COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD HX64 104630 QP801 .K12 Biochemical studies QPZOI K/2 Columbia atotoetsitp intljeCitpoflrttigork (Eallwjr of ^yairtana anb Buv^ans Separimeni of ptjostoloiuj iPurrljajB?& bg ifjp •^ ' l * rredericS. L88, Oolnmbia IMrvsity, HevYoric. Biochemical Studies of Sulfocyanates DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIRE- MENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF PURE SCIENCE OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. BY MAX KAHN, M.A., M.D. NEW YORK CITY 1912 Easton, Pa. : eschenbach printing company. 1912. Biochemical Studies of Sulfocyanates DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIRE- MENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF PURE SCIENCE OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. BY MAX KAHN, M.A., M.D. NEW YORK CITY 1912 Easton, Pa.: Eschenbach Printing Company. 1912. Ql?20| K\2- TO MY MOTHER. CONTENTS. Tace. Acknowledgment ..,. 6 Chapter I. History 7 Chapter II. The ferric chloride -ether test 33 Substances that give a similar color with ferric chloride or that may interfere, in general, with the sulfo- cyanate test 39 Conclusions 46 Chapter III. Comparison of quantitative methods 47 Chapter IV. The distribution of sulfocyanates in the animal body. . 52 Study of the sulfocyanate content of the salivary and other glands of the body 57 Conclusions 58 Chapter V. Metabolism and excretion of sulfocyanates 59 Effect of administration of sulfur 60 Effect of administration of sodium sulfide 63 Effect of administration of taurine 65 Effect of administration of thiourea 67 Effect of administration of acetonitrile 69 Effect of administration of thioacetic acid 69 Effect of administration of alanin 70 Effect of administration of glycocoll 71 Effect of preventing saliva from entering the stomach, upon the sulfocyanate output 71 Effect of fasting upon the output of sulfocyanates. . 72 General conclusions 73 Chapter VI. Toxic effects of potassium sulfocyanate on animals and plants 74 On frogs 74 On guinea pigs 75 On dogs 76 On white lupins 77 On timothy grass seed 78 Bactericidal properties of potassium sulfocyanate .... 79 Effect of KSCN on yeast fermentation 80 Effect of KSCN on the souring of milk 80 Conclusions 81 Bibliography 82 Biographical 84 Publications 85 ACKNOWLEDGMENT. Professor William J. Gies has suggested the experiments embodied in this dissertation; he has aided me constantly in my work with his cheering encouragement and fruitful suggestions. For his interest in my labors and for his ma- terial aid, I wish now to express to him my very sincere gratitude. By his designation I have enjoyed the use of a grant, for the purpose of this research, from the Dental Society of the State of New York. I wish to thank the entire staff of the Department of Biological Chemistry for the courtesy and cordiality which they have always shown me. M. K. Laboratory of Biological Chemistry, College op Physicians and Surgeons, Columbia University, New York, May 18, 1912. CHAPTER I. HISTORY. The slight tha,t Immanuel Kant, the great German phil- osopher, had cast upon chemistry as an inexact science has long ago been proven to be undeserved; for, of all sciences, chemistry (together with physics) stands preeminently forth as the most exact and most mathematical branch of natural philosophy. Nevertheless, if one were to take a superficial glance over the literature of certain topics in this science, one would at first assuredly become impressed with the fact that uncertainty is the fundament of this study. Particularly striking is the history of the experimentation and theorizing that have been laboriously undertaken in the study of sulfocyanate in the animal organism. Denials, refutations, contradictions and abnegations can be found for every positive statement made on this subject. To every Yea there is an emphatic Nay, which is in turn followed by a corroboration and again by doubt and interrogation. In fact over the whole mass of literature on this subject one can but make a very large question mark — and sometimes several exclamation points and asterisks. One is sometimes amazed at the ridiculous claims and un- founded deductions that certain authors make for the sulfo- cyanates. Ofttimes, the reader would fain laugh loudly at the absurd statements and pedantic phraseology that he meets with in going over certain papers on this subject. I confess that I have frequently been broadly amused at the literary antics and scientific contortions that supposedly learned men deign to adopt to prove what they consider an important point. Like the schoolmen of old, many of the contributors to this subject are industriously attempting an answer to the scientific counterpart of that most "live" of all questions: "How many angels can comfortably dance on the point of a needle." However, let us say with the old Roman — Nihil h u m a n i a m e alien u m /> uto. The history of saliva 1 is in itself a very interesting chapter 1 Hays: Med. News, 1904, lxxxiv, p. 582. 8 in the study of the development of medicine. In the days of Hippocrates, nothing almost was known of physiology, and the salivary secretions were thought to be excrementitious. Galen supposed that the saliva was carried to the mouth by certain veins. Most of the physicians of those days agreed, that saliva was composed of water, salt and spirit. It was used as a medicament in certain ailments. We are told that Vespasius, when visiting Alexandria, was requested by one of the inhabitants to deign and expectorate into his eye, in order to be relieved of blindness. The saliva was used as a balm in sores and pains and in order to remove the pits of small-pox. The athletes of ancient Greece used to soak their plams in saliva in order to attain grace and suppleness of the hands. Even now when a husky laborer wants to do an especially hard piece of work, he spits into his palms, and seems to derive much benefit therefrom. In the small villages of Russia when a child is suffering from a cutaneous disease of the face, it is taken to an "old woman" who mum- bles some words and spits several times into the mouth of the child — a very valuable and efficacious remedy, and highly aesthetic ! Perhaps, the first investigator to attempt the analysis of saliva scientifically was Hapel de la Chenaye, 1 who in 1780 published his results. In 1790, Berzelius, the great Swedish chemist, described the properties of the enzyme ptyalin, which was more fully studied by Leucho in 1828. In 1 8 14, Gottfried Rheinhold Treviranus 2 reported his results of the study of the chemistry of saliva. "I have found," he writes, 3 "in the saliva two substances which doubtlessly play an important function The second I call blood acid (Blutsaure) , which is found also in the blood and which has the following characteristics : With a saturated solution of iron in nitric acid or in dilute sulfuric acid, it reacts to give a scarlet, blood-like coloration. This reaction is also obtained upon dropping the iron solution upon the 1 Chenaye: Mem. de la Soe. Roy. de Medicine de Paris, 1780, lxxxi, p. 325- 2 Treviranus: Biologie oder Physiologic der lebenden Natur fur Naturforscher und Aerzte, 1814, iv, p. 330. 3 Treviranus: My translation. saliva. If alkalies are added to the mixture of blood acid with iron, an orange-colored precipitate is produced. With silver nitrate a black-brown precipitate forms. Potassium cyanide produces no effect. Upon this acid depends the color of the blood." The empirical name of "Blood-acid" was disliked by the chemists of those days who desired to know the structure and properties of this substance. When Porret 1 in 1820 discovered Rhodanwasserstoffsdure, the scientists endeavored to ascertain whether this acid was present in the animal organism. Tiedemann and Gmelin 2 in 1826, confirmed lire's 3 finding that the "blood acid" in the saliva was identical with the sulfocyanic acid discovered by Porret. They recommended that its presence should be tested for in all the secretions and excretions of the body. They were not sure whether urine contained it, but strongly urged further researches along that line, advising that even the perspiration be examined for that substance. Berzelius 4 at first expressed doubt, but later gave an unenthusiastic confirmatory report as to its presence in the saliva. Physiological chemists began to investigate whether the salts of hydrosulfocyanic acid are localized only in the salivary secretions, or are of wide-spread occurrence in the animal body. Researches were instituted to determine the absence or presence of this substance in the secretions of the lower animals. Rough quantitative results were published. Mitscherlich 5 had the opportunity of collecting the saliva from the parotid gland alone, in a subject who had an ex- ternal fistulous opening of that gland. He reported that the amount of potassium sulfocyanate in the parotid secretion was three parts per ten thousand. The parotid of dog, horse and sheep, he affirmed, also contained the sulfocyanate. 1 Porret: Gilbert's Ann., 1820, liii, p. 184. 1 Tiedemann and Gmelin: Die Verdauiing nach Versuchen, 1826, i, p. 9. 3 Ure: Hays, Loc. cit. 4 Berzelius: Jahresbericht, 1827, i, p. 48. 8 Mitscherlich: Chcm. Centralblatt, 1833, p. 514. IO Oehl 1 using a colorimetric method {i. e., comparing standard amounts of sulfocyanate which had been treated with ferric chloride, with the saliva which had been similarly treated) obtained figures almost identical with those of Mitscherlich. Succeeding observers found that the salt under discussion was present in various secretions and tissues. Maly 2 found traces of it in the submaxillary gland ; Longet 3 reported positive results in the sublingual secretion . Ellenberger and Hof meister * did not find the acid in the tissues of the horse, cow, sheep, hog, goat. Various laboratory workers obtained varying figures for the quantity of sulfocyanate in the mixed human saliva. Wright 5 in his extensive studies on the physiology and pathology of the saliva found 0.51-0.98 per cent, of the salt. In 1846, Frerich 8 stated that the figures of the English observer were too high; he obtained only 0.01 per cent, of potassium sulfocyanate in the saliva of a healthy person. Tillanus 7 confirmed the findings of Frerich. In 1850, Jacubowitsch 8 carried out an extensive study of saliva. His method for the quantitative determination of the sulfo- cyanates was to oxidize the sulfur to the sulfate ion and then to precipitate with barium and weigh the precipitated barium sulfate. His procedure in detail was as follows: He took 750 cc. saliva, and extracted several times with alcohol. The extract was filtered, and the alcohol was driven off by evaporation from the filtrate. The remaining portion was distilled with phosphoric acid, and the distillate was mixed with barium hydrate and barium nitrate and heated for some time. The barium sulfate was filtered off on an ashless filter 1 Oehl: La saliva umana studiata colla siringazione dei condotti gheandolari, 1864, p. 177; also Constatt's Jahresbericht, 1864. 2 Maly: Hermann's Handb. d. Physiol., v, p. 14. 3 Longet: Compt. rend. hebd. des seances de 1'Academie des Sciences, 1856, p. 480. ' Ellenberger and Hofmeister: Physiologie der Haussaugethiere, 1890, p. 495. * Wright: Lancet, 1841-42, pp. 5, 72, 214, 262, 535, 563, 628, 672, 813, etc. 6 Frerich: Wagner's Handworterbuch, 1846, iii, p. 766. ' Tillanus: Constatt's Jahresbericht, 1849, p. 105. * Jacubowitsch: "De saliva," Inaugural Dissert., Dorpat, 1850. 1 1 paper, dried, ignited and weighed. Jaeubovvitsch found in the mixed saliva 0.006 per cent, of sulfoeyanate. Like Mitscherlich, he found this substance in the parotid secretion of man, dog, hocse and sheep. Bidder and Schmidt 1 corrob- orated the findings of their pupil Jaeubovvitsch. Yierodt 2 used the spectroscope in order to determine the amount of potassium sulfoeyanate in the saliva. He found in one thousand parts of the secretion 0.16 parts of the salt. Leh- man 3 obtained 0.0064-0.009 per cent, of the substance under discussion in the human saliva. On the other hand, Tiegerstedt 4 found somewhat higher quantities of the acid in the saliva — 0.1 part per one thousand. Bruylants, an industrious Belgian scientist, found the subject of sulfo- cyanates in the saliva in a very vague state. His researches will be discussed later (p. 18). He found on the average 0.0374 part of the acid in a liter of saliva. Immanuel Munk 6 in 1 869 began a study of the sulfocyanates in the human body. His method for the quantitative deter- mination of the acid in the saliva, I shall give here in some detail: The saliva was filtered, and soda was added to the filtrate. The filtrate was evaporated on a water bath to small bulk and was extracted several times with alcohol. The extract was again filtered and the filtrate was evaporated to dryness. The dry residue was dissolved in some water and was filtered. The filtrate was acidified with dilute nitric acid, and enough silver nitrate was added to complete pre- cipitation. The precipitate that fell out was composed of silver chloride and silver sulfoeyanate. This was collected on an ashless filter paper, washed with water carefully and dried at ioo° C. It was then fused with soda and sodium nitrate in a silver crucible. The excess of nitric acid was gotten rid of by adding hydrochloric acid and evaporating. 1 Bidder and Schmidt: Die Verdauungssafte und der Stoffweehsel, 1852. a Vierodt: Die Anwendbarkeit des Spektralapparates, 1873, P- J 50. 3 Lehman: Zeitschr. f. Physiol. Chem., 1881, v, p. 302. * Tiegerstedt: Lehrbueh der Physiologie des Mensehen, 1891, i, p. 2 2 1 . 5 Bruylants: Bull, de l'Academie de Medicine Bclgique, 1888, p. 21. • Munk: Virehow's Archiv., 1869, p. 350. 12 The remainder was dissolved in water, filtered, and the filtrate precipitated with barium chloride. After allowing the precipitate to settle for twenty-four hours, the barium sulfate was collected on an ashless filter paper. This was dried, ignited and weighed. His researches were confirmed in 1877 and his figures 1 for the amount of sulfocyanates in the saliva were ten to forty times the amount that Bruylants reported in 1888. Further studies 2 were made by Munk and, indeed, he is the most reliable and scientific investigator in this field. Before proceeding further with the literature on the analysis of saliva, it is advisable to pause and look into the negative side of this question. In 1843, Blondlot 3 criticized the findings of Tiedemann and Gmelin. I shall quote him verbally in his own language: "Que penser de 1' assertion emisse par M M. Tiedemann et Gmelin, relativement a la presence dans la salive de l'acide hydrosulfocyanique? D'abord le fait sur lequel ces auteurs s'appuient pour avancer cette opinion etrange, savoir, la coloration de la salive en rouge par le perchlorure de fer, n'a peutetre reproduit par la plupart des auteurs qui ont voulu le verifier, et je puis affirmer, pour mon propre compte, que jamais cette experience ne m'a reussi, ce qui porte a croire que les savantes professeurs de Heidelberg ont ete induits en erreur par quelques circonstances . accidentelles ou pathologiques. Quant au procede qu'ils ont mis en usage a l'effet d'isoler ce principe, il suffit, pour toute critique, de dire quil consiste a distiller de l'extrait alcoholique de salive avec de l'acide phosphorique mediocrement con- centre." Another scientist to state that sulfocyanates were not a normal constituent of saliva, was Schiff. He advanced the theory 4 that the presence of the rhodanate is only a test- tube experience, and that the substance does not occur in the living body. When the saliva, he said, is exposed 1 Munk: Deut. Med. Woch., 1877, p. 46. 2 Munk: Arch. f. d. ges. Physiol. Bonn, 1895, lxi, p. 620. 3 Blondlot: Traite Analytique de digestion, 1843, p. 123. 1 M. Schiff: Lecons sur la physiologie de la digestion, 1867, i, p. 147. *3 to the air, it undergoes spontaneous decomposition. The saliva from animals was collected, and put into a number of test tubes. He tested the liquids for potassium sulfocyanate at twenty minutes' interval of exposure. Those which were exposed longer, gave, according to him, very much higher results: "En effet, chez des animaux enrages, l'experi- mentation n'est pas facile, on ne s'approche pas volontiers d'eux pour recueillir leur salive au fur et a mesures qu'elle se forme, mais Ton se contents de leur appliquer, une fois pour toutes, un collecteur, on il s'en remasse une certaine quantite que Ton examine apres quelque temps." The great physiologist Claude Bernard propounded the theory that the presence of sulfocyanates in the saliva was a simple result of tobacco smoking, and that those that did not indulge in the use of the tobacco leaves, had no rhodanates in their body. Later on 1 he withdrew his absolute denial, but said that smokers have very much more of this acid and salts in the saliva than non-smokers. The latter statement was taken exception to by very many observers; preeminent among whom was Hoppe-Seyler. 2 He flatly denied that smoking had any effect upon the amount of sulfocyanate excreted in the saliva. He also observed that the dog's tissues contained no traces of this acid or its salts, thus dis- proving such authorities as Mitscherlich, Gmelin and Jacubo- witsch. But lately, Bunting 3 put the very pertinent query whether the substance that gives the red color with ferric chloride is really potassium sulfocyanate, or whether it is some other substance, whose structure and properties have not yet been described. I shall discuss this author's paper more fully in a later chapter. (p. 33). It may be advisable at this point to review the several attempts that have been made to affect the qualitative deter- mination of the salts of sulfocyanic acid. The characteristic test that was discovered by Treviranus was for many years 1 Bernard: Lceons sur les effcts des substances toxiques et medi- camentenses, 1857, p. 386. 1 Hoppe-Seyler: Lehrbuch d. Physiol. Chem., 1881, p. 186. 3 Bunting: Dental Cosmos, 1910, Hi, p. 1346. the only one adopted by chemists in their search for the thiocyanates. In fact, with the discovery of Porret, this acid began to be used in testing for very slight traces of iron. Several other reactions were subsequently reported, and claims were made that they were typical only of the rhodanates. The ferric chloride test 1 modified somewhat since the days of Treviranus, is carried out as follows: To a little saliva in a small porcelain crucible or dish, add a few drops of dilute ferric chloride and acidify slightly with hydro- chloric acid. Red ferric thiocyanate forms. To show that the red coloration is not due to iron phosphate add a drop of mercuric chloride, when a colorless mercuric thiocyanate forms. In 1872, Bottger 2 recommended the adoption of the fol- lowing test as corroborative of the presence of sulfocyanates in the saliva. He took strips of filter paper and soaked them in a tincture of guaiacum. The strips were then dried and drawn through a 1 : 2000 solution of copper sulfate. When a drop of saliva is added to this paper, the latter be- comes blue, if the thiocyanates are present. This test seems to have been neglected, and the majority of modern physiological chemistry text-books make no mention of it. Solera 3 in 1877, searching for a new test for the rhodanates, made the following discovery: Thiocyanates liberate iodine when acting upon iodic acid. He prepared a test paper according to these directions: Saturate a good quality of filter paper with 0.5 per cent, starch paste to which has been added sufficient iodic acid to make a 1 per cent, solution of iodic acid, and allow the paper to dry in the air. Cut it in strips of suitable size and preserve for use. If a piece of this starch paste-iodic acid paper is moistened with a little saliva, it will assume a blue color if thiocyanates are present, due to the liberation of iodine and the subsequent formation of the so-called iodide of starch. Richard Gscheidlen 4 whose labors in this field will soon be 1 Hawk: Practical Physiological Chemistry, 1910, p. 56. * Bottger: Zcitschr. f. anal. Chem., 1872, xi, p. 350. 5 Solera: Maly's Jahresber., 1877, vii, p. 256. 4 Gscheidlen: Maly's Jahresbd. Tierchemie, 1874, iv, p. 91. 15 fully considered, adopted the Treviranus test, and, three years before Solera, also prepared a thioeyanate test paper. Strips of filter paper are soaked in a mixture of ferric chloride and dilute hydrochloric acid, and dried in the air. Upon this a drop of saliva is let fall from the mouth. The paper will become red if the test is positive. I shall briefly describe several other tests for the identifica- tion of sulfocyanates in the saliva. Colosanti 1 found that if a very dilute solution of thiocyanic acid or its potassium or sodium salt is treated with a few drops of copper sulfate solution, it will assume a beautiful green color. He cautioned, however, that before applying the test to saliva, the latter must be freed of mucin by alcohol, and after filtering, the filtrate should be concentrated strongly on a water bath and then redissolved in a little water. This solution is then tested as above. In the same year, the same author 2 following out his in- vestigations of 1887-1888 3 reported a new reaction of sulfo- cyanic acid. He made use of the finding of Agostini 4 on the behavior of sugar when treated with gold chloride. Colosanti's discovery hinges upon the fact that when a very dilute solution of thioeyanate is made alkaline with sodium or potassium hydroxid, it will give a very pretty violet coloration if treated with a 1 : 1000 solution of auric chloride. If the sulfocyanate is not very dilute, the reaction will take place in the cold, otherwise it is necessary to warm the reagents. In 1903, Ganassini 6 in his article " Complementa al mctodo Solera e nuovi metodi per la ricerca delV acido sulj ocianico ," gave several new reactions for the rhodanates, and modified Solera's test. The modification consisted in this; that the author in analyzing saliva for the thiocyanates, not only determined the amount of iodine liberated, but also the quantity of cyanogen iodide that resulted according to the 1 Colosanti: Maly's Jahresber. d. Tierchemie, 1890, xix, p. 72. 2 Colosanti: Maly's Jahresber. d. Tierchemie, 1890, xix, p. 73. 3 Colosanti: Bull, della R. Acad. Medic, di Roma, 1887, xiv, p. 184. 4 Agostini: Ann. di chim. e farmac, 1886, iv, pp. 3, 228. 8 Ganassini: Biochcmisches Centralblatt, 1904, ii, p. 361. i6 equation: 5KSCN + 7HIO3 = 5 KHS0 4 + 5CNI + I 2 + H 2 0. The several new tests that he recommended were : (1) The blue coloration that thiocyanate gives with ammonium molybdate and hydrogen sulfide. (2) The formation of lead sulfate and hydrocyanic acid upon treatment with lead peroxide. (3) The black coloration with lead tartrate. There was also suggested by the same author a micro- chemical method for the determination of the salts of sulfo- cyanic acid. With mercurous cyanide, the rhodanate forms a double combination, which, upon the addition of iodic acid, is transformed to mercurous iodide. Polacci in his report of the researches from the chemical and toxicological in- stitute of Pavia made the following test for thiocyanates in the saliva: To some pure calomel in a crucible add 10-12 drops saliva, and stir well. Metallic mercury separates if the test is positive. Of the quantitative analytical methods for the thiocyanates there are several which should be discussed, some briefly and a few quite extensively. The color reaction that the rhodanates give with the chloride of iron suggested a colorimetric scheme for the quantitative determination of the acid and its salts. Oehl 1 in 1864 invented a color scale for the measuring of the amount of sulfocyanic acid in the saliva. Each color in the scale was compared to a standard thiocyanate solution which had been treated with iron chloride, and the amount noted on the scale. The saliva, after being treated according to Treviranus, was then com- pared with the scale, and the amount found and recorded. Mitscherlich 2 who worked almost simultaneously with Oehl, and also used a colorimetric method, obtained results nearly identical with those of the Italian observer. The method of Oehl was made use of by most observers, among whom can be mentioned Gscheidlen 3 and Pehl 4 in their search for thio- 1 Oehl: La saliva umana studiata colla siringazione dei condotti ghlandolari, 1864, p. 177. 2 Mitscherlich: Loc. cit. 5 Gscheidlen: Maly's Jahr. d. Tierchemie, 1876, vi, p. 140. 4 Pehl: Maly's Jahr. d. Tierchemie, 1877, v "> P- 2 °5- 17 cyanates in the tissues and fluids of the body. Vierodt 1 in 1873 made use of the spectroscope to determine the amount of thiocyanates in a fluid. About twenty-five years later, Wroblewski 2 in his analytical study of saliva, also came to the conclusion that it is very feasible to use the spectroscope for the determination of the amount of sulfo- cyanate in the salivary secretions. The results of both the above observers were denied by Kriiss, 3 who stated that it is quite impracticable to apply the spectroscope to this pur- pose. The method of Colosanti 4 also lends itself to colorimetric application, similar to the scheme of Oehl, except that copper sulfate is used instead of ferric chloride. Albert 5 went one step further in this colorimetric process. He caused glasses to be colored various shades of red, by comparing with standard thiocyanate solutions which had been treated with dilute ferric chloride. He then applied a Fleischman hemoglobinometer to his purpose, and thus had a very efficient method, as he states, for the quantitative analysis of hydrosulfocyanic acid or its salts. The Dental Society of America recommended the following test : 8 "Take 1 cc. of the specimen in tube A, and 1 cc. of 1 : 2000 NH 4 SCN in tube B. Add two drops of a 5 per cent, solution of ferric chloride to each tube; add water to tube B until its color matches that of the specimen. Read the scale in thousandths and ten thousandths. Care must be taken to have the bottom of the meniscus on the line. If these tubes are introduced in the colorimeter, the readings can be made more accurately. If later, diacetic acid is found, a correction is made in the finding." The gravimetric methods have quite a short history. 1 Vierodt: Loc. cit. 1 Wroblewski: Chem. Zentralblatt, 1897, ii, p. 532. 3 Kriiss: Maly's Jahresb. d. Tierchemie, 1897, xx\'ii, p. 368. 4 Colosanti: Loc. eit. 5 Albert: Lancet, 1898, i, p. 494. • Ferris and Schiadieck: Dental Cosmos, 191 1, liii, p. 1297. i8 Jacubowitsch 1 in his inaugural dissertation employed a gravimetric process which has already been described. Munk 2 also used a similar procedure. Bruylants 3 wanted to prove positively the presence of sulfocyanic acid in the saliva. He seemed to have strong doubts on this question. He carried out the following ex- periments: He collected iooo cc. of saliva and added some chloroform as a preservative and then he made it alkaline in reaction. After evaporating to 200 cc, he added 25 cc. hydrochloric acid and then extracted several times with ether. The collected ether extract was shaken with 15 cc. water and a little ferric chloride — -enough to give the maximum red color to the water layer, i. e., upon further adding the iron halide no increase in color was to be noticed. The red, aqueous layer was acted upon by ammonia until it be- came completely decolorized; it was then warmed and filtered, and the nitrate divided into two parts. Part one was evaporated to dryness and dissolved in ab- solute alcohol; the alcohol was then driven off, and the re- mainder dissolved in water and precipitated with lead acetate. After allowing to stand for twenty-four hours, the crystalline precipitate was filtered off and dried at 105 ° C. and weighed. Part two was evaporated to 10 cc. and distilled with 2 cc. concentrated hydrochloric acid. The distillate was collected under water and was treated with zinc and sulfuric acid at 30 ° C. In the gases produced he found, hydrocyanic acid, hydrogen sulfid and methylamin. Bruylants also advocated a rapid though rough method for the cyanate determination : A portion of the saliva was treated with a few drops of chloroform, and filtered. To about 10-15 cc. of the clear filtrate, he added one to two drops of concentrated hydrochloric acid and an equal number of drops of ferric chloride solution. After several hours' standing, he compared the color with a standard solution of ammonium sulfocyanate, according to the method of Pehl. 4 1 Jacubowitsch : Loc. cit. 2 Munk: Loc. cit. 3 Bruylants: Maly's Jahresbericht d. Tierchemie, 1888, xviii, p. 134. * Pehl : Loc. cit. 19 The Belgian scientist was very emphatic in denying that the rhodanates were typical of the saliva, since he contended that they are to be universally found in the animal tissues. As has been said before, however, his figures for the thio- cyanate content of the saliva, were very markedly lower than those of Munk or Gscheidlen. I might here just mention the method of Volhard' which he reported in his paper entitled " Die Anwendung des Sehwefelcyanammonium in der Massanalyse." Perhaps, the most exact of the methods for the quantitative determination of the thiocyanates is the iodometric titrating process. Rupp and Schied 2 and later Rupp 3 alone wrote on this method. The underlying principle of this method is the fact that sulfocyanate solutions, treated with sodium bicarbonate, decolorize large amounts of iodine, cyanogen iodide being formed, according to the following equation: CNSK + 4 I 2 + 4 H 2 - H 2 S0 4 + 6HI + KI + CNI. The process is finished in four hours at ordinary tempera- ture. Upon carefully acidifying with hydrochloric acid, the potassium iodide is changed to potassium chloride and hydriodic acid is formed; the latter acts on the cyanogen iodide to form hydrocyanic acid. The whole process can be expressed thus: CNSK + 31, + 4 H 2 - H 2 S0 4 + 5HI + KI + HCN that is, one molecule of sulfocyanic acid is equivalent to six molecules of iodine. I shall briefly describe the procedure for the analysis of sulfocyanates according to this method. 4 The following reagents are necessary: (a) Dilute nitric acid 1 : 100; (b) silver nitrate 3 per cent. ; (c) clean infusorial earth which has been washed in acid; (d) sodium bicarbonate, chemically pure; (e) potassium iodide; (/) N 1/10 iodine solution; (g) N 1/10 1 Volhard: Liebig's Ann. d. Chem. u. Pharm., 1883, cxc, p. 24. a Rupp and Schied: Bcr. deut. chem. Ges., 1902, xxxv, p. 2 191. 3 Rupp: Arch. d. Pharm., 1905, ccxxxxiii, p. 358; also in Chem. Central., 1905, ii, p. 1228. 4 Neuberg: "Der Ham," 1911, i, p. 542. 20 sodium thiosulfate solution; (h) hydrochloric acid solution 10 per cent.; (t) starch solution 2 per cent. The analysis is conducted in the following way: The fluid to be examined is filtered, and the filtrate heated to throw down any albumin, etc., and again filtered. Acidify with very dilute nitric acid and add an excess of silver nitrate. In order to cause complete separation add some infusorial earth and stir and warm ten minutes. Add a little more silver nitrate to see that precipitation is complete. Filter under diminished pressure on a filter paper stuck in a platinum cone. Make sure that the filtrate is clear. Wash several times with nitric acid. Transfer to a wide-necked glass container with water, and add 3 grams sodium bi- carbonate to alkaline reaction. Add 3 grams potassium iodide and shake slightly until the solution is clear. Add N 1 / 10 iodine solution until it assumes a brown color. Shake slightly and let stand in a dark place for four hours. The solution is carefully acidified with 10 per cent, hydrochloric acid. Add a few cubic centimeters of the starch solution. Titrate with A 7 1/10 sodium thiosulfate until a lemon-yellow color is obtained. Kabdebo 1 , a Hungarian observer, in his study of the origin and fate of the sulfocyanates in the body, used the iodometric method of Rupp and Schied in his analyses. Hydrosulfocyanic acid was at first supposed to be typical of the salivary secretion, and it was claimed that this acid is not found in the other fluids of the body. However, later on, observers found the acid in almost every organ and fluid of the body. Funke 2 attempted unsuccessfully to determine the origin of the thiocyanates in the human body. The only conclusion that he came to was that the rhodanates were a constant component of the salivary secretions. Carpenter 3 states that the substance is absent in the saliva and urine of herbivora. Treviranus suggested that it is present in the blood, and Tiedemann and Gmelin remarked that it is 1 Kabdebo: Jahresber. der Tierchem., 1907, xxxvii, p. 401. 2 Funke: Lehrb. d. Physiol., 1858, i, p. 220. 3 Carpenter: Human Physiology, 1848, p. 134. 21 present in most of the secretions of the body. Gscheidlen 1 found it in the urine of most subjects. Lea 2 says that it occurs only in the urine of those animals which excrete their nitrogen chiefly as urea. In 1 869, Leared 3 found it in the blood. In the first months of life of human beings, Pribram 4 did not find any thiocyanates. Nencki 8 examined the gastric juice of a dog for this acid and found 0.005 gram per liter of the secretion. He made sure that the gastric juice was free from saliva, or else his results would not have been of any scientific value. In 1900, Muck 5 reported that he had discovered traces of the rhodanates in the nasal and conjunctival secretions. Musso 7 in his researches on sulfur in milk, discovered that traces of hydrosulfocyanic acid was also present in this fluid. This was corroborated by Bruylants 8 who reported that he found 0.0016 gram per 1000 grams of milk. The latter observer also analyzed ox-gall, and gave the following figure, 0.01 gram per 1000. In the cystic fluid of the abdomen he found 0.0007 gram per 1000; in the fluid of a hydrocele 0.00055 gram per 1000. He did not find this substance in the urine of individuals suffering from podagra. Various and conflicting results have been reported as to the presence of the thiocyanates in the fluids of the lower animals. As was stated before, Mitscherlich, Gmelin and Jacubowitsch found it in the saliva of man, dog, horse and sheep. Ellenberger and Hofmeister did not find it in the secretions of horse, cow, sheep, hog, or goat. De Souza 9 found it in the blood, saliva, pancreatic juice and bile of human beings. Bruylants stated that the thiocyanates were absent in reptiles or fowls. Hoppe-Seyler also found none of this 1 Gscheidlen: Loe. cit. a Lea: Chemical Basis of the Animal Body. 3 Leared: Proc. Roy. Soc. London, 1869, xviii, p. 16. 1 Pribram: Jahrb. d. Physiol, u. Pathol, d. ersten Kindes alters, 1868, i, p. 148. s Nencki: Berichte der deut. chcm. Ges., 1895, xxviii, p. 1318. • Muck: Munch, mediz. Wochenschr., 1900, xlvii, p. 1168. 7 Musso: Berich. f. Physiol. Chem., 1877, vii, p. 168. 8 Bruylants: Loe. cit. • De Souza: Journal Physiology, 1907, xxxv, p. 332. 22 substance in dogs. In a calf, twenty-two days old, Bayer 1 found the rhodanates. The presence of the salts of hydrosulfocyanic acid in the living beings, gave rise to speculations as to its origin and function and fate. We will neglect here the suggestion made by Claude Bernard that the rhodanates are really a foreign substance introduced into the human body through the use of tobacco, and the other theory advanced by Schiff that the thiocyanates are really absent in the saliva, but are produced there on exposure, due to spontaneous decomposition. Richard Gscheidlen 2 demonstrated to his own satisfaction that the thiocyanates are first produced in the saliva, and then it passes into the stomach, and via the blood, to all tissues of the body. He did the following researches upon living animals: He caused the ducts of the salivary glands to empty themselves outside of the mouth. He then daily collected the saliva and tested for the thiocyanates, and al- ways obtained positive results. When, however, he ex- amined the blood of the animal or its urine, he always found negative results. This proved to him conclusively that the thiocyanates are first formed in the salivary glands. His attempts to isolate sulfocyanates from the urine, were suc- cessful according to his report, only when the dogs swallowed their saliva. But Thudichum 3 wrote in 1877 that he could not duplicate the results of Gscheidlen, even though he followed his methods. The latter, never- theless, reported that in spite of Thudichum's findings, he could still isolate the KSCN. The paper in which Gscheidlen refutes the English observer is entitled: " Wiederlegung der von Hern Thudichum erhobenen Einwande." The work of Gscheidlen 4 would find some corroboration in the studies of Longet 5 a summary of whose findings, I 1 Bayer: Maly's Jahresber. d. Tierchemie, 1876, vi, p. 172. 2 Gscheidlen: Pfluger's Arch., 1877, xiv, p. 401. 'Thudichum: Maly's Jahr. d. Tierchemie, 1877, vii, p. 205. * Gscheidlen: Loc. cit. 8 Longet: Compt. rend. hebd. des seances de l'Academie des sciences, 1856, p. 480. 23 shall give here: (a) Potassium sulfocyanate is a normal and constant component of saliva. (6) It is present not only in the mixed saliva, but also in the secretions of the parotid, submaxillary, and sublingual individually, (c) The presence of potassium thiocyanate is characteristic of the saliva, because it is not found in the other animal secretions, (d) The amount of thiocyanate is only proportional to the concentration of saliva. In 1901, Grober 1 refuted the findings of Schiff, 2 agreed somewhat with Longet, and advanced another theory as to the origin of the substance under discussion. He also found that in human beings, KSCN is only present in the saliva. Unlike Schiff, he not only did not find an increase in the thio- cyanate content of saliva upon exposure, but in fact, he noticed that the amount of KSCN became less with the duration of its separation. He was emphatic in denying that it had anything to do with smoking tobacco or using nicotine. He noticed an increase, however, when prussic acid was taken in very minute doses by his subjects. The excretion of KSCN, he suggested, depended only on the accumulation and disintegration of proteids in the body. Fen wick, 3 in a laborious attempt to discover whether the sulfocyanates had any specific relation to various diseases, did not come to any conclusions on this point, as some later writers have done on this continent. He, however, found that there exists a relationship between the bile and the KSCN in the body. He found that the sulfur in the thio- cyanate was derived from some sulfur compound of the bile. When he diverted the bile from the alimentary canal, he failed to discover any rhodanate in the saliva. De Souza 4 negated the results of Gscheidlen. He found on the contrary, that instead of the KSCN passing from the saliva into the blood, it travelled in the other direction. He stated that the sulfocyanates pass out from the blood into 1 Grober: Deut. Arch. f. clin. Med., 1901, lxix, p. 243. 3 Schiff: Loe. cit. 3 Fenwick: Medic. Chirur. Transactions, 1882, Ixi, p. 118. * De Souza: Loc. cit. 2 4 the saliva, pancreatic juice, bile and urine. In the sub- maxillary saliva, pancreatic juice and bile the concentration is always less than, and appears to depend upon, the con- centration in the blood. The concentration in the urine, on the other hand, he said, may be greater or less than the concentration in the blood, and is diminished by sodium sulfate diuresis. He also noticed that sulfocyanates in the food are readily absorbed and remain as such in the body for a considerable time. After injection or feeding, the parotid saliva contains less sulfocyanate than the sub- maxillary, and still less than the blood serum. Brubaker 1 seems to agree with Gscheidlen, but is more certain than the latter author, for he states in his text book that the sulfocyanates in the body were derived from the secretions of the parotid glands only. Some authors have thought it possible that the rhodanates in the saliva were a kind of vicarious excretion, similar to the excretion of urea in saliva in cases of nephritis. 2 What is the chemical origin of the sulfocyanates in the living body? This indeed is a question the answer to which it is very difficult to give. Theories galore have been ad- vanced and objected to on this question. Funke in 1858 3 gave up in despair the attempt to solve this problem. Florian 4 essayed, but also was unable to give a satisfactory answer. The only thing that he became convinced of finally was that the thiocyanates are a constant and normal com- ponent of the saliva. Bruylants 5 endeavored to prepare the sulfocyanic acid from proteids. He took egg albumin and serum albumin as his starting materials. His researches were crowned with success. The procedures that he followed were three in number: (a) Dry distillation of the albumin gave him a yield of 0.205-0.224 per cent. (6) Fusion with potassium hydroxide or (c) boiling with the same hydrate, gave somewhat better results. It must be noticed here 1 Brubaker: "Text Book of Physiology," 1908, p. 156. 2 Leube: Deutsche Arch. f. Clin. Med., 1904, lxvi, p. 80. 3 Funke: Lehrbuch d. Physiol., 1858, p. 220. * Florian: Gaz. medicalle de Paris, 1884, p. 354 and 1889, p. 317. '■ Bruylants : Loc. cit. 25 that these were test tube experiments, and not at all to be taken as conclusive evidence that similar processes are at work in the quick body. As one of my old professors used to say: "Two and two in the laboratory are not the same two and two in the living being." Studies were undertaken by various chemists 1 to determine the fate of certain nitriles when fed to animals. Aceto-, propio-, butyro-, and capro-nitriles were especially investi- gated, with the hope of finding that they may be the cause of the origin of certain substances as the thiocyanates ; but the results were unsatisfactory. The toxic effects of these compounds will be spoken of in due time. Kossel 2 found that adenin yielded hydrocyanic acid upon heating. Gautier 3 reported that upon hydration of xanthin, prussic acid was formed. These attempts and successes for the cyanide give us hope that we will soon discover the forerunner of the sulfocyanate. Martinotti 4 reviews some- what superficially the literature on this subject. Willianen, 5 a Russian scientist, found that the urine of rabbits, which ordinarily contains no trace of rhodanates, will give a positive reaction for that substance if the animals are fed glycocoll, creatinin and adenin (weak). It also seems apparent to the author that the amino acids as well as the other named substances, which on decomposition give hydrocyanic acid, are also the source of the thiocyanates in the living organism. Upon oxidation of albumins, Plimmer 6 produced hydro- cyanic acid. G. Kabdebo 7 in his studies in this field, discovered that the administration of acetonitrile lessened the oxidation of sulfur in the body. He also reported that the neutral sulfur 'Lang: Arch. f. exper. Path. u. Pharm., 1894, xxxiv, p. 247; and Giacosa: Zeit. f. Physiol. Chem., 1883, viii, p. 95. 2 Kossel: Berliner Klin. Woch., 1889, No. 19. 3 Gautier: Chimic biologique, Paris, 1892, p. 230. 4 Martinotti: Central f.. Bakteriol. u. Parasitkunde, 1896, xix, p. 142. 8 Willianen: Biochem. Centrall., 1906, v, p. 477. * Plimmer: Jour, of Physiology, 1903-1904, xxxi, p. 65 and 1904, xxxii, p. 51. 7 Kabdebo: Jahresber. der Tierehemie, 1907, xxxvii, p. 401. 26 of the body unites with the acetonitrile to form hydro- sulfocyanic acid. De Souza in his observations 1 on the elimination of the sulfocyanates from the blood, and their supposed formation in the salivary glands, found that upon feeding the acetonitrile to dogs (which according to him have no rhodanates in their fluids) , sulfocyanates were found not only in the urine but also in the saliva and blood serum. The question, however, to my mind, is far from settled. It is interesting at this point to know what normal or pathologic conditions effect the secretion of KSCN in the body in general and in the saliva, in particular. As Kriiger 2 remarks there are three theories concerning the thiocyanates : i . That KSCN is absent altogether. 2. That KSCN is present, but that it is a product of de- composition of saliva. 3. That KSCN is a normal constituent of the saliva. Supposing that the third theory is accepted, what causes an increase in the output of KSCN in the saliva? Claude Bernard, 3 as has been stated before, reported that smoking tobacco causes an increase in the amount of thio- cyanic acid in the saliva. This was contradicted by Hoppe- Seyler 3 who stated that smoking had no effect at all on the rhodanates content of the saliva. Gscheidlen 3 found the urine of smokers richer in KSCN than the urine of non- smokers. Grober 3 stated that the amount of thiocyanates in the saliva does not depend upon smoking or using nicotine in any form. Nevertheless, Kriiger 3 is quite positive that tobacco smoking increases the amount of KSCN in the saliva two to three times. Dr. Lothrop tells me that in his studies with Professor Gies 4 he did not notice any variation in the reaction for sulfocyanates in smokers and non-smokers. Experimentally, KSCN can be diminished in the saliva by prolonged stimulation of the salivary glands. 5 In his 1 De Souza: Loc. cit. 2 Kriiger: Zeitsch. f. Biologie, 1899, xxxvii, p. 6. 3 Loc. cit. 4 Lothrop and Gies: Journal Allied Dental Soc, 1910, v, p. 4; 1911, vi, p. 65. 6 Schneider: Am. Jour. Physiol., 1901,- v, p. 274. 27 experiments, Schneider uniformly found that the parotid saliva is always richer in KSCN than the submaxillary secretion, collected from the same individual at the same time. Grober 1 found that feeding of hydrocyanic acid causes an increase. Many other circumstances have been accredited as the influencing factors of the presence or absence of the thio- cyanates in the saliva. The variety of food, according to Bruylants, 1 has no effect upon the thiocyanates, which is formed in the organism. Is the condition of the teeth a causative factor for the in- crease or decrease of thiocyanates? Longet 1 investigated this question and came to the conclusion that the sick or healthy condition of the teeth has nothing at all to do with the presence or absence or amount of the potassium thio- cyanate in the saliva. In 1899, Kriiger 1 gave a very emphatic affirmation of the finding of Longet. Lately, however, Low 2 and Waugh 3 found that most patients who have caries of the teeth have no thiocyanates in their saliva. Lothrop and Gies 4 were unable to corroborate these findings. Various diseases that attack the organism in general, are said to cause changes in the potassium sulfocyanate output in the saliva. Some pseudo-scientists have gone so far as to say that by analyzing the saliva in various illnesses one could make diagnoses of those affections : Fenwick 5 analyzed the saliva for KSCN in numerous disturbances of health, but could come to no conclusion, except that he found variations in the amount of thiocyanates in the saliva. Kyle 8 advocates the examination of saliva (sialosemiology) in various diseases, as for example hay fever, and assures us that positive inferences can be drawn from the analyses. In 1908, there appeared a very comical article 7 on this so-called sialo- 1 Loc. cit. a Low: Dental Cosmos, 191 1, liii, p. 1269. 3 Waugh: Dental Cosmos, 1910, Hi, p. 170 and 420. * Lothrop and Gies: Loc. cit. 8 Fenwick: Loc. cit. • Kyle: Jour. Am. Med. Assoc, 1907, xlix, p. 402. ' Lcroy: N. Y. Med. Jour., 1908, lxxxxvii, p. 448. 28 semiology. The author makes the following "humorous" statements: Absence of potassium thiocyanate indicates various nervous lesions, as for example, epilepsy, paralysis and dementia praecox( !) . The reappearance of sulfocyanate in typhoid fever is hailed as a very evident sign of the return of good health. Excess of this marvelous chemical indicates (sic) heart, brain or kidney lesions. Boding good to no one is the phenomenon of the following misbehavior of the rhodanate: In cases where the reaction for sulfocyanate gives a dark brown color instead of a brick-red color, and if this reaction is noticed time and again in the same patient, you may notify the friends of a fatal issue(!?). The great Russian physiologist, Pavlov, 1 studied the effect of psychic stimulation upon the secretion of the various fluids of the body. His brilliant researches which were crowned with remarkable success do not form a part of this paper. But it is in this connection that one may mention the finding of Eberle, 2 that in states of excitement, worry or anger there is a marked increase in the amount of potassium sulfocyanate in the saliva. The English observer Davidson 3 in 1841 found that patients treated with mercury do not give the thiocyanate reaction in the saliva. This, of course, is due to the formation of a colorless mercury thiocyanate. He also observed that in certain febrile diseases, he obtained a negative test for the rhodanate. In diabetes, as well, the presence of the sugar might cause, he said, a diminution or a complete absence of the sulfocyanate. Longet 4 found that the amount of KSCN is not dependent on the age, sex, diet or the condition of the nervous system. In salivation, the same author con- tinues, it would sometimes appear that potassium thio- cyanate is absent, but this is really not the case, because the saliva only needs concentration to get a positive result. 1 Pavlov: Die Arbeit der Verdauungsdrusen, 1898. 2 Eberle: Physiologie der Verdauung, 1838, p. 32. 3 Davidson: London Med. Gazette, 1841, xxix, p. 338. * Longet: Loc. cit. Bruylants 1 did not find any thiocyanates in the secretions of gouty patients Kriiger 1 confirmed the findings of Longet. Grober' stated that those patients that suffer from cachexia from any cause show very little hydrosulfocyanic acid in the saliva. The toxic effects of the sulfocyanates have been given some attention; but the opinions advanced are very con- flicting. Wohler 2 and Frerichs 3 were of the opinion that the salts were not at all toxic in quite large doses. Claude Bernard, 4 Setschenow 5 and Podcopaew 8 reported individually that the potassium thiocyanate has some toxic action. Nysten 7 quotes Littre and Charles Robin as saying that the sulfocyanate of potassium is very toxic. Paschkis 8 was also of the opinion that the toxic influence of this substance is to be reckoned with. Guareschi 9 found that the rhodanate precipitates many organic bases and alkaloids, and thus acts as a kind of prophylactic in cases of poisoning. When Chouppe 10 attempted to grow plants using saliva as a watering agent, he found that the growth rapidly faded and withered. Raulin 11 noticed that in growing the Aspergillus fumigatus, it was necessary to add iron to the spraying water to neutralize a poison in the plant, which tended to prevent its growth. This deleterious substance was hydrosulfocyanic acid. The toxicologist Witthaus 12 in his discussion of sulfocyanic acid states that it is poisonous; very much more so than its salts. 1 Loc. cit. I Wohler: Gilbert's Ann., 1829, lxii, p. 271. 3 Frerichs: Wagner's Handworterbuch der Phys., 1964, iii, p. 766. 4 Bernard : Lecons sur les effets des substances toxiques et medica- mentenses Paris, 1857, p. 386. * Setschenow: Virchow's Archiv., 1857, xiv, p. 356. ' Podcopaew: Virchow's Archiv., 1878, xxxiii, p. 505. 7 Nysten: Sulfocyanure, 1878, p. 15. 8 Paschkis: Wiener mediz. Jahresber., 1885, p. 531. •Guareschi: Introduzione alio studio degli alcoloidi. Torino, 1892, P- 33- 10 Chouppe: Florian, Gaz. med. dc Paris, 1884, p. 354. II Raulin: Gamier and Schlagdenhauffen; Fremy's Enclyclopedie- chimique, 1892, ix, p. 195. 11 Witthaus: "Organic and Inorganic Chemistry," 1905, p. 302. 30 Lauder Brunton 1 affirms that in mollusca, hydrosulfo- cyanic acid has the following actions: It diminishes reflex actions and quickens the heart beat; large doses arrest the heart in systole. Lately 2 a series of experiments was performed showing the effects of sodium thiocyanate on the output of saliva. I shall quote the following verbatim : ''Experiment X. — A series of experiments was then in- stituted to show the effects of i grain of sodium thiocyanate taken internally. Specimens were taken before, and 36 and 80 minutes after. "The subject was conscious of a slightly stimulating effect, and showed an increased blood pressure. There was a decrease in the enzyme, but the last specimen was taken an hour after a noon meal. "One of the most noteworthy findings of this plot, which has been verified by a number of other findings, is the de- crease in the amount of mucin; but the increase in the centri- fuged sediment was found to be true in the examination of ten other specimens, as this drug has a physiological effect in reducing sediment of all kinds in the saliva as well as the urine." The antiseptic action of saliva in general, and of the thio- cyanates specifically, was studied by many scientists. In 1833, Kletzinsky 3 suggested that the potassium thiocyanate functionated in the saliva as an antiseptic. Gamier and Schlagdenhauffen 4 reported that the thiocyanates have some bactericidal properties. Edinger 5 suggested the theory that sulfocyanic acid builds various compounds with the different aromatic radicals in the body, and is thus enabled to protect the body against infectious diseases. He found that KSCN was antiseptic for diphtheria bacillus, cholera bacillus, and the staphylococcus pyogenes aureus. Another observer, 1 Brunton: Pharmacology, 1878, p. 114. 2 Dental Cosmos, 191 1, liii, p. 1297. Report of the committee. 3 Heller's Archiv., 1833, P- 39- * Gamier and Schlagdenhauffen: Loc. cit. •Edinger: Ber. d. Freiburger naturforsch. Gesel., 1894, ix, p. 27. 3i Martinotti, 1 did not wish to come to a positive conclusion whether potassium rhodanate inhibits the development of tuberculosis in the living organism. An Italian bacteri- ologist, Sanarelli 2 , found that the saliva rapidly destroys cultures of streptococcus pyogenes, staphylococcus pyogenes aureus, micrococcus tetragenus, and the cholera bacillus, but it has no effect upon the diphtheria bacillus and the pneumococcus. Some dentists, especially, have found that the saliva due to its KSCN content has antiseptic and bactericidal prop- erties. It has been thought by some of the members of this profession that the potassium sulfocyanate prevents the growth of plaques on the teeth and thus is prophylactic for dental caries. Michel 3 considers the rhodanates as a natural protective agent against tooth decay. Low 4 noticed that when the thiocyanates are present, the teeth are almost always free from caries; patients who have caries have no KSCN or the very faintest traces in their saliva; and he further reports that, clinically, when he fed his patients KSCN, the caries disappeared. He expressly wishes it to be under- stood, however, that the thiocyanate in his opinion has no antiseptic action. Kirk 5 has fully criticized Low in a recent article pointing out some of the contradictory phases of Low's theory. The latter, however, has found support in the work of Waugh 6 and Roberts who found that potassium thio- cyanate restricts the growth of the dental plaque. There have been not a few observers, on the other hand, who have found exactly opposite results to the ones reviewed above. Miller 7 stated that KSCN does not possess any appreciable antiseptic influence in the greatest strength in which it is found in the human saliva. Likewise, Hugen- schmidt 8 considered the bactericidal property of saliva very slight indeed, if it is at all present. Two French scientists, 1 Martinotti: Centralbl. f. Bakt. u. Parasitkunde, 1896, xix, p. 142. 2 Sanarelli: Arch. ital. di. clinic med., 1891, iii, p. 230. 3 Michel: Deut. Monatsch. f. Zahnheilk, 1911, xxix, p. 507. 4 Low: Loc. cit. 6 Kirk: Dental Cosmos, 1911, liii, p. 1345. * Waugh: Dental Cosmos, 1910, Hi, p. 420. 7 Miller: Dental Cosmos, 1903, xlv, p. 689. 8 Ilugenschmidt: Dental Cosmos, 1896, xxxviii, p. 881. 32 Nicholas and Dubief 1 did not find the thiocyanate nor the saliva to have any antiseptic properties. Barnes 2 obtained data that contradicted somewhat the results of Sanarelli and Edinger. He reported that the saliva has no bactericidal action on the pneumococcus, streptococcus pyogenes, staphylococcus pyogenes albus, and the influenza bacillus. Wounds in the mouth heal rapidly, he observed, due to the leucocytes that are present in the salivary secretion. Black, 3 the discoverer of the dental plaque, denied ab- solutely that the saliva had any antiseptic action. Oppen- heim 4 contrary to the findings of Smith, 4 Auftrecht, 4 Michel 5 and Gruber, 4 recorded that 0.5 per cent., 1 per cent, and 2 per cent, solution of the thiocyanate had no sterilizing in- fluence upon bacteria, especially of the fermenting kind. Seaman and Gies 6 in 1910 did not find that biological pro- portions of the sulf ocyanates had any retarding influence upon the production of plaques, or upon the growth of bacteria. H. P. Pickerill in his Cartwright Prize Essay (191 2) on the Prevention of Dental Caries and Oral Sepsis, presents a very brief and very incomplete review of the literature on the sulfo- cyanates. He concludes the chapter rather vaguely, and not on the basis of his findings, with the following words: "On the whole, we may conclude that while undoubtedly sulfocyanate of potassium is a beneficial element in saliva, and one making for freedom from disease, yet it can not be regarded as the most important or only factor in producing a natural immunity to dentalcaries or oral sepsis." 7 There are many authors whose papers I have not re- viewed because they simply stated what was well known or else they merely sided with one or another laboratory worker on general principles. I shall, however, give a list of them, without any comment whatever at the end of this dissertation. 1 Nicholas and Dubief: Jour, de Physiol., 1878, i, p. 979. 2 Barnes: Trans. Chicago Path. Soc, 1907-1909, vii, p. 249. 3 Black: Dental Digest, 1909, xv, p. 603. * Oppenheim: Hecht Dental Cosmos, 1909, li, p. 1275. 6 Michel: Loc. cit. • vSeaman and Gies: Dental Cosmos, 191 o, lii. ' This dissertation was already in print when Pickerill's book became available. CHAPTER II. THE FERRIC CHLORIDE — ETHER TEST. Few investigators have doubted the reliability of the qualitative test for the thiocyanates discovered by Trcvi- ranus. It is known that certain substances give a similar coloration when treated with the chloride of iron; but their occurrence in the saliva is so rare that these substances have been excluded from any consideration. Recently, Bunting 1 advanced the theory that the substance in the saliva that gives the red color with ferric chloride is usually not a sulfo- cyanate, but some other substances whose presence, properties and reactions have not yet been described. His conclusions are based on a series of experiments that he has performed. I shall give a brief summary of his methods of analysis. A few cubic centimeters of each saliva were placed in each of two 25 cc. Erlenmeyer flasks; to one of these flasks he added 0.1 cc. of Njioo KSCN solution. The salivas were then evaporated to dryness under suction over a hot water bath. Bunting found that upon treating these dried salivas with a few drops of ferric chloride and then shaking with ether, he invariably obtained a reddish coloration of the ether in all the control salivas (■/. c, in those to which he had added some KSCN), but he usually did not get any tinting of the ether when he had not put in some thiocyanate. I herewith attach a table of his results and his remarks: Water solutior 1. Ether solution. No. Color. Per cent. Test. Control I Light red O.OO36 + + 2 Straw O . OO I 6 + 3 Straw O.OOO7 + 4 Light red O.OO35 + 5 Dark lemon 0.002I + 6 Amber red O.OO46 Faint + 7 Dark red O.OO51 + + 8 Dark lemon O.OO32 Trace + 9 Dark lemon O.OO26 Trace + 10 Lemon O.OOO9 + 1 1 Red O.OO43 Faint + 12 Light red OOO34 Faint + 1 Bunting: Dental Cosmos, 1910, Hi, p. 1346. 34 " From these results, a wide discrepancy in the two methods is obvious. Of the five cases giving weak or doubtful tests in water, but one gave any test in ether. It must be remem- bered that ferric sulfocyanate shows a much more delicate color test in ether than in water, so that KCNS, if present at all, should be discernible in the ether solution. Of the re- maining seven which gave a positive distinctly red color reaction, there were but two that gave a like reaction in ether. These two were distinctly red in their reaction and more pronounced than in the water solution. The remaining five gave a negative or very faint reaction." Experimental. i. When KSCN solution is treated with a few drops of dilute hydrochloric acid and a few drops of 3 per cent, solu- tion of ferric chloride, a brilliant red coloration is obtained, This when shaken with ether, gives a cherry-red tint to the ethereal layer above the water. If the thiocyanate solution is diluted so as to give a light red color, there will be no red coloration of the ether. Upon shaking the dilute thio- cyanate with the ether, the following facts are to be noted : A. That the ether is not tinted. B. That the color of the ferric thiocyanate in the watery layer becomes much paler. The red color in the ethereal solution of ferric thiocyanate can be readily caused to disappear by the addition of a few drops of water. If a few cubic centimeters of saliva be put into a test tube and treated with some dilute HC1 and FeCl 3 , a reddish colora- tion is invariably obtained. Quite infrequently one sees the straw, lemon or dark lemon hues of which certain authors speak. The reddish tint may be light or quite dark. Upon shaking with ether, no matter what the color may be, the color becomes markedly paler without imparting any rosy tint to the ethereal layer. 2. Upon twenty-five specimens of saliva from the mouths of healthy persons as well as diseased (from the wards of Mt. Sinai Hospital), I carried out the following modification 1 of the ferric chloride test for KCNS in saliva in ether solution: 1 Bunting: Loc cit. 35 Color of ether. Pink Pink Pink Five cc. of saliva were placed in a round watch glass and evaporated to dryness over a slowly steaming water bath. To the dried residue, there were added two drops of water and one or two drops of ferric chloride. This was then well stirred to make a thick paste. Five cc. of ether were then added and stirred well. The color of the ether was then noted. Table I. Saliva No. Color with FeCl 3 . i Deep red 2 Deep red 3 Light red 4 Light red 5 Red 6 Light red 7 Light red 8 Light red 9 Light red io Dark red Pink 1 1 Light red 1 2 Light red 13 Red Pink 1 4 Light red 15 Deep red Pink 16 Light red 1 7 Light red 18 Red Pink 19 Light red 20 Light red 2 1 Red Pink 22 Light red 23 Light red 24 Light red 25 Light red Of the twenty-five cases examined by the watch glass method, eight gave a reddish tint to the ethereal layer. The other seventeen gave absolutely no color to the ether. All of them, upon being treated with ether became lighter in hue, even though in the great majority of cases the ethereal layer did not become colored. 3. Twenty-four of these salivas were tested for the thio- cyanates by the Erlenmeyer flask-suction method, as rec- 36 ommended by Bunting. The exact method of procedure was as follows : Into each of two 25 cc. Erlenmeyer flasks were put 5 cc. of saliva. To one of these flasks, there was added 0.1 cc. of a N/ioo KSCN solution. The flasks were provided with rubber stoppers through which passed glass tubing, bent at right angles outside the flasks. These glass tubes were connected to a suction pump, and the flasks heated in a water bath whose temperature was kept at 40 ° C. The salivas were evaporated to dryness and treated as follows: To each flask were added one drop of water and one to two drops of FeCl 3 solution and shaken with 5 cc. of ether. The color of the ether of the two flasks containing the saliva was then compared. The results as they were noted are given in the accompanying table. Table II. Saliva No. Color of water solution. Ether so Test. lution. Control. I Deep red + 4- 2 Light red — 3 Light red — 4 Red + + 5 Light red — 6 Light red — 7 Light red 4-* 8 Light red — 9 Deep red + + 10 Light red — 11 Light red — 12 Red + 4- 13 Light red — 14 Deep red + + 15 Light red — 16 Light red — 17 Red + 4- 18 Light red — 19 Light red + * 20 Red + + 21 Light red — 22 Light red — 23 Light red — 24 Light red — *See page. 37 Of the twenty-four salivas examined with the suction and control methods, seven gave positive results without the addition of any thiocyanate. Fifteen specimens of saliva gave not the faintest tint to the ether, either in the test flask or in the control flask. Cases No. 7 and 19 behaved somewhat out of the ordinary. These two salivas resemble the speci- mens examined by Bunting. Nos. 7 and 19 upon being dried under suction at a tempera- ture of 40 C, and then treated with a drop of water and two drops of FeCl 3 gave no color to the ether upon being shaken with the latter. These same salivas gave a faint pink color to the ether in the control flask, i. e. y in that flask to which 0.1 cc. N/100 KSCN had been added. Correlation and Explanation oj the Foregoing Findings. Undissociated ferric thiocyanate is soluble in ether. The dissociation of any substance increases with the dilution, ■j. c, the more dilute a solution, the stronger is the dissocia- tion and the less the undissociated portion that is present. At a certain dilution, a substance is completely dissociated. When a dilute watery solution of ferric sulfocyanate (pale red) is shaken with ether, the ether does not become colored because there is no undissociated thiocyanate to enter the ether. If we increase the concentration of the thiocyanate in the water, the ether is immediately tinted rose-red. There is, therefore, a certain level or limit of dilution or concentra- tion of ferric thiocyanate in water, below which no un- dissociated thiocyanate remains and below which, therefore, the ether will not be colored. The salivas that I have examined behaved quite similarly with the dilute solutions of KSCN. All the salivas, upon being treated with dilute acid and a few drops of ferric chloride, gave red colors varying in shade and intensity. None of the salivas (without drying) imparted any tint to the supernatant ethereal layer. That is to say, the amount of KSCN in the saliva is so slight that, it is completely dis- sociated. Eight of the salivas recorded in Table I gave, upon drying 38 and treatment with FeCl 3 and ether, a pink coloration to the ether. These salivas had evidently more KSCN than the other seventeen eases. The solution formed by the addition of the few drops of water and ferric chloride was not of sufficient dilution to completely dissociate the iron sulfo- cyanate, and therefore, some of it went into the ether and caused it to become red. This explanation will hold for the seven specimens of saliva that gave positive results with the suction method. Seventeen of the saliva specimens as recorded in Tables I a ad II gave no reddish tint to the ether. Evidently the thiocyanate was present in such very minute quantities, that the addition of a few drops of water is quite capable of completely dissociating it and thus preventing the ether from assuming any rosy color. Specimens Nos. 7 and 19 are particularly interesting. These salivas, upon evaporation on a watch glass and testing with ferric chloride and ether, gave no red color to the ether. The result was also negative upon evaporating under suction. But when 0.1 cc. N/100 KSCN solution was added and the saliva then dried under lessened atmospheric pressure, a positive result was obtained. The explanation of these facts seems quite plain. The amount of sulfocyanate present in the salivary secretion was just at the limit of complete dissociation. The amount of KSCN that was added was quite enough to increase the con- centration of the thiocyanates and thus allow some of the substance to remain undissociated. The fact, therefore, that frequently we do not get a colora- tion of the ether when shaken with dried saliva which had been treated with FeCl 3 solution is no evidence of the absence of thiocyanates. In fact, according to the light of the disso- ciation theory, it is somewhat a corroborative evidence of its presence. The absence of the ether coloration is in a way a quantitative test of the amount of the sulfocyanate in the saliva. For it is evident that slight amounts of thiocyanates will give a negative result, while greater quantities will cause a positive reaction. 39 4. Substances that will give a similar color with ferric chloride, or that may interfere, in general, with the sulfocyanatc test. (a) Neutral Formates. 1, — When a neutral formic salt is treated with dilute ferric chloride, a deep red coloration is produced. This color is still retained when the medium is just acid, i. e., upon addition of a few drops of formic or acetic acid. One or two drops of 10 per cent. HC1 may be added without changing the color, but a few drops more of this acid dispels the red hue. I made a solution of the neutral formate which when treated with dilute neutral ferric chloride gave a red coloration of the same intensity as the average saliva when similarly treated. Of this solution of the neutral formate (untreated by FeCl 3 ), 1 added known amounts to saliva. I then treated the saliva together with the formate with FeCl 3 solution, and noticed the effect produced. I examined three salivas. I put 2 cc. of each saliva into each of six tubes. Tube No. i, I treated with 2 drops of HC1 and several drops of FeCl.,. Tubes Nos. 2, 3, 4, 5, 6 were each respectively treated with 2, 5, 8, 10 and 15 drops of the neutral formate solution, and then tested with the dilute ferric chloride. The salivas examined were of those tested by Bunting's suction process. Table III. Salivas to which has been Added Neutral Formate. on tfi a a o o •a t3 Bunting method. a CO a e* ■0 a 01 73 a « a "o (Ng *j s OJ •— OS O £ - ~ r. £ 8 £ "£~ % I Acid + _L Light red + -f 2 Acid Light red + 4- 3 Neutral + T Light red + + + Dark red + + + + Dark red + + + Dark red (6) Neutral Acetate. — The same experiments were per- formed with the acetates as with the formate on five samples of saliva. The following table records the results. A plu- sign indicates a darkening of color. 1 Weston: Identification of Carbon Compounds, 1907, p. 15. 1 The reaction in each case was tested by means of litmus paper. 40 Table IV. Salivas to which has been Added Neutral Acetate. Bunting method. a a o o a o ■o»j -S _o "o (N^2 lO o at u s§ i Pi CD * & * Alkaline Light red + + Alkaline Light Light red red + Neutral + + Light Light Light red red red Acid + + Light red + + Acid — + Light red + + + + + + + + + + + + + + + + + + + + Dark red + + + + (c) The neutral trichlor acetate has the same effect as the neutral formate or neutral acetate. Ethyl acetate is not colored red by FeCl 3 . (d) Pyrogallic acid gives a very dark red color in quite dilute solution, when treated with ferric chloride. The accompanying table will explain itself. The effect of this substance upon the thiocyanate test in the saliva is only of theoretical importance, since practically this polyphenol never occurs in the oral secretions. When an excess of potassium hydroxide is added, the color becomes quite black. Table V. Salivas to which has been Added Pyrogallol. Bunting method. Pi Alkaline Alkaline Neutral Neutral Neutral H O u o -w O — Light red + + + + + + + + + + + a o + + + + + + dark dark -t- ■+■ -t- -r T "I" T f UcJXK + + + + + + + + + dark + ++ + + + + + dark + + + + + + + dark (e) Certain substances give a violet or purplish coloration upon treatment with ferric chloride. These chemical bodies, 4' should they he present in the saliva (as they sometimes un- doubtedly are), will cause a darkening of the color formed in the sulfocyanate test, and the amount of the rhodanate will consequently seem excessive. Phenol and salicylic acid were especially examined. Either of these substances upon addition to saliva, caused a darkening of the hue, so that the color seemed tawny red. A dilute solution of the sodium salicylate was used in these experiments. Table VI. Saliva to which has been Added Salicylate. Bunting c ui method. J3 n &2 00 % BO a a a o a o u C o H -s •a T3 T3 o r. "o h o (N o ■* >o M > u *; & So A% JS J3 J2 J3 a ■ tn C CD u .— in *j *j ♦j *J <2 V £ £ s: ^ ^ '^ I Acid Pale red Pale red + _)_ + 4- + 4- 2 Neutral 4- + Pale red Pale red + 4- 4- + + 3 Neutral Pale red Pale red Pale red 4- + 4- + 4 Acid — — Pale red Pale red + + + 4- + + 5 Alkaline Pale red Pale red + + + + 4- The addition of a few drops of HC1 caused a paling of the color, due to the disappearance of the salicylate shade. The cresols give color reactions upon treatment with ferric chloride. Orthocresol gives a light green color to a dark olive green, depending upon the dilution. Para cresol gives a pale blue color. Pyrocatechol gives with FeCl ;! a dark green color which changes to violet. Orsinol gives a violet-blue color. Creosote, which has been used in the treatment of tuber- culosis, and is used now by dentists as a dental germicide contains phenol, cresol, creasol and guaiacol. With ferric chloride it gives a brown-red color. With a 2 per cent, phenol solution, nearly similar results were obtained. Resorcin should also be mentioned here, as a possible complicating substance. Upon shaking the colored phenol solution with ether, it becomes decolorized. (/') Benzoates and Succinates. — The neutral salts of these acids give a red precipitate with ferric chloride. If the 42 saliva is viscid, mucinous — "ropy" — the precipitate will not settle out and will appear to be diffuse. These substances, though presumably present in slight amounts in the saliva, should be reckoned with, because many of the vegetable sauces and canned goods are rich in the benzoates. The color that a very thick saliva assumes if a few drops of the neutral benzoate is added, and if it be then treated with iron chloride, is a dark brick-red. The addition, however, of several drops of a 10 per cent. HC1 tends to lighten the color. (g) Care must be taken to prevent the alkaline carbonates or hydrates from interfering with the thiocyanate test. With ferric chloride these substances give an orange-red to a brick- red precipitate. Usually their presence is so slight that no fear need be had of their complicating effect. The addition of dilute HC1 causes this precipitate to dissolve and vanish. (h) Meconic acid, 1 C 5 H0 2 .(OH).(COOH) 2 , is a substance which is peculiar to opium in which it exists in combination with a part, at least of the alkaloids. It crystallizes in small pris- matic needles; is acid and astringent to taste, loses its water of crystallization at 120 C, is quite soluble in water and alcohol, sparingly soluble in ether. With ferric chloride, it forms a blood-red color which is not discharged by mercuric chloride or by dilute acids, but is discharged by stannous chloride and by alkaline hypochlorides. This acid may, theoretically, be present in salivas of patients suffering from acute opium poisoning or chronic morphinomania. Table VII. Salivas to which has been Added Meconic Acid. a Bunting method. c .0 3 O in a U 09 S ■a 10 a a ■s > 3 O V) C V a ~8 CO 5 £ 1 Neutral Yellow-red Yellow-red Yellow-red + + + 2 Neutral ■ Pale red Pale red Pale red + + + 3 Acid Pale red Pale red Pale red + + + 4 Alkaline + + Dark red Dark red Dark red + + + 5 Acid Pale red Pale red Pale red + + + 1 Witthaus: Inorganic and Organic Chemistry, 1905. 43 In the above tests a very dilute solution of meconie acid was used — one that with FeCl 3 gave a color reaction which resembled a i : 5000 solution of KSCN. The quantities added to each test tube are indicated in the accompanying table. The addition of a few drops of the acid caused a deepening of shade. (0 Aceto-acetic acid or diacetic acid, CH,.CO.CH 2 .COOH, occurs in urine of febrile diseases and in advanced cases of diabetes. It is conceivable, a priori, that in such conditions of disease, this acid may be also excreted in the saliva. Bunt- ing gave this acid as an example of a substance that might conflict with the thiocyanate test. Jones 1 without giving any authority for his opinion, states that diacetic acid is "ever present." This is a somewhat strange "finding" for it is known that this substance, when it does occur in the human organism, is accompanied by the severest symptoms of metabolic disturbance. Together with be taoxy butyric acid and acetone, it is considered in the clinical pathology of urine, as a sign of grave and unfavorable prognostic im- port. One can not imagine that this volatile substance should calmly remain in the saliva after Bunting's drastic treatment. Diacetic acid 2 with ferric chloride gives a violet-red or Bordeaux-red color, which disappears upon addition of HC1. It is soluble in ether to which it gives a yellowish red color not at all similar to the ferric thiocyanate in ether. The color in the ether disappears spontaneously in twenty-four to forty-eight hours. The fact that its color persists upon addition of hydrochloric acid, is enough to differentiate it from sulfocyanate. It was thought advisable however to examine ten salivas for diacetic acid by the Arnold- Lipliawsky reaction. The reagent consists of two solutions (a) a 1 per cent, solution of potassium nitrite, (6) 1 gram of /?-aminoacetphenon dis- solved in 1 00 cc. distilled water and about 2 cc. of HC1 (con- centrated) added drop by drop until the solution, which is at first yellow, becomes colorless. Before using, a and b are mixed in the ratio of 1 : 2. 1 Jones: Dental Review, 1911, xxv, p. 1167. * Hawk: Physiological Chemistry, 1910. 44 The test is conducted as follows : Place 5 cc. of filtered saliva and an equal volume of the reagent in a test tube; add a few drops of concentrated ammonia, and shake the tube vigorously. A brick-red color is produced. Take i cc. of this colored solution, add 10-20 cc. HC1, 3 cc. chloroform, and 2-4 drops of ferric chloride solution, and carefully mix the liquids without shaking. If the chloroform assumes a blue or violet color, the test is positive for diacetic acid; if this acid is absent the color may be yellow or light red. In ten cases of saliva, tested by this method, not a trace of this acid was found. In five cases of diabetic saliva, no diacetic acid was found by this test. The addition of a dilute pure solution of this acid to saliva causes a darkening of color. Table VIII. Saliva to which has been Added Diacetic Ester. n > Si i Bunting method. "o «S * m a V h< V) *J CD u xn a . •5-3 a * JA a 00 i I Acid — — Pale red + + + + + + 2 Neutral — — Pale red + + + + + + 3 Acid — — Yellow-red + + + + + + 4 Alkaline — — Yellow-red + + + + + + 5 Alkaline — — Pale red T + + + + + (j) Antipyrin and Phenacetin. — Two coal tar products in common use as analgesics and antipyretics give a red color when treated with the ferric chloride solution. Experi- mentally, upon addition of several drops of solution of these substances to saliva, and then testing with ferric chloride, a deepening of shade was noticed, even with very dilute solu- tions. Salophen ma)' here be mentioned as well. (k) Certain quinolin homologues — as thallin, ethyl thallin and kairin have been used in medicine as antiperiodics and antipyretics. If these substances are excreted unchanged in the saliva, they may complicate the thiocyanate test, for they also give a red coloration with iron chlorid solution. 45 (/) Sodium thiosuljatc — in weak solution gives a very transient violet-red color upon treatment with dilute ferric chloride solution, the thiosulfate being oxidized to the sulfate state. (m) Glycocoll — aminoacetic acid — CH 2 .NH 2 COOH — is one of the amino acids produced by disintegration and decomposi- tion of proteins. In dilute solutions with neutral ferric chloride solution it gives an intense red color. It is not soluble in ether. Upon adding varying quantities of this substance (in dilute solutions) to several samples of saliva, and then testing with ferric chloride, no deepening of the red shade was noticed, except in cases in which the salivary thiocyanate content was very weak or else when the amount of glycocoll added was comparatively excessive. (n) Amidol. — /»-Diamidophenol hydrochloride, is a synthetic product used in developing photographic plates. In extremely dilute solutions, it gives an intense red color with ferric chloride. The addition of one or two drops of a very dilute solution of this chemical to a few cubic centi- meters of saliva, and then treating with the iron chloride induces an extreme red coloration. (o) Patients undergoing a long course of treatment with mercury may not give the thiocyanate test in their saliva. 1 This is no evidence that they completely lack the rhodanate. It may be that the mercury (especially in cases of salivation) so effects the thiocyanate as to produce the colorless mercury sulfocyanate. 5. Spontaneous disappearance 0} the red color in the ethereal layer. (a) In all the salivas that gave positive results by the Bunting's suction test for thiocyanate (t. e., those cases in which the ether was colored pink) , the following phenomenon was observed The pink ethereal layer, upon being drawn off into a clean test tube and being allowed to stand from 5-30 minutes was spontaneoulsy, decolorized in each case. (b) An ethereal solution of thiocyanate was made by shaking some absolute ether with a concentrated ferric 1 Davidson: London Medical Gazette, 1841, xxix, p. 338. 4 6 thiocyanate solution. The ethereal layer was then drawn off and diluted with ether, until the pink color produced resembled closely the color of the ethereal extracts obtained in the positive cases of saliva by Bunting's method. This pale red ethereal solution was put in several clean test tubes, each well stoppered, and allowed to stand four to twenty- four hours. A paling of color was noticed in all of the tubes, and some tubes were completely colorless. Upon several repetitions, I was able to obtain as a starting point a very pale rose-red ether which upon being allowed to stand from one to two hours was entirely decolorized spon- taneously. CONCLUSIONS. i. The ferric chloride colorimetric test for thiocyanates in saliva is inexact and unreliable. 2. A negative result by the Bunting suction method, is no evidence of the absence of the sulfocyanate in saliva. 3. A positive result by the Bunting suction method is evidence of a comparatively large amount of sulfocyanate in the saliva. 4. Various medicinal substances and various chemical compounds that are the result of decomposition of proteids and carbohydrates may, if excreted in the saliva, give a very marked red coloration when treated with ferric chloride, and thus convey the impression that the amount of thiocyanate is very large. 5. There is a spontaneous disappearance of the pink color in the ethereal layer in the positive cases to the Bunting suction method. CHAPTER III. COMPARISON OF QUANTITATIVE METHODS. I shall eschew an extensive discussion of the colorimetric methods for the quantitative determination of the thio- cyanates. A resume of such proceedings as have been in vogue during the last century was given in the review of the literature on thiocyanates. I am convinced that to rely upon colorimetric analyses will lead one to very inaccurate results. There is too much of the personal element in the comparison of the intensity of two shades of color. There are two procedures for the rhodanate determination, that, though tedious and requiring precise accuracy in manipulations, will give exact figures. These methods are (i) gravimetric, (2) iodometric. Jacubowitsch 1 whose extensive studies on saliva were con- sidered classical was one of the first to suggest a gravimetric method for the determination of thiocyanates. He was especially interested in the salivary secretions, but his method can be used, with slight modification, in examining most of the tissues and fluids of the body. He collected 750 cc. saliva, and extracted several times with alcohol. The extract was filtered and the filtrate evaporated. The nearly dry residue was distilled with phosphoric acid. In this way he oxidized all the sulfur to the sulphate ion, and he then precipitated this with barium hydrate and barium nitrate. The sulfate of barium was collected on an ashless filter paper, filtered, dried, ignited and weighed. Munk, 2 modified this method by first precipitating the thiocyanate with silver nitrate and then oxidizing it by fusion with soda and the nitrate of sodium. This method will be described in detail a little later (vide -infra). The gravimetric method suggested by Bruylants 3 seems to me to be very unwieldy and not as accurate as the other 1 Jacubowitsch: "De Saliva," Inaugural Dissert., Dorpat, 1850. J Munk: Deut. Med. Woch., 1869, lxix, p. 427; 1877, lxxvi, p. 350; also Arch. f. d. ges. Physiol., 1895, lxi, p. 620. 3 Bruylants: Maly's Jahres. d. Tierchem., 1888, xviii, p. 134. 4 8 methods. I shall simply mention it here; it has been described with some detail in the first chapter. In 1894, Lang 1 suggested a titration method for the de- termination of the thiocyanates. Two processes of titration are requisite by this method. The first one determines the amount of chloride plus thiocyanates according to Volhard; 2 the second determines the amount of chlorides alone, ac- cording to Mohr. 3 The difference between these quantities gives the thiocyanate figure. The iodometric method was first suggested by Rupp and Schied 4 and was improved by Thiel 5 and Rupp. 6 In 1906 Edinger and Clemens 7 applied this method in the analyses of biological fluids and tissues. The method, as I followed it, is detailed by Paul Meyer 8 in his extensive treatise on the analysis of urine. Of all these methods, those of Munk and Rupp and Schied were chosen as giving the most accurate results. A com- parison of their degree of accuracy was then attempted. Description of Methods. 1. Munk's Gravimetric Method. 9 — The fluid to be examined is filtered. If the substance is solid, it is extracted with water for twenty-four hours, and the extract then filtered. The solid portion is washed several times on the filter paper with warm water. The filtrate is treated with some soda and then evaporated on the water bath. This is then extracted several times with alcohol, and filtered. The filtered alcoholic extract is evaporated, and the residue dissolved in water and again filtered. The filtrate is acidified with dilute nitric acid and the thiocyanate precipitated with silver nitrate. The solid silver chloride and silver thiocyanate are collected 1 Lang: Arch. f. exp. path. u. pharm., 1894, xxxiv, p. 253. 2 Volhard: Liebig's Ann., 1877, cxc, p. 1. 3 Paul Meyer: In C. Neuberg's "Der Harn," 1911, i, p. 651. 4 Rupp and Schied: Ber. deut. chem. Ges., 1902, xxxv, p. 219. 8 Thiel: Ber. deut. chem. Ges., 1902, xxxv, p. 2766. 8 Rupp: Chem. Zentral., 1905, ii, p. 1288. 7 Edinger and Clemens: Zeitsch. f. klin. Med., 1906, lix, p. 223. 8 Meyer: In C. Neuberg's "Der Harn," 1911, i, p. 651. 8 Borcher: Rcpcrt. d. anal, chem., 1881, iv, p. 130. 49 on an ashless filter paper, washed carefully and dried at iOO° C. The precipitate together with the filter paper are fused in a silver crucible with soda and sodium nitrate. The excess of nitric acid is gotten rid of by adding hydro- chloric acid solution and evaporating. The remainder is dissolved in water, filtered and the filtrate precipitated with barium chloride. After allowing the precipitate to sediment for twenty-four hours, it is collected on an ashless filter paper, dried, ignited and weighed. From the amount of barium sulphate, thus obtained, one can easily calculate the quantity of thiocyanate present. 2. Rupp, Schied and Thicl lodomelric Method. — The principle of this method consists in the fact that sulfocyanate solutions treated with bicarbonate, decolorize large amounts of iodine, cyanogen iodide being formed, CNSK + 4l 2 + 4H 2 = H 2 S0 4 + 6HI + KI + CNI. The process is finished in four hours at ordinary tempera- ture. Upon acidifying with hydrochloric acid solution carefully, the potassium iodide is changed to potassium chloride and hydriodic acid and this acts on the cyanogen iodide to form hydrocyanic acid. The whole process can be expressed in the following equation: CNSK + 3 I 2 + 4 H 2 = H 2 S0 4 + 5HI + KI + CNH, i. e., one molecule of thiocyanate is equivalent to six mole- cules of iodine. The analysis is conducted as follows: Reagents used: 1. Nitric acid, 1 per cent. 2. Silver nitrate, 3 per cent. 3. Infusorial earth, clean, washed in acid. 4. Sodium bicarbonate, C. P. 5. Potassium iodide, C. P. 6. iVi/10 iodine solution. 7. Hydrochloric acid, 10 per cent. 8. N 1/10 sodium thiosulphate solution. 9. Starch solution, 2 per cent. The liquid to be examined is filtered. If it is solid, it is macerated and extracted with water for twenty-four hours. 5Q The extract is then filtered. In order to remove any albu- mins that may be present, the extract is heated and the precipitated proteins filtered off. The clear filtrate is acidified with nitric acid and an excess of silver nitrate is added. In order to cause complete sedimentation, some infusorial earth is added. It is now filtered under suction on a filter paper stuck in a perforated platinum cone. Care must be observed that the filtrate is clear. The collected precipitate is transferred to a wide-necked glass container by means of water. 3 grams of the bicarbonate of soda are added to alkaline reaction. Then 3 grams of solid potas- sium iodide are added and the solution is shaken slightly until it is clear. N 1/10 iodine solution is then added until a permanent brown color is formed. This is then shaken slightly and allowed to stand in a dark place for four hours. After acidifying very carefully, with 10 per cent, hydrochloric acid solution a few cubic centimeters of the starch solution are added, which is freshly prepared. This is finally titrated with N 1 j 10 sodium thiosulfate solution until the blue color just disappears. The two methods were tested in duplicate on pure solutions of potassium sulfocyanide and on biological tissues and fluids to which had been added known amounts of the thiocyanate. The following is a list of the substances upon which analyses were conducted: 1. A pure solution of potassium thiocyanate. 2. Urine to which was added a known amount of the thio- cyanate. 3. Fifty grams of meat to which was added a known amount of thiocyanate. 4. Saliva (250 cc.) to which was added a known amount of thiocyanate. The data found are set forth in the accompanying table (Table I). In my hands the iodometric method gave better results. It is not as long nor as complicated as the gravi- metric method; the sources of error and the amount of error are less; it takes less time to carry it out; four or five analyses can be easily run simultaneously. 5i Table I. Comparison of Results by Iodometric and Gravimetric Analyses. Substance examined to Wt. Iodometric Gravimetric. which had been added a in Amount Amount known amount of KSCN. gins. found. Error. found. Error. Distilled water 500 O . O408 0.0002 O . O4065 O.OOO35 Meat 50 O.O3814 O.OO296 OO3765 O OO345 Meat 50 OO375 O.OO36 OO3823 O.OO287 Meat 50 OO395 O.OO16 O.O3844 O.OO266 Meat 50 O.O3782 O.OO328 OO3727 O.OO383 Urine 500 O . O45 I O.OO4* O • O463 O.OO52* Urine 500 OO432 0.002I* O.O458 O.OO47* Saliva 250 O.O447 O.OO36* OO432 0.002I* I analyzed the following biological tissues and fluids for thiocyanates to determine whether the rhodanates are normally present and if so, to what extent: 1. A liter of human urine. 2. 500 grams of bovine liver. 500 cc. of human saliva. 500 grams of beef meat. Table II. KSCN in Various Biologic Fluids and Tissues. Weight. Tissues. Grams. Amount of KSCN.f Urine IOOO O.O262 Bovine liver 500 O.OO47 Human saliva 500 O.OI28 Beef meat 500 O.O I have found, as many other investigators have previously done, that the urine and saliva contain varying amounts of the rhodanate. The bovine liver, according to my analysis contains 4.7 mg. per 500 grams of the tissue. Beef meat does not contain any thiocyanate. ♦Increase. fin all the analyses recorded in this dissertation, the quantity of thio- cyanate* was always calculated and recorded as potassium thiocyanate. CHAPTER IV. THE DISTRIBUTION OF THE THIOCYANATES IN THE ANIMAL BODY. The salts of sulfocyanic acid have been found in fluids of the animal body, other than in the saliva. Musso 1 found the thiocyanates in milk, Leared 2 in the blood, Gscheidlen 3 in the urine, Nencki 4 in the gastric juice, and Muck 5 in the nasal and conjunctival secretions. De Souza 6 stated that it is present in the blood, pancreatic juice and bile. Fen- wick 7 also implied that the bile contains some rhodanate. Grober 8 and Longet 9 on the other hand, denied that the thiocyanates are present in fluids other than saliva. Bruy- lants 10 found the salts of sulfocyanic acid in milk, ox gall, in the fluid of a hydrocele and in cystic fluid of the abdomen. No investigator has ever systematically determined the distribution of thiocyanic salts in the various organs and tissues of the body. Vague statements are present in the literature on this subject, as to the rhodanate content of various species of animals, but I have met no exact figures relating to the amount of this substance in the different animal tissues. It was deemed advisable, therefore, before proceeding any further, to determine the amount of sulfo- cyanates present in the various organs of a dog. General Description of Experiments. — The organs and tissues of six dogs were analyzed. The first four dogs used had been experimented on by other workers in the labora- 1 Musso: Berichte f. physiol. Chem., 1877, vii, p. 168. 2 Leared: Proc. Roy. Soc. London, 1869, xviii, p. 16. 3 Gscheidlen: Arch. f. d. ges. Physiol., 1877, xiv, p. 401. 4 Nencki: Berichte d. d. chem. Ges., 1895, xxviii, p. 1318. 5 Muck: Munch, med. Woch., 1900, xlvii, p. 1168. • De Souza: Journal of Physiol., 1907, xxxv, p. 332. 7 Fenwick: Brit. Med. Jour., 1882, i, p. 397. • Grober: Deut. Arch. f. klin. Med., 1901, lxix, p. 243. • Longet: Traitc de Physiologie, 1868, i, p. 191. 10 Bruylants: Bull, de l'Acad. de Medicin Belgique, 1888, xxi, p. 147. 53 tory. The experiments, however, that were performed on these animals could have produced no radical changes in the animals. The first two dogs had several ounces of blood taken away from them, and the second pair of animals had been transfused with blood taken from other dogs. The fifth and sixth dogs were perfectly normal animals. Special care was taken, as will be seen later on, to determine the health condition of these last two dogs. The dogs were bled to death from the right femoral arteries. Cocaine was injected over the site of this blood vessel. A general anaesthetic was never used. The animal showed no signs of suffering. All the blood was collected in a wide mouthed, glass container, and it was defibrinated as soon as collected. Particular care was taken to exsanguinate the animals completely. The pancreas, spleen, kidneys, liver, gall-bladder and bile, heart, brain, salivary glands, testicles and muscle of thigh were taken out in the order named, and were put separately in stout, glass-stoppered, wide-mouthed glass bottles. Care was observed that the bile did not ooze out and vitiate the results. Rubber gloves were worn and were rinsed in a running stream of distilled water after the removal of each organ. Analysis of all the tissues was begun immediately after their removal from the body. Experiment i. — A female dog, weighing 15.5 kilos, was used in this experiment. It was noticed that the dog was nervous and irritable. It was bled to death on January 11, 1912, from the right femoral artery. The blood clotted very slowly. The exsanguination was complete. No points of bleeding were noticed upon opening the abdomen. The various tissues enumerated above were removed. The heart, when opened, contained in its right ventricular com- partment, two whip shaped worms four or five inches long. They were carefully preserved and sent to the pathologist who pronounced the parasites to be the filaria hematis. The amount of potassium sulfoeyanide found in the various tissues are charted in Table I. 54 Table I. Weight. Amt. of KSCN Tissue. Grams. Mg. Bile 25 None Blood 955 52.86 Brain 79 None Heart 92 None Kidneys 87 None Liver 393 29.84 Muscle 200 None Pancreas 30 None Salivary glands 11 None Spleen 32 None Experiment 2. — A young male pup (9-10 months old), weighing 7.48 kilos, was bled to death on Jan. 31, 1912. The dog had not finished his first dentition and his testicles were as yet undescended. The bleeding was quite complete. The accompanying table contains the analytic data (Table II) . Table II. Weight. Amt. of KSCN Tissue. Grams. Mg. Bile 12 None Blood 598 27.32 Brain 72 None Heart 53 None Kidneys 5i None Liver 332 17.82 Muscle 200 None Pancreas 21 None Spleen 22 None Experiment 3. — A male dog weighing 9.34 kilos was bled to death on February 5, 191 2. The procedure was identical with the ones described above. The animal had been trans- fused several times with the blood of another dog. The same precautions were observed and the same organs removed and immediately analyzed. It was thought advisable to tie off the small and large intestines, to remove them separately and to analyze these organs and their contents for sulfo- cyanate. The small intestine was tied off at the duodenum and ileo-caecal junction. Of the large intestine, the portion between the caecum and rectum was used. 55 The analytical data are given in Table III. Table III. Tissue. Bile Blood Brain Heart Small intestine Large intestine Kidneys Liver Muscle Pancreas and spleen Experiment 4. — The various organs and tissues, enumerated in the preceding experiments (together with the small and large intestines and stomach) were removed from a male dog weighing 11.3 kilos on February 13, 1912. The dog was thoroughly exsanguinated from the left femoral artery under local cocaine anaesthesia. The analytical findings are as follows : Table IV. Weight. Ann. of KSCN, Grams. Mg. 37 4-3 752 32.56 63 None 69 None 635 7-4 235 5 8 53 None 217 19. 2 200 None 60 None Weight. Amt. of KSCN. Tissue. Grams. Mg. Bile 28 7 65 Blood 65O 4234 Brain 87 None Heart 65 None Intestine (small) 67O 22 . 72 Intestine (large) 285 14 97 Kidneys 66 None Liver 278 39 5 Muscle 200 None Pancreas and spleen 79 None Stomach and contents 210 None Experiment 5. — In this experiment and the following, an attempt was. made to determine the normality of the dogs before their organs were analyzed. The dogs selected were to all appearance full grown, vigorous and healthy. They were kept in cages devised by Prof. Gies. 1 The urine daily voided was collected and analyzed for sulfocyanates. The same was done with the feces. 1 Gies: Amer. Jour. Physiol., 1905, xv, p. 403. 56 The dogs were fed daily at 10 a.m. the following mixture: Meat 1 15 grams, cracker meal 4 grams, lard 3 grams, bone-ash 1 gram, and water 35 cc. per each kilo of weight of the dog. The dogs were kept in the cages for six days, until we were satisfied that they were healthy, normal dogs. On the seventh day the animals were bled to death. The first dog was a female weighing 12. 1 kilos. It was bled to death on February 27, 1912, from the right femoral artery. The analytical results are the folio wing : (Table V). Table V. Weight. Amt. of KSCN. Tissue. Grams. Mg. Bile 9 3-0 Blood 765 17.8 Brain 58 None Heart 65 None Intestine (small) 589 96 Intestine (large) 175 5-5 Kidneys 73 None Liver 280 10. 7 Muscle 200 None Pancreas and spleen 64 None Stomach and contents 128 None Experiment 6. — The preceding experiment was duplicated on a male dog. weighing 11.75 kilos. It was also bled to death on February 27, 1912, after a feeding period of seven days. The analytical figures are as follows : (Table VI). Table VI Weight. Amt. of KSCN, Tissue. Grams. Mg. Bile 4 None Blood 635 20.35 Brain 61 None Heart 67 None Intestine (small) 595 9-4 Intestine (large) H7 8.7 Kidneys 52 None Liver 230 12.2 Muscle 200 None Pancreas and spleen 58 None Stomach contents 172 None s: Amer. Jour. Physiol., 1901, V, P- 235- 57 Study of the Sulfocyanate Content of the Salivary and Olln t Glands oj the Body. According to some authorities, all the rhodanates in the animal organism are produced in the salivary glands. Gscheidlen 1 caused a drainage of the saliva away from the mouth and found that, while the secreted saliva still contained sulfocyanate, the blood and the urine ceased to show the presence of this substance. Brubaker 2 localized the forma- tion of the thiocyanate in the parotid gland; he states that the submaxillary and sublingual bodies have nothing to do with the formation of this substance. I analyzed the salivary glands of the ox and the dog. In 250 grams of ox gland tissue, I found only a slight amount of sulfocyanate, viz., 10.23 m g- It * s quite impossible to state the amount of thiocyanate in the salivary bodies of the dog. The total weight of the glands in a dog is very small, so that I was unable to obtain a figure for one dog. The expedient was suggested of collecting the glands of five or six dogs and analyzing en masse. Various other glands — duct and ductless — of the animal organism were analyzed. The ox salivary glands, the ox thyroid, the ox liver, the calf thymus, the dog's salivary glands, the dog's spleen, the dog's pancreas, the dog's liver and the dog's testicles were each quantitatively analyzed for potassium sulfocyanide. In the case of the salivary glands and testicles of dogs, an analysis was run on the col- lected glands from six dogs. The data of these analyses are given in Table VII. Table VII. Weight. Amt. of KSCN Tissue. Animal. Grams. Mg. Liver Dog 393 28.94 Liver Ox 500 4-7 Pancreas Dog 30 None Salivary glands Dog 65 None Salivary glands Ox 250 10.23 Spleen Dog 32 None 1 Gscheidlen: Loe. eit. 2 Brubaker: Text Book of Physiology, 1908, p. 156. 58 Table VII (Continued) . Weight. Amt. of KSCN. Tissue. Animal . Grams. Mg. Thymus Calf 370 None Thyroids Ox 605 None Testicles Dog IO7 None Blood Ox 5OO 7.6 Bile Ox 500 9-7 Feces Man 235 12.8 Table VIII shows the location of the thiocyanates in the vari- ous tissues and fluids of the animal body. Table VIII. Salivary Saliva Glands Man + Dog — Ox . . . . + Blood + + Bile + + + + Intestine Urine Feces + + + + + CONCLUSIONS. i. The salts of sulfocyanic acid are found in certain fluids and tissues of the body outside of the saliva and salivary glands. 2. The liver seems to be the gland in the body which con- tains most of the thiocyanate. 3. The thiocyanate of the liver is excreted through the bile into the small and large intestines, where quite perceptible quantities were found. The stomach contents showed no trace of the sulfocyanate in dogs normally fed. When how- ever, sodium sulfide is given the stomach contents showed traces of sulfocyanate. 4. The blood contains appreciable quantities of the rhodanate. 5. Of the glands of the body, the liver and the salivary bodies (ox) show the presence of the thiocyanate. I was unsuccessful in my attempt to demonstrate the presence of sulfocyanate in dog's salivary glands. The spleen, the pancreas, the thymus, the thyroid and the testicles do not contain any thiocyanate. CHAPTER V. THE METABOLISM AND EXCRETION OF SULFOCYANATES. Having determined the distribution of the thioeyanates in the animal organism, a study was made of the excretion and metabolism of. this substance. It was surmised by Grober 1 that potassium sulfocyanate is produced in the organism by decomposition of proteins. De Souza 2 found that after feeding acetonitrile to dogs, the sulfocyanic salts were present in large amounts in the urine, saliva and serum. Kabdebo 3 concluded from his investiga- tions that acetonitrile lessens the oxidation of sulfur, and that the neutral sulfur unites with the acetonitrile to form sulfocyanic acid. Willianen 4 demonstrated to his own satisfaction that upon feeding glycocoll and other amino acids to rabbits, he caused an excretion of sulfocyanic acid in the animals' urine, which otherwise failed to show the presence of sulfocyanate. Diena 5 recently studied the effects of the ingestion of rhodalzid (an albumen-sulfocyanate compound prepared by Nerking) upon the excretion of sulfocyanates in the saliva, gastric juice, pancreatic juice and bile. He made fistulae to these organs in animals and collected the juices after giving tablets of rhodalzid. He found that the saliva showed very marked traces after the administration of the substance. The pancreas and stomach showed smaller traces. He reported the findings of Kondo (Japan) that the rhodalzid causes increase in purin bases output. Having these results in mind, it was proposed to study first the normal excretion of thioeyanates in the dog. Dogs V and VI were fed as has been previously described, their urine and feces were daily collected, and the amount of thiocyanate determined. I found that the urine contained small amounts of sulfo- cyanate, while the feces were quite rich in this substance. 1 Grober: Deut. Arch. f. klin. Med., iooi, lxix, p. 243. 2 De Souza: Journal of Physiology, 1901, xxxv, p. 332. 3 Kabdebo: Maly's Jahr. d. Tierchemie, 1907, xxxvii, p. 403. * Willianen: Bioehem. Centralbl., 1906, v, p. 477. * Diena: Bioehem. Zeit., 1912, xxxix, p. 13. 6o The animals were fed on meat, cracker meal, lard and bone ash. Several samples of a mixture of these substances were analyzed for sulf ocyanates ; none was found. The accompanying tables record the quantities of thio- cyanates found respectively in the urine and feces. Date. Feb. 2 1 22 23 24 25 26 27 Date. Feb. 21 23 24 25 26 27 Table i.— Dog V. Urine. Cc. 49O 450 460 3IO 275] 500 \ 350 J Amount of KSCN in urine. Mg. 8-3 7-4 11. 4 Table 2.— Dog VI. Amount of KSCN in urine. Mg. 7-5 63 Amount of KSCN in feces. Mg. 15-7 15-2 29.8 Amount of KSCN in feces. Mg. 12-5 12 .6 17.6 27-5 Effect of the Administration of Powdered Sulfur upon the Excretion of Sulf ocyanates. Two male dogs, full grown and normal to all appearances were used in these experiments. The dogs were put in their cages on February 28, 19 12 and were fed daily at 9.30 a.m. Their meal consisted of meat 15 grams, cracker meal 4 grams, lard 3 grams, bone ash 1 gram and water 35 cc. per each kilo of weight of the animals. Their excreta were collected daily, weighed and measured, and then analyzed for the thiocyanate. On March 4, 191 2, after having observed the dogs for five days and found them normal, the feeding of sulfur was begun. The first day each of the dogs was given 6i 2 grams of flowers of sulfur, administered in a ball of meat. Great care was taken that none of the sulfur should be lost. If the animal refused to take the meat-ball from the hand, its mouth was opened and the meat with the sulfur was forcibly pushed down the pharynx of the animal. On March 5th, 3 grams were given to each animal; on March 6th, 4 grams; on March 7th, 5 grams; March 8th, 6 grams; March 9th, 7 grams; March 10th, 8 grams. The feces and urine were daily analyzed for thiocyanates. The results are to be found in Tables 3 and 4. Table 3- —Dog VII.- -Weight 11.36 Kilos. Amount of Amount of Amount of S given. Urine. KSCN in urine. Feces. KSCN in feces. Date. Grams. Cc. Mg. Grams Mg. Feb. 29 550 3 4 42 78 Mar. 1 450 3 7 45 8 5 2 3IO 3 2 49 8 9 3 560 4 1 40 8 •7 4 540 4 3 36 9 . 1 5 2 280 4 3 47 8 •7 6 3 47O 3 9 85 9 5 7 4 42O 4 2 105 12 3 8 5 370 3 2 62 10 . 2 9 6 350 4 4 58 10 4 10 7 42O 3 7 55 10 .0 1 1 8 380 4 3 59 11 •5 Table 4 —Dog VIII. — Weight 8.65 Kilos. Amount of Amount of Amount of S given. Urine. KSCI Feces. KSCN in feces. Date. Gram: Cc. Mg. Grams. Mg. Feb. 29 220? 37 8.4 Mar. i 34oi 5-7 44 7 9 2 3 320/ 2 70 s 5-9 39 4i 10 9 2 7 4 300 32 42 9 5 5 2 170 3-7 38 10. 2 6 3 320 3 5 47 9 6 7 4 250 3 9 65 9 7 8 5 340 3 6 57 9 3 9 6 37o 3 6 55 8. 8 10 7 330 3 8 49 10. 5 1 1 8 320 3 5 47 9 4 On March 11, 191 2, dogs VII and VIII were bled to death 62 from their left femoral arteries under local cocaine anesthesia. The organs and tissues were removed and analyzed for sulfo- cyanate. The analytical data for the organs of the two dogs, are given in Tables 5 and 6. Table 5.— Dog VII. Weight. Amount of KSCN. Tissue. Grams. Mg. Bile 17 3-2 Blood 653 27.6 Brain 63 None Heart 51 None Intestine (small) 253 10.3 Intestine (large) 127 8.7 Kidneys 63 None Liver 258 25.8 Muscle 200 None Spleen 22 None Pancreas 12 None Stomach contents I IO None Table 6.- -Dog VIII. Weight. Amount of KSCN. Tissue. Grams. Mg. Bile 22 4.O Blood 580 22.5 Brain 60 None Heart 73 None Intestine (small) 295 n. 2 Intestine (large) 55 5-4 Kidneys 62 None Liver 297 21 .6 Muscle 200 None Pancreas 22 None Spleen 27 None Stomach contents 128 None It will be seen from the Tables 3-6 that the feeding of elementary sulfur has no effect upon the excretion of the sulfocyanate substances. The average daily output of sulfo- cyanic salts in the urine and feces was the same before and after the administration of flowers of sulfur. Neither did these experiments show that sulfur produces an increase in the (so to speak) stored up thiocyanates in the various organs and tissues that contain this substance. 63 Effect of Administration of Sodium Sulfide upon the Elimina- tion of Sulfocyanatcs, Two dogs whose excreta were analyzed daily for five and eleven days respectively were fed according to their weight with meat, lard, cracker meal, bone ash and water. Before beginning to administer the sulfide to these animals, an attempt was made to determine which dosage will produce the least amount of discomfort to the animals, and especially which dosage will not cause vomiting; for, when the sodium sulfide enters the stomach, it is acted upon by the hydro- chloric acid of the gastric juice and sulfuretted hydrogen is produced which will cause emesis. A third dog was taken and 0.5 gram sodium sulfide was given him in a gelatin capsule. He promptly vomited. Next day before feeding this dog a capsule containing 0.2 gram sodium sulfide was given. The dog licked up his food. After half an hour there was noticed marked eructation of hydrogen sulfide. The vomiting was very slight. It was decided, therefore, to use 0.15 gram of sodium sulfide for the dogs IX and X. On March 20, 191 2, dogs IX and X were each given 0.15 gram sodium sulfide in gelatin capsules. They ate their food heartily and did not seem to suffer any discomfort. They were fed daily in like manner, 0.15 gram sodium sulfide for five days. The urine and feces were collected daily and analyzed for sulfocyanate. The daily urine and feces analyses are given in Table 7 and 8. Table 7. — Dog IX. — Weight 6.94 Kilos. Aim Date. Na2S given. Gram. Urine. Cc. KSCN in urine. Mg. Feces. Grams. KSCN in feces Mg. iar. 16 " 17 " 18 320l 340 2IO J 8-3 27 I 25 t 9-6 19 20 220 \ 250 j 5 5 24 1 27 \ 30 J 14 7 21 O I 5 I90 2.9 35 7.2 " 22 O.I5 3IO 46 32 7-5 23 O.I5 370 4 4 30 7.8 24 O.I5 300 3 9 33 8.3 " 25 O.I5 220 4.2 29 8-5 6 4 Table 8. — Dog X. — Weight 8.2 Kilos. Amt. Na2S given. Urine. KSCN in urine. Feces. KSCN in feces Data. Gram. Cc. Mg. Grams. Mg. Mar. 10 250 ' 25] II 27O 7-4 22 > 18.7 ' 12 29O J 27 J ' 13 30O 32 1 ' 14 280 8.2 39 16.3 ' 15 330. 45 J ' 16 ' 17 ' 18 340 370 330 I2.3 32? 4i5 37 X 32 i IO.5 12.2 19 220 20 2IO 39 6.1 21 O. 15 36O 4-3 43 6.7 22 O. 15 340 4-7 37 7-5 23 O. 15 3IO 4-5 35 7-4 ' 24 O. 15 29O 4-5 28 7.8 ' 25 O. 15 280 4-7 32 7.6 On March 25, 1912, the two dogs were bled to death and their tissues and organs analyzed for thiocyanate. The usual precautions described previously were taken. It was found in both cases necessary to bleed the animals from both femoral arteries, because the blood clotted very easily before complete exsanguination. The blood was quite dark. The results of the analyses are reported in Tables 9 and 10. Table 9. — Dog IX. Tissue. Weight. Grams. Amount of ] Mg. Bile Blood Brain 29 508 70 3-7 38.8 None Heart 62 None Small intestine 328 20.6 Large intestine Kidneys Liver Muscle 152 65 236 200 16.2 None 27.0 None Pancreas 24 None Spleen 28 None vStomach contents 131 3-5 65 Tahle io. — Dog X. Weight. Amount of KSCN Tissue. Grams. Mg. Bile 25 None Blood 467 4238 Brain 56 None Heart 63 None Small intestine 289 17-5 Large intestine 172 10.2 Kidneys 75 None Liver 267 22. 7 Muscle 200 None Pancreas 30 None Spleen 28 None Stomach contents 126 4,3 The feeding of sodium sulfide to dogs produces no very marked effects upon the thiocyanate content or excretion. It was noticed, however (see Tables 7 and 8), that there was a slight rise in the sulfocyanate excretion in the feces, espe- cially in the fourth and fifth day of the feeding. The stomach contents in both dogs showed traces of the thiocyanate. This may have been caused by a regurgitation of duodenal contents into the stomach. One should be very sceptical in deducing any conclusion from this finding purporting to show that sodium sulfide is changed in the stomach to thio- cyanate. On the contrary the stomach as well as the intestine should be considered the excretory mechanism for the thio- cyanate salts. Effect of the Administration of Taurine upon the Elimination and Distribution of Thiocyanatcs. On March 28, 191 2, dogs XI and XII weighing respectively 8.4 kilos and 8.55 kilos were put in separate cages and were fed daily as described previously for a period of seven days. On April 4, 191 2 each dog was given 0.1 gram taurine in a gelatine capsule. On succeeding consecutive days, the dogs were given 0.2 gram, 0.4 gram, 1.0 gram, 1.0 gram. The dogs were bled to death from the right femoral arteries on April 9, 191 2. The usual precautions were adopted and the various tissues and organs removed and analyzed. 66 The analytic data for the daily urine and feces are given in Tables n and 12. Apr. Date. Mar. 29 30 3i 1 2 3 4 5 6 7 8 Table ii. — Dog XI. — Weight 8.4 Kilos Amt. of taurine. Urine. KSCN in urine. Fee Grams. Cc. Mg. Gra O. I 0.2 O.4 O.8 I .O 11. 4 12. 1 8-3 6.2 5-5 KSCN in feces. Mg. 19-5 22.5 16.4 8-5 8.2 9 1 9 7 Table 12. — Dog XII. — Weight 8.55 Kilos. Date. Mar Amt. of taurine. Urine. Grams. Apr. 29 30 31 I 2 3 4 5 6 7 o. 1 O. 2 O.4 0.8 I O KSCN in urine. Mg. 7-5 I2.0 149 4-5 4-7 5-3 5 5 KSCN in feces. Mg. 16.9 17.4 14.2 9-4 9 7 9.0 10.4 Table 13. — Dog XI. Weight. Amount KSCN. Tissue. Grams. Mg. Bile 18 None Blood 475 28.5 Brain 64 None Heart 73 None Small intestine 305 15-4 67 Table 13. — Dog XI {Continued). Tissue. Weight. Grams. Amount KSCV Mg. Large intestine l62 9 7 Kidneys 74 None Liver 275 '5 1 Muscle 200 None Pancreas 27 None Spleen 30 None Stomach contents 207 None Table 14.- -Dog XII. Weight. Amount KSCN. Tissue. Grams. Mg. Bile 28 None Blood 505 24.6 Brain 70 None Heart 68 None Small intestine 327 17. 2 Large intestine 155 7-5 Kidneys 69 None Liver 244 12.7 Muscle 200 None Pancreas 25 None Spleen 28 None Stomach contents 195 None Taurine, an amino sulfonic acid produces no noticeable effect upon the excretion or distribution of sulfocyanate in the animal organism. In this respect it behaves altogether unlike glycocoll (according to Willianen). The bile in both dogs failed to reveal the presence of sulfocyanate; the in- testines and liver contained relatively the usual amount of the rhodanate. Effect of the Administration of Thiourea upon the Excretion of Sulfocyanates. Two dogs were kept in the cages several days to determine their normal sulfocyanate output. Thiourea was then administered to them in meat balls. The doses given were 5.0 grams, 5 grams, 7.5 grams, 10 grams and 15 grams on consecutive days. The dogs were bled to death on April 15, 19 1 2. The results will be found in Tables 15-17 68 Table Date. Apr. 10 ii 12 13 14 15 15 Thiourea. Grams. 5-0 7-5 10. o Dog XIII.— Weight 7.92 Kilos. Urine. Cc 290) 340$ 285} 240$ 2Io), 200^ KSCN iu urine. Mg. 5-3 4-7 Feces. KSCN in feces. Mg. l6.2 4-9 19.4 On April 15th the dog was given 15 grams of thiourea. Table 16. — Dog XIV. — Weight 8.04 Kilos. Thiourea. Urine. KSCN in urine. Feces. KSCN in fe Date. Grams. Cc. Mg. Grams. Mg. Apr. 10 270' 36] II 5-0 360 \ 8.7 32 17.4 12 5-0 380. 28 J 13 5-0 27O 1 37 I " 14 7-5 l6o 7-5 54 20.2 " 15 10.0 I70. 52 J On April 15th dog was given 15 grams thiourea. The data for the analysis of the tissues of dogs XIII and XIV will be found in Table 17. Table 17. Dog XIII. Dog. XIV. Weight. Amt. KSCN. Weight. Amt. KSCN Tissue. Grams. Mg. Grams. Mg. Bile 30 4.6 20 3-7 Blood 470 20. 7 465 I9.4 Brain 69 None 72 None Heart 67 None 65 None Small intestine 285 20.3 305 17.6 Large intestine l6o 7-1 I46 8-3 Kidneys 72 None 63 None Liver 263 17-5 185 22.8 Muscle 200 None 200 None Pancreas 21 None 17 None Spleen 27 None 20 None Stomach contents I70 None 222 None Thiourea under the conditions of these experiments does not produce any effect on the amount of thiocyanate eliminated from or distributed through the body. 6 9 Effect of the Administration oj Acetonitrile upon the Excre- tion of Sulfocyanatcs. The feeding to a dog, weighing 10.77 kilos, of 0.5 gram of acetonitrile daily, caused a marked exacerbation in the sulfo- cyanate output in the urine. The blood also upon analysis, gave a very high thiocyanate figure. The results of the acetonitrile experiment will be found in Tables iS and 19. Table 18. — Dog XV. — Weight 10.77 Kilos. Amt. KSCN Amt. KSC Acetonitrile. Urine. in urine. Feces in feces. Date. Grams. cc. Mg. Grams Mg. Apr. 15 2 20 2.8 52 IO.7 16 24O 3-4 28 8.0 " 17 5 230 3-3 17 8.2 18 5 270 4-4 48 9 1 " 19 0-5 300 63 42 8.9 " 20 OS 380 6.7 43 8-5 " 21 5 24O 8.9 27 8.4 Table 19. —Dog XV. Weight. Amt. KSCN. Tissue. Grams. Mg. Bile 16 6-5 Blood 560 47.O Brain 72 None Heart 75 None Small intestine 340 21 5 Large intestine 144 10.6 Kidneys 64 None Liver 287 18.4 Stomach contents 142 None Effect of the Administration of Thioacetic Acid upon the Distri- bution of Sulfocyanate in the Animal Body. Dog XVI, male, weighing 13. 45 kilos, was used in this ex- periment. Thioacetic acid is very evil smelling and very poisonous. The dog was given daily 6 to 8 drops of thioacetic acid in a gelatin capsule. The dog vomited, but he always licked up the vomitus so that none of the acid was lost. He was bled to death on the fifth day. The analysis of the organs did not show any variation from the usual amount of sulfocy- anates. 70 Effect of Administration of Alanin upon the Excretion of Sulfo- cyanates. An attempt was made to corroborate the findings of Willianen who stated that amino acids, like glycocoll, caused an increase in the sulfocyanate output in the urine. The effect of the ad- ministration of alpha- amino-propionic acid will first be discussed. A dog, weighing 7.85 kilos, was fed from 2 to 5 grams alanin from April 24 to 29. It was found that there was a marked increase in the sulfocyanate elimination in the \ rine. The amount of thiocyanate in the feces remained constant. Table 20 shows the daily KSCN output in the urine and feces. Tabus 20.— Dog XVII.— Weight 7.85 Kilos. Date. Alanin. Grams. Urine. Cc. Amt. KSCN in urine. Mg. Feces. Grams. Amt. KSCN in feces. Mg. Apr. 19 220 } 28] 20 225 f 7-7 29 J 12.8 ' 21 2IO J 42 J ' 22 270] 37] ' 23 .... 230 \ 8-3 43 13 -7 ' 24 2 270 J 35 J ' 25 3 250 2-5 30 5-5 ' 26 4 245 5-7 32 5-3 ' 27 5 360 5-2 4i 6.2 ' 28 5 270 8.4 40 5-o ' 29 5 340 8.8 57 5-4 On April 29, 1912, the dog was bled to death. The analyses of the tissues and organs (see Table 21) did not reveal any in- crease in the sulfocyanates in the tissues. Table 21. —Dog XVII. Weight. Amt. KSCN Tissue. Grams. Mg. Bile 21 6.2 Blood 4IO 21.8 Brain 79 None Heart 58 None Small intestine 348 25-4 Large intestine 61 10.3 Kidneys 45 None Liver 243 18.7 Muscle 200 None Pancreas and Spleen 48 None Stomach contents 105 None 7i Effect of the Administration of Glycocoll upon th* Excretion of Sulfocyanates. Willianen used 5-10 grams of amino acetic acid in his ex- periments, and he reported a marked increase in the sulfocy- anate elimination. I used only 0.5 to 1.5 grams, and have found that with this dosage no effect was produced on the thiocyanate excretion or distribution. The results found are reported in Tables 22 and 23. Table 22 — Dog XVIII. — Weight 9.24 Kilos. Glycocoll. Urine. Amt. KSCN in urine. Amt. KSCN Feces. in feces. Date. Grams. Cc. Mg. Grams. Mg. Apr. 16 0.50 2IO 30 34] II 17 O.75 200 3 4 22 [ 20.5 a l8 I OO 420 2.9 55 J H 19 1.25 340 4-5 11} ■'« II 20 I . 50 370 3-5 Table 23 — Dog XVIII. Weight Amt. KSCN Tissue. Grams. Mg. Bile None Blood 5IO 29 3 Brain 56 None Heart 71 None Kidneys 75 None Small intestine 385 27-5 Large intestine 163 17.2 Liver 302 No Analysis Spleen 19 None Pancreas 24 None Muscle 200 None Stomach contents 170 None The Effect of Preventing the Saliva from entering the Stomach, upon the sulfocyanate output. A male bull dog weighing 10.4 kilos was kept in a cage for several days to determine his state of health. He was found to be entirely normal. On April 20, 191 2, the dog was put under ether anaesthesia, and, using all aseptic and antiseptic precautions, an incis on 72 was made* in the left side of the neck, the esophagus was ex- posed and was cut transversely. The upper and lawer portions were sutured to the skin. The wound did not suppurate. The siliva was collected on cotton swathings tied around the dog's neck; no saliva could possibly enter the stomach. The dog was fed twice daily through a stomach tube inserted through the opening in the neck ; the food consisted of milk, cracker meal, bone ash, beef extract, Witte's peptone and gelatin. The dog was daily put in a holder and the urine was collected when voided. In this way 825 cc. of urine were collected in a period of eight days. The analysis showed that even with the absence of saliva from the stomach, the urine still showed the normal traces of sulfocyanate. In 370 cc. urine there were 4.2 mgs. KSCN; in 230 cc. urine, 2.7 mgs. KSCN: in 225 cc. urine 2.3 mgs. KSCN. The dog died on May 2, 1912. The blood, bile and liver were analyzed. In 340 grams of blood I found 15.5 mgs. KSCN; in 272 gms. of liver there were 17.8 mg. KSCN, and in 27 grams bile there were 5.4 mg. KSCN. The saliva which was collected through the opening in the gullet, did not con- tain any rhodanate. Effect of Fasting upon the output of Sulfocyanates. A male dog, weighing 8.46 kilos was deprived of food for a period of seven days. He was given water ad libidum. His urine was daily collected and analyzed for sulfocyanates. The daily amount of rhodanate eliminated did not vary from the average for the other dogs. On the seventh day of fasting, the dog was exsanguinated from the left femoral arteries. The analysis of the tissues shows that fasting diminishes the amount of sulfocyanates in the body. The Intestinal tract did not contain any rhodanate. (See Table 24.) * I wish to express my indebtedness to Dr. Morris Hirsch Kahn of Mount Sinai Hospital for the performance of this operation and for his cooperation in sending me numerous specimens of saliva from his ward patients. 73 Table 24.— Doc. XX. Weight. Tissue. (".111. Amt. KSCN Bile 19 Blood 455 Brain 55 4-7 153 None Heart 60 None Small intestine 180 None Large intestine 50 Kidneys 45 Liver 180 None None 12.4 Pancreas and Spleen 32 Stomach contents 85 None None CONCLUSIONS. 1. The sulfocyanates are normally present in various parts of the animal body. 2. The sulfocyanates are excreted in the urine and feces 3 The production and elimination of sulfocyanates in the urine are not dependent upon the amount of thiocyanate pres- ent in the saliva. Dog saliva apparently is free from sulfo- cyanate while the urine invariably contains it. 4. The ingestion of amino acids (like alanin), and of nitriles (like acetonitrile) increases the excretion and distribu- tion of thiocyanates in the body. 5. Thiocyanate is apparently produced from protein in the body. The results for the fasting dog harmonize with this conclusion. 6. The ingestion of sulfur, sodium sulfide, thioacetic acid, thiourea and taurine did not increase the output of sulfo- cyanates. CHAPTER VI. THE TOXIC EFFECTS OF POTASSIUM SULFOCYANATE- Lauder Brunton 1 found that solutions of potassium sulfo- cyanate, when administered to mollusca, diminish the reflex actions, but have little effect on the excitability of the nerves. A small dose of this salt somewhat quickens the cardiac action, a large dose stops the heart in diastole, and if it is directly applied to the heart, the stoppage is permanent. Kletzinski 2 suggested quite some time ago that the sulfo- cyanate of potassium is toxic to bacterial life, and its function in the saliva is bactericidal. Nysten quotes Litre and C. Robin that potassium thiocyanate is very toxic. Wurtz, on the other hand, mentions Woehler and Frerichs as his authorities for the statement that potassium sulfocyanate is not at all poisonous. Claude Bernard, Setschenow and Podcopaew were of the opinion that this thiocyanate is very slightly toxic; Paschkis, however, thinks that its toxicity is quite noticeable. Nerking 3 has synthesized an albumen rhodanate which he called rhodalzid. Lehman 4 and Diena 5 studied this com- pound and found it not at all toxic. The following experiments were performed in the study of the toxic properties of potassium sulfocyanate. 8 The details of procedure will be given with each experiment. The Effect of Injections of Various Amounts of Potassium Sulfocyanate into the Dorsal Lymph Sacs of Frogs. (a) Injected into the dorsal lymph sac of a frog weighing 25.5 grams, 1 cc. of a 5 per cent, solution of potassium sulfo- 1 Brunton: Pharmacology, Therapeutics and Materia Medica, 1893, p. 114. 2 Kletzinski: Heller's Archiv., 1833, p. 39. 3 Nerking: Med. Klinik, 191 1, cit. after Diena. 4 Lehman: Arch. f. Zahnheilk., 1911, cit. after Diena. 5 Diena: Biochem. Zeitschr., 1912, xxxix, p. 13. 8 The KSCN used was Kahlbaum's. In order to make certain that no cyanide was present, several large portions were tested according to the directions of A. A. Noyes, (Jour. Amer. Chem. Soc, xxxiv, 1912, p. 609). No trace of cyanide was found. 75 cyanate (50 mg.). Frog had violent strychnine convulsions with marked opisthotonus. Died in two minutes. Heart stopped in systole. (6) Injected into dorsal lymph sac of frog (weight 27 grams) 1 cc. of 4 per cent, solution of potassium sulfocyanate (40 mg.). Frog went into spasms; opisthotonus; died in 35 minutes. Heart stopped in systole. (c) Injected into dorsal lymph sac of frog (weighing 21.5 grams) 1 cc. of 30 per cent, potassium sulfocyanate solution (30 mg.). Frog had spasms; died in tetany in 45 minutes. Heart stopped in systole. (d) Injected into dorsal lymph sac of frog (weight 38.8 grams) 1 cc. of 2 per cent, potassium sulfocyanate solution. Frog sickened, but had no spasms. Died in 4 1 , hours. These experiments were repeated several times with practically the same results. (e) Injected into dorsal lymph sac of frog (weight 19.5 grams) 7.5 mg. potassium sulfocyanate. Died in opisthotonus in fourteen hours. (/) Injected into dorsal lymph sac of frog (21.5 grams) 7.5 mg. of potassium sulfocyanate. Frog died in opisthotonus in sixteen hours. (g) Injected into dorsal lymph sac of frog (weight 20.5 grams) 5 mg. of potassium sulfocyanate on April 2, 191 2. Frog was hypersensitive, went into extreme tetanic contrac- tion upon the slightest stimulation. The hypersensitiveness lasted for three days. On April 9, 191 2, frog still well, has lost its irritability. Seems normal. (h) Exact duplication of experiment g. Frog weighed 18.5 grams. Dose was 5 mg. of potassium sulfocyanate. Recovered completely. Effect on Subcutaneous Injections of Solutions of Potassium Sulfocyanate on Guinea Pigs. Experiment 1. — Injected subcutaneously into a guinea pig, weighing 430 gms. 500 mgs. of KSCN. The pig died in one hour in marked convulsions. This dose was also given hypo- dermically to another pig, weighing 412 gms. This animal died in violent convulsions in two hours after injection. 7 6 Experiment 2. — Into a guinea pig, weighing 350 gms., I in- jected subcutaneously 1 cc. of a 20% solution KSCN (200 mgs.) The pig was found dead next morning. Experiment 3. — Injected into a guinea pig, weighing 385 gms., 1 cc. of a 10% solution KSCN (100 mgs.) The animal expired next morning in very violent tetanic convulsions. Experiment 4. — A hypodermic injection of 1 cc. of a 5% solution KSCN (50 mgs.) had a sickening effect upon the guinea pig, the animal had diarrhoea and did not eat its food, After 24 hours it began to have convulsions and finally died. Animal weighed 335 grams. Experiment 5. — The injection of 25 mgs. produced no visible effects upon a guinea pig weighing 415 grams. Effect on the Administration of Potassium Sulfocyanate, by Mouth. Experiment 1. — A dog, weighing 8.1 kilos, was given 0.61 gram KSCN in a meat ball on each of three consecutive days. Calculated, the amount given is equal to 75 mgs. KSCN per kilo of dog, or about 1V4 grains. After the second dose, the dog did not partake of his food, vomited, had tremors all over body. The dog died in convulsions twenty-four hours after the third dose. The analysis of the urine showed that most of the sulfocyanate was excreted by the kidneys. Experiment 2. — Administered to a dog, weighing 10.8 kilos, 50 mgs. KSCN per kilo of the animal. The dog became de- pressed, vomited and had diarrhoea. The condition did not seem to become worse after five findings. Experiment 3. — To a dog, weighing 6.12 kilos, I gave by mouth 200 mgs. KSCN in a meat ball on each of five consecu- tive days. The dog suffered from depression, tremors, diar- rhoea, vomiting. Recovered upon cessation of administration of the sulfocyanate. Experiment 4. — A dose of 25 mgs. per each kilo of weight of a dog, weighing 9.4 kilos produced no toxic effects that were discernible. It may be mentioned here that Breton, Bruyant and Mezie 1 recently reported that they injected 0.03 gm. NH 4 SCN into the 1 M. Breton, L. Bruyant and A. Mczie: Compt. rend. soc. biol., LXXII, 1912, p. 400. 77 jugular veins of dogs. They recovered the thiocyanate in one half hour in the urine. They do not refer to the toxic effects. The Effects of Solutions of Potassium Sulfocyanide upon //(. Germination and Growth of Plants. Raulin found that in growing the Aspergillus fumigaius, it was necessary to add iron salts to the spraying water to neutralize a certain poison in the plant which prevents the growth of the plant. This toxic principle he found to be sulfocyanic acid. Chouppe found saliva fatal to vegetable life. He de- termined this fact by watering seedlings with saliva only. He described this deleterious influence to the sulfocyanate content of the saliva. Experiment to Determine Toxic Effects of Potassium Thio- cyanate on White Lupins. Selected white lupin beans (Lupinus alba) were soaked in distilled water over night. They were then carefully planted in moss which had been soaked in water and washed clear of all visible impurities. When the roots had sprouted to be from three-quarters of an inch to an inch in length they were taken from the moss and treated as follows: Jena glass beakers (400 cc. volume) were carefully washed with soap and water and then with solutions of acid and alkali. They were then rinsed with distilled water. Into each beaker were placed 200 cc. of various dilutions of potassium sulfo- cyanate. The beans were taken out from the moss, were carefully wiped off on a smooth soft cloth and the shells were carefully peeled. Upon the root of each seedling a mark was placed 15 mm. from the tip. India-ink was used for the marking. The bean was then carefully speared upon a sharp glass rod which was supported by a cork plate covering the beaker. The rod was lowered so that only the root of seedling was in the liquid, the body did not come in contact with the solution. The solutions used were as follows: 5 per cent., 4 per cent., 3 per cent., 2 per cent., 1 per cent., 3 / 4 per cent., l /i P er cent., l f t per cent., 2 / 5 per cent., ' s per 78 cent., V 10 per cent., V20 P er cent -> 1 Im P er cent -> and V100 P er cent. Control tests were made with distilled water and tap water. The following observations were made daily: The length of the root, the amount of chlorophyl formation, the growth of side roots, the growth of tufts, the amount of mold growth in the solutions. Concentrations of potassium sulfocyanate from 0.2-5.0 per cent, are completely toxic to these plants. The roots do not grow, they assume a white, waxy, oedematous ap- pearance, no side roots are formed, and not tufts make their appearance. Chlorophyl formation though inhibited, is still present in all the beans. The solution showed very heavy growths of various fungi and protozoa. The dilute solutions of the thiocyanate from 0.01-0.1 per cent, inhibit the growth of the roots proportionately to the amount of the noxious substance present. The roots grow to a considerable length with the 0.01 per cent, solution, side roots are formed in various number, chlorophyl formation is present, and the growth of molds is very much less. Tuft growth is quite marked in the 0.01 per cent., 0.02 per cent., 0.025 per cent., 0.05 per cent, solutions and very slight in the 0.1 per cent, solution. Compared with the two controls of distilled water and tap water even the 0.0 1 per cent, solution of potassium thio- cyanate is quite toxic; for, the growth of the main root and side branches, as well as the growth of tufts, is strongly in- hibited by the 0.0 1 per cent, dilution. Experiments to Determine Toxic Effects of Potassium Thio- cyanate on Timothy Grass Seedlings. Disks of white blotting paper were cut two inches in diameter. They were allowed to soak in distilled water for five minutes. Each disk of paper was put in a flat petri dish and sprinkled evenly with timothy grass seed. The disk was then covered with a glass beaker, and was sprayed daily with pure water until the grass blades were about Via inch in height. 79 Solutions of potassium sulfocyanate were made of various dilutions, o.oi-io.o per cent. A separate grass plot was used for each dilution. A control was kept with distilled water. Each disk was watered twice daily with five drops of the respective solutions. The toxic effects of the sulfo- cyanate were noticed in two days. The grass blades did not increase in size, became yellowish green in color, and finally after seven days, shrivelled and blackened. A profuse growth of molds was noticed in all the plots which had been sprayed with potassium sulfocyanate. The control plot showed an exuberant growth of delicate, slender, elongated blades almost two inches in length, without any mold growth, Several repetitions were made of these experiments, and almost identical results were obtained. The experiment was then varied. Instead of watering the plant daily with the sulfocyanate solution it was only sprayed six times with five drops of the toxic substance. After the third day distilled water was used. The objection to spraying the plant daily with the thiocyanate solution is that as the water evaporates the sulfo- cyanate remains at the roots of the plant and the daily concentration of this salt becomes steadily greater. However, even with the precaution just now detailed, the toxic effects of the sulfocyanate was quite evident. The growth was stunted, and mold growth was prolific in direct proportion to the amount of potassium sulfocyanate. The Effect of KSCN Solutions on the Germination of Seeds. White Lupins that were allowed to soak for eighteen hours in solutions of greater concentration than 0.02% did not germi- nate upon being transferred to beakers containing distilled water. The growth of molds in these beakers was very ex- uberant. A 0.01% solution of KSCN did not prevent the germination of the beans. Upon timothy seedlings potassium sulfocyanate, even in dilutions of 0.01%, completely inhibits germination. Bactericidal Properties of Potassium Sulfocyanate. Tubes containing gelatin and gelatin-peptone media were inoculated with bacteria. To each tube was added a different 8o amount of potassium sulfocyanate. Control tubes were also kept. The tubes were allowed to stand at room tem- perature for several days. The growth in each tube was noticed. It was observed that culture media containing less than 3% KSCN did not inhibit the growth of bacteria. A 6%, 7%, 8%, 9% and 10% KSCN gelatin seemed to retard but did not com- pletely inhibit the growth of dust bacteria. The growth of molds in every tube was very luxuriant. Though the growth of the bacteria was quite marked in every case, still the retar- dation in growth is directly proportional to the amount of sulfo- cyanate present. Effect of Potassium Sulfocyanate on Yeast Fermentation. A 5 per cent, glucose solution was made up and put into a number of Einhorn saccharometers. To the various saccharometers I added various amounts of potassium sulfo- cyanate. A control tube to which no sulfocyanate had been added, was also made. A uniform suspension was prepared of the best yeast in distilled water. To each saccharometer I added 1 cc. of this suspension. The amount of fermentation was determined by the quantity of carbon dioxide produced in each saccha- rometer. No inhibition in the amount of fermentation was noticed. In fact, if anything, the growth of the yeast in the sugar solu- tions seemed to be stimulated by the presence of high amounts of potassium thiocyanate. Effect of KSCN on the Souring of Milk. To a number of test tubes containing milk, I added varying amounts of potassium sulfocyanate and a few drops of litmus solution. Solutions containing 0.5%, 1%, 2%, 3% and 4% of KSCN prevented the coagulation of milk even after four days. Lower amounts of sulfocyanate in milk were without noticeable effect in this regard. The amount of acid production is inversely proportional to the amount of potassium sulfocyanate present. Upon titrating five cc. of filtered milk from each of six tubes to which varying 8i amounts of KSCN were added, I found that, compared with the control to which no KSCN was added there was a marked reduction in the quantity of acid formation. In these titrations NaOH solution (i cc. = 0.0079 S ras - NaOH) was used, phenol phthalein being the indicator. The results are recorded in the accompanying table. Amount of Acid Formation in Milk upon adding varying amounts of KSCN. Per cent. Amt. NaOH Number. Amt. of Milk. KSCN. cc. Control 5 cc. O.O 1.4 I 5 cc O.06 I .2 II 5 cc. O. 12 I .2 III 5 cc. O.24 I . I IV 5 cc. O.48 O.9 V 5 cc. 095 O.8 VI 5 cc. I .90 0.3 (Several experiments along these lines are in progress, and will be described later. When this dissertation went to press, the available data of the later experiments were not sufficiently complete for insertion.) conclusions. 1. Potassium sulfocyanate is toxic to both plants and animals. Its toxicity is so marked that indiscriminate dispen- sation of the substance to patients is dangerous. 2. The growth of molds is enhanced by potassium sulfo- cyanate. 3. Small biological quantities of KSCN have no inhibiting influence on bacteria. 4. Yeast fermentation is uninhibited or stimulated by potas- sium sulfocyanate. 5. The souring of milk is inhibited by large (relatively) amounts of the thiocyanate. ADDENDUM. Bibliography not specifically alluded to in the body of this disserta tion. i. Archetti, Chem. Centralblatt, 1904, i, pp. 318, 371 and 796. 2. Acr'ee and Hickins, Dental Review, xxii, p. 219. 3. Bujuid, Virchow's Arch., xci, p. 190. 4. Benson, Jahresb. d. fort. d. Chem., 1852, p. 439. 5. Berkson, The Odontologist, 191 1, vii, p. 15. 6. Beach, Dental Cosmos, 1908, 1, p. 469. 7. Bentley and Le Roy, N. Y. Med. Jour., 1908, Ixxxix, p. 210. 8. Buttazzoni, Stomatol. Milano, 1902, i, p. 764. 9. Bergman, Therapie der Gegenwort, 1903, p. 200. 10. Claisse and Dupre, Compt. rend. soc. de Biol., 1894, vi, p. 55. 11. Chittenden and Richards, Am. J. Phys., 1898, i, p. 461. 12. Carlson and McLean, Am. Jour. Physiol., 1908, xx, p. 457. 13. Duclaux, Le Microbe et la Maladie, Vol. iv. 14. Doubleday, Dental Cosmos, 1909, li, p. 412. 15. G. Deniges, Jour. d. Pharm., 1898, xxv, p. 88. 16. Edinger and Clemens, Z. /. klin. Med., 1906, lix, p. 223. 17. Goadley, Brit. Med. Jour., 1910, p. 770. 18. Gmelin, Handb. d. Chem. 19. Goodman, Proc. Path. Soc. Philadel., 1909, xxii, p. 9. 20. Gautrand, Du chimisme salivaire, Lyon, 1895. 21. Grawitz and Steffen, Berl. Klin. Woch., 1894, xxxi, p. 419. 22. Hofbauer, Arch. f. d. ges. Physiol., 1896, lxv, p. 503. 23. Jappeli, Zeit. f. Biologie, 1908, li, p. 127. 24. Jamison, Ann. d. Chem. u. Pharm., lviii, p. 264. 25. Klason, Jour, prakt. Chem., xxxv, p. 407. 26. Liebig, Ann. d. Chem. u. Pharm., lxi, p. 126. 27. Lidow, Jour. Russ. Chem. Soc, xvi, p. 271. 28. Langley, Trans. Roy. Phil. Soc, 1880, clxxx, p. 109. 29. Marcantonio, Rejorma Med. Napoli, 1897, xiii, p. 254. 30. Mislawsksky and Smirnow, Arch. f. Physiol. Leipzig, 1896, p. 93. 31. Mensel, Die Quellkraft d. Rhodanate, 1886. 32. Nencki, Ber. deut. chem. Ges., xxvii, p. 1318. 33. Nollner, Jahresb. d. Fortsch. d. Chem., 1856, p. 443. 34. Neumeister, Physiol, d. Menschen, p. 844. " 35. Ostwald, Jour, prakt. Chem., xxxii, p. 305. 36. Pribram, Jahr. d. Phys. u. Path., 1868, i, p. 148. 37. Pettenkofer, Chem. Centralbl., 1846, p. 231. 38. Pozzi, Chem. Centralbl., 1904, ii, p. 331. 39. Radiszewski, Ann. d. Chem. u. Pharm. 40. Sticker, Munch, med. Woch., 1896, xliii, p. 1041. «3 4i. Schoumow-Simanowsky, Arch. j. exp. path. u. Pharm., 1891, xxxiv, p. 332. 42. Sertoli, Virchow's Hirsch Jahresb., 1869, >i P- '04. 43. Schiff, Ann. d. Chem. u. Pharm., cvi, p. 116. 44. Tozner, Arch. int. Physiol., 1905, ii, p. 153. 45. Thiel, Ber. deut. chem. Ges., 1902, xxxv, p. 2766. 46. Triolo, Jahresber. d. Tierchem., 1897, xxvii, p. 813. 47. Ville and Mestrezat, Compt. rend. Soc. d. Biol., 65, 66. 48. Volckel, Jahr. d. Fort. d. Chem., 1853, p. 406. 49. Von Beustl, Brit. Dent. Jour., 1911, xxxii, p. 65. 50. Van Setten, Muller Arch. d. Anat., 1838, p. 164. 51. Volhard, Ann. d. chem. u. Pharm., cxc, p. 24. 52. Warner, Chem. Centralbl., 1903, i, p. 985. 53. Weber, Jahresb. d. Tierchem., 1892, xxii, p. 245. 54. Zeise, Ann. d. Chem. u. Pharm., xlvii, p. 36. 55. Zimmerman, Ann. d. Chem. u. Pharm., excix, p. 1. BIOGRAPHICAL. Max Kahn was born on March 17, 1887. In 1905 he ob- tained a scholarship in Cornell University which he held until 1 9 10, when he was granted the degree of Doctor of Medicine from that institution. During the years 1910-1912, he pursued graduate work in Columbia University. In June. 191 1, he received the degree of Master of Arts, his major subject being Organic Chemistry, his thesis being entitled 'The Chemistry of the Xaphthodiazines." He held the University Scholarship in Columbia University for the year 1911-1912. during which time he pursued studies in Biological Chemistry for the degree of Doctor of Philosophy. He has lately been appointed Director of the Chemical and Physiological Laboratories of Beth Israel Hospital, New York City. PUBLICATIONS. 1. Shakespeare's knowledge of medicine. New York Medical Journal, 1910, xcii, p. 957. 2. Moliere and the physician. Johns Hopkins Hospital Bulletin, 1911, xxii, p. 344. 3. The importance of the colloidal nitrogen in the urine, ia the diagnosis of cancer. American Journal of Gastroenterology, 191 1, i, p. 11. 4. Ueber den Wert des colloidalen Stickstoffs im Urin bei der Krebs- diagnose. Archiv. fur Verdauungs Krankheiten, 1911, xvii, p. 557. 5. On the absorption and distribution of aluminium from aluminized food. Biochemical Bulletin, 1911, i, p. 235. 6. The colloid nitrogen content of normal and cancerous dog's urine. (In press.) 7. The chemistry of renal and cystic calculi. (In press.) 8. History of the lithotomy operation. (In press.) 9. Rambam the physician. (In press.) 10. Therapeutic superstitions and vulgar specifics. Medical Record. (In press.) COLUMBIA UNIVERSITY LIBRARY This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, as provided by the rules of the Library or by special ar- rangement with the Librarian in charge. DATE BORROWED DATE DUE DATE BORROWED DATE DUE lQAfl Jv» ol t9W Ufc r & \&5& C2S(23S)M100 QP802 Kahn K12 c y»nate s - tu d lee of 8 ulf . y£-v,r