r 
 
 QP 
 
 2LZ 
 
 UC-NRLF 
 
LIBRARY 
 
THE FATTY ACID RADICALS 
 
 OF 
 LIVER LECITHIN 
 
 Arr 
 
 BY 
 
 HENRY S. SIMMS 
 
 TED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
 DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF 
 PURE SCIENCE OF : COLUMBIA UNIVERSITY 
 
 NEW YORK 
 1922 
 
THE FATTY ACID RADICALS 
 
 OF 
 LIVER LECITHIN 
 
 HENRY S. SIMMS 
 
 \v 
 
 Introduction 
 
 Part 1. The Liver Lecithin 
 
 Part 2. The Unsaturated Fatty Acids of Liver Lecithin 
 
 SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE 
 
 DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF 
 
 PURE SCIENCE OF COLUMBIA UNIVERSITY 
 
 REPRINTED FROM 
 
 THE JOURNAL OF BIOLOGICAL CHEMISTRY 
 VOL. XLVIII, No. 1, SEPTEMBER, 1921, and VOL. LI, No. 1, MARCH, 1922 
 
BXCHANO* 
 
Introduction. 
 
 Numerous investigators in medicine and biology have come to 
 consider lipoids as highly essential constituents of the animal 
 organism, in that they appear to be involved in certain necessary 
 functions of the tissues. 
 
 The lipoid known as lecithin occurs, as do the other lipoids, in 
 all animal tissues. The following structural formula has been 
 generally accepted : 
 
 CH 2 O-CO-Ri 
 
 CH O-CO-R 2 
 
 O 
 
 II 
 CH 2 O P O C 2 H 4 N= (CH 3 ) 3 
 
 I I 
 
 OH OH 
 
 Lecithin is composed of the five radicals, choline, phosphoric 
 acid, glycerol, and two- fatty acid radicals ( O-CO-Ri and 
 -O-CO-R 2 ). 
 
 In the attempt to complete our knowledge of the chemical 
 constituents of lecithin, it seemed advisable to ascertain the 
 nature of these fatty acid radicals with more care and accuracy 
 than had previously been attempted. As a result of the investi- 
 gations which are reported in the following pages, it has been 
 found that the lecithin from livers gives on hydrolysis four fatty 
 acids. Two of them, stearic and palmitic, are saturated; while 
 two, oleic and arachidonic, are unsaturated. The proportion in 
 which the unsaturated acids occur has been found to vary. Since 
 the molecules were shown to exist in monomolecular form it is 
 evident that lecithin is a mixture of different molecules which 
 vary from each other in the particular fatty acid radicals which 
 they contain. 
 
 5075D6 
 
[Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY, September, 1921, Vol. xlviii, 
 
 No. 1, pp. 185-196.] 
 
 THE LIVER LECITHIN. 
 
 BY P. A. LEVENE AND H. S. SIMMS. 
 (From the Laboratories of The Rockefeller Institute for Medical Research.) 
 
 (Received for publication, July 7, 1921.) 
 
 The older work on the unsaturated lipoids of the liver has been 
 reviewed in the paper of Levene and Ingvaldsen. In the same 
 paper a new analysis of the liver lecithin was reported. The new 
 facts reported in that paper related principally to the nature of the 
 fatty acids. In the course of the work reported by Levene and 
 Ingvaldsen 1 two fatty acids were found, one saturated, and the other 
 unsaturated, which analyzed for a polyunsaturated arachidic acid. 
 Since an acid of that structure has never before been described in 
 connection with lecithin, further corroboration of the finding seemed 
 desirable. Furthermore, in the course of the present year it was 
 shown by Levene and Rolf 2 that the egg lecithin and that of the brain 
 contained two saturated fatty acids. This result was obtained by 
 means of fractional distillation of the esters of the combined saturated 
 acids. In light of this observation it became necessary to reinvesti- 
 gate all lecithins in regard to the number of fatty acid radicles, 
 saturated and unsaturated, present in their molecule. In regard to the 
 liver lecithin it was now found that it contained two saturated and 
 two unsaturated acids. 3 
 
 The saturated acids are palmitic and stearic. They were isolated 
 and identified by the same procedure as described by Levene and Rolf. 
 
 The unsaturated acids are: one, unsaturated stearic, and the 
 other, unsaturated arachidic. On reduction one is converted into 
 stearic and the other into arachidic. The exact degree of unsaturation 
 of either one of the two acids is as yet not known. There are, however, 
 indications that one (arachidic) may be tetra-unsaturated. On the 
 
 1 Levene, P. A., and Ingvaldsen, T., /. Biol. Chem., 1920, xliii, 359. 
 
 2 Levene, P. A., and Rolf, Ida P., /. Biol. Chem., 1921, xlvi, 193, 353. 
 
 3 Evidence has recently been obtained in this laboratory that egg lecithin 
 also contains the two unsaturated acids. 
 
 273 
 
274 LIVER LECITHIN 
 
 addition of bromine a substance was obtained which analyzed for an 
 octobromide of arachidic acid. However, it will require a larger 
 quantity of material to establish the degree of unsaturation of each 
 of the two acids with certainty. The presence of several acids in 
 the liver lecithin again emphasizes the question of the existence oi 
 more than one lecithin. 
 
 It was attempted to answer this question by the molecular weight 
 estimation of the hydrolecithin. The hydrolecithin from the liver 
 lecithin has been prepared essentially according to Paal's procedure. 
 
 The molecular weight of the substance was found 810 and 700 
 (in two estimations). The theory of a monophosphatide requires 
 809, that of a diphosphatide 1600. Consequently, liver lecithin 
 consists of a mixture of monolecithins. 
 
 In the course of the present work the process of preparation of pure 
 free lecithin from its cadmium chloride salt has been improved so that 
 analytically pure substance is prepared in good yield; namely, about 
 50 gm. of free lecithin from 100 gm. of the cadmium chloride salt. 
 
 The procedure in the main is as follows: The salt of lecithin is 
 dissolved in chloroform and this solution is transferred into a solution 
 of dry ammonia gas in dry methyl alcohol. The resulting lecithin is 
 purified from the slight quantity of impurities by the acetic acid 
 process developed by Levene ana Ingvaldsen. The details of the 
 procedure are given in the experimental part. 
 
 EXPERIMENTAL. 
 
 /. Preparation of Pure Lecithin. 
 
 Various attempts to produce pure liver lecithin without converting 
 it into the cadmium chloride salt met with little success. The 
 following method proved to be the easiest and most efficient. 
 
 The liver, in 100 pound lots, is minced, dried, and extracted, first 
 with acetone, second with ether, and last with alcohol. These ex- 
 tracts are treated separately as follows : 
 
 Acetone Extract. This is allowed to stand at 0C. over night. 
 A precipitate of fat is deposited, which is removed by filtration. The 
 filtrate is concentrated (if necessary) and the lecithin precipitated by 
 adding a saturated solution of cadmium chloride in alcohol until no 
 further precipitate is formed. 
 
P. A. LEVENE AND H. S. SIMMS 275 
 
 The residue from the above filtration is suspended in alcohol and 
 warmed until the fat is melted. The mixture is then cooled over 
 night. The fat precipitated on standing is again filtered from the 
 alcoholic solution and again treated with alcohol as before. This 
 extraction is repeated until the mother liquor no longer gives with 
 cadmium chloride a precipitate of lecithin cadmium salt. The latter 
 is recognized by the fact that on dissolving in a small amount of 
 moist ether it is again precipitated by the addition of an excess of 
 acetone. From three to seven extractions may be required. The 
 alcoholic mother liquors are then precipitated with cadmium chloride. 
 
 Ether Extract. This is concentrated to a small volume and allowed 
 to stand at 0C., when a precipitate consisting of fat and cerebrosides 
 is formed. The precipitate is extracted with ether. The ethereal 
 extract is added to the original filtrate and cooled once more to per- 
 mit the separation of the cerebrosides which the solution may still 
 contain. After filtering, the combined mother liquors are concen- 
 trated and treated with alcohol to separate the lecithin from cephalin. 
 The alcoholic liquors are then treated with cadmium chloride. 
 
 Alcoholic Extract. This is likewise concentrated and cooled to 
 remove cerebrosides, the mother liquor being decanted if possible, 
 otherwise filtered, or centrifuged if necessary. 
 
 The cerebrosides are again extracted with warm ether. The 
 extract is cooled and centrifuged. The alcoholic and ethereal liquors 
 are then treated with cadmium chloride. 
 
 Treatment of Cadmium Chloride Salts. 
 
 It is necessary to allow the cadmium chloride precipitate of lecithin 
 to stand at least half an hour until it is sufficiently coagulated to 
 permit filtration. The filtered material, which is not quite dry, is 
 transferred to a large beaker or precipitating jar and stirred up with 
 a large volume of cold acetone. If the acetone liquor turns dark 
 from dissolved material the suspension is allowed to settle, the liquor 
 decanted off, and more cold acetone is added. Finally the material 
 is filtered by suction. 
 
 This material is purified in two steps: the one is the "ether crys- 
 tallization," the other is the " toluene-ether" process. 1 It is a matter 
 
276 LIVER LECITHIN 
 
 of judgment as to which shall be used first and the number of times 
 which each should be repeated. The aim is to obtain a white granular 
 material which filters quickly. 
 
 The ether crystallization consists in dissolving the cadmium chloride 
 salt in warm ether, water being added, a few drops at a time, until the 
 suspended material goes into solution. An excess of water hinders 
 the solution of the larger particles. The solution is allowed to stand 
 over night, or longer, at 0C. The substance should separate in a 
 granular form, easily filterable by suction. If it forms a pasty solution 
 not easily filtered, time and material will be wasted in attempting 
 a filtration. Another precipitation with acetone should remove 
 impurities which interfere with the process. 
 
 This purification removes not only the fats and oils but also takes 
 out most of the cephalin present. Since the cadmium chloride salt 
 is itself slightly soluble in cold ether, some of the material may be 
 lost in purification. Hence the following precautions are necessary. 
 1. Excess of ether is to be avoided. With very impure material it is 
 more advisable to repeat the purification several times with small 
 quantities of solvent than to use a large excess at one time. The 
 amount of ether filtered off should not be more than twice the volume 
 of the residue. 2. The filtered material should not be washed with 
 ether, but should be filtered as quickly as possible until the solvent 
 runs very slowly. 3. In case the material fails to filter properly, it 
 should be transferred to a beaker, warmed slightly until dissolved, 
 precipitated with acetone, purified by the toluene ether method, 
 and subsequently passed through the ether crystallization process. 
 4. The filtration should be carried out in the cold. 
 
 The toluene-ether process consists in dissolving the cadmium chloride 
 salt in a minimum volume of toluene (adding a slight amount of 
 water if necessary). If the toluene fails to dissolve all the material 
 the residue should be centrifuged off. The solution is then treated 
 with 4 volumes of ether containing 1 per cent water. The solution is 
 cooled to 0C. over night and filtered. 
 
 The latter method gives larger yields but removes less of the 
 cephalin and other impurities. It probably removes impurities not 
 taken out by the former method, hence the cadmium chloride com- 
 pound should be purified by both methods. 
 
P. A. LEVENE AND H. S. SIMMS 277 
 
 Experience shows that in the case of liver lecithin the toluene- 
 ether method should precede the ether crystallization method of 
 purification of the cadmium chloride salt in order to obviate difficulty 
 in filtering from the ether. 
 
 One purification by each method should be sufficient to give almost 
 white dry material with an amino content of less than 3 per cent of 
 the total nitrogen present. Such a product may be converted into 
 free lecithin. 
 
 Conversion of the Cadmium Chloride Compound into Free Lecithin. 
 
 The cadmium chloride salt is dissolved in chloroform and is con- ' 
 verted into free lecithin by means of a solution of ammonia in methyl 
 alcohol. 100 gm. of the cadmium chloride salt are dissolved in 300 cc. 
 of warm chloroform and poured into 400 cc. of methyl alcohol con- 
 taining 20 gm. of ammonia gas. This is added slowly with rapid 
 stirring. The product of reaction is allowed to stand a short time 
 before filtering. The precipitate may be filtered off through a folded 
 filter paper. The chloroform methyl alcohol solution of lecithin is 
 then concentrated under diminished pressure. Near the end of the 
 concentration the material foams considerably for a short time and 
 then the foaming subsides. The vacuum concentration should be 
 carried out at a low temperature. If during the operation a precipi- 
 tate of fat settles out this should be filtered off. The remaining leci- 
 thin is practically free from solvent. It is dissolved in a minimum 
 (5 to 10 cc.) of glacial acetic acid. This is poured into 800 cc. of 
 boiling hot acetone, stirred, and allowed to cool to room temperature. 
 A very small dark precipitate (1 to 2 gm.) settles out. The superna- 
 tant liquid is decanted or filtered. The precipitate is slightly soluble 
 in ether and insoluble in acetone but somewhat soluble in ethyl 
 alcohol and more soluble in methyl alcohol. 
 
 No. 126. 
 
 0.0154 gm. of substance gave on combustion 0.0954 gm. 6f H;0, 0.2315 
 gm. of C0 2 , and 0.0114 gm. of ash. 
 
 0.1910 gm. of substance used for Kjeldahl nitrogen determination required 
 3.90 cc. of 0.1 N acid corresponding to 0.00546 gm. of N. 
 
 0.2865 gm. of substance gave 0.0390 gm. of Mg 2 P 2 07. 
 
278 LIVER LECITHIN 
 
 C 4 4Hg 7 O 9 NP. Calculated. C 65.59, H 10.89, N 1.74, P 3.86. 
 Found. C 61.16, H 10.34. N 2.92, P 3.88. 
 
 It 'contains 10 per cent amino nitrogen. 
 
 No. 124. 
 
 1.5 gm. of substance were hydrolyzed with HC1, neutralized, concentrated, 
 and made up to 15 cc. 
 
 5 cc. of this solution required for Kjeldahl nitrogen determination 5.60 cc. of 
 0.1 N HC1. 
 
 2 cc. of this solution for Van Slyke determination gave 0.57 cc. of N 2 at T = 
 27, P = 762.2 mm. 
 
 Amino N 10 
 Total N " 100 
 
 The liquors are then cooled in a freezing mixture to 5C. Fre- 
 quently at this phase a second small precipitate settles out. A sample 
 of this material analyzed as follows: 
 
 No. 122. 
 
 0.1024 gm. of substance gave on combustion 0.1098 gm. of H^O, 0.2216 gm. 
 of CO 2 , and 0.0118 gm. of ash. 
 
 0.1832 gm. of substance for Kjeldahl nitrogen determination required 3.10 cc. 
 of 0.1 N acid corresponding to 0.00434 gm. of N. 
 
 0.2748 gm. of substance gave 0.0464 gm. of Mg 2 P2O 7 . 
 
 C44H 8 7O 9 NP. Calculated. C 65.59, H 10.89, N 1.74, P 3.86. 
 Found. C 59.50, H 12.10, N 2.37, P 4.75. 
 
 The mother liquors are concentrated under diminished pressure 
 until all the ether and most of the acetic acid are removed. Water is 
 added a little at a time and the material is shaken or stirred until a 
 thick emulsion of a light brown color is formed. This is poured into 
 800 to 1,000 cc. of acetone, chilled down to 5C. It is carefully 
 stirred and allowed to stand at to 5C. over night, when it is 
 transferred to a crystallizing dish and washed free from excess water 
 by stirring with cold dry acetone. The acetone is decanted off and the 
 lecithin dried in a vacuum desiccator. 
 
 From 40 to' 45 gm. of pure material may be obtained from 100 gm. 
 of cadmium chloride salt. (Theoretical yield, 81 to 82 gm.) Several 
 samples have been analyzed. They differed little one from another in 
 their elementary composition. The analysis of one of thejn is as 
 follows: 
 
P. A. LEVENE AND H. S. SIMMS 279 
 
 No. 119. 
 
 0.0996 gm. of substance gave on combustion 0.0994 gm. of H 2 0, 0.2768 gm. 
 of CO 2 , and 0.0090 gm. of ash. 
 
 0.1798 gm. of substance required 2.40 cc. of 0.1 N acid, corresponding to 0.00336 
 gm. of N. 
 
 0.2697 gm. of substance gave 0.0390 gm. of Mg 2 P 2 7 . 
 
 C44H 87 O 9 NP.* Calculated. C 65.59, H 10.89, N 1.74, P 3.86. 
 Found. C 64.83, H 11.16, N 1.87, P 4.03. 
 
 * This formula represents material consisting of equal parts of two lecithins, 
 each one containing two of the four fatty acids. 
 
 II. The Fatty Acids of Lecithin. 
 
 For the preparation of fatty acids from lecithin, the material was 
 hydrolyzed 8 to 15 hours with 10 parts of 10 per cent HC1. The 
 fatty acids on cooling appeared as a semisolid cake. They were 
 dissolved in methyl alcohol and precipitated in the presence of am- 
 monium hydroxide with a nearly equal weight of lead acetate dissolved 
 in a minimum quantity of water. After freezing, the mother liquors 
 were filtered off. The lead salts which contained both the saturated 
 and unsaturated fatty acids were extracted repeatedly with boiling 
 ether until further extraction produced only slight precipitate with 
 hydrochloric acid. 
 
 The ether solution then contained the lead salts of the unsaturated 
 acids while the ether-insoluble material consisted of the lead salts of 
 the saturated acids. Both fractions were decomposed with HC1, 
 dissolved in ether, washed with water, dried, and the solvent evapo- 
 rated off. 
 
 A lot of 528 gm. of the lecithin cadmium chloride free from amino 
 nitrogen was hydrolyzed with 10 per cent solution of hydrochloric 
 acid. The yield of fatty acids was 223 gm. 
 
 Unsaturated Fatty Acids. 
 
 These were obtained by extracting the lead salt first by means of 
 acetone and then by means of ether. Each extract was worked over 
 separately. The acetone extract was concentrated and the residue 
 thus obtained extracted with ether. From both of these fractions the 
 acids were liberated and reconverted into the lead salts. These were 
 
280 LIVER LECITHIN 
 
 again purified and again converted into the free acids. A sample of 
 the acids gave the following iodine and hydrogen values: 
 
 0.2907 gm. of substance absorbed 0.435 gm. of iodine by the Wijs method. 
 
 0.5141 gm. of substance reduced by Paal's method absorbed 67 cc. of H2 
 in 3 hours at 17C., 759 mm. pressure, or 1.103 gm. of H 2 per 100 gm. of 
 substance. 
 
 Ci 8 H 34 O2. Calculated. Iodine value 91, Hydrogen number 0.721. 
 
 Ci 8 H 3 20 2 . Calculated. " " 182, " " 1.447. 
 
 Found. " " 154, " 1.103. 
 
 It was later found that this material consisted of two fatty acids, 
 hence it is possible that one was a singly unsaturated, and that the 
 other contained two or more double bonds. 
 
 The free fatty acids were finally reduced by Paal's method. The 
 samples of reduced acids obtained from each fraction analyzed as 
 follows : 
 
 No. 84 (material obtained from the acetone extract of the lead salts). 
 
 0.1020 gm. of substance gave on combustion 0.1198 gm. of H^O and 0.2830 
 gm. of CC>2. 
 
 No. 85 (material obtained from the ether extract of the lead salts) 
 
 0.1012 gm. of substance gave on combustion 0.1190 gm. of H 2 O and 0.2828 
 gm. of CO 2. 
 
 No. 84. C 75.96, H 13.19. 
 " 85. C 76.21, H 13.15. 
 
 Since the two fractions proved practically of identical elementary 
 composition they were combined and converted into the methyl esters. 
 These were freed from adhering sulfuric acid by washing with water 
 and finally by recrystallization from methyl alcohol. They were then 
 fractionated by distilling at a pressure of 1 to 2 mm. 
 
 The following fractions were obtained. 
 
 A 182-185C. 
 
 B '. 175-185C. 
 
 C 182-195C. 
 
 D 185-203C. 
 
 Fractions A and D were redistilled and the following fractions were 
 obtained. 
 
P. A. LEVENE AND H. S. SIMMS 281 
 
 From A Ai 158-165C. 
 
 A 2 170-182C. 
 
 From D D x 182-192C. 
 
 D 2 187-197C. 
 
 For identification, the esters were saponified with an alcoholic solu- 
 tion of sodium hydroxide. The acids were liberated and converted 
 into the lead salts. The acids were again liberated from the lead salts 
 and analyzed. Fraction A a corresponded apparently to pure stearic 
 acid. 
 
 Analysis 101. 
 
 0.1024 gm. of substance gave on combustion 0.1186 gm. of H^O and 0.2860 
 gm. of CO 2. 
 
 0.8950 gm. of substance in a molecular weight determination required 6.50 cc. 
 of 0.5 N NaOH. 
 
 Ci 8 H 36 O2. Calculated. C 75.93, H 12.76. 
 Found. C 76.16, H 12.96. 
 
 Molecular weight was 275, that of stearic acid is 284. 
 
 The substance melted at 70.5-71.C, stearic acid melts at 70-7lC. 
 
 When this was mixed with some very pure stearic acid melting at 
 74C., the mixture melted. at 74C. 
 Fraction D 2 apparently corresponded to pure arachidic acid. 
 
 Analysis 100. 
 
 0.1000 gm. of substance gave on combustion 0.1166 gm. of H 2 and 0.2822 
 gm. of CO 2. 
 
 0.9760 gm. of substance neutralized 6.75 cc. of 9.5 N NaOH. 
 
 C 20 H 4 oO2. Calculated. C 76.95, H 12.91. 
 Found. C 76.97, H 13.24. 
 
 Molecular weight was 314, that of arachidic acid is 313. 
 
 The substance melted at 75.5-76C., arachidic acid melts at 75-77C. 
 
 When this was mixed with some pure arachidic acid melting at 
 75C., the mixture melted at 75C. 
 
 Saturated Fatty Acids. 
 
 The lead salts which were insoluble in acetone and ether were con- 
 verted into free acids. These were twice esterified with methyl 
 alcohol. The mixture of methyl esters thus obtained was distilled at 
 a pressure of 1 to 2 mm. into the following fractions: 
 
282 LIVER LECITHIN 
 
 a 160-163C. 
 
 b ; 159-167C. 
 
 c 158-1 72C. 
 
 d 170-180C. 
 
 and residue. 
 
 Fractions a and d were redistilled as follows. 
 
 From a at 156-162C. 
 
 a 2 Residue. 
 
 From d di 180-183C. 
 
 d 2 182-188C. 
 
 Fraction ai apparently corresponded to pure palmitic acid. 
 
 Analysis 92. 
 
 0.1009 gm. of substance gave on combustion 0.1220 gm. of H 2 and 0.2802 
 gm. of CO 2. 
 
 0.8168 gm. of substance neutralized 6.14 gm. of 0.5 N NaOH. 
 
 CieHssO*. Calculated. C 74.92, H 12,58. 
 Found. C 75.09, H 12.98. 
 
 Molecular weight was found to be 266, palmitic acid had a molecular weight 
 of 256. 
 
 The melting point was 62C., palmitic acid melts at 63-64C. 
 
 When this was mixed with some pure palmitic acid melting at 
 64C., the mixture melted at 63C. 
 
 Fraction d 2 apparently corresponded to pure stearic acid. 
 
 Analysis 94. 
 
 0.1009 gm. of substance gave on combustion 0.1220 gm. of H 2 and 0.2802 
 gm. of CO 2 . 
 
 0.6686 gm. of substance neutralized 4.82 cc. of 0.5 N NaOH. 
 
 C 18 H 8 6O 2 . Calculated. C 75.93, H 12.76. 
 Found. C 75.72, H 13.53. 
 
 Molecular weight was 278, stearic acid had a molecular weight of 284. 
 The substance melted at 7lC., stearic acid melts at 70-7lC. 
 
 When this was mixed with a sample of very pure stearic acid melting 
 at 74C. the mixture melted at 74C. 
 
 ///. Bromine Addition Products of the U maturated Acids. 
 
 An attempt was made to separate and to characterize th:e individ- 
 ual unsaturated acids by preparing the bromine addition products. 
 
P. A. LEVENE AND H. S. SIMMS 283 
 
 40 gm. of pure lecithin, which had been prepared from the cadmium 
 chloride salt as described above, were used. 
 
 No. 119. 
 
 0.0996 gm. of substance gave on combustion 0.0994 gm. of H 2 0, 0.2768 gm. 
 of CO 2 , and 0.0090 gm. of ash. 
 
 0.1798 gm. of substance required 2.40 cc. of 0.1 N acid, corresponding to 0.00336 
 gm. of N. 
 
 0.2697 gm. of substance gave 0.0390 gm. of Mg 2 P 2 O 7 . 
 
 C 4 4H 8 7O 9 NP. Calculated. C 65.59, H 10.89, N 1.74, P 3.86. 
 Found. C 64.83, H 11.16, N 1.87, P 4.03. 
 
 This was hydrolyzed with a 10 per cent solution of hydrochloric 
 acid, the acids were dissolved in ether, washed with water, dried, 
 and the ether evaporated off. The iodine number of the mixed 
 acids was 91. 
 
 0.2457 gm. of substance absorbed 0.232 gm. of iodine by the Wijs method. 
 
 Average molecular weight of 280. Calculated. Iodine value 91. 
 
 Found. " " 91. 
 
 The acids were converted into the lead salts, the unsaturated acids 
 extracted with ether and converted into the free acids. These were 
 dissolved in 18-30 petrolic ether and brominated at 0C. with 3 cc. 
 of bromine dissolved in petrolic ether. 
 
 On freezing to -10 a precipitate was obtained. The mother liquor 
 was concentrated and again cooled to 10. The combined precipi- 
 tate was recrystallized from petrolic ether and then recrystallized 
 from ethyl ether. 
 
 This gives three fractions: A, the petrolic ether-soluble fraction: 
 B, the fraction insoluble in petrolic ether but soluble in ethyl ether; 
 and C, the fraction insoluble in both solvents. This last fraction 
 contains the material having most bromine (namely, the hexabromides 
 and octobromides, if present). The first fraction should be largely 
 dibromides while the tetrabromides should predominate in the fraction 
 insoluble in petrolic ether but soluble in ethyl ether. 
 
 Fraction C (the material insoluble in both solvents) was recrystal- 
 lized from ethyl ether; the yield was 1 gm. In an open tube melting 
 point determination it darkened, turning black at 200C. It con- 
 tracted at 240C. and decomposed at 243C. In a closed tube it con- 
 tracted at 239C. and melted without decomposition at 243C. This 
 analyzed as follows: 
 
284 LIVER LECITHIN 
 
 No. 129. 
 
 0.2012 gm. of substance gave 0.2936 gm. of AgBr. 
 
 This would indicate a hexabromide. 
 
 C2oH 3 4O 2 Br 6 . Calculated for hexabromarachidic acid. i.O. 
 Ci8H 3 oO2Br 8 . Calculated for hexabromstearic acid. 63.2. 
 Found. 62.11. 
 
 The material was recrystallized from ether. On heating in an 
 open tube it darkened at 180-200C., contracted at 240C., and 
 decomposed at 244C. 
 
 It analyzed as follows : 
 
 Analysis 130. 
 
 0.1068 gm. of substance gave 0.1670 gm. of AgBr. 
 
 This corresponds more closely to an octobromide. 
 
 CaoH^OaBrg. Calculated for octobromarachidic acid. 67.80. 
 Found. 66.55. 
 
 There was not sufficient material for further treatment. 
 IV. Hydrolecithin from Liver Lecithin. 
 
 For the preparation of hydrolecithin 10 gm. of pure liver lecithin 
 free from amino nitrogen were used (Analysis 119 given above). 
 This was reduced by Paal's method. The hydrolecithin produced was 
 recrystallized twice from acetone and once from methyl ethyl ketone. 
 
 This analyzed as follows: 
 
 No. 128. 
 
 0.1074 gm. of substance gave on combustion 0.1082 gm. of HgO, 0.2554 gm. 
 of C0 2 , and 0.0098 gm. of ash. 
 
 0.1926 gm. of substance for Kjeldahl nitrogen determination required 2.40 cc. 
 of 0.1 N acid, corresponding to 0.00336 gm. of N. 
 
 0.2839 gm. of substance gave 0.0400 gm. of Mg 2 P20 7 . 
 
 C44H 3 iO 9 NP. Calculated. C 65.30,.H 11.33, N 1.73, P 3.84. 
 Found. ' C 65.03, H 11.29, N 1.74, P 3.86. 
 
 A molecular weight determination was made as follows: 
 
 1.036 gm. of substance raised the boiling point of 16 gm. methyl alcohol 0.07lC. 
 0.964 gm. of substance raised the boiling point of 16 gm. methyl alcohol 0.077C. 
 
 C4 4 H 9 iONP. Calculated. Molecular weight 809. 
 
 Found. First .determination 810 
 
 Second " 700. 
 
Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY 
 Vol. LI, No. 1, March, 1922 
 
 THE UNSATURATED FATTY ACIDS OF LIVER LECITHIN. 
 
 BY P. A. LEVENE AND H. S. SIMMS. 
 
 (From the Laboratories of The Rockefeller Institute for Medical Research.} 
 
 (Received for publication, January 3, 1922.) 
 
 In previous communications on liver lecithin results were 
 reported showing that the unsaturated fatty acids obtained on 
 hydrogenation gave stearic and arachidic acids. It was further 
 reported that the iodine number of the mixed unsaturated fatty 
 acids was 154, and that the mixed unsaturated acids on bromina- 
 tion formed a bromide, which on the basis of its bromine content 
 and of its melting point seemed to be the octobromarachidic 
 acid. 
 
 From these findings it was concluded that liver lecithin con- 
 tained more than one unsaturated fatty acid, and that the indi- 
 vidual acids differed in the degree of their unsaturation. It then 
 seemed suggestive that the acid of higher unsaturation was the 
 tetra unsaturated arachidic acid. Since there was no accurate 
 knowledge either as to the number of the unsaturated acids present 
 in the mixture or as to the degree of their unsaturation, there was 
 no indication as to the ratio of the individual acids. In order to 
 complete our knowledge of the unsaturated fatty acids, it became 
 necessary to find: (1) the number of the unsaturated fatty acids 
 present in the liver lecithin; (2) the exact nature of the acids of 
 higher unsaturation; and (3) the ratio of the individual fatty 
 acids. 
 
 In the course of the present work it was found that liver lecithin 
 yields only two unsaturated fatty acids, oleic and arachidonic 1 
 acids, and of these oleic acid predominates. 
 
 1 Since the tetra unsaturated arachidic acid is called arachidonic acid by 
 Lewkowitsch (Lewkowitsch, J., Chemical technology and analysis of oils, 
 fats and waxes, London, 4th edition, 1909, i, 211) this name will be adopted 
 by the present writers. 
 
 285 
 
286 Fatty Acids of Liver Lecithin 
 
 In connection with the finding of arachidonic acid as a con- 
 stituent of liver lecithin, it is interesting to recall the work of 
 Hartley, 2 done under the direction of Leathes. On oxidation of 
 the unsaturated acids from liver fat, including phosphatides, this 
 author isolated an unsaturated fatty acid which was oxidized 
 to a tetrahydroxyarachidic acid. Furthermore, on bromination 
 of the same fatty acids Hartley obtained a bromide, having a 
 bromine content only 0.7 per cent higher than that required by 
 theory for octobromarachidic acid. 
 
 The present writers, 8 and Levene and Ingvaldsen, 4 have pre- 
 viously reported that on hydrogenation of the unsaturated acids 
 of liver lecithin, arachidic acid was isolated. They have also 
 obtained from liver lecithin a bromide, which on analysis gave a 
 bromine value about 1 per cent lower than that required by theory 
 for the octobromarachidic acid. In the course of the present work 
 an octobromarachidic acid was isolated which gave correct analyt- 
 ical values. This acid was then reconverted into a tetra unsatu- 
 rated arachidic acid, which on hydrogenation was converted into 
 arachidic acid. From the product of bromination of the unsatu- 
 rated fatty acids, fractions were obtained which analytically 
 resembled the hexa- and tetrabromides. On purification of these 
 fractions only the octobromide was isolated in pure form. Hence 
 present day evidence does not permit us to assume the presence of 
 more than one poly unsaturated fatty acid in lecithin. On the other 
 hand, from the more soluble fraction of the bromination product 
 a substance was obtained which yielded unsaturated fatty acids 
 with an iodine number of 107. This is not far removed from 
 that required by oleic acid which has an iodine number of 90. 
 From this fraction on hydrogenation pure stearic acid was isolated. 
 
 Assuming that only two unsaturated acids, arachidonic and 
 oleic acids, are present in the lecithin fraction, and taking into 
 consideration the fact that the iodine number of the mixed un- 
 saturated acids of this lecithin is 196, it becomes easy to calculate 
 the ratio of the two acids from the equation 90z + 335?/ = 196 
 (x -f- y). It follows that the ratio is approximately 1.3 parts of 
 oleic acid to 1 part of arachidonic acid. 
 
 1 Hartley, P., J. Physiol., 1908-09, xxxviii, 353. 
 
 1 Levene, P. A., and Simms, H. S., J. Biol. Chem., 1921, xlviii, 185. 
 
 4 Levene, P. A., and Ingvaldsen, T., J. Biol. Chem., 1920, xlifr, 359. 
 
P. A. Levene and H. S. Simms 287 
 
 The lecithin referred to above was obtained from the acetone 
 extract of the liver. From the ethereal extract there was ob- 
 tained a lecithin of lower unsaturation. The iodine number of 
 the unsaturated acids was 136. This corresponds to 4.3 parts of 
 oleic acid to 1 part of arachidonic acid. 
 
 Bearing in mind the molecular weight estimation of dihydro- 
 lecithin reported in the previous communication, namely 810, 
 one is justified in concluding that the liver contains several leci- 
 thins and that the oleyl lecithins predominate. 
 
 EXPERIMENTAL. 
 
 Preparation of Material. 
 
 Beef livers in 100 Ib. lots were minced, dried, and extracted, 
 first with acetone, then with ether, and last with alcohol. These 
 extracts were treated by the methods described in an earlier paper. 
 A cadmium chloride salt of pure lecithin was obtained which was 
 free from amino nitrogen. 
 
 Analysis 5. 1.0 gin. substance was hydrolyzed with HC1, neutralized, 
 concentrated, and made up to 15 cc. 
 
 2 cc. of this solution: no N 2 (Van Slyke). 
 
 5 " " " " : 1.95 cc. of 0.1 N HOI (Kjeldahl). 
 
 Amino N 
 
 X 100 = per cent ammo nitrogen. 
 
 Total N 
 
 The Unsaturated Fatty Adds. 
 
 765 gm. of the above mentioned material were hydrolyzed with 
 10 per cent HC1 for 15 hours. The fatty acids which separated 
 were dissolved in ether and washed with water until free from 
 mineral acid. The ether solution was dried and concentrated to 
 about 1.5 liters. This solution was allowed to stand at 0C. over 
 night. A white precipitate settled out. This was filtered off 
 and recrystallized twice from ether and once from dry acetone. 
 It analyzed as follows: 
 
 Analysis 15. 0.1019 gm. substance: 0.1184 gm. H 2 O and 0.2834 gm. CO 2 . 
 C 18 Hj 8 2 . Calculated. C 75.93, H 12.76. 
 Found. "75.84, " 13.00. 
 
288 Fatty Acids of Liver Lecithin 
 
 The ether mother liquors from which this stearic acid had been 
 separated were then concentrated to dryness under diminished 
 pressure. The residue was dissolved in methyl alcohol, treated 
 with lead acetate solution, and made alkaline with ammonium 
 hydroxide. On cooling this solution to 5C. the lead salts of the 
 fatty acids settled out. These were filtered off and extracted 
 repeatedly with ether until ether would no longer extract material 
 giving a precipitate with hydrochloric acid. 
 
 The combined ether extracts of the lead salts were treated with 
 HC1 to convert the salts into free fatty acids. The ether solution 
 was then washed with water until free from mineral acid and 
 dried with sodium sulfate. The ether was removed by distilla- 
 tion under diminished pressure. The residue of unsaturated 
 fatty acids weighed 160 gm. The iodine number was obtained. 
 
 0.2281 gm. of substance absorbed 0.453 gm. of iodine by the Wijs method. 
 
 Average molecular weight 290. 
 
 Calculated (two double bonds) . Iodine number 175. 
 
 Found. " " 196. 
 
 These unsaturated acids were dissolved in glacial acetic acid and 
 brominated with a 25 per cent solution of bromine in glacial 
 acetic acid. The temperature was kept as low as possible with- 
 out freezing the acetic acid. 
 
 The bromine solution was added from a special vacuum jacket 
 burette constructed for the purpose. This consisted of two 
 tubes of Pyrex glass sealed together at both ends, with a stop- 
 cock attached. The lower end of the inner tube consisted of 
 spiral capillary tubing to take up the thermal expansion. The 
 space between the outer and inner tubes was evacuated and sealed. 
 Bromine solution in this burette could be kept cool longer than in 
 an ordinary burette. 
 
 The acids were brominated in 5 to 10 gm. lots and allowed to 
 stand over night with a slight excess of bromine. A yellow pre- 
 cipitate formed which was filtered off. 
 
 The precipitate (Fraction A) was expected to contain the higher 
 bromides and the acetic acid mother liquor (Fraction B), the 
 lower bromides. 
 
P. A. Levene and H. S. Simms 289 
 
 Fraction A, the Fraction Insoluble in Glacial Acetic Acid. 
 
 Identification of Arachidonic Acid. The precipitate was ex- 
 tracted repeatedly with ether until it no longer contained any 
 ether-soluble material. This residue analyzed as follows: 
 
 Analysis 17. 0.1066 gm. substance: 0.0356 gm. H 2 O and 0.0998gm.CO z . 
 
 0.1996 " " :0.3166gm. AgBr. 
 
 C 2 oH 32 O2Br 8 . Calculated. C 25 43, H 3.42, Br 67.72. 
 
 Found. " 25.53, " 3.73, " 67.57. 
 
 In melting point determination this material darkened, melted, 
 and decomposed at 245C. (A slightly less pure sample of this 
 material previously obtained contracted at 240C. and decom- 
 posed at 244C. while it began to darken below 200C.) 
 
 20 gm. of the above material were obtained. 10 gm. of this 
 were suspended in dry methyl alcohol and reduced with zinc 
 dust and dry hydrochloric acid gas. The methyl alcohol was 
 filtered, diluted with water, and shaken repeatedly with gasoline. 
 The gasoline solution was then shaken with water, dried, and 
 evaporated to dryness under diminished pressure. The iodine 
 number of the resulting material was obtained. 
 
 0.0492 gm. substance absorbed 0.150 gm. iodine by the Wijs method. 
 C 2 oH 32 O 2 . Calculated. Iodine number 335. 
 Found. " " 305. 
 
 This unsaturated material was reduced with hydrogen in al- 
 coholic solution by the method of Paal. The solution, after being 
 filtered, was concentrated and cooled to 5C. The reduced 
 acid separated out. This was recrystallized three times from dry 
 acetone. It analyzed as follows: 
 
 Analysis 34. 0. 1034 gm. substance: 0. 1176 gm.H 2 O and 0. 2912 gm. CO-,. 
 CzoEUOa. Calculated. C 76.85, H 12.91. 
 Found. " 76.81, " 12.72. 
 
 Identification of Oleic Acid. It was stated above that the 
 octobromarachidic acid was extracted repeatedly with ether to 
 remove the lower bromides. The ether mother liquors from 
 this extraction were concentrated to 500 cc. and cooled to 0C. 
 A white precipitate settled out. This was separated by filtration 
 and will be referred to as Fraction I. 
 
290 Fatty Acids of Liver Lecithin 
 
 The ethereal solution was evaporated to dryness under dimin- 
 ished pressure and cooled to 0C. Two oily layers formed. These 
 were separated and dissolved separately in methyl alcohol. Each 
 on cooling formed a sticky sediment. These were separated. 
 The one from the upper layer will be referred to as Fraction 
 II, that from the lower as Fraction III. 
 
 The methyl alcohol mother liquors from Fractions II and III 
 were then combined and reduced with zinc dust and hydrochloric 
 acid as described above. The resulting material weighed 30 gm. 
 It gave an iodine value as follows: 
 
 0.2776 gm. substance absorbed 0.296 gm. iodine by the Wijs method. 
 Ci8H 34 O2. Calculated. Iodine number 90. 
 Found. " " 107. 
 
 10 gm. of this material were reduced with hydrogen by Paal's 
 method and recrystallized twice from dry acetone. In order to 
 decompose any methyl ester which might have formed during the 
 zinc reduction the material was saponified with NaOH in methyl 
 alcohol solution. The soap was precipitated in acetone, dried, 
 and converted into free acid with hydrochloric acid in the presence 
 of ether. The ether solution, freed from inorganic material by 
 shaking with water, was dried and evaporated. After three more 
 recrystallizations from dry acetone the acid analyzed as follows: 
 
 Analysis 33. 0. 0994 gm. substance: 0. 1154 gm.H 2 O and 0.2776 gm. CO 2 . 
 CigH^Oj. Calculated. C 75.93, H 12.76. 
 Found. " 76.15, " 12.99. 
 
 Melting point of stearic acid 70-71 C. 
 Found. 68C. 
 
 Attempt to Isolate Other Unsaturated Acids than Arachidonic and 
 Oleic Acids. Fraction I, referred to above, was a grayish brown 
 dry powder, difficulty soluble in ether. For purification it was 
 extracted with hot methyl alcohol and filtered hot. The insoluble 
 material analyzed as follows: 
 
 Analysis 21. 0.1062 gm. substance: 0. 0374 gm.H 2 O and 0. 1030 gm. CO 2 . 
 
 0.2041 " " :0.3030gm. AgBr. 
 
 Ci 8 H so O 2 Br 4 . Calculated. C 28.49, H 3.99, Br 63.26. 
 
 C 20 H 82 O 2 Br 8 . " 25.43, " 3.42, " 67.72. 
 
 Found. " 26.46, " 4.04, " 65.26. 
 
P. A. Levene and H. S. Simms 291 
 
 The alcohol extracted only a small portion of the material. 
 When this extract was cooled to 20C. a precipitate formed which 
 was too small for further treatment. On cooling the mother 
 liquor to 5C. a second precipitate formed. This was separated 
 and analyzed. 
 
 Analysis 22. 0.1062 gm. substance: 0.0408 gm.H 2 O and 0.1 108 gm. COi 
 
 0.2026 " " :0.3018gm. AgBr. 
 
 Ci 8 H3oO 2 Br 6 . Calculated. C 28.49, H 3.99, Br 63.26. 
 Found. " 28.45, " 4.29, " 63.39. 
 
 In a melting point determination this material softened at 
 130C., darkening up to 170C. when it partially melted and 
 partially decomposed. At 240C. it became liquid and decom- 
 posed in the manner of octobromarachidic acid. 
 
 Since the analysis of the material approached that of a hexa- 
 bromide-, the substance was extracted six additional times with 
 a large volume of methyl alcohol. In a melting point determina- 
 tion the residue became black at 200C. and decomposed at 
 230C. The combined extracts on cooling to 0C. formed a pre- 
 cipitate which analyzed as follows: 
 
 Analysis 31. 0.2016 gm. substance: 0.3146 gm. AgBr. 
 C 18 H 3 oO 2 Br 6 . Calculated. Br63.26. 
 C 2 oH 3 2O 2 Br8. " " 67.72. 
 
 Found. " 66.41. 
 
 In a melting point determination this material softened at 
 140C., darkening with rise of temperature, and melting with 
 decomposition at 243C. 
 
 The methyl alcohol mother liquors were concentrated to 100 
 cc. and cooled to 0C. A second precipitate formed which melted 
 as follows. It softened at 140C., partially decomposed at 180C., 
 and melted with decomposition at 243C. 
 
 Thus each fraction obtained from Fraction I on purification ap- 
 proached the character of the octobromide, hence it is justifiable 
 to assume that this fraction consisted mainly of the octobromide. 
 
 It was next attempted to isolate a tetrabromide from the 
 material referred to as Fractions II and III (page 290). Each 
 was dissolved in excess of ether and treated with 5 parts of gasoline. 
 
 Precipitates were formed in each and were separated. These 
 had a sticky consistency which indicated that they were not pure. 
 
292 Fatty Acids of Liver Lecithin 
 
 They were obtained in a quantity too small for analysis. The 
 combined gasoline mother liquors were concentrated to small 
 volume and again treated with gasoline. A precipitate was 
 formed which analyzed as follows: 
 
 Analysis 25. 0.1003 gm. substance: 0. 0408 gm. H 2 O and 0.1118 gm.CO 2 . 
 
 0.1972 " " :0.2806gm. AgBr. 
 
 Ci 8 H 32 O 2 Br4. Calculated. C 36.01, H 5.38, Br 53.28. 
 C 18 H, O 2 Br 6 . " " 28.49, " 3.99, " 63.26. 
 
 Found. " 30.39, " 4.55, " 60.56. 
 
 The mother liquors from this precipitate were again concen- 
 trated, dissolved in dry methyl alcohol, and reduced with zinc 
 dust and hydrochloric acid. The reduced material had an iodine 
 number as follows: 
 
 0.1990 gm. substance absorbed 0.264 gm. iodine by the Wijs method. 
 Found. Iodine number 123. 
 
 The reduced material was dissolved in glacial acetic acid and 
 rebrominated. This bromine will be referred to as Fraction 
 IV. 
 
 The acetic acid solution was concentrated to dryness under 
 diminished pressure. The residue was dissolved in hot absolute 
 alcohol and cooled to 5C. A precipitate formed which was 
 again dissolved in hot absolute alcohol and reprecipitated by 
 cooling to 5C. After reprecipitating eight times from absolute 
 alcohol and once from methyl alcohol, a melting point determina- 
 tion was made. It softened at 150C. and melted with decom- 
 position at 240C. Apparently this is an impure octobromide. 
 
 The mother liquors were concentrated and cooled to 5C. A 
 precipitate was formed which, after several reprecipitations from 
 absolute alcohol and from methyl alcohol, melted as follows: 
 At 130C. it softened and became black. At 230C. it melted with 
 decomposition. This also appears to be an impure octobro- 
 mide. 
 
 In order to ascertain whether a tetrabromide was present in the 
 mother liquors from the purification of the last substance they 
 were concentrated to dryness under diminished pressure and dis- 
 solved in gasoline. Since no precipitate formed even on cooling 
 to 5C., it was concluded that no tetrabromide was-present. 
 
P. A. Levene and H. S. Simms 293 
 
 The three precipitates obtained from Fractions II and III were 
 combined and also reduced with zinc dust and hydrochloric acid. 
 The reduced material gave an iodine number as follows: 
 
 0.2815 gm. substance absorbed 0.488 gm. iodine by the Wijs method. 
 Found. Iodine number 180. 
 
 This material was also rebrominated in glacial acetic acid 
 solution. It will be referred to as Fraction V. The bromide 
 solution was treated in the same way as Fraction IV. It yielded 
 similar results. Purification of the insoluble material gave an 
 octobromide. 
 
 Fraction B, the Fraction Soluble in Acetic Acid. 
 
 This consists of the acetic acid mother liquors from which 
 Fraction A was filtered. The liquors were concentrated under 
 diminished pressure and treated with gasoline. An oily layer 
 settled out. This was separated, dissolved in methyl alcohol, 
 and reduced with zinc and hydrochloric acid. 20 gm. of reduced 
 material were formed having an iodine number as follows : 
 
 0.1974 gm. substance absorbed 0.356 gm. iodine by the Wijs method. 
 Found. Iodine number 180. 
 
 The acids were rebrominated in glacial acetic acid. These 
 bromides (Fraction VI) were treated in the same manner as 
 Fractions IV and V and yielded similar material. Had there 
 been any tetrabromides they would have been found in Fractions 
 IV, V, and VI. 
 
 Lecithin Containing a Smaller Percentage of Highly Unsaturated 
 
 Acids. 
 
 A sample of lecithin was obtained from the alcohol-insoluble 
 portion of the ether extracts of liver. This was purified by 
 dissolving in acetic acid and treating with alcohol. The solu- 
 tion after being separated from the precipitate which formed 
 was concentrated under diminished pressure, emulsified with 
 water, and precipitated with acetone. This precipitate was 
 dissolved in alcohol and cooled to 0C. A precipitate formed. 
 The solution was separated, concentrated under diminished 
 pressure, dissolved in ether, and precipitated with acetone. 
 
294 Fatty Acids of Liver Lecithin 
 
 This precipitate was converted into the cadmium chloride 
 salt and purified once by the ether crystallization method. The 
 purified compound was converted into free lecithin by means of 
 ammonia in methyl alcohol solution. The lecithin was again 
 converted into the cadmium chloride salt and again purified by 
 the ether crystallization process, using a large volume of ether. 
 This material was free from amino nitrogen. 
 
 About 1.0 gm. of material was dissolved in 15 cc. of glacial acetic acid. 
 
 2 cc. of this solution: no N (Van Slyke). 
 
 5 " " " " : 1.35 cc. 0.1 N HC1 (Kjeldahl). 
 
 This cadmium chloride salt of lecithin was then hydrolyzed with 
 10 per cent HC1. The iodine number was determined on the fatty 
 acids after they had been freed from mineral matter and dried. 
 
 0.2120 gm. substance absorbed 0.256 gm. iodine by the Wijs method. 
 Found. Iodine number 65. 
 
 These acids were then converted into the lead salts and extracted 
 with ether. The ether-soluble lead salts were converted into 
 free acids with HC1. Their iodine number was determined. 
 
 0.2672 gm. substance absorbed 0.363 gm. iodine by the Wijs method. 
 Found. Iodine number 136. 
 
Vita. 
 
 HENRY S. SIMMS was born in South Manchester, Connecticut, 
 on May 26, 1896. He attended public schools in Middlebury, 
 Vermont, and Braintree, Massachusetts. He attended the 
 Braintree High School for two years and graduated from the 
 high school of Gorham, Maine. 
 
 In 1914 he entered the University of Maine where he studied' 
 chemistry for two years. After an interim of one year, he con- 
 tinued his studies there for another year. In 1918 he trans- 
 ferred to the Massachusetts Institute of Technology where he 
 studied for two years, acting as assistant in the department of 
 Inorganic Chemistry during the latter year. 
 
 In 1920 he received the degree of Bachelor of Science from the 
 Massachusetts Institute of Technology. As partial fulfillment of 
 the requirements for this degree he submitted a thesis which has 
 since appeared in two publications: 
 
 Microanalytical Methods in Oil Analysis. 
 
 Augustus H. Gill and Henry S. Simms, 
 
 Jour. Ind. and Eng. Chem., 1921, xiii, 547. 
 and 
 Refractive Indices of Oils, 
 
 Henry S. Simms, 
 
 Jour. Ind. and Eng. Chem., 1921, xiii, 546. 
 
 In 1920 he became a Fellow of The Rockefeller Institute for 
 Medical Research, where, under the direction of Dr. P. A. Levene, 
 he began his research on lipoids. 
 
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