Relationship between embryonic and tumor lipids: I. Changes in the neutral lipids of the developing chick brain, heart and liver

Relationship between Embryonic and Tumor Lipids: I. Changes in the Neutral Lipids of the Developing Chick Brain, Heart and Liver RANDALL WOOD, Division of Gastroenterology, Departments of Medicine and Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65201

ABSTRACT

Organ dry weight, per cent total lipid, per cent neutral lipid, per cent phospholipid, and neutral lipid class composition of embryonic and mature brain, heart and liver were determined at 10, 13, 16, 19, 22, 27 and 53 days after incubation was initiated. All three tissues showed an increase in total lipids from the 10th day to hatching (21st day). The 10th day brain showed relatively high levels of sterol esters which decreased with increased development while free sterol levels increased. Heart free sterol and sterol ester percentages decreased with increased time, while triglyceride levels increased dramatically after the I6th day. Liver showed a massive accumulation of neutral lipid after the 17th day. The neutral lipid was not triglyceride, as might have been expected, but sterol ester. Liver sterol, sterol ester and triglyceride levels were approximately equal at the 10th and I3 th days, after which time sterol ester rose rapidly to more than 90% of the total neutral lipids by the 19th day. The neutral lipid class distributions were characteristically different for each tissue throughout embryonic development. The relative high sterol ester levels in each of the tissues early in development suggests that the high level of sterol esters in neoplastic tissue may be related to the growth process of increasing cell numbers. On the other hand, the absence or the presence of only trace amounts of glyceryl ether diesters in any of the embryonic tissues suggests that the elevated levels of this lipid class in most tumors may be related to the neoplastic process or to conditions resulting from neoplasia.

INTRODUCTION

Over the last few years 1 have studied the structure and metabolism of lipids of neoplastic cells, primarily Ehrlich ascites carcinoma cells, to ascertain whether differences exist between normal and neoplastic tissue. The data have

indicated a number of basic differences in lipid structure and metabolism between the two tissues (1-5). Numerous other investigators have also observed differences between the lipid metabolism of normal and neoplastic tissue (6-10). Errors in the lipid metabolism of neoplastic cells could affect membrane structure, lipid dependent enzyme systems and other biological processes involving lipids. However not all differences observed between normal and neoplastic tissue can be attributed to errors in lipid metabolism. In addition to any differences in lipid metabolism that may be associated with neoplasia, there are differences associated with rapid cell growth or proliferation. Despite the lack of research in this area, there is adequate evidence that demonstrates the differ- ence in lipid metabolism between the developing embryo and the adult organism (11-13).

There is an accumulation of data that demonstrates the similarities between embryonic tissue and neoplastic tissue. Abelev in 1963 (14) found that hepatomas synthesized and secreted into blood a specific embryonic a-globulin which has been confirmed in primary liver tumors of rats, mice and humans (15-16). Isoenzymes of numerous enzymes that pre- dominate in fetal tissue, but are absent or present at very low levels in normal tissue, have been shown to be present at relatively high levels in neoplastic tissue (17-19). The architec- ture of the surface membranes of embryonic and neoplastic cells exhibit similarities in that they are readily agglutinated by certain carbo- hydrate-binding proteins, whereas normal adult cells are unaffected (20-22). It has also been shown that the amino acid sequence of the C-terminal portion of glycine-arginine-fich his- tones isolated from bovine lymphosarcoma, Novikoff hepatoma, fetal calf thymus and pea seedlings are identical (23). Although space does not permit an exhaustive review of the similarities between embryonic and neoplastic tissues, the examples cited are sufficient to demonstrate that many similarities exist.

Comparative lipid data, both metabolic and structural, from neoplastic and embryonic tissue is very limited. However, despite the scar- city of data, some similarities between neoplastic and embryonic tissue lipid metabolism

596

E M B R Y O N I C T I S S U E N E U T R A L L I P I D S 597

have been noted. Desmosterol, a sterol first reported in the developing chick brain by Stokes et al. (24) and in rat brain by Kritchev- sky and Holmes (25), has been found to occur also in fibroblasts and undifferentiated brain tumors (26,27), but is not a detectable component in the adult brain. The rate of liver cholesterol biosynthesis is decreased by exogenous dietary cholesterol in the adult (28); however all hepatomas examined thus far (6,7) fail to respond to dietary cholesterol, as has recently been found with fetal rat liver (29).

This is the first in a series of papers on the structure and metabolism of lipids of embryonic tissues. It is hoped that the comparison of the lipid structure and metabolism data from embryonic tissues, normal mature tissue and neoplastic tissue will enable us to determine whether the differences observed between the lipid metabolism of normal and neoplastic tissue are associated with growth or neoplasia. These studies will also broaden our knowledge about the involvement and physiological role of lipids during embryonic development. This re- port compares the neutral lipid class composition of brain, heart and liver at various stages of the chick's development.

M A T E R I A L S A N D METHODS

Twenty-eight dozen Standard Northwest Arkansas Broiler strain eggs were selected at random and placed in a large atmospherically controlled incubator. Brains, hearts and livers were excised from embryos and chicks 10, 13, 16, 19, 22, 27 and 53 days after incubation was inflated. Chicks were fed a standard maize base starter ration. The 53 day group was placed on a standard broiler diet ca. 10 days after hatching. Excised organs were held in isotonic saline in an ice bath during collection.

The pooled tissue of each time period was lyophilized, and the total lipids were extracted by the Bligh and Dyer procedure (30). A second extraction was performed by readjusting chloroform and methanol concentrations to again give a single phase. Neutral lipids were separated from the phospholipid fraction by silicic acid chromatography (31). Percentages of neutral lipid and phospholipid fractions were determined gravimetrically. The neutral lipid fraction was separated into classes by thin layer chromatography (TLC) on adsorbent layers of Silica Gel G developed in a solvent system of hexane-diethyl ether-acetic acid 80:20:1 v/v. TLC plates used for qualitative and quantitative estimations were sprayed with sulfuric acid, charred, and documented by photography. Pre- parative TLC used to isolate each class for

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quantitation and further analysis was visualized by spraying with Rhodamine 6G and viewing under UV light. The purity and identity of each class were checked by TLC in other solvent systems and by gas liquid chromatography (GLC) of the intact compounds and their hydrolysis products.

Quantitation was achieved by gas liquid chromatography (GLC) analysis of intact classes and their hydrolysis products containing a known quantity of an appropriate internal standard. Each lipid class was eluted quantita- tively from the adsorbent, scraped from a developed chromatoplate (5 x 20 cm) that had been streaked with a known quantity (ca. 2.0 mg) of a neutral lipid fraction. One-half of the sterol ester and triglyceride fraction and all of the free fatty acid fraction were converted to methyl esters; a known quantity of methyl arachidate was added, and each analyzed before and after hydrogenation. Cholestane was added to the free sterol fractions and also analyzed by GLC. From the responses of the standards relative to the quantities added, the quantities of fatty acids and cholesterol present were calculated. The remaining 50% of the sterol ester and triglyceride fractions were hydrogen- ated; then a known quantity of cholesteryl heptadecanoate and dihexadecanoyl hexadecyl glyceryl ether was added to the respective fractions, and they were analyzed intact by high temperature GLC. The quantity of triglyceride

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and cholesteryl esters determined in this man- ner served as a check on the percentages calculated from the methyl ester data. Dupli- cate samples usually agreed within +5% for major components and _+10% for minor components by either method. Agreement between percentages calculated from hydrolysis products and intact lipid classes was -+10%. Analyses of standard mixtures of varying concentrations agreed within _+5% for high levels and _+10% for low concentrations of compounds. Since the attributes of GLC as an analytical tool are well known, the only drawbacks of this approach are limited to technical skill of the operator to make quantitative manipulations and the time and error involved in measuring chromatographic peak areas. The latter is virtually eliminated by the use of a digital integrator.

Gas liquid chromatographic analyses were carried out with an Aerograph Model 2100 instrument. Methyl ester analyses were carried out using a 180 cm x 2 mm (ID) pyrex column (packed with 10% EGSS-X coated on 100-120 mesh Gas Chrom-P) temperature programed from 140-200 C at 4 C/min. Free sterols were quantified using a 180 c m x 2 mm (ID) pyrex column (packed 1% OV-17 coated on 100-120 mesh Gas Chrom-Q) operating at an isothermal temperature of 215 C. Analysis of intact triglycerides and sterol esters was performed on a 75 cm x 2.5 mm (ID) pyrex column packed with 1% OV-17 coated on 100-120 mesh Gas Chrom-Q. Column temperatures were pro- grained from 200-300 and 350 C at 4 C/min for analysis of sterol esters and triglycerides, respectively.

Methyl esters were prepared by the time- honored 2% sulfuric acid-anhydrous methanol procedure. Esterification was carried out either by refluxing for 2 hr with a large excess of reagent or heating in a boiling waterbath in sealed screw cap culture tubes. Neutral lipid standards (purity 99%) were purchased from Nu-Chek-Prep, Inc., Elysian, Minn. All solvents were glass-distilled and obtained from Burdick and Jackson Lab., Muskegon, Mich. Other chemicals and reagents used were reagent grade or better.

RESULTS

The increased dry weight of each tissue with increased development time is shown in Figure 1. Brain and heart showed a gradual and sustained increase over the measured time period. Liver exhibited a pronounced accumulation of dry weight between the 17th and 22nd days after incubation was initiated. Hatching occurred on the 21st day, and it is assumed that

LIPIDS, VOL. 7, NO. 9

EMBRYONIC TISSUE NEUTRAL LIPIDS 599

the increased liver weight occurred before that time, which indicates a ca. 300% increase in fiver weight over a 3-4 day period.

Total Phospholipid and l~leutral Lipid

The number of embryos and chicks, percentage total lipid and percentage neutral lipid and phospholipid for each tissue at each time period is given in Table I. All three tissues showed an increased accumulation of total lipids from the 10th day of incubation to hatching. Total lipid (relative to dry weight) of brain continued to increase from 14 to 33% while the neutral lipid and phospholipid fractions remained constant at approximately one-third and two-thirds, respectively. Heart total lipids increased from 8% at 10 days to 15% at hatching, and, like brain the phospholipid to neutral lipid ratio was approximately two for the same time period. At 27 days after incubation was initiated the heart phospholipid to neutral lipid ratio was down to one, but was back to three at the 53 days. Liver total lipids showed a 400 to 500% increase from the 10th day to hatching, which paralleled the increase in liver mass (Fig. 1). Neutral lipids and phospholipid percentages were approximately equal at the 10th day, but at hatching there was eight times more neutral lipid than phospholipid. After hatching, liver neutral lipids decreased, and at 53 days the phospholipid to neutral lipid ratio was two. Egg neutral lipid to phospholipid ratio was four.

A clearer picture of the lipid changes that occur during development is seen for each tissue in Figure 2, which shows the micrograms of both lipid fractions per milligram of dry weight with time. Except for liver neutral lipid and brain phospholipid fractions, the values for each fraction were bunched in a narrow region between ca. 25 and 75/~g/mg dry weight for all time periods. The liver neutral lipid fraction also fell within this range on the 10th, 13th and 53rd days. Figure 2 shows that the tremendous accumulation of liver lipids was due to neutral lipid fraction and not phospholipid fraction, which remained relatively constant. It can also be seen that the accumulation of total lipids in the brain with development represents primarily an increase in phospholipids.

Brain Sterol esters, triglycerides, free fatty acids

and sterols were present at all time periods. The quantitative data shown in Table II indicate a marked decrease in the sterol esters with increased development. Sterols, the major neutral lipid class, showed a small percentage but a definite increase until hatching. Free fatty acids and triglycerides decreased with increased time (Table II). A trace component between the

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sterols and free fatty acids, most prevelant at the earlier time periods, was not characterized. The calculated recovery percentage shown in Table II indicates less than 5% trace components.

Heart

The neutral lipid class distributions of heart at various times is shown in Figure 3. Sterol esters, triglycerides and sterols represent the major neutral lipid classes. Triglycerides represent about one-third of the heart neutral lipid at the 10th day, which decreases to half this level by the 16th day and then increases to 75% by the 27th day. Sterol esters showed the reverse picture, increasing when triglyceride levels fell and decreasing when triglyceride levels rose. Cholesterol, the major sterol, represented ca. 50% of the heart neutral lipids through the 16th day and then decreased to ca. 15% by the 53rd day. Free fatty acids were present only as a trace component and was not quantified. A component above the triglyceride band at day 53 was identified as a glyceryl ether diester. A spot below the triglyceride band at the same time period was not characterized.

Liver

The most dramatic changes occurred in the neutral lipid classes of the developing liver, as shown in Figures 4 and 5. Cholesterol, triglycerides and sterol esters each represented approximately one-third of the liver neutral lipids at days 10 and 13. However after the 13th day


600 R. WOOD

TABLE II

Percentage of Neutral Lipid Classes at Various Stages of Development in the Chick Embryo Brain a

Per Cent of total neutral lipids

Days after incubation Free Sterol

initiated Sterols fatty acids Triglycerides esters Recovery

10 83.3 2.6 3.1 10.0 99 13 83.8 2.3 1.6 7.1 95 16 85.6 1.4 1.6 6.3 95 19 90.6 1.8 2.1 2.8 97 22 91.1 1.9 1.4 3.1 97 27 91.3 0.7 0.9 2.3 95 53 91.2 1.0 b 1.0 b 2.0 b 95

apercentages represent the mean of duplicate analysis. bAs determined by thin layer chromatography.

cho les te ro l and t r iglycer ide levels decreased rapid ly to a low level wh ich pers is ted un t i l a f te r the 2 7 t h day. The decreased cho les te ro l and t r iglycer ide levels were o f f se t by an accumula- t ion of cho les te ro l es ters t h a t r eached 90% of the neu t r a l l ipids by the 19 th day and r ema ined at the level un t i l a f te r the 2 7 t h day. Af te r m a t u r i t y (53 days a f te r i n c u b a t i o n was init i- a ted) liver t r iglyceride values r ep resen t ca. 75% of the neu t ra l lipids, and cho les te ro l es ter values were down be low 5%.

Egg

Triglycer ides r ep resen t more t h a n 90% of the egg neu t ra l l ipids (Fig. 5). Choles te ro l and choles te ro l esters were the on ly o t h e r egg l ipid classes p resen t in a m o u n t s greater t h a n 1% of the neu t ra l l ipid f rac t ion .

DI SC USSI ON

The l i te ra ture on the lipids of the developing chick e m b r y o t h r o u g h 1967 has been covered by R o m a n o f f (32) . A review of the work covered by R o m a n o f f and a search of the l i t e ra tu re since revealed t h a t a quan t i t a t i ve d e t e r m i n a t i o n of all the neu t ra l l ipid classes had no t been d e t e r m i n e d at var ious d e v e l o p m e n t a l stages for the chick hea r t or bra in . The neu t ra l l ipid classes of liver, the m o s t ex tens ive ly e x a m i n e d chick e m b r y o tissue, have been q u a n t i t a t e d for var ious d e v e l o p m e n t a l stages; bu t mos t of the s tudies were carr ied ou t us ing co lumn c h r o m a t o g r a p h y and gravimetr ic tech- niques , wh ich have since been rep laced by more accura te m e t h o d s . The p resen t s tudy , in addi- t ion to add ing to our knowledge where da ta are lacking, allows compar i s ons of changes in the neut ra l l ipid classes wi th in the same s tudy , of neura l , muscu la r and digestive tissue, each of which exh ib i t s d i s p r o p o r t i o n a t e g r o w t h rates occur r ing at d i f fe ren t stages of d e v e l o p m e n t .

Brain

The chick e m b r y o b ra in has been used as a mode l by n u m e r o u s inves t igators to s tudy cho les t e ro l me t abo l i sm . Aside f rom cho les te ro l , the o t h e r b ra in neu t r a l l ipids have gone a lmos t uns tud ied , par t icu la r ly in ear ly stages of devel- o p m e n t . The data (Table II) show tha t free fa t ty acids, t r ig lycer ides and sterol esters are p resen t in subs tan t i a l a m o u n t s , in add i t ion to free s tero ls at 10 days of i ncuba t i on . All th ree l ipid classes decreased w i t h increased incuba- t ion t ime, and free s terol values increased. The levels and changes in the free f a t ty acid and t r iglycer ide values at var ious d e v e l o p m e n t a l t imes of the chick b ra in do no t appear to have been r epo r t ed previously . Ind i rec t evidence for the p resence of cho les te ro l esters in the develop ing chick brain was r epo r t ed by Mandel et al. (33) in 1949. Adams and Davison (34) r epo r t ed the p resence of es ter i f ied choles tero l in the developing h u m a n ne rvous sys tem and the chick, b u t the i r da ta on the chick b ra in was i n c o n s i s t e n t and inconclus ive . Wells and D i t tme r (35) r epo r t ed s ignif icant levels of s terol esters in the th ree -day-o ld ra t tha t decreased to u n d e t e c t a b l e levels a f te r six days.

E x a m i n a t i o n of the hydrolys is p r o d u c t s and the i n t a c t s terol esters by GLC has es tab l i shed the i d e n t i t y and presence of this neu t ra l l ipid class in the brain b e y o n d any reasonable d o u b t . The decrease in s terol es te r c o n t e n t of the b ra in wi th increased m a t u r i t y suggests it may play a role in cell division or ma tu r a t i on . I f the level of s terol esters is r e la ted to ra te of b ra in deve lopmen t , s terol es ter levels a round the 5 th day of i n c u b a t i o n (when b ra in d e v e l o p m e n t is mos t rap id [32] ) shou ld be still h igher t han the 10 th day values r e p o r t e d here.

Heart

The lipids of the deve lop ing chick hea r t



apparently have not been reported previously. Total lipids of heart were lower than either brain or liver, and unlike the other two tissues (which showed an increase in total lipids each successive time period) heart decreased from the 13th to the 16th day of incubation (Table I). The decreased total lipid level at this time period corresponds to one of the two self- inhibiting growth retardation periods that are known to occur on the 9th and 16th days of incubation (32). During the growth retardation period at the 16th day, which is the most pronounced, metabolic processes such as ad- sorption, storage and oxidation of organic matter also show cyclic changes (32). Heart triglyceride levels reached a minimum, and cholesterol ester values reached a maximum, also at this time (Fig. 3). It should also be pointed out that this was the time period when fiver sterol ester levels started to increase dramatically (Fig. 2) and when brain sterol esters showed the largest decrease to the next time period (Table II). All of this information seems to show a very active metabolism of sterol esters at this time and suggests further that the observed decrease in total neutral lipids of the heart at the 16th day of incubation, relative to the 13th and 19th days, is a real phenomenon and is not due to technical error.

Heart and liver triglyceride percentages at 10, 13 and 16 days of incubation were similar, but after the 16th day liver triglyceride levels continued to fall while heart triglyceride levels began to rise. At present, triglycerides from all three tissues are being examined in detail to determine whether de novo synthesis or accumulation of preformed yolk triglyceride occurs in these tissues.

Liver

The rapid increase of liver dry matter after the 16th day of incubation (Fig. 1) has been observed previously by a number of investigators, and these data have been analyzed from several viewpoints by Romanoff (32). The high percentage lipid of the dry liver weight (Table I) is in good agreement with previously reported values (36,37). Accumulation of cholesterol esters in the neutral lipids (Figs. 4 and 5) was somewhat surprising, since the egg neutral lipids contained more than 90% triglycerides and less than 1% cholesterol esters (Fig. 5). Tsuji et al. (38) first observed the increased cholesterol ester of whole embryos with increased incubation time. Moore and Doran (37), the first to examine the neutral lipids of the developing embryo in detail, found that the cholesterol esters accumulated in the liver, which has since been confirmed by Noble and

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Moore (36), Feldman and Grantham (39) and the present study. Recalculation of previous data expressed in terms of wet weight (39), few time periods (37) and incomplete class analysis (36) made comparison of present class percentages with those reported previously difficult. However in general the comparisons agreed reasonably well. The present study, which examined both earlier and later developmental periods than previous studies (36,37,39), shows that at 10 days of incubation cholesterol, cholesterol ester and triglyceride values are approximately equal, and that when the liver rapidly accumulates large amounts of cholesterol esters before hatching, the level remains high at least 6 days after hatching. The eventual fall in cholesterol ester values is accompanied by a concomitant rise in triglyceride level. The storage of lipids in the chick liver in the form of cholesterol esters as opposed to triglycerides (which are most abundant in the egg) poses some most interesting questions about biosynthesis, transport and storage of lipids during development.

Embryonic vs. Tumor Lipids

A prime objective in initiating this series of studies on the developing embryonic tissue lipids was to determine whether observed ab- normalities in the lipid metabolism of tumors are associated with neoplasia or rapid cell growth and proliferation. The determination of the neutral lipid class composition, a basic but necessary operation, was not expected to pro- vide any insight into this question. However the high level of sterol esters observed early in all three developing tissues at least opens the door to speculation.


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FIG. 4. Thin layer chromatoplate depicting the distribution of sterol esters (A), triglycerides (B), free fatty acids (C) and free sterols (D) of the embryonic chick liver at 10, 13, 16, 19, 22, 27 and 53 days after incubation was initiated (arranged chronologically in lanes 1-7). Lane 8 represents a standard mixture, and the origin and solvent front are indicated by 0 and S, respectively. Chromatography was carried out on adsorbent layers of Silica Gel G developed in a hexane-diethyl ether-acetic acid 80:20:1 v/v solvent system. The standard triglyceride (triarachidin) moved just ahead of the sample trigtycerides because of the higher molecular weight.

Sterol ester levels of neoplasms are generally higher than most normal tissue. Boyd et al. (40) reported higher sterol ester levels in Walker 256 carcinosarcoma than any of five normal host tissues examined. Elevated levels of sterol esters have been reported for five transplantable mouse tumors (41), Ehrlich ascites ceils (43) and in the blood of patients with cancer (43). In minimal deviation hepatomas, where a valid comparison can be made with normal liver, the sterol ester level has been found to be signifi- cantly higher (44). However not all rapidly proliferating cells contain high levels of cholesteryl esters, as data from a number of "normal" cells grown in culture have indicated (45). This observation does not, however, rule out the possible involvement of sterol esters in cell proliferation, because most "normal" cells grown in culture require for growth serum which contains sterol esters. Rothblat et al. (45) have shown that lyrnphoblast cells (L5178Y) grown on media containing serum incorporate exogenous serum cholesteryl esters. Bailey (46) has shown that a strain of non- tumorgenic lymphoblast cells (MB III) grown in culture do not contain significant levels of cholestryl esters but do not replicate on media without serum, Whether serum sterol esters are

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required for MB III cell replication remains to be determined. It has been suggested that the levels of sterol esters in cultured cells may be regulated by rates of uptake and hydrolysis of exogenous cholesteryl esters (45). In this light, elevated levels of sterol esters in embryonic tissue may represent a lack of hydrolysis. However high sterol ester levels in the developing liver, heart and even brain, relative to the adult values, may suggest that the elevated sterol ester levels in neoplasms are related to the growth process. If sterol esters are involved in cell proliferation as a structural component, a regulator or even a source of energy, their controlled biosynthesis and transport in a neo- plasm could have significant therapeutic value. However the full significance of the observation will have to await more detailed metabolic studies.

The first characterization and identification of glyceryl ether diesters from neoplastic tissue was carried out on lipids isolated from Ehrlich ascites cells (1), and the presence of glyceryl ether diesters in a large number of transplantable rat and mouse tumors has been established more recently (47). Glyceryl ether diesters are not characteristic of neoplastic tissue. They have been isolated from human perinephric fat (48) and beef heart lipids (49), and indirect evidence suggests that they occur in most mammalian tissues (50,51); but based upon indirect quantitative data (52,53) it appears



t h a t genera1!y n e o p l a s t i c t i s sue c o n t a i n s ele- v a t e d levels o f g l y c e r y l e t h e r d i e s t e r s .

T h e a b s e n c e o r p r e s e n c e o f o n l y t r ace q u a n t i t i e s o f g l y c e r y l e t h e r d i e s t e r s in all t h r e e e m b r y o n i c t i s sue s a t all t i m e p e r i o d s , e x c e p t 53 d a y h e a r t , s u g g e s t s t h a t t h e e l e v a t e d levels o f g l y c e r y l e t h e r d i e s t e r s f o u n d in n e o p l a s m s are n o t d u e to t h e g r o w t h p r o c e s s , b u t in f ac t m a y be r e l a t e d t o t h e n e o p l a s t i c p r o c e s s o r c o n d i - t i on r e s u l t i n g f r o m n e o p l a s i a . R e c e n t l y H o w a r d a n d Ba i l ey (54 ) r e p o r t e d a c o r r e l a t i o n b e t w e e n t he e l e v a t e d g lyce ry l e t h e r c o n t e n t a n d de- c r e a s e d ac t i v i t y o f t h e e n z y m e c~-glycerol p h o s - p h a t e d e h y d r o g e n a s e fo r b o t h h e p a t o m a s a n d c u l t u r e d cells . T h e s e f i n d i n g s s u g g e s t t h a t t h e e l e v a t e d levels o f g l y c e r y l e t h e r d i e s t e r s m a y be d u e t o a l t e r e d e n z y m a t i c ac t iv i t i e s w h i c h r e s u l t in b u i l d - u p o f p r e c u r s o r s f o r t h e i r b i o s y n t h e s i s .

ACKNOWLEDGMENTS

N. Holman of the Poultry Husbandry Dept. carried out the egg incubations, and J. Falch gave technical assistance.

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46. Bailey, J.M., in "Lipid Metabolism in Tissue Culture Cells," Mongraph #6 Edited by G.H. Rothhlat and D. Kritchevsky, Wistar Inst i tute Press, Philadelphia, 1967, p. 85.

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LIPIDS, VOL. 7. NO. o

Documents

Relationship between embryonic and tumor lipids: I. Changes in the neutral lipids of the developing chick brain, heart and liver