COLUMBIA LIBRARIES OFFSITE HX00030015 RECAP THE UNIVERSITY OF CHICAGO intNDKIl BV JOHN n, KOCK F.»' EI.L j: R The Decennial Publications rilE LFXITHANS WALDEMAR KOCH COLUMBIA UW^FW^ DEPARTMENT OF PHtSiPlOGY COtLtO* or PHV*C4AH« *"0 SU«K»£CI«« 4(7 WUT f rrv ■tMHTM 8T»«T HEW VOMK Q^HSl. X^ Columbia ^nitJer^ttp CoUcgc of ^fjpstciansi anb ^urgeonss Hibrarp Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/lecithanstheirfuOOkoch THE UNIVERSITY OF CHICAGO rOURDED BY JOHN D. BOCEEFELLER The Decennial Publications THE LECITHANS THEIK FUNCTION IN THE LIFE OF THE CELL BY WALDEMAR KOCH ASSISTANT IN PHAEMACOLOOT PRINTED FROM VOLUME X CHICAGO THE UNIVERSITY OF CHICAGO PRESS 1902 K11 Copyright 1902 BY THE UNIVEESITY OF CHICAGO PRINTED NOVEMBEH 1, 1902 THE LECITHANS THEIR FUNCTION IN THE LIFE OF THE CELL Waldemar Koch The ash left on the incineration of tissues obtained from various parts of the body, especially the brain, has long been known to contain phosphorus. Of the chemical combination in which this phosphorus was present in the original tissues nothing was known until Gobley' carefully studied an organic phosphorus-containing body, which he isolated from eggs and called lecithin. He obtained as splitting products glycero- phosphoric acid and some of the fatty acids. Diaconow^ continued this work at the suggestion of Hoppe Seyler, and isolated as splitting products glycerophosphoric acid, stearic and oleic acids, and a base which he identified with Baeyer's neurin and the neurin obtained by Liebreich'' from his protagon by decomposition with barium hydrate. From the ease with which his lecithin could be split up Diaconow con- cluded that it was a neurin salt of distearyl glycerophosphoric acid. This view was, however, disproved by Hundeshagen^ on account of the fact that the body prepared by the union of neurin and distearyl glycerophosphoric acid in alcohol solution would not give the characteristic myelin forms, although it possessed all the other properties of lecithin. Strecker*^ brought confusion into this subject by identifying the base obtained by him from lecithin with the cholin he had isolated from bile.° Thudichum' pointed out the difference between the body derived from bile and the base isolated from lecithin, and identified the latter as neurin by a number of analyses made with carefully purified material. In the book above referred to Thudichum also records some other important observations. Among the large number of compounds isolated by him from the brain there are some which do not contain glycerin; as he always finds phosphorus in the form of orthophosphoric acid, he concludes to call these bodies " Pho.sphatids " and consider them rather as derivatives of orthophosphoric acid than glycerophosphoric acid, as previously accepted. His formulse resemble the types of the Typentheorie of Gerhardt and Wurtz, in leaving the exact building up of the molecule a matter of doubt. From a study of the fatty acids in his various compounds Thudichum concludes, contrary to Diaconow, that there are no i^hosphatids with only one fatty acid, but that each one contains either palmitic, stearic, or mar- I Gobley, Journal de pharmacie et de chimie (1850), « Hdndeshaqen, /oumaI/«r profc««c7ie CAemie (1SS3), Vol. XVII, p. 401, and Vol. XVIII, p. 107. Vol. XXVIII, p. 219. ^Diaconow, Hoppe SeyJers medicinUch^hemische Un- sStreckee, Liebig-, Annalen der Chemie und Fhar- tersuchungen,lS66,\o\.Il, p. 221; also Centralblatt far die macie (186S) CXLVIII p 18 medicinischen WUsenschaften (1868), Vol. VI, pp. 2, 97, ' ■ P. • and 434. ' Ibid. (1862), Vol. CXSIII, p. 353. 'LiEBREICH, Licbig's Annalen der Chemie und Fhar- 'Thttbichcm, Die chcmische Konstitution des GeMms macie (1865), Vol. CXXXIV, p. 34. des Menschen und der Tiere, 1901. p. 123. 93 The Leoithans gearic as one of the constituents; which, however, gives no character to the molecule, while the other acid — oleic for brain lecithin, kephalinic for kephalin — gives to the molecule its distinctive properties. Without considering further the important question of the structure of these com- pounds, I would propose to classify them under the general term "Lecithans." The introduction of the word " lecithan " as a group name seems preferable to the use of an entirely new and unfamiliar term like " phosphatids," as proposed by Thudichum. At the same time, the change of the last syllable of lecithin to an gives sufficient variation to prevent any such confusion as attended the generalization of the word "albumen." The lecithans, then, are substances containing in the molecule phos- phoric acid, fatty acids, nitrogen, and, in most cases, glycerin. They resemble each other very closely in their physical appearance, being waxy, non-crystalline, and very hygroscopic. Toward water they all show the same behavior, although their solubility or the solubility of their salts in organic solvents may vary. The very general distribution of the lecithans in all forms of living tissues speaks for their value in the life of the cell. A more careful study of these compounds indi- cates that they are valuable in two ways: first, on account of their physical properties; and, secondly, on account of their chemical behavior. PHYSICAL PEOPEKTIES The behavior of the lecithans with water seems of especial interest, and can be watched under the microscope. A waxy piece of brain lecithin placed in water first swells up and then gives off long filaments (called myelins) which sometimes resemble a shepherd's crook, at other times a mass of twisted skein. If allowed to stand for some time, with frequent shaking, a perfect emulsion is finally formed. A lecithan which has been part of the living tissues, such as brain lecithin, gives a much more perfect emulsion than egg lecithin, which is merely stored food material. If such an emul- sion is the substratum of the living cell — and there seems good reason to consider it so — it may explain some of the physical properties of living protoplasm. The study of this emulsion is especially interesting in connection with the changes in the physical conditions of the living cell brought about by electrolytes, as shown by the recent work of J. Loeb and his school. Action of electrolytes on an emulsion of brain lecithin. — Four g. of brain lecithin (free from calcium, and containing less than 1 per cent, sodium or potassium) are treated with one liter distilled water. The resulting emulsion is sufficiently trans- parent for purposes of study, can be filtered unchanged, has a neutral reaction to litmus, and remains unaltered for weeks, especially after sterilization by boiling. The results of my experiments with this emulsion may be classified as follows: Univalent kations. — Salts of Na, K, NH4, Li, Ag, even in very concentrated solu- tion, give no precipitate and have apparently no effect on the emulsion. The hydro- gen ion is an exception, in the case of acids which are sufficiently dissociated. A 94 Waldemar Koch concentration of ^ttk sulphuric will give a precipitate. Carbonic acid is not BuflSciently soluble to give any precipitate. Divalent kations.—Mg, Ca, Sr, Ba, Co, Ni, Fe", Zn, Cd, Cu, and Pb all give a precipitate which, similar to the one with acids, is flocculent, gelatinous, and settles to the bottom in less than one hour, leaving the supernatant liquid perfectly clear. The concentrations which will just give the precipitate vary somewhat and have been found in the case of Ca, Sr, and Ba to be jrr^ , tt: , and stj j respectively. Trivalent kations. — Fe'", Al, Au give no precipitate and behave like monovalent kations. Cr gives unsatisfactory results. Au, after standing for several hours, is pre- cipitated in the metallic state. Am'ons. — CI, Br, I, SO4", oxalate, citrate, and ferrocyanide (K4Fe(CN)5) give no precipitate, and even in concentrated solution have no apparent effect on the emul- sion. OH is an exception, causing the emulsion to clear up. Non-electrolytes. — Albumins, peptones, glucose, urea, alkaloids, and narcotics like urethan and chloral give no precipitation reactions and leave the emulsion apparently unchanged. Chloroform has a tendency to be emulsified by the emulsion, a reaction which Thudichum had already observed with ether. The precipitations above observed with the hydrogen ion and divalent kations seem to be of an entirely physical nature because: 1. They are independent of the concentration of the lecithin. An nnnr. lecithin emulsion will begin to precipitate with about the same concentration of the divalent kation as an ^j=j7; emulsion. Stronger emulsions are not sufficiently transparent for observation. 2. Removal of the supernatant liquid by decantation and the addition of water will cause the precipitates to redissolve. Cadmium, copper, and other salts of various lecithans have been prepared in alcohol solution and analyzed, but they are readily broken up on the addition of water, and belong to a class of physical compounds even more unstable than ordinary double salts. It would seem, then, that when a certain limiting concentration of the divalent kation is reached, the emulsion can no longer exist and the lecithin is precipitated, carrying with it possibly some of the salt. Very interesting, on account of the possi- bility of furnishing an explanation of such results as Loeb' obtains with Fundulus, are the antagonistic effects of univalent kations in preventing the precipitation. Near the limits at which Ca will just give a precipitate, a very small amount of Na will suffice to prevent this precipitate ; as more Ca is added, relatively more Na is needed. A direct comparison of my results with J. Loeb's is not possible, because, in the first piece, the amounts of Ca, Na, and lecithin in the Fundulus egg are not known, and, in the second place, the reaction between the solution and the egg does not come about as 8L0EB, American Journal of Physiology (1902), Vol. VI, p. ill. 95 The Lecithans After Three Hours - Immediate ppt. Ppt. settled - No ppt. No ppt. - No ppt. No ppt. - Immediate ppt. Ppt. settled - No ppt. No ppt. - No ppt. No ppt. - No ppt. No ppt. directly as in my case. The following table gives the data obtained with an emulsion of brain lecithin: I. 5 CO. =i^ emul. + 5 c.c. water + 5 c.c. j^ Ca(N03)2 - ■5c.c.5mNaCl+ 5 c.c. |^ Ca(N03)2 - I emul. + 5 c.c. ^ NaCl + 1.5 c.c. ^ Ca(N03)2 - emul. + 5 c.c. jg NaCl + 5 c.c. jg Ca(N03)2 - emul. + 5 c.c. ^ NaCl +3.5 c.c. ^ Sr(N03)2 - VI. 5 c.c. ^ emul. + 5 c.c. 2hn KC1+ 5 c.c. - Ca(N03)2 - VII. 5 c.c. ^ emul. + 5 c.c. ^ Fed, + 5 c.c. ^ Ca(N03)2 - VIII. 5 c.c. qTjp; emul. + 5 c.c. urea concentrated solution + 5 c.c.:^ Ca(N03)2 Ppt. formed slowly Ppt. settled IX. 5 c.c. ^^ emul. + 5 c.c. glucose concentrated solution + 5 c.c. yq Ca(N03)2 Ppt. formed slowly Ppt. settled We may conclude, then, that the precipitation of lecithin by divalent kations is a physical phenomenon probably of an electrical nature, because: 1. Non-electrolytes do not prevent the precipitation (I, VIII, IX). 2. The trivalent kation Fe'" is much more efficient in preventing the precipita- tion than a monovalent one like Na (II, VII). 3. The precipitate is formed independent of the concentration of the lecithin and can be redissolved by the addition of water. The application of these observations to Loeb's results must be postponed until other lecithans have been more carefully studied. CHEMICAL PROPERTIES The chemical properties of the lecithans depend on two groups in the molecule: first, the fatty acids, and, second, the complex of which the nitrogen is a part. The phosphoric acid, although the nucleus and very important in the building up of the molecule, does not seem to enter into any reaction, except on the complete destruction of the lecithan ; as Halliburton^ has found the phosphorus to decrease in degenerating nerves only after the eighth day. Each lecithan contains, according to Thudichum, two fatty acids in the molecule: one — either palmitic, stearic, or margaric — does not impart any particular property to the compound; the other — oleic in the case of lecithin, kephalinic in the case of kephalin — gives to the molecule its distinctive character. This distinctive group is always unsaturated, will therefore add iodine, and bring about the reduction of osmic acid. Upon this group, then, depends the use of osmic acid as a stain for nervous 9 W. D. Hallibueton, The Cliemical Side of Nervous Activity, 1901, p. 87. 96 Waldemar Koch tissues in histological technique. The value of osmic acid as a general test for fats depends on the fact that all fats in the body contain some oleates. Pure stearates and palmitates will not give the test. The darkening of the lecithans on exposure to the air is also dependent on this group. In the case of kephalin, the change on exposure to the air takes place so rapidly as to suggest an autoxidizable substance capable of activating oxygen. The guiac-blue reaction, however, gives a negative result ; and Thudichum'" has shown that kephalin exposed to an atmosphere of oxygen in a eudi- ometer will not decrease the volume of the gas. The change is probably due to an internal rearrangement in the molecule, and takes place within the molecule of the fatty acid itself; as Thudichum" obtained from kephalin an acid by saponification (kephalinic acid) which exhibited the same changes as the mother-substance. Less apparent, but nevertheless important, are the changes which the molecules of the lecithans undergo in the complex which contains the nitrogen. Thus Hallibur- ton'" has found the cholin to increase in the cerebro-spinal fluid as the result of general paralysis. For the quantitative investigation of the cholin or neurin the methyl groups attached to the nitrogen seem especially useful, as Herzig and Meyer" have devised a method by which such groups can be accurately determined. The descrip- tion of the method is not easily accessible. I will therefore repeat it here, with such modifications as have been found useful, before going on to describe the results obtained with lecithans from various sources. HEEZIG AND MEYER S DETERMINATION OF METHYL ATTACHED TO NITROGEN The apparatus consists of a double glass bulb, 4 cm. wide at the largest diameter and 2^ cm. at the narrowest diameter, and 12 cm. high. The bulb (a) is connected to (6) by a glass tube which runs to the bot- tom of (6). The double bulbs are placed in an iron sand bath with double bottom and a par- l tition (cd) so that (a) can •^"'/'""^ be heated in sand, while (b) remains compara- tively cool. C is a flask for catching the distillation products from the bulbs, and D is a condenser kept at a tem- \j ,, , ^^ j^ perature of from 40° to 50° C. E is "" ^ '' a beaker into which the water enters at G, is heated to a temperature of from 40° to 50° C. by a Bunsen burner, Q and is drawn off by means of a siphon 10 Op. cit, p. 128. n Ibid., p. U9. 12 Op. cit., p. 50. 13 Herzig and Ueyee, Monatshefteiar Chemie, Vol. XV, p. 613. 97 The Lbcithans at H, to enter the condenser D and keep it at tlie proper temperature. By regulating the flow of the water and the height of the flame, the temperature of the water can easily be kept within the required limits. F are Geissler bulbs for absorbing every- thing but the methyl iodide, which is absorbed in K and L. The analysis is carried on as follows: 0.2 g. of the substance to be analyzed is placed in (a) with 2 g. of dry ammoniun iodide and enough hydriodic acid (sp. gr. 1.6) to half fill the lower bulb. In (6) is placed 1 g. of ammonium iodide. The part A of the sand bath is filled with sand and a thermometer reading to 360° C. placed in the sand. A stream of dry COg is allowed to enter at M, and when all the air is displaced, a triple burner is lighted under the sand bath. In the meanwhile the water must be started and kept running through the condenser D at a temperature of from 40° to 50° C. As the temperature of 200° C. is reached in the sand bath, methyl iodide begins to split off and is carried over by the COg mixed with hydriodic acid and iodine. Most of the iodine and hydriodic acid is condensed at D and collected in C. Some passes over and is absorbed in F, which contains the following solution : Sodimn carbonate - . . . \ -paxi Potassium arsenite 1 part Water 10 parts The methyl iodide passes on and is collected in K, which contains 2 g. of silver nitrate dissolved in 5 c.c. water and 45 c.c. absolute alcohol. The methyl iodide dissolves in the alcohol, and is decomposed by the silver nitrate with the formation of silver iodide. After some time the temperature in the sand bath gradually rises to 240° C, and after a little while longer methyl iodide ceases to come over, as can be seen by the liquid in -K" becoming perfectly clear. L, which also contains silver nitrate, is used merely as a guard. The solution can be removed at this point, and the silver iodide collected corresponds to all the kephalin and one methyl group of the lecithin. Fresh silver nitrate is placed in K, another burner placed under the sand bath, and the temperature raised to 300° 0. The remaining two methyl groups of lecithin come over, while kephalin gives off no more, or only a trace, of methyl iodide. The second part of the sand bath, B, is now filled with sand and heated to 800° to decompose anything which may have escaped previous heating. The two alcoholic solutions containing the silver iodide are diluted with much water and warmed for several hours on a steam bath to remove alcohol. Strong nitric acid is then added, and the silver iodide filtered into a Gooch crucible and weighed. In case we are not dealing with a mixture of lecithans, all the silver iodide can be weighed in one portion. PEEPAEATION AND ANALYSES OF VAEIOUS LECITHANS Egg lecitMn. — The yolks of ten eggs are allowed to stand with 600 c.c. ether over night, 1 liter alcohol added, the solution filtered and evaporated on water bath. The residue is dissolved in 200 c.c. cold ether and 1 liter acetone added. The precipitated 98 Waldemar Kooh 9 lecithin is treated over night with 1 liter cold alcohol, the solution filtered and evapo- rated. The residue is once more dissolved in ether, precipitated with acetone, and dried over sulphuric acid in a vaccuum desiccator. I." 0.8113 g. of the substance gave 0.1136 g. MgoPaO,; i. e., 3.91 per cent. P. II. 0.9150 g. of the substance gave 0.1278 g. MgaPzO,; i. e., 3.90 per cent. P. III. 0.330 g. of the substance gave 0.3001 g. Agl; i. e., 5.80 per cent. CH3. IV. 0.325 g. of the substance gave 0.2960 g. Agl; i. e., 5.81 per cent. CH3. V. 0.320 g. of the substance gave, below 240° C, 0.0760 g. Agl; i. e., 153 pei cent. CII3. The methyl iodide given off above that temperature was lost on account of an accident. In III and IV the methyl iodide came over at 220° C. and 300° C. The two portions were not separated. *hosphorus as 3.97 calculated for III Found rv V 3CH3: 5.66 5.80 5.81 1 CH3: 1.88 1.53 Brain lecithin and kephalin. — One kilo sheep's brains is minced in a meat- chopper and freed from water and extractives by boiling with 1 kilo acetone for eight hours. The solution is filtered cold, and the remaining acetone removed from the brains by gentle heating at 50° C. Seven hundred c.c. cold ether are now added and allowed to stand for three days, the solution is filtered, and another portion of ether added, and again allowed to stand. The ether filtrates are united and slowly evapo- rated to one-fourth their original volume in a tall beaker. The solution is then care- fully removed by means of a pipette from the white precipitate, which has settled to the bottom, and 1.5 kilo alcohol is added to the solution. Kephalin. — The precipitated kephalin is extracted five times with boiling alcohol, dissolved in ether, precipitated with acetone, again dissolved in ether, and allowed to settle in a long, narrow, closed test-tube. The clear ether solution is removed by decantation, evaporated, and the residue recrystallized twice from hot acetic ether. The resulting kephalin is very hygroscopic and must be dried over sulphuric acid for analysis. It agrees perfectly in all its properties with the kephalin described by Thudichum (p. 127). On analysis it gave the following results: I. 0.2469 g. of the substance gave 0.5388 g. CO, and 0.2159 g. H2O; i. e., 59.5 per cent. C and 9.7 per cent. H. II. 0.415 g. of the substance neutralized 5.2 c.c. yV acid; i. e., 1.78 per cent. N. Ill 0.9644 g. of the substance gave 0.1330 g. Mg^PoO-,; i. e., 3.85 per cent. P. IV. 0.7235 g. of the substance gave 0.0990 g. MgoP^O-,; i. e., 3.82 per cent. P. V. 0.3488 g. of the substance gave, below 240° C, 0.0945 g. Agl; /. e., 1.73 per cent. CH3. - VI. 0.490 g. of the substance gave, below 240° C, 0.1312 g. Agl; ;". c. 1.71 per cent. CH3. i*For phosphorus determinations the very excellent method of Neumann was used (Eiije/manii'j ^rc/iit' /