Tumor Lipids: Carbon Number Distribution of Triglycerides and Glyceryl Ether Diesters RANDALL WOOD 1 and R.D. HARLOW,Medical Division 2, Oak Ridge Associated Universities, Oak Ridge, Tennessee 37830

ABSTRACT

T r i g l y c e r i d e s and glyceryl ether diesters were isolated from a number of transplantable rat and mouse tumors and analyzed intact by gas liquid chromato- graphy (GLC). The carbon number distri- butions of the triglycerides were dis- t inctly different from those of the glyceryl ether diesters. Both glyceride c l a s ses con ta ined abnormally high molecular weight species. The data support earlier evidence that indicated diglycerides used for the biosynthesis of phosphatides are also precursors of trigly- cerides in neoplasms. Glycerides isolated from three centrifuged fractions of Ehrlich ascites cells exhibited similar carbon number distributions. The results indicate a random distribution of trigly- c e r i d e and glyceryl ether diester m o l e c u l a r s p e c i e s among cellular organeiles. Triglycerides, glyceryl ether diesters and neutral plasmalogens of the same carbon number were partially resolved by GLC.

INTRODUCTI ON

The occurrence of an unidentified neutral lipid in tumor tissue was first reported by Bollinger (1) and Cheng et al. (2) and later con- firmed (3,4). The lipid, isolated from Ehrlich ascites cells, was subsequently identified as a glyceryl ether diester (GEDE) (5). Reduction of total neutral lipids from several transplantable rat and mouse tumors with l i thium aluminum hydride (6) has revealed relatively high levels of alkyl ethers of glycerol in these neoplasms (7). However, free alkyl glyceryl ethers and alkyl acyl glycerides (8), in addition to GEDE, con- tribute to the total percentage of glyceryl ethers obtained from the neutral lipids by hydrogenolysis. Thin layer chromatography (TLC) of the neutral lipids of the rat and mouse tumors indicated the presence of GEDE (7), but class identi ty and purity of GEDE, and even triglycerides, are difficult to establish by

1 Present address: Neuropsyehiatrie Research Laboratory, V.A. Hospital, Hines, I!1. 60141.

2Under contract with the U.S. Atomic Energy Commission.

adsorption TLC. Baumann et al. (9) have demonstrated the similarities in the TLC Rf values of diacyl and alkyl acyl glycerides of diols (ethane and propane) and that of GEDE and triglycerides. The occurrence of diol lipids is covered in a review by Bergelson (10).

The use of high temperature gas liquid chro- matography (GLC) to identify and quantify intact GEDE, triglycerides, alkyl acyl, diacyl and dialkyl glycerides has been well established (5,8,11). This report describes the GLC analysis and comparison of carbon numbers of triglycer- ides and GEDE isolated from several trans- plantable rat and mouse tumors.

EXPERIMENTAL PROCEDURES

Lipid Material

Transplantable rat and mouse tumors were grown and harvested as described previously (7). A non-transplantable fibroadenoma that occurs in rats approximately one year after exposure to 800 R total body irradiation was also examined. Ehrlich ascites cells were dis- rupted in a French pressure cell and separated into three fractions [ 15,000 g(10 min), 34,000 g(3 hr), and supernate] by centrifugation. The term fraction is used instead of names of specific organeiles usually sedimented at these centrifugation forces because of considerable contamination from other cell organelles and cell fragments. However, the impure nature of these fractions does not affect the validity of the data. The total lipids were extracted according to the Folch et al. procedure (12). Neutral lipids were separated from the phos- pholipids by silicic acid chromatography (13), and the triglycerides and GEDE were isolated by preparative TLC (5,8). Both lipid classes were purified by rechromatography in a hexane-diethyl ether 90:10 v/v solvent system. Quantification was carried out on hydrogenated samples (14), which prevented peak broadening caused by highly unsaturated glyceride species.

Gas Liquid Chromatography

The satisfactory and reliable high tempera- ture chromatographic system used for the quantitative GLC analysis of intact triglycerides and related high molecular weight compounds was constructed from an Aerograph Model 204

776

TUMOR LIPIDS 777

liO0 MIXTURE OF GLYCERYL ETHER DIESTERS AND TRIGLYCERIDES COLUMN: 70CM X 4MM O.D. (2.5MM I.D.) PACKED WITH I%OV-/ TEMP: 90

54 80

70 z 0

60 56 52 :

40 8

5e 50 30 ~ ? k , 0

24 21 18 is i~ ~ ~ i o MINUTES

FIG. 1. A chromatogram tracing depicting the partial resolution of triglycerides from glyceryl ether diesters of the same carbon number. Analysis was carried out on a 70 cm x 4 mm O.D. (2.5 mm I.D.) Pyrex column packed with 1% OV-1 and manually temperature-programmed from 200-350 C. The large solvent peak was due to a large amount of benzene solvent.

chromatograph. The modified chromatograph has been used to analyze a number of high molecular weight lipids (8,11,14-16), but the m o d i f i c a t i o n s necessary for quantitative analysis have not been described.

The column inlet, flash heater and column exit were modified to accept a 4 mm O.D. (2.5 mm I.D.) glass column (70 cm), fitted with 3/16 in. Kovar Pyrex to metal seals described by Radin (17) and Litchfield et al. (18). Extension of the column through the flash heater to the injection septum allows on- column sample injection without exposure to metal surfaces. Unpacked columns were installed while being heated to the softening point to allow tightening of the Swagelock fittings without breaking the column. Gas pres- sure was used to pack and unpack the installed column. The column was packed with either 1% OV-1 or 1% OV-17 coated on 100-120 mesh Gas Chrom Q. The temperature was manually programmed from approximately 200-335 C at approximately 5 C/min.

Detector sensitivity and stability was improved by the following modifications: (a) installation of a ceramic flame tip (0.03 in. I.D.); (b) the use of oxygen instead of air; (c) installation of oxygen and hydrogen gas pre- heaters (20 ft x 1/16 in., 0.04 in. I.D., stainless steel tubing) and pressure regulators (Brook Model 8743); and (d) grounding the positive electrode to the flame tip. Spurious peaks and excessive baseline drift were eliminated by pre- conditioning septums at 300 C under vacuum for 4 hr. Temperatures of the flash heater and

detector were maintained at 325 C and 350 C. Helium carrier gas, hydrogen and oxygen flow rates were I00, 50 and 300 ml/min, respectively.

Peak areas were measured by the triangula- tion method (mean of three measurements) or with a Datex Model DIR-1 digital integrator. A standard triglyceride mixture (trilaurin, tri- myristin, tripalmitin, tristearin, triarachidin and tribehenin) was used to calibrate the instrument daily and the mole percentage given has been corrected for slight losses of the high molecular glycerides unless noted otherwise.

Materials

Purified triglycerides were purchased from The Hormel Institute, Austin, Minn. Glass-dis- tilled solvents were purchased from Burdick and Jackson Laboratories, Inc., Muskegon, Mich. Other chemicals were reagent grade or better and were used without further purifi- cation.

RESULTS A N D DISCUSSION

Resolution of Triglycerides, GEDE and Neutral Plasmalogens by GLC

The large quantity of triglycerides relative to the quantity of GEDE and the marginal TLC resolution of GEDE and the neutral plasma- logens enhanced the possibility of cross con- tamination between these lipid classes. There- fore, before quantitative analyses of the trigly- cerides and GEDE could be carried out, we had to demonstrate that such class contamination

LIPIDS, VOL. 5, NO. 9

778 RANDALL WOOD AND R.D. HARLOW

HYDROGENATED NEUTRAL PLASMALOGEN

*54 GEDE

D 58 56 54

6 6 2 ~ ~ 52

/ !

NEUTRAL PLASMALOGEN *54TG. *54 GEDE

58 5454 o . n

60 ! 52

6Z I /

NEUTRAL PLASMALOGEN

i"i sO

52

62 / /

NEUTRAL PLASMALOGEN +54 GEDE

B i 5654

so ~

/ /

FIG. 2. Chromatogram tracings that illustrate the elution order of neutral plasmalogens relative to the elution order of a triglyceride and glyceryl ether diester of the same carbon number. The ehromato- grants represent A, neutral plasmalogens isolated from ratfish liver oil (18); B, neutral plasmalogens spiked with a standard carbon number 54 glyceryl ether diester; C, neutral plasmalogens co-ehromatographed with carbon number 54 glyceryl ether diester and tri- stearin; and D, hydrogenated neutral plasmalogen sample spiked with standard glyceryl ether diester. Chromatographic conditions were the same as noted in Figure 1.

GLYCERYL ETHER DIESTERS

58

TRIGLYCERIDES

5 6

L ~-3 M,N-4

TIME

56 5 4

5 2

k..J 5z ,~ g

5O

L FIG. 3. Representative chromatogram showing the

carbon number distribution of triglycerides and glyceryl ether diesters isolated from several trans- plantable rat and mouse tumors. These chromatograms are of glycerides isolated from Friend virus leukemia mouse tumors. Chromatographic conditions were the same as indicated in Figure 1 except that the liquid phase was OV-17, instead of OV-1.

could be detected. Figure 1 shows the partial resolution of a mixture of triglycerides and GEDE. The GEDE were eluted immediately ahead of the corresponding carbon number, instead of directly between triglyceride carbon numbers as might have been expected, since they differed in molecular weight by only 14. The OV-17 liquid phase gave slightly improved resolution of the mixture of triglycerides and GEDE, but the slight retarded elution of the GEDE relative to the corresponding triglyceride was still observed. Complete resolution of tri- glycerides and GEDE was not obtained on either liquid phase but was sufficient to enable detection of triglycerides contaminating GEDE and vice versa.

The chromatograms shown in Figure 2 illus- trate the elution order of neutral plasmalogens relative to standard GEDE and triglycerides. Although the alk-l-enyl ether bond of the neutral plasmalogen is generally considered very labile, the neutral plasmalogens were able to withstand the high GLC temperatures without any apparent degradation (Fig. 2A). The neutral plasmalogens were eluted ahead of GEDE of the same carbon number (Fig. 2B).

Triglycerides and neutral plasmalogens of two higher carbon numbers were eluted as a single peak (Fig. 2C). Figure 2D shows that upon hydrogenation neutral plasmalogens give rise to GEDE. The early elution of the neutral plasma- logens relative to the GEDE of the same carbon number enable contamination from this class to be detected by GLC analysis of the GEDE frac- tion before hydrogenation. Triglyceride and GEDE samples that showed any contamination with the analogues were repurified by TLC.

Despite the slight difference between the tri- glyceride, GEDE and neutral plasmalogen structures, they can be resolved by adsorption chromatography (I 9). The present data demon- strate that the effect of the alkyl and alk-l-enyl ether bonds permit GLC to partially resolve tri- glycerides, GEDE and neutral plasmalogens of the same carbon number.

Triglycerides and GEDE Isolated From Various Tumors

The lipids of each neoplasm examined con- tained glyceryl ether diesters. Only triglycerides and GEDE were found in the respective frac- tions, as determined by the absence of other

LIPIDS, VOL. 5, NO. 9

TUMOR LIPIDS

TABLE I

Rat and Mouse Tumor Triglycerides and Glyceryl Ether Diesters

779

Per cent carbon number (mole) c Tumor a Lipid b tissue class 46 48 50 52 54 56 58 60 62 64

T G --- W a l k e r 256 GEDE ---

TG --- R-3259 GEDE ---

TG 1.6 TBI GEDE 0.4

TG 0.4 EAC GEDE 0.8

TLT TG --- GEDE 0.7

FVL TG --- GEDE ---

S-180 TG --- GEDE ---

T-241 TG --- GEDE ---

TG --- B-16 GEDE 1.9

TG --- E-0771 GEDE ---

1.1 7.0 20.6 29.1 20.2 4.2 5.3 1.6 0.7 0.6 4.3 15.2 25.9 29.9 6.4 4.7 2.1 0.9

2.5 18.2 42.6 0.7 10.7 24.9

4.6 16.5 30.6 4.4 23.0 40.4

1.3 6.9 21.7 6.3 18.2 24.4

0.9 12.6 42.4 4.1 16.3 31.5

0.8 8.5 30.6 0.8 6.4 17.6

31.8 3.3 0.9 0.5 . . . . . . 24.8 24.5 11.9 2.3 0.4 Trace

24.6 13.0 5.0 1.5 0.7 --- 14.3 11.1 4.9 1.3 0.2 ---

32.4 19.1 3.2 3.8 1.1 --- 20.1 17.6 9.2 2.5 1.1 ---

41.0 2.0 0.8 0.4 0.1 --- 23.5 13.4 7.4 2.3 0.7 0.2

28.8 15.3 8.9 3.3 1.7 1.3 21.8 25.2 17.4 6.6 3.4 0.7

1.1 11.1 37.9 45.0 3.0 1.3 0.6 . . . . . . 1.8 12.2 32.4 20.9 17.5 11.4 2.6 0.8 0.4

0.3 5.7 38.1 53.1 2.0 0.5 0.2 . . . . . . 2.0 11.0 26.1 22.1 21.3 13.5 3.0 0.7 0.2

0.7 13.1 43.6 39.5 2.6 0.4 0.1 . . . . . . 8.3 22.0 29.5 18.1 13.3 5.5 1.1 0.2 ---

1.8 19.8 42.1 34.5 1.4 0.4 . . . . . . . . . 2.0 14.0 26.0 22.5 23.1 10.1 1.7 0.4 ---

aThe tumor abbreviations are Walker 256, Walker carcinosarcoma 256 (rat); R-3259, R3259/96A sarcoma (rat); TBI, non-transplantable fibroadenoma occurring in rats approximately one year after 800R total body irradiation; EAC, Ehrlich ascites carcinoma cells (mouse); TLT, taper liver tumor (mouse); FVL, Friend virus leukemia (mouse).; S-180, sarcoma 180 (mouse); T-241, sarcoma T241 (mouse); B-16, melanoma B16 (mouse); and E-0771, adenocarcinoma E0771 (mouse).

bTG, triglyceride; GEDE, glyceryl ether diester. Cpercentages represent the mean of duplicate analyses of two samples. Agreement between percentage of

major components was + 5% and for minor components -+ 10%.

peaks f r o m the c h r o m a t o g r a m s , i nd ica t ing the absence o f diol l ipids (20 ) in t he se f r ac t ions . T h e c a r b o n n u m b e r d i s t r i b u t i o n o f b o t h t r igly- cerides and G E D E s h o w e d very h igh m o l e c u l a r we igh t spec ies fo r each lipid class o f each neo - p l a s m (Tab le I). Typ ica l c h r o m a t o g r a m s s h o w n in F igure 3 i l lus t ra te t he r e s o l u t i o n and wide c a r b o n n u m b e r d i s t r i b u t i o n o f t he t u m o r t r igly- cerides and G E D E . C a r b o n n u m b e r p e r c e n t a g e s o f t r ig lycer ides and G E D E obtained f o r t en t u m o r s are given in Table I. C a r b o n n u m b e r p e r c e n t a g e ( m o l e ) o f t r ig lycer ides d i f f e r ed grea t ly f r o m the G E D E pe rcen t ages , b u t u sua l ly the range o f c a r b o n n u m b e r s was s imi lar fo r b o t h lipid classes der ived f r o m the s ame neo- p lasm. C a r b o n n u m b e r 52 and 54 w e r e the m a j o r t r ig lycer ide species, w h e r e a s a m a j o r per- cen tage o f t h e G E D E species was d i s t r i b u t e d m o r e even ly f r o m c a r b o n n u m b e r 50 to 58

(Table I). T h e d i ss imi la r i ty o f c a r b o n n u m b e r s b e t w e e n t he se t w o l ipid classes s u p p o r t s t he p a t h w a y s p r o p o s e d earl ier (8 ) w h i c h sugges ted i n d e p e n d e n t r o u t e s o f b i o s y n t h e s i s . Genera l ly the c a r b o n n u m b e r p e r c e n t a g e s o f the G E D E f r o m the va r i ous t u m o r s s h o w e d a c loser agree- m e n t t h a n t h e a g r e e m e n t b e t w e e n t r ig lycer ide pe rcen tages . T h e c a r b o n n u m b e r p e r c e n t a g e s o f t r ig lycer ides f r o m s o m e o f t he n e o p l a s m s agreed r a t h e r c losely . A d e n o c a r c i n o m a E 0 7 7 1 , R 3 2 5 9 / 9 6 A s a r c o m a , m e l a n o m a B16, s a r c o m a 180 and t a p e r liver t u m o r t r ig lycer ide per- cen tages were r e a s o n a b l y close. T h e t r ig lycer ide p e r c e n t a g e s o f Walker c a r c i n o s a r c o m a 256 and Ehr l i ch asc i tes c a r c i n o m a were s imilar . The G E D E p e r c e n t a g e s o f R 3 2 5 9 / 9 6 A s a r c o m a , a d e n o c a r c i n o m a E 0 7 7 1 , s a r c o m a 180 and s a r c o m a T241 agreed, as did Eh r l i c h asci tes c a r c i n o m a , m e l a n o m a B16 and t a p e r liver

LIPIDS, VOL. 5, NO. 9

780 RANDALL WOOD AND R.D. HARLOW

TABLE II

Comparison of Triglyceride and Glyceryl Ether Diester Dis tr ibut ion According to Carbon Number in Various Ehrlich Ascites Cell Fractions

Per cent carbon number (weight) a Glyceride

Fraction type 48 50 52 54 56 58 60 62

TG Trace 1.2 19.9 41.8 21.4 14.1 1.3 0.2 Homogenate GEDE 0.8 11.4 21.4 24.4 26.3 13.5 2.0 0.2

15,000 x g TG Trace 1.0 21.9 42.9 19.5 12.5 1.0 0.1 (10 min) GEDE 1.0 11.9 22.8 24.1 23.9 12.5 1.7 0.2

34,000 x g TG Trace 1.2 23.1 45.0 19.5 10.4 0.8 0.1 (3 hr) GEDE 1.4 14.8 24.3 24.1 23.8 10.4 0.9 Trace

TG Trace 1.9 24.5 42.1 19.0 11.7 0.8 Trace Supernate GEDE 0.4 8.0 23.7 25.9 26.2 11.6 1.3 Trace

aThe percentages represent the mean of duplicate analyses on three separate samples and have not been corrected for slight losses of the higher molecular weights.

t umor percentages of GEDE. Detai led analyses of tr iglycerides and gly-

ceryl e ther diesters of Ehrlich ascites cells earlier revealed that bo th lipid classes exhibi ted a 1-random-2-random-3-random distr ibut ion, and that 1,2-diacyl and 1,2-alkyl acyl glycerides derived f rom phosphat idylchol ine were similar to the corresponding glycerides derived f rom triglycerides and GEDE (8). These observations suggested that the selectivity of diglycerides for the biosynthesis of tr iglycerides and phospha- t idylchol ine that occurs in normal tissue was absent in Ehrl ich ascites cells, moreover the absence of diglyceride selectivity might be c o m m o n to o ther neoplasms. The high molecular weight species of tr iglycerides found in the neoplasms of the present s tudy indicated that the lack of diglyceride selectivity in trigly- ceride biosynthesis may represent a significant difference be tween the lipid metabol i sm of normal and neoplastic tissue. Carbon numbers of tr iglycerides of most mammal ian tissues analyzed thus far have not contained significant quanti t ies of tr iglyceride species longer than carbon number 54 (21-23). However , high molecular weight triglycerides are not restr icted to neoplasms. Marine oil tr iglycerides have been shown to exhibi t a wide carbon number distri- bu t ion (18,23).

Glyceryl ether diesters of neoplasms were first ident i f ied in Ehrl ich ascites cells (5). The present data demonst ra tes the occurrence of GEDE in eight addi t ional t ransplantable rat and mouse tumors , impl icated earlier by indirect evidence (7). The significance of glyceryl ether diesters in neoplast ic tissue is not known, but they may occur as a result of increased concen- t rat ions of alkyl glyceryl ether precursors in the neoplast ic cell. Glyceryl ether diesters have been isolated f rom human perinephric fat (24),

beef heart lipids (25), and indirect evidence sug- gests that they occur in most mammal ian tissues (6,26).

Carbon Number Distribution of Triglycerides and GEDE From Ehrlich Ascites Cell Fractions

The carbon number percentages (weight) of the homogena te tr iglycerides and GEDE s h o w e d small deviat ions f rom the cor- responding Ehrlich ascites cells values given in Table I. The differences are probably due to different cell harvesting t imes after t umor trans- plantat ion. The values in Table I were obta ined f rom seven day cells, whereas the percentages in Table II were obtained f rom cells harvested five days after t ransplantat ion.

The fractions obta ined f rom the disrupted Ehrl ich ascites cells by centr i fugat ion contained different quanti t ies of GEDE as est imated f rom the quant i t ies isolated by TLC. The 15,000 g f ract ion contained approx imate ly three t imes the quant i ty of GEDE as the 34,000 g and supernate fractions. Table II gives the carbon number percentages of tr iglycerides and GEDE derived f rom the various fractions. As expected, based upon prior analyses, the carbon number dis t r ibut ion of the GEDE of each fract ion was dis t inct ly different f rom the tr iglyceride distri- but ion. More impor tan t ly , the carbon number percentages of the tr iglycerides and GEDE of one fract ion were not significantly different f rom the corresponding percentages o f the o ther fractions. This similari ty be tween the carbon number percentages of the fract ions for bo th lipid classes suggests a random distr ibut ion of tr iglycerides and G E D E molecular species among cellular componen t s , a l though the cellular components may differ significantly in the percentage of each lipid class present. Similar carbon number distr ibutions in the

LIPIDS, VOL. 5, NO. 9

TUMOR LIPIDS 781

v a r i o u s cell f r ac t ions could have resu l ted f r o m r a n d o m d i s t r ibu t ion of p r e f o r m e d glycer ides or b io syn thes i s of the glycer ides in the com- p o n e n t s of t h e cell f r ac t ions f r o m a c o m m o n source of precursors . Bar t ley (27) has r epo r t ed t ha t lipids of var ious cell organel les ob t a ined f rom the same t issue have a p p r o x i m a t e l y the same f a t t y acid c o m p o s i t i o n . Our resul ts , wh i ch go a s tep fu r t h e r , ind ica te a r a n d o m distri- b u t i o n of t r ig lycer ide and GEDE molecu la r species a m o n g the cellular c o m p o n e n t s . Such a d i s t r ibu t ion of p h o s p h o g l y c e r i d e mo lecu la r species m a y also occur in n o r m a l and neop las t i c t issue.

ACKNOWLEDGMENTS

E.A. Cress prepared the Ehrlich ascites cell fractions, and Arlene Wood typed and reviewed this manuscript. Synthetic GEDE (glyceryl 1,2-dipalmitoyl-3-octadecyl and glyceryl 1,2-distearoyl-3-hexadecyl ethers) and the neutral plasmalogens (alk-l-enyl 2,3-diacyl glyceryl ethers) were gifts from H.K. Mangold, W.J. Baumann and H.H.O. Schmid of the Hormel Institute.

REFERENCES

1. Bollinger, J.N., USAEC Report ORINS-53, p. 21 (1965).

2. Cheng, S., C. Piantiadosi and F. Snyder, Ibid. p. 23 (1965).

3. Snyder, F., E.A. Cress and N. Stephens, Lipids 1:381-386 (1966).

4. Bollinger, J.N., Ibid. 2:143-148 (1967). 5. Wood, R., and F. Snyder, J. Lipid Res. 8:494-500

(1967). 6. Wood, R., and F. Snyder, Lipids 3:129-135

(1968). 7. Snyder, F., and R. Wood, Cancer Res. 28:972-978

(1968). 8. Wood, R., and F. Snyder, Arch. Biochem. Bio-

phys. 131:478-495 (1969).

9. Baumann, W.J., H.H.O. Schmid, H.W. Ulshofer and H.K. Mangold, Biochim. Biophys. Acta 144:355-365 (1967).

10. Bergelson, L.D., in "Progress in the Chemistry of Fats and Other Lipids," Edited by R.T. Holman, Pergamon Press, Ltd., Oxford, 10:239 (1969).

11. Wood, R., W.J. Baumann, F. Snyder and H.K. Mangold, J. Lipid Res. 10:128-131 (1969).

12. Folch, J., M. Lees and G.H. Sloane Stanley, J. Bi.ol. Chem. 226:497-509 (1957).

13. Borgstrom, B., Acta Physiol. Scand. 25:101-110 (1952).

14. Wood, R., Cancer Res. 30:151-154 (1970). 15. Wood, R., and R.D. Harlow, Lipids 4:159-162

(1969). 16. Harlow, R.D., R. Lumb and R. Wood, Comp. Bio-

chem. Physiol. 30:761-769 (1969). 17. Radin, N.S., J. Chromatog. 20:392-394 (1965). 18. Litchfield, C., R.D. Harlow and R. Reiser, Lipids

2:363-370 (1967). 19. Schmid, H.H.O., and H.K. Mangold, Biochim. Bio-

phys. Acta 125:182-184 (1966). 20. Bergelson, L.D., V.A. Vaver, N.V. Prokazova,

A.N. Ushakov and G.A. Popkova, Ibid. 116:511-520 (1966).

21. Litchfield, C., R.D. Harlow and R. Reiser, JAOCS 42:849-857 (1965).

22. Wood, R., and R.D. Harlow, Arch. Biochem. Bio- phys. 131:495-501 (1969).

23. Kuksis, A., in "Lipid Chromatographic Analysis," Edited by G.V. Marinetti, Marcel Dekker, Inc., New York, 1:239-337 (1967).

24. Schmid, H.H.O., and H.K. Mangold, Biochem. Z., 346:12-25 (1966).

25. Schmid, H.H.O., and T. Takahashi, Biochim. Bin- phys. Acta 164:141-147 (1968).

26. Gilbertson, J.R., and M.L. Karnovsky, J. Biol. Chem. 238:893-897 (1963).

27. Bartley, W., in "Metabolism and Physiological Sig- nificance of Lipid," Edited by R.M.C. Dawson and D.N. Rhodes, John Wiley and Sons, Ltd., London, 1964, p. 369-381.

[.Received April 7, 1970]

LIPIDS, VOL. 5, NO. 9