white flocculent precipitate formed is collected at the pump, suspended in hot water, and decomposed by hydrogen sulphide ; after neutralising and boiling off the hydrogen sulphide the solution is filtered and then furnishes a strongly active secretine solution. The active substance can further be precipitated from this solution by excess of picric acid, but attempts to obtain it chemically pure have so far been unsuccessful. 8 * CHAPTER VIII. (APPENDIX.) PRACTICAL CHEMICAL METHODS AND DETAILS. A. GENERAL METHODS FOR THE SEPARATION AND ISOLATION OF BASES. WITH few exceptions the simple natural bases are readily soluble in water, but not in ether or chloroform. As a rule they cannot therefore be extracted from alkaline solution by shaking with organic solvents, and the methods of Stas and DragendorfT, employed for the isolation of vegetable alkaloids and based on the use of solvents immiscible with water, are therefore not applicable. The earliest work on putrefaction bases, therefore, suffered from too close adherence to the methods used for alkaloids ; amylamine and phenyl-ethylamine which are readily soluble in ether and in chloroform, and p-hydroxy-phenyl-ethylamine which dissolves in amylalcohol, are among the few simpler bases which can be isolated in this manner. In general, therefore, the isolation of these bases is effected by means of an insoluble salt or other derivative, a method which in the case of putrefaction bases was first extensively used by Brieger, with conspicuous success. The simplest (aliphatic) monamines are volatile with steam and can therefore easily be separated by steam distillation , first from acid solution in order to remove non-basic volatile products and subsequently from alkaline solution. Some non-volatile bases, particularly betaines, are decomposed by strong alkalies with evolution of trimethylamine ; if such bases are present the solution should only be made alkaline with magnesium oxide and the distillation should be carried out at a low temperature under reduced pressure. This precaution is for instance important in the estimation of trimethylamine in urine. When bases have to be isolated from a complex mixture such as a tissue extract, it is necessary to remove first proteins and peptones as far as possible. The oldest method employed for this purpose is to evaporate the aqueous extract to a small bulk and add alcohol which precipitates the proteins, but leaves the salts of organic bases in solution. The separation is, however, not very complete ; in some cases it may be improved by using acetone instead of alcohol. The aqueous solution 116 GENERAL METHODS FOR ISOLATING BASES 117 containing proteins and bases is evaporated to a thin syrup, and this is mixed with sand and then ground up under acetone. Dry acetone does not dissolve the salts of most organic bases, but enough water remains behind in the aqueous extract to prevent precipitation of the salts by acetone. The preliminary purification of a tissue extract after removal of coagulable protein is, however, best effected by means of lead acetate or by tannin. In the former case the solution is first treated with normal lead acetate and then with the basic salt ; the joint precipitate of these reagents is then filtered off and the excess of lead is removed from the filtrate as sulphide, sulphate, or phosphate. The tannin method has been largely employed by Kutscher and his pupils ; it completely removes peptones and proteoses, but bases are also carried down by the bulky precipitate ; according to Krimberg the yield of bases from meat extracts is much smaller after purification with tannin than with lead acetate. Many bases form tannates insoluble in neutral solution, so that the reaction before precipitation should be made distinctly acid by adding phosphoric acid, if necessary. A 20 per cent, aqueous tannic acid solution is then added until no further pre- cipitation occurs ; at this stage the precipitate ceases to be milky and flocculates ; a considerable excess of tannic acid must be avoided since it redissolves the precipitate (it is a case of the mutual precipitation of two colloids). On standing overnight the bulky precipitate shrinks to the consistency of pitch and the clear supernatant solution can easily be poured off. In order to remove the excess of tannin, a warm saturated baryta solution is added until, after stirring, the surface of the liquid shows a reddish or purple colour. The barium tannate is filtered off at the pump, the filtrate is acidified with sulphuric acid, and without removing the barium sulphate formed, freshly prepared lead hydroxide, suspended in distilled water, is stirred in. This removes the last traces of tannin and the excess of sulphuric acid, and now, after filtration, the solution should contain at most only traces of lead and should be alkaline to litmus. The last operations illustrate the general principle that as far as possible no ions should be introduced into the solution which cannot afterwards be removed, for the separation of bases from inorganic salts is often difficult. Kossel and Weiss [1910] use a solution containing 70 grm. of tannic acid, 100 grm. of sodium chloride and 50 c.c. of glacial acetic acid per litre for the precipitation of peptones. The solution of bases which has been purified by one or other of u8 THE SIMPLER NATURAL BASES the above methods is now evaporated to a small volume, when on standing some bases, such as creatine, may crystallise out. Generally, however, they are too soluble in water and must be separated by some general precipitant. The most important reagent for this purpose is phosphotungstic acid, introduced into physiological chemistry by Drechsel. The acid is readily soluble in ether, in acetone and in water. It precipitates all nitrogen bases from their aqueous solution if the latter contains 5 per cent, by weight of sulphuric acid. Ammonia is also precipitated and should therefore be expelled, if present in quantity. It is important to employ a good preparation of phospho- tungstic acid, such as that of Kahlbaum, which dissolves in water with hardly any opalescence. A method for preparing the acid has been given by Winterstein (" Chemiker Zeitung," 1 898, p. 539). In order to obtain the bases from an aqueous solution, sulphuric acid is added to the latter to make 5 per cent, and a concentrated aqueous solution of phosphotungstic acid, which should also contain 5 per cent, of sulphuric acid, is added until no further immediate precipitation occurs. After standing for a day the precipitate is filtered off at the pump and thoroughly washed with 5 per cent, sulphuric acid. Often the pre- cipitate is partially or wholly soluble in acetone, and more readily in a mixture of acetone and water. (Compare Wechsler, below.) By pouring the solution of the precipitate into a large bulk of 5 per cent, sulphuric acid, the phosphotungstates of the bases are reprecipi- tated and in this way they can be purified more readily than by washing at the pump. In synthetic work, and when only one or two bases are present, a phosphotungstate may occasionally be crystallised from a large volume of boiling water (for instance in the case of iminazolyl-propionic acid). The bases are again liberated from their phosphotungstates by means of baryta, finely powdered or dissolved in water. For this purpose the phosphotungstate precipitate must be carefully suspended in water in as fine a state of division as possible ; where possible it is much quicker to dissolve the precipitate in dilute acetone and then add an aqueous baryta solution. Wechsler [1911] recommends a mixture of three volumes of acetone with four volumes of water ; this dissolves arginine phosphotungstate to the extent of 120-130 per cent, and of the histidine salt even 160 per cent, of its own weight, but albumose phosphotungstates only to the extent of 2-7 per cent. The precipitate of barium phosphotungstate and sulphate settles down rapidly. Several drops of the clear supernatant fluid are sucked up into a capillary pipette and tested on a glass plate. When they no GENERAL METHODS FOR ISOLATING BASES 119 longer give a precipitate with baryta, but precipitate both with sul- phuric acid and with sodium carbonate solutions, enough baryta has been added to liberate the bases. The barium phosphotungstate is then filtered off on the pump and washed out thoroughly with hot water until the washings no longer give a precipitate with a phos- photungstic-sulphuric acid solution. The excess of barium is at once removed from the filtrate and washings by passing carbon dioxide through them ; on filtration and evaporation the organic bases are obtained either in the free state or as carbonates. Should it be necessary to remove the excess of phosphotungstic acid from the filtrate, after precipitation of bases as phosphotungstates, this can be done either by precipitation with excess of baryta, or, ac- cording to Jacobs [1912], by extracting the acid solution with amyl- alcohol, which may be conveniently mixed with up to four parts of ether. This method may also be used for decomposing the phos- photungstates of bases if they are soluble in hot water. Mercuric chloride is next in importance to phosphotungstic acid as a precipitant of bases. It is not so universal a precipitant and is most frequently used after phosphotungstic acid to separate the re- covered bases into several fractions. With suitable precautions mer T curie chloride may, however, often replace phosphotungstic acid altogether. It was first used extensively by Brieger for isolating putrefaction bases, before phosphotungstic acid had come into general use. Mercuric chloride is generally used in saturated alcoholic solution which is added to an alcoholic or sometimes to an aqueous solution of the bases to be precipitated. Some bases are precipitated from neutral solution, but others only after the solution has been made slightly alkaline. In aqueous solution sodium carbonate is used, in alcoholic solution fused sodium acetate, dissolved in alcohol, is added, or the solution is saturated with powdered sodium acetate. If such a solution is afterwards also saturated with powdered mercuric chloride, very few bases escape precipitation. Generally the mercuric chlorides are much more soluble in hot water than in alcohol ; Brieger extracted the precipitate formed in alcoholic solution with boiling water, when the mercuric chloride compounds of peptones remained undissolved. On filtration and cooling choline mercurichloride crystallised out. Another example of the use of mercuric chloride is the preparation of histidine from blood, by Frankel's method. After the blood (or haemo- globin) has been hydrolysed by boiling with concentrated hydrochloric acid, most of the acid is distilled off and the residue, after being nearly 120 THE SIMPLER NATURAL BASES neutralised with sodium hydroxide, is filtered. The filtrate is then made alkaline with sodium carbonate and the histidine is precipitated by adding alcoholic mercuric chloride solution. Engeland has worked out a method for separating the bases of meat extract in which all the bases are first precipitated by the alternate addition of cold saturated solutions of mercuric chloride and of sodium acetate. The precipitate dissolves for the most part in hot water acidulated with hydrochloric acid and is freed from mercury by means of hydrogen sulphide. After evaporation of the aqueous filtrate the residue is dissolved in alcohol and alcoholic mercuric chloride is added ; finally the solution is satur- ated with the powdered salt. This precipitates neosine, carnitine and vitiatine as mercurichlorides which are removed by filtration. Alcoholic sodium acetate solution is now added and precipitates the mercury salts of histidine, methyl guanidine and /3-alanine. Cf. also p. 114. Silver nitrate is principally used to precipitate bases containing an imino-group and is of great value for their separation. As in the case of mercuric chloride, the degree of acidity or alkalinity of the solution is the determining factor. In the presence of (nitric) acid only purine bases are precipitated as insoluble silver compounds ; in a slightly alkaline solution, i.e. after the addition of a limited quantity of baryta, the silver compounds of histidine and allied bases are thrown down ; excess of baryta then precipitates the silver compound of arginine. The separation of arginine and histidine in this manner may be rendered quantitative and if silver sulphate is used instead of the nitrate, the process affords a means of estimation by determination of the nitrogen in the various fractions (see Plimmer's " Chemical Constitu- tion of the Proteins," Part I, pp. 35-8). The practical details in the application of silver nitrate may be illustrated by a description of Kutscher's method for the isolation of bases from meat-extract. After purification by means of tannin, as described above, and concentration to a small volume, creatine and some creatinine crystallise out. Then, after filtration, the solution is acidified with sulphuric acid and the resulting precipitate of lead sulphate is filtered off. Now a 20 per cent, silver nitrate solution is added to the filtrate and this causes the pre- cipitation of the purine bases (as compounds with silver nitrate), together with a little silver chloride. After standing for some time this pre- cipitate is filtered off and enough silver nitrate is added to the solution to enable the whole of the bases capable of forming silver compounds to be precipitated as such by subsequent addition of baryta. Enough silver nitrate has been added for this purpose when a drop of the solution, mixed on a watch glass with cold saturated baryta water, GENERAL METHODS FOR ISOLATING BASES 121 shows no longer a white precipitate (silver compound of bases) but at once a brown precipitate (of silver oxide). The addition of barium hydroxide in excess would now precipitate both the histidine and the arginine fraction, but a separation of these may be effected by utilising the fact that histidine silver is precipitated by an ammoniacal silver solution but arginine silver is not. Hence, after adding enough silver nitrate, baryta is added in small quantities until a drop of the clear supernatant or filtered solution no longer gives a white precipitate with a reagent which is prepared by adding ammonia to 10 per cent, silver nitrate until the silver oxide has just dissolved. The histidine fraction, which is thus precipitated by baryta, is filtered off, and the precipitate, after washing, is suspended in water in as fine a state of division as possible. If a suitable centrifuge is available this means of separation is greatly to be preferred. The silver is then removed with hydrogen sulphide, or with hydrochloric acid, a little sulphuric acid being first added to precipitate adherent baryta. The barium sulphate formed can be readily filtered off with the silver sulphide or chloride. Baryta in excess is now added to the filtrate of the " histidine " fraction, and precipitates the silver compounds of the " arginine " frac- tion, which are treated in the same way. The former fraction may contain histidine, /3-iminazolyl-ethylamine, carnosine and creatinine, the latter arginine, agmatine and methyl- guanidine. The separation is not always quite sharp, however. Thus Reuter found adenine (a purine base) in the histidine fraction of the bases from Boletus edulis and trimethyl-histidine in the arginine fraction from this same fungus. In Kutscher's examination of mushroom extract trimethyl-histidine altogether escaped precipitation by silver and appeared in the lysine fraction. After the silver precipitate of the arginine fraction has been filtered off, the solution may still contain various bases constituting the so- called " lysine " fraction. The excess of baryta is removed by sul- phuric acid and that of silver by hydrochloric acid ; then the bases remaining in solution are precipitated by phosphotungstic acid, and after recovery from the phosphotungstic precipitate, they are separated by mercuric chloride or by other means. Potassium bismuth iodide and potassium tri-iodide are more or less general precipitants for bases and have been chiefly used in investi- gations on plant alkaloids, but only to a slight extent for the separation of animal bases. Potassium bismuth iodide (Dragendorff's reagent, modified by Kraut) gives brick red and generally amorphous precipitates 122 THE SIMPLER NATURAL BASES with organic bases. The reagent is prepared by dissolving 80 grm. of bismuth subnitrate in 200 c.c. of pure nitric acid of density n8, and pouring this solution slowly, with stirring, into a concentrated aqueous solution of 227 grm. of potassium iodide. A precipitate forms and dissolves on stirring to a deep orange solution. This is cooled strongly to allow potassium nitrate to crystallise out as far as possible. The clear solution is poured off and made up to I litre; the more concen- trated solution may also be employed. The reagent should be kept in the dark. Kossel and Weiss [1910] recommend a solution of 50 grm. sodium iodide and 100 grm. bismuth iodide in 100 c.c. of 0*5 per cent, aqueous hydriodic acid. To regenerate the bases, the precipitate caused by addition of Dragendorff's reagent is ground up with freshly precipitated lead hydroxide, which is transformed to lead oxyiodide. After filtration the last traces of lead are removed by hydrogen sulphide ; the solution is then concentrated to a syrup, which is extracted with alcohol. To precipitate bases as periodides a concentrated solution of iodine in potassium iodide is employed (compare the estimation of choline and betaine by Stanek's method). The periodides may be decomposed by sodium bisulphite or thiosulphate, but this introduces into the solu- tion a good deal of inorganic matter. It is better to grind up the per- iodide in warm water with finely divided copper, so-called " molecular copper," prepared by Gattermann's method, as follows : Zinc dust is added through a sieve to a cold saturated solution of copper sulphate in a porcelain dish, until the solution is only faintly blue. The pre- cipitated copper settles down and is repeatedly washed by decantation. To remove traces of metallic zinc, the copper is placed under several times its volume of distilled water and quite dilute hydrochloric acid is added until no more hydrogen is evolved and the copper is no longer carried up to the surface of the solution but remains quietly at the bottom. The copper is then collected on a filter at the pump, washed until neutral and kept in a well-stoppered bottle in the moist state. It is very easily oxidised. For the isolation of individual bases from the fractions obtained by any of the above methods, it is necessary to prepare a crystalline derivative. Bensoylation is occasionally resorted to (in the case of diamines from urine, p-hydroxyphenyl-ethylamine, etc.) but generally a salt of the base is crystallised. The hydrochlorides of putrescine and of betaine are almost insoluble in alcohol, in contradistinction to the corresponding cadaverine and choline salts. The nitrates of some bases (guanidine, methylguanidine, arginine, hypaphorine, certain GENERAL METHODS FOR ISOLATING BASES 123 purine bases) can be readily crystallised from water and are particu- larly little soluble in dilute nitric acid. Much more frequently picrates are prepared. The picric acid is added in aqueous and also in alcoholic solution ; the precipitated picrate is recrystallised from water, from dilute or from strong alcohol. Often, on cooling a hot solution, it separates first in oily drops which only become definitely crystalline on standing. Ammonium salts, when present, may sometimes lead to confusion owing to the forma- tion of ammonium picrate, which is not very soluble in water and forms long thin pale yellow needles ; these have no proper melting point, but decompose suddenly on heating. When a base is insol- uble in ether (as is the case with most of the simpler natural bases) it can be readily recovered from its picrate by dissolving the latter in hot dilute hydrochloric acid and, after cooling, extracting the picric acid with ether or with benzene. On the large scale most of the picric acid generally separates and can be filtered off. The estimation of picric acid in picrates can be carried out very conveniently and with enough accuracy by means of the " nitron " reagent of Busch [1905]. This process has the further advantage over a combustion that the base is recovered unchanged. Picrolonates are much less soluble than picrates and generally crystallise well, but to some extent this advantage is neutralised by the slight solubility in water of picrolonic acid itself. An alcoholic solution of the acid is generally added to an aqueous solution of the base. The precipitate is at first often amorphous, but readily crystal- lises from hot water in some cases. The high molecular weight of picrolonic acid renders the melting points and analyses of picrolonates of less significance than those of picrates. Platinic chloride is used in concentrated aqueous or (more frequently) alcoholic solution. The platinichlorides of the simplest bases are often readily soluble in water, but not in alcohol, and may be crystal- lised from dilute alcohol. Gold chloride is generally used in a 30 per cent, aqueous solution. Aurichlorides sometimes partially decompose on recrystallisation, gold being set free. In order to avoid this and obtain a gold salt of normal composition, the salt should be recrystallised from -j- - I per cent, hydrochloric acid to which a little gold chloride has been added. In special cases zinc chloride or cadmium chloride are used for forming double salts in alcoholic solution, or the base is isolated as chrornate, perchlorate or metaphosphate. 124 THE SIMPLER NATURAL BASES B. SPECIAL METHODS. PROPERTIES OF INDIVIDUAL BASES AND OF THEIR SALTS. 4 Bases Volatile with Steam. Methyl-, dimethyl-, and trimethylamine, isobutyl- and the amyl- amines can all be readily distilled by passing steam into their alkaline solutions. The last two can be separated from the others by extract- ing an alkaline solution with chloroform or ether and distilling ; isobutylamine boils at 68, isoamylamine at 95. The separation of the first three bases from one another can be accomplished in various ways. Delepine [1896, Ch. I] dissolves the mixture of their salts in cold concentrated formaldehyde solution. An equal volume of potassium hydroxide is added and the solution is distilled. Trimethylamine passes over as such, dimethylamine forms CH 2 [N(CH 3 )J 2 and CH 2 (OH)N(CH 3 ) 2 , b.p. 80-85, and monomethylamine yields (CH 2 : NCH 3 ) 2 , b.p. 166. For the quantitative determination of trimethylamine and ammonia, Budai (Bauer) [1913] has worked out a titration method with for- maldehyde. The neutral aqueous solution of the mixed hydrochlorides is treated with an excess of formalin (10 c.c.), previously neutralised to phenolphthalein. The solution is then titrated with standard potassium hydroxide until pink with phenolphthalein ; this gives the amount of ammonia present. The solution, together with the hexa- methylene tetramine formed from the ammonia, is strongly acidified with concentrated hydrochloric acid and boiled down to one-third of its original volume. It is then distilled with excess of potassium hydroxide. This gives ammonia + trimethylamine ; the latter is estimated by difference. The quantitative separation of ammonia, mono-, di-, and trimethylamine is carried out by processes due to Bresler [1900], Bertheaume [1910, i, 2], and Francois [1907, I, 2] and is chiefly based on the fact that trimethyl- and dimethylamine hydrochloride alone are soluble in boiling chloroform. 1-2 grm. of the mixed hydrochlorides are dried at 1 10, weighed out, dissolved in a little very dilute hydro- chloric acid, mixed with at least 20 grm. of pure silver sand, dried in vacuo over sulphuric acid, and extracted with hot chloroform in a small funnel tube over glass wool. The chloroform is evaporated, the residue is weighed and dis- solved in 2000 parts of water ; 200-300 c.c. of the solution are measured, cooled to o and for every 100 c.c. of solution taken, at least 30 c.c. of an ice cold solution of 127 grm. of iodine and 15 grm. of potassium iodide in 100 c.c. of water are added. After one hour the APPENDIX TO CHAPTER I AMINES 125 crystals of the periodide of trimethylamine are sucked off on to glass wool, washed with 3-4 c.c. of a mixture of one part of the above potas- sium tri-iodide solution with three parts of water. The crystals are then dissolved in sodium thiosulphate solution, and after adding excess of sodium hydroxide, the trimethylamine is distilled ; the distillate is titrated with acid. The mother liquor of the crystals of trimethyl- amine periodide yields by a similar treatment the dimethylamine on distillation. The separation of ammonia and monomethylamine, which are left behind as hydrochlorides mixed with the sand, is effected by Frangois's process, of which the following is an example : 70 grm. of methylamine + 7 grm. of ammonia (both in the free state) in 2000 c.c. of water are shaken for one hour with 200 grm. of yellow mercuric oxide. The solution is decanted and the precipitate is washed. The filtrate and washings contain all the methylamine, but almost the whole of the ammonia is in the mercury precipitate. To remove the remainder, 40 c.c. of caustic soda and 40 c.c. of saturated potassium carbonate solution are added, together with 100 grm. of mercuric oxide. The solution now only contains monomethylamine. Methylamine can be distinguished from ammonia by means of Nessler's reagent ; the amine gives a cream-coloured precipitate, ammonia a brown one. The estimation of small quantities of amines in the presence of much ammonia has been described by Bertheaume [1910, 2]. Fleck [1896] recommends the separation of trimethylamine from ammonia by means of the sulphates, rather than the chlorides. Ammonium sulphate is insoluble in absolute alcohol, in which am- monium chloride is distinctly soluble ; trimethylamine salts dissolve readily in alcohol. de Filippi [1906] has estimated trimethylamine in urine by destroy- ing ammonia, primary and secondary amines by means of sodium hypo- bromite ; this reagent leaves tertiary amines intact. Doree and Golla [1910] by a slightly modified method found 0*014 P er cent, trimethy- lamine in urine. They state that this amine cannot bedistinguished from choline by the alloxan test, nor by the bismuth iodide or periodide test. Melting points and solubility of trimethylamine salts : Hydrochloride 271-275 soluble in boiling chloroform. Picrate . . 216 soluble in 77 parts of cold water. Picrolonate . 250-252 in 1121 parts of cold and 166 parts of boiling water, 794 of cold and 233 of boiling alcohol. Aurichloride . 228 yellow monoclinic crystals, readily soluble in hot alcohol, slightly in water. Platinichloride 240-245 regular orange crystals, little soluble in boiling alcohol. 126 THE SIMPLER NATURAL BASES Isobutylamine hydrochloride does not melt at 160, as stated in Beilstein, but at 177-178 (Thorns and Thiimen [1911]). The platinichloride forms golden yellow crystals, very soluble in alcohol and in water, decomposing at 224-225 and melting at 230- 232. Isolation of isoamylamine from putrid horse meat. The material had undergone putrefaction anaerobically for eight to ten days at 37. The proteins were coagulated, the filtrate was evaporated to a syrup, mixed with sand and extracted with acetone. After distilling off the acetone, hydrochloric acid was added to the residue, which was washed with chloroform to remove fatty acids, etc., and then rendered alkaline and again extracted with chloroform. After evaporation of the solvent the base was distilled and converted into the crystalline oxalate. Isoamylamine hydrochloride forms deliquescent crystals ; the hydro- bromide is non-deliquescent. The acid oxalate C 5 H 13 N, H 2 C 2 O 4 is ob- tained by mixing ethereal solutions of oxalic acid and of the base ; m.p. 169; it slowly loses amylamine at 100 and should be dried in vacuo. ^^platinichloride forms golden yellow leaflets, readily soluble in hot water. Isolation and Separation of Putrescine and Cadaverine. Both bases are very common in putrefaction. They are not readily volatile with steam, nor can they readily be extracted from aqueous solution by ether or by chloroform. They can be precipi- tated by phosphotungstic acid, and after treatment with silver nitrate and baryta they are found in the lysine fraction (see above). From this they can be precipitated by mercuric chloride in alcoholic solution, or they may be precipitated directly by this reagent, as was done by Brieger, without previous use of phosphoturigstic acid. He precipi- tated both bases from an alcoholic extract of a putrefaction mixture by means of alcoholic mercuric chloride and afterwards fractionally crystallised the platini- and aurichlorides (putrescine aurichloride is the less soluble in water). It is, however, more convenient to separate the hydrochlorides, that of putrescine being but little soluble in 96 per cent, alcohol, whereas the corresponding cadaverine salt dissolves readily. From urine Udranszky and Baumann [1888, I, 1889] separated both bases as dibenzoyl compounds by shaking with benzoyl chloride in sodium hydroxide solution ; this process is quantitative even in a I : 10,000 solution of the base. The benzoyl derivatives are washed with APPENDIX TO CHAPTER I AMINES 127 water and dissolved in a little boiling alcohol. After concentration the alcoholic solution is poured into thirty volumes of water when the benzoyl compounds crystallise. The concentrated alcoholic solution of the crystals is then poured into twenty volumes of ether when dibenzoyl putrescine separates and the cadaverine compound remains dissolved. Another method is due to Loewy and Neuberg [1904]. After filtering off the cystine the bases in the urine are precipitated with phosphotungstic acid and after regeneration are treated in alkaline solution with phenylisocyanate. The precipitated com- pounds of the diamines are very little soluble in most organic solvents, and are boiled out with alcohol, dried and dissolved in warm pyridine. On adding dry acetone the putrescine compound crystallises at once, the cadaverine compound only on standing. Properties and Compounds of Putrescine. The base is obtained synthetically by reduction of ethylene di- cyanide (succino-nitrile), but more conveniently by reduction of succindialdoxime (Willstatter and Heubner [1907]). Putrescine is a liquid of semen-like odour; m.p. 27-28; b.p. 158- 160; slightly volatile with steam; very soluble in water, miscible with alcohol, very little soluble in ether. The dihydrochloride, C 4 H 12 N 2 . 2HC1, crystallises in leaflets and needles and is insoluble in absolute alcohol. On destructive distilla- tion it yields pyrrolidine (rigid proof of the constitution) (Ackermann [1907, I])- The platinichloride, C 4 H 12 N 2 . H 2 PtCl 6 , needles or six-sided plates, is sparingly soluble in water (Brieger [1885, 2, p. 26]). The aurichloride, C 4 H 12 N 2 . 2HAnCl 4 . 2H 2 O, is less soluble than the cadaverine salt (Brieger [1886, I, p. 51]). The mercur i chloride is readily soluble in water, but not in alcohol. The dipicrate, C 4 H 12 N 2 . 2C 6 H 8 O 7 N 3 , silky needles, hardly soluble in cold water, decomposes at 250. The dipicrolonate, C 4 H 12 N 2 . 2C 10 H 8 O 5 N 4 , dissolves in 13,157 parts of cold and 65 3 parts of boiling water, and in 17,857 parts of cold and 954 parts of boiling alcohol ; decomposes at 263 (Otori [1904, 3]). The dibenzoyl derivative, C 4 H 8 (NHCOC 6 H 5 ) 2 , crystallises in long needles ; m.p. 178 ; almost insoluble in ether ; sparingly in cold, readily in hot alcohol. The phenylisocyanate, C 4 H 8 (NH . CO. NH . C 6 H 6 ) 2 , forms sheaves of needles from pyridine acetone ; m.p. 240 (corr.). Insoluble in water and most organic solvents ; hardly soluble in boiling alcohol. 128 THE SIMPLER NATURAL BASES Properties and Compounds of Cadaverine. Cadaverine or pentamethylene diamine was obtained by Ladenburg [1886] by the reduction of trimethylene dicyanide, but is now most easily obtained from potassium phthalimide and pentamethy- lene dichloride ; the latter compound is readily formed from benzoyl piperidine and phosphorus pentachloride, by von Braun's method [1904]. Cadaverine is also formed in small quantity by the destructive distillation of lysine (Neuberg [1905]). Cadaverine is a liquid with the odour of semen and of piperidine ; b.p. 178-179 ; somewhat volatile with steam, readily soluble in water and in alcohol, hardly in ether ; is precipitated by alkaloidal reagents. The dihydro chloride, C 5 H 14 N 2 . 2HC1, needles, non-deliquescent according to Gulewitsch [1894], is readily soluble in 96 per cent, alcohol, sparingly in absolute alcohol. On destructive distillation it yields piperidine. ^\\Q. platinichloride, C 5 H 14 N 2 . H 2 PtCl 6 , forms orange coloured rhom- bic prisms, somewhat resembling ammonium platinichloride (for details see Brieger [1885, 2, p. 37]) ; they blacken at 195 and decompose at 215; soluble in 70*8 parts of water at 21 (Gulewitsch [1894]), in 113 to 114 parts of water at 12 (Udranszky and Baumann). The aurichloride, C 5 H 14 N 2 . 2HAuCl 4 , forms long needles and also flat prisms; m.p. 186-188; fairly readily soluble in water and contain- ing water of crystallisation. The mercurichloride, C 5 H 12 N . 2HC1 . 4HgCl 2 , prepared with excess of mercuric chloride, crystallises from hot water and melts at 214*5 (Gulewitsch [1894]). It already loses mercuric chloride at 95. Soluble in 32-5 parts of water at 21; not appreciably soluble in alcohol. The dipicrate, C 5 H 14 N 2 . 2C 6 H 3 O 7 N 3 , forms long needles ; m.p. 221 ; sparingly soluble in hot water, hardly at all in boiling alcohol. The dipicrolonate, C 5 H 14 N 2 . 2C 10 H 8 O 5 N 4 ^ darkens at 220 and melts at 250; soluble in 7575 parts of cold water and 357 parts of boiling water, 5952 parts of cold and 475 parts of boiling alcohol (about twice as soluble as the putrescine salt) (Otori [1904, 3]). The dibenzoyl derivative, C 5 H 10 (NHCOC 6 H 5 )2, long needles, hardly soluble in ether, melts at 135. The phenylisocyanate, C 5 H 10 (NHCONHC 6 H 5 ) 2 , is somewhat more soluble in pyridine acetone than the putrescine compound and melts at 207-209 (corr.). APPENDIX TO CHAPTER I AMINES 129 Tetramethyl-putrescine, C 8 H 20 N 2 . This base occurs along with hyoscyamine, in Hyoscyamus muticus. It is a strongly alkaline liquid, boiling at 169 and miscible with water, alcohol and ether in all proportions. Pharmacologically it is inert (0-05 grm. given as salt hypodermically to frogs and 0-5 grm. intra- venously to rabbits was without effect). The dihydro chloride, m.p. 273, is neutral and deliquesces in moist air ; the dipicrate is fairly readily soluble in water; m.p. 198. ^hzplatinichloride, C 8 H 2() N2 . H 2 PtCl 6 . 2H 2 O, is readily soluble in hot, but much less in cold water; m.p. 234. The aurichloride, of similar solubility in water, dissolves very readily in acetone and forms golden yellow anhydrous prisms decomposing at 206-207. The constitution (CH 3 ) 2 : N . CH 2 . CH 2 . CH 2 . CH 2 . N : (CH 3 ) 2 was established by syn- thesis (Willstatter and Heubner [1907]). Agmatine. On treatment with silver nitrate and baryta, in the way described in section A of this chapter, this base is precipitated in the arginine fraction. Agmatine salts. The sulphate, C 5 H U N 4 . H 2 SO 4 , forms long needles, m.p. 229 ; the dipicrate, C 5 H U N 4 . 2C 6 H 3 O 7 N 3 , forms crystals melting at 238 and decomposing at 244; the aurichloride, C 5 H N 4 . 2HAuCl 4 , crystallises in yellow needles. The carbonate separates from aqueous solution on concentration as a chalky mass. Phenyl-ethylamine. From a putrefaction mixture this base is best isolated in the manner described above for isoamylamine, from which it is separated by its much higher boiling point. Phenyl-ethylamine and its salts. The base is easily obtained synthetically, by the reduction of benzylcyanide ; the highest recorded yield by this reaction is 53 per cent, of the theory (Wohl and Berthold [1910]). It is also obtainable from phenyl acetic acid, via the amide, by Hofmann's reaction and via the hydrazide and urethane, by Curtius's method ; it is further one of the products of the destructive distillation of phenylalanine. The synthetic base is a liquid of slight amine-like odour and readily absorbs carbon dioxide from the air, forming the crystalline carbonate. The boiling point of the base is 196 at 747 mm., 197- 198 at 754 mm. ; it is somewhat lighter than water, and dissolves in 24 parts of water at 20 ; it is miscible with alcohol and with ether. 9 130 THE SIMPLER NATURAL BASES The hydrochloride, C 8 H U N . HC1, is soluble in alcohol and melts at 217 ; with mercuric chloride a sparingly soluble crystalline compound is formed. Other salts are the acid oxalate, C 8 H U N . C 2 H 2 O 4 , m.p. 1 8 1 ; the normal oxalate, (C 8 H n N) 2 C 2 H 2 O 4 , m.p. 2 1 8 ; and ttizpicrate, C 8 H n N. C 6 H 3 O 7 N 3 , tetragonal prisms, m.p. 171-174, readily soluble in warm water. The benzoyl derivative, C 6 H 5 . CH 2 . CH 2 . NH . CO . C 6 H 5 , melts at 114. p-Hydroxy-phenyl-ethylamine. Small quantities of this amine are most readily prepared by heat- ing tyrosine under reduced pressure in test tubes dipping into a bath of fusible metal at 260-270 ; the amine sublimes ; the yield is 50 per cent. (cf. F. Ehrlich and Pistschimuka [1912]). For the isolation from complex mixtures such as are obtained in putrefaction, the base can be precipitated with phosphotungstic acid, but the phosphotungstate is rather soluble. On fractionation with silver and baryta, the base is obtained as platinichloride from the lysine fraction. A better way is to utilise its phenolic properties by washing its solution in *5N sodium hydroxide with amyl alcohol, neutralising, add- ing sodium carbonate and extracting the amine with amyl alcohol. After distilling off the solvent with steam, the dibenzoyl derivative is obtained by the Schotten-Baumann method. In sufficient quantity p-hydroxy-phenyl-ethylamine is best purified by distillation ; it boils at 161-163 at 2 mm - an( * 175-181 at 8 mm. It is also readily purified by crystallisation from boiling xylene in which it is very sparingly soluble. It forms colourless hexagonal leaflets melting at 161, soluble in 95 parts of water at 15 and in about 10 parts of boiling ethyl alcohol. The base is fairly soluble in amyl alcohol, but hardly at all in ether or chloroform. It gives Millon's and Morner's reaction for tyrosine, but no coloration with triketo- hydrindene hydrate. The hydrochloride, C 8 H n ON . HC1, is very soluble in water and may be crystallised from concentrated hydrochloric acid; m.p. 268. The phosphate, C 8 H n ON . H 3 PO 4 . i|H 2 O, forms white prisms, readily soluble in water; m.p. 209-210. The picrate, C 8 H n ON . C 6 H 3 O 7 N 3 , forms short prisms; m.p. 200. The platinichloride, (C 8 H n ON) 2 HJ?tCl 6 , forms six-sided leaflets. The N-monobenzoyl derivative crystallises from alcohol in hexagonal plates; m.p. 162. The dibenzoyl derivative, C 6 H 5 CO.O.C 6 H 4 .CH 2 CH 2 .NH.CO.C 6 H 5 , APPENDIX TO CHAPTER I AMINES 131 is the most useful and characteristic derivative of the base. Formed by the Schotten-Baumann reaction, it crystallises readily from alcohol and melts at 170; this derivative gives Morner's reaction, but not Millon's. Yeast transforms p-hydroxy-phenyl-ethylamine to the correspond- ing alcohol, tyrosol, OH . C 6 H 4 . CH 2 . CH 2 OH (Ehrlich and Pistschimuka [1912]). p-Hydroxy-phenyl-ethylamine is attacked by various oxidases and converted to pigments, but does not always behave in the same way as its parent substance tyrosine. Thus Neuberg [1908, Ch. VI] found that a ferment from a melanoma at- tacked the amine, but not the amino-acid, whereas an extract of the ink-bag of Sepia acts on tyrosine more readily than on the amine. Compare also J. Chem. Soc., Abstr., 1908, 94, i., 236. Hordenine. Gaebel's process of isolation was as follows : The extract of 3 kilos, of malt germs with 95 per cent, alcohol was evaporated to a syrup and extracted with I litre of water. After filtration the aqueous extract was made alkaline with sodium carbonate, shaken once with a little ether to remove a colouring matter, and then ten times with large quantities of ether. The concentrated ethereal extract was dried with potassium carbonate and evaporated, when the residual syrup soon crystallised. On recrystallisation from dry ether, with charcoal, the pure base is obtained; the yield is O'2 per cent, of the air dry germs. Properties: Hordenine forms colourless crystals melting at II7'8 (corr.) and boiling at 173-174 and 1 1 mm. Distillation under reduced pressure is the most convenient method of purification. The base dissolves readily in alcohol and in chloroform, and fairly readily in ether and in water ; it is hardly soluble in benzene. Hordenine gives Millon's and Piria's reactions for tyrosine ,and reddens phenolphtha- lein ; it is not coloured by concentrated sulphuric acid, but reduces potassium permanganate in the cold and ammoniacal silver nitrate on warming. Ite sulphate, (C 10 H 15 NO) 2 . H 2 SO 4 . H 2 O, the hydrochloride and the hydrobromide are sparingly soluble in alcohol. The quaternary iodide, hordenine methiodide, obtained by the action of methyl iodide in methyl alcoholic solution on hordenine (or on p-hydroxy-phenyl-ethyl- amine), forms large glassy prisms, sparingly soluble in cold water ; m.p. 230-231. 9* I 3 2 THE SIMPLER NATURAL BASES Indolethylamine. The free base, on recrystallisation from a mixture of alcohol and benzene, forms long colourless needles, melting at 145-146. It is readily soluble in alcohol and in acetone, but is almost insoluble in water, ether, benzene and chloroform. It gives very intensely Hopkins and Cole's reaction with glyoxylic and sulphuric acids, characteristic of tryptophane ; the bluish-violet coloration is still obtainable with the base in a dilution of I : 300,000. Unlike tryptophane, aminoethyl- indole is not coloured by bromine water, nor does it react with triketohydrindenehydrate. The hydrochloride > C 10 H 12 N 2 . HC1, forms thin prisms melting at 246 and is soluble in about 12 parts of water at 18. The picrate is the most characteristic salt of the base. It has the composition C 10 H 12 N 2 . C 6 H 3 O 7 N 3 and is obtained by adding a cold saturated solution of picric acid to a solution of the hydrochloride in water ; the mixture at once becomes turbid and orange-red in colour, and dark red crystals, consisting of fern-like aggregates of needles or prisms (resembling in shape those of ammonium chloride) rapidly separate. This picrate is almost insoluble in water and very sparingly so in alcohol and most organic solvents, but dissolves readily in acetone ; it melts and decomposes at 242-243. The picrolonate crystallises readily from hot water in deep chrome- yellow prisms melting at 231. The monobenzoyl derivative of 3-/3-amino-ethylindole is difficult to crystallise, and therefore not suitable for characterising the base ; it forms stout prisms melting at 137-138. /3-Iminazolyl-ethylamine. Bacterial Preparation. Ackermann [1910, l] dissolved 49 grm. of histidine hydrochloride in 4 litres of water, added 10 grm. of Witte peptone, 20 grm. of glucose, a few drops of magnesium sulphate and sodium phosphate solutions, and excess of calcium carbonate to keep the reaction alkaline. After inoculation with putrid pancreas the solution was kept fifty- two days at 35. It yielded 6 1 '6 grm. of iminazolyl- ethylamine dipicrate which is 42 per cent, of the theoretical ; a very small quantity of iminazolyl-propionic acid was also formed. When working with small quantities of histidine and pure cultures of certain bacteria one can occasionally obtain solutions of which the physiological activity indicates an almost complete conversion. How- APPENDIX TO CHAPTER I AMINES 133 ever, it seems generally impossible to isolate more of the amine than Ackermann obtained and the yield is often very much less. The mode of action of one and the same organism seems to depend on conditions which areas yet imperfectly understood, so that this method is rather uncertain. Mellanby and Twort [1912] have isolated a bacillus of the typhoid-coli group from the intestine of various mammals (from the duodenum downwards) which is capable of decarboxylating histidine. The best yields of the amine were obtained by inoculating histidine solutions with adequate quantities of a vigorous twenty-four hours' culture of the organism on glycerine-agar and incubating for one week at 37. The solutions contained histidine I per cent., ammonium tartrate I per cent., dipotassium phosphate O'l per cent, magnesium sulphate O'O2 per cent., calcium chloride 0*01 per cent., but no peptone. Solutions containing cri per cent, histidine give a better yield. See also patents by Hoffmann, La Roche & Co. [1912] and papers by Berthelot and Bertrand [1912, i, 2; 1913, I, 2] and by Bertrand and Berthelot [1913] describing the isolation of Bacillus aminophilus intes- tinalis, a Gram-negative capsulated organism, resembling B. lactis aerogenes and the bacillus of Friedlander, but differing from these in its great power of decarboxylating amino-acids. For isolation of the organism they used o '2 grm. K 2 SO 4 , 0'2 grm. MgSO 4 , 0*5 grm. K 2 HPO 4 , 0*25 grm. KNO 3 , 0*02 grm. CaCl 2 and I -5 grm. histidine hydrochloride per litre. To isolate /3-iminazolyl-ethylamine when pure histidine has been submitted to putrefaction, it is hardly necessary to precipitate with phosphotungstic acid. Instead one can precipitate at once with picric acid, having removed ammonia, and recrystallise the picrate. For the isolation of the base from complex mixtures such as ergot, it is neces- sary to fractionate with silver nitrate and baryta. The base is then found in the histidine fraction. Its hydrochloride is conveniently separated from inorganic salts by extraction with methyl alcohol. Salts of $-iminazolyl-ethylamine. The dihydrochloride, C 5 H 9 N 3 . 2HC1, is extremely soluble in water and sparingly soluble in ethyl alcohol; it crystallises in prisms; m.p. 240. The dihydrobromide has similar solubilities and forms stout prisms sintering at 265 and melting at 284 (corr.). The acid phosphate, C 5 H 9 N 3 . 2H 3 PO 4 , is some- what less soluble in water and crystallises very well ; it decomposes indefinitely at 120-140. r I\\Qplatmichloride t C 5 H 9 N 3 . H 2 PtCl 6 , orange coloured prisms readily soluble in hot water and hardly at all in alcohol, blackens and de- 134 THE SIMPLER NATURAL BASES composes between 200 and 240 without melting. The aurichloride, C 5 H 9 N 3 .(HAuCl 4 ) 2 , melts with decomposition at 200-210. The dipicrate, C 5 H 9 N 3 . (C 6 H 3 O 7 N 3 ) 2 , is the most convenient salt for purposes of isolation. It forms deep yellow rhombic leaflets, melting at 238-242 (corr.) according to the rate of heating. It is very spar- ingly soluble in cold water and can be recrystallised from hot water. The monopicrate, C 5 H 9 N 3 . C 6 H 3 O 7 N 3 , m.p. 233-234, forms bunched, slightly curved, pointed needles. The dipicrolonate, C 5 H 9 N 3 (C 10 H 8 O 5 N 4 ) 2 , dissolves in about 450 parts of boiling water, from which it crystallises in sheaves of needles, melt- ing at about 264. Reactions of $-iminazolyl-ethylamine. In common with histidine, this amine gives Pauly's reaction with p-diazobenzene sulphonate ; a very distinct rose pink coloration is still obtainable at a dilution of I : 10,000. It also gives Knoop's histidine reaction, a claret colora- tion, on boiling with bromine water. It is precipitated by ammoniacal silver oxide, by mercuric chloride in the presence of potassium hydroxide, and by phosphotungstic acid. On the other hand, it is distinguished from histidine in not giving the biuret reaction, nor Ruhemann's reaction with triketohydrindenehydrate, and it further behaves differently on benzoylation. When shaken with benzoyl- chloride in potassium hydroxide solution, the glyoxaline ring is rup- tured and tribenzoyl-butentriamine is formed, of the following con- stitution : CH . NH . CO . C 6 H 6 C . NH . CO . C 6 H 6 CH 2 . CH 2 . NH . CO . C 6 H 5 . Histidine, on the other hand, yields a monobenzoyl derivative. BASES OF CHAPTER II. /9-Alanine. The substance may be obtained synthetically in several ways, the best being from succinimide (Holm [1904]); I mol. of succinimide in 10 per cent, potash solution containing 6 mol. of KOH and I mol. of KOBr is warmed for two hours to 50-60. The resulting /3-alanine is purified by esterification. Abderhalden and Fodor [1913] isolated /3-alanine according to Fischer's ester method. The free ester boils at 54 and 10 mm. The hydrochloride melts at 64. On distilling the ester at ordinary pressure it gives the pungent smell of ethyl acrylate which is a good mode of recognition of /3-alanine. Synthetic yS-alanine forms prisms, melting at 206-207 and decom- posing into acrylic acid and ammonia. The hydrochloride melts at 1 22 -5. The sulphate, (C 3 H 7 O 2 N) 2 H 2 SO 4 , decomposes at I 50. The platinichloride, (C 3 H 7 O 2 N) 2 H 2 PtCl 6 , crystallises from water or hydrochloric acid in deep yellow needles, m.p. 180; it is soluble in alcohol (Engeland [1908, i]). The copper salt, (C 3 H 6 O 2 N) 2 Cu + 6H 2 O, forms azure crystals (Holm [1904]). 7-Aminobutyric Acid. According to Engeland and Kutscher [1910, 3, Ch. Ill, buty- robetaine] ry-aminobutyric acid is precipitated in dilute solution by phosphotungstic acid and also by mercuric chloride in the presence of sodium acetate, but not by mercuric chloride alone. These properties it shares with histidine and methyl-guanidine, from which it may be separated by silver nitrate and baryta, when it appears in the lysine fraction. It can also be separated by distillation of its ester, prepared by Fischer's method. ^-Amino-butyric acid was first obtained by Schotten [1884] by oxidising piperidylurethane with fuming nitric acid and subsequently 135 136 THE SIMPLER NATURAL BASES hydrolysing the oxidation product (for details see Abderhalden and Kautzsch [1912]). The free acid forms leaflets melting at 183-184 XCH 2 . CH 2 with conversion into the anhydride pyrrolidone, NH V | ^CO . CH 2 . The kydrochloride crystallises in stout prisms ; m.p. 135- The platinichloride forms orange prisms; m.p. 220. The aurichloride crystallises in glistening plates; m.p. 138. The ethyl ester boils at 75-77/ 12 mm. -Amino-valeric Acid. E. and H. Salkowski obtained this substance from putrid blood fibrin by evaporating the mixture repeatedly with water, adding barium chloride to remove some fatty acids as soaps, acidifying the nitrate, washing with ether, evaporating to dryness, and extracting the residue with alcohol. On standing for a long time in a desiccator the residue from the alcoholic solution gave the crystalline hydrochloride of S- amino-valeric acid, from which the platinichloride and finally the aurichloride was isolated. Formation from proline. Ackermann [1911, 2] obtained 3-6 grm. of S-amino-valeric acid aurichloride from 34 grm. of proline after putrefaction for nine days with glucose, peptone and salts. Neuberg isolated the acid by means of a-naphthylisocyanate and obtained at the same time n-valeric acid, which would result from the deamina- tion of S-amino-valeric acid. Neuberg [1911, I] used a I percent, proline solution, made and kept alkaline by repeated addition of sodium bicarbonate, and containing a few drops of saturated magnes- ium sulphate, potassium chloride and sodium phosphate solutions, but no glucose or peptone; from 23 grm. of proline I2T grm. was re- covered unchanged, together with 27 grm. S-amino-valeric acid hydro- chloride, and 2 -3 grm. of silver n-valerate. S-Amino-valeric acid crystallises in pearly leaflets, extremely soluble in water, and melting at 1 57-1 58 when they undergo transformation to piperidone. The aqueous solution is faintly acid and has an astringent taste. The substance is precipitated in dilute solution by phospho- tungstic acid, but not by cupric acetate or ammoniacal silver solution. The hydrochloride, C 5 H n O 2 N . HC1, forms rhombic leaflets which on heating distil for the most part without change. The platinichloride, (C 5 H n O 2 N)2H 2 PtCl 6 , forms long rhombic leaf- lets, readily soluble in hot water but only slightly in cold water and in alcohol. APPENDIX TO CHAPTER II w-AMINO-ACIDS 137 The normal aurichloride, C 5 H n O 2 N . HAuCl 4 . H 2 O, crystallises in monoclinic orange coloured crystals ; m.p. 86-87 ; an abnormal auri- chloride, C 5 H U O 2 N . AuCl 3 , is also known; it forms pale yellow crystals decomposing at 130 and is transformed to the more deeply coloured normal salt by recrystallisation from dilute hydrochloric acid. Benzoyl--amino-valeric acid is formed by the oxidation of benzoyl piperidine with potassium permanganate and by the benzoylation of S-amino-valeric acid. It melts at 94 and at 105. S-Amino-valeric acid does not yield a blue copper salt on boiling with cupric oxide or on adding cupric acetate. /3-Iminazolyl-propionic Acid. This substance was isolated by Ackermann from the filtrate of the /9-iminazolyl-ethylamine picrate [1910, i] obtained in the putrefac- tion of histidine. The picric acid was removed from this filtrate, the solution was evaporated, the residue was extracted with alcohol and to the alcoholic solution platinic chloride was added. A slight pre- cipitate was filtered off and the alcoholic solution was evaporated to dryness. The residue, dissolved in a minimum quantity of boiling water, deposited the crystals of the platinichloride of /3-iminazolyl- propionic acid. /3-Iminazolyl-propionic acid is readily soluble in water, less so in alcohol and crystallises from dilute acetone ; m.p. 208-209. The nitrate, C 6 H 8 C>2N2 . HNO 3 , readily soluble in methyl alcohol, forms elongated six-sided leaflets; m.p. 143-148. The platinichloride, (C 6 H 8 O 2 N 2 ) 2 . H 2 PtCl 6 , melts at 209. The phosphotungstate crystallises from hot water in characteristic rectangular leaflets, decomposing above 300. The copper salt forms blue needles. Carnosine (Ignotine). Carnosine is obtained from the regenerated phosphotungstic acid precipitate (after neutralisation with nitric acid) by means of silver nitrate and excess of baryta. After decomposing the silver precipitate with hydrogen sulphide and removing the baryta by carbon dioxide, the solution is neutralised with nitric acid and concentrated ; carnosine nitrate crystallises out after the addition of alcohol. Krimberg, by Gulewitsch's method, obtained 15-3 grm. of the free base from I Ib. of Liebig's extract, or 3-4 per cent.; by Kutscher's process he only obtained 3 grm. of carnosine from the same quantity 138 THE SIMPLER NATURAL BASES of meat extract, and Kutscher himself obtained 3 grm. of ignotine from I Ib. of Liebig's extract. The free base, C 9 H U O 3 N 4 , crystallises in needles, soluble in 3-2 parts of water at 25 and appreciably so in alcohol ; m.p. 248-5 - 250 ; [a] D 2 c = 21, independent of the dilution. A 2*5 per cent, aqueous solution gives no precipitate with platinic chloride, but it causes turbidity with picric acid and a precipitate with gold chloride and potassium bismuth iodide. The nitrate, C 9 H U O 3 N 4 . HNO 3 , melts at 219 and dissolves in 1*04 parts of water at 25; [a D 20 ] in 1-48 per cent, solution = +24-2, in 8 per cent, solution = +22 '8; excess of nitric acid lowers the rotation [Gulewitsch, 1913]. The copper salt, C 9 H U O 3 N 4 . CuO, forms deep blue six-sided plates, resembling cystine crystals in shape. It is sparingly soluble in hot water and results when carnosine is boiled with copper carbonate. Carnosine yields a sparingly soluble dipicrolonate, of which Mauthner [1913] has attempted to use the mono-sodium salt as a means of estimating carnosine in the histidine fraction of muscle extracts. Carnosine resembles arginine and differs from histidine in requir- ing a fixed alkali for its precipitation as silver compound from a solu- tion of carnosine nitrate containing an equimolecular amount of silver nitrate. With silver nitrate in excess the silver compound is also precipitated by careful addition of ammonia, but is soluble in excess. Demjanowski [1912, Ch. V, methyl-guanidine] gives the following limits of precipitation in aqueous solution : mercuric chloride, I : 2000 ; mercuric sulphate, I : 100,000 ; mercuric nitrate, I : 100,000 ; 25 per cent, phosphotungstic acid, I : 20,000. Urocanic Acid. Preparation. Jaff obtained the substance by a very simple method. The urine was evaporated to a syrup and the latter was extracted with hot alcohol ; after evaporation of the alcohol the residue was acidified with sulphuric acid ; after washing with ether to remove impurities the urocanic acid crystallised from the aqueous layer. Hunter used phosphotungstic acid for the isolation of urocanic acid and this is probably also the most certain method of obtaining it from urine. The amount when present in urine is not inconsider- able ; Jaff6 obtained 2-3 grm. per day and Siegfried found the urine to contain O'i8 per cent, of the substance. Jaffe gave the formula C 12 H 12 O 4 N 4 , 4H 2 O to the free acid, but this APPENDIX TO CHAPTER II w-AMlNO-ACIDS 139 must be halved. The free acid is slightly soluble in cold water (0-15 per cent, at 18 according to Siegfried) and readily soluble in hot water. The melting point depends greatly on the rate of heating ; after crystallisation from dilute acetone Barger and Ewins found 2 35-236 (uncorr.). Hunter gives 231-232 (corn), Jaffe 212-213, Siegfried 229. Hunter obtained the acid in slender, beautifully iri- descent needles or tetragonal prisms. With sodium p-diazobenzene sulphate it gives the red coloration of histidine. The acid is pre- cipitated from solution by silver nitrate ; the precipitate dissolves in excess of ammonia and in nitric acid. The barium salt, (C 6 H 5 O 2 N 2 ) 2 Ba . 8H 2 O, crystallises in needles and loses 6H 2 O at 100 and the rest at 150. The nitrate, C 6 H 6 O 2 N 2- HNO 8 , is the most characteristic salt. It is sparingly soluble in dilute nitric acid and crystallises in small sickle-shaped plates frequently united to cross- or rosette-shaped aggre- gates (figured by Hunter, p. 541); m.p. 198 with explosive decom- position (Barger and Ewins). The picrate, C 6 H 6 O 2 N 2 . C 6 H 3 O 7 N 3 , forms golden yellow prisms; m.p. 213-214, 224-225 (corr.). The picrolonate, C 6 H 6 O 2 N 2 . C 10 H 8 O 5 N 4 , crystallises from dilute alcohol ; m.p. 268 (corr.). The phosphotungstate forms small rectangular plates from dilute acetone or from hot water. Kynurenic Acid. To obtain kynurenic acid, Kretschy [1881] fed a dog of 34 kilos, weight daily with I kilo, of horse meat, 70 grm. of bread and I litre of water. At first the daily production of the acid was cri grm. but after I month O'8 grm. The best method, however, is to give tryptophane by the mouth. The urine is acidified and the pre- cipitate formed in twenty-four hours is filtered off and purified by dissolving in ammonia, acidifying slightly with acetic acid and leaving for twenty-four hours to allow a brown impurity to precipitate. After filtration the solution is acidified with 4 per cent, hydrochloric acid. Adherent uric acid may be removed by Hopkins's method and the kynurenic acid may be finally recrystallised from 800 parts of boiling alcohol (Homer [1913]). The pure acid forms long glistening needles, of the formula C 10 H 7 O 3 N, H 2 O. The water of crystallisation is given off at 140-145. The highest melting point obtained by Miss Homer was 288-289 (uncorr.). The acid is practically insoluble in cold water and 100 parts of boiling water only dissolve 0*09 parts ; I 4 o THE SIMPLER NATURAL BASES 100 c.c. of boiling alcohol dissolve cri grm. The following salts are crystalline: C 10 H 6 O 3 NK + 2H 2 O, (C 10 H 6 O 3 N) 2 Ba + 4iH 2 O, (C 10 H 6 O 3 N) 2 Ca + 2H 2 O and (C 10 H 6 O 3 N) 2 Cu + 2H 2 O. The barium salt is fairly soluble in hot water, but the copper salt is almost in- soluble in it. The crystalline hydrochloride C 10 H 7 O 3 N, HC1 easily loses hydrochloric acid (Brieger [1879]); the basic properties of the substance are further evident from its precipitation by phosphotungstic acid (Hofmeister [1880, Ch. V, creatine]). Kynurine> formed in a 90 per cent, yield by heating kynurenic acid to 253-258, is little soluble in cold water, more so in alcohol The hydrated substance C 9 H 7 ON, 3H 2 O melts at about 52, the anhy- drous substance at 202. It is a feeble base yielding a platinichloride (C 9 H 8 ON) 2 PtCl 6 + 2H 2 O and a crystalline hydrochloride ; with bro- mine the substance C 9 H 4 Br 3 ON is formed (Brieger [1879]). Jafffs reaction for kynurenic acid '[1883]. A solution of the acid is evaporated on the water bath with hydrochloric acid and potassium chlorate ; the red residue becomes brownish green with ammonia, soon changing to an intense emerald green ; the chief product is tetrachloro- oxykynurine, C 9 H 3 O 2 NC1 4 . A convenient method of estimation has been described by Capaldi [1897,2]. BASES OF CHAPTER III BETAINES. Betaine (Acetobetaine). The isolation by Schulze's method is described along with that of choline (p. 150) as is also StaneVs method of estimation (p. 151). For the estimation in crude sugar and in molasses Stanek [1904] dissolves 20-30 grm. of the former or 3-5 grm. of the latter in 50 c.c. of I o per cent, sulphuric acid previously saturated with sodium chloride. This yields in either case a 1-3 per cent, solution of betaine which is completely precipitated by the potassium tri-iodide reagent (if the precipitate is oily, it may be rendered filterable by adding finely powdered iodine); the nitrogen is determined in the precipitate as described in the section on choline (p. 151). For the estimation of betaine in plants Stanek and Domin [1910] may also be consulted. In order to prepare betaine from molasses Stanek [1901-2] utilises the great stability of the base by mixing the molasses with an equal volume of concentrated sulphuric acid and heating for three hours to 130. After neutralisation with lime, evaporation to dryness and ex- traction of the residue with alcohol, the alcoholic extract is treated with charcoal, concentrated to a syrup and saturated with gaseous hydrogen chloride, when betaine chloride crystallises out. A method of isolating betaine from the desaccharified strontium liquors as the phosphate is given by Andrlik [1903-4] and as the chloride by Stoltzenberg, German patent No. 243332 and [1912], The last-named method is similar to that given by Urban [1913], but the best method of all is apparently that due to Ehrlich [1912 and D.R.P. 157173 of 1904], From the desaccharified residue (" Melasse Schlempe ") the betaine is extracted as base by means of 96 per cent, alcohol, and after evaporation of the alcohol, the free base is converted into the chloride which is crystallised. The commercial product acidol is prepared according to this method. Chemical Properties and Derivates of Betaine. Betaine crystallises from alcohol in deliquescent crystals containing one molecule of water which is lost at 100. The hydrated substance 141 142 THE SIMPLER NATURAL BASES probably has the constitution (CH 3 ) 3 N(OH) . CH 2 . COOH, of which the other substance is a cyclic anhydride. Betaine and its isomeride, the methyl ester of dimethyl-ammo- acetic acid, are interconvertible at temperatures between 135 (the boiling point of the ester) and 293 ; over this range betaine is the more stable and it is formed in good yield by heating the ester in a sealed tube to 200. On the other hand a 50 per cent, yield of the ester is obtainable by heating betaine to 300, when the ester distils out. At or above 293 betaine begins to be decomposed into tri- methylamine and other substances (Willstatter [1902, I]). Betaine is a very feeble base, forming a series of stable salts. The salts with mineral acids have a strongly acidic reaction, and for this reason the chloride is sold as a solid substitute for hydrochloric acid under the name " acidol ". The chloride^ C 5 H 12 O 2 NC1, forms leaflets, melting and decomposing at 227-228 (243); it is very soluble in water and differs from the hydrochlorides of most organic bases in being almost insoluble in absolute alcohol (i grm. dissolves in 365 c.c. of absolute alcohol at room temperature; Schulze [1909, Ch. IV, choline]). The iodide, C 5 H 12 O 2 NI, non-deliquescent crystals; m.p. 188-190; very soluble in hot alcohol, but little in cold (Willstatter [1902, i]). The periodide, C 5 H 12 O 2 NI . I 5 , loses iodine on exposure to the air [Stanek, 1912]. Compounds with potassium iodide of the formulae C 5 H U 2 N . KI . 2H 2 O and (C 5 H n O 2 N) 2 . KI . 2H 2 O have also been de- scribed (see Willstatter [1902, i]). The/^ is formed by heating glycocyamine hydrochloride to 160-170 ; small quantities are more readily prepared by heating I grm. of this hydrochloride with 5 c.c. of concentrated hydrochloric acid to 140 in a sealed tube. The free base is obtained by boiling the resulting hydrochloride with freshly precipitated lead hydroxide. An alcoholic solution (but not an aqueous solution) of glycocyamidine hydrochloride gives with alcoholic zinc chloride a crystalline salt (C 3 H 5 ON 3 ) 2 ZnCl 2 . The picrate, C 3 H 5 ON 3 . C 6 H 3 O 7 N 3 , forms yellow needles ; m.p. 206-210. The normal aurichloride is very soluble and easily changes to the less soluble gold salt C 3 H 6 ON 3 . AuCl 3 ; m.p. I53-I54 (Korndorfer [1905]). n * 1 64 THE SIMPLER NATURAL BASES Glycocyamidine, like creatinine, gives Weyl's and Jaffa's reactions ; there is, however, this point of difference, that whereas the red or yellow coloration produced by creatinine, sodium nitroprusside, and caustic soda is discharged by acetic acid or changed to green on boiling (formation of Prussian blue), glycocyamidine yields with acetic acid a stable burgundy red coloration. Guanidine. Guanidine is a strong base, absorbing atmospheric carbon dioxide to form the well crystallised carbonate, (CH 5 N 3 ) 2 . H 2 CO 3 , soluble in water but not in alcohol. Of the salts with mineral acids the nitrate CH 5 N 3 . HNO 3 is among the least soluble ; it forms large plates, melt- ing at 214. The picrate> CH 5 N 3 . C 6 H 3 O 7 N 3 , when pure forms characteristic ir- regular aggregations of leaflets ; m.p. 315, on rapid heating up to 320. The solubility in cold water is I : 2630 at 9 and the salt may be used for the estimation of guanidine (Emich [1891]). From complex mix- tures, particularly when arginine is present, guanidine is not so readily precipitated by picric acid ; the arginine should first be precipitated by alcoholic picrolonic acid solution, and then, after removal of the excess of picrolonic acid from the filtrate, the guanidine may be pre- cipitated by aqueous picric acid (Kutscher and Otori [1904]). The picrolonate, CH 5 N 3 . C 10 H 7 O 5 N 4 , dissolves in excess of alcoholic picrolonic acid solution (separation from arginine, above). With aqueous picrolonic acid an amorphous precipitate is formed, which crystallises from hot water in clusters of thin needles; m.p. 272-274 (Schenck [1905, 2]). The aurichloride, CH 5 N 3 . HAuCl 4 , forms deep yellow needles, little soluble in water. With alcoholic cadmiumchloride a double salt CH 5 N 3 . HC1 . 2CdCl 2 results; m.p. 390-395 (Schenck [1904]). Guanidine is precipitated in the " arginine" fraction by silver nitrate and baryta as a silver compound CH 5 N 3 . Ag 2 O which may be crystallised (Kutscher and Otori [1904]). Guanidine salts in con- centrations down to O'Oi per cent, give a white or pale yellow precipi- tate with Nessler's reagent ; arginine gives a similar precipitate. Methylguanidine. Methylguanidine may be synthesised by heating cyanamide and methylamine hydrochloride in alcoholic solution to 60-70. It forms deliquescent crystals. The nitrate^ C 2 H 7 N 3 . HNO 3 , forms rhombic APPENDIX TO CHAPTER V 165 leaflets, melting at 150 (i$5), not very soluble in cold alcohol, and less in water and particularly in dilute nitric acid. The picrate C 2 H 7 N 3 . C 6 H 3 O 7 N 3 , m.p. 201-5, crystallises in two modifications ac- cording to Gulewitsch [1906] and is more soluble than guani- dine picrate. The picrolonate, C 2 H 7 N 3 . C 10 H 7 O 5 N 4 , dissolves in 4000 parts of cold water; m.p. 291 (Wheeler and Jamieson [1907]). The aurichloride C 2 H 7 N 3 . HAuCl 4 , m.p. 198, is soluble in ether. The platinichloride (C 2 H 7 N 3 ) 2 . H 2 PtCl 6 forms monoclinic prisms and dissolves in 1 4-3 parts of water at 18-19. Benzene-sulphonyl-methyl-guanidine, C 2 H 6 N 8 . SO 2 . C 6 H 5 , m.p. 184, soluble in 2500 parts of cold water, is suitable for the isolation (Acker- mann [1906]). Aqueous mercuric chloride does not precipitate the nitrate of methylguanidine even in 5 per cent solution ; mercuric sulphate precipitates a I per cent, solution, phosphotungstic acid a solution of i : 9000 (Demjanowski [1912]). Dimethylguanidine- The aurichloride, C 3 H 9 N 3 . HAuCl 4 , melts at 144, decomposes at 150 and forms thin leaflets or plates. IL\\Q ptcrolonate, C 3 H 9 N 3 . C 10 H 7 O 5 N 4 , m.p. 275-278, was probably obtained from human urine by Kutscher and Lohmann [1906, 3, 4] and forms four-sided prisms. The picrate, C 3 H 9 N 3 . C 6 H 3 O 7 N 3 , forms small pointed needles or branch-like growths ; m.p. 224 (Wheeler and Jamieson [1907]). BIBLIOGRAPHY. REFERENCES TO CHAPTER I. AMINES. ABELOUS, J. E., et E. BARDIER (1909). De Vaction hypotensive et myotique de I'urine normale. Compt. rend., 48, 1471-72 ; also Compt. rend. Soc. de Biol., 66, 876-77. ABELOUS, J. E., et H. RIBAUT (1908). Sur la substance hypertensive qu'on peut extraire de muscle putre fie. Compt. rend. Soc. de Biol., 64, 907-98. ABELOUS, J. E., H. RIBAUT, A. SOULI&, et G. TOUJAN (1906, i). Sur la presence dans les macerations de muscles putrefies de substances elevant la pression arterielle. Compt. rend. Soc. de Biol., 58, 463. ABELOUS, J. E., H. RIBAUT, A. SOULIE", et G. TOUJAN (1906, 2). Sur la presence dans les macerations de muscles putrefies d^une ptomaine elevant la pression arterielle. Compt. rend. Soc. de Biol., 58, 530-32. ACKERMANN, D. (1907, i). Notiz zur Kenntniss des Putrescins. Zeitschr. physiol. Chem., 53, 545-46. ACKERMANN, D. (1907, 2). Bin Beitrag zur Chemie der Faulnis. Zeitschr. physiol. Chem., 54, 1-31. ACKERMANN, D. (1908, i). Ein Fdulnisversuck mit Arginin. Zeitschr. physiol. Chem., 56, 305-15. ACKERMANN, D. (1909, i). Ueber die Entstehung von Fdulnisbasen. Zeitschr. physiol. Chem., 60, 482-501. ACKERMANN, D. (1909, 2). Ein Faulnisversuch mit lysinfreiem Eiweiss. Zeitschr. physiol. Chem., 64, 91-94. ACKERMANN, D. (1910, i). Ueber den bakteriellen Abbau des Histidins. Zeitschr. physiol. Chem., 64, 504-10. ACKERMANN, D. (1910, 2). Die Isolierung der Fdulnisbasen. Handbuch der biochemischen Arbeitsmethoden. II. Band, 1002-43. Urban und Schwarzenberg. Berlin und Wien. ACKERMANN, D. (1910, 3). Ueber ein neues, auf bakteriellem Wege gewinnbares Apo- rrhegma. Zeitschr. physiol. Chem., 51, 323-33. ACKERMANN, D., und F. KUTSCHER (1910, i). Untersuchungen ueber die physiologische Wirkung einer Secale-base und des Imidazoly lathy lamins. Zeitschr. f. Biol., 54, 387-94. ACKERMANN, D., und F. KUTSCHER (1910, 2). Ueber die Aporrhegmen. Zeitschr. physiol. Chem., 69, 265-72. ACKERMANN, D., und F. KUTSCHER (1911). Ueber das Vorkommen von Lysin im Harn bei Cystinurie. Zeitschr. f. Biol., 57, 355-59. ACKERMANN, D., und H. SCHUTZE (1910). Ueber die Bildung von Trimethylamin durch Bacterium prodigiosum. Zentralbl. f. Physiol., 24, 210-11. ACKERMANN, D., und H. SCHUTZE (1911). Ueber Art und Herkunft der fiuchtigen Basen aus Kulturen des Bacterium prodigiosum, Archiv f. Hygiene, 73, 145-52. ARONSON, H. (1912). Welter e Untersuchungen iiber Anaphylatoxin und Bakteriengift. Berl. klin. Wochenschr., 49, 642-46. 167 1 68 THE SIMPLER NATURAL BASES BAEHR, G., und E. P. PICK (1913, l). Pharmakologische Studien an der Bronchialmuskulatnr der iiberlebenden Meerschweinchenlunge. Arch. exp. Path. Pharm., 74, 41-64. BAEHR, G., und E. P. PICK (1913, 2). Beitrdge zur Pharmakologie der Lungengefdsse. Arch. exp. Path. Pharm., 74, 65-72. BAIN, W. (1909). Pressor bases in normal urine and their diminished excretion in gout. Lancet, ii., 365-67. BAIN, W. (1910). Further work on the pressor bases of urine. Lancet, i., 1190-94. BARBOUR, H. G. (1913). Note on the action of histamine upon surviving arteries. ]. Pharmacol. Exp. Therap., 4, 245-50. BARGER, G. (1909, i). Isolation and synthesis of p-hydroxyphenylethylamine, an active principle of ergot, soluble in water. J. Chem. Soc., 95, 1123-28. BARGER, G. (1909, 2). Synthesis of hardening, the alkaloid from barley. J. Chem. Soc., 95, 2193-97. BARGER, G., and H. H. DALE (1909). The active principles of ergot. J. Physiol., 38, Proc., Ixvii-lxxix. BARGER, G., and H. H. DALE (1910, i). Chemical structure and sympatho-mimetic action of amines. J. Physiol., 41, 19-59. BARGER, G., and H. H. DALE (igio, 2). A third active principle in ergot extracts (Preliminary note). Proc. Chem. Soc., 26, 128-29. BARGER, G., und H. H. DALE (1910, 3). Die physiologische Wirkung einer Secale-base und deren Identifizierung als Imidazolyldthylamin. Zentralbl. f. Physiol., 24, 885-889. BARGER, G., and H. H. DALE (1910, 4). ^-ft-Amino-ethylglyoxaline (0-Iminazolyl-ethyl- amine) and the other active principles of ergot. J. Chem. Soc., 97, 2592-95. BARGER, G., and H. H. DALE (1911). &-Iminazolylethylamine, a depressor constituent of intestinal mucosa. J. Physiol., 41, 499-503- BARGER, G., and G. S. WALPOLE (1909, i). Isolation of the pressor principles of putrid meat. J. Physiol., 38, 343-52. BARGER, G., and G. S. WALPOLE (1909, 2). Further syntheses of p-hydroxyphenylethyl- amine. J. Chem. Soc., 95, 1720-24. BERTHEAUME, J. (igio, i). Sur une nouvelle methode de dosage des trois methylamines et de V ammoniaque melangees. J. Pharm. Chim., [vii.], 2, 259-64. BERTHEAUME, J. (1910, 2). Sur le dosage des methylamines dans une grande masse d' ammoniaque. J. Pharm. Chim., [vii.], 2, 302-6; also Compt. rend., 150, 1063. BERTHELOT, A., et D. M. BERTRAND (1912, i). Recherches sur la Jlore intestinale. Isole- ment d'un microbe capable de produire de la fi-imidazolcthylamine anx depcns de rhistidine. Compt. rend., 154, 1643-45. BERTHELOT, A., et D. M. BERTRAND (1912, 2). Sur quelques proprieth biochimiques du Bacillus aminophilus intestinalis. Compt. rend., 154, 1826-8. BERTHELOT, A., et D. M. BERTRAND (1912, 3). Contribution d Vetude de la toxicite de la $-imidazolethylamine. Compt. rend., 155, 360-2. BERTHELOT, A., et. D. M. BERTRAND (1913, i). Recherches sur laflore intestinale. Sur la production possible de ptomaines en milieu acide. Compt. rend., 156, 1027-30. BERTHELOT, A., et D. M. BERTRAND (1913, 2). Recherches sur la Jlore intestinale. Sur I'action pathogene d'une association microbienne : Proteus vulgaris et Bacillus amino- philus intestinalis. Compt. rend., 156, 1567-70. BERTRAM, J., und H. WALBAUM (1894). Ueber das ResedawurzelBl. J. prakt. Chim., 50, 555-5 61 - BIBLIOGRAPHY OF CHAPTER I 169 BERTRAND, D. M., and A. BERTHELOT (1913). Ptomaine-producing bacteria in the hitman intestinal flora. Lancet, 84, 523-24. BICKEL, A., und M. PAWLOW (1912). Untersuchungen zur pharmakologischen Wirkung des p-Oxyphenylathylamins. Biochem. Zeitschr., 47, 345-54. BIEDL, A., und R. KRAUS (1912). Die Kriterien der anaphylaktischen Vergiftung. Zeitschr. f. Immunitatsforsch. exp. Therap. (Originale), 15, 447-474. BIENSTOCK (1899). Untersuchungen uber die Aetiologie der Eiweissfaulnis. Archiv f. Hygiene, 36, 355-8g. BIENSTOCK (1901). Untersuchungen uber die Aetiologie der Eiweissfaulnis. II. Milch- fdulnis, Verhinderung der Faulnis der Milch, Darmfaulnis. Archiv f. Hygiene, 39, 390-427. BISSEGGER, W., und L. STEGMANN (1908). Zur Kenntniss der bei der Verdauung des Caseins auftretenden Produkte. I. Mitteilung. Zeitschr. physiol. Chem., 58, 147-52. BOCKLISCH, O. (1885). Ueber Fdulnisbasen (Ptomaine) aus Fischen. II. Ber. d. deutsch. chem. Gesellsch., 18, 1922-27. BRADFORD, J. R., and H. P. DEAN (1894). The pulmonary circulation. J. Physiol., 16, 34-96 (p. 85). BRAUN, J. VON (1904). Ueber eine neue Methode zur Aufspaltung cyclischer Amine. Ber. deutsch. chem. Gesellsch., 37, 2915-22. BRAUN, J. VON, und H. DEUTSCH (1912). Synthesen in der fett-aromatischen Reihe (VII Phenol Basen). Ber. deutsch. chem. Gesellsch. , 45, 2504-2522. BRESLER (1900). Der deutsche Zucker, 42 and 43. (Quoted by Bertheaume.) BRIEGER, L. (1882). Zur Kenntniss der Faulnisalkaloide. Zeitschr. physiol. Chem., 7, 274-81. BRIEGER, L. (1883). Zur Kenntniss der Faulnisalkaloide. Ber. d. deutsch. chem. Gesellsch., 16, 1186-91; 1884, 17, 515-17, 1137-39. BRIEGER, L. (1884). Ueber basische Produkte (Ptomaine) aus menschlichen Leichen. Ber. d. deutsch. chem. Gesellsch., 17, 2741-42. BRIEGER, L. (1885, i). Ueber Ptomaine. Berlin, August Hirschwald. BRIEGER, L. (1885, 2). Weitere Untersuchungen uber Ptomaine. Berlin, August Hirschwald. BRIEGER, L. (1886, i). Untersuchungen uber Ptomaine. Dritter Theil. Berlin, August Hirschwald. BRIEGER, L. (1886, 2). Die Quelle des Trimethylamins im Mutterkorn. Zeitschr. physiol. Chem., II, 184-85. BUDAI (BAUER), KOLOMAN (1913). Methode zur quantitativen Bestimmung des Ammoniaks und Trimethylamins. Zeitschr. physiol. Chem., 86, 107-121. BURMANN, J. (1912). Sur un nouveau principe actif de V ergot de seigle. Schweiz. Wochenschr. f. Chem. u. Pharm., 50, 85-89. CAMMIDGE, P. J., and A. E. GARROD (1900). On the excretion of di amines in cystinuria. J. Path. Bact, 6, 327-33- CAMUS, L. (1906). L'hordenine, son degre de toxicite, symptomes de V intoxication. Compt. rend., 142, 110-13 ; see also Arch, intern, de Pharm., et de The"rap., 1906, 16, 43. CLARK, A. (1910). The clinical application of ergot amine (tyramine). Biochem. J., 5, 236-42. CRAWFORD, A. C. (1911). The pressor action of an American mistletoe. J. Amer. Med. Assoc., 57, 865-68. CZAPEK, F. (1903). Der Stickstoffwechsel der Pflanze. Ergebnisse der Physiol. (Bioch.), 2, 639-72. DALE, H. H., and W. E. DIXON (1909). The action of pressor amines produced by putrefaction. J. Physiol., 39, 25-44. DALE, H. H., and P. P. LAIDLAW (1910). The physiological action of&-iminazolylethylamine. J. Physiol., 41, 318-44. i;o THE SIMPLER NATURAL BASES DALE, H. H., and P. P. LAIDLAW (1911). Further observations on the action of &-imind- zolylethylamine. J. Physiol., 43, 182-95. DELEPINE, M. (1896). Stir une nonvelle methodc de separation des methylamincs. Compt. rend., 122, 1064-66. DESSAIGNES (1856). Trimethylamine obtenue de Vurine humaine. Compt. rend., 43, 670-71. DIXON, W. E., and F. E. TAYLOR (1907). Physiological action of the placenta. Brit. Med. J., ii., 1156. DOREE, C., and F. GOLLA (1910). Trimethylamine a normal constituent of human blood, urine, and cerebrospinal fluid. Biochem. J., 5, 306-23. EHRENBERG, A. (1887). Ueber einige in einem Falle von sogenannter " Wurstvergiftung " aus dem schadlichen Material dargestellte Faulnisbasen. Zeitschr. physiol. Chem., n, 239-56. EHRLICH, F. (1912). Synthese des Tyrosols und seine Umwandlung in Hordenin. Ber. deutsch. chetn. Gesellsch., 45, 2428-37. EHRLICH, F., und P. PISTSCHIMUKA (1912). Ueberfuhrung von Aminen in Alkohole durch Hefe- und Schimmelpilze. Ber. deutsch. chem. Gesellsch., 45, 1006-12. ELLINGER, A. (1900). Die Constitution des Ornithins und des Lysins. Zugleich ein Beitrag zur Chemie der Eiweissfaulnis. Zeitschr. physiol. Chem., 29, 334-48. EMERSON, R. L. (1901). Ueber das Auftreten von Oxyphenylathylamin bei Pankreas- verdauung und iieber fermentative CO^-Abspaltung. Baitr. chem. Physiol. Pathol., I, 501-6. EMMERLING, O. (1896). Beitrag zur Kenntniss der Eiweissfaulnis. Ber. deutsch. chem. Gesellsch., 29, 2721-26. EMMERLING, O. (1897). Die Zersetzung von Fibrin durch Streptococcen. Ber. deutsch. chem. Gesellsch., 30, 1863-68. ENGEL, H. (1912). Chemotherapeutische Versuche mit Adrenalin und dhnlich konsti- tuierten Stoffen bei tumorkranken Tieren. Zeitschr. f. exp. Path. u. Therap., n, 9-39. ENGELAND, R. (1908, 3). Ueber den Nachweis organischer Basen im Harn. Zeitschr. physiol. Chem., 57, 49-65. ENGELAND, R., und F. KUTSCHER (1910, i). Ueber eine zweite wirksame Secale-base. Zentralbl. f. Physiol., 24, 479-80. ENGELAND, R., und F. KUTSCHER (1910, 2). Ueber einige Bestandteile des Extractum Secalis cornuti. Zentralbl. f. Physiol., 24, 589-91. ERDMANN, C. C. (1910). On the alleged occurrence of trimethylamine in urine. J. Biol. Chem., 8, 57-60. ERMENGEM, E. VAN (1897). Ueber einen neuen anaeroben Bacillus und seine Beziehungen zum Botulismus. Zeitschr. f. Hygiene Infektionskrankh., 26, 1-56. ERMENGEM, E. VAN (1912). Der Bacillus botulinus und der Botulismus. Kolle und Wassermann's Handbuch der pathogenen Mikro-organismen, 2e Auflage, Bd. iv. 909-938. EWINS, A. J. (1911). The synthesis of 5- ft-aminoethylindole. J. Chem. Soc., 99, 270-73. EWINS, A. J., and P. P. LAIDLAW (1910, 2). The synthesis of 3-fi-aminoethylindole and its formation from tryptophan (Preliminary note}. Proc. Chem. Soc., 26, 343. EWINS, A. J., and P. P. LAIDLAW (1910,3). The fate of parahydroxy-phenylethylamine in the organism. J. Physiol., 41, 78-87. EWINS, A. J., and P. P. LAIDLAW (1913). The fate of indolethylamine in the organism. Biochem. J., 7, 18-25. EWINS, A. J., and F. L. PYMAN (1911). Experiments on the formation 0/4 (or 5)-/3 aminoethylglyoxaline from histidine. J. Chem. Soc., 99, 339-44- BIBLIOGRAPHY OF CHAPTER I i;i FILIPPI, F. DE (1906). Das Trimetkylamin ah normales Produkt des Stoffwechsels, nebst einer Methode fur dessen Bestimmung im Harn und Kot. Zeitschr. physiol. Chem., 49, 433-56. FINDLAY, L. (1911). The systolic pressure at different points of the circulation in the child and the adult. Quart. J. Med., 4, 489-97. FLECK, H. (1896). The separation of trimethylamine from ammonia. J. Amer. Chem. Soc., 18, 670-72. FRANOIS, M. (1907, i). Sur une methode exacts de separation de Vammoniaque et de la monomethylamine. J. Pharm. Chim., [vi.], 25, 517-22. FRANCOIS, M. (1907, 2). Recherche et dosage de V ammoniaque dans la monomethylamine et les amines grasses tres volatiles. J. Pharm. Chim., [vi.], 25, 523-28 ; also Compt. rend., 1907, 144, 857-59. FRIEDBERQER, E., und A. MORESCHI (1912). Ueber Anaphylatoxin. Berl. klin. Wochenschr., 49, 741-44. FROHLICH, A., und E. P. PICK (1912). Die Folgen der Vergiftung durch Adrenalin, Histamin, Pituitrin, Pepton, sowie die anaphylaktische Vergiftung in Bezug auf das vegetative Nervensystem. Arch. exp. Path. Pharm., 71, 23-61. GAEBEL, G. O. (1906). Ueber das Hordenin. Arch. Pharm., 244, 435-4 1 - GARCIA, S. A. (1892-93, 1-4). Ueber Ptomaine welche bei der Fdulnis von Pferdejleisch und Pankreas entstehen I. -IV. Zeitschr. physiol. Chem., 17, 543-95. GARROD, A. E. (1909). Inborn errors of metabolism. London (Chapter V). GARROD, A. E., and W. H. HURTLEY (1906). Concerning cystinuria. J. Physiol., 34, 217-23. GAUTIER, A. (1896). Les toxines microbiennes et animates. Paris, pp. vii + 617. GAUTIER, A. (1882). Sur la decouverte des alcaloides derives des matures prot'eiques animates. Compt. rend., 94, 1119-24. GAUTIER, A. (1906). Sur les tyrosamines. Bull. Soc. Chim., [iii.], 35, 1195-97. GAUTIER, A., et A. F^TARD (1882). Sur le mecanisme de la fermentation putride des matures prot'eiques et sur les alcaloides qui en resultent. Compt. rend., 94, 1598-1601. GAUTIER, A., et A. ETARD (1883). Sur les produits derives de la fermentation bacterienne des albuminoides. Compt. rend., 97, 263-67. GAUTIER, A., et L. MOURGUES (1888). Sur les alcaloides de Vhuile de foie de morue. Compt. rend., 107, 110-12. GUARESCHI, J., und A. Mosso (1883, 1-2). Die Ptomaine, chemische, physiologische und gerichtlich-medicinische Untersuchungen. J. prakt. Chem., 27, 425-32 ; 28, 504-12. GUGGENHEIM, M. (1912). Zur Kenntniss der Wirkung des p-Oxyphenyldthylamins. Therapeut. Monatsh., 26, November. GULEWIT-CH, WL. (1894). Ueber Cadaverin und Cholin aus faulem Pferdejleisch. Zeitschr. physiol. Chem., 20, 287-305. HANDOVSKY, H., und E. P. PICK (1913). Untersuchungen uber die pharmakologische Beeinflussbarkeit des peripheren Gefasstonus des Frosches. Arch. exp. Path. Pharm., 71, 89-101. HARDEN, A., and H. MACLEAN (1911). On the alleged presence of an alcoholic enzyme in animal tissues and organs. J. Physiol., 42, 64-92. HARVEY, W. H. (1911). Auto-intoxication and experimental nephritis in rabbits. J. Pathol., 16, 95-105. HASEBROEK, K. (1887). Ueber das Schicksal des Lecithins im Korper und eine Beziehung zum Sumpfgas im Darmkanal. Zeitschr. physiol. Chem., 12, 148-62. 172 THE SIMPLER NATURAL BASES HEIMANN, E. (1912). Chemisch-physiologische nnd klinische Studien uber Systogen, ein synthetisches Sekale-Ersatzprdpara t. Muench. med. Wochenschr., 59, 1370-72. HENZE, M. (1913). p-Oxypheny lathy lamin, das Speicheldriisengift der Cephalopoden. Zeitschr. physiol. Chem., 87, 51. HOFFMANN, LA ROCHE & Co., BASLE (1912). Patents on the bacterial preparation of ft-iminazolylethylamine from histidiue. D.R.P. 252872, 252873, 252874, 256116. HOFMANN, A. W. (1874). Ueber das dtherische Oel von Nasturtium officinale. Ber. deutsch. chem. Gesellsch., 7, 520-23. HUSEMANN, TH. (1876). Beitrcige zur Wirkung dss Trimethylamins und der Ammoniak- salze. Arch. exp. Path. Pharm., 6, 55-77. JEANNERET, J. (1877). Untersuchungen uber die Zersetzung von Gelatin und Eiweiss durch die geformten Pankreasfermente bei Luftausschluss. J. prakt. Chem., 15, 353-89. KIESEL, A. (igi i). Ueber den ferment ativ en Abbau des Arginins in den Pflanzen. Zeitschr. physiol. Chem., 75, 169-96. KIKKOJI, J. (1909). Beitrage zur Kenntniss der Autolyse. Zeitschr. physiol. Chem., 63, 109-35. KINOSHITA, J. (1910, i). Ueber das Auftreten und die quantitative Bestimmung des Trimethylamins im menschlichen Harn. Zentralbl. f. Physiol., 24, 776-79. KOSSEL, A. (1910, i). Uber das Agmatin. Zeitschr. physiol. Chem., 66, 257-61. KOSSEL, A. (1910, 2). Synthese des Agmatins. Zeitschr. physiol. Chem., 68, 170-72. KUTSCHER, F. (igio, i). Die physiologische Wirkung einer Secale-base und des Imidazoly lathy lamins. Zentralbl. f. Physiol., 24, 163-65. KUTSCHER, F., und A. LOHMANN (1905). Die Endprodukte der Pankreasselbstverdauung. Zeitschr. physiol. Chem., 44, 381-87. LADENBURG, A. (1886). Ueber die Identitat des Cadaverins mit dem Pentamethylendiamin. Ber. dautsch. chem. Gesellsch., 19, 2585-86. LAIDLAW. P. P. (1911). The physiological action of indolethylamine. Biochem. J., 6, 141-50. LANGSTEIN, L. (1901, 1902). Zur Kenntniss der Endprodukte der peptischen Verdauung I. Beitr. chem. Physiol. Path., I, 507-23 ; //. ibid., 1902, 2, 229-37. LAWROW, D. (1901). Zur Kenntniss des Chemismus der peptischen und tryptischen Ver- dauung der Eiweisskorper. Zeitsch. physiol. Chem., 33, 312-28. LEGER, E. (1906, i). Sur Vhordenine : alcalo'ide nouveau retire des germes, dits touraillons, de Vorge. Compt. rend., 142, 108-10. LEGER, E. (1906, 2, 3 ; 1907). Sur la constitution de Vhordenine. Compt. rend., 143, 234-36, 916-18 ; 144, 488-91. LEPRINCE, M. (1907). Contribution a Vetude chimique du Gui (Viscum album}. Compt. rend., 145, 940-41. LOEWY, A., und C. NEUBERG (1904). Zur Kenntniss der Diamine. Zeitschr. physiol. Chem., 43, 355-57. MELLANBY, E. (1911). A short chemical study of a case of cyclic vomiting, with some remarks on creatinuria and acidosis. Lancet, 89, ii., 8-12. MELLANBY, E., and F. W. TWORT (1912). On the presence of &-imidazolylcthylamine in the intestinal wall ; with a method of isolating a bacillus from the alimentary canal which converts histidine into this substance. J. Physiol., 45, 53-6o. MODRAKOWSKI, G. (1912). Ueber die Grunderscheinungen des anaphylaktischen Shoks. Arch. exp. Path. Pharm., 69, 67-68. ,-M6"RNER, C. TH. (1896). Ueber ein eigenthiimliches Nahrungsmittel, nebst einigen Beobachtungen uber darin angetroffene Fdulnisbasen. Zeitschr. physiol. Chem., 22, 514-21. BIBLIOGRAPHY OF CHAPTER I 173 MULLER, A. (1857). Ueber die Faulnisprodukte der Hefe. J. prakt. Chem., 70, 65-69. NENCKI, M. (1876). Ueber die Zersetzung des Gelatins und des Eiweisses bei der Fattlnis tnit Pankreas. Festschrift zum 4O-jahrigen Jubilaum des Professors Valentin. Bern. (Quoted by Nencki, 1882.) NENCKI, M. (1882). Zur Geschichte der basischen Faulnissprodukte. J. prakt. Chem., 26, 47-52. NENCKI, M. (1889). Untersuchungen uber die Zersetzung des Eiweisses durch anaerobe Spaltpilze. Monatsh., 10, 506-25. NEUBAUER, O. (1911). Abbau der Aminosduren durch Fdulnisbakterien und andere Pilze. Abderhalden's Biochemisches Handlexikon, IV Band. Berlin. NEUBERG, C. (1905). Zur Kenntniss der Diamine II Mitteilung. Eine neue Synthese der Diatnine. Zeitschr. physiol. Chem., 45, 110-20. NEUBERG, C. (1906). Ueber die Entstehung optisch-aktiver Fettsduren in der Natur. Biochem. Zeitschr., I, 368-73. NEUBERG, C. (1911, l). Biochemische Umwandlung von a-Pyrrolidincarbonsdure in n-Valeriansdure und S-Aminovaleriansdure. Biochem. Zeitschr., 37, 490-500. NEUBERG, C., und E. ASCHER (1907). Notiz uber das Desaminocystin und Aminodthan- disulfid. Biochem. Zeitschr., 5, 451-55. NEUBERG, C., und L. KARCZAG (1909). Verhalten von d. 1. a-Aminoisovaleriansdure (d. 1. Valin) bei der Fdulnis. Biochem. Zeitschr. 18, 435-39. NEUBERG, C., und E. ROSENBERG (1907). Ueber die bei der Eiweissfdulnis auftretenden Fettsduren, sowie ueber die optisch-aktive Valeriansdure und Capronsaure. Biochem. Zeitschr., 7, 178. OECHSNER DE CONINCK (1886). Sur les produits de la fermentation bacterienne des poulpes marins. Association fran9. pour 1'avanc. d. Sciences. C.R. xvieme session, Nancy, ie partie, pp. 112-13. OECHSNER DE CONINCK (1888, 1889, 1890, 1895). Contribution d V etude des ptomaines. Compt. rend., 106, 858-61, 1604-5; 108, 58-59, 809-10; no, 1339-41; 117, 1097-98. OECHSNER DE CONINCK (1891). Sur les ptomaines. Compt. rend., 112, 584-85. OEHME, C. (1913). Ueber die Wirkungsweise des Histamins. Arch. exp. Path. Pharm., 72, 76-96. OHTA, K. (1910). Ueber das Verhalten des Organfettes bei der Autolyse und antiseptischem Aufbewahren. Biochem. Zeitschr., 29, 1-12. ORNSTEIN, O. (1913). Bin Fall von Botulismus. Zeitschr. f. Chemotherapie (Originale), I. OTORI, J. (1904, 3). Die Pikrolonate einiger physiologisch wichtiger Verbindungen. Zeitschr. physiol. Chem., 43, 305-15. PFEIFFER, H. (1911). Richtigstellung der " Bemerkungen " von A. Biedl und R. Kraus zu meiner in Bd. 10, No. 5-6, dieser Zeitschrift erschienenen Arbeit " Ueber Eiweisszer- fallstoxikosen ". Zeitschr. f. Immunitatsforsch. u. exp. Therap. (Originale), n, 133-42. PICTET, A., und G. COURT (1907). Ueber einige neue Pflanzenalkaloide. Ber. deutsch. chem. Gesellsch., 40, 3771-83. POPIELSKI, L. (1910, 2). Erscheinungen bei direkter Einfiihrung von chemischen Korpern in die Blutbahn. Zentralbl. f. Physiol., 24, 1102-4. PYMAN, F. L. (1911). A new synthesis of 4 (or 5)-0-Aminoethylglyoxaline, one of the active principles of ergot. J. Chem. Soc., 99, 668-82. RETTGER, L. F. (1906). Studies on putrefaction. J. Biol. Chem., 2, 71-86. 174 THE SIMPLER NATURAL BASES RETTGER, L. F. (1907). Further studies on putrefaction. J. Biol. Chem., 4, 45-55- RETTGER, L. F., and C. R. NEWELL (1912). Putrefaction with special reference to the Proteus group. J. Biol. Chem., 13, 341-46. REUTER, C. (1912). Beitrage zur Kenntniss dcr stickstoffhaltigen Bestandteile der Pilze. Zeitschr. physiol. Chem., 78, 167-245. RIELANDER (1908). Einige neue Bestandteile des Extraktum Secalis cornuti. Sitzungber. Gesellsch. Naturw. Marburg., 5 Aug., No. 7. Roos, E. (1892). Ueber das Vorkommen von Diaminen bei Krankheiten. Zeitschr. physiol. Chem., 16, 192-200. ROSENHEIM, O. (1909). The pressor principles of placental extracts. J. Physiol., 38, 337-42. ROSENMUND K. W. (1909). Ueber p-Oxyphenyldthylamin. Ber. deutsch. chem. Gesellsch., 42, 4778-83. ROSENMUND, K. W. (1910). Die Synthese des Hordenins, eines Alkaloids aus Gerstcnkei- men, und iiber (a)-p-Oxyphenyldthylamin. Ber. deutsch. chem. Gesellsch., 43, 306-13. ROTHMANN, A. (1908). Ueber das Verhalten des Kreatins bei der Autolyse. Ill Mitteilung. Zeitschr. physiol. Chem., 57, 131-42. SALKOWSKI, E. (1909). Bemerkungen uber Autolyse und Konservierung. Zeitschr. physiol. Chem., 63, 136-42. SCHENCK, M. (1905, i). Ueber Selbstverdauung einiger Hefearten (obergdrige Hefe, Brennereihefe, Kahmhefe). Wochenschr. f. Brauerei, 22, 221-27. SCHITTENHELM, A., und W. WsiCHARDT (1912). Ueber die Rolle der Ueberempfindlichkeit bei der Infektion und Immunitdt III. Muench. med. Wochenschr., 59, 67-69. SCHMITT, R., und O. NASSE (1865). Beitrag zur Kenntniss des Tyrosins. Liebig's Annalen, 133, 211-16. SCHULZE, E. (1906). Neue Beitrage zur Kenntniss der Zusammensetzung und det Stoff- wechsels der Keimpflanzen. Zeitschr. physiol. Chem., 47, 507-69. SCHUMM, O. (1905-6). Beitrage zur Kenntniss der Autolyse. Beitr. z. chem. Physiol. u. Path., 7, 175-203. SPIRO, K. (1901). Die aromatische Gruppe des Leims. Beitr. z. chem. Physiol. u. Path., I, 347-50. SUGIMOTO, T. (1913). Pharmakologische Untersuchungen am uberlebenden Meerschwein- chenuterus. Arch. exp. Path. Pharm., 74, 27-40. TAKEDA, K. (1909). Der Nachweis von Trimethylamin im Harn. Pfluger's Archiv, 129, 82-88. TORQUATI, TORQUATO (1910). The formation of hordenine during the germination of barley. J. Chem. Soc., 1911, 100, Abstr. i., 523. THOMS, H., und F. THDMEN (1911). Ueber das Fagaramid t einen neuen stickstoffhaltigen Stoff aus der Wurzelrinde von Fagara xanthoxyloides Lam. Ber. deutsch. chem. Gesellsch., 44, 3717-30. TRENDELENBURG, P. (1912). Physiologische und pharmakologische Untersuchungen an den isolierten Bronchialmuskulatur. Arch. exp. Path. Pharm., 69, 79. TRIER, G. (1912, 3). Ueber einfache Pflanzenbasen und ihre Beziehungen zum Aufbau der Eiweissstoffe und Lecithine. Berlin, pp. iv + 117. UDRANSZKY, L. VON, und E. BAUMANN (1888, i). Das Benzoylchlorid als Reagenz. Ber. deutsch. chem. Gesellsch., 21, 2744-51. UDRANSZKY, L. VON, und E. BAUMANN (1888, 2). Ueber die Identitdt des Putrescins und des Tetramethylendiamins. Ber. deutsch. chem. Gesellsch., 21, 2938-41. UDRANSZKY, L. VON, und E. BAUMANN (1889). Ueber das Vorkommen von Diaminen, sogenannten Ptoma'inen bei der Cystinurie. Zeitschr. physiol. Chem., 13, 562-94. BIBLIOGRAPHY OF CHAPTER I 175 UDRANSZKY, L. VON, und E. BAUMANN (1890). Weitere Beitrage zttr Kenntniss der Cystinurie. Zeitschr. physiol. Chem., 15, 77-92. SLYKE, L. L. VAN, and E. B. HART (1903). The relation of carbon dioxide to proteolysis in the ripening of Cheddar cheese. Amer. Chem. J., 30, 8-24. VOSWINCKEL, H. (1912). Ueber einc neue Synthese des Hordenins. Ber. deutsch. chem. Gesellsch., 45, 1004-6. (See also D.R.P. 248385.) WERIGO, B. (1892). Ueber das Vorkommen des Pentamethylendiamins in Pankreasinfusen. Pfluger's Archiv, 51, 362-66. WILLSTATTER, R., und W. HEUBNER (1907). Ueber eine neue Solanaceenbase. Ber. deutsch. chem. Gesellsch., 40, 3869-75. WINDAUS, A., und H. OPITZ (1911). Synthese einiger Imidazolderivate. Ber. deutsch. chem. Gesellsch., 44, 1721-25. WINDAUS, A., und W. VOGT (1907). Synthese des Imidazoly lathy lamins. Ber. deutsch. chem. Gesellsch., 40, 3691-95. WINTERSTEIN, E., und A. KtfNG (1909). Ueber das Auftreten von p-Oxypheny lathy lamin im Emmenthaler Kdse. Zeitschr. physiol. Chem., 59, 138-40. WINTERSTEIN, E., und J. THO"NY (1902). Beitrage zur Kenntniss der BestandteiU des Emmenthaler Kdses. Zeitschr. physiol. Chem., 36, 28-38. WOHL, A., und E. BERTHOLD (1910). Ueber die Darstellung der aromatischen Alkohole und ihrer Acetate. Ber. deutsch. chem. Gesellsch., 43, 2175-85. YOSHIMURA, K. (1909). The chemical composition of Tamari-Shoyu. J. Coll. Agric. Tokyo, I, 89-96. YOSHIMURA, K. (1910). Ueber Faulnissbasen (Ptomaine) aus gefaulten Soyabohnen (Glycine hispida). Biochem. Zeitschr., 18, 16-22. REFERENCES TO CHAPTER II. w-AMINO-ACIDS. ABDERHALDEN, E., und K. KAUTZSCH (1912). Fdulnisversuche mit d. Glutaminsdure und Studien uber die y-Aminobuttersdure. Zeitschr. physiol. Chem., 8l, 294-314. ABDERHALDEN, E., und A. FODOR (1913). Versuche uber die bei der Fdulnis von l-Aspara- ginsdure entstehenden Abbaustufen. Eine neue methode zum Nachweis von ft-Alanin. Zeitschr. physiol. Chem., 85, 112-30. ABDERHALDEN, E., G. FROMME, und P. HIRSCH (1913). Die Bilduug von y-Aminobutter- saure aus d. Glutaminsdure unter den Einfluss von Mikroorganismen. Zeitschr. physiol. Chem., 85, 131-35. ABDERHALDEN, E., und A. SCHITTENHELM (1907). Studien uber den Abbau raccmischer Aminosduren im Organismus des Hundes unter verschiedenen Bedingungen. Zeitschr. physiol. Chem., 51, 323-33. ACKERMANN, D. (1907, 2). Ein Beitrag zur Chemie der Fdulnis. Zeitschr. physiol. Chem., 54, 1-31. ACKERMANN, D. (igio, i). Ueber den bakteriellen Abbau des Histidins. Zeitschr. physiol. Chem., 64, 504-510. ACKERMANN, D. (1910, 3). Uebcr ein neues, auf baktericllem Wegegewinnbares Aporrhegma. Zeitschr. physiol. Chem., 69, 273-81. ACKERMANN, D. (1911, 2). Die Sprengung des Pyrrolidinringes durch Bakterien. Zeitschr. Biol., 57, 104-11. ACKERMANN, D. (1911, l). Ueber das &-Alanin als bakterielles Aporrhegma. Zeitschr. Biol., 56, 87-90. ENGELAND, R. (1908, i). Ueber Liebig's Fleischextract. Zeitschr. Unters. Nahr. Genussm., 16, 658-64. FISCHER, E., und G. ZEMPLEN (1909). Neue Synthese von Amino-oxysduren und von Piperidinderivaten. Ber. deutsch. chem. Gesellsch., 42, 4878-92. GABRIEL, S., und W. ASCHAN (1891). Ueber die Natur eines Productes der Eiweissfdulniss. Ber. deutsch. chem. Gesellsch., 24, 1364-66. HOLM, F. H. (1904). Ueber das &-Alanin. Arch. Pharm., 242, 590-612. KNOOP, F., und A. WINDAUS (1906). Die Konstitution des Histidins. Beitr. chem. Physiol. Pathol., 7, 144-47. LEY, H. (1909). Beitrdge zur Theorie der inneren Komplexsalze. Ber. deutsch. chem. Gesellsch., 42, 3S4-7 6 - MICRO, K. (1905). Hydrolyse des Fleischextraktes. Zeitschr. Unters. Nahr. Genussm., 10, 393-415. NEUBERG, C. (1911, i). Biochemische Umwandlung von a-Pyrrolidincarbonsdure in n-Valeriansdure und b-Aminovaleriansdure. Biochem. Zeitschr., 37, 490-500. NEUBERG, C. (1911, 2). Wird d. Ornithin bei der Fdulnis racemisiert ? Biochem. Zeitschr., 37, 507-9. SALKOWSKI, E. und H. (1883). Ueber basische Fdulnisprodukte. Ber. deutsch. chem. Gesellsch., 16, 1191-95. SALKOWSKI, H. (1898). Ueber Aminovaleriansdure. Ber. deutsch. chem. Gesellsch., 31, 77 6 ' 8 3- SCHOTTEN, C. (1884). Ueber die Oxydation des Piperidins. Ber, deutsch, chem. Gesellsch., 17, 2544-47. 176 BIBLIOGRAPHY OF CHAPTER II 177 CARNOSINE. GULEWITSCH, WL., und S. AMIRADZ!BI (1900, i). Zur Kenntniss der Extraktivstoffe der Muskeln. Zeitschr. physiol. Chem., 30, 565-73. GULEWITSCH, WL., und S. AMIRADZIBI (1900, 2). Ueber das Carnosin, eine neue organische Base des Fleischextraktes. Ber. deutsch. chem. Gesellsch., 33, 1902-3. GULEWITSCH, WL. (1906). Zur Kenntniss der Extraktivstoffe der Muskeln. VI. Ueber die Identitdt des Ignotins mit dem Carnosin. Zeitschr. physiol. Chem., 50, 204-8. GULEWITSCH, WL. (1907). Zur Kenntniss der Extraktivstoffe der Muskeln. VIII. Ueber die Bildung des Histidins bei der Spaltung von Carnosin. Zeitschr. physiol. Chem., 50, 535-37. GULEWITSCH, WL. (1911). Zur Kenntnis der Extraktivstoffe der Muskeln. XII. Ueber die Konstitution des Carnosins. Zeitschr. physiol. Chem., 73, 434-46. GULEWITSCH, WL. (1913). Zur Kenntniss der Extraktivstoffe der Muskeln. XIV. Ueber das Carnosin und Carnosin Nitrat. Zeitschr. physiol. Chem., 87, i-n. KRIMBERG, R. (1906, i). Zur Kenntniss der Extraktivstoffe der Muskeln. IV. Uber das Vorkommen des Carnosins, Carnitins und Methylguanidins int Fleisch. Zeitschr. physiol. Chem., 48, 412-18. KUTSCHER, F. (1905). Ueber Liebig's Fleischextract. Zeitschr. Unters. Nahr. Genussm., 10, 528-37. MAUTHNER, M. (1913). Uber den Karno singe halt von Saugetiermuskeln. Monatsh., 34, 883-900. SMORODINZEW, Z., (1913). Zur Kenntniss der Extraktivstoffe der Muskeln. XV. Uber das Vorkommen des Carnosins, Methylguanidins und Carnitins im Pferdefleisch. Zeitschr. physiol. Chem., 87, 12-20. UROCANIC ACID. BARGER, G., and A. J. EWINS (1911). The constitution of Ergothioneine, a betaine related to histidine. J. Chem. Soc., 99, 2336-41. ENGELAND, R. (1908, 3). Ueber den Nachweis organischer Basen im Harn. Zeitschr. physiol. Chem., 57, 49-65. HUNTER, A. (1912). On urocanic acid. ]. Biol. Chem., u, 537-45. JAFFE, M. (1874). Ueber einen neuen Bestandtheil des Hundeharns. Ber. deutsch. chem. Gesellsch., 7, 1669-73. JAFFIJ, M. (1875). Ueber die Urocaninsdure. Ber. deutsch. chem. Gesellsch., 8, 811-13. SIEGFRIED, M. (1898). Ueber Urocaninsdure. Zeitschr. physiol. Chem., 24, 399-409. KYNURENIC ACID. ABDERHALDEN, E., E. S. LONDON, und L. PINCUSSOHN (1909). Ueber den Ort der Kynurensdurebildung im Organismus. Zeitschr. physiol. Chem., 62, 139-41. BRIEGER, L. (1879). Zur Kenntniss der Kynurensdure. Zeitschr. physiol. Chem., 4, 89-92. CAMPS, R. (1901, i). Ueber Liebig's Kynurensdure und das Kynurin. Constitution und Synthese beider. Zeitschr. physiol. Chem., 33, 390-411. CAMPS, R. (1901, 2). Von der Amido-phenylpropionsdure zur Kynurensdure und deren Verwandten. Ber. deutsch. chem. Gesellsch., 34, 2703-18. CAPALDI, A. (1897, i). Zur Kenntniss der Kynurensdure. Zeitschr. physiol. Chem., 23, 87-91. CAPALDI, A. (1897, 2). Bin Verfahren zur quantitativer Bestimmung der Kynurens&ure, Zeitschr. physiol. Chem., 23, 92-98. 12 1 78 THE SIMPLER NATURAL BASES ELLINGER, A. (1904, i). Ueber die Constitution der Indolgruppe im Eiweiss (Synthese der sogen. Skatolcarbonsaure) und die Quelle der Kynurensaure. Ber. deutsch. chem. Gesellsch., 37, 1801-8. ELLINGER, A. (1904, 2). Die Entstehung der Kynurensaure. Zeitschr. physiol. Chem., 43, 325-37. GLAESSNER, K., und L. LANGSTEIN (1902). Zur Kenntnis der Entstehung der Kynuren- saure im Organismus. Beitr. chem. Physiol. Path., I, 34-43. HAUSER, A. (1895). Untersuchungen ueber die Kynurensaurebildung im Organismus. Arch. exp. Pathol. Pharm., 36, 1-7. HOMER, A., (1913). A note on the constitution of kynurenic acid. J. Physiol., 46, xviii.-xix. and Ixii. (Proc. Physiol. Soc.). JAFFE, M. (1883). Eine empfindliche Reaktion auf Kynurensaure. Zeitschr. physiol. Chem., 7, 399-402. KRETSCHY, M. (1881). Untersuchungen ueber Kynurensaure. Monatsh., 2, 57-85. KRETSCHY, M. (1883). Ueber die Oxydation von Kynurin und von Kynurensaure. Monatsh., 4, 156-61. KRETSCHY, M. (1884). Untersuchungen ueber Kynurensaure. II. Oxydation der Kynuren- saure. Monatsh., 5, 16-32. LIEBIG, J. (1853). Ueber Kynurensaure. Liebig's Annalen, 86, 125-26. SCHMIEDEBERG, O., und O. SCHULTZEN (1872). Untersuchungen ueber die Kynurensaure und das Zersetzungsprodiict, das Kynurin. Liebig's Annalen, 164, 155-59. SOLONIN, P. (1897). Z ur Kenntniss der Kynurensaure. Zeitschr. physiol. Chem., 23, 497-504. WENZEL, F. (1894). Synthese des Kynurins. Monatsh., 15, 453-68. REFERENCES TO CHAPTER III. BETAINE (TRIMETHYLGLYCINE). ACKERMANN, D., und F. KUTSCHER (1907, 1-4). Ueber Krabbenextrakt I.-IV. Zeitschr. Unters. Nahr. Genussm., 13, 180-84, 610-13, 613-14; 14, 687-91. ANDRLIK, K. (1903-4). Gewinnung von Retain aus den Abfallaugen von der Melasse- entzuckerung mitt els Strontian. Zeitschr. Zuckerindustr. Bdhmen, 28, 404-6. ANDRLIK, K., A. VELICH, und VL. STANEK (1902-3). Ueber Betain in physiologisch- chemischer Beziehung. Zeitschr. Zuckerindustr. Bohmen, 27, 161-80. BEBESCHIN, K. (1911). Zur Kenntniss der Extraktivstoffe der Ochsennieren. Zeitschr. physiol. Chem., 72, 380-86. DELEANO, N. T., und G. TRIER (1912). Ueber das Vorkommen von Betain in griinen Tabakblattern. Zeitschr. physiol. Chem., 79, 243-46. EHRLICH, F. (1912). Uber die Gewinnung von Betainhydrochlorid aus Melasse. Schlempe. Ber. deutsch. chem. Gesellsch., 45, 2409-13. EHRLICH, F., und F. LANGE (1913). Uber die biochemische Umwandlung vcn Betain in Glykolsaure. Ber. deutsch. chem. Gesellsch., 46, 2746-52. EWINS, A. J. (1912). The constitution and synthesis of damascenine, the alkaloid of Nigella damascena. J. Chem. Soc., 101, 544-52. FISCHER, E. (1902). Ueber Betainchloraurat, Ber. deutsch. chem. Gesellsch., 35, 1593-95. GRIESS, P. (1875). Ueber eine neue Synthese des Betains (Oxyneurin). Ber. deutsch. chem. Gesellsch., 8, 1406-7. HENZE, M. (1910). Ueber das Vorkommen des Betains bei Cephalopoden. Zeitschr. physiol. Chem., 70, 253-55. HUSEMANN, A. (1875). Identitdt der PJlanzenbasen Lycin und Betain. Arch. Pharm., 206, 216-19. HUSEMANN, A., und W. MARM (1863). Vorlaufege Mittheilung uber Lycin, ein neues Alkaloid in Lycium barbarum L. (gemeiner Teufelszwirti). Liebig's Annalen, II Supplementsb., 383-87. HUSEMANN, A., und W. MARM (1864). Ueber Lycin. Liebig's Annalen, III Supplementsb., 245-49. KOHLRAUSCH, A. (1909). Ueber das Verhalten von Betain, Methylpyridyl-am- moniumhydroxydund Trigonellin im tierischen Organismus. Zentralbl. f. Physiol., 23, 143-47. KOHLRAUSCH, A. (1911). Untersuchungen uber das Verhalten von Betain, Trigonellin utid Methylpyridylammoniumhydroxyd im tierischen Organismus. Zeitschr. f. Biol., 57, 273-308. KUTSCHER, FR. (1909). Notiz zu der Arbeit der Herren U. Suzuki und K. Joshimura: Ueber die Extraktivstoffe des Fischfleisches. Zeitschr. physiol. Chem., 63, 104-5. KUTSCHER, FR. (1910, 2). Die Extraktivstoffe einiger Seetiere. Sitzungsber. Ges. Naturw. Marburg, p. 47 (quoted by Kohlrausch). KUTSCHER, FR. (1910, 3). Ueber einige Extraktivstoffe. Sitzungsber. Ges. Naturw. Marburg, p. 93 (quoted by Kohlrausch). LIEBREICH, O. (1869, 2). Ueber das Oxyneurin. Ber. deutsch. chem. Gesellsch., 2, 167-68. 179 12 * i8o THE SIMPLER NATURAL BASES LIEBREICH, O. (1870). Ueber die Identitdt des Oxyneurin mit dem Betain. Ber. deutsch. chem. Gesellsch., 3, 161-63. RITTHAUSEN, H., und F. WEGER (1884). Ueber Betain aus Pressriickstanden der Baumwollensamen. J. prakt. Chem., [ii.j, 30, 32-37- SCHEIBLER, C. (1866). Ueber ein im Rubensafte vorkommendes leichtlosliches Alkaloid. Jahresber. Untersuch. Fortschr. Zuckerfabr., 6, 172. SCHEIBLER, C. (1869). Ueber das Betain, eine im Safte der Zuckerruben (Beta vnlgaris) vorkommende Pflanzenbase. Ber. deutsch. chem. Gesellsch., 2, 292-95. SCHEIBLER, C. (1870). Ueber das Betain und seine Constitution. Ber. deutsch. chem. Gesellsch., 3, 155-61. SCHULZE, E. (1910). Ueber das Vorkommen -von Betain in den Knollen des Topin- amburs (Helianthus tuberosus). Zeitschr. physiol. Chem., 65, 293-94. SCHULZE, E., und N. CASTORO (1904). Beitrage zur Kenntnis der in ungekeimten Pflanzensamen enthaltenen Stickstojfverbindungen. Zeitschr. physiol. Chem., 41, 455-73. SCHULZE, E., und G. TRIER (1910, i). Ueber die in den Pflanzen vorkommenden Betaine. Zeitschr. physiol. Chem., 67, 46-58. SCHULZE, E., und G. TRIER (1912, i). Untersuchungen uber die in Pflanzen vorkom- menden Betaine. II Mitteilung. Zeitschr. physiol. Chem., 76, 258-90. SCHULZE, E., und G. TRIER (1912, 2). Untersuchungen uber die in den Pflanzen vorkommenden Betaine, III Mitteilung'. Zeitschr. physiol. Chem., 79, 235-42. STANEK, VL. (1901-1902). Verbesserte Methode zur Darstellung von Betain. Zeitschr. Zuckerind. Bbhmen, 26, 287-89. STANEK, VL. (1904). Ueber das Betainperjodid und ueber die quantitative Bestim- mung des Betains durch eine Losung von Jod in jfodkalium. Zeitschr. Zuckerind. Bbhmen, 28, 578-83. STANK, VL. (1911, i). Ueber die Lokalisation von Betain in Pflanzen. Zeitschr. physiol. Chem., 72, 402-9. STANEK, VL. (1911, 2). Ueber die Wanderung von Betain in Pflanzen bei einigen Vegetationsvorgangen . Zeitschr. physiol. Chem., 75, 262-71. STANK, VL. (1912). Uber Darstellung groszer Betainperjodidkrystallen. Zeitschr. Zuckerind. Bohmen, 36, 577. STANK, VL., und K. DOMIN (1910). Ueber das Vorkommen von Betain in den Chenopodiaceen. Zeitschr. Zuckerind. Bbhmen, 34, 297-3,04. STANK, VL., und O. MisKOVSKf (1907). Kann Betain als Stickstoffn'dhrsubstanz der Hefe betrachtet werden? Zeitschr. f. d. ges. Brauwesen, 30, 567-68. STOLTZENBERG, H. (1912). Ein neues Verfahren zur Gewinnung von Betainhydro- chlorid aus Melasseschlempe. Trennung von Glykokoll, Betain und Glutaminsiiure. Nichtvorkommen von Betain unter den Spaltprodukten einiger Eiweisskorper. Ber. deutsch. chem. Gesellsch., 45, 2248-52 ; see also D.R.P. 243332. SUWA, A. (1909, i). Untersuchungen uber die Organextrakte der Selachier. I. Die Mus- kelextraktstoffe des Dornhais (Acanthias vnlgaris). Pflxiger's Archiv, 128, 421-426. SUZUKI, U., und K. JOSHIMURA (1909). Ueber die Extraktivstoffe des Fischfleisches. Zeitschr. physiol. Chem., 62, 1-35. TRIER, G. (1913, 5). Weitere Beitrage zur Kenntniss einfacher Pflanzenbasen. Zeitschr. physiol. Chem., 85, 372-91. URBAN, K. (1913). Uber die Darstellung von Betain aus Melasseabfallaugen. Zeitschr. Zuckerind. Bohmen, 37, 339-41. VELICH, A. (1904-1905). Bemerkungen zum Studium des Betains. Zeitschr. Zuckerind. Bbhmen, 29, 14-25. VELICH, A., und VL. STANEK (1904-1905). Ueber das Betain in fysiologisch-chemischer Beziehung. Zeitschr. Zuckerind. Bbhmen, 29, 205-19. BIBLIOGRAPHY OF CHAPTER III 181 VOLTZ, W. (1907). Unter suchung en ilber die Verwertnng des Betains durch den Wiederkaiier (Schaf). Pfluger's Archiv, 116, 307-33. WALLER, A. D., and R. H. A. PLIMMER (1903). The physiological action of betaine extracted from beet-sugar. Proc. Roy. Soc., 72, 345-52. WILLSTATTER, R. (1902, i). Ueber Betaine. Ber. deutfcch. chem. Gesellsch., 35, 584-620. WILLSTATTER, R. (1902, 2). Ueber Beta'inchloraurat. Ber. deutsch. chem. Gesellsch., 35, 2700-3. STACHYDRINE. ENGELAND, R. (1909, 2). Ueber Hydrolyse von Casein und den Nachweis der dabei entstandenen Mono-aminosauren . Ber. deutsch. chem. Gesellsch., 42, 2962-69. ENGELAND, R. (1909, 3). Die Konstitution des Stachydrins. Arch. Pharm., 247, 463-66. ENGELAND, R. (1910, i). Bemerkung zu den Arbeiten von E. Schulze und G. Trier, Ueber die in den Pflanzen vorkommenden Betaine und uber das Stachydrin. Zeitschr. physiol. Chem., 67, 403-4. JAHNS, E. (1896). Vorkomtnen von Stachydrin in den Blcittern von Citrus vulgaris. Ber. deutsch. chem. Gesellsch., 29, 2065-68. PLANTA, A. vov (1890). Ueber einige stickstoffhaltige Bestandtheile der Wurzel- knollen von Stachys tuberifera. Ber. deutsch. chem. Gesellsch., 23, 1699-1700. PLANTA, A. VON, und E. SCHULZE (1893, i). Ueber Stachydrin. Ber. deutsch. chem. Gesellsch., 26, 939-42. PLANTA, A. VON, und E. SCHULZE (1893, 2 )- Ueber die organischen Basen der Wurzel- knollen von Stachys tuberifera. Arch. Pharm., 231, 305-13. SCHULZE, E., und G. TRIER (1909, i). Ueber das Stachydrin. Zeitschr. physiol. Chem., 59, 233-35. SCHULZE, E., und G. TRIER (1909, 2). Ueber die Konstitution des Stachydrins. Ber. deutsch. chem. Gesellsch., 42, 4654-59. SCHULZK, E., und G. TRIER (1910, 2). Ueber das Stachydrin und uber einige neben ihtn in den Stachys knoll en und in den Orangenblattern enthaltene Basen. Zeitschr. physiol. Chem., 67, 59-96. TRIER, G. (1910). Ueber die Umwandlung des Stachydrins in den isomeren Hygrin- sauremethylester. Zeitschr. physiol. Chem., 67, 324-31. WILLSTXTTER, R. (1900). Synthese der Hygrinsdure. Ber. deutsch. chem. Gesellsch., 33, 1 160-66. WILLSTATTER, R., und F. ETTLINGER (1903). Synthese der Hygrinsaure und der a- Pyrrolidincarbonsdure. Liebig's Annalen, 326, 91-128. YOSHIMURA, K., und G. TRIER (1912). Weitere Beitrage uber das Vorkommen von Betainen im Pflanzenreich. Zeitschr. physiol. Chem., 77, 290-302. BETONICINE AND TURICINE. KUNG, A. (1913). Die Synthese des Betonicins und Turicins. Zeitschr. physiol. Chem., 85, 217-24. KUNG, A., und G. TRIER (1913). Ueber Betonicin und Turicin. Zeitschr. physiol. Chem., 85, 209-16. TRIMETHYLHISTIDINE AND ERGOTHIONEINE. BARGER, G., and A. J. EVVINS (1911). The constitution of ergothioneine, a betaine related to histidine. J. Chem. Soc., 99, 2336-41. 1 82 THE SIMPLER NATURAL BASES BARGER, G., and A. J. EWINS (1913). The identity of trimethylhistidine (histidine- betaine) from various sources. Biochem. J., 7, 204-6. ENGELAND, R., und F. KUTSCHER (1912, i). Versuche zur Synthese des Herzynins. Zentralbl. f. Physiol., 26, 569-70. ENGELAND, R., und FR. KUTSCHER (1912, 2). Die Methylierung von Histidin, Arginin, Lysin. Zeitschr. Biol., 59, 415-19. KUTSCHER, FR. (1910, 4). Die basiscken Extractstoffe des Champignons (Agaricus campestris). Zentralbl. f. Physiol., 24, 775-76. TANRET, CH. (1909, i). Sur une base nouvelle retiree du seigle ergote, Vergothioneine. J. Pharm. Chim., [vi.], 30, 145-53- HYPAPHORINE. GRESHOFF, M. (1898). Mededeelingen uit 's Lands Plantentuin. XXV. Batavia The Hague. ROMBURGH, P. VAN (1911). Hypaphorine en het verband dezer stof met Tryptophaan. Verslag Kon. Akad. Wetensch., Amsterdam, 1250-53. ROMBURGH, P. VAN, and G. BARGER (1911). Preparation of the betaine of tryptophan and its identity with the alkaloid hypaphorine. J. Chem. Soc., 99, 2068-71. TRIGONELLINE. ACKERMANN, D. (1912, i). Ueber das Vorkommen von Trigonellin und Nikotinursdure im Harn nach Verfutterung von Nikotinsdure. Zeitschr. f. Biol., 59, 17-22. HANTZSCH, A. (1886). Ueber Ammoniumderivate von Saureathern des Pyridins und Chinolins. Ber. deutsch. chem. Gesellsch., 19, 31-40. JAHNS, E. (1885). Ueber die Alkaloide des Bockhornssamens. Ber. deutsch. chem. Gesellsch., 18, 2518-23. JAHNS, E. (1887). Die Alkaloide des Bockshornsamens. Arch. Pharm., 225, 985-9?- SCHULZE, E., und E. WINTERSTEIN (1910). Studien uber die Proteinbildung in reifenden PJlanzensamen. Zeitschr. physiol. Chem., 65, 431-76. OTHER PYRIDINE BASES. ACHELIS, W., und FR. KUTSCHER (1907). Der Nachweis organischer Basen im Pferde- harn. Zeitschr. physiol. Chem., 52, 91-94. BERTRAND, G., et G. WEISWEILLER (1913). Sur la composition de Vessence de cafe ; pre- sence de la Pyridine. Compt. rend., 157, 212-213. His, W. (1887). Ueber das Stoffwechselproduct des Pyridins. Arch. exp. Path. Pharm., 22, 253-60. KUTSCHER, FR., und A. LOHMANN (1907). Das Vorkommen von Pyridinmethylchlorid im menschlichen Harn und seine Beziehungen zu den Genussmitteln Tabak und Kajfee. Zeitschr. Unters. Nahr. Genussm., 13, 177-79. 7-BUTYROBETAINE AND CARN1TINE. DOMBROWSKI, S. (1902). Stir la mannite, les azotates et les alcaloides des urines normales. Compt. rend., 135, 244-46. ENGELAND, R. {1908, i). Ueber Liebig's Fleischextract. Zeitschr. Unters. Nahr. Genussm., 16, 658-64. ENGELAND, R. (1909, i). Zur Kenntniss der Bestandteile des Fleischextraktes. Ber. deutsch. chem. Gesellsch., 42, 2457-62. ENGELAND, R. (1910, 2). Zur Kenntniss des Carnitins ; die Synthese der ft-Oxy-y- trimethylamino-buttersdure. Ber. deutsch. chem. Gesellsch., 43, 2705-7. BIBLIOGRAPHY OF CHAPTER III 183 ENGELAND, R. (1908, 2). Das Verhalten des Carnitins im tierischen Stoffwechsel. Zeitschr. Unters. Nahr. Genussm., 16, 664-66. ENGELAND, R., und FR. KUTSCHER (igio, 3). Ueber ein methyliertes Aporrhegma des Tierkorpers. Zeitschr. physiol. Chem., 69, 282-85. FISCHER, E., und A. GODDERTZ (1910). Synthese der y-Amino-a-oxybuttersaure und ihres Trimethy Iderivats. Ber. deutsch. chem. Gesellsch., 43, 3272-80. GULEWITSCH, WL., und R. KRIMBERG (1905). Zur Kenntniss der Extraktivstoffe der Muskeln. II. Ueber das Carnitin. Zeitschr. physiol. Chem., 45, 326-30. KRIMBERG, R. (1906, 2). Zur Kenntniss der Extraktivstoffe der Muskeln. V. Zur Frage uber die Konstitution des Carnitins. Zeitschr. physiol. Chem., 49, 89-95. KRIMBERG, R. (1907, i). Zur Kenntniss der Extraktivstoffe der Muskeln. VII. Ueber einige Verbindungen des Carnitins. Zeitschr. physiol. Chem., 50, 361-73. KRIMBERG, R. (1907, 2). Zur Kenntniss der Extraktivstoffe der Muskeln. IX. Zur Frage uber die Konstitution des Carnitins. Zeitschr. physiol. Chem., 53, 514-25. KRIMBERG, R. (1908, i). Zur Kenntniss der Extraktivstoffe der Muskeln. X. Ueber die Identitdt des Novains mit dem Carnitin. Zeitschr. physiol. Chem., 55, 466-80. KRIMBERG, R. (1908, 2). Zur Kenntniss der Extraktivstoffe der Muskeln. XI. Ueber die Beziehung des Oblitins zum Carnitin. Zeitschr. physiol. Chem., 56, 417-24. KRIMBERG, R. (1909). Bemerkung zum Aufsatz des Hrn. R. Engeland uber Bestandteile des Fleisch-extraktes. Ber. deutsch. chem. Geselisch., 42, 3878-80. KUTSCHER, FR. (1905). Ueber Liebig's Fleischextract. Zeitschr. Unters. Nahr. Genussm., 10, 528-37. KUTSCHER, FR. (1906, 2). Die Spaltung des Oblitins durch Bakterien. Zeitschr. physiol. Chem., 48, 331-33- KUTSCHER, FR. (1907, 2). Der Nachweis toxischer Basen im Harn. IV. Zeitschr. physiol. Chem., 51, 457-63. KUTSCHER, FR., und A. LOHMANN (1906, i). Die physiologische Wirkung einiger aus Rindermuskeln gewonnenen organischen Basen. Pfliiger's Archiv, 114, 553-68. KUTSCHER, FR., und A. LOHMANN (1906, 2-4). Der Nachweis toxischer Basen in Harn. Zeitschr. physiol. Chem., 48, 1-8 ; 48, 422-24; 49, 81-87. ROLLETT, A. (1910). Synthese einiger Oxybetaine. II. Mitteilung : Synthese des y- Trimethyl ft-oxybutyrobetains (inactiven Isocarnitins). Zeitschr. physiol. Chem., 69, 60-65. TAKEDA, K. (1910). Untersuchungen uber einige nach Phosphorvergiftung im Harn auftretende Basen. Pfliiger's Archiv, 133, 365-96. MYOKYNINE. ACKERMANN, D. (1912, 2). Ueber einen neuen basischen Bestandteil der Muskulatur des Hundes und seine Beziehung zum Hexamethylornithin. Zeitschr. f. Biol., 59, 433-40. ACKERMANN, D. (1913, i). Writer e Beitrdge zur Kenntniss des Myokynins. Zeitschr. f. Biol., 6l, 373-78. REFERENCES TO CHAPTER IV. CHOLINE. ABDERHALDEN, E., und FR. MULLER (1910). Die Blutdruckwirkung des reinen Cholins. Zeitschr. physiol. Chem., 65, 420-30. ABDERHALDKN, E., und FR. MULLER (ign). Weitere Beitrage uber die Wirkung des Cholins (Cholinchlorhydrat) auf den Blutdruck. Zeitschr. physiol. Chem., 74, 253-72. ALLEN, R. W. (1904). Choline, a new method of testing for its presence in the blood and cerebro-spinal fluid, Proc. Physiol. Soc., Ivi-lviii, J. Physiol., 31. ALLEN, R. W., and H. FRENCH (1903). Some observations upon the te>t for choline in human blood. Proc. Physiol. Soc., xxix-xxx, J. Physiol., 30. BABO, L. VON, und M. HIRSCHBRUNN (1852). Ueber das Sinapin. Liebig's Annalen, 82, 10-32. BASKOFF, A. (1908). Ueber das Jecorin und andere lecithinartige Produktc der Pferdcleber. Zeitschr. physiol. Chem., 57, 395-460. BOCARIUS, N. (1901). Zur Kenntniss der Substanz welche die Bildung von Florcnce- 'schen Krystallen bedingt. Zeitschr. physiol. Chem., 34, 339-46. BODE, J. (1892). Ueber einige AbkommUnge des Neurins und Cholins. Liebig's Annalen, 267, 268-99. BOEHM, R. (1885, 2). Ueber das Vorkommen und die Wirkungen des Cholins und die Wirkungen der kiinstlichen Muscarine. Arch. exp. Path. Pharm., 19, 87-100. BUSQUET, H., et V. PACHON (1909). Sur faction vaso-constrictive de la choline. Compt. rend. Soc. de Biol., 67, 218-21. CERVELLO (1886). Sur V action physiologique de la neurine. Arch. ital. de Biol., 7, 172-97. CLAUS, A., und C. KEESE (1867). Ueber Neurin und Sinkalin. J. prakt. Chem., 102, 24-27. CORIAT, I. H. (1904). The production of cholin from lecithin and brain tissue. Amer. J. Physiol., 12, 353-64. COUSIN, H. (1907). Sur la nature des produits azotes formes dans la decomposition de la cephaline. J. Pharm. Chim., [vi.], 25, 177-80. CRAMER, W. (1904). On protagon, cholin, and neurin. J. Physiol., 31, 30-37. DIAKONOW (1868). Ueber die phosphorhaltigen Kdrper der Huhner- und Storeier. Zeitschr. f. Chem., n, 154. DONATH, J. (1903). Das Vorkommen und die Bedeutung des Cholins in der Cerebro- spinalflussigkeit bei Epilepsie und organischen Erkrankungen des Nervensy stems, nebst weiteren Beitragen zur Chemie desselben. Zeitschr. physiol. Chem., 39, 526-44. DONATH, J. (1905-1906). Detection of choline in the cerebro-spinal fluid by means of the polarisation microscope. J. Physiol., 33, 211-24. DUNHAM, E. K., und C. A. JACOBSON (1910). Ueber Carnaubon : Bin glycerinfreies Phosphatid, lecithindhnlich konstituiert mit Galaktose als Kern. Zeitschr. physiol. Chem., 64, 302-15. DYBKOWSKY, W. (1867). Ueber die Identitat des Cholins und des Neurins. J. prakt. Chem., 100, 153-64. 184 BIBLIOGRAPHY OF CHAPTER IV 185 ERLANDSEN, A. (1907). Untersuchungen ueber die lecithittartigen Substanzen des Myocardium* und der quergestreiften Muskeln. Zeitschr. physiol. Chem., 51, 71-155. FLORENCE, A. (1897). Neues Verfahren zum Nachweis von Samenflecken. Chem. Zentralbl., ii., 1161, from Repert. de Pharm., 1897, 388. FRANKEL, S. (1909). Ueber Lipoide. VII. Ueber Kephalin von E. NEUBAUER. Biochem. Zeitschr., 21, 321-36. FRANKEL, S. (1910). Ueber Lipoide. IX. Ueber das Sahidin atis Menschenhirn von K. LlNNERT. Biochem. Zeitschr., 24, 268-76. FRANCHINI, G. (1908). Untersuchungen ueber Lecithin, Cholin, und Ameisensaure. Arch. d. Farmacol. sperim., 7, 371-89 ; Chem. Zentralbl., ii., 1785. FUNK, C. (1911, l). On the chemical nature of the substance which cures polyneu- ritis in birds induced by a diet of polished rice. J. Physiol., 43, 395-4- FURTH, O. VON, und C. SCHWARZ (1908). Ueber die Natur der blutdruckerniedrigcnden Substanz in der Schilddruse. Pfluger's Archiv, 124, 361-68. GAUTRELET, J., et L. THOMAS (1909). Action hypotensive du serum de chien priv'e de surrenales. Compt. rend., 149, 149-50. GRIESS, P., und G. HARROW (1885). Ueber das Vorkommen des Cholins im Hopfen. Ber. deutsch. chem. Gesellsch., 18, 717-19. GULEWITSCH, WL. (1898, i). Ueber Cholin und einige Verbindungen desselben. Zeitschr. physiol. Chem., 24, 513-41. GULEWITSCH, WL. (1899). Ueber die Leukomatine des Ochsengehirns. Zeitschr. physiol. Chem., 27, 50-82. HALLIBURTON, W. D. (1905). Die Biochemie der peripheren Nerven. Ergebnisse der Physiologic, 4, 24-83. HANDELSMAN, J. (1908). Experimentelle und chemische Untersuchungen ueber das Cholin und seine Bedcutung fur die Entstehung epileptischer Krdmpfe. Deutsch. Zeitschr. f. Nervenheilk., 35, 428-52. HOESSLIN, H. VON (1906). Ueber den Abbau des Cholins im Tierkorper. Beitr. chem. Physiol. Pathol., 8, 27-37. HOFMANN, K. A., und K. HOBOLD (1911). Die Perchlorate der Cholin- und Neuringruppe : Nachweis von Cholin und Neurin. Ber. deutsch. chem. Gesellsch., 44, 1766-71. HOFMANN, K. A., R. ROTH, K. HOBOLD, und A. METZLER (1910). Beziehung zwischen Konstitution und Verhalten gegen Wasser bei den Ammonium- und Oxoniumper- chloraten. Ber. deutsch. chem. Gesellsch., 43, 2624-30. HUNT, R. (1899-1900). Note on a blood pressure lowering body in the suprarenal gland. Proc. Amer. Physiol. Soc., xviii-xix, Amer. J. Physiol., 3. HUNT, R., and R. DE M. TAVEAU (1911). The effects of a number of derivatives of choline and analogous compounds on the blood-pressure. Bulletin No. 73 of the Hy genie Laboratory of the Public Health and Marine Hospital Service. Washington. JAHNS, E. (1885). Ueber die Alkaloide des Bockhornssamens. Ber. deutsch. chem. Gesellsch., 18, 2518-23. JAHNS, E. (1890). Ueber die Alkaloide der Arecanuss. II. Ber. deutsch. them. Gesellsch., 23, 2972-78. JAHNS, E. (1893). Vorkommen von Betain und Cholin im Wurmsamen. Ber. deutsch. chem. Gesellsch., 26, 1493-96. JOESTEN, J. (1913). Experimentelle Untersuchungen uber die Florence' sche Reaktion. Vierteljahrschrift fur gerichtl. Medizin, 45, 323-50. KAJURA, S. (1908). Is choline present in the cerebro-spinal fluid of epileptics ? Quart. J. exp. Physiol., I, 291-96. KAUFFMANN, M. (1908). Ueber den angcblichen Bcfund von Cholin in der Lumbal- flussigkeit. Neurol. Zentralbl., 27, 260-62. 1 86 THE SIMPLER NATURAL BASES KAUFFMANN, M. (1910). Ueber das angebliche Vorkommen von Cholin in pathologischer Lumbalfliissigkeit. Zeitschr. physiol. Chem., 66, 343-44. KAUFFMANN, M. (1911). Ueber den Befund von Cholin in Ochsengehirn. Zeitschr. physiol. Chem., 74, 175-78. KAUFFMANN, M., und D. VORLANDER (1910). Ueber den Nachweis des Cholins nebst Beitrdgen zur Kenntniss des Trimethylamins. Ber. deutsch. chem. Gesellsch., 43, 2735-43. KIESEL, A. (1907). Versuche mit dem Stanck'schen Verfahren zur quantitativen Bestim- mung des Cholins. Zeitschr. physiol. Chem., 53, 215-39. KINOSHITA, T. (1910, 2). Ueber den Cholingchalt tierischer Gewebe. Pfliiger's Archiv, 132, 607-31. KOBERT, R. (1892). Ueber Pilzgifte. Sitzb. Dorp. Naturf. Vers., 9, Heft 3, Chem. Zentralbl., ii., 929. KOCH, W. (1902). Zur Kenntniss des Lecithins, Kephalins und Cerebrins aus Nerven- substanz. Zeitschr. physiol. Chem., 36, 134-40. KRAFT, F. (1906). Ueber das Mutterkorn. Arch. d. Pharm., 244, 336-59- KRUGER, M., und P. BERGELL (1903). Zur Synthese des Cholins. Ber. deutsch. chem. Gesellsch., 36, 2901-4. KUNZ, H. (1885). Ueber den Alkaloidgehalt des Extractum Belladonnae Pharm. Germ. II. Arch. Pharm. 223, 701-9. KUNZ, H. (1887). Beitrdge zur Kenntniss des Emetins. Arch. d. Pharm., 225, 461-79. KUTSCHER, FR. (1910, 4). Die basischen Extraktstoffe des Champignons (Agaricus compestris). Zentralbl. f. Physiol., 24, 775-76. KUTSCHER, FR., und A. LOHMANN (1903). Die Endprodukte der Pankreas- und Hefeselbst- verdauung. Zeitschr. physiol. Chem., 39, 159-64. KUTSCHER, FR., und A. LOHMANN (1906, 2). Der Nachweis toxischer Basen in Harn. Zeitschr. physiol. Chem., 48, 1-8. LIEBREICH, O. (1865). Ueber die chemische Beschaffenheit der Gehirnsubstanz. Liebig's Annalen, 134, 29-44. LIEBREICH, O. (1869, i). Ueber die Oxidation des Neurins. Ber. deutsch. chem. Gesellsch., 2, 12-13. LIPPMANN, E. O. VON (1887). Ueber einige organische Bestandtheile des Rilbensaftes. Ber. deutsch. chem. Gesellsch., 20, 3201-8. LOHMANN, A. (1907). Cholin, die den Blutdruck erniedrigende Substanz der Nebenniere. Pfliiger's Archiv, 118, 215-27. LOHMANN, A. (1908). Ueber die antagonistische Wirkung der in den Nebennieren enthaltenen Substanzen Suprarenin und Cholin. Pfliiger's Archiv, 122, 203-9. LOHMANN, A. (1911). Ueber einige Bestandteile der Nebennieren, Schilddrusen und Hoden. Zeitschr. f. Biol., 56, 1-31. MACLEAN, H. (1908). Weitere Versuche zur quantitativen Gewinnung von Cholin und Lecithin. Zeitschr. physiol. Chem., 55, 360-70. MACLEAN, H. (1909). On the nitrogen-containing radicle of lecithin and other phosphatides. Biochem. J., 4, 38-58. MANSFELD, G. (1904). Ueber den Donath'schen Nachweis von Cholin in Fallen von Epilepsie. Zeitschr. physiol. Chem., 42, 157-64. MENDEL, L. B., and F. P. UNDERBILL (1910). The physiological action of cholin. Zentralbl. f. Physiol., 24, 251-53. MENDEL, L. B., F. P. UNDERBILL, and R. R. RENSHAW (1912). The action of salts of choline on arterial blood pressure. J. of Pharmacol. and Exp. Therap., 3, 649-60. MENGE, G. A. (1911). Some new compounds of the choline type. J. Biol. Chem., 10, 399-406. BIBLIOGRAPHY OF CHAPTER IV 187 MODRAKOWSKI, G. (igo8). Ueber die physiologische Wirkung des Cholins. Pfliiger's Archiv, 124, 601-32. MORUZZI, G. (1908). Versuche zur quantitativen Gewinnung von Cholin aus Lecithin. Zeitschr. physiol. Chem., 55, 352-59. MOTT, F. W., and W. D. HALLIBURTON (1899). The physiological action of choline and neurine. Phil. Trans., B, 191, 211-68. MULLER, FR. (1910). Beitrdge zur Analyse der Cholinwirkung. Pfliiger's Archiv, 134, 289-310. NJEGOVAN, V. (1911). Beitrdge zur Kenntniss der p flan zlichen Phosphatide. Zeitschr. physiol. Chem., 76, 1-26. PAL, J. (1910). Zur Kenntniss der Cholinwirkung. Zentralbl. f. Physiol., 24, 1-2. PAL, J. (1911). Ueber die Wirkung des Cholin und des Neurin. Zeitschr. f. exper. Pathol. u. Therap., 9, 191-206. POLSTORFF, K. (1909, i). Ueber den Gehalt einiger essbaren Pilze an Cholin. Festschrift Otto Wallach, 579-83. POLSTORFF, K. (1909, 2). Ueber das Vorkommen von Betainen und von Cholin in Coffein- und Theobromin-enthaltenden Drogen. Festschrift Otto Wallach, 569-578. POPIELSKI, L. (1910, i). Ueber die Blutdruckwirkung des Cholins. Zeitschr. physiol. Chem., 70, 250-52. RENSHAW, R. R. (1910). Preparation of choline and some of its salts. J. Amer. Chem. Soc., 32, 128-30. RIEDEL, J. D. (1908). Preparation of choline from lecithin. D.R.P. 193449- ROSENHEIM, O. (1905-6). New tests for choline in physiological fluids. J. Physiol., 33, 220-24. ROSENHEIM, O. (1907). Choline in the cerebro-spinal fluid. J. Physiol., 35, 465-72. RUCKERT, A. (1908). Ueber die Einwirkung von Oiditim lactis und Vibrio cholera auf Cholin Chlorid. Arch. Pharm., 246, 676-91. SAMELSON, S. (1911). Ueber gefdssverengerende und erweiterende Substanzen. Arch. exp. Path. Pharm., 66, 347-51. SCHMIDT, E. (1891, 1904, i). Ueber Cholin, Neurin und verwandte Verbindungen. /, //. Liebig's Annalen, 267, 249-318; 337, 37-121. SCHMIDT, E. (1904, 2). Ueber die Beziehungen zwischen chemischer Konstitution und physiologischer Wirkung einiger Ammoniumbasen. Arch. d. Pharm., 242, 705-14. SCHMIDT, F. W. (1907). Ueber Cholincadmiumchlorid. Zeitschr. physiol. Chem., 53, 428. SCHULZE, E. (1887). Ueber das Vorkommen von Cholin in den Keimpflanzen. Zeitschr. physiol. Chem., II, 365-72. SCHULZE, E. (1888). Ueber einige stickstoffhaltige Bestandtheile der Keimlinge von Soya hispida. Zeitschr. physiol. Chem., 12, 405-15. SCHULZE, E. (1890). Ueber basische Stickstoffverbindungen aus den Samen von Vicia sativa und Pisum sativum. Zeitschr. physiol. Chem., 15, 140. SCHULZE, E. (1892, i). Ueber einige stickstoffhaltige Bestandtheile der Keimlinge von Vicia sativa. Zeitschr. physiol. Chem., 17, 193-216. SCHULZE, E. (1896). Untersuchungen ueber die zur Klasse der stickstoffhaltigen organ- ischen Basen gehorenden Bestandteile einiger landwirtschaftlich benutzten Satnen, Olkuchen und Wurzelknollen, so wie einiger Keimpflanzen. Landwirtsch. Versuchs-Stat., 46, 23-77. SCHULZE, E. (1904). Ueber das Vorkommen von Hexonbasen in den Knollen der Kartoffel (Solatium tuberosum) und der Dahlie (Dahlia variabilis). Landwirtsch. Versuchs-Stat., 59, 331-43. 1 88 THE SIMPLER NATURAL BASES SCHULZE, E. (1909). Ueber die zur Darstellung von Cholin, Betain und Trigonellin aus Pflanzen verwendbai'en Methoden und uber die quantitative Bestimmung diescr Basen. Zeitschr. physiol. Chem., 60, 155-79. SCHULZE, E., und S. FRANKFURT (1893). Ueber das Vorkommen von Betain und Cholin in Malzkeimen und im Keim des Weizenkorns. Ber. deutsch. chem. Gesellsch., 26, 2151-55. SCHULZE, E., and U. PFENNINGER (1911). Untersuchungen uber die in den Pflanzen vorkommenden Betaine. I. Mitteilung. Zeitschr. physiol. Chem., 71, 174-85. SCHULZE, E., und G. TRIER (1912, 3). Ueber die allgemeine Verbreitung des Cholins. Zeitschr. physiol. Chem., 81, 53-58. SCHWARZ, C., und R. LEDERER (1908). Ueber das Vorkommen von Cholin in der Thymus, in der MHz und in den Lymphdrusen. Pfluger's Archiv, 124, 353-60. STANEK, VL. (1905). Ueber das Cholinperjodid und die quantitative Fdllung von Cholin durch Kaliumtrijodid. Zeitschr. physiol. Chem., 46, 280-85. STANEK, VL. (1906, i). Ueber die quantitative Trennung von Cholin und Betain. Zeitschr. physiol. Chem., 47, 83-87. STANEK, VL. (1906, 2). Ueber die quantitative Bcstlmmung von Cholin und Betain in pjlanzlichen Stoffen und einige Bemerkungen iiber Lecithine. Zeitschr. physiol. Chem., 48, 334-46. STANFORD, R. V. (1913). Vergleichende Studien uber Ccrebrospinalflussigkeit bei Geisteskrankheiten. II. Stickstoff. Quantitative Bestimmung von kleinen Mcngen Stickstoff. Zeitsch. physiol. Chem., 86, 219-33. STRECKER, A. (1849). Beobachtungen uber die Galle verschiedener Thieve. Liebig's Annalen, 70, 149-97. STRECKER, A. (1862). Ueber einige neue Bestandtheile der Schiveinegalle. Liebig's Annalen, 123, 353-60. STRECKER, A. (1868). Ueber das Lecithin. Liebig's Annalen, 148,. 77-90. STRUVE, H. (1902). Beobachtungen uber das Vorkommen und uber verschiedene Eigen- schaften des Cholins. Zeitschr. analyt. Chem., 41, 544-50. STRUVE, H. (1904). Cholin in pjlanzlichen und thierischen Gebilden. Liebig's Annalen, 330, 374-77. THOMS, H. (1898, i). Ueber das Vorkommen von Cholin und Trigonellin in Strophanthus Samen und uber die Darstellung von Strophanthin. Ber. deutsch. chem. Gesellsch., 31, 271-77. THOMS, H. (1898, 2). Cholin und Trigonellin in den Samen von Strophanthus Kombe. Ber. deutsch. chem. Gesellsch., 31, 401- TOTANI, G. (1910, i). Ueber das Vorkommen von Cholin in Stierhoden. Zeitschr. physiol. Chem., 68, 86-87. TOTANI, G. (1910, 2). Ueber die basischen Bestandteile der Bambusschdsslinge. Zeitschr. physiol. Chem., 70, 388-90. TRIER, G. (1912, 3). Ueber einfache PJlanzcnbasen und ihre Beziehungen zum Aufbau der Eiweissstoffe und Lecithine. Berlin, pp. iv + 117. TRIER, G. (1913, 5). Weitere Beitrdge zur Kenntniss einfacher Pflanzenbasen. Zeitschr. physiol. Chem., 85, 372-91. UTZ, F. (1905). Beitrdge zur Kenninis giftiger Pilze. Apotheker Zeitung, 20, 993. VINCENT, S., and W. CRAMER (1904). The nature of the physiologically active substances in extracts of nervous tissues and blood, with some remarks on the methods of testing for choline. J. Physiol., 30, 143-54- WALLER, A. D., and S. C. M. SOWTON (1903). The action of choline, neurine, muscarine, and betame on isolated nerve and upon the excised heart. Pror. Roy. Soc., 72, 320-44. WEBSTER, W. (1909). Choline in animal tissues and fluids. Biochem. J., 4, 117-26. BIBLIOGRAPHY OF CHAPTER IV 189 WINTERSTEIN, E. (1904). Ueber einige Bestandteile des Emmentalerkdses. II. Zeitschr. physiol. Chem., 41, 485-504. WINTERSTEIN, E M und G. TRIER (1909). Die Alkaloide. Berlin, pp. vii + 340. WURTZ, A. (1867). Synthese de la nevrine. Compt. rend., 65, 1015-18. YOSHIMURA, K. (1910). Beitrage zur Kenntniss der Zusammensetzung der Malzkeime. Biochem. Zeitschr., 31, 221-26. AMINO-ETHYL ALCOHOL (COLAMINE). BAUMANN, A. (1913). Uber den stickstoffhaltigen Bestandtell des Kephalins. Biochem. Zeitschr., 54, 30-39. RENALL, M. H. (1913). Uber den stickstoffhaltigen Bestandteil des Kephalins. Biochem. Zeitschr., 55, 296-300. THUDICHUM, J. L. W. (1884). A treatise on the chemical constitution of the brain. London. THUDICHUM, J. L. W, (1901). Die chemische Konstitution des Gehirns des Menschen und der Tiere. Tubingen. TRIER, G. (1911). Aminodthylalkohol, ein Produkt der Hydrolyse des "Lecithins" (Phosphatids) der Bohnensamen. Zeitschr. physiol. Chem., 73, 383-88. TRIER, G. (1912, i). Uber die Gewinnung von Aminodthylalkohol aus Eilecithin. Zeitschr. physiol. Chem., 76, 496-98. TRIER, G. (1912, 2). Ueber die Umwandlung von Aminodthylalkohol (Colamin) in Cholin. Zeitschr. physiol. Chem., 80, 409-11. TRIER, G. (1913, 1-4). Uber die nach den Methoden der Lecithindarstellung aus Pjian- zensamen erhaltlichen Verbindungen. I-IV. Zeitschr. physiol. Chem., 86, 1-32, 141-42, 153-73, 407-14. NEURINE. BAEYER, A. (1866). Synthese des Neurins. Liebig's Annalen, 140, 306-13. BAEYER, A. (1869). Ueber das Neurin. Liebig's Annalen, 142, 322-26. BRIEGER, L. (1885, i). Ueber Ptomaine. Berlin, August Hirschwald. GULEWITSCH, WL. (1898, 2). Ueber Neurin und einige Verbindungen desselben. Zeitschr. physiol. Chem., 26, 175-88. HOFMANN, A. W. (1858). Rech'erches pour servir a rhistoire des bases organiques. V. Action du bibromure d'ethyllne sur la trimethylamine. Ann. de Chim. et Phys., [Hi.], 54, 356-62. LOHMANN, A. (1909). Nertrin, ein Bestandteil der Nebennieren. Pfliiger's Archiv, 128, 142-44. MUSCARINE. ABEL, J. J., and W. W. FORD (1906). On the poison of Amanita phalloides, a glucosidal nitrogenous haemolysin. J. Biol. Chem., 2, 273-88. BERLINERBLAU, J. (1884). Ueber Muscarin. Ber. deutsch. chem. Gesellsch., 17, 1139-45. BOEHM, R. (1885, i). Beitrage zur Kenntniss der Hutpilze in chemischer und toxiko- logischer Beziehung. Arch. exp. Path. Pharm., 19, 60-86. BOEHM, R. (1885, 2). Ueber das Vorkommen und die Wirkungen des Cholins und die Wirkungen der kunstlichen Muscarine. Arch, exp. Path. Pharm., 19, 87-100. BRABANT, C. (1913). Uber das Homologe des Muscarins der C 3 Reihe. Zeitschr. physiol. Chem., 86, 206-14. 190 THE SIMPLER NATURAL BASES FISCHER, E. (1893, 1894). Ueber den Amido-acetaldehyd 77, ///. Ber. deutsch. chem. Gesellsch., 26, 464-71 ; 27, 172. FUHNER, H. (1908, i). Ueber das Schicksal des synthetischen Muskarins im Tierkorper. Arch. exp. Path. Pharm., Schmtedeberg Festschrift, 208-13. FUHNER, H. (1909). Ueber das Verhalten des synthetischen Muskarins im Tierkdrper. Arch. exp. Path. Pharm., 6l, 283-96. HARMSEN, E. (1903). Zur Toxicologie des Fliegenschwammes. Arch. exp. Path. Pharm., 50, 361-452. HARNACK, E. (1875). Untersuchungen uber Fliegenpilzalkaloide. Arch. exp. Path. Pharm., 4, 168-90. HONDA, J. (1911). Ueber Fliegenpilzalkaloide und das kunstliche Muscarin. Arch. exp. Path. Pharm., 65, 454-66. NOTHNAGEL, G. (1893). Ueber das Muscarin. Ber. deutsch. chem. Gesellsch., 26, 801-6. SCHMIEDEBERG, O., und R. KopFE (1869). Das Muscarin. Leipzig. SCHMIEDEBERG, O., und E. HARNACK (1877). Ueber die Synthese des Muscarins und uber muscarinartig wirkende Ammoniumbasen. Arch. exp. Pathol. Pharm., 6, 101-12. TRIMETHYLAMINE-OXIDE. SUWA, A. (1909, l). Untersuchungen uber die Organextrakte der Selachier. I. Die Muskelextraktstoffe des Dornhais (Acanthias vulgaris). Pfluger's Archiv, 128, 421-26. SUWA, A. (1909, 2). Untersuchungen uber die Organextrakte der Selachier. II. Ueber das aus den Muskelextraktstoffen des Dornhais gewonnene Trimethylaminoxide. Pfliiger's Archiv, 129, 231-39. HOMOCHOLINE AND NEOSINE. ACKERMANN, D., und FR. KUTSCHER (1908). Zur Konstitutionsermittelung des Neosins. Zeitschr. physiol. Chem., 56, 220-22. BERLIN, E. (1910, i). Vorlaufige Mitteilungen. I. Ueber die Wirkung des Homo- ckolins. II. Glykokoll aus Krabbenextrakt. Zentralbl. f. Physiol., 24, 587-89. BERLIN, E. (1910, 2). Eine neue Synthese des y-Homocholins. Zentralbl. f. Physiol., 24, 779-80. BERLIN, E. (1911). Homocholin und Neosin. Zeitschr. f. Biol., 57, 1-74. ENGELAND, R. (1908, i). Ueber Liebig's Fleischextrakt. Zeitschr. Unters. Nahr. Genussm., 16, 658-64. MALENGREAU, F., und A. LEBAILLY (1910). Ueber die synthetischen Homocholine. Zeitschr. physiol. Chem., 67, 35-41. REFERENCES TO CHAPTER V. A. CREATINE AND CREATININE. ACKERMANN, D. (1913). Uber den ferment ativ en Abbatt des Kreatinins. Zeitschr. f. Biol., 62, 208-16. AMBERG, S., and W. P. MORRILL (1907). On the excretion of creatinin in the new- born infant. J. Biol. Chem., 3, 311-20. ANTONOFF, N. (1906-7). Ueber kreatininbildende Bakterien. Zentralbl. f. Bakteriol., I Abt., 43, 209-12. BAUR, E., und H. BARSCHALL (1906). Beitrage zur Kenntniss des Fleischextraktes. Arbeiten kaiserl. Gesundheitsamte, 24, 522-75. BEKER, J. C. (1913). Die Verteilung des Kreatins im Sdugetierkorper. Zeitschr. physiol. Chem., 87, 21-37. BENEDICT, F. G., and A. R. DIEFENDORF (1907). The analysis of urine in a starving woman. Amer. J. Physiol., 18, 362-76. BENEDICT, F. G., and V. C. MYERS (1907, i). The elimination of creatinine in women. Amer. J. Physiol., 18, 377-96. BENEDICT, F. G., and V. C. MYERS (1907, 2). The determination of creatin and creatinin. Amer. J. Physiol., 18, 397-405. BROWN, T. GRAHAM, and E. P. CATHCART (1909). The effect of work on the creatine content of muscle. Biochem. J., 4, 420-26. CABELLA, M. (1913). Ueber den Gehalt an Kreatin der Muskeln verschiedener Tiere und in den verschiedenen Arten des Muskelgewebes. Zeitschr. physiol. Chem., 84, 29-38. CATHCART, E. P. (1907). Ueber die Zusammensetzung des Hungerharns. Biochem. Zeitschr., 6, 109-48. CATHCART, E. P. (1909). The influence of carbohydrates and fats on protein metabolism. J. Physiol., 39, 311-30. CATHCART, E. P., and M. Ross TAYLOR (1910). The influence of carbohydrates and fats on protein metabolism. II. The effect of phloridzin glycosuria. J. Physiol., 41, 276-84. CHAPMAN, A. C. (1909). On Jaffe's colorimetric method for the estimation of creatinine. Analyst, 34, 475-83. CHEVREUL (1835). Sur la composition chimique du bouillon de viandes (Extrait d'un rapport fait a VAcademie des Sciences par M. Chevreul). J. de Pharm., 21, 231-42. CHISHOLM, R. A. (1912). The creatin content of muscle in malignant disease and other pathological conditions. Biochem. J., 6, 243-47. CLOSSON, O. E. (1906). The elimination of creatinin. Amer. J. Physiol., 16, 252-67. COOK, F. C. (1909). Factors which influence the creatinine determination. J. Amer. Chem. Soc., 31, 673-93. CZERNECKI, W. (1905). Zur Kenntniss des Kreatins und Kreatinins im Organismus. Zeitschr. physiol. Chem., 44, 294-308. DAKIN, H. D. (1906). The formation of glyoxylic acid. J. Biol. Chem., i, 271-78. DAKIN, H. D. (1907). The action of arginase upon creatin and other guanidin deri- vatives. J. Biol. Chem., 3, 435-441. 191 192 THE SIMPLER NATURAL BASES DENIS, W. (1912). Metabolism studies on cold-blooded animals. I. The urine of thef.sk. J. Biol. Chem., 13, 225-32. DESSAIGNES (1854). Recherche sur quelques products de transformation de la creatine. Compt. rend., 38, 839-43. DESSAIGNES (1855). Nouvelles recherches sur la metliyluramine et sur ses derives. Compt. rend., 41, 1258-1261. DORNER, G. (1907). Zur Bildung von Kreatin und Kreatinin in Organismus, besonders der Kaninchen. Zeitschr. physiol. Chem., 52, 225-78. EDLEFSEN, G. (1908). Zur Frage der quantitativen Bestimmung des Kreatinins im Harn. Munch, med. Wochenschr., 55, 2524-26. EMMETT, A. D., and H. S. GRINDLEY (1907). Further studies on the application of Folin's creatin and creatinin method to meat and meat extracts. J. Biol. Chem., 3, 491-516. ENGELAND, R. (1908, 4). Destination von Kreatinin. Zeitschr. physiol. Chem., 57, 65-66. FIEBIGER, J. (1903). Ueber Kreatinin im Harn verschiedener Haustiere. Zentralbl. f. Physiol., 17, 33-34. FOLIN, O. (1904). Beitrag zur Chemie des Kreatinins und Kreatins im Harne. Zeitschr. physiol. Chem., 41, 223-42. FOLIN, O. (1905, i). Laws governing the chemical composition of urine. Amer. J. Physiol., 13, 66-115. FOLIN, O. (1905, 2). A theory of protein metabolism. Amer. J. Physiol. 13, 117-38. FOLIN, O. (1906). The chemistry and bio-chemistry of kreatin and kreatinin. Festschr. f. Olof Hammarsten, III. FOLIN, O., and F. C. BLANCK (1910). The preparation of creatinine from urine. J. Biol. Chem., 8, 395-97. FOLIN, O., and W. DENIS (1910). The preparation of creatinine from creatin. J. Biol. Chem., 8, 399-400. FOLIN, O., and W. DENIS (1912). On creatine in urine of children. J. Biol. Chem., u, 253-56. FORSCHBACH, J. (1908). Kreatininausscheidung bei Krankheiten. Archiv exp. Path. Pharm., 58, 113-40. FOSTER, N. B., and H. L. FISHER (1911). Creatin and creatinin metabolism in dogs with Eck fistula. J. Biol. Chem., 9, 359-62. FUNARO, R. (1908). Ueber den Kreatiningehalt des Sauglingsharns. Biochem. Zeitschr., 10, 467-71. FURTH, O. VON (1900). Ueber den Stoffwechsel der Cephalopoden. Zeitschr. physiol. Chem., 31, 353-80. GOTTLIEB, R., und R. STANGASSINGER (1907 ; 1908, i). Ueber das Verhalten des Kreatins bei der Autolyse. Zeitschr. physiol. Chem., 52, 1-41 ; 55, 295-321. GOTTLIEB, R., und R. STANGASSINGER (igo8, 2). Ueber die Bildung itnd Zersetzung des Kreatins bei der Dnrchblutung uberlebender Organe. Zeitschr. physiol. Chem., 55, 322-37. GREGOR, A. (1900). Beitrage zur Physiologic des Kreatinins. Zeitschr. physiol. Chem., 31, 98-118. GREGORY, W. (1848). Ueber den Gehalt einiger Fleischarten an Kreatin. Liebig's Annalen, 64, 100-8. GRINDLEY, H. S., and H. S. WOODS (1906). Methods for the determination of creatinin and creatin in meats and their products. J. Biol. Chem., 2, 309-15. * HARDEN, A., and D. NORRIS (1911). The diacetyl reaction for proteins. J. Physiol., 42, 332-36. HEINTZ, W. (1849). Beitrage zur Kenntniss des Kreatins und Kreatinins. PoggendorfPs Annalen der Physik und Chemie, 74, 125-42. HOFMEISTER, F. (i88o). Ueber die durch Phosphorwolframsaurefallbaren Substanzen des Harns. Zeitschr. physiol. Chem., 5, 67-74. BIBLIOGRAPHY OF CHAPTER V 193 HOOGENHUYZE, C. J. C. VAN, und H. VERPLOEGH (1905). Beobachtungen uber die Krea- tininausscheidung beim Menschen. Zeitschr. physiol. Chem., 46, 415-71. HOOGENHUYZE, C. J. C. VAN, und H. VERPLOEGH (1908). Weitere Beobachtungen uber die Kreatininausscheidung beim Menschen. Zeitschr. physiol. Chem., 57, 161-266. HORBACZEWSKI, J. (1885). Neue Synthese des Kreatins. Abstract in Maly's Jahrbuch fur Tierchemie, 86-87. INOUYE, K. (1912). Ueber die Entstehung des Kreatins im Tierkorper. Zeitschr. physiol. Chem., 8l, 71-79. JAFFE M. (1886). Ueber den Niederschlag welchen Pikrinsaure in normalem Harn erzeugt und uber eine neue Reaction des Kreatinins. Zeitschr. physiol. Chem., 10, 391-400. JAFFE, M. (1906). Untersuchungen uber die Entstehung des Kreatins im Organismus. Zeitschr. physiol. Chem., 48, 430-68. JAKSCH, R. VON (1881). Studien uber den Harnstoffpilz. Zeitschr. physiol. Chem., 5, 395-421. JOHNSON, G. S. (1892). On the bases (organic] in the juice of flesh. Part I. Proc. Roy. Soc., 50, 287-302. KLERCKER, Kj. O. AF (1907). Beitrag zur Kenntnis des Kreatins und Kreatinins im Stoffwechsel des Menschen. Biochem. Zeitschr., 3, 45-87. KOCH, W. (1905). Relation of creatinin excretion to variation in diet. Amer. J. Physiol., 15, 15-29. KORNDORFER, G. (1904, i). Ueber das Isokreatinin. Arch. Pharm., 242, 373-79- KORNDORFER, G. (1904, 2). Ueber das Kreatinin. Arch. Pharm., 242, 641-48. KRAUSE, R. A. (1910). The excretion of creatine in diabetes. Quart. J. exp. Physiol., 3, 289-96. KRAUSE, R. A. (1911). On the urine of women under normal conditions with special re- ference to the presence of creatine. Quart. J. exp. Physiol., 4, 293-304. KRAUSE, R. A. (1913). On age and metabolism and on the significance of the excretion of creatine. Quart. J. exp. Physiol., 7, 87-101. KRAUSE, R. A., and W. CRAMER (igio). The occurrence of creatin in diabetic urine. Proc. Physiol. Soc., Ixi-lxii; J. Physiol., 40. KRUKENBERG, C. F. W. (1881). Untersuchungen der Fleischextracte verschiedener. Fische und Wirbellosen. Abstract in Maly's Jahrbuch I. Tierchemie, II, 340-44. KUTSCHER, F. (1905, 1906). Ueber Liebig's Fleischextrakt. Zeitschr. Unters. Nahr. Genussm., 10, 528-37; II, 582-84. LEATHES, J. B. (1907). On the excretion of nitrogen, creatinine, and uric acid in fever. J. Physiol., 35, 205-14. LEFMANN, G. (1908). Beitrdge zum Kreatininstoffwechsel. Zeitschr. physiol. Chem., 57, 476-514. LETSCHE, E. (1907). Beitrdge zur Kenntnis der organischen Bestandteile des Serums. Zeitschr. physiol. Chem., 53, 31-112. LEVENE, P. A., and L. KRISTELLER (1909). Factors regulating the creatinin output in man. Amer. J. Physiol., 24, 45-65. LIEBIG, J. (1847). Ueber die Bestandtheile der Fliissigkeiten des Fleisches. Liebig's Annalen, 62, 257-369. LONDON, E. S., und N. BOLJARSKI (1909). Zur Frage uber den Anteil der Leber am Kreatininstoffwechsel. Zeitschr. physiol. Chem., 62, 465-67. LYMAN, J. F. (1908). A note on the chemistry of the muscle and liver of reptiles. J. Biol. Chem., 5, 125-27. MALY, R. (1871). Einfache Darstellung von salzsaurem Kreatinin aiis Ham. Liebig's Annalen, 159, 279-80. 13 194 THE SIMPLER NATURAL BASES MASCHKE, O. (1878). Ueber eine neue Kreatinin Reaction. Zeitschr. analyt. Chem., 17, 134-41. MAYERHOFER, E. (1909). Einiges zur Esbach'schen quantitativen Eiweissbestimmung und ilber eine neue Kreatininverbindung. Wien. klin. Wochenschr., 22, 90-92. MELLANBY, E. (1908). Creatin and creatinin. J. Physiol., 36, 447- 8 7- MELLANBY, E. (1913). The metabolism of lactating women. Proc. Roy. Soc., B. 86, 88-109. MENDEL, L. B., and W. C. ROSE (1911, l). Experimental studies on creatine and creatinine. J. Biol. Chem., 10, 213-54. MENDEL, L. B., and W. C. ROSE (1911, 2). Experimental studies on creatine and creatinine. II. Inanition and the creatine content of muscle. J. Biol. Chem., 10, 255-64. MICRO, K. (1910). Ueber die Isolierung des Kreatinins aus Extrakten. Zeitschr. Unters. Nahr. Genussm., 19, 426-34. MULDER, E., und N. MOUTHAAN (1869). Kreatin und Aldehyde. Zeitschr. f. Chem., 12, 341. MYERS, V. C., and M. S. FINE (1913. *) The creatin content of muscle under normal conditions. Its relations to the urinary creatinine. J. Biol. Chem., 14, 9-26. MYERS, V. C., and M. S. FINE (1913, 2). The influence of starvation on the creatine con- tent of muscle. J. Biol. Chem., 15, 283-304. MYERS, V. C., and M. S. FINE (1913, 3). The influence of the administration of creatine and creatinine on the creatine content of muscle. J. Biol. Chem., 16, 169-86. MYERS, V. C., and G. O. VOLOVIC (1913). The influence of fever on the elimination of creatinine. J. Biol. Chem., 14, 489-508. NEUBAUER, C. (1861, I, 2). Ueber Kreatinin. Liebig's Annalen, 119, 27-52 ; 120, 257-68. NEUBAUER, C. (1863). Ueber quantitative Kreatin- und Kreatininbestimmung im Muskel- fleisch. Zeitschr. analyt. Chem., 2, 22-34. NEUBAUER, C. (1866, i). Ueber Kreatinin und Kreatin. Liebig's Annalen, 137, 288-97. NEUBAUER, C. (1866, 2). Ueber einige Verbindungen des Kreatins mit Metallsalzen. Liebig's Annalen, 137, 298-301. OKUDA, Y. (1912). Quantitative determination of creatine, creatinine and mono-amino acids in certain flshes, Mollusca and Crustacea. J. Coll. Agric. Tokyo, 5, 25-31. PATON, D. N. (1910). Creatin excretion in the bird and its significance. J. Physiol., 39, 485-504. PATON, D. N., and W. C. MACKIE (1912). The liver in relation to creatine metabolism in the bird. J. Physiol., 45, 115-18. PEKELHARING, C. A. (1911). Die Kreatininausscheidung beim Menschen unter den Einfluss vom Muskeltonus. Zeitschr. physiol. Chem., 75, 207-15. PEKELHARING, C. A., und C. J. C. VAN HOOGENHUYZE (1909). Die Bildung des Kreatins im Muskel beim Tonus und bei der Starre. Zeitschr. physiol. Chem., 64, 262-93. PEKELHARING, C. A., und C. J. C. VAN HOOGENHUYZE (1910). Die Ausscheidung von parenteral zugefuhrtem Kreatin bei Saugetieren. Zeitschr. physiol. Chem., 69, 395-407. PETTENKOFER, M. (1844). Vorlaufige Notiz uber einen neuen stickstoffhaltigen Korper im Harne. Liebig's Annalen, 52, 97-100. PLIMMER, R. H. ADERS, M. DICK and C. C. LIEB (1909). A metabolism experiment with special reference to the origin of uric acid. J. Physiol., 39, 98-117. BIBLIOGRAPHY OF CHAPTER V 195 POULSSON, E. (1904). Ueber das " Isokreatinin " und dessen Identitdt mit Kreatinin. Arch. exp. Pathol. Pharm., 51, 227-38. PRICE, D. S. (1851). Creatine a constituent of the flesh of the Cetacea. Quarterly J. Chem. Soc., 3, 229-31. RIESSER, O. (1913). Theoretisches und Experimentelles zur Frage der Kreatinbildung im tierischen Organismus. Versuche iiber Kreatinbildung aus Betain und Cholin. Zeitschr. physiol. Chem., 86, 415-53. RONA, P. (igio). Notiz zur Kreatininbestimmung. Biochem. Zeitschr., 27, 348. ROSE, W. C. (1911). Experimental studies in creatine and creatinine. III. Excretion of creatine in infancy and childhood. J. Biol. Chem., 10, 265-70. ROTHMANN, A. (1908). Ueber das Verhalten des Kreatins bei der Autolyse. III. Zeitschr. physiol. Chem., 57, 131-42. SAIKI, T. (1909). A study of the chemistry of cancer. II. Purin bases, creatin, and creatinin. ]. Biol. Chem., 7, 21-26. SALKOWSKI, E. (1879). Zur Kenntniss des Kreatinins. Zeitschr. physiol. Chem., 4, 133. SALKOWSKI, E. (1886). Ueber die Neubauer'sche Methode zur Bestimmung des Kreatinins im Harn. Zeitschr. physiol. Chem., 10, 113-20. SALKOWSKI, E. (1890). Beitrdge zur Chemie des Harns. Zeitschr. physiol. Chem., 14, 471-90. SALKOWSKI, E. (1911). Ueber das Vorkommen von Traubenzucker und Kreatinin im Huhnerei. Biochem. Zeitschr., 32, 335-41. SCAFFIDI, V. (1913). tJber das Verhalten des Muskelkreatins bei der Ermudung. Biochem. Zeitschr., 50, 402-17. SCHLOSSBERGER (1844). Chemische Untersuchung der Muskeln eines Alligators. Liebig's Annalen, 49, 341-46. SCHLOSSBERGER (1848). Kreatin als ein Bestandtheil der menschlichen Muskeln nachge- wiesen. Liebig's Annalen, 66, 80-83. SCHMIDT, E. (1912). Ueber das Kreatinin und dessen Oxime. Arch. d. Pharm., 250, 330-81. SEEMANN, J. (1907). Beitrag zur Frage der Kreatininbildung. Zeitschr. f. Biol., 49, 333-44. SHAFFER, P. (1908). The excretion of kreatinin and kreatin in health and disease. Amer. J. Physiol., 23, 1-22. SHOREY, E. C. (1912). The isolation of creatinine from soils. J. Amer. Chem. Soc., 34, 99-107. SKINNER, J. J. (1912). Beneficial effect of creatinine and creatine on growth. Botanical Gazette, 54, 152-63. SPRIGGS, E. I. (1907). The excretion of creatinin in a case of pseudo-hypertrophic muscular dystrophy. Biochem. J., 22, 206-16. SULLIVAN, M. X. (1911). The origin of creatinine in soils. ]. Amer. Chem. Soc., 33, 2035-42. SUZUKI, U., und MITARBEITER (1912). Uber die Extraktivstoffe des Fischfleisches und der Muscheln. J. Coll. Agric. Tokyo, 5, 1-24. TAYLOR, A. E. (1910). The sources of error in the Folin method for the estimation of creatinine. J. Biol. Chem., 9, 19-20. TAYLOR, M. Ross (1910). Creatin and creatinin excretion in diabetes mellitus. Biochem. J., 5, 362-77. THESEN, J. E. (1898). Ueber Isokreatinin. Zeitschr. physiol. Chem., 24, 1-17. THOMPSON, W. H., T. A. WALLACE and H. R. S. CLOTWORTHY (1913). Observations on the use of the Folin method for the estimation of creatine and creatinine. Biochem. J., 7, 445-65. 13 i 9 6 THE SIMPLER NATURAL BASES TOPPELIUS, M., und H. POMMEREHNE (1896). Ueber Kreatinine verschiedenen Ursprungs. Arch. d. Pharm., 234, 380-97. TOWLES, C., and C. YOEGTLIN (igii). Creatine and creatinine metabolism in dogs during feeding and inanition, with especial reference to the function of the liver. J. Biol. Chem., 10, 479-97- TWORT, F. W., and E. MELLANBY (1912). On creatin-destroying bacilli in the intestine and their isolation. J. Physiol., 44, 43-49- URANO, F. (1907). Ueber die Bindungswcise des Kreatins im Muskel. Beitr. chem. Physiol. Pathol., 9, 104-15. VANDEVELDE, G. (1884). Studien zur Chemie des Bacillus subtilis. Zeitschr. physiol. Chem., 8, 367-90. VAN HOOGENHUYZE, C. J. C., und H. VKRPLOEGH. See under letter H. VOIT, CARL (1868). Ueber das Verhalten des Kreatins, Kreatinins und Harnstoffs im Thierkorper. Zeitschr. f. Biol., 4, 77-162. VOLHARD, J. (1868). Ueber die Synthese des Kreatins. Sitzungsber. kon. bayer. Akad. d. Wissensch., II, 472-79. WALPOLE, G. S. (1911). The direct determination of creating in pathological urine. J. Physiol., 42, 301-8. WEBER, S. (1908). Physiologisches zur Kreatininfrage. Arch. exp. Path. Pharm., 58, 93-112. WEYL, TH. (1878). Ueber eine neue Reaction auf Kreatinin und Kreatin. Ber. deutsch. chem. Gesellsch., n, 2175-77. WORNER, E. (1899). Beitrdge zur Kenntniss des Kreatinins. Zeitschr. physiol. Chem., 27, 1-13. WOLF, C. G. L. (1911). Creatine and creatinine metabolism. J. Biol. Chem., 10, 473-78. WOOD, J. K. (1903). The affinities of some feebly basic substances. J. Chem. Soc., 83, 568-78. B. GLYOCYAMINE AND GLYCOCYAMIDINE. KORNDORFER, G. (1905). Glycocyamin und Glycocyamidin. Arch. Pharm., 242, 620-40. NENCKI, M., und N. SIEBER (1878). Ueber eine neue Synthese des Glykocyamins. J. prakt. Chem., 125, 477-80. RAMSAY, H. (1908). Neue Darstellung der Glykocyamine oder Guanidosduren. Ber. deutsch. chem. Gesellsch., 41, 4385-93. C. GUANIDINE. BE"CHAMP (1857). Essai sur les substances albumino'ides et leur transformation en uree. J. Pharm. Chim., [iii.], 31, 32-45. CAMIS, M. (1909). Physiological and histological observations on muscle, chiefly in relation to the action of guanidine. J. Physiol., 39, 73-97- EMICH, F. (1891). Notizen uber das Guanidin. Monatsh., 12, 23-28. FtiHNER, H. (1908, 2). Die periphere Wirkung des Guanidins. Arch. exp. Pathol. Pharm., 58, 1-49. GERGENS, E., und E. BAUMANN (1876). Ueber das Verhalten des Guanidins, Dicyan- diamidin und Cyanamid im Organismus. Pfliiger's Archiv, 12, 205-14. KUTSCHER, F., und J. OTORI (1904). Der Nachweis des Guanidins unter den bei der Selbstverdauung des Pankreas entstehenden Korpern. Zeitschr. physiol. Chem., 43, 93-108. KUTSCHER, F., und M. SCHENCK (1905). Die Oxydation von Eiweissstojfen mit Cal- ciumpermanganat (Die Oxydation von Leim). Ber. deutsch. chem. Gesellsch., 38, 455-59. BIBLIOGRAPHY OF CHAPTER V 197 KUTSCHER, F., und SEEMANN (1903). Die Oxydation der Thymusnucle'insdure mit Calciumpermanganat. Ber. deutsch. chem. Gesellsch., 36, 3 2 3-27- LIPPMANN, E. O. VON (1896). Ueber stickstoffhaltige Bestandteile der Rubensdften. Ber. deutsch. chem. Gesellsch., 29, 2645-54. LOSSEN, F. (1880). Guanidin ein Oxydationsproduct des Eiweisses ; Beitrag zur Frage der Harnstoffbildung. Liebig's Annalen, 201, 3 6 9"7 6 - OTORI, J. (1904, l). Die Spaltung des Pseudomucins durch starke, siedende Sduren. Zeitschr. physiol. Chem., 43, 74-85. OTORI, J. (1904, 2). Die Oxydation des Pseudomucins und Caseins mit Calciumper- manganat. Zeitschr. physiol. Chem., 43, 86-92. SCHENCK, M. (1904). Zur Kenntniss einiger physiologisch wichtiger Substanzen. Zeitschr. physiol. Chem., 43, 72-73. SCHENCK, M. (1905, 2). Ueber das Guanidinpikronolat. Zeitsch. physiol. Chem., 44, 427. SCHULZE, E. (1892, 2). Ueber das Vorkommen von Guanidin im PJlanzenorganismus. Ber. deutsch. chem. Gesellsch., 25, 658. SCHULZE, E. (1892, 3). Zum Nachweis des Guanidins. Ber. deutsch. chem. Gesellsch., 25, 658. D. AND E. METHYL- AND DIMETHYLGUANIDINE. ACHELIS, W. (1906). Ueber das Vorkommen von Methylguanidin im Harn. Zeitschr. physiol. Chem., 50, 10-20. ACKERMANN, D. (1906). Benzolsulfomethylguanidin. Zeitschr. physiol. Chem., 48, 382. BOCKLISCH, O. (1887). Ueber Ptomaine aus Reinculturen von Vibrio Proteus (Finkler und Prior). Ber. deutsch. chem. Gesellsch., 20, 1441-46. BRIEGER, L. (1886, l). Untersuchungen uber Ptomaine. Dritter Theil. Berlin, August Hirschwald. DEMJANOWSKI, S. (1912). Zur Kenntnis der Extraktivstoffe der Muskeln. XIII. Ueber die Fdllbarkeit einiger stickstoffhaltigen Extraktivstoffe durch Phosphorwolframsaure und Quecksilberoxydsalze. Zeitschr. physiol. Chem., 80, 212-17. ENGELAND, R. (1908, 3). Ueber den Nachweis organischer Basen im Harn. Zeitschr. physiol. Chem., 57, 49-65. GULEWITSCH, WL. (1906). Zur Kenntniss der Extraktivstoffe der Muskeln. III. Ueber das Methylguanidin. Zeitschr. physiol. Chem., 47, 471-75. HEYDE, M. (1911). Ueber den Verbrennungstod und seine Beziehungen zum an- aphylaktischen Shok. Zentralbl. f. Physiol., 25, 441-44. HEYDE, M. (1912). Weitere Untersuchungen uber die Beziehungen der Guanidine und Albumosen zum parenteralen Eiweisszerfall und anaphylaktischen Schock. Zentralbl. f. Physiol., 26, 401-4. KOCH, W. F. (1912). On the occurrence of methylguanidin in the urine of para- thyroidectomized animals. J. Biol. Chem., 12, 313-15. KRIMBERG, R. (1906, i). Zur Kenntnis der Extraktivstoffe der Muskeln. IV. Ueber das Vorkommen des Carnosins, Carnitins, und Methylguanidins im Fleisch. Zeitschr. physiol. Chem., 48, 412-18. KUTSCHER, F. (1905, I ; 1907, i). Zur Kenntniss von Liebig's Fleischextrakt. Zentralbl. f. Physiol., 19, 504-8 ; 21, 33-35. KUTSCHER, F., und A. LOHMANN (1906, 3, 4), Der Nachweis toxischer Basen im Harn. Zeitschr. physiol., 48, 422-24 ; 49, 81-87. 198 THE SIMPLER NATURAL BASES LOEWIT, M. (1913). Die anaphylaktische und anaphylaktoide Vergiftung beim Meer- schweinchen. Arch. exp. Path. Pharm., 73, 1-32. SMORODINZEW, J. (1912). Ueber die stickstoffhaltigen Extraktivstoffe der Lcber. Zeitschr. physiol. Chem., 80, 218-31. WHEELER, H. L., and G. S. JAMIESON (1907). On some picrolonates : guanidins. J. Bio^ Chem., 4, 111-17. REFERENCES TO CHAPTER VI. ADRENALINE. A. GENERAL. BAYER, G. (1910). Die normale und pathologische Physiologic des chromaffinen Gewebes der Nebennieren. Sonderabdruck aus Lubarsch-Ostertag, Ergebnisse der pathologischen Anatomic, 14, 1-132. BIEDL, A. (1913). Inner e Sekretion, ihre physiologische Bedeutungfur die Pathologic. Urban und Schwarzenberg, Berlin und Wien, 2nd ed., pp. 313-521. FURTH, O. VON (1904). Sammelreferat : Neuere Untersuchungen uber die chemisette Zusammensetzung der gefdssverengernden Substanz in der Nebenniere. Biochem. Zentralbl., 2, 1-9. GOLDZIEHER (1911). Die Nebennieren. Wiesbaden (quoted by Borberg). LANGLOIS, J. P. (1897). Les Capsules surrenales. These de Paris, chez Felix Alcan. (Full account of earlier literature.) ROLLESTON, H. D. (1895). The Goulstonian Lectures on the suprarenal bodies. Brit. Med. J., L, 629-34, 687-91, 745-48 (with bibliography of 178 references). VINCENT, SWALE (1910). Innere Sekretion und Drusen ohne Ausfuhrungsgang. Die innere Sekretion der Nebennieren. Ergebnisse der Physiologie, 9, 509-86. B. SPECIAL. ABDERHALDEN, E., und P. BERGELL (1904). Zur Kenntniss des Epinephrins (Adren- alins). Ber. deutsch. chem. Gesellsch., 37, 2022-24. ABDERHALDEN, E., und M. GUGGENHEIM (1908). Weitere Versuche uber die Wirkung der Tyrosinase aus Russula delica auf tyrosinhaltige Polypeptide und auf Suprarenin. Zeitschr. physiol. Chern., 57, 329-31. ABDERHALDEN, E., und K. KAUTZSCH (1909). Weitere Studien uber das physiologische Verhalten von I. und d. Suprarenin, Zeitschr. physiol. Chem., 61, 119-23. ABDERHALDEN, E., K. KAUTZSCH, und FR. MULLER (1909). Weitere Studien uber das physiologische Verhalten von I. und d. Suprarenin. Zeitschr. physiol. Chem., 62, 404-9. ABDERHALDEN, E., und FR. MULLER (1908). Ueber das Verhalten des Blutdruckes nach intravenoser Einfuhrung von I., d., und dl. Suprarenin. Zeitschr. physiol. Chem., 58, 185-88. ABDERHALDEN, E., und SLAVU (1909). Weitere Studien uber das physiologische Ver- halten von /., d., und dl. Suprarenin. Zeitschr. physiol. Chem., 59, 129-37. ABDERHALDEN, E., und F. THIES (1909). Weitere Studien uber das physiologische Ver- halten von 1., d., und dl. Suprarenin. Zeitschr. physiol. Chem., 59, 22-28. ABEL, J. J. (1898). further observations on the chemical structure of the active principle of the suprarenal capsule. Johns Hopkins Hospital Bulletin, 9, 215-18. 199 200 THE SIMPLER NATURAL BASES ABEL, J. J. (1899). Ueber den blutdruckerregenden Bestandtheil der Nebenniere, das Epinephrin. Zeitschr. physiol. Chem., 28, 318-62. ABEL, J. J. (1901, i). Further observations on eplnephrin. Johns Hopkins Hospital Bulletin, 12, 80-84. ABEL, J. J. (1901, 2). The behaviour of epinephrin to Fehling's solution and other characteristics of this substance. Johns Hopkins Hospital Bulletin, 12, 337-43- ABEL, J. J. (1902, i). On a simple method of preparing epinephrin and its compounds. Johns Hopkins Hospital Bulletin, 13, 29-36. ABEL, J. J. (1902, 2). On the elementary composition of adrenalin. Proc. Amer. Physiol. Soc., xxix. ; Amer. J. Physiol., 8. ABEL, J. J. (1902, 3). On the behaviour of extracts of the suprarenal gland towards Fehling's solution. Proc. Amer. Physiol. Soc., xxx. ; Amer. J. Physiol., 8. ABEL, J. J. (1902, 4). On the oxidation of epinephrin and adrenalin with nitric acid. Proc. Amer. Physiol. Soc., xxxi. ; Amer. J. Physiol., 8. ABEL, J. J. (1903, i). Weitere Mettheilungen uber das Epinephrin. Ber. deutsch. chem. Gesellsch., 36, 1839-47. ABEL, J. J. (1903, 2). On epinephrin and its compounds, with especial reference to epinephrin hydrate. Amer. J. Pharm., 75, 301-25. ABEL, J. J. (1904). Darstellungen und Eigenschaften eines Abbauproductes des Epine- phrins. Ber. deutsch. chem. Gesellsch., 37, 368-81. ABEL, J. J., and A. C. CRAWFORD (1897). On the blood-pressure raising constituent of the suprarenal capsule. Johns Hopkins Hospital Bulletin, 8, 151-57. ABEL, J. J., and D. I. MACHT (1911). The poison of the tropical toad, Bufo Agua. J. Amer. Med. Assoc., 56, 1531-36. ABEL, J. J., and D. I. MACHT (1912). Two crystalline pharmacological agents ob- tained from the tropical toad, Bufo Agua. J. Pharm. and Exp. Therap., 3, 319-77. ABEL, J. J., and R. DE M. TAVEAU (1905). On the decomposition products of epine- phrin hydrate. J. Biol. Chem., i, 1-32. ABELOUS, J. E., A. SouLiE 1 , et G. TOUJAN (1905). Dosage colorimetrique de Vadrenaline. Compt. rend. Soc. de Biol., 57, 301-2. ADDISON, THOMAS (1849). Communication at a meeting of the South London Medical Society, 6 March, 1849. In London Medical Gazette, 8, 517-18; see also A collection of the published writings of Thomas Addison. New Sydenham Society, 1868, pp. 211-39. ALDRICH, T. B. (1901). A preliminary report on the active principle of the suprarenal gland. Amer. J. Physiol., 5, 457. ALDRICH, T. B. (1902). Is adrenalin the active principle of the suprarenal gland ? Amer. J. Physiol.., 7, 359-68. ALDRICH, T. B. (1905). Adrenalin, the active principle of the suprarenal gland. J. Amer. Chem. Soc., 27, 1074-91. ANREP, G. VON (1912). On the part played by the suprarenals in the normal vascular reactions of the body. J. Physiol., 45, 307-17- ARGYLL CAMPBELL, J. (1911). The effects of certain animal extracts upon the blood- vessels. Quart. J. Exp. Physiol., 4, 1-17. ARNOLD, J. (1866). Ein Beitrag zu der feineren Structur und dem Chemismus der Nebennieren. Virchow's Archiv f. path. Anat. u. Physiol., 35, 64-107. ASHER, L. (1912). Die innere Sekretion der Nebenniere und deren Innervation. Zeitschr. f. Biol., 58, 274-304. BIBLIOGRAPHY OF CHAPTER VI 201 BARGER, G. (1908). The action of phosphorus pentachloride on the methylene ethers of catechol derivatives. Part III. The cyclic carbonates of dichloro-ethyl- and-propyl- catechol. J. Chem. Soc., 93, 2081-85. BARGER, G., and H. H. DALE (1910, i). Chemical structure and sympathomimetic action of amines. J. Physiol., 41, 19-59. BARGER, G., and A. J. EWINS (1906). Note on the molecular weight of epinephrine. Proc. Chem. Soc., 22, 38-39- BARGER, G., and H. A. D. JOWETT (1905). The synthesis of substances allied to epine- phrine. J. Chem. Soc., 87, 967-74. BATELLI, F. (1902). Dosage colorimetrique de la substance active des capsules surrenales. Compt. rend. Soc. de Biol., 54, 571-73- BAYER, G. (1909). Methoden zur Verscharfung von Adrenalin- und Brenzcatechinreak- tionen. Biochem. Zeitschr., 20, 178-88. BERTRAND, G. (1904, i). Sur la composition chimique et laformule de V adrenaline. Bull. Soc. Chim., [iii.], 31, 1188-93. BERTRAND, G. (1904, 2). Sur les characteres physiques de V adrenaline. Bull. Soc. Chim., [iii.], 31, 1289-92. BIBERFELD, J. (1908). Synthetic suprarenin. Pharm. J., [iv.], 26, 626. BLUM, F. (1901). Ueber Nebennierendiabetes. Deut. Arch. f. klin. Med., 71, 146-67. BOAS, K. (1909). Zur Methodik des Adrenalinnachweises. Zentralbl. Physiol., 22, 825-26. BOTTCHER, K. (1909). Eine neue Synthese des Suprarenins und verwandter Verbindungen. Ber. deutsch. chem. Gesellsch., 42, 253-66. BORBERG, N. C. (1912). Das Adrenalin und der Nachweis desselben. Skand. Arch. f. Physiol., 27, 341-420. BORUTTAU, H. (1899). Erfahrungen uber die Nebennieren. PflUger's Archiv, 78, 97-128. BRODIE, T. G., and W. E. DIXON (1904). Contributions to the physiology of the lungs. Part II. On the innervation of the pulmonary blood-vessels and some observations on the action of suprarenal extracts. J. Physiol., 30, 476-502. BROWN-SEQUARD, E. (1856, i, 2). Recherches experimentales sur la physiologie et la patho- logic des capsules surrenales. Compt. rend., 43, 422-25; ibid. 542-46. BROWN-S&QUARD, E. (1857). Nouvelles recherches sur ^importance des fonctions des capsules surrenales. Compt. rend., 45, 1036-39. BRUNNER (1892). Schweiz. Wochenschr. f. Pharmacia, 30, 121-23. (Quoted by Abel.) CAMERON, I. D. (1906). On the methods of standardising suprarenal preparations. Proc. Roy. Soc., Edinb., 26, 157-71. CANNON, W. B., and D. DE LA PAZ (1911). Emotional stimulation of adrenal secretion. Amer. J. Physiol., 28, 64-70. CANNON, W. B., and R. G. HOSKINS (1911-12). The effect of asphyxia, hyperpncea and sensory stimulation on adrenal secretion. Amer. J. Physiol., 29, 274-79. CANNON, W. B., and H. LYMAN (1913). The depressor effect of adrenalin on arterial pressure. Amer. J. Physiol., 31, 376-98. CANNON, W. B., A. T. SHOHL, and W. S. WRIGHT (1911-12). Emotional glycosuria. Amer. J. Physiol., 29, 280-87. CANNON, W. B., J. C. AUB, and C. A. L. BINGER (1912). A note on the effect of nicotine injection on adrenal secretion. J. Pharm. Exp. Therap., 3, 379-85. CEVIDALLI, A. (1908). Di alcune reazoni delV adrenalina. Lo Sperimentale, 62, 787-89. 202 THE SIMPLER NATURAL BASES COMESSATTI, G. (1909). Bcitrag zum chemischen Nachweis des Adrenalins im Blutserum. Berl. klin. Wochenschr., 48, 356-58. Cow, D. (1911). Some reactions of surviving arteries. J. Physiol., 42, 125-43. CRAWFORD, A. C. (1907). The use of suprarenal glands in the physiological testing of drug plants. U.S. Depart, of Agric., Bureau of Plant Industry, Bulletin No. 112. Washington. CUSHNY, A. R. (1908). The action of optical isomers. III. Adrenalin. J. Physiol., 37, 130-38. CUSHNY, A. R. (1909). Further note on adrenalin isomers. J. Physiol., 38, 259-62. CYBULSKI, N. (1895). Weitere Untersuchungen iiber die Funktion der Nebenniere. Bull, intern, de 1'Acad. des Sciences de Cracovie ; Classe d. sc. math, et nat., 82-91. DAKIN, H. D. (1905, i). The physiological action of synthetical substances allied to adrenalin. Proc. Physiol. Soc., 25 February, xxxiv.-xxxvi., J. Physiol., 32. DAKIN, H. D. (1905, 2). The synthesis of a substance allied to adrenalin. Proc. Roy. Soc., ser. B, 76, 491-97. DAKIN, H. D. (1905, 3). On the physiological activity of substances indirectly related to adrenalin. Proc. Roy. Soc., ser. B, 76, 498-503. DALE, H. H. (1906). On some physiological actions of ergot. J. Physiol., 34, 163-206. DALE, H. H., and P. P. LAIDLAW (1912, 2). The significance of the suprarenal capsules in the action of certain alkaloids. J. Physiol., 45, 1-26. DIXON, W. E., and F. RANSOM (1912). Broncho-dilator nerves. J. Physiol., 45, 413-28. EHRMANN, R. (1905). Ueber eine physiologische Wertbestimmung des Adrenalins und seinen Nachweis im Blut. Arch. exp. Path. Pharm., 53, 97-111. ELLIOTT, T. R. (1905). The action of adrenaline. J. Physiol., 32, 401-67. ELLIOTT, T. R., and H. E. DURHAM (1906). Subcutaneous injections of Adrenalin. J. Physiol., 34, 490-98. ELLIOTT, T. R. (1912). The control of the suprarenal glands by the splanchnic nerves. J. Physiol., 44, 374-409. ELLIOTT, T. R. (1913). Notes on the quantitative estimation of adrenaline. J. Physiol., 46, xv-xvii ; Proc. Physiol. Soc. EMBDEN, G., und O. VON FURTH (1904). Ueber die Zerstorung des Suprarenins (Adre- nalins) im Organismus. Beitr. z. chem. Physiol. Path., 4, 421-29. EWINS, A. J. (1910). Some colour reactions of adrenine and allied bases. J. Physiol., 40, 317-26. EWINS, A. J., and P. P. LAIDLAW (1910, i). The alleged formation of adrenine from tyrosine. J. Physiol., 40, 275-78. FALTA, W., und L. Ivcovic* (1909). Ueber die Wirkungsweise des Adrenalins bei verschiedener Applikation und das Auftreten desselben im Harn. Wiener klin. Wochenschr., 22, 1780-83. FARBWERKE VORM. MEISTER, Lucius UND BRUNING (1904). D.R.P. 152814 applied for 15 August, 1903. Chem. Zentralbl., ii., 270. FENGER, F. (1912). On the presence of active principles in the thyroid and suprarenal glands before and after birth. J. Biol. Chem., u, 489-92 ; 12, 55-59- FLACHER, F. (1908). Ueber die Spaltung des synthetischen dl-Suprarenins in seine optisch aktiven Komponenten. Zeit. physiol. Chem., 58, 581-89. FoA, P., e P. PELLACANI (1884). Sul fermento fibrigeno e sulle azioni tossiche esercitate da alcuni organi freschi. Archivio per le scienze mediche, 7, 113-66 (157-66). Abstracted in Archives italiennes de biologic, 1883, 4, 56-63. BIBLIOGRAPHY OF CHAPTER VI 203 FOLIN, O., and W. DENIS (1912). On phosphotungstic-phosphomolybdic compounds as colour reagents. J. Biol. Chem., 12, 239-43. FOLIN, O., W. B. CANNON, and W. DENIS (1913). A new colorimetric method for the determination of epinephrin. J< Biol. Chem., 13, 477' 8 3- FRANKEL, A. (1909). Ueber den Gehalt des Blutes an Adrenalin bei chronischer Nephritis und Morbus Basedowii. Arch. exp. Path. Pharm., 60, 395-407. FRANKEL, S., und R. ALLERS (1909). Ueber eine neue, charakteristische Adrenalin- reaktion. Biochem. Zeitschr., 18, 40-43. FRIEDMANN, E. (1904). Zur Kenntniss des Adrenalins (Suprarenins). Beitr. chem. Physiol. Pathol., 6, 92-93. FRIEDMANN, E. (1906). Die Konstitution des Adrenalins. Beitr. chem. Physiol. Pathol., 8, 95-120. FROHLICH, A. (1909). Eine neue physiologische Eigenschaft des d. Suprarenins. Zentralbl. f. Physiol., 23, 254-56. FORTH, O. VON (1898, I, 2 ; 1900). Zur Kenntniss der brenzkatechindhnlichen Substanz in den Nebennieren. /., //., ///. Zeit. physiol. Chem., 24, 142-58 ; 26, 15-47 '> 29* 105-23. FURTH, O. VON (1901). Zur Kenntniss des Suprarenins. Beitr. z. chem. Physiol. Path., I, 243-51. FURTH, O. VON (1903). Zur Kenntniss des Suprarenins (Adrenalins). Monatsh., 24, 261-90. GASKELL, J. F. (1912). The distribution and physiological action of the suprarenal medullary tissue in Petromyzonfluviatilis. J. Physiol., 44, 59-67. GESSARD, C. (1904). Sur le pigment des capsules surrenales. Compt. rend., 138, 586-88. GOLLA, F. L., and W. L. SYMES (1913). The reversible action of adrenaline and some kindred drugs on the bronchioles. J. Pharmac. exp. Therapeutics, 5, 87-103. GOTTLIEB, R. (1897). Ueber die Wirkung der N ebennierenextrakte auf Herz und Blut- druck. Arch. exp. Path. Pharm., 38, 106-12. GOTTLIEB, R., und J. M. O'CONNOR (1912). Ueber den Nachweis und die Bestimmung des Adrenalins im Blute. Handbuch der biochemischen Arbeitsmethoden, Band VI., 585-603. Urban und Schwarzenberg, Berlin und Wien. GUGGENHEIM, M. (1913). Dioxyphenylalanin, eine neue Aminosdure aus Vicia Faba. Zeitschr. physiol. Chem., 88, 276-84. GUNN, J. A. (1911). Adrenin-like actions of cobra venom. Quart. J. Exp. Physiol., 5, 67-81. GUNN, A., and E. F. HARRISON (1908). The coloration of adrenine solutions. Pharm. J., [iv.], 26, 5i3- I 4- GURBER, A. (1897). Zur Kenntniss der wirksamen Substanzen der Nebenniere. Sitzungsber. d. physikal-medicin. Gesellsch. in Wiirzburg, 4, 54-57. HALE, WORTH, and ATHERTON SEIDELL (1911). Colorimetric and physiological estimation of the active principle of the suprarenal gland. Amer. J. Pharm., 83, 551-58. HALLE, W. L. (1906). Ueber die Bildung des Adrenalins im Organismus. Beitr. chem. Physiol. Pathol., 8, 276-80. HANDOVSKY, H., und E. P. PICK (1912). Ueber die Entstehung vasokonstriktorischer Substanzen durch Verdnderung der Serum kolloide. Arch. exp. Path. Pharm., 71, 62-68. HOLM, F. (1867). Ueber die chemischen Bestandtheile der Nebennieren. J. prakt. Chem., i, 150-52. HOSKINS, R. G. (iqn). A consideration of some biologic tests for epinephrin. J. Pharm. Exp. Therap., 3, 93-99. 204 THE SIMPLER NATURAL BASES HOUGHTON, E. M. (1901). The pharmacological assay of preparations of the suprarenal glands. Amer. J. Pharm., 73, 531-35. HOUGHTON, E. M. (1902). The pharmacology of the suprarenal gland and a method of assaying its products. J. Amer. Med. Assoc., 38, 150-53. HUNT, REID (1906). The comparative physiologic activity of some commercial suprarenal products. J. Amer. Med. Assoc., 47, 790-92. ITAMI, S. (1912). The action of carbon dioxide on the vascular system. J. Physiol., 45, 338-43. JACKSON, D. E. (1909). The prolonged existence of adrenaline in the blood. Amer. J. Physiol., 23, 226-45. JACKSON, D. E. (1912). The pulmonary action of the adrenal gland. J. Pharm. Exp. Therap., 4, 59-74. JANUSCHKE, H., und LEO POLLAK (1911). Zur Pharmakologie der Bronchialmusku- latur. Arch. exp. Path. Pharm., 66, 205-20. JOWETT, H. A. D. (1904). The constitution of epinephrine. ]. Chem. Soc., 85, 192-97. KEHRER, E. (1907). Physiologische und pharmakologische Untersuchungen an den uberlebenden und den lebenden inneren Genitalien. Arch. f. Gynakologie, 8l, 160-210. KEHRER, E. (1908). Der uberlebende Uterus als Testobjekt fur die Wertigkeit der Mutterkornprdparate. Arch. exp. Path. Pharm., 58, 366-85. KRAUSS, L. (1909). Die Jodsaurereaktion des Adrenalins. Biochem. Zeitschr., 22, 131. KRETSCHMER, W. (1907). Dauernde Blutdmcksieigerung durch Adrenalin und uber den Wirkungsmechanismus des Adrenalins. Arch. exp. Path. Pharm., 57, 423-40. KRUKENBERG, C. FR. W. (1885). Die farbigen Derivate der Nebennierenchromogene. Virchow's Archiv f. path. Anat. u. Physiol., 101, 542-61. LAWEN, A. (1903-4). Quantitative Untersuchungen uber die Gefasswirkung von Suprarenin. Arch. exp. Path. Pharm., 51, 415-40. LANGLEY, J. N. (1901). Observations on the physiological action of extracts of the suprarenal bodies. J. Physiol., 27, 237-56. LESAGE, J. (1904, i, 2). Toxidte de I'adrenaline- en injection intraveineuse pour le chien. Compt. rend. Soc. de Biol., 56, 632-34 ; . . . pour le chat, ibid., 56, 665-66. LEWANDOWSKY, M. (1908). Ueber eine Wirkung des Nebennierenextractes auf das Auge. Zentralbl. f. Physiol., 12, 599-600. LEWANDOWSKY, M. (1899). Ueber die Wirkung des Nebennierenextractes auf die glatten Muskeln,im Besonderen des Auges. Arch. f. Anat. u. Physiol. (Physiol. Abteil.), 360-66. LEWANDOWSKY, M. (1900). Wirkung des N ebennierenextraktes auf die glatten Muskeln der Haut. Zentralbl. f. Physiol., 14, 433-35. LOEWI, OTTO, und HANS MEYER (1905). Ueber die Wirkung synthetischer, dem Adrenalin verwandter Stoffe. Arch. exp. Path. Pharm., 53, 213-26. MANNICH, C. (1910). Studien in der Reihe des Adrenalins. Arch. Pharm., 248, 127-71. MELTZER, S. J., and CLARA MELTZER-AUER (1904, i). The effect of suprarenal extract upon the pupils of frogs. Amer. J. Physiol., II, 449-54. MELTZER, S. J., und KLARA MELTZER-AUER (1904, 2). Ueber den Einfluss der Nebennieren- extrakte auf die Pupille des Frosches. Zentralbl. f. Physiol., 18, 317-18. BIBLIOGRAPHY OF CHAPTER VI 205 METZGER (1897). Zur Kenntniss der wirksamen Substanzen der Nebenniere. Diss. Wiirzburg (quoted by Abel). MEYER, HANS M. (1904). Zur Konstitution und Synthese des Suprarenins (Adrena- lins). Zentralbl. f. Physiol., 18, 501. MEYER, O. B. (1906). Ueber einige Eigenschaften der Gefassmuskulatur mit besonderer Berucksichtigung der Adrenalinwirkung. Zeitschr. f. Biol., 48, 352-97. MOORE, B. (1895). On the chemical nature of a physiologically active substance occurring in the suprarenal gland. Proc. Physiol. Soc., 16 March, xiv.-xvii. ; J. Physiol., 17. MOORE, B. (1897). On the chromogen and on the active physiological substance of the suprarenal gland. J. Physiol., 21, 382-89. MtJHLMANN, M. (1906). Zur Physiologic der Nebenniere. Deutsch. med. Wochenschr., 22, 409-12. MULON, P. (1905). Sur la reaction osmique de la medullaire des surrenales. Compt. rend. Soc. de Biol., 57, 757-58. NEUBERG, C. (1908). Enzymatische Umwandlung von Adrenalin. Biochem. Zeitschr., 8, 383-86. O'CONNOR, J. M. (1911). Ueber Adrenalinbestimmung im Blute. Muench. med. Wochenschr., 58, 1439-42. O'CONNOR, J. M. (1912, i). Ueber den Adrenalingehalt des Blutes. Arch. exp. Path. Pharm., 67, 195-232. O'CONNOR, J. M. (1912, 2). Ueber die Abhangigkeit der Adrenalinsekretion vom Splanch- nicus. Arch. exp. Path. Pharm., 68, 383-93. OGAWA, S. (1912). Beitrage zur Gefasswirkung des Adrenalins. Arch. exp. Path. Pharm., 67, 89-110. OLIVER, G., and E. A. SCHAFER (1894, l8 95 *) On ine physiological action of extracts of the suprarenal capsules. Proc. Physiol. Soc., 10 March, i.-iv., J. Physiol., 16; ibid. 16 March, ix.-xiv., J. Physiol., 17. OLIVER, G., and E. A. SCHAFER (1895, 2). The physiological effects of extracts of the suprarenal capsules. J. Physiol., 18, 230-76. PANCRAZIO, F. (1909). 77 persolfato di sodio rivelatore delV adrenalina. Gazz. degli ospedali e delle cliniche, 30, 1513-14. PANCRAZIO, F. (1910). Pel dosaggio delV adrenalina. Padova, Tip. Elzeviriana, 1899. Quoted in Gazz. degli ospedali e delle cliniche, 31, 190. PATON, D. NOEL (1903). On the nature of adrenalin glycosuria. J. Physiol., 29, 286-301. PATON, D. NOEL (1904). The effect of adrenalin on sugar and nitrogen excretion in the urine of birds. J. Physiol., 32, 59-64. PAULY, H. (1903). Zur Kenntniss des Adrenalins. Ber. d. deutsch. chem. Gesellsch., 36, 2944-49. PAULY, H. (1904). Zur Kenntniss des Adrenalins. II. Ber. d. deutsch. chem. Gesellsch., 37, 1388-1401. PAULY, H. (1909). Bemerkungen zu der Abhandlung des Herrn Bottcher. Eine neue Synthese des Suprarenins und verwandter Verbindungen. Ber. d. deutsch. chem. Gesellsch., 42, 484-85. PAULY, H., und K. NEUKAM (1908). Ueber einige Derivate des Athylbrenzcatechins. Ber. d. deutsch. chem. Gesellsch. 41, 4151-61. PELLACANI, P. (1879). Intorno agli effetti tossici delle diluzioni acquose degli organi freschi introdotte nelV organismo di alcuni animali. Archivio per le Scienze mediche, 3, No. 24, 1-32. POLLAK, L. (1909). Experimented Studien ueber Adrenalin Diabetes. Arch. exp. Path. Pharm., 6l, 149-73. 206 THE SIMPLER NATURAL BASES POLLAK, L. (1910). Zur Frage der Adrenalingewbhnung. Zeitschr. physiol. Chem., 68, 69-74. SCHULTZ, W. H. (1909, i). Quantitative pharmacological studies : Adrenalin and adrenalin- like bodies. Hygienic Laboratory Bulletin, No. 55. Washington. SCHULTZ, W. H. (1909, 2). Experimental criticism of recent results in testing adrena- lin. J. Pharm. Exp. Therap., I, 291-302. SCHULTZ, W. H. (1910). Quantitative pharmacological studies : Relative physiological activity of some commercial solutions of epinephrin. Hygienic Laboratory Bulletin, No. 61. Washington. SCHUR, H. (1909). Ueber eine neue Reaktion im Hani. Wiener klin. Wochenschr., 22, 1587-88. SOLLMAN, T., and E. D. BROWN (1906). The comparative physiologic activity of some commercial suprarenal preparations. J. Amer. Med. Assoc., 47, 792-93. STEWART, G. N. (1912). The alleged existence of adrenalin (epinephrin) in pathological sera. J. Exp. Med., 15, 547-69. STOLZ, FR. (1904). Ueber Adrenalin und Alkylaminoacetobrenzcatechin. Ber. d. deutsch. chem. Gesellsch., 37, 4149-54. STRAUB, W. (1909). Mechanismus der Adrenalinglykosurie. Muench. med. Wochenschr., 56, 403-4. SZYMONOWICZ, L. (1895). Ueber die Erscheinungen nach der Nebennierenexstirpation bet Hunden und uber die Wirkung der Nebennierenextracte. Bull, intern, de 1'Acad. des Sciences de Cracovie. Classe d. sc. math, et nat., 56-58. SZYMONOWICZ, L. (1896). Die Funktion der Nebenniere. Pfluger's Archiv, 64, 97-164. TAKAMINE, J. (1901, i). English Patent No. 1467 0/22 Jan. 1901. J. Soc. Chem. Ind., 20, 746. TAKAMINE, J. (1901, 2). Adrenalin, the active principle of the suprarenal glands and its mode of preparation. Amer. J. Pharm., 73, 523-31. TAKAMINE, J. (1901, 3). The isolation of the active principle of the suprarenal gland. Proc. Physiol. Soc., 14 Dec., xxix.-xxx. ; J. Physiol., 27. TAKAMINE, J. (1901, 4). The blood-pressure raising principle of the suprarenal glands a preliminary report. Therapeutic Gazette, 25, 221-24. TRENDELENBURG, P. (1910). Bestimmung des Adrenalingehaltes im normalen Blut sowie beim Abklingen der Wirkung einer einmaligen intravenosen Adrenalininjektion mittels physiologischer Messmethode. Arch. exp. Path. Pharm., 63, 161-76. VINCENT, SWALE (1897-98). On the general physiological effects of extracts of the supra- renal capsules. J. Physiol., 22, ni-20. VIRCHOW, R. (1857). Zur Chemie der Nebennieren. Virchow's Archiv f. path. Anat. u. Physiol., 12, 481-83. VULPIAN (1856, i). Notesur quelques reactions propres a la substance des capsules surrenales. Compt. rend., 43, 663-65. VULPIAN (1856, 2). Note sur quelques reactions propres au tissu des capsules surrenales chex les Reptiles. Compt. rend. Soc. de Biol., 3, 223. WATERMAN, N. (1909). Ueber einige Versuche mit Rechtssuprarenin. Zeit. physiol. Chem., 63, 290-94. WATERMAN, N. (1911). Zur Frage der Adrenalinimmunitat. Zeitschr. physiol. Chem., 74, 273-81. WEIDLEIN, E. R. (1912). Epinephrin in the whale. J. Industr. and Engin. Chem., 4, 636-45 ; Chem. Zentralbl., 1913, i., 816. WEISS, O., und J. HARRIS (1904). Die Zerstorung des Adrenalins im lebenden Tier. Pfluger's Archiv, 103, 510-14. BIBLIOGRAPHY OF CHAPTER VI 207 WIGGERS, C. J. (1909). The action of adrenalin on the pulmonary vessels. J. Pharm. Exp. Therap., I, 341-48. ZAK, E. (1910). Experimented und klinische Beobachtungen uber Storungen sympathischer Innervationen (Adrenalin Mydriasis] und uber intestinale Glykosurie. Pfliiger's Archiv, 132, 147-74. ZANFROGNINI, A. (1909). Bine neue kolorimetrische Methode der Adrenalinbestimmung Deutsch. med. Wochenschr., 35, 1752-53. REFERENCES TO CHAPTER VII. SPERMINE. LADENBURG, A., und J. ABEL (1888). Ueber das Aethylenimin (Spermin ?). Ber. d. deutsch. chem. Gesellsch., 21, 758-66. MAJERT, W., und A. SCHMIDT (1890). Ueber das Piperazin (Hofmann's Didthylendiamin* Ladenburg's Aethylenimin, Schreiner's Spermin). Ber. d. deutsch. chem. Gesellsch., 23, 3718-23. POEHL, A. (1891). Ueber Spermin. Ber. d. deutsch. chem. Gesellsch., 24, 359-60. SCHREINER, P. (1878). Ueber eine neue organische Basis in thierischen Organismen. Liebig's Annalen, 194, 68-84. MUSCLE-, URINE-, AND PUTREFACTION BASES. ACKERMANN, D. (1908, 2). Ueber eine neue Base aus gefaultem Pankreas. Zeitschr. physiol. Chem., 57, 28-29. ACKERMANN, D. (1907, 2). Bin Beitrag zur Chemie der Fdulnis. Zeitschr. physiol. Chem., 54, 1-31. BAUM, FR. (1903). Ueber ein neues Produkt der Pankreasselbstverdauung. Beitr. chem. Phys. Path., 3, 439-41. KRIMBERG, R., und L. IZRAILSKY (1913). Zur Kenntniss der Extraktivstoffe der Muskeln* Uber das Kreatosin, eine neue Base des Fleischextraktes. Zeitschr. physiol. Chem., 88, 324-30. KUTSCHER, FR. (1906). Bemerkungen zu unserer ersten Mitteilung : Der Nachweis toxischer Basen im Harn. Zeitschr. physiol. Chem., 49, 88. KUTSCHER, FR. (1907). Zur Kenntniss von Liebig's Fleischextract II. Zentralbl. f. Physiol., 21, 33-35. SWAIN, R. E. (1903). Weiteres uber Skatosin. Beitr. chem. Physiol. Path., 3, 442-45. THE PITUITARY ACTIVE PRINCIPLE. BELL, W. BLAIR (1909). The pituitary body and the therapeutic value of the infundi- bular extract in shock, uterine atony and intestinal paresis. Brit. Med. J., ii., 1609-13. DALE, H. H. (1909). The action of extracts of the pituitary body. Biochem. J., 4, 427-47. DALE, H. H., and P. P. LAIDLAW (1912, i). A method of standardising pituitary (infundi- bular) extracts. J. Pharm. Exp.Therap., 4, 75-95. ENGELAND, R., und F. KUTSCHER (1911). Ueber einige physiologisch wichtige Substanzen A. Die physiologisch wirksamen Extraktstoffe der Hypophyse. Zeit. f. Biol., 57, 526-33. FRANKL-HOCHWART, L. VON, und A. FROHLiCH (1910). Z ' ur Kenntniss der Wirkung des Hypophysins (Pituitrins, Parke, Davies &> Co.), auf das sympathische und autonome Nervensystem. Arch. exp. Path. Pharm., 63, 347-56. FROHLICH, A., UND E. P. PICK (1913). Zur Kenntniss der Wirkung der Hypophysen- praparate. I-III. Arch. exp. Path. Pharm., 74, 92-106, 107-13, 114-18. Fi?HNER, H. (1912). Das Pituitrin und seine wirksame Bestandteile. Munch, med. Wochenschr., 59, 852-53. 208 BIBLIOGRAPHY OF CHAPTER VII 209 FtJHNER, H. (1913). Pharmakologische Untersuchungen uber die wirksamen Bestandteile der Hypophyse. Zeitschr. f. d. ges. exp. Medizin., i, 397-443. GUGGENHEIM, M. (1913). Proteinogene Amine. Peptamine : Glycyl-p-oxyphenylathylamin, Alanyl-p-oxypheny lathy lamin. Glycyl-&-imidazoly lathy lamin. Biochem. Zeitschr., 51, 369-87. HAMMOND, J. (1913). The effect of pituitary extract on the secretion of milk. Quart. Journ. exp. Physiol., 6, 311-38. HERZBERG, S. (1913). Klinische Versuche mit den isolierten wirksamen Substanzen der Hypophyse. Deutsch. med. Wochenschr., 39, 207-10. HOUGHTON, E. M., and C. H. MERRILL (1908). The diuretic action of adrenalin and the active principle of the pituitary gland. J. Amer. Med. Assoc., 51, 1849-54. MAGNUS, R., and E. A. SCHAFER (igoi). The action of pituitary extracts upon the kidney. Proc. Physiol. Soc., 20 July; J. Physiol., 27, ix. OLIVER, G., and E. A. SCHAFER (1895, 3)* On the physiological action of extracts of the pituitary body and certain other glandular organs. J. Physiol., 18, 277-79. OTT, J., and J. C. SCOTT (1911). The action of animal extracts upon the secretion of the mammary gland, Therap. Gazette, 35, 689-91. PANKOW, O. (1912). Ueber Wirkungen des Pituitrins (Parke, Dames &> Co.), auf KreislauJ und A tmung. Pfluger's Archiv, 147, 89-99. PATON, D. N., and A. WATSON (1912). The actions of pituitrin, adrenalin, and barium on the circulation of the bird. J. Physiol., 44, 413-24. SCHAFER, E. A. (1913). On the effect of pituitary and corpus luteum extracts on the mammary gland in the human subject. Quart. J. exp. Physiol., 6, 17-19. SCHAFER, E. A., and P. T. HERRING (1906). The action of pituitary extracts upon the kidney. Phil. Trans. Roy. Soc., 199, B, 1-29. SCHAFER, E. A., and K. MACKENZIE (1911). The action of animal extracts on milk secretion. Proc. Roy. Soc., 84, 16-22. VITAMINE, ORYZANIN, TORULIN. COOPER, E. A. (1913). Tnc preparation from animal tissues of a substance which cures polyneuritis in birds induced by diets of polished rice. Biochem. J., 7, 268-74. EYKMAN, C. (1897). Eine Beri Beri-ahnliche Krankheit der Huhner. Virchow's Archiv, 148, 523-32. EDIE, E. S., W. H. EVANS, B. MOORE, G. C. E. SIMPSON, and A. WEBSTER (1912). The antineuritic bases of vegetable origin in relationship to beri-beri, with a method of isolating torulin, the antineuritic base of yeast. Biochem. J., 6, 234-42. FUNK, C. (1911). On the chemical nature of the substance which cures polyneuritis in birds induced by a diet of polished rice. J. Physiol., 43, 395-400. FUNK, C. (1912, i). The preparation from yeast and certain foodstuffs of the substance the deficiency of which in diet occasions polyneuritis in birds. J. Physiol., 45, 75-81. FUNK, C. (1912, 2). Further experimental stiidies on beri-beri. The action of certain purine and pyrimidine derivatives. J. Physiol., 45, 489-92. FUNK, C. (1913). Studies on beri-beri. VII. Chemistry of the vitamine fraction from yeast and rice polishings. J. Physiol., 46, 173-79- HOPKINS, F. G. (1912). Feeding experiments illustrating the importance of accessory factors in normal dietaries. J. Physiol., 44, 425-60. 14 210 THE SIMPLER NATURAL BASES SCHAUMANN, H. (1912, i). Ueber die Darstellung und Wirkungsweise einer der in der Reiskleie enthaltenen, gegen experimentelle Polyneuritis wirksamen Substanzen. Arch. f. Schiffs- und Tropenhygiene, 16, 349-61. SCHAUMANN, H. (1912, 2). Zu dem Problem der Beri-beri Atiologie. Arch. f. Schiffs- und Tropenhygiene, 16, 825-37. SUZUKI, U., T. SHIMAMURA, und S. ODAKE (1912). Ueber Oryzanin, ein Bestandteil der Reiskleie und seine physiologische Bedeutung. Biochem. Zeitschr., 43, 89-153. SEPSINE. FAUST, E. S. (1903, 1904). Ueber das Faulnisgift Sepsin. Arch. exp. Path. Pharm., 51, 248-69. FORNET, W., und W. HEUBNER (1908, 1911). Versuche iiber die Entstehung des Sepsins. Arch. exp. Path. Pharm., Schmiedeberg Festschrift, 176-80, and 65, 428-53. SECRETINE. DALE, H. H., and P. P. LAIDLAW (1912, 3). A method of preparing secretin. Proc. Physiol. Soc., 18 May; J. Physiol., 44, xi.-xii. REFERENCES TO CHAPTER VIII. BUSCH, M. (1905). Gravimetrische Bestimmung der Salpetersaure. Ber. deutsch. chem. Gesellsch., 38, 861-66. JACOBS, W. A. (1912). A note on the removal of phosphotungstic acid from aqueous solutions. J. Biol. Chem., 12, 429-30. KOSSEL, A., und F. WEISS (1910). Vber die Einwirkung von Alkalien auf Proteinstoffe. Zeitsch. physiol. Chem., 68, 165-69. E. WECHSLER (1911). Zur Technik der Phosphorwolframsdurefdllungen. Zeitschr. physiol. Chem., 73, 138-43. 211 14* INDEX. ACETYL choline in ergot, 63. Acids produced in putrefaction, 8. Addison's disease, 81, 89. Adrenal gland, see Suprarenal Gland. Adrenaline, 81-105. Agmatine, 16, 29, 129. /3-Alanine, 34, 36, 135. Alkaloid, definition of, 6. Amanitine, 65. Amphicreatinine, 70. Amino-acids, behaviour of, in putrefaction, 7-io, 33. 7-Amino-butyric acid, 34, 135. e-Amino-caproic acid, 35. Amino-ethyl alcohol, 58, 59, 155. disulphide, 13. glyoxaline, see Iminazolyl-ethyl- amine. indole, see Indolethylamine. 8 -Ami no- valeric acid, 35, 136. Amylamines, 13, 126. Anaphylactic shock, 30, 31, 80. Aporrhegmata, 3. Arginine fraction of bases, 121. Arterenol, 86, 87, 91. Arteriosclerosis, 27, 97. Aurichlorides of abnormal composition, 123, 137, 143, 145, 147. Autolysis, difficulty of securing sterility in, io, 15, 77. BACILLUS aminophilusintestinalis,?, 25, 133. botulinus, 5. liquefaciens, 12. putrificus, 8. vulgatus, 12. Bacterium prodigiosum, 11, 12. sepsinogenes, 114. Base, definition of, 5. Betaine, 12, 40-43, 77, 78, 141-143, 150-152. Betaines, general properties of, 39, 40. Betonicine, 44, 144. Bilineurine, 54. Blood pressure, action of adrenaline on, 96, 97, 98, 102. of amines on, 26-32. of choline and neurine on, 62-64. of pituitary extracts on, no. persistent high, 25, 27. Botelus edulis, bases in, io, 13, 15, 19, 45. Bronchioles, affected by adrenaline, 99. affected by j8-iminazolyl-ethylamine, 29 32. Bufo agua, adrenaline in, 94. Butylamine, 12, 126. 7-Butyrobetaine, 39, 49, 147, 148. CADAVERINE, 14-16, 126-128. Carnitine, 50, 51, 148, 149. Carnosine, 36, 137, 138. '.phalopoda, p-hydroxy-phenyl-ethylamine in salivary gland of, 20, 28. creatine absent from, 71. creatinine absent from, 72. Cerebro-spinal fluid, alleged choline content of, in disease, 56. Cheese, bases in, 16, 19. Choline, n, 12, 54-64, 78, 150-155. physiological action, 61-63. of acetic acid ester, 63, 68. of nitrous acid ester, 68. nitric acid ester, 68, 153, 156. Chromaffin or chromophil tissue, 94. Chromogen of suprarenal gland, 81. Chrysocreatinine, 70. Cod liver oil, bases in, 12, 13, 18. Colamine, 58. Collidine, 17. Crangitine, 107. Crangonine, 107. Creatine and creatinine, 69-78, 157-163. Creatosine, 107. Curare action, 49, 65. Cyclic vomiting, 25. Cystine, amine from, 13. Cystinuria, 15. DEAMINIZATION, 8, 33, 35. Decarboxylation, conditions favouring, 7, 9, 12, 14, 16, 25. by bacteria, 7, 8, io. by ferments, io. Dimethylamine, n, 124, 125. Dimethylguanidine, 80, 165. Dissociation constant, of amino-acids, 33. of creatine, 158. of creatinine, 159. Dragendorff's reagent, 121. EPINEPHRIN, see Adrenaline, 81. Epinephrin hydrate, 82, 83. Epinine, 87, 91. Ergamine, see #-iminazolyl-ethylamine. Ergot, io, 13, 15, 16, 19, 28, 46, 63. Ergotoxine, cause of vaso-motor reversal, 98. Estimation of adrenaline, colorimetri , 92. physiological, 101-105. of amino- ethyl alcohol, 58. of betaine in the presence of choline, 150, 151- in crude sugar and molasses, 141. in plants, 141. of carnosine, 138. 213 2I 4 THE SIMPLER NATURAL BASES Estimation of choline, 150, 151. of creatine directly, 161. indirectly, as creatinine, 163. of creatinine, 70, 161-163. of guanidine, 164. of kynurenic acid, 140. of the methylamines, 124, 125. of the pituitary active principles, HI. of trigonelline, 147, 151. Ethylamine, 11. GADININE, 50. Germination, formation of betaine, 43, 55. of choline, 55 ; of guanidine, 79. of hordenine, 21. absence of primary amines, 15, 18. Glycocyamine, 78, 79, 163, 164. Glycocyamidine, 78, 79, 163, 164. Guanidine, 79, 164. Gynesine, 107. HERCYNINE, 45. Herring spawn, 16. Histamine, see Iminazolyl-ethylamine. Histidine, decarboxylation of, by bacteria, 132, 133- formation of, from carnosine, 36. fraction of bases, 121. in human urine, 37. lower homologue of, in human urine, 37. polypeptide of, in human urine, 37. preparation of, from blood, 119. Homobetaine, 51, 68. Homocholine, 68. Homomuscarine, 67. Homorenon, 87, 91. Hordenine, 20, 21, 131. physiological action, 28. p-Hydroxy-phenyl-ethylamine, 18-20, 130- 131- physiological, action, 26-28. p-Hydroxy-phenylacetic acid, 27. Hypaphorine, 47, 146. Hypophysis cerebri, 108. IGNOTINE, see Carnosine. Imidazolyl-ethylamine, see Iminazolyl-ethyl- amine. Iminazolyl-acrylic acid, 36, 46, 138, 139. Iminazolyl-ethylamine, 22-24, 132-134. physiological action, 29-32. Iminazolyl-methylamine, 24. Iminazolyl-propionic acid, 35, 137. Indolaceturic acid, 29. Indolethylamine, 21, 22, 132. physiological action, 28, 29. Iso-amylamine, 13, 126. physiological action, 26. Isobutylamine, 12, 126. Isocreatinine, 70. KRAUT'S reagent, 121. Kynosine, 107. Kynurenic acid, 37, 139, 140. Kynurine, 37. LEUCOMAINES, 6. Lycine, 40. Lysine, destructive distillation of, 128. fraction of bases, 121. in cystinuric urine, 15. in putrefaction, 14, 35. MARCITINE, 108. Meat, bases in, 107. Mercuric chloride, use in the isolation of bases, 49, 113, 114, 119, 145, 150, 158. Metchnikoffs sour milk treatment, 25. Methylamine, n, 124, 125. formation from choline by putrefaction, 153. Methylation by the animal organism, 48, 49, 77, 78, 79- Methylguanidine, 69, 79, 159, 164, 165. Methylhydantoin, 159, 160. Methylpyridinium hydroxide, 48, 49, 61. Methylpyrroline, 13. Mingine, 107. Muscarine, 64-67, 68. Muscle, bases in, 107. Mydine, 19. Myokynine, 52. NEOSINE, 68. Neurine, 54, 60, 61, 155, 156. physiological action, 64. Nicotinic acid, 48, 112. Nitric acid ester of choline, 153, 156. Nitrosocholine, nitrous acid ester of choline, 63, 68. Novaine, see Carnitine, 50, 149. DBLITINE, 51, 148, 149. Ornithine, behaviour in putrefaction, 14, 35. methylation, 52. Oryzanine, 112. Ox-ethylamine, 58, 60. Oxyneurine, 40. Oxyproline, 44, 45. PARAGANGLION aorticum, 93. Pentamethylene diamine, see Cadaverine. Peptamines, no. Periodides, 122, 125. 142, 145, 151, 152 155. Phenyl-ethylamme, 16-18, 129. physiological action, 26. Phosphotungstic acid, 6, 118, 119, 150. y-Picoline, 49. Picric acid, 123. Picrolonic acid, 123. Pituitary active principle, 108-111. Placental extracts, supposed activity of, 19. Potassium bismuth iodide, 121, 122. tri-iodide, 121, 122. Preparation of bases, general methods, 116- 123. special methods, 84, 106, 109, 113, 114, 124-165. Proline, 13, 33, 35, 44. Proteus vulgaris, 12, 25. Proto-alkaloids, 13. Pseudo-muscarine, 63, 65-67, 68. Ptomaines, 2, 5, 6, see also Putrefaction Bases. Putrefaction, 7-9, and Ch. I ; 33-35, 61, 67. bases, Ch. I; 33-35, 49, 50, 54, 61, 67, 79, 108, 113. INDEX 215 Putrescine, 14-16, 126, 127. Putrine, 108. Pyridine bases, 17, 48, 49. Pyrrolidine, 13. REDUCTION by putrefaction, 8, n, 33, 67, 153- Reductonovaine, 51. SALIVARY gland, action of adrenaline, 99. of -y-butyrobetaine, 50. of choline, 63. of p-hydroxyphenyl-ethylamine, 27. of j8-iminazolyl-ethylamine, 32. of muscarine, 66. secretes p-hydroxy-phenyl-ethylamine in Cephalopoda, 20, 28. Sarcosine, 69, 78, 159. Secretine, 114. Sepsine, 113. Silver nitrate method of separating bases, 120, 121. Sinkaline, 54. Skatosine, 108. Spermine, 106. Stachydrine, 43, 44, 143, 144. Streptococcus, production of amines by, n, J 7- Suprarenal gland, 81, 82. adrenaline content of, 92-95. Suprarenin, 82-84 ; see Adrenaline. Sympathomimetic action, 26, 98. Synthetic amines, physiological action of, 26, 28, 87. TANNIN method for purifying extracts con- taining bases, 117. Tetramethylene diamine, see Putrescine. Tetramethyl putrescine, 16, 129. Toruline, 112. Toxins of bacteria, 4-6. Trigonelline, 47, 48, 147, 150, 151. Trimethylamine, n, 12, 41, 124, 125. oxide, 67. Trimethylhistidine, 45, 46, 144, 145. Trimethyltryptophane, 47, 146. Tryptophane, bases from, 21, 22, 47. Turicine, 45, 144. Typhotoxine, 50. Tyramine, see p-Hydroxy-phenyl-ethylamine. Tyrosamines, 18. Tyrosol, 131. URINE, adrenaline in, 89. list of bases from, 107. Urocanic acid, 36, 138, 139. Urohypertensine, 27. Urohypotensine, 30. Uterus, action of adrenaline, 97, 103, 104. of agmatine, 29. of p-hydroxy-phenyl-ethylamine, 27. of j8-iminazolyl-ethylamine, 29. of pituitary, in. VASO-DILATIN, 30. Vaso-motor reversal, 63, 98. Viridine, 108. Vitamine, 111-113. Vitiatine, 107. XANTHOCREATININE, 70. YEAST, action on amines, 25, 131, betaine, 43. ABERDEEN : THE UNIVERSITY PRESS THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW BOOKS REQUESTED BY ANOTHER BORROWER ARE SUBJECT TO RECALL AFTER ONE WEEK. RENEWED BOOKS ARE SUBJECT TO IMMEDIATE RECALL LIBRARY, UNIVERSITY OF CALIFORNIA, DAVIS D4613 (12/76) I I Illll SHM