STUDIES ON A METHOD FOR THE QUANTITATIVE ESTIMATION OF CERTAIN GROUPS IN

PHOSPHOLIPINS.

BY MARY LOUISE FOSTER.

(From the Hull Laboratbries of the University of Chicago, Chicago.)

(Received for publication, January 11, 1915.)

In 1903 Baskoff’ published a method for the quantitative estimation of glycerol and the nitrogen complex. He dissolved lecithin in absolute alcohol and saturated this with hydrochloric acid gas. The solution was heated with a reflux condenser until hydrolysis and esterification of the fatty acids had taken place. The alcohol was then almost entirely removed by heating, the solution diluted with water, and the esters of the fatty acids were shaken out with ether. The solution at this stage contained the nitrogen complex, glycerophosphoric acid, free phosphoric acid, and glycerol. A portion of this was treated according to the method of Stanek-Kiese12 with potassium triiodide, and the nitrogen of the precipitate was determined by Kjeldahl. It gave 1.09 per cent, which represents 11.07 per cent choline. A second portion was evaporated to dryness and extracted with absolute alcohol to remove the choline hydrochloride. The residue was treated with water and again taken down to dryness, a process repeated several times. It was finally transferred to a Fanto-Zeise13 apparatus where it was concentrated to 5 cc., 15 cc. of hydriodic acid were added, and it was heated to boiling. At the same time a stream of CO2 was passed through the whole apparatus. The vapor of isopropyl iodide which escaped from the upright cooler was led for purification from hydriodic acid vapor through a wash bottle containing red phosphorus under water, and finally

1 A. Baskoff: Ztschr. f. physiol. Chem., Ivii, p. 435, 1908. 2 V. Stanek: ibid., xlvi, p. 28.3, 1905. 3 S. Zeisel and R. Fanto: Ztschr. f. anal. Chem., xlii, p. 549, 1903.

403

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404 Quantitative Estimation of Phospholipins

into an alcoholic solution of silver nitrate. The silver iodide was weighed, and by calculation gave the amount of glycerol. Baskoff found in lecithin 10.61 per cent, calculated as C3HG(OH)3; the theoretical amount is 11.8 per cent as C&H,(OH)3, 5.2 per cent as C3H6, on Trier’s* formula.

The above work suggested the possibility of splitting off the glycerol group and the methyl groups attached to the nitrogen in one operation, but at different temperatures, by means of hydri- odic acid. Preparations of phospholipins, i.e., lecithin, the alcohol- soluble compound, and kephalin, the alcohol-insoluble compound, were made according to Waldemar Koch’s5 method, except that care was taken to use only anhydrous solvents. By this method, after repeated purification, the alcohol-soluble compound gave by Arnold-Gunning Kjeldahl for N = 1.91 per cent, and by the Neumann method for P = 3.05 per cent weighed as Mg,P207; the alcohol-insoluble preparation gave N = 1.89 per cent; P = 3.73 per cent.

The following table shows the results obtained by various investigators of these substances :

KEPHALIN Thudichum. ............. W. Koch5 ............... Neubauere. .............

My preparation. .............

........

........

.........

. . . . . . .

peLkt ......... 1.68 ....... ..1.7 8 ....... ..1.65 ........ .1.91

LECITHIN Diakonow7.. . . ...... ....... ..1.8 Thierfelders. . . ...... ...... .:.2.08 McLean9.. . . ...... ....... ..1.8 8 Eppler . . . . . . . . . . . ....... ..2.09 Thudichum. ...... ....... ..2.0 3 W. Koch.. . . . . . ...... ......... 1.80 Erlandsen’o. . , . . . ...... ........ .1.87

My preparation.. . . ...... ......... 1.89

3.8 3.97 3.95 3.95 4.29 3.79 3.95 3.73

4 G. Trier: Ueber einfache Pflanzenbasen und ihre Beziehungen rum Aufbau der Eiweissstofle und Letithine, Berlin, 1912.

6 W. Koch: Am. Jour. Physiol., xi, p. 319, 1904. 6 E. Neubauer: Biochem. Ztschr., xxi, p. 321, 1909. TDiakonow: Med. them. Untersuch., 1867, p. 21. *H. Thierfelder: Ztschr. f. physiol. Chem., xlvi, p. 518, 1905. 9H. McLean: ibid., lvii, p. 297, 1908.

10 A. Erlandsen: ibid., li, p. 71, 1907.

P pm cent 4.27 3.84 3.45 3.05

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M. L. Foster 405

While these figures vary considerably among themselves, the ratio 1 : 1 between N and P is fairly well maintained and shows that these phospholipins, although obtained by sundry methods of extraction, nevertheless belong to the class of mono-amino- mono-phosphatides, as stated by Erlandsen.

Before applying the method for determining the glycerol in these preparations, controls were run with pure glycerol. The glycerol (Merck’s preparation) was so diluted that 0.5 cc. con- tained 0.2 to 0.3 gram of glycerol. This was placed in a small round-bottomed flask with 1 gram of ammonium iodide and 15 cc. of hydriodic acid, and connected with an upright 15 inch condenser having a thermometer and a side tube, leading to a Geissler bulb which contained 2 per cent solutions of sodium carbonate and potassium arsenite, as recommended by Klinger and Kreutz.” The flask containing the glycerol was heated in a glycerine bath to 112’ to 113”, at which temperature a cloud appeared in the silver nitrate solution. The heating was con- tinued at this temperature until the precipitate became crystal- line and settled, leaving the supernatant liquid perfectly clear. The reaction probably takes place according to the equations:

CH20HCHOH-CH20H + 3HI = CH,ICHICH,I + 3H,O CH21CHICH21 + 2HI = (CH&CHI + 21Z.

gm. 0.2466 0.2292 0.2306 0.0719

-~ 8”.

0.6323 0.5296 0.5167 0.1800

0.2479 loo.4 0.2077 90.62 0.2026 87.8 0.0704 98.4

PER CENT

The time required for completion of the reaetion was two to three hours, the temperature remaining constant at 112” to 113”. The temperature was finally allowed to rise to 120°, at which point if no further precipitation took place, the process was dis- continued. The weight of silver iodide multiplied by the factor

1’ H. Klinger and A. Kreutz: Ann. d. Chem., ccxlix, p. 147, 18%.

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406 Quantitative Estimation of Phospholipins

0.3922 gives the weight of glycerol .found. This method was a combination of the Fanto-Zeisel and Hewitt and MooreI methods.

A blank gave negative results and warranted the conclusion that the Geissler bulb was holding back all hydriodic acid and iodine.

The phospholipins were then tested in the apparatus. Keph- alin was tested first and gave a precipitate at 112’, corresponding in every detail to that obtained with glycerol. Even on heating a second time no turbidity appeared in the silver nitrate solution.

HEPahLIN WT. OF GLYCEROL NO. USED AgI. C*LcUL*TED GLYCEROL

gm. 1. 0.2181 0.&7 = og.bN =“;YIY”;“” 2. 0.7260 0.1517 = 0.0595 = 8.21 3. 0.3242 0.0628 = 0.0246 = 7.62

On raising the temperature no further precipitation in a fresh silver nitrate solution took place. This indicates that no met,hyl group was liberated.

x0. gm. gm. !Jm. per cent 1. 0.2438 lecithin 0.0563 AgI at 112” = 0.0220 glycerol = 9.05 1 a. 0.2438 lecithin 0.0368 AgI at 180” = 0.00235 methyl = 0.96 2. 0.2235 lecithin 0.0548 AgI at 112” = 0.0215 glycerol = 9.61 3. 0.2615 lecithin 0.0668 AgI at 112” = 0.0262 glycerol = 10.01 4. 0.4972 hydrolyzed

lecithin 0.1214 AgI at 112” = 0.0476 glycerol = 9.57 5. 0.4972 hydrolyzed

lecithin 0 .I135 AgI at 112” = 0.0445 glycerol = 8.95 6. 0.4972 hydrolyzed

lecithin 0.1182 AgI at 112” = 0.0464 glycerol = 9.32

Average for Nos. 1, 2, and 3, unhydrolyzed = 9.55 per cent; for 4, 5, and 6, hydrolyzed according to Baskoff’s method = 9.38 per cent. It is evident that this process is unnecessary, the hy- driodic acid alone being sufficient.

In every instance after apparently complete precipitation of iodide from the glycerol had taken place, the temperature was run up slowly to 300”, but no further clouding of the fresh silver nitrate solution took place except in 1 a, where a precipitate was obtained. This was, however, too much to be wholly glycerol and too little for three methyl groups. Two facts had become evident: the glycerol group in the phospholipins under consider-

I2 J. T. Hewitt and T. S. Moore: Jour. Chem. Sot., lxxxi, p. 320, 1902.

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M. L. Foster

ation is very easily and almost quantitatively split off by hydri- odic acid; and, second, either the method is ineffective where imide methyl groups are concerned; or there is no methyl in the substance. The first fact will necessitate the recalculation of Waldemar Koch’s results. In the case of kephalin:

0.3488 gm. substance; 0.0945 gm.AgI = 0.0378 gm. glycerol = 10.8 per cent.

instead of

0.3488 gm. substance; 0.0945 gm. AgI = 0.006036 gm. CHa = 1.73 per cent.

This admits of no methyl group in kephalin and agrees with Winterstein’sL3 finding of 10.2 per cent glycerol, or 1.63 per cent CHS, as given by this method. In the case of egg lecithin Koch got a precipitate of 0.0760 gram of silver iodide from 0.320 gram of the substance below 240”. Calculating this as glycerol, the yield ‘is 9.5 per cent, and leaves for methyl only 4.3 per cent instead of 5.80 per cent, or two methyl groups instead of three.

These irregularities and inconsistencies suggested the necessity of testing choline, which has so often been called the alkaloidal base of lecithin. Choline chloride was prepared by the method of Wurtz,‘” as modified by Renshaw.15

Attempts were made to determine the methyl groups in this preparation by the use of the Fanto-Zeisel method given above, but no adequate cleavage of the methyl group took place. Accord- ing to Decker,l’j dry heating is necessary and this is the chief advantage of the double barrel distilling flask used in the Herzig and Meyer method. By using separate cups filled with paraffin the temperature can be absolutely controlled, as is not the case in the sand bath, where the temperature about the immersed flask was found to vary 40’ to 50” in spots a few inches apart. The tube connecting the two flasks was bent into an inverted U, so that the flasks were immersed nearly to the stoppers in paraffin. The temperature was allowed to rise slowly to 140°, where it was held for half an hour, then allowed to rise again to 180” to

1s E. Winterstein and 0. Hiestand: Ztschr. f. physiol. Chem., liv, p. 300, 1907-0s.

11 A. Wurtz: Ann. d. Chem., Supplement, vi, p. XM, 1868. 15 R. R. Renshaw: Jour. Am. Chem. Sot., xxxii, p. 128, 1910. I8 H. Decker: Ber. d. deutsch. chem. Gesellsch., xxxvi, p. 2895, 1903.

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408 Quantitative Estimation of Phospholipins

185”, at which point a precipitate was usually formed in the silver nitrate solution. Here the temperature was held until the reaction was over, a process lasting four to five hours. The precipitate was then treated as with glycerol.

The reaction takes place in two stages. According to Wurtz, the change of the alkyl groups of choline into’the iodide form occurs between 120” to 150”. The boiling point of hydriodic acid is 127’, slightly raised in t,his instance by the presence of the ammonium iodide.

N(CH,),.CH,.CH,.OH.Cl + 2HI = C2H,I.N(CH,),I + H,O + HCI

This reaction is slow and requires time. It seems probable that it is because this reaction has been incomplete that repeated successive heatings are necessary. No way of knowing when this reaction is complete has been discovered, however. At a higher temperature dissociation takes place, and methyl iodide, which decomposes less readily than ethyl iodide, is split off, and passes over into the alcoholic silver nitrate, where it is precipitated.

Besides the substitution of a paraffin bath for the sand bath, some other modifications were introduced: a current of water heated to 40” to 50” ran through the condenser while the Geissler bulb containing 2 per cent solutions of sodium carbonate and potassium arsenite was immersed in water heated to 50” to 60“; a second flask containing the arsenite solution, also heated, was introduced as a precautionary measure; and finally the gases were bubbled through a tall cylinder of alcoholic silver nitrate by means of a modified Folin tube. This effected a more com- plete precipitation. A second, third, and even fourth heating increased the yield, although the evidence of the settling of the precipitate in each previous heating had been taken as sufficient proof that no more iodide was forming. This reheating was made by turning back into the first bulb the hydriodic acid which had collected in the flask connected directly with the condenser, and then repeating the process exactly as in the first instance. Much more uniform results were obtained after the slow digestion was introduced, but a white smoke which appeared as a constant factor was not satisfactorily explained.

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M. L. Foster 409

TOTAI. CHa NO.

gm.

0.1000 0.0431 0.0762 0.0418 0.0311 0.0416 0.0401 0.0655

gm.

0.4286 0.1575 0.2907 0.1805 0.1451 0.1964 0.1895 0.3075

__- -_ sm. per cent

0.0274 27.4 0.01006 23.3 0.01857 24.4 0.01153 i I 27.87 0.00927 i 29.8 0.01255 1 30.01 0.012105 ’ 30.18 0.01964 1 29.68

c

per cent

35.1 46.3 39.3 32 -27 37.3 42.6 40.01 36.99

The slow digestion first used on No. 4 brought about fairly uniform results for the first yield, but although applied also in the succeeding heatings the final yield shows considerable variation. The factor 0.06388 was used to convert the silver iodide into methyl which should amount theoretically to 32.2 per cent of choline chloride. If the hydroxyethyl group is also liberated and calculated as methyl, the total per cent of methyl would be 42.9 per cent.

A mixture of glycerol and choline chloride was then tried. The separation of glycerol was sharp and complete at 112” to 120°, no more precipitate appearing in the silver nitrate solution as the temperature was raised. The second precipitate began to appear usually at 175” and was most abundant at 185”. -~.-

NO. PEMPERA- WEIGHT 01

PURE &I

. - ~~- gm. per cent

0.1495 0.0359

Glycerol I 92.2 CHg 21.3

0.0738. Glycerol, 102.6 0.0351 CH3 j 35.1 0.0698 Glycerol ) 97.2

0.0562 CH3 / “1’

cm.

0.1620 0.1682 0.0719 0.1000 0.0719 0.1322

c. ; gm. 112-12O”i 0.3811

:Kl,oj 0”:~::: 183” j 0.5486 112-120”: 0.1782 160-243” 0.8927

1 1 Glycerol.. Choline Cl. Glycerol.. Choline Cl.. Glycerol.. Choline Cl..

2 i

3. j

These figures represent the total yield obtained after several heatings.

With the improved apparatus analyses of the phospholipins were repeated.

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410 Quantitative Estimation of Phospholipins

NO.

__

1

2

3

4

5

6

Lecithin. . . . .

Lecithin. . . . .

Egg lecithin..

Lecithin HCI

Kephalin.. . . .

Lecithin HCl .

L

AYOUNT USEm

/tn. 0.3642

0.4927

0.2945

0.2868

0.1613

0.1513

_--._

rEMPIE*- WmIcIHT OF CM..C!UL.4TED Tlnm Ad TO

______ c. gm. gm.

112O 0.1012 0.0397 Glyperol 180-185” 0.0766 0.0049 CH8 112” 0.1238 0.0496 Glycerol 160-240” 0.1122 0.0672 CHs 112O 0.0658 0.0258 Glycerol 180-190” 0.1065 CHa 112O 0.0850 0.0333 Glycerol 180-190” 0.2743 0.01752 CHa lJ2O ’ 0.0338 0.01326 Glycerol 180” 112O 0.039 0.1529 Glycerol 180-190” 0.3474 0.02219 C&

-- 1 06-r cent 10.9 1.34

10.05 1.45 8.75 3.62

11.6 6.10 8.21

9.96 15.1

These results for glycerol are slightly higher than those obtained by the use of the simple apparatus. If any reliance is to be placed on this method for the determination of the methyl groups, it is evident that lecithin prepared from sheep’s brains by the use of acetone, alcohol, and ether contains only one methyl group, t.heratioof N:P:CHa=1.89:3.73: 1.45=0.12:0.09. Koch found, allowing 9.5 per cent. glycerol as indicated by the precipitate below 240°, 3.80 per cent for CHI, or a ratio of N : P : CHS = 1.80 : 3.79: 3.80 = 1: 1: 2. The egg lecithin gives a slightly lower figure for glycerol and a much higher result for methyl, while the lecithin hydrochloride prepared by treatment of the above lecithin with cadmium chloride and removal of the cadmium with hydrogen sulphide as prescribed by Thudichum gives still higher results. The result would indicate for this preparation three methyl groups. Kephalin gave no precipitate at the higher temperature, indicating no methyl groups. A comparison of Nos. 4 and 6 shows widely varying results for samples from the same bottle. This is probably due to the different amounts of time for which each was tested; No. 4 was given two heatings lasting about seven hours, while No. 6 was given three heatings amounting to about ten hours in all. Neither was heated long enough to give final negative results. It is evident that pro- longed heating int,roduces errors-a fact already noticed by StritaMue to the formation of various alkyl iodides by the hydriodic acid.

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M. L. Foster 411

These varying results suggested the necessity of testing the method on some of the constituents of lecithin other than choline.

LIrlIBBTANCE AMOUNT &I TAmm YIELDED PXJND: C*LCUL*hTED:

gm. CP. pel cent per cent

Palmitic acid.. . . . . 0.1198 0.010 at 190” 0.5 CH3 Oleic acid.. . . . . . . . . . . . . . 0.3194 0.0137 “ 190=’ 0.2 CH3 Mono-ethylaminehydro-

chloride.. . . . . . . . . . . . . 0.1149 0.0939 9.7 CzHs 35.6 CzHs Trimethyl amine.. . , . . . . 0.0513 0.432 53.79 CH3 47.17 CH3

A sixth heating of the trimethyl amine hydrochloride gave negative results in the silver nitrate solution. White smoke was a constant accompaniment of the experiment except in the sixth heating, where there was none. Smoke and precipitate began to appear at 150”, a much lower temperature than in the analysis of choline chloride or ethyl amine hydrochloride. The total time of heating was about seventeen hours. It is difficult to account for this excessive yield.

Some methyl iodide was repeatedly fractionated and a portion boiling between 42” to 43” was introduced into a stoppered bottle and dropped into the bulb of the apparatus containing hydriodic acid and given the usual treatment. The precipitate appeared in the silver nitrate solution almost immediately, the stopper having been forced out by expansion. The temperature was, however, raised to 180” and the heating continued till the solution had become clear. 0.2057 gram of methyl iodide yielded 0.3229 gram of silver iodide = 94.84 per cent of the total. Some methyl iodide in a similar stoppered bottle was dropped directly into some alcoholic silver nitrate solution. 0.1505 gram of methyl iodide yielded 0.2440 gram of silver iodide = 97.97 per cent. The analytical method with the apparatus gives then a yield of 96.7 per cent. It is evident from this experiment that the methyl iodide is not lost in transit, but comes over nearly quantitatively, if the direct precipitation be taken as the standard. The only possible explanation for the high yield in the experiment with trimethyl amine hydrochloride would seem to be that hydriodic acid forms unsaturated compounds which yield more than one methyl iodide equivalent. Work by Dr. Nef suggests this as possible and probable.

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412 Quantitative Estimation of Phospholipins

From these results it is evident that the method in its present shape is not applicable to the analysis of phospholipins for the purpose of determining their formulae. Hydriodic acid is a powerful reducing agent and attacks both methyl and ethyl groups. Kahanl’ found that methyl iodide heated to 265’ for three hours gave no gas, but heated to 270” it went over into methane and ethylene. At 185’ methyl iodide is fairly stable, giving only slight evidence of gas. This is not true of ethyl iodide, which goes over into ethane, ethylene, and hydrogen. I found that the ethyl amine hydrochloride gave the sublimate and smoke so characteristic of all the choline chloride determi- nations. The gas or smoke was collected and found to be an unsaturated hydrocarbon. Goldschmiedt’8 has studied many compounds containing the methyl group in various positions, using this method. He finds that the splitting off of the alkyl group is dependent upon the structure of the compound, the nature and the position of the groups, and that it is impossible quantitatively to differentiate NCHI and OCH,. The methyl group often seems to wander from nitrogen to oxygen. It. is possible that longer digestion below the boiling point of hydriodic acid may result in a wider separation by temperature of the methyl and ethyl iodide and admit of a fractional separation of these two alkyl groups.

SUMMARY.

1. By the use of a paraffin bath, the temperature of which can be controlled, it is possible to obtain by a modified Herzig and Meyer method a sharp separation of the glycerol and alkyl groups in certain phospholipins, the former reacting at 112” and the latter at 180”-190°C.

2. The glycerol is obtained in almost theoretical quantity. 3. The ethyl and methyl groups are not so quantitatively

determined and differentiated. 4. Thudichum’s preparation of lecithin shows the three methyl

‘7 Z. Kahan: Jour. Chem. Sot., xciii, pt. i, p. 132, 1908. 18 G. Goldschmiedt and A. Kirpal: Monatschr. f. Chem., xvii, p. 491,

1896. G. Goldschmiedt: ibid., xxvii, p. 849, 1906. G. Goldschmiedt and 0. Hbnigschmid: ibid., xxiv, p. 681, 1903.

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M. L. Foster 413

groups commonly attributed to lecithin; but our preparations show less.

5. The method shows no methyl or ethyl groups in kephalin.lg 6. The analysis of synthetic choline chloride shows that hydri-

odic acid does not split off the methyl groups quantitatively. 7. Ethyl amine hydrochloride gives many of the phenomena,

viz., the white smoke and the sublimation in the U tube, noted in the course of the reduction of the choline chloride. These are not so marked in the case of trimethyl amine hydrochloride.

I wish to express here my thanks to Professor A. P. Mathews and to Professor F. C. Koch, to whom I am indebted for many helpful suggestions.

Smith College, Northampton, Mass.

‘9MacArthur announces in the Jour. Am. Chem. Sot., xxxvi, p. 2397, 1914, that kephalin contains neither choline nor neurine.

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Mary Louise FosterCERTAIN GROUPS IN PHOSPHOLIPINS

QUANTITATIVE ESTIMATION OF STUDIES ON A METHOD FOR THE

1915, 20:403-413.J. Biol. Chem. 

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