Human vascular endothelial cells synthesize and release 24- and 26-carbon polyunsaturated fatty acids

Biochimica Elsevier

et Biophysics

171

Acta, 795 (1984) 171-178

BBA 51712

HUMAN VASCULAR ENDOTHELIAL CELLS SYNTHESIZE 26CARBON POLYUNSATURATED FATTY ACIDS MIRIAM D. ROSENTHAL Departmenr

of Biochemistry,

* and JENNY Eastern

AND RELEASE

24- AND

R. HILL

Virginia Medical School, P.O. Box 1980, Norfolk,

VA 23501 (U.S.A.)

(Received January 25th, 1984)

Key words: Polyunsaturated

fatty acid; Arachidonic

acid metabolite;

(Human endothelial cell)

Vascular endothelial cells from human umbilical vein readily incorporate [ r4Cjeicosatrienoate (20: 3 (n - 6)) and desaturate it to synthesize [ “C]arachidonate (20 : 4) and I r4C]docosatetraenoate (22 : 4). Both substrate and metabolites are extensively esterified in cellular phospholipids and triacylglycerol. After these cells are incubated for 24-48 h with 4.5 FM [ 14C]20: 3 in culture medium plus 10% fetal bovine serum, the medium contains a number of radiolabeled free fatty acids. In addition to arachidonate and docosatetraenoate, these include still longer-chain polyunsaturated fatty acids. We have identified these as 24 : 4, 24 : 5, 26 : 4 and 26 : 5 by both radio-gas chromatography and HPLC. Although the 24- and 26-carbon polyunsaturated fatty acids represent a negligible percentage of cellular r4C-labeled fatty acids, they are each present in the medium at a concentration of lo-40 nM, whereas [‘4C]arachidonate is 60-100 nM. In particular, products of A4 desaturation are a significant component of radiolabeled polyunsaturated fatty acids in medium but not in the cells. Since docosapolyenoic fatty acids have recently been shown to give rise to biologically active oxygenated derivatives, the selective release and possible subsequent metabolism of even longer polyunsaturated fatty acids warrants further investigation.

Introduction Linoleic acid (18 : 2) provided in the mammalian diet is utilized to synthesize longer-chain (n - 6) polyunsaturated fatty acids [l]. These include 8,11,14-eicosatrienoate (20 : 3) and arachidonate (5,8,11,14-20 : 4) which are the immediate precursors of the l- and 2-series of prostaglandins and a variety of lipoxygenase products [2,3]. Many tissues, including testis, brain, and adrenal, also contain significant amounts of 7,10,13,16-docosatetraenoate (22 : 4) and 4,7,10,13,16-docosapentaenoate (22 : 5) [4]. Al* To whom correspondence should be addressed. Abbreviations: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Fatty acids are designated by the number of carbons: : number of double bonds, i.e., 22 : 4, docosatetraenoate. 0005-2760/84/$03.00

0 1984 Elsevier Science Publishers B.V.

though 22-carbon dihomo-prostaglandin metabolites can be produced in the kidney [5], the functions of the longer chain polyunsaturated fatty acids have not been fully elucidated. Studies in our laboratory have shown that vascular endothelial cells derived from human umbilical vein synthesize both arachidonate and docosatetraenoate from [ l4 Cllinoleate; these radiolabeled polyunsaturated fatty acids are then found esterified in both cellular phospholipids and triacylglycerols [6]. Endothelial cell cultures also readily incorporate exogenous arachidonate, and, when stimulated with thrombin, bradykinin, or calcium ionophore, release previously incorporated arachidonate and synthesize prostacyclin [7]. In order to determine whether these cells also released endogenously elongated and desaturated polyunsaturated fatty acids, we examined the spent

172

culture medium after cells were grown in medium supplemented with [‘4C]eicosatrienoate. Surprisingly, we found that the medium contained not only [‘4C]arachidonate and [‘4C]docosatetraenoate, but also radiolabeled 24- and 26-carbon tetraenoic and pentaenoic fatty acids. Traces of these very-long-chain polyunsaturated fatty acids are also identifiable in cellular glycerolipids. This report presents the initial characterization of the synthesis of these tetracosaand hexacosapolyenoic acids by vascular endothelial cells and their selective release into the culture medium. Materials and Methods Hepes-buffered Medium 199, fetal bovine serum and other cell culture reagents were obtained from GIBCO, Grand Island, NY, collagenase (type 1A) from Sigma Chemical Co., St. Louis, MO., and gelatin from J.T. Baker, Phillipsburg, NJ. Endothelial cell growth supplement was obtained from Collaborative Research, Waltham, MA. [14C] Eicosatrienoate (54.9 mCi/mmol) and [i4C]arachidonate (51.6 mCi/mmol) were obtained from New England Nuclear and stored in hexane at -20°C under nitrogen. Purity (greater than 98%) was verified by both thin-layer and gas-liquid chromatography. All solvents were reagent grade. Ceil culture Endothelial cells were obtained from human umbilical veins according to the method of Jaffe [8]. Cultures were maintained as described previously [6]; medium with 10% fetal bovine serum was used throughout. Homogeneity of the endothelial cell cultures was verified by morphological criteria and by the presence of factor VIII antigen [8]. For each experiment, aliquots of i4C-labeled fatty acid solutions were gently evaporated to dryness under nitrogen and redissolved in 95% ethanol which was added directly to fetal bovine serum before addition of the latter to the culture medium [9]. The final concentration of 14C-labeled fatty acid in medium was usually 4.5 PM. The fetal bovine serum, when used at 10% contributed 23 PM free fatty acid to the culture medium. Lipid extraction Culture media were extracted

in 3 vol. acetone/

ethyl acetate (1 : 2, v/v) as described by Slayback et al. [lo]. In some experiments, duplicate samples of medium were extracted by a modification of the method of Bligh and Dyer [ll] after acidification with formic acid [12]. 10 pg heptapentadecanoic acid (17: 0) was added to each 4 ml of culture medium as a carrier. Endothelial cells were harvested by trypsinization and extracted with acetone/ethyl acetate as described previously [6]. All lipid extracts were stored under nitrogen at - 20 o C. Medium lipid extracts were separated on Silica Gel H thin-layer chromatographic plates developed in petroleum ether/diethyl ether/acetic acid (82: 18 : 1, v/v). Neutral lipid standards in parallel lanes were visualized with a few drops of methanolic iodine. Free fatty acids were then extracted from the silica gel in chloroform/ methanol/diethyl ether (1 : 1 : 1, v/v). Aliquots of each medium lipid extract were separated on a second thin-layer plate and neutral lipid and phospholipid spots scraped for scintillation counting [91. Gas-liquid chromatography Free fatty acid samples were methylated using BCl, in methanol. Fatty acid methyl esters were prepared from cellular lipid extracts using methanolic base. Samples were analyzed by gasliquid chromatography as described previously [6]. The distribution of 14C-labeled fatty acid methyl esters was determined with a Packard 894 Gas Flow Proportional Counter [6]. High-performance liquid chromatography (HPLC) Samples of both the free fatty acids from the medium and fatty acid methyl esters were dissolved in acetonitrile and separated on an HPLC system (Waters Assoc.) consisting of 6000A pumps, U6K injector, 660 gradient programmer and a 7.8 mm x 30 cm reversed-phase column (~Bondapak C,,). Fatty acid methyl esters were eluted using a non-linear gradient (No. 5 on the Waters 660) of 60-100% acetonitrile (solvent B) in 15% methanol/85% aqueous 3 mM H,PO, (solvent A) for 1 h at 2 ml/mm [16]. The gradient was started 10 min after injection. Free fatty acids were eluted similarly using gradient No. 7 and starting with 55% acetonitrile. The effluent was monitored for absorbance of polyunsaturated fatty acids at 215

173

nM on a Schoeffel SF 770 detector. Fractions were collected at 0.5 min intervals using a Pharmacia FRAC-100 fraction collector. Aliquots of each fraction were used for liquid scintillation counting. The fractions containing radiolabeled fatty acids were acidified with HCl, diluted 3-fold with water, and extracted three times with petroleum ether. The fatty acids were then methylated and analyzed by radio-gas chromatography. Results

When human umbilical vein endothelial cells in the presence of [14C] are incubated eicosatrienoate, the culture medium contains a mixture of radiolabeled free fatty acids (Fig. 1). These include arachidonate, docosatrienoate and the desaturation-elongation docosatetraenoate, products of eicosatrienoate which have been characterized in the endothelial cell phospholipids and triacylglycerol [6,7]. Vascular endothelial cell cultures also demonstrate some P-oxidation of 14Clabeled fatty acids and reutilization of radio-

Fig. 1. Radio-gas chromatogram of fatty acid methyl esters prepared from free fatty acids from culture medium of endothelial cells incubated with [‘4C[eicosatrienoate. Confluent primary cultures of human endothelial cells were subcultured with a 1 : 3 split ratio. Two days later, the culture medium was replaced with incubation medium containing 10% fetal bovine serum and 18.2 nmol/4 ml [l-‘4C]eicosatrienoate(l nCi/flask). After 48 h the culture medium was extracted with acetone/ethyl acetate. The free fatty acids were purified by thin-layer chromatography, and methylated. Gas-liquid chromatography was performed as described in Materials and Methods. The radioactivity tracing was obtained using an on-line Packard Gas Flow Proportional Counter; the simultaneous mass tracing is not shown.

labeled carbon in fatty acid synthesis. This is reflected in the measurable amounts of [‘4C]myristate and [‘4C]palmitate in both the medium and cells. The unanticipated aspect of the radio-gas chromatogram shown in Fig. 1 is the presence of 14C-labeled fatty acid methyl ester peaks eluting after docosatetraenoate. Based on their retention times and the fact that our substrate was [‘4C]eicosatrienoate, these late 14C-labeled fatty acid methyl ester peaks appeared to represent 24and 26-carbon polyunsaturated fatty acids. These radioactive fatty acids are consistently produced by different sets of endothelial cell cultures (n = 14 to date). We have obtained similar results when the culture medium is acidified and extracted with chloroform/methanol in lieu of the acetone/ethyl acetate procedure. In both cases, thin-layer chromatography was used to purify free fatty acids from the medium lipid extract prior to their methylation. Analyses of medium incubated for 48 h in the absence of cells produced radio-gas chromatograms with only a single peak corresponding to the initial substrate. Very-long-chain polyunsaturated fatty acids have been described in testicular tissues [13]. Grogan et al. [14,15] have investigated the accumulation of 22 : 5 (n - 6) in triacylglycerol of condensing spermatids of mice and rats and characterized synthesis of 24: 4 and 24: 5 in these preparations. Recently, Grogan has characterized 26-, 28- and 30-carbon polyunsaturated fatty acids as well [16]. Chain length of each of these longer polyunsaturated fatty acids has been verified by gas chromatographic retention time after catalytic hydrogenation to the corresponding saturated fatty acid. The number of double bonds in each was also determined spectrophotometrically after alkaline isomerization. The peaks of radioactivity in our samples, tentatively identified as 24 : 4 and 24 : 5, had retention times identical to those obtained for radiolabeled 24- and 26-carbon polyunsaturated fatty acids purified after intratesticular injections of [l14C1arachidonate. To confirm the identifications, several batches of endothelial cells were incubated with [i4C]eicosatrienoate and the resulting culture media extracted and separated by thin-layer chromatography to provide a large pooled sample of radiolabeled free fatty acids from culture medium.

174

A very small aliquot of this sample was methylated, mixed with nonradioactive fatty acid methyl esters prepared from rat testis, and cochromatographed by reversed-phase HPLC [16]. Polyunsaturated fatty acid methyl esters were detected by absorbance at 215 nM. Upon analysis of the collected fractions, radioactive peaks were found to coincide with the known mass peaks of the fatty acid methyl esters of 20 : 4, 20 : 3 plus 22 : 4 (not fully resolved), 24 : 5, 24 : 4, 26 : 5 and 26 : 4. The large pooled sample of radiolabeled free fatty acids from culture medium was then separated by reversed-phase HPLC (Fig. 2). An aliquot of each fraction was used for scintillation counting and peaks initially identified by comparison of retention time with those of free fatty acids prepared from mouse testis. The fractions corresponding to each peak of radioactivity were then

20:4

extracted and methylated. The identification of each fraction was verified by gas-liquid chromatography with both flame ionization and radioactivity detection. As seen in Fig. 2, the hydrophobic reversed-phase column separates fatty acids of equal chain length with the more polar fatty acids eluting earlier, i.e., 20 : 4 before 20 : 3. Thus, reversed phase HPLC of both free fatty acids and fatty acid methyl esters confirmed the normal phase gas chromatographic identification of radioactive 24 : 4, 24 : 5,26 : 4 and 26 : 5 in culture media of human endothelial cells incubated with [‘4C]eicosatrienoate (20 : 3 (n - 6)). The HPLC separation also permited identification of small amounts of [t4C]24 : 3. The time-course of changes in radiolabeled polyunsaturated fatty acids in the culture medium is shown in Table I. Uptake of the substrate was relatively rapid with 34% of the 14C-labeled fatty acid esterified in cellular lipids by 6 h. Although 20% of the cellular t4C-labeled acyl groups was arachidonate at this time (data not shown), analysis of the free fatty acids in the culture medium

TABLE

I

ACCUMULATION OF RADIOLABELED POLYUNSATURATED FATTY ACIDS IN THE CULTURE MEDIUM AFTER INCUBATION OF ENDOTHELIAL CELLS WITH [t4C]EICOSATRIENOATE

IJi 2414

1(1:0

14:o

1, 0

10

20

30

Fraction

40

50

60

Number

Fig. 2. Reversed-phase HPLC elution profile of free fatty acids from culture medium of endothelial cells incubated with [t4C]eicosatrienoate. Human endothelial cells were incubated with [t4C]eicosatrienoate and the culture medium extracted as described in Fig. 1. A pooled sample of free fatty acids from several sets of cultures was purified by thin-layer chromatography, dissolved in acetonitrile, and separated on a reversed-phase column as described in Materials and Methods. Starting at 45 min, 1 ml fractions were collected, and 50 ~1 of each used for scintillation counting. Subsequent analysis of peaks by radio-gas chromatography is described in the text.

Replicate flasks of first-passage endothelial cells were incubated with 18.2 nmol/4 ml [I-t4C]eicosatrienoate. At each time point, free fatty acids were extracted from the culture medium and analyzed by radio-gas chromatography. The results (expressed in pmol/4 ml) are the mean of triplicate determinations. tr, trace. “C-labeled

fatty acid

Incubation 6

time (h): 24

48

72

20:3 20:4 2213 22:4 22:5 24~4 24~5 26~4 26:5

7440

885 483 103 138 tr 67 38 -

264 391 71 177 48 170 I67 27 52

212 559 43 206 183 183 180 52 93

Total ’

7590

1810

1450

1780

’ Radiolabeled 14 : 0, 16 : 0, 18 : 0 and 18 : 1 are included total but not tabulated.

in the

175

indicated only one radioactive peak. By 24 h, the full spectrum of desaturation and elongation products of [‘4C]eicosatrienoate was present in the medium. Further accumulation of very-long-chain polyunsaturated fatty acids occurs through 48 h, but only very slow, if at all, after that time. Earlier studies [6,7] on A6 and A5 desaturation of 14C-labeled fatty acids by endothelial cells characterized extensive esterification of the resultant [i4C]arachidonate and [‘4C]docosatetraenoate in cellular phospholipids and triacylglycerols, but provided no evidence for the presence of 24- and 26-carbon polyunsaturated fatty acids in cellular lipids. Analysis of acyl profiles of the total glycerolipids of unsupplemented cells indicated 3.5% 20 : 3, 17.9% 20 : 4 and 3.9% 22 : 4(n - 6); 24and 26-carbon polyunsaturated fatty acids were not detectable. Based upon our analyses of radiolabeled polyunsaturated fatty acids in culture media, we reexamined 14C-labeled fatty acid

methyl esters prepared from cellular lipids, employing injections of much larger samples into the gas chromatograph. Fig. 3 shows that, using this approach, small quantities of [14C]24 : 4 and [ 14C]24 : 5 are identifiable in cellular lipids. Table II compares the distribution of radiolabeled polyunsaturated fatty acids in cellular glycerolipids and free fatty acids in the medium after a 48 h incubation with [i4C]eicosatrienoate. Interestingly, when expressed as pmol/culture flask, the total amounts of radiolabeled 24- and 26-carbon polyunsaturated fatty acids in the cells and the medium are of a similar oder of magnitude. Most radiolabeled acyl groups are, however, esterified in cellular lipids with a medium/cell ratio of 0.17 for the total mixture of radiolabeled fatty acids and still lower ratios for 20 : 3, 20 : 4, and 22 : 4. By contrast, the total amount of each of the 24- and 26-carbon polyunsaturated fatty acids is greater in the medium than in cellular glycerolipids. All of the above experiments utilized subconfluent first-passage subcultures. Confluent monolayers of vascular endothelial cells have been found to differ markedly from actively mitotic cells in

L 2

2.1

TABLE

II

COMPARISON OF RADIOLABELED POLYUNSATURATED FATTY ACIDS OF ENDOTHELIAL CELLS AND CULTURE MEDIUM Endothelial

cells were incubated for 48 h with 18.2 nmol/4 ml 14C-Labeled polyunsaturated fatty acid of cellular glycerolipids and free fatty acids in the were obtained as in Figs. 1 and 3.

[ I4Cleicosatrienoate. profiles medium

I4 C-Labeled fatty acid

TIME(min)

Fig. 3. Radio-gas chromatogram of fatty acid methyl esters prepared from cellular glycerolipids after incubation with [‘4C]eicosatrienoate. Endothehal cells were incubated for 48 h with 18.2 nmol/4 ml [‘4C]eicosatrienoate as in Fig. 1. The cells were then harvested and their lipids extracted. Thin-layer chromatographic separation of an aliquot indicated that 97.3% of the radioactivity in the cellular lipids extract was in phospholipid and triacylglycerol. Fatty acid methyl esters were prepared from the remainder of the extract using methanolic base. Radio-gas chromatography as in Fig. 1.

20:3 20:4 22~3 22~4 22:5 24:4 2415 26~4 26~5 Total a

Medium

Cells

(pmol/flask)

(pmol/flask)

264 391 71 177 48 170 167 27 52

2465 3883 206 1581 26 137 43

1450

8590

26

Ratio: medium ~ cells 0.11 0.10 0.34 0.12 1.8 1.2 3.9 _ 2.0 0.17

* Radiolabeled 14 : 0, 16 : 0, 18 : 0 and 18 : 1 are included total but not tabulated.

in the

176

their synthesis of both angiotensin-converting enzyme [17] and prostacyclin [18]. We therefore supplemented confluent primary endothelial monolayers with [‘4C]eicosatrienoate (20 : 3 (n - 6)) and found that the resultant culture medium contained radiolabeled very-long-chain polyunsaturated fatty acids with a profile qualitatively similar to that obtained from actively growing cells (data not shown). Cells supplemented with [‘4C]arachidonate also produced radiolabeled 24- and 26-carbon tetraenoic and pentaenoic fatty acids. It appeared that addition of [‘4C]arachidonate rather than [‘4C]eicosatrienoate resulted in somewhat greater accumulation of the very-long-chain fatty acids. To investigate this further we increased the concentrations of exogenous [i4C]eicosatrienoate, thus substantially increasing the pool of radiolabeled acyl groups in cellular lipids. Much of the incorporated [ 14C]eicosatrienoate was desaturated. Use of 60 ,uM rather than 5 FM [‘4C]20: 3 increased intracellular [ l4 Cl20 : 4 and [ I4 Cl22 : 4 from 4.07 and 1.37 to 25.0 and 8.4 pmol/flask, respectively. Analysis of the resultant culture medium (Table III) indicates a marked increase in [14C]20 : 4 and TABLE

III

EFFECT OF INCREASED CONCENTRATIONS OF EXOGENOUS EICOSATRIENOATE ON ACCUMULATION OF RADIOLABELED POLYUNSATURATED FATTY ACIDS IN THE MEDIUM Subconfluent first-passage endothelial cells were incubated for 48 h with 1 pCi/flask [‘4C]eicosatrienoate plus sufficient nonlabeled 20: 3 to provide the indicated concentrations. Radiolabeled free fatty acids were then extracted from the culture medium and analyzed as in Table I. Results are expressed as pmol fatty acids per 4 ml medium. 14C-Labeled fatty acid 20:3 20:4 22:3 22:4 24:3 24:4 24:5 26:4 26:5 Total

Initial concentration 20

5 182 356 89 145 117 74 16 47

718 902 264 398 65 190 159 127 99

1090

3111

of [14C]20: 3 (PM): 60 6269 3860 1760 1050 371 448 309 262 46 15444

[14C]22 : 4 with increased initial concentrations of [ 14C]20 : 3. There is also a very large increase in [14C]22:3, reflecting direct elongation of the increased eicosatrienoate without prior desaturation. By contrast, the increases in radiolabeled 24: 4 and 24 : 5 are relatively modest. These results suggest that, unlike 22: 3, the 24- and 26-carbon polyunsaturated fatty acids are normal metabolites of vascular endothelial cells grown with low concentrations of eicosatrienoate or arachidonate. Discussion These results provide evidence that cultures of endothelial cells from human umbilical vein further desaturate and elongate exogenous polyunsaturated fatty acids to produce 24- and 26-carbon tetraenoic and pentaenoic acids. Relative to other polyunsaturated fatty acids, these very-long-chain fatty acids are disproportionately released into the culture medium, so that with time they each represent 5-15s of the radiolabeled free fatty acid pool. We have also found that cells previously labeled with [ “C]eicosatrienoate or [ i4C] arachidonate continue to release these radiolabeled tetracosa- and hexacosa-polyenoic acids into nonsupplemented culture medium. Synthesis of 24- and 26-carbon tetraenoic and pentaenoic fatty acids has been characterized in testis where these polyunsaturated fatty acids accumulate in sizeable amounts, especially in triacylglycerol and cholesterol esters [14,15]. Although fatty acid composition appears to play an important role in normal differentiation of the germinal cells, the biochemical roles of very-longchain polyunsaturated fatty acids remains to be clarified. Small amounts of 24: 4 have also been described in triacylglycerol of muscle cells and fibroblasts fed very high levels (120 PM) of 22:4 [19]. To our knowledge, this is the first report of synthesis of tetracosapolyenoic fatty acids in a cell system where the products are extensively released as extracellular free fatty acids rather than primarily remaining esterified in cellular glycerolipids. Although we have characterized the accumulation of 24- and 26-carbon polyenoic fatty acids in culture medium, the present study does not permit estimation of net rates of synthesis. Since these very-long-chain polyunsaturated fatty acids can be

177

esterified in cellular glycolipids, there may be a dynamic equilibrium between cellular incorporation and release similar to that found for other free fatty acids in medium containing serum protein [20]. 7,10,13,16-Docosatetraenoate can be chainshortened to provide arachidonate [4]; data based on the use of radiolabeled 20: 3 or 20 : 4 cannot determine the extent of retroconversion of 24- and 26-carbon polyunsaturated fatty acids in our system. There is also measurable P-oxidation of fatty acids in endothelial cell cultures as reflected by appearance of radiolabeled carbon in palmitate and stearate, and in non-lipid material. P-Oxidation of the very-long-chain polyunsaturated fatty acids might also serve to limit their accumulation in the culture medium. Perhaps the most interesting metabolic fate for the very-long-chain polyunsaturated fatty acids would be conversion to more polar derivatives with possible biologic activity. Sprecher et al. [S] have shown that, in the kidney medulla, 7,10,13,16-docosatetraenoic acid can be catabolized by fatty acid cyclooxygenase into a full complement of 22-carbon prostaglandins and thromboxane. The dihomoPGE, was then shown to stimulate adenylate cyclase in the interstitial cells that synthesize it. Furthermore, a wide variety of polyunsaturated fatty acids are substrates for platelet lipoxygenase [21]. Thus, Aveldano and Sprecher [22] have recently suggested that, after release from tissue lipids, all naturally occurring polyenoic acids may serve as substrates for conversion to oxygenated metabolites with the potential to modify cell function. Further investigation will be necessary to characterize the mixture of more polar radiolabeled metabolites in endothelial cell culture media. The present findings are also of interest with respect to products of A4 desaturation. Most cells in culture have been reported to lack measurable A4 desaturase activity [20]. Studies with endothelial cells have usually detected only trace amounts of radiolabeled 4,7,10,13,16-docosapentaenoate in cellular lipids [6,7]. Spector et al. [23] have, however, recently reported synthesis of [r4C]22 : 6 from [‘4C]20 : 5( n - 3). Our studies of medium free fatty acids also indicate that vascular endothelial cells do exhibit A4 desaturase activity producing both 22 : 5 and longer pentaenoic fatty acids. Tetra-

cosapentaenoate shows the highest observed medium/cell ratio of any radiolabeled fatty acid. Additional studies will be required to determine whether, in these cells, A4 desaturation of longchain polyunsaturated fatty acids is directly linked to their release from the cell. Acknowledgements We wish to thank Dr. W. McLean Grogan for his invaluable assistance in the identification of the 24- and 26-carbon polyunsaturated fatty acids. He kindly supplied a radio-gas chromatography standard of radiolabeled polyunsaturated fatty acids from rat testicular lipids and provided the facilities and expertise for the HPLC separations. We also thank the staff and nurses at the Labor and Delivery Unit of Norfolk General Hospital for providing the umbilical cords. This work was supported by a Grant-in-Aid from the American Heart Association with funds contributed in part by the AHA, Virginia Affiliate. References 1 Jeffocat, R. (1979) Essays Biochem. 15, 1-36 2 Moncada, S. and Vane, J.R. (1979) N. Engl. J. Med. 300, 1142-1147 3 Samuelsson, B. (1981) Prog. Lipid Res. 20, 23-30 4 Sprecher, H. (1981) Prog. Lipid Res. 20, 13-22 5 Sprecher, H., Van Rollins, M., Sun, F., Wyche, A. and Needleman, P. (1982) J. Biol. Chem. 257, 3912-3918 6 Rosenthal, M.D. and Whitehurst, M.C. (1983) Biochim. Biophys. Acta 750, 490-496 7 Spector, A.A., Kaduce, T.L., Hoak, J.C. and Fry, G.L. (1981) J. Clin. Invest. 68, 1003-1011 8 Jaffe. E.A. (1980) Transplant Proc. 12, Suppl. 1, 49-53 9 Rosenthal, M.D. (1981) Lipids 16, 173-182 10 Slayback, J.R.B., Cheung, L.W.Y. and Geyer, R.P. (1977) Anal. Biochem. 83, 872-384 11 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37,911-917 12 Daniel, L.W.. King, L. and Waite, M. (1981) J. Biol. Chem. 256, 12830-12835 13 Davis, J.T. and Coniglio, J.G. (1966) J. Biol. Chem. 241, 610-612 14 Grogan, W.M. and Lam, J.W. (1982) Lipids 17, 605-611 15 Grogan, W.M. and Huth, E.G. (1983) Lipids 18, 275-284 16 Grogan, W.M. (1984) Lipids 19, 341-346 17 DelVecchio, P.J. and Smith, J.R. (1981) J. Cell. Physiol. 108, 337-345 18 Eldor, A., Vlodavsky, I., Hy-Am, E., Atzmon, R., Weksler, B.B., Raz, A. and Fuks, Z. (1983) J. Cell. Phys. 114, 179-183

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19 Gavino, V.C., Miller, J.S., Dillman, J.M., Mile, G.E. and Cornwell, D.G. (1981) J. Lipid Res. 22, 57-62 20 Spector, A.A., Mathur, S.N., Kaduce, T.L. and Hyman, B.T. (1981) Prog. Lipid Res. 19, 155-186 21 LeDuc, L.E., Wyche, A., Sankarappe, S.K., Sprecher, H. and Needleman, P. (1982) Mol. Pharmacol. 19, 242-247

22 Aveldano, M.I. and Sprecher, H. (1983) J. Biol. Chem. 258, 9339-9343 23 Spector, A.A., Kaduce, T.L., Figard, P.H., Norton, K.C., Hoak, J.C. and Czervionke, R.L. (1983) J. Lipid Res. 24, 1595-1604