lipids of cultured hepatoma cells: ii. effect of media lipids on cellular phospholipids

Lipids of Cultured Hepatoma Cells: II. Effect of Media Lipids on Cellular Phospholipids RANDALL WOOD and JOHN FALCH, Division of Gastroenterology, Departments of Medicine and Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65201

ABSTRACT

Minimal dev ia t ion h e p a t o m a cells we re c u l t u r e d in a m o d i f i e d S w i m ' s 77 medium s u p p l e m e n t e d w i t h dec rea s ing a m o u n t s o f s e r u m , l ipid-free s e r u m , and l ipid-free s e r u m c o n t a i n i n g added pa lmi t i c or l inoleic acids. Cellular p h o s p h o l i p i d s were e x t r a c t e d and the class d i s t r i b u t i o n deter- m i n e d quan t i t a t i ve ly . The f a t t y acid c o m - p o s i t i o n o f each p h o s p h o l i p i d class was determined, and the p e r c e n t a g e s f r o m cells g r o w n o n each o f the va r ious m e d i a were c o m p a r e d . Cellular p h o s p h o l i p i d class and f a t t y acid c o m p o s i t i o n s d i f fe red f r o m media compositions, i nd ica t ing t h a t i n t ac t s e r u m p h o s p h o l i p i d s are n o t incor - p o r a t e d i n t o cel lular s t r u c t u r e s . P h o s p h a - t i dy l cho t ine p e r c e n t a g e s dec reased as the m e d i a s e r u m and l ipid levels decreased , whi le p h o s p h a t i d y l i n o s i t o l and p h o s p h a -

t i d y l e t h a n o l a m i n e p e r c e n t a g e s increased . S p h i n g o m y e l i n o f cells g r o w n in m e d i u m c o n t a i n i n g added l inoleic acids c o n t a i n e d a h igh level o f a 24 :2 acid. All classes, e x c e p t s p h i n g o m y e l i n , c o n t a i n e d e levated levels o f !8 :1 acid and decreased levels o f p o l y u n s a t u r a t e d f a t t y acids, relat ive to n o r m a l ra t liver. Cells c u l t u r e d o n lipid- free m e d i u m did n o t c o n t a i n inc reased c o n c e n t r a t i o n s o f 20 :3 acid, sugges t ing t h a t th is h e p a t o m a cell c a n n o t d e s a t u r a t e m o n o e n o i c acids. P h o s p h o g l y c e r i d e s o f cells, g r o w n on l ipid-free m e d i u m , had the h ighes t m o n o e n e f a t t y acid c once n - t r a t ion , w h e r e a s t h o s e cells g r o w n on m e d i a c o n t a i n i n g added l inoleic acid had the l o w e s t c o n c e n t r a t i o n s , sugges t ing t h a t l ino lea te m a y i nh ib i t or regu la te m o n o - eno i c acid b i o s y n t h e s i s in th is cell. These mass da ta also d e m o n s t r a t e t h a t m o n o -

TABLE I

Phospholipid Percentages of Minimal Deviation Hepatoma Cells Grown in Culture on Media Containing Varying Amounts of Lipid a

Percentages b Basal medium supplement Orig. Lyso-PC SPH PC PI PS X 1 PE X 2 SF c

20% Bovine serum + 5% fetal calf serum 0.6 1.7 8.8 41.4 8.4 6.6 -- 26.4 2.0 4.4



5% Fetal calf serum 0.5 1.2 7.4 32.8 11.8 ...,_~ 6.1 -- 33.8 2.2 4.2 5% Bovine serum 0.8 1.4 7.5 28.8 19.3 - - 35.9 1.7 4.6 2.5% Fetal calf serum 0.6 2.3 7.4 29.1 12.8 5.7 1.6 33.9 1.8 4.8 5% Lipid-free fetal

calf serum 1.0 3.0 7.9 27.1 11.6 6.0 1.8 34.8 1.8 5.0 5% Lipid-free fetal calf

serum + palmitic acid d 6.9 3.4 7.6 27.9 14.3 1.7 -- 30.3 3.4 4.5 5% Lipid-free fetal calf

serum + linoleic acid d 1.1 3.6 12.0 26.1 14.2 5.1 - - 28.3 3.8 5.9 Fetal calf serum 1.2 21.7 25.1 50.2 . . . . . . . 2.0 Bovine serum 0.7 10.8 18.3 67.0 . . . . . . 0.9

aorig. = origin, Lyso-PC = lyso-phosphatidylcholine, SPH = sphingomyelin, PC = phosphatidylcholine, PI = phosphatidylinositol, PS = phosphatidylserine, X 1 = unidentified phosphorous containing compound migrating between phosphatidylserine and phosphatidylethanotamine, PE = phosphatidylethanolamine, X 2 = unidentified phosphorous containing compound migrating between phosphatidylethanolamine and the solvent front, and SF = solvent front.

bpercentages represent the mean of duplicate phosphorous analyses, and values have not been corrected for differences in tool vet of lipid classes.

CSolvent front material consists primarily of diphosphatidylglycerol but also contains minor amounts of other phosphorous and nonphosphorous containing compounds.

d37.5 #g/ml of medium.

702

HEPATOMA PHOSPHOLIPIDS 703

enoic fatty acid biosynthesis in this cultured hepatoma cell responds to dietary changes.

INTRODUCTION

The importance of lipids in many biological processes and the advantages of using minimal deviation hepatoma cells (HTC), grown in tissue culture, to study the structure and metabolism of lipids to determine their possible direct or indirect involvement in neoplasia have been discussed previously (1). To the authors' knowl- edge, the phospholipids of HTC cells grown in tissue culture have not been studied in any detail. Results from this laboratory (1) have shown that phospholipids of HTC cells, cultured in media containing various amounts of serum and lipids, showed little qualitative dif- ference in class distributions and the same approximate total phospholipid fatty acid compositions.

The quantitative determination of phospholipid class compositions and fatty acid compositions of individual phosphoglycerides derived from HTC cells, grown on media containing varying levels of serum and lipids, is reported in this paper.

MATERIALS AND METHODS

Minimal deviation hepatoma 7288C cells were grown in roller cultures on a modified Swim's 77 medium supplemented with various levels of serums and lipids (see Table I for supplements), as described previously (1). Cells were harvested, lyophilized, extracted, and the lipids fractionated into neutral lipids and phospholipids as described earlier (1). Phospholipid classes were resolved by thin layer chromatography (TLC) on adsorbent layers of Silica Gel HR, developed in a solvent system of chloro- f o r m - m e t h a n o l - a c e t i c acid-0.9% saline, 50:25:8:4, v/v. Quantitation of individual classes resolved by TLC was determined by phosphorous analysis, according to the proce- dure of Rouser et al. (2). Chromatoplates were sprayed with sulfuric acid and charred for visualization and documentation by photog- raphy. Class identification was based upon the use of phosphorous and ninhydrin spray rea- gents (3), gas liquid chromatography (GLC) of methyl esters, and cochromatography with standards. Methyl esters of the phospholipids were prepared by acid catalyzed esterification and analyzed by GLC, as previously described (4). Identification of methyl esters is based upon relative retention times before and after hydrogenation and coehromatography with commercially available esters of known s truc-

ture. Except for 24:0 and 24:1 acids, acids with retention times longer than 20:4 must be considered as tentatively identified, since there are a large number of esters with similar retention times possible in this area of the chromatogram. The use of classical names for unsaturated fatty esters does not imply that the double bond positions and configurations have been determined.

RESULTS

Phospholipid Distribution

The percentage distribution of the phospholipids, derived from HTC cells cultured on various media and from serums, is given in Table I. Serums, the only media component containing lipids, were characterized by high levels of lyso-phosphatidylcholine, sphingomyelin, and phosphatidylcholine, which differed dramatically from the cellular level of these phospholipids. Cellular phosphatidylcholine levels decreased significantly as the level of serum decreased to ca. 5%. The decrease of phosphatidylcholine was offset by increases in the concentrations of phosphatidylinositol and p ho sp ha t idylethanolamine. Sphingomyelin, phosphatidylserine, and other minor compo- nents showed little if any change as the lipid level of the media changed. Except for an increased level of sphingomyelin, when linoleic acid plus lipid-free fetal calf serum supplemented the medium, the addition of fatty acids to the medium had little effect upon the phospholipid composition relative to that of the ceils grown in lipid-free medium.

Phosphatidylchol ine

This lipid class, one of the major compo- nents of cells and serums, showed a percentage change in some of the fatty acids present in this phospholipid, as tile serum and lipid levels of the media changed (Table I1). The fatty acid compositions of cellular and serum phospha- tidylcholines were different. Except where fatty acids were added to the media, palmitic and stearic acid percentages remained relatively constant, whereas 18:1 and 20:1 acids increased and 18:2 and 20:4 acids decreased with a decrease in media serum and l ipid levels. Table II shows that linoleic acid, added to medium containing lipid-free fetal calf serum, increased cellular 18:2 and 20:4 acid levels, whereas added palmitic acid did not elevate phosphatidylcholine palmitate concentrations.

Phosphatidylethanolamine

The fatty acid compositions of phospha-

LIPIDS, VOL. 8, NO. 12

704 R. WOOD AND J. FALCH

TABLE 11

Comparison of Fatty Acid Composition of Phosphatidylcholine Derived from Minimal Deviation Hepatoma Cells Cultured in Media Containing Varying Amounts of Serum and Lipid

Basal medium Fatty acid percentagesa, b

supplement 16:0 16:1 18:0 18:1 18:2 20:1 20:3 20:4 22:2

20% Bovine serum + 5% fetal calf serum 27.8 4.4 9.7 30.8 9.0 1.6 1.5 12.2 1.2



5% Fetal calf serum 28.0 7.3 9.4 44.5 3.7 2.4 T c 3.2 0.6 5% Bovine serum 20.6 6.8 8.1 39.6 9.6 2.6 1.2 7.2 0.9 2.5% Fetal calf serum 23.1 8.5 6.5 46.8 5.4 2.8 0.9 3.8 1.1 5% Lipid-free fetal calf sernm 20.5 8,6 8.3 52.8 3.5 3.4 T 1.7 T

5% Lipid-free fetal calf serum + palmitic acid d 25.0 S.0 12.4 41.8 6.7 1.8 0.6 5.0 1.2

5% Lipid-free fetal calf serum + linoleic acidd, e 28.3 2.8 14.8 11.4 26.5 T T 10.6 T

Bovine serum f 15.3 0,9 26.6 19.2 14.7 --- 5.9 7.1 -- Fetal calf serumg 22.2 1.1 23.9 26.4 1.2 -- 2,8 7.8 --

aDifference between sum of fatty acid percentages in each row and nents not given in the table.

bpercentages represent the mean of duplicate analyses. CT = detectable levels but less than one half per cent. d37.5 ~zg/ml of medium. eContained 3.6% C-20:2 fatty acid. fSample also contained 2.9 and 0.5% of 22:5 and 22:6 respectively. gSample also contained 6.6 and 4,9% of 22:5 and 22:6 respectively.

100% represents sum of minor compo-

t i d y l e t h a n o l a m i n e , a ma jo r cel lular p h o s p h o - lipid bu t ab sen t f r o m the se rums , are given in Table I l l . Th is p h o spho l i p id class c o n t a i n e d m o r e stearic and less pa lmi t ic acids t han phospha t idy lcho l ine ; but , as wi th p h o s p h a t i d y l c h o - line, the c o n c e n t r a t i o n of b o t h of these acids r ema ined relat ively c o n s t a n t as s e rum lipid levels decreased. The pe rcen tages of t8 :1 increased and 20 :4 decreased as s e rum and lipid levels o f the m ed i a decreased. The pe rcen t ages of acids in p h o s p h a t i d y l e t h a n o l a m i n e wi th re- t en t i on t imes longer t h a n 2 0 : 4 were too small to a t t a c h any s ignif icance to the observed changes in these acids. The add i t ion of l inoleic acid to the m e d i u m resu l ted in increased levels of 18:0, 18:2, and 20 :4 acids, a c c o m p a n i e d by a decrease in 18:1 levels, whereas the add i t ion of pa lmi t ic acid to the m e d i u m caused on ly a rise in s tear ic acid levels bu t n o t palmit ic .

Phosphatidylinositol and Pbosphatidylserine

The f a t t y acid c o m p o s i t i o n of p h o s p h a t i d y l - inosi to l , p h o s p h a t i d y l s e r i n e , and, in some cases, of these two classes c o m b i n e d tha t was derived f r o m HTC cells grown on var ious med i a is s h o w n in Table IV. These cellular p h o s p h a t i d e s , absen t f r o m the se rums , were charac te r i zed by the h ighes t levels of stearic acid of all the

phospha t i de s . P h o s p h a t i d y l i n o s i t o l c o n t a i n e d h igher pe rcen tages o f 18:0 and 20 :4 and lower pe rcen tages o f 18:1 t h a n phospha t i dy l s e r i ne . Decreased s e r u m and lipids in the med ia resu l ted in increased levels of 18:1 and decreased levels of 20 :4 for b o t h p h o s p h a t i d e classes. Phospha t idy l se r ine c o n t a i n e d h igher pe rcen t - ages o f acids wi th r e t e n t i o n t imes longer than 20 :4 than did p h o s p h a t i d y l i n o s i t o l , bu t the values were too low to al low m u c h s ignif icance to be a t t a c he d to th is observa t ion . The level of 18:2 in these two lipid classes was general ly low; however , w h e n l inoleic acid was added to the m e d i u m , 18:2 and 20 :4 va lues increased at the expense of 18:1. F a t t y acids added to the med i a did n o t alter pa lmi t a t e and s teara te levels of these c o m b i n e d p h o s p h a t i d e f ract ions .

Diphosphatidylglycerol

When the analy t ica l TLCs were developed for p h o s p h o r o u s analys is (Table I), d iphospha - t idylglycerol was n o t resolved su f f i c ien t ly f r om the so lven t f ron t to pe rmi t separa te quant i f i - ca t ion; bu t , w h e n prepara t ive TLC was used to isolate indiv idual p h o s p h o l i p i d classes, a rea- sonab ly pure d i p h o s p h a t i d y l g l y c e r o l f rac t ion was ob ta ined in m o s t cases. The f a t ty acid c o m p o s i t i o n of the d iphospha t i dy lg lyce ro l frac-



TABLE III

Comparison of Fatty Acid Composition of Phosphatidylethanolamine Derived from Minimal Deviation Hepatoma Cells Cultured in Media Containing Varying Amounts of Serum and Lipid

Basal medium Fatty acid percentagesa, b supplement 16:0 16:1 18:0 18:1 18:2 20:1 20:3 20:4 22:2 22:5 22:6

20% Bovine serum + 5% fetal calf serum 5.2 2.1 17.6 39.1 4.5 1.9 1.4 20.1 0.9 2.8 2.6

10% Bovine serum + 5% fetal calf serum 7.0 2.3 18.6 42.7 7.7 1.7 T c 14.5 0.7 1.6 1.5

5% Bovine serum + 5% fetal calf serum 7.5 2.9 18.2 44.2 %3 1.5 1.6 12.4 1.3 1.2 1.6

5% Fetal calf serum 7.2 3.5 19.1 49.2 3.4 1.9 0.8 9.6 0.8 0.8 1.9 5% Bovine serum 5.4 2.7 17.4 45.9 8.3 2.9 0.9 10.4 0.7 2.1 0.8 2.5% Fetal catf serum 5.6 3.4 14.0 52.4 5.2 2.8 l , l 10.6 1.4 0.9 2.2 5% Lipid-free fetal calf serum 5.1 4.3 15.3 56.9 3.9 2.9 0.8 6.3 0.5 2.1 T

5% Lipid-free fetal calf serum + palmitic acid d 5.5 2.5 22,3 47.8 4.9 1.8 0.8 9.0 1.1 1.3 1.8

5% Lipid-free fetal calf serum + linoleic acidd, e 6.6 1.0 28.5 14.6 21.6 0.5 1.2 15.8 T 2.0 1.2

aDifference between sum of fatty acid percentages in each row and 100% represents the sum of minor com- ponents not given in the table.

bpercentages represent the mean of duplicate analyses. CT = detectable levels but less than one half per cent. dContained 5.2% C-20:2 fatty acid. e37.5/~g/ml of medium.

tion, ob ta ined f rom cells grown on various media, is given in Table V. This class was character ized by low levels o f s tearate and palmita te and higher concen t r a t ions of 14:0, 16: 1, 18: 1, and 18:2 acids than found in o the r phosphat ides . The concen t r a t ion of m o n o e n o i c acids increased, while 18:2 levels decreased, as media serum lipid levels decreased. Palmitic acid added to m ed ium conta in ing lipid-free fetal calf serum had li t t le inf luence on the compos i t i on of d iphosphat idy lg lycero l , whereas added l inoleic acid greatly elevated 18:2 percentages, while 18:1 levels decreased.

Sphingomyelin

The fa t ty acid compos i t i ons of sphingomyelin, derived f rom HTC cells grown on various media and f rom bovine and fetal calf serums, are shown in Table VI. Bovine serum sphingomyel in con ta ined a high percentage of 16:0 and 23:0 acids, whereas fetal calf serum sphingomyel in con ta ined a high percentage of 22:0 and 24:1 acids. The compos i t i ons of the serums di f fered f rom the sph ingomyel in com- posi t ion of the cells. Al though some dif ferences in sph ingomyel in fa t ty acid compos i t i on were observed, they did n o t appear to be related to type of serum or a m o u n t of lipid in the medium. Palmit ic acid, added to med ium containing l ipid-free fetal calf serum, did no t elevate sph ingomyel in palmi ta te but caused an increase in 18:0 and 24 :0 percentages. Cells grown on

med ium conta in ing added linoleic acid did no t conta in significant levels of 18:2 in the sphingomyel in acids bu t con ta ined a high level of 24:2 acid.

Unidentified Material

A c o m p o u n d of the cellular phosphol ip ids , absent f rom the serums, having an Rf value be tween phospha t i dy l e thano l amine and diphosphat idylg lycerol and represen t ing ca. 2% of the phosphol ip id p h o s p h o r o u s was analyzed for fa t ty acids. The fa t ty acid compos i t i on of this c o m p o u n d differed f rom the o the r phospholipid classes. The major acids were 16:0, 18:0, and 18:1. The m o n o e n o i c acid peaks were broadened , indicat ing the presence of more than one c o m p o n e n t , bu t the small sample size p revented fu r ther character iza t ion . Variabil i ty of the major fa t ty acid percentage seemed to be unre la ted to se rum and l ipid levels, excep t when l inoleic acid was added to the medium.

Under preparat ive TLC condi t ions , material migrating near the solvent f ron t usually was resolved f rom diphosphat idylg lycero l . The fa t ty acid compos i t i on of the solvent f ron t material , conta in ing a high level o f 24:0 and 24:1 acids, suggests tha t ceramide was a major c o m p o n e n t . However, e levated levels o f 18: 1, relative to the percentage found in sphingomyel in , indica ted the presence of o the r c o m p o n e n t s . Excep t when l inoleic acid was added to the growth med ium, ident i f iable changes in the fa t ty acid


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HEPATOMA PHOSPHOLIP1DS

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DISCUSSION

The class compositional data of the phospholipids, derived from HTC cells grown on various media, demonstrate that the percentages of some phosphoglycerides can be affected by media serum and lipid levels, while others are unaffected. The change is not restricted to the lipids found in media since phosphatidylethanolamine and phosphatidylinositol, absent .~"z from bovine and fetal calf serum, were two of ;5 three classes showing the most change. These data indicate that care must be exercised when comparing phospholipid data, even from the ~ .~ same cell line, with that obtained in other q . ~ ' )

laboratories, especially when the growth condi- ~ tions are not known. .~

The phospholipid class compositions of the ~ HTC cells grown on various media did not agree "~ with compositions reported for rat liver (5-7); ~'~ however, the composition of the cells cultured _~'7, on 25% serum more nearly agreed. Even when . ~ the media contained high levels of serum, ~ phosphatidylcholine, the major serum phospho- ~.~ lipid, was lower in HTC cells than rat liver; and ~ phosphatidylethanolamine, phosphatidylserine, > ~ and phosphatidylinositol, absent from serum, ~ were elevated above rat liver percentages. Ber- ~, ~ gelson et al. (5) have shown that host grown ~ < ~ hepatoma 27 contained elevated levels of phos- ~.~ phatidylserine, phosphatidylethanolamine, and ~ sphingomyelin and a lower level of phospha- ~ - tidylcholine, relative to rat liver values. Morris .. ~) hepatoma 3924A, a fast growing hepatoma, ~-~ was shown to contain decreased levels of =c) phosphatidylcholine and higher amounts of ~

o phosphatidylinositol (8). The same investigators ~ also reported that the level of phosphatidyl- = choline in the Ruber H-35 hepatoma was lower .~ 7: than rat liver levels; however, the latter analysis ~ o was complicated by a high percentage of a polar ~ .~ phosphorous containing compound. These corn- ~ parisons indicate that cultured HTC cells have -~ an altered phospholipid composition, relative to fi rat liver, similar in some respects to solid hepatomas grown in host animals.

Each phospholipid class from HTC cells, like that of liver, exhibited a characteristic fatty acid composition that changed with changes in media serum and lipid levels. Except for sphingomyelin, phosphatidylcholine had the highest level of palmitate. Phosphatidylcholine of the cultured cells contained reduced levels of 18: 0, 20:4, and 22:6 acids and a much higher concentration of 18:1, relative to values reported for rat liver lecithin (9-12). Increased concentrations of oleic acid in phosphatidyl-

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708 R. WOOD AND J. FALCH

TABLE VI

Comparison of Fatty Acid Composition of Sphingomyelin Derived from Minimal Deviation Hepatoma Cells Cultured in Media Containing Varying Amounts of Serum and Lipid

Basal medium Fatty acid percentagesa, b supplement 16:0 18:0 18:1 22:0 22:1 23;0 24:0 24:1 24:2

20% Bovine serum + 5% fetal calf serum 30.8 6.1 1.1 9.4 1.4 4.3 23.6 20.2 T c

10% Bovine serum + 5% fetal calf serum 27.0 6.3 2.2 8.9 0.5 2.7 22.9 27.6 T

5% Bovine serum + 5% fetal calf serum 39.2 5.9 1.3 7.2 1.6 1.5 18.3 23.2 ---

5% Fetal calf serum 35.3 7.1 1.7 6.9 0.9 1.0 24.4 21.6 -- 5% Bovine serum 28.4 4.7 3.3 5.6 2.6 2.2 13.7 34.3 -- 2.5% Fetal calf serum 33.0 4.5 2.8 5.9 2.8 t.0 17.0 31.5 - - -

5% Lipid-free fetal calf serum 30.7 5.6 3.6 5.8 3.7 0.6 17.4 30.5 --

5% Lipid-free fetal calf serum + palmitic acid d 28.7 11.2 4.3 7.3 T -- 26.0 21.6 --

5% Lipid-free fetal calf serum + linoleic acid d 30.3 7.2 1.2 4.7 . . . . . 10.2 25.7 23.0

Bovine serum 47.3 11.8 1.3 6.4 -- 12.8 9.6 7.5 0.5 Fetal calf serum e 29.1 10.6 1.2 15.9 1.5 2.4 9.8 23.8 1.2

aDifference between sum of fatty acid percentages in ponents not given in the table.

bpercentages represent the mean of duplicate analyses. CT = detectable levels but less than one half per cent. d37.5 ~tg/ml of medium. eSample contained 2.6% of 20:0.

each row and 100% represents the sum of minor corn-

chol ine f rom hepa tomas and h e p a t o m a cell f ract ions have been r epo r t ed previously by Ruggieri and Fallani (13), Bergelson and Dyat lovi tskaya (14), and van Hoeven and E m m e l o t (15).

Rat liver phospha t i dy l e thano l amine usually conta ins 40-50% satura ted acids (9,11,12) , bu t the sa tura ted acids of the HTC cells, part icu- larly palmit ic acid, were r educed greatly. This class had the highest levels o f 20:4 and 22:6, but the values were m u c h lower than percent - ages of these acids in rat liver phospha t idy le th - anolamine (9,11,12) . The decreased levels of po lyunsa tu ra ted acids and palmit ic acid were replaced wi th oleic acid. van Hoeven and E m m e l o t (15) have shown tha t phospha t idy l - e thano lamine derived f rom h e p a t o m a 484A plasma m e m b r a n e s had a m u c h lower stearic/ oleic rat io than normal membranes , but they did n o t see a decreased rat io in two mouse hepatomas , no r did Ruggieri and Fallani (16) observe a significant increase in oleic acid of hepa toma 5123 phospha t idy le thano lamine .

Phosphat idylser ine and phospha t idy l inos i to l were charac ter ized by a high level of stearic acid, which is typical of rat liver (11,17). The significant reduc t ions in the levels o f 20:4 in phospha t idy l inos i to l and 20:4 and 22:6 in phospha t idy lse r ine , relative to the concent ra- tions found in these phospho l ip id classes of rat liver (17), are c o m p e n s a t e d for by increased

oleic acid levels. Both of these phospha t ides , isolated f rom plasma membranes of a rat hepa toma , have been shown to conta in a decreased stearic/oleic acid ratio, but two mouse hepa tomas did no t (15).

D i p h o s p h a t i d y l g l y c e r o t con ta ined the highest level of 18:2, but the concen t r a t ions were reduced greatly f rom the levels found in rat liver (17). The lower levels o f 18:2 were offse t by increased percentages of bo th 16:1 and 18:1. It is easy to see h o w a n u m b e r of mi tochondr ia l en zy mes tha t require d iphospha- t idylglycerol for activity could be af fec ted by a change in the molecular species of this class.

The character is t ic compos i t i on of rat liver sph ingomyel in (17) also prevailed in the HTC cells, excep t for a s o m e w h a t higher level o f palmit ic acid. This was the only phospho l ip id class of HTC cells that did n o t show an elevated level o f oleic. I t is o f in teres t tha t , when linoleic acid was added to the med ium, a very high percentage of 24:2 appeared, while only traces of this acid were found when cells were grown on media conta in ing 20% bovine serum, which con ta ined twice the level o f ester if ied linoleic acid. We have shown previously (18) that sphingomyel in f rom Ehrl ich ascites cells contained a high level of this acid, which is absent , or present in only trace amounts , in normal mature tissue. The enzyme sys tem responsible for the synthesis o f 24:0 and 24:1 may have



lost some of i ts specif ici ty in neop lasms and, there fore , is able to accep t and e longate l inoleic acid. The presence of this long chain d ienoic acid in the g lycosphingol ip ids of the surface m e m b r a n e s cou ld have far - reaching e f fec t u p o n the cell.

General ly , all p h o s p h o l i p i d classes, excep t sph ingomyel in , s h o w e d a decrease in po lyunsa t - u ra t ed acids, especial ly 20 :4 , and an increase in m o n o e n o i c acids, par t icu la r ly oleic acid, as the se rum and l ipid levels decreased. The decreased cel lular levels o f 18:2 and 20 :4 acids were expec ted , since m a m m a l i a n cells requi re exog- enous sources of these acids, bu t the replace- m e n t of these acids wi th only m o n o e n o i c acids was u n e x p e c t e d . Bailey et al. (19) have s h o w n t h a t an increase in the levels of 16:1, 18:1, 20 :3 , and, in some ins tances , 22 :3 occur red when several cell l ines were grown u n d e r cond i t ions where an essent ia l f a t ty acid defi- c iency exis ted. The absence of increased levels of 20 :3 or 22 :3 in e i the r the neu t r a l l ipids (1) or phospho l ip ids , even when the cells were cu l tu red on l ipid-free m e d i u m , suggests t ha t these cells may n o t be able to desa tu ra te m o n o e n e f a t t y acids fur ther . We have s h o w n in a separate pub l i ca t i on (20) t ha t these cells can desa tura te s a tu ra t ed acids to m o n o e n e s , and the da ta in th is s t udy suggest t ha t , to a l imi ted e x t e n t , l inoleic acid can be conve r t ed i n to a rach idonic acid.

The c o n c e n t r a t i o n of oleic acid in all the phospha t i de s was the h ighes t when the cells were grown on l ipid-free m e d i u m and was the lowes t when the cells were grown on m e d i u m con ta in ing added free l inoleic acid or 25% serum. The possibi l i ty t h a t free f a t ty acids, wh ich also were p resen t in serum, migh t have caused the decrease in the cel lular concen t r a - t ions of oleic acid is nega ted by the fact t ha t palmit ic acid added to the m e d i u m had l i t t le e f fec t on oleic acid levels. A n o t h e r fac to r c o m m o n to b o t h med ia was the h igh concen- t ra t ions of l inoleic acid: ca. 68 p g / m l of the 20% bovine serum-5% fetal calf serum m e d i u m and 37.5 /~g/ml of the m e d i u m c o n t a i n i n g added l inoleic acid. The m u c h greater e f fec t of the free l ino lea te p r o b a b l y is re la ted to i ts greater avai labi l i ty to the cells. These da ta suggest t ha t the level of l inoleic acid m ay inh ib i t or regulate m o n o e n o i c acid b iosyn thes i s in these cu l tu red ra t h e p a t o m a cells. The inf luence on m o n o e n o i c acid m ay be only appa ren t and actual ly m ay af fec t all endoge- nous ly b io syn thes i zed fa t ty acids. It has been shown previously (21-23) tha t mouse hepa t i c l ipogenesis, as measu red by the rate of 14C_ac e_ ta te i n c o r p o r a t i o n i n to lipid, is i n f luenced more by die tary l inolea te t han o t h e r f a t t y acids.

Unl ike the mouse , ra t l iver hepa t i c f a t ty acid b iosyn thes i s is less sensi t ive to d ie ta ry l ipids; however , r ecen t evidence ind ica tes t h a t d ie tary saf f lower oil ( t r i l ino le in) also inh ib i t s liver f a t ty acid synthes i s in th is species (23) . The decreased fa t ty acid b io syn thes i s in cu l tu red cells, when se rum is added (19 ,24) to the m e d i u m , may be d u e to l ino lea te in the serum. Recen t work by Jacobs et al. (25) , w i th cu l tu red p r imary skin f ib rob las t s ind ica tes t h a t whole serum or l ipid de f ic ien t serum, plus f a t ty acids, reduces the level of ace ty l -CoA carboxylase . In the i r e x p e r i m e n t s , l inoleic acid was m u c h less effect ive than pa lmi t i c acid in r educ ing the a m o u n t of radioac t ive ace ta te i n c o r p o r a t e d i n to to ta l lipids.

One of the more i m p o r t a n t aspects of these data is the d e m o n s t r a t e d response of m o n o - enoic acid b iosyn thes i s to d ie tary l ipids in these cells. Previous s tudies have s h o w n tha t lipo- genesis, as d e t e r m i n e d by 14C-aceta te incorpo- r a t ion in to f a t t y acids, in mouse and ra t h e p a t o m a was n o t a f fec ted by d ie tary fat (22 ,26-28) . The a p p a r e n t d i sc repancy b e t w e e n the mass data f rom these cu l tu red h e p a t o m a cells and the r ad io i so tope data f rom hos t g rown h e p a t o m a s is n o t u n d e r s t o o d bu t is the sub jec t of f u r t he r invest igat ions .

ACKNOWLEDGMENTS

This work was supported by grant USPH 5-R01 CA 12973 from the National Cancer Institute.

REFERENCES

1. Wood, R., Lipids 8:690 (1973). 2. Rouser, G., A.M. Siakotos and S. Fleischer, Ibid.

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7t0 R. WOOD AND J. FALCH

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

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Biophys. 135:272 (1969). 18. Wood, R., and R.D. Harlow, Ibid. 141:183

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Tillman, Lipids 7:125 (1972). 20. Wiegand, R.D., and R. Wood, In press. 21. Allmann, D.W., and D.M. Gibson, J. Lipid Res.

6:51 (1965). 22. Abraham, S., Amer. J. Clin. Nutr. 23:1120

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Acta 280:258 (1972). 24. Rothblat, G.H., Advan. Lipid Res. 7:135 (1969). 25. Jacobs, R.A., W.S. Sly and P.W. Majerus, J. Biol.

Chem. 248:1268 (1973). 26. Sabine, J.R., S. Abraham and H.P. Morris, Cancer

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Biochim. Biophys. Aeta 116:407 (1966). 28. Sabine, J.R., S. Abraham and I.L. Chaikoff,

Cancer Res. 27:793 (1967).

[Received July 6, 1973]


lipids of cultured hepatoma cells: ii. effect of media lipids on cellular phospholipids

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