Hepatoma, host liver, and normal rat liver phospholipids as affected by diet

Hepatoma, Host Liver, and Normal Rat Liver Phospholipids as Affected by Diet RANDALL WOOD, Division of Gastroenterology, Departments of Medicine and Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65201

ABSTRACT

Individual phospholipid classes derived from hepatoma, host liver, and normal liver of rats maintained on chow and fat free diets were examined in detail and the s p h i n g o m y e l i n and phosphoglyceride structures compared. The concentration of hepatoma sphingomyelin was higher while phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and diphosphatidylglycerol were only one-fourth to one-half normal liver concentrations, irrespective of diet. Hepatoma phosphatidylcholine, phosphatidylethanolamine, phosphatidyl- s e r ine , and phosphatidylinositol contained higher percentages of 18:1 and, except phosphatidylinositol, much lower percentages of most polyunsaturated fatty acids than liver. The 1-position of host liver phosphatidylcholine and phosphatidylethanolamine, normal liver phosphatidylcholine and phosphatidylethanolamine, and hepatoma phosphatidylcholine from animals on both diets had the same approximate fatty acid composi-

tion, but the percentage of 16:0 in hepa- t o m a phospha t idy le thano lamine was reduced dramatically. The low percentage of 16:0 at the 1-position of both phosphatidylethanolamine and triglycerides suggests that the 1-position fatty acids of these two classes may have a similar ori- gin. The fat free diet reduced the percentage of 18:2 in liver diphosphatidylglycerol 3-fold and the decrease was offset by increased percentages of 16:1 and 18:1; whereas the very low percentage of 18:2 in hepatoma diphosphatidylglycerol was offset by increased percentages of 18:0 and 16:0. Liver phosphatidylinositol and phosphatidylcholine from the animals fed the fat free diet contained the highest percentage of 20:3, which replaced 20:4. Hepatoma sphingomyelin contained a much higher concentration of 24:0 and 24:1 than liver. The hepatoma sphingomyelin also contained a C-24 dienoic acid, which was not detected in host and normal liver. Host liver contained a higher percentage of 22:6 than normal liver. The

digiycerides derived from host liver PC contained a significantly higher percentage of carbon number 38 than normal liver. Diglycerides derived from hepatoma phosphatidylcholine and phosphatidylethanolamine exhibited a 1-random-2- random distribution of fatty acids, whereas diglycerides from liver phosphatidylcholine and phosphatidylethanolamine showed pairing of specific fatty acids.

INTRODUCTION

The occurrence of hyperlipemia and loss of body lipid in tumor bearing animals (1) indicate that lipid metabolism in the host is affected by the tumor. On the other hand, the correlation between dietary fat and the incidence of some tumors (2-4) suggest that host lipids may affect the tumor. The few scattered reports that de- scribe the change or lack of change that occurs in the lipids of tumor bearing animals have been reviewed in the first paper of this series (5). This is the third in a series of papers that de- scribes the results of experiments designed to investigate the relationship between host and tumor lipid metabolism. The data reported here compare quantitatively the phospholipids derived from hepatoma 7288CTC, normal rat liver, and host fiver of animals maintained on normal and fat free diets. Preliminary reports of these studies have appeared (6,7).

METHODS AND MATERIALS

Hepatoma 7288CTC, livers from normal rats, and livers of rats bearing hepatoma 7288CTC (denoted throughout this report as host liver) were obtained from groups of three or more animals (175-225 g) maintained on chow and fat free diets for four to five weeks as described previously (5). Lipids were extracted by the Bligh and Dyer procedure (two extractions) (8), fractionated into neutral lipid and phospholipid fractions by silicic acid chromatography (9), and weighed. Phospholipids were resolved into individual classes by thin layer chromatography (TLC) (10) and quantified by the phosphorus method of Rouser, et al., (11). Individual phospholipid classes on preparative chromatoplates were visualized under UV light after spraying

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6G. The localized bands were scraped from the chromatoplate, etuted from the absorbent, and checked for purity. Samples that were not >95% pure were repurified by chromatography. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were subjected to phospholipase A hydrolysis as described previously (12) to obtain the fat ty acid compositions of the 1- and 2-positions.

The fat ty acids released by hydrolysis of PC and PE, the lysophosphatides, and other phospholipid classes were converted to methyl esters and analyzed by gas liquid chromatography (GLC) on polar columns as described earlier (13). PC and PE were hydrolyzed to diglycerides with Ctostridiurn welchii phospholipase C (14), diglyceride acetates were prepared by the p-toluenesulfonic acid catalyzed acetylation procedure (15), hydrogenated, and analyzed intact by high temperature GLC (13).

Diglyceride and fat ty acid percentages rep- resent the mean of two or more determinations on the pooled sample for each tissue and dietary condition. Agreement between determined percentages was usually +5% for major components and +10% for minor components. Identi- ties of fat ty acids were based uoon cochroma- tog raphy with standards before and after hydrogenation. Positions of double bond in C-16, C-18, and C-20 monoenes were determined and are reported in a companion paper.

Crotalus atrox venom (phospholipase A) was purchased from Ross Allen's Reptile Institute (Silver Springs, FL). Phospholipase C (el. w e l c h i i ) was o b t a i n e d from Calbiochem (LaJolla, CA). Diglyceride and phospholipid standards were supplied by Applied Science Lab (State College, PA) and Supelco, Inc. (Bellefonte, PA). All solvents were glass dis- tilled and obtained from Burdick and Jackson Labs (Muskegon, MI).

R ESU LTS

Class Composition

The percentages of major phospholipid classes from hepatoma, host liver, and normal fiver of animals maintained free diets are given in Table positions of normal and host tially the same, and diet had Hepatoma contained less PC

on chow and fat I. The class corn- livers were essen- little or no effect. and much higher

percentages of sphingomyelin than liver, and, as in the case of liver, diet had no effect on the hepatoma phospholipid class composition.

Concentrations (mg/g wet wt) of the various phosphotipid classes can be calculated from the total phospholipid values given in the first column of Table I. Most phospholipid classes

from normal livers of animals on both diets and from host livers of chow fed rats exhibited the same approximate concentrations, but host livers from rats fed the fat free diet contained ca. 50% or less PC, phosphatidylinositol (PI), phosphatidylserine (PS), and diphosphatidylglycerol (DPG). Hepatoma sphingomyelin concentrations were higher than those of liver, whereas all other phospholipid class levels were lower. Hepatoma PC and PE concentrations were only 25 to 50% of liver levels.

Positional Distribution of Fatty Acids

The fatty acid composit ion of the 1- and 2-positions of PC and PE derived from hepatoma, host liver, and normal liver of rats maintained on chow and fat free diets is given in Table II. The 1-position of all hepatic tissue PC e x h i b i t e d t h e same compos i t ion : 16:0, 4 1 + 2 . 8 % and 18:0, 56 .6+4%. Host and normal liver PE were similar at the 1-position and contained primarily palmitate and stearate. In sharp contrast, the 1-position of hepatoma PE, affected little by diet, contained only 25 to 35% as much palmitate as liver PE. The decrease in palmitate was offset by an increase in stearate percentages.

Polyunsaturated fat ty acids dominated the 2-position of PC and PE of livers but not the hepatoma. Generally, the 2-position of PC in host and normal liver from animals on the same diet was similar. This was also true in the case of PE. The exception to this generalization was the usually higher percentages of 18:2 and 22:6 observed in host livers. The fat free diet reduced 18:2 and 20:4 and increased 20:3, 18: 1, and 16:1 percentages at the 2-position of liver and hepatoma PC. Hepatoma PC exhibited elevated percentages of 16:0 and 18:1 at the 2- position compared to liver PC. The 2-position of all PE samples contained twice as much 18:0 and, with the exception of hepatoma, less than one-half the percentage of 18:1 as the corre- sponding position of PC. The fat free diet reduced 18:2 percentages at the 2-position of liver and hepatoma PE, but 20:4 and 18:1 percentages showed little change. Hepatoma PE contained elevated 18:1, and reduced 20:4 and 22:6 percentages relative to the 2-position of liver PE.

Fatty Acid Composition of PS, PI and DPG

The fatty acid compositions of PS, PI, and DPG isolated from hepatoma, host liver, and normal liver of rats maintained on chow and fat free diets are shown in Table III. DPG used for these composit ional analyses was purified by rechromatography in a less polar solvent system (12). All hepatic tissue PS was characterized by


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TABLE III

Fatty Acid Composition of Phosphoglycerides Derived from Rat Liver, Host Liver, and Hepatoma Tissue of Animals Maintained on Normal and Fat Free Diets

Fatty acid percentages a

Hepatic tissue 16:0 16:1 18:0 18:1 18:2 20:3 20:4 22:6

Liver, normal, chow diet 6.3 66.9 Liver, normal, fat free diet 6.4 61.9 Liver, hepatoma host, chow diet 5.4 45.8 Liver, hepatoma host, fat free diet 4.3 65.3 Hepatoma 7288CTC, chow diet 3.5 47.6 Hepatoma 7288CTC, fat free diet 4.5 T b 53.4

Liver, normal, chow diet 7.9 Liver, normal, fat free diet 4.7 Liver, hepatoma host, chow diet 5.7 Liver, hepatoma host, fat free diet 4.7 Hepatoma 7288CTC, chow diet 3.2 Hepatoma 7288CTC, fat free diet 4.3

Liver, normal, chow diet 3.9 2.1 Liver, normal, fat free diet 5.6 15.5 Liver, hepatoma host, chow diet 6.1 1.0 Liver, hepatoma host, fat free diet 7.2 10.8 Hepatoma 7288CTC, chow diet 16.0 1.2 Hepatoma 7288CTC, fat free diet 13.6 0.9

Phosphatidylserine

2.8 1.1 18.3 3.6 6.2 T 1.2 20.7 1.8 3.2 3.9 24.2 15.8 5.5 0.7 1.7 14.6 3.7

29.7 6.6 4.4 1.9 24.4 1.7 1.4 2.4 T

Phosphatidyllnositol

56.7 2.7 1.7 29.9 63.3 1.6 11.0 19.4 45.8 2.2 3.6 1.1 37.0 3.7 65.3 3.2 0.6 11.5 12,6 1.7 47,6 15.7 7.9 21.3 1.3 53.4 15.4 3.4 4.1 16.2 1.0

Dlphosphatidylglycerol

1.8 16.2 75.9 1.8 45.4 25.4 4.5 1.7 3.3 14.3 70.6 1.0 0.9 7.8 31.1 33.5 2.6 3.7 1.5

25.1 26.0 16.8 7.3 4.3 35.8 29.1 7.1 6.1 T

aThe difference between the sum of the percentages in any row and 100% represents the sum of other fatty acids present in small amounts and not given in the table. The phospbolipid classes from the hepatomas of animals on both diets contained 1.0-4.0% of each of the following acids: 20:1, 20:2, and 22:4. Normal liver and host liver did not contain more than trace levels of these acids.

bT = Detectable levels of < 0.5%.

a h igh level of s tearate . H e p a t o m a PS exh ib i t ed a severalfold increase in the pe rcen tage of 18:1 and a m u c h decrease pe rcen tage of 2 0 : 4 relat ive to liver. The effects of the fat free diet were marginal in b o t h h e p a t o m a and liver PS. Host liver PS c o n t a i n e d an increased percen tage of 22:6 .

B o t h h e p a t o m a an d liver PI, like PS, con- t a ined a high percen tage of 18:0. H e p a t o m a PI 18:1 was several fold h igher t h a n liver; bu t , un- like h e p a t o m a PS, the pe rcen tage of 2 0 : 4 was no t t oo d i f fe ren t f rom liver. The fat free diet

r educed 18:2 and 2 0 : 4 percen tages in l iver and h e p a t o m a PI and e levated the pe rcen tage of 20 :3 . Again, as in PS, the pe rcen tage of some p o l y u n s a t u r a t e d fa t ty acids increased in the hos t liver PI.

High leve]s of 18:2 charac te r i zed the DPG of hos t and n o r m a l liver, bu t no t the h e p a t o m a . The percen tage of 18:2 was r educed 55 to 65% in b o t h hos t and n o r m a l liver DPG of animals fed the fat free diet relat ive to chow fed animals. The 18 :2 was replaced wi th 16:1 and 18:1. Only 18:2 and 18:0 o f h e p a t o m a DPG r e s p o n d e d to the fat free diet . H e p a t o m a DPG c o n t a i n e d b e t w e e n 40 and 50% sa tu ra t ed f a t t y

acids, whereas n o r m a l liver DPG c o n t a i n e d 6-8% and hos t l iver 10-15%.

The fa t ty acid c o m p o s i t i o n of sph ingomye- l in f r o m h e p a t o m a and liver as a f fec ted b y diet as shown in Table IV. Genera l ly , the effects of the fat free diet on hepa t i c t issue sph ingomye- l i n f a t t y acid c o m p o s i t i o n were min imal . Palmit ic acid percen tages increased in hos t liver sph ingomye l in relat ive to n o r m a l liver. Ligno- ceric acid pe rcen tages were lower in hos t liver sph ingomye l ins t han in n o r m a l liver. The percentage of ne rvon ic acid in h e p a t o m a sphingo- myel in was a p p r o x i m a t e l y doub le t ha t of liver. t l e p a t o m a s p h i n g o m y e l i n c o n t a i n e d a C-24 d ienoic acid which was no t de tec tab le in liver sph ingomye l in .

Carbon Number Distributions of PC and PE Diglycerides

The c a r b o n n u m b e r d i s t r ibu t ions of diglycerides derived f rom PC and PE of h e p a t o m a , hos t liver, and n o r m a l l iver f rom animals fed c h o w and fat free diets are given in Table V. l - r a n d o m - 2 - r a n d o m d i s t r i bu t ion data ca lcula ted wi th f a t t y acid percen tages f rom Table II are also given in Table V for compar i son . Diglycer- ides f rom norma l liver PC of an imals fed the fat


H E P A T O M A AND L I V E R PHOSPHOLIPIDS

free diet contained a higher percentage of lower tool wt species than liver PC of chow fed ani- mats. Host liver PC of both chow and fat free diet fed rats contained a higher percentage of higher tool wt species than either normal liver PC or hepatoma PC. The carbon number distribution of hepatoma PC, unaffected b y diet, agreed more closely with normal liver PC percentages than with host liver PC percentages. Determined and calculated carbon number percentages of liver PC did not agree; however, a rather close agreement was obse rved for hepatoma PC.

The carbon number distribution of diglycerides derived from PE was dramatically different from PC diglycerides of both liver and hepatoma. In contrast to liver PC, the fat free diet caused an elevation in the higher mol wt species of both normal and host liver PE relative to chow fed noraml and host liver PE. Hepatoma PE, unaffected by diet, exhibited a carbon number distribution very different from liver PE. As observed for liver PC, the determined and calculated carbon number distributions for liver PE were not comparable. In contrast, hepatoma PE showed agreement between determined and calculated carbon number percentages.

DISCUSSION

Class Composition

Phospholipid class percentages were affected little by feeding the fat free diet or by the presence of the hepatoma. This is in general agreement with previously reported data (12, 16-18). The combination of hepatoma and fat free diet reduced host liver phospholipid mass dramatically (5), and not all classes were affected equally. Both percentage and mass data showed an elevation of hepatoma sphingomyelin. Bergelson, et al., (17) have shown that hepatoma 27, hepatoma 22, zajdela ascites hepatoma, and cellular fractions from these neoplasms contained higher percentages of sphingomyelin than liver. Transformed diploid cells grown in culture (19), cultured hepatoma cells (10), and a number of transplantable rat and mouse tumors (20) also have been shown to contain higher percentages of sphingomyelin than nontransformed cells and normal liver. Decreases in the other hepatoma phospholipid classes relative to normal liver may be more significant than the elevated concentrations of sphingomyelin. A number of different neoplasms examined show lower levels of phospholipids than do normal tissues, whether ex- pressed on the basis of wet wt, protein, or total nitrogen (18,19,21). This decreased concentra-

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HEPATOMA AND LIVER PHOSPHOLIPIDS 743

tion of phospholipids may be a characteristic of most neoplasms that has not been recognized because most data have been reported as relative percentages.

Phosphatidylcholine

Positional analysis of liver PC, the most studied phospholipid of hepatic tissue, confirms earlier reports (22-24) that indicated the 1-position, composed primarily of palmitate and stearate, was affected only slightly by diet. Likewise, the 1-position was not affected in hepatoma. The similarities in the composit ion of 1-position of PC in hepatoma and fiver (Table II) have been reported in hepatoma 5123C (25), whereas other hepatomas (25,26) have elevated 18:1 percentages. The 2-position fat ty acid percentages of normal and host liver PC from animals on both diets generally agreed well with reported percentages obtained from animals fed regular diets (22,25-28) and essen- tial fa t ty acid deficient diets (22). The higher percentage of 16:0 and lower percentage of C-20 and C-22 polyunsaturated fat ty acids at the 2-position of hepatoma PC relative to liver are in agreement with published data for other hepatomas (25,26) and Ehrlich ascites ceils (14).

The similarities in the fat ty acid composit ion at the 1- and 2-positions of normal and host liver PC of chow fed animals (Table II) might lead one to conclude that the hepatoma had no effect on host liver PC species, but comparison of determined diglyceride carbon number percentages (Table V) indicated that pairing of fat ty acids yielding higher tool wt species had occurred. Higher tool wt species of PC in the host liver of the fat free fed animals was also observed. The lack of agreement between determined and calculated carbon number percentages for normal and host liver PC diglycerides illustrates, as shown previously (28), the prefer- ential pairing of fat ty acids. In contrast, the agreement between determined and calculated percentages of hepatoma PC diglycerides indicates lack of specificity in fat ty acid pairing. The diglycerides derived from PC of Ehrlich ascites ceUs examined previously (14) also exhibited a 1-random-2-random distribution.

Phosphatidylet hanola mine

The 1-position of fiver PE was affected to a small degree by both diet and the tumor. 1-position percentages of liver PE from rats bearing Yoshida and Morris 5123 hepatomas (29) agreed well with the chow fed host liver PE percentages. Percentages previously reported for the 1-position of normal liver PE (28) were similar to the fat free f e d - h o s t fiver PE per-

centages. Although the 1-position percentages for normal liver PE reported previously and the present percentages differ in the ratio of palmi- rate to stearate, it is of interest that in each case the 1-position compositions for PE and PC are similar. This similarity suggests that the fat ty acids at the 1-positions of PC and PE arise from the same source. In contrast to the 1-position percentages, which were similar for liver and hepatoma PC, hepatoma PE contained only one-third to one-fourth the palmitate of liver PE. The low levels of pa lmi ta te at the 1-posi- t ion of hepatoma PE (Table II) and hepatoma tfiglyceride (30) suggest that the 1-position fatty acids of PE and TG arise from the same source, whereas the 1-position fat ty acids of normal rat liver PC and PE appear to arise from the same source. Ehrlich ascites cells (14), Yoshida hepatoma (29), and Morris 5123 hepatoma (29) all contained lower percentages of palmitate at the 1-position of PE than fiver PE.

The level of C-20 and C-22 polyunsaturated fat ty acids at the 2-position of hepatoma PE was much reduced in comparison to liver. This decreased level was offset by a rise in the percentage of 18:1, following the trend of two hepatomas (29) and Ehrlich ascites cells (14).

The molecular species of liver PE were affected differently than liver PC; normal liver PE from the animals fed the fat free diet contained an elevated percentage of higher mol wt species, and the hepatoma did not affect the host fiver PE carbon number distribution. It is of interest that both hepatoma PC and PE diglycerides showed only very small peaks on the chromatograms that could have resulted from odd carbon numbered diglycerides or ether linked diglycerides. These data demonstrate that the high level of ether l inked phospholipids found in some neoplasms (31) is not characteristic of all neoplasms.

Phosphatidylserine, Phosphatidylinositol, and Diphosphatidylglycerol

Liver and hepatoma PS and PI were characterized by a high percentage of stearate, as reported previously for rat liver (12) and Ehr- fich ascites cells (32). The fat free diet had only marginal effects on the percentage of 20:4 in liver PS, whereas the percentage of 20:4 in liver PI was reduced. The reduced percentage of 20:4 in PI appeared to be replaced with 20:3. If the 20:3 acid was esterified at the 2-position of PI as was the case for PC, then PI contained the highest quant i ty of 20:3 per mole, followed by PC and DPG. The percentage of 20:4 in hepatoma PS was reduced dramatically, whereas the percentage of this acid in hepatoma PI was


744

within the range of liver values. Both PS and PI of Ehrlich ascites cells exhibited similar types of fatty acid profiles (30). The high percentage of 18:2, characteristic of liver DPG (12), was partially replaced with 16:1 and 18:1 fatty acids in liver of animals fed fat free diet. The percentage of 18:2 in hepatoma DPG, lower than liver DPG from animals fed the fat f ree diet, was not replaced with monoenoic acids but with stearate and palmitate.

Sphingomyelin

The fatty acid profile of liver sphingomyelin, characterized by a high percentage of C-22, C-23, and C-24 saturated and monoenoic fatty acids (32), was not greatly affected by the fat free diet. The lower percentages of 18:0, 20:0, 22:0, 23:0, and 23:1 in hepatoma sphingomyelin were replaced with a higher percentage 24:1 (Table IV). Actually, the percentages do not reflect the real pictures because of various tissue levels of sphingomyelin. Concentrations (mg/g wet wt) calculated from the data in Table IV show that 24:0 and 24:1 hepatoma sphingomyelin species greatly exceed liver concentrations. Hepatoma sphingomyelin also contained a C-24 dienoic acid shown previously to be present in Ehrlich ascites cells (32), cultured hepatoma cells (10), and a large number of transplantable rat and mouse tumors (20), but absent or present in only trace amounts of sphingomyelin of normal tissue (12,20,32).

Effect of Hapatoma on Host Lipids

Some phospholipid classes of host liver showed an increase in the percentage of 18:2 and 20:4 over normal liver percentages, whereas all host liver phospholipid classes, except sphingomyelin, showed an increase in the percentage of 22:6. The increased percentage of 22:6 would only be apparent if the phospholipid species containing polyunsaturated fatty acids were being conserved at the expense of other class species. However, in a number of instances the increased percentages could have only occurred due to a net increase in the concentration of host liver 22:6. Neifakh and Lankin (33) have reported that the concentrations of 18:1, 18:2, and 20:4 were higher in total lipids derived from rats bearing Walker carcinoma and hepatoma 22 at 14 and 11 days, respectively, than normal liver. They (33) apparently did not observe or measure the concentration of host liver 22:6, which showed the most pronounced increases in the present study.

The increased percentage of carbon number 38 of diglycerides derived from host liver PC (Table V) indicates an apparent effect of the

RANDALL WOOD

tumor on the distribution of molecular species. It would appear that the tumor can affect phospholipid biosynthesis or can be instrumental in affecting the turnover of some molecular species in the host liver.

Phosphoglyceride Molecular Species

If one accepts the concept that specific molecular species of phosphoglycerides are vital cellular components and serve specific functions, then it is not surprising that liver and hepatoma behave differently. Not only does the hepatoma cell have to function with only one- fourth to one-half the amount of PC, PE, PS, and DPG of normal liver cells, but it has to manage with different molecular species of phosphoglycerides than found in liver. The C-20 and C-22 polyunsaturated fatty acids of the aforementioned classes are dramatically reduced, which means the quantity of molecular species containing these fatty acids is further reduced. In addition, the hepatoma has to con- tend with a 1-random-2-random distribution of fatty acids in PC and PE that further reduces the quantity of some molecular species. All of these abnormalities in the lipids of the hepatoma make it clear that cellular components and functions which require specific molecular species containing polyunsaturated fatty acids will be severely affected. Because of the abnormalities in the lipid constituents of neo- plastic ceils, it is not surprising that they function differently than normal cells.

ACKNOWLEDGMENTS

This work was supported by Public Health Service Research Grant No. CA 12973 from the National Cancer Institute. J. Falch provided technical assist- ance.

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3. Gammal, E.B., ILK. Carroll, and E.R. Plunkett, Ibid. 27:1737 (1967).

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Physiol. 37:911 (1957). 9. BergstriJm, B., Aeta Physiol. Scand. 25:101

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1:85 (1966). 12. Wood, R., and R.D. Harlow, Arch, Biochem.

Biophys. 135:272 (1969).


HEPATOMA AND LIVER PHOSPHOLIPIDS 745

13. Wood, R., and J. Falch, Lipids 9:979 (1974). 14. Wood, R., and F. Snyder, Arch. Biochem. Bio-

phys. 131:478 (1969). 15. F r i t z , J.S., and G.H. Schenk, Anal. Chem,

31:1808 (1959). 16. Rouser, G., G.J. Nelson, S. Fleischer, and G.

Simon in "Biological Membranes ," Edited by D. Chapman, Academic Press, New York, NY, 1968, p. 5.

17. Bergelson, L.D., E.V. Dyatlovitskaya, T.I. Tork- hovskaya , I.B. Sorokina, and N.P. Gorkova, Biochim. Biophys. Acta 210:287 (1970).

18. Ruggieri, S., and A. Fallani, Lo Sperinaentale 118:483 (1968).

19. Howard, B.V., J.D. Butler, and J.M. Bailey, in "Tumor Lipids: Biochemistry and Metabolism," Edited by Randall Wood, AOCS, Champaign, IL, 1973, p. 200.

20. Wood, R., Lipids 9:830 (1974). 21. White, H.B., Jr., in " T u m o r Lipids: Biochendstry

and Metabolism," Edited by Randall Wood, AOCS, Champaign, IL, 1973, p. 75.

22. Van Golde, L.M.G., and L.L.M. Van Deenen, Biochim. Biophys. Acta 125:496 (1966).

23. Van Golde, L.M.G., W.A. Pieterson, and L.L.M.

Van Deenen, Ibid. 152:84 (1968). 24. Kramer, J.ICG., Lipids 8:641 (1973). 25. Ruggieri, S., and A. Fallani, in " T u m o r Lipids:

Biochemistry and Metabolism," Edited by Ran- dall Wood, AOCS, Champaign, IL, 1973, p. 89.

26. Bergelson, L.D., and E.V. Dyatlovitskaya, Ibid. p. 111.

27. Kuksis, A., L. Marai, W.C. Breckenridge, D.A. Gornall, and O. Stachnyk, Can. J. Physiol. Phar- mar 46:511, 1968.

28. Wood, R., and R.D. Harlow, Arch. Biochem. Biophys. 131:495 (1969).

29. Ruggieri, S., and A. Fallani, Lo Sperimentale 118:503 (1968).

30. Wood, R. Lipids 10:404 (1975). 31. Snyder, F., and R. Wood, Cancer Res. 28:972

(1968). 32. Wood, R., and R.D. Harlow, Arch. Biochem.

Biophys. 141:183 (1970). 33. N e i f a k h , Y.A., and V.Z. Lankin, Biofizika

12:1085 (1967).

[Received April 11, 1975]

A Guide for Authors is Located in Lipids 10(January):60 (1975)


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Hepatoma, host liver, and normal rat liver phospholipids as affected by diet