Incorporation of [3H]acetate into L-cell lipids Dependence on method of linoleate administration and timing of the pulse label

Biochimica et Biophysics Acta, 665 (1981)

Elsevier/North-Holland

531-53’7

531

Biomedical Press

BBA 57841

INCORPORATION

OF [‘H] ACETATE INTO L-CELL LIPIDS

DEPENDENCE ON METHOD OF LINOLEATE

ADMINISTRATION

AND TIMING OF THE PULSE LABEL

STANLEY M. LIFFMANN and ROBERT D. LYNCH * Department

of Biological Sciences,

University of Lowell, I University Avenue, Lowell, MA 01854 (U.S.A.)

(Received November 25 th, 1980) (Revised manuscript received May lst, 1981) Key words: Lipid analysis; Linoleate supplement;

Acetate incorporation:

(L-cell)

An aqueous solution containing 5 ~01 of sodium linoleate was supplied to cultures of L-fibroblasts by two methods: (a) direct addition to the culture as a single dose at the start of a 48 h incubation period (method I); or (b) infusion into the culture at a constant rate throughout the same time period (method II). At 23 h and 47 h the cells were subjected to a 1 h pulse of [ 3H]acetate. At 23 h method I was 1.2-times as effective as method II in reducing incorporation of radioactive label into cell sterol. Conversely, the decrease in utilization of [ 3H]acetate for acyl group biosynthesis was 1.7~times greater by method II than by method I. At 47 h both methods had similarly reduced [ 3H]acetate incorporation into both fractions by 85%. Regardless of the method used for supplementation, the large decline in biosynthesis from [3H]acetate noted at 47 h was not accompanied by any corresponding decrease in the total amount of sterol synthesized. Additional data suggest that SC% of more of the decrease in [3H]acetate incorporation at this time may be attributable to metabolites derived from the unlabeled exogenous fatty acid, which act to dilute the radioactively labeled tracer and/or to inhibit endogenous lipid biosyn-

Introduction When mammalian cells in culture are supplied with or are deprived of exogenous sources of fatty acids, the de novo synthesis of their lipids may be regulated at least in part by the following two mechanisms: (a) long-term adjustments in the amount of enzyme protein per cell [l] ; (b) short-term direct inhibition of enzyme activity, by such intermediates as palmitoylCoA [2,3]. Both mechanisms probably play a role in determining the utilization of radioactively labeled acetate for lipid biosynthesis by cells in culture which have been exposed to various types and amounts of exogenous fatty acids [4,5]. Studies on the mechanisms responsible for the inhibition of radioactively labeled acetate incorpora-* To whom correspondence

should be addressed.

OOOS-2760/81/0000-0000/$02.50

o 1981 Elsevier/North-Holland

tion into lipids of L-fibroblasts by fatty acids have yielded results which differ depending on the nature of the added fatty acid. The addition of palmitate to these cells, for example, resulted in a parallel decline in [ 1 J4C] acetate incorporation into both the sterol and acyl group fractions [6]. When linoleate was used, however, incorporation of radioactive label was diminished only in the acyl group fraction [4]. While such differences may be significant, it is often difficult to compare the results from one laboratory to another. Large variations exist not only in the growth phase of the cells used, but also in the length of time of exposure of cells to radioactively labeled tracer, and the method of supplementation with exogenous fatty acid [4-71. The importance of this latter factor has been demonstrated recently using strain L fibroblasts [S] and human diploid cells (IMR-90) [9]. In both cases Biomedical Press

532

the adverse effects of exogenous fatty acids on growth of cells and the accumulation of triacylglycerol can be decreased greatly if fatty acids are added by constant infusion instead of as a single dose at zero time. These studies monitored only growth and triacylglycerol accumulation, which are readily observed responses to a large influx of fatty acids. More subtle changes in the biology and biochemistry of the cells subsequent to this influx of fatty acid, however, are not so easily measured. The objective of the present study was two-fold: (a) to compare the utilization of [3H]acetate for lipid biosynthesis during a 1 h pulse period after cells had received fatty acids either as a single dose or by slow infusion for either 24 or 48 h; (b) to demonstrate that factors other than the added fatty acid itself are responsible for a large part of the depression in radioactively labeled acetate incorporation into lipids by cells incubated for 48 h with exogenous fatty acid.

Materials and Methods Cell culture Suspension cultures of L-fibroblasts were routinely grown in the absence of antibiotic in 50 ml Waymouth’s MB 752/t medium (Associated Biomedic Systems, Inc., Buffalo, NY) in 125 ml Erlenmeyer culture flasks. The growth medium was supplemented with 0.025% Methocel, 15Hz, (Dow Chemical Co., Indianapolis, IN), 19 6 mg/l CaC12, 30 FM thymidine and 2.5% horse serum (Grand island Biological CO., Grand Island, NY) 90-95% delipidized by a modification [lo] of the method of Albutt [ 113. The suspension cultures were agitated at 120 rev./min on a rotary platform shaker (New Brunswick Scientific Co., New Brunswick, NJ). Cell numbers were monitored with a Coulter Electronic Counter (Coulter Electronics, Hialeah, FL), and were maintained in the log phase of growth between 1 .O . 10’ and 1.2 . lo6 cellsfml by d~ution with fresh medium. Ceils grown by this method maintained a doubling time of approx. 18-21 h. Fatty acid supplementation * Linoleic acid (18 : 2(9,12)) (Applied Sciences, Inc., State College, PA) was dissolved in heptane un* Fatty acids are designated by the number of carbons: number of double bonds.

der nitrogen gas to a concentration of 50 mg/ml and stored in liquid nitrogen. Just before use, the fatty acid was diluted to a convenient concentration with heptane and washed free of peroxides with 2 vol. methanol/water (3 : 1, v/v) [12]. The heptane phase was collected and dried in a stream of nitrogen gas. The resulting fatty acid residue was promptly redissolved in 0.12% NaOH and sterilized by passage through a 0.22 pm M~lipore fdter. The fatty acid solution was administered by continuous infusion [7] or as a single dose bound to albumin [6]. In the infusion system, the fatty acid solution was delivered through sterile Teflon tubing by a syringe pump (SAGE, model 355, Orion Research Inc., Cambridge, MA) at a rate of 9.1 . lo-’ ml/h. Fatty acid-albumin complexes (4 : 1 molar ratio) were prepared by addition of the fatty acid salt solution to 100 ml of complete cell culture medium containing albumin which had been heated to 56°C 161. After rapid cooling on ice, the medium was filtered through a sterile 0.22 pm Millipore filter and then stored at 4°C. By either method the change in volume of culture medium after supplementation was less than 10%. Whether the fatty acid was added as a single dose or by infusion, the initial cell densities were adjusted to approx. 2 . lO’/ml at the start of the experiment. Radioactive labeling of lipids Radioactive sodium [jH]acetate (70 Ci/mol) (New England Nuclear, Boston, MA) was sterilized as an aqueous salt by passage through a 0.22 pm Millipore filter. At different times after supplementation of culture with fatty acid, either by the infusion or the single dose method, a I h pulse of 20 I.tCi [3H]acetate was added to the cells. The labeling period was terminated by addition of a loo-fold excess of unlabeled sodium acetate in a volume of 0.1 ml. Cells were rapidly chilled on ice and removed from the radioactively labeled medium by centrifugation. Cell harvesting and lipid extraction A known number of cells ((5-10) - 106) was centrifuged at 600Xg for 5 min at 4”C, and resuspended in 15 ml harvesting solution containing 5 mM glucose, 0.14 M NaCl, 9.4 mM KCI, 1.8 mM NaHCO,, 0.5 mM NaaEDTA, 10 mM Hepes and 5 mM NaOH. After recentrifuging, the supernatant was removed and the tubes were drained well. In some cases 25 c(g

533

cholestane (Applied Sciences, Inc.) or 25 pg triheptadecanoin (Applied Sciences, Inc.) were added to the cell pellet as an internal standard to quantitate, respectively, the amount of desmosterol [13] or triacylglycerol [14]. Total cell lipids were extracted three times with chloroform/methanol (2 : 1, v/v) as described by Marinetti [15], and the volume of extract was adjusted to 7.5 ml. In some experiments 5-ml aliquots of the cell free medium, obtained at the end of the radioactive pulse period, were lyophilized and the lipids extracted as described above. An upper phase wash mixture [ 161, methanol/water/chloroform (48 : 47 : 3, v/v), was then added and the two phases were mixed vigorously for 30 s. After a brief centrifugation the bottom phase was removed and the upper phase back-washed with 2.5 ml chloroform. During extraction of the medium the chloroform phase was washed twice with 5 ml water to remove contaminating Methocel. The chloroform layers were dried under nitrogen gas and redissolved in 4 ml of chloroform/methanol (1 : 1, v/v). Measured aliquots were saved for determination of total radioactivity and total sterol; the remaining extract was used for separation of lipid classes by thin-layer chromatography . Neutral lipid classes were separated from the phospholipids by chromatography on 0.45 mm thick silica gel H thin-layer plates impregnated with 0.11% ammonium sulfate [ 171. The chromatograms were developed in a solvent system of hexane/ether/glacial acetic acid (60 : 39 : 1, v/v) and separated into the following components, which are listed in order of increasing RF: (1) phospholipid, (2) sterol, (3) diacylglycerol, (4) fatty acid, (5) triacylglycerol, (6) sterol ester. The developed chromatograms were visualized either by brief exposure to iodine vapor or by spraying with a 0.1% solution of 8-anilino-1-naphthalene sulfonic acid [18]. Once identified by comparison with standards, lipid classes were scraped into scintillation vials for radioactive counting, or into 15 ml screw-top tubes for methylation. Radioassay Total lipid radioactivity was determined by dissolving 1 ml of the total lipid extract in 10 ml of scintillation-grade toluene containing 0.4% Omnifluor (New England Nuclear). The same scintillation solution was used to assay the radioactivity in the neutral

lipid classes adsorbed to silica gel. Samples containing phospholipid adsorbed to silica gel were mixed with 1 ml water [19] before addition of 0.4% Omnifluor in toluene/Triton X-100 (2 : 1, v/v). All radioactive measurements were made with a Beckman Model LS 230 liquid scintillation system. Corrections for quenching were made by using an external standard. Gas chromatography Methyl esters of phosphoglycerides and triacylglycerols were prepared by alkaline methanolysis according to Glass [20] and then concentrated in 5 ~1 of heptane. Between 0.3 and 0.5 ~1 were injected directly onto a 6-ft glass column of 10% SP-222PS (Supelco Inc. Bellefonte, PA) packing material at 180°C. Fatty acids were identified by comparing their retention times with those of commercial standards (Applied Science Laboratories, Inc.). Aliquots of the total lipid extract taken for sterol determination, were dried under Nz gas and resuspended in 5 ~1 heptane. An aliquot of 0.5 ~1 was injected onto a 3-ft glass column of 3% SP-2250 (Supelco Inc.) at 250°C to quantitate the sterol fraction [13]. The columns were mounted in a Varian Series 2440 gas chromatograph equipped with a flame ionization detector and a Hewlett’Packard 3380 A integrator. Results Added as a single dose at the start of an experiment or by infusion at a constant rate throughout the incubation period, 5 pmol 18 : 2 caused a marked reduction in [3H]acetate incorporation into the lipids of L-fibroblasts when the [3H]acetate was added as a 1 h pulse at the 23rd or 47th h of incubation. The depression of [3H]acetate incorporation into the sterol fraction at 24 h was somewhat greater in cells receiving the 18 : 2 as a single dose than it was when cultures were infused with 18 : 2 (Table I). By contrast, the decrease in utilization of [3H]acetate for acyl group biosynthesis was more pronounced when the fatty acid was supplied by infusion. This occurred despite the fact that at 24 h these cells had been exposed to only half the amount of fatty acid supplied to the parallel cultures receiving a single dose of 18 : 2 at the start of the incubation period. At 48 h, when the entire 5 pmol of 18 : 2 had been infused into the culture, the pattern of isotope incorporation

534 TABLE 1 TIME-DEPENDENT EFFECT OF DIFFERENT METHODS OF ADMINISTERING LINOLEATE ON THE UTILIZATION 01: 13H]ACETATE FOR LCELL LIPID SYNTHESIS Cuitures of strain L fibroblasts (1.1 10’ cells in 50 ml culture media) received 5.0 gmol linoleate either by infusion over 48 11,or complexed to albumin and added as a single dose at zero time. At 23 and 47 h after the start of supplementation, cultures were pulse labeled for I h with 20 @i [3H]acetate. Cells were then harvested, and the lipid was cxtrncted and separated as described in Methods and Materials. All values are expressed as the percentage of the [sH]acetate incorporated into control cultures which had been incubated in parallel, but without fatty acid. Data represent mean f S.D. of 3-6 determinations. At 24 h, methodrelated differences in isotope incorporation into total acyl and sterol fractions were significant at the P < 0.001 and P < 0.05 level, respectively (Student’s f-test). Time of pulse label [aWlAcetate incorporation as a precent of control -(h) Single dose method Infusion method _ ____Total lipid

Total acyl groups

Sterol

Total lipid

Total acyl groups

stero1

41.3 + 4.2 16.0 k 2.1

65.5 + 1.6 16.6 t 2.1

27.6 + 4.1 13.4 t 1.5

40.2 + 2.6 17.5 t 1.5

41.3 t 2.4 18.8 + 2.4

31.8 _t 4.3 12.2 10.8

-23-24 41-48 ~-___~__

was the same as that of cells which had received the single dose of 18 : 2 48 h earlier. By either method of delivery the incorporation of [jH]acetate was depressed to a minimum value 85% below that of control cells in both the sterol and acyl group fractions. Cultures presented with 5 gmol linoleate by either method maintained the same doubling time as the cultures incubated without exogenous fatty acid. The large reduction of [3H]acetate incorporation into sterol occurred without depressing cell growth. This was ~consistent with earlier observations by others that a decrease in the sterol levels of L-fibroblasts was accompanied by cessation of cell growth [2 11. Chromatographic analysis of the lipid extract from cells after 48 h of exposure to 18 : 2 showed, however, that despite an 80% decrease in t3H]acetate incorporation into the sterol fraction, the desmosterol content of the cells from supplemented cultures (3.8 pg,/106 cells) was not significantly different from control cells (3.6 pg/106 cells). These data suggest that a reduced rate of sterol biosynthesis in supplemented cultures could not account for the decline in [3H]acetate incorporation into sterol at 24 and 48 h after the start of supplementation with fatty acid. There are at least two possible explanations for these anomalous results. (a) A more rapid efflux or exchange of newly synthesized sterol with non-labeled sterol in the medium during the radioactive pulse period could, at least in

part, account for the maintenance of constant sterol levels despite an apparent decrease in [3H]acetate incorporation. This explanation is consistent with observations by others who found that the egress of lipids from mouse L-fibroblasts was enhanced in the presence of a variety of exogenous lipid sources [22]. (b) Fatty acids or metabolites derived from their oxidation may have inhibited acetyl-CoA synthetase, thereby simultaneously reducing the incorporation of [3H]acetate into both sterol and fatty acid fractions. Alternatively, the unlabeled products of fatty acid oxidation may have diluted radioactive lipid precursor pools utilized for sterol and acyl group synthesis. Since growth was unaffected and incorporation was the same at 48 h regardless of the method of adding fatty acid, the rel~aining studies were carried out with cells which had received a single dose of the fatty acid at the start of a 48 h incubation period. To test whether there was a greater loss of newly synthesized radioactively labeled lipids from cells cultured in the presence of 18 : 2, the lipid-soluble fraction from the cells and medium were analyzed after being pulsed with [3H]acetate from the 23rd to 24th and the 47th to the 48th after the start of incubation with exogenous fatty acid. The sterol and acyl group radioactivity present in the medium of 18 : 2-supplemented cultures was much less than that in the medium of control cultures (Table II). Moreover, the ratio of lipid-soluble radioactivity in the medium to

53s TABLE II THE EFFECTS OF 18 : 2 SUPPLEMENTATION ON THE EXCHANGE OR LOSS OF NEWLY SYNTHESIZED LIPID FROM LFIBROBLASTS INTO THE GROWTH MEDIUM. Cultures of L-fibroblasts were radioactively labeled, harvested and extracted as described in Table I, except that the supplemented flasks received only a single dose of 18 : 2 for 48 h. Aliquots of medium were separated from the cells by centrifugation and the lipid from cells and medium isolated and counted as described in Materials and MethodsThe data for supplemented cells represent the mean + 1 S.D. of three determ~ations. Data from non-supplemented cells are from a single experiment. 18 :2 (+I-)

+ + -

Time of pulse label (h)

23-24 47-48 23-24 47-48

dpm/ 1O6 cells or medium from lo6 cells Total lipid Total acyl groups --

Sterol

Cells

Media

Cells

Media

Cells

Media

14 627 + 840 42492370 29660 31894

2 044 t 287 383+ 56 3 880 3 I34

9675 i 868 2513 i 193 15 080 17615

1858 -+276 292 + 42 3 447 2401

4 022 It 143 1632rt174 14520 14 002

Ill? 14 292 _+42 330 733

that of the cells was similar in supplemented and unsupplemented cultures. Together, these data show that although the exogenous fatty acid depressed Z3H]acetate incorporation into newly synthesized lipid, the fraction of lipid soluble radioactivity lost to the medium was the same as that of control cells. Although results from previous studies using radio-

actively labeled fatty acids suggested that there was little oxidation to COs by L~broblasts (231, there were no data presented to exclude the possibility that these ceils oxidize fatty acids to the level of acetylCoA, or that metabolites such as acetoacetate and /3-hydroxybutyrate were synthesized. The following experiment was designed to deter-

TABLE III DEPRESSION OF [aH]ACETATE INCORPORATION INTO CELL LIPIDS BY FACTORS PRESENT IN MEDIUM OF CELLS PREINCUBATED WITH LINOLEATE FOR 48 h Four cultures of L-fibroblasts each received 5 rmol linoleate as a single dose; four others served as controls. After 47 h of incubation all eight cultures were centrifuged. The medium from two control cultures was removed and replaced with the medium from two 18 : 2-supplemented cultures. Conversely, the 18 : 2-supplemented cells were resuspended in medium derived from nonsupplemented cells. The other four cultures served as controls and were resuspended in the same medium from which they had been separated. After 30 min of re~quilibration at 37’C, the cultures were pulse labeled for 1 h with faH]acetate and harvested, and their lipids were isolated and counted as described in Materials and Methods. The results represent the mean of two determinations. Individual values did not deviate more than 10% from the mean values. Expt. No,

Source of cells and medium during the l-h pulse period

dpm/l O6 cell a Total lipid

Cells 18 : 2

TotaI acyl groups

Sterol

23 626 (100%) 6955 (29%) 7746 (33%) 3135 (13%:)

12 620 (100%) 3495 (28%) 5 006 (40%) 1 051 (8%)

Medium Control

18 12

Control

I

_

+

_

+

2 3 4

+ _ +

f -

_ + t

+ -

36 630 10 780 13 042 4321

(lOO’&) (29%) (36%) (12%)

a k’igures in parenthesis are dpm/lOe cells as a percent of values in Expt. No. 1.

536 TABLE IV LIPID COMPOSITION OF NON-SUPPLEMENTED CELLS AFTER INCUBATION IN MEDIUM FROM CULTURES GROWN FOR 48 h WITH 18 : 2 Eight cultures of L-fibroblasts were treated as described in Table IV. The lipids from cells in Expts. 1 and 3 (see Table 111)were extracted and isolated as described in Materials and Methods. The methyl esters prepared from the phospholipid and triacylglycerol fraction were injected into a gas chromatograph and their peaks were identified by comparison of their retention time to known standards. 25 pg of triheptadecanoin was added during lipid extraction to serve as an internal standard for triacylglycerol qLlantitation. The results are the mean of two determinations. Expt. No.

Phospholipid fatty acid composition (wt.%)

Triacylglycerol Gg/ 106 cells)

14 :o

16 :o

16 : 1

18 : 0

18 : 1

18 : 2

trace trace

17.3 16.0

4.0 2.1

10.9 11.9

66.3 66.7

1.5 2.7

___-1 3

mine whether molecules such as these accumulating in the medium of cells receiving exogenous fatty acid were responsible for the observed decrease in [3H]acetate incorporation into lipids. Eight cultures were grown simultaneously. To each of four of these was added a single dose of 5 nmol albumin sodium linoleate; the other four served as untreated control cultures and received only albumin. After 47 h of growth the medium from two of the cultures supplemented with 18 : 2 was isolated by centrifugation and used to resuspend cells similarly harvested from two non-supplemented cultures. The cell-free control medium was used to resuspend cells which had previously been supplemented with 18 : 2. The other four cultures were also centrifuged, and the cells were resuspended in the medium from which they had been separated. All cultures were then pulse labeled for 1 h with [3H]acetate. The results from this experiment showed clearly that medium recovered from cells incubated with 18 : 2 immediately depressed the incorporation of [jH]acetate into both the sterol and acyl group classes. of control cells (Table III). That this decline was not due to the presence of linoieate or other fatty acids was demonstrated by analyzing the growth media of supplemented cells. Fatty acids were extracted from acidified medium with butanol. Heptadecanoic acid was added as an internal standard. At the start of the incubation 50 ml medium was found to contain 4.5 rt: 0.1 pmol linoleate and less than 0.1 pmol of all other fatty acids. After 48 h of cell growth the medium

1.52 1.62

contained only 0.25 + 0.01 pmol linoleate, with little change in the concentration of other fatty acids. Furthermore, analysis of the lipids from control cells incubated with medium from cells previousIy treated with 18 : 2 revealed no change in phospholipid fatty acid composition or triacylglycerol content (Table IV). A significant fraction of the inhibition of [3H]acetate incorporation into cell lipids, therefore, results from metabolites which diffuse into the medium during exposure to exogenous fatty acids. These may regulate enzymes required for [3H]acetate utilization or merely dilute the radioactively labeled precursor pools. Failure of the medium from non-supplemented cells to restore fully f3HJacetate incorporation into lipids of previously supplemented cells to the level of control cells may result from high intracellular levels of these metabolites. The latter would be maintained by oxidation of fatty acids continuously released from triacyl~ycerol droplets within the cytoplasm. Preliminary experiments show that the addition of @-hydroxybutyrate and acetoacetate to cultures of L-fibroblasts resulted in a very rapid reduction of acetate incorporation into sterol and acyl groups to 50% of control levels. Discussion Studies with mammalian cells in vitro have shown that most of their fatty acids and sterol are derived from the serum used to supplement the medium in which they are cultured [25]. However, when grown

531 in either serum-free

medium or delipidized serum all or most of the lipids are synthesized from endogenous acetyl-CoA [6,23]. Under these latter conditions the addition of purified fatty acids causes alterations in both the cell lipid acyl group composition, and the rate of synthesis of sterol and fatty acids from radioactively labeled acetate [1,6,7]. It was shown in the present study that the manner in which the fatty acids are supplied to the cells affects the amount of [3H]acetate incorporated into lipids during a l-h pulse. Administration of 18 : 2 by continuous infusion of 24 h reduced the incorporation of [3H]acetate into fatty acyl groups to a greater extent than the single dose method. During the same time period however, cultures supplemented with fatty acid using the single dose method incorporated slightly less [3H]acetate into sterol than did cultures receiving fatty acid by infusion. At 24 h the labeling pattern was, therefore, sensitive to the method of administration of the fatty acid, and the effect on the endogenous biosynthesis of acyl groups and sterol were distinguishable. By 48 h both methods of administering 18 : 2 resulted in an equivalent inhibition of [3H]acetate incorporation into lipids. Interestingly, the radioactivity incorporated into sterol and acyl groups declined by nearly the same percentage. This parallel decrease had been noted previously when confluent monolayers of L-929 cells were grown in the presence of palmitic acid or cholesterol [6]. In that study, using homogenates prepared from cells exposed to fatty synthetase was shown to be acid, acetyl-CoA inhibited. A role for fatty acid (derived from exogenous lipid) as an allosteric inhibitor was proposed [6]. In the present study, however, with little exogenous fatty acid remaining, the medium from cultures grown with 18 : 2 for 48 h when transferred to control cells, caused a 65% reduction in the incorporation of [3H]acetate into their lipids within 1.5 h of the medium swap. It appears, therefore, that metabolites derived from the oxidation of 18 : 2 rather than 18 : 2 itself either inhibited acetylCoA synthetase or diluted the radioactively labeled acetate. That these same mechanisms may also operate intracellularly was supported by the observation that 1.5 h after the addition of control medium to 18 : 2 supplemented cells [3H]acetate incorporation had not returned to control levels.

Acknowledgements This work was supported by USPHS Grant AG00747. The authors thank Dr. Eveline E. Schneeberger for her critical reading of the manuscript. References 1 Albert% A.W., Ferguson, K., Hennessy, S. and Vagelos, P.R. (1974) J. Biol. Chem. 249,5241-5250 McGee, R. and Spector, A.A. (1975) J. Biol. Chem. 250, 5419-5425 Goodridge, A.B. (1973) J. Biol. Chem. 248,4318-4326 Tsai, P.Y. and Geyer, R.P. (1977) Biochim. Biophys. Acta 489,381-389 5 Majerus, R.A. and Majerus, P.W. (1973) J. Biol. Chem. 248,8392-8401 6 Howard, B.V., Howard, W.J. and Bailey, J.M. (1974) J. Biol. Chem. 249,7912-7921 7 Mulligan, J.J., Lynch, R.D., Schneeberger, E.E. and Geyer, R.P. (1977) Biochim. Biophys. Acta 470,92-103 8 Lynch, R.D. and Liffman, S.M. (1981) Proc. Sot. Exp. Biol. Med., in the press 9 Lynch, R.D. (1980) Lipids 15,412-420 10 Lynch, R.D., Schneeberger, E.E. and Geyer, R.P. (1976) Biochemistry 15,193-200 11 Albutt, E.C. (1966) J. Med. Lab. Technol. 23,61-82 12 Christophersen, B.O. (1968) Biochim. Biophys. Acta 164, 35 -46 13 Ishikawa, T.T., MacGee, J., Morrison, J.A. and Glueck, C.J. (1974) J. Lipid Res. 15,286-291 14 Schneeberger, E.E., Lynch, R.D. and Geyer, R.P. (1971) Exp. Cell Res. 69,193-206 15 Marinetti, H.V., Erblad, J. and Stotz, E.J. (1958) J. Biol. Chem. 233,562-565 16 Folch, J., Lees, M. and Stanley, G.H.S. (1957) J. Biol. Chem. 226,497-509 17 Kaulen, H.D., (1972) Anal. Biochem. 45,664-667 18 Gitler, C. (1972) Anal. Biochem. 50,324-325 19 Nebb, R.A. and Mettrick, D.F. (1972) J. Chromatgr. 61,75-80 20 Glass, R.L. (1971) Lipids 6,919-925 21 Kandutsch, A., Chen, H.W. and Heiniger, H.J. (1978) Science 201,498-501 22 Chau, I.Y. and Geyer, R.P. (1978) Biochim. Biophys. Acta 542,214-221 23 Geyer, R.P. (1967) in Lipid Metabolism in Tissue Culture Cells (Rothblat, G.H. and Kritchevsky, D., eds.), pp 3% 47, The Wistar Institute Symp. Monogr. No. 6, The Wistar Institute Press, Philadelphia 24 Daae, L.N.W. and Bramer, J. (1970) Biochim. Biophys. Acta 210,92-104 25 Geyer, R.P., Bennett, A. and Rohr, A. (1962) J. Lipid Res. 3,80-83