Metabolism of 8,11,14,17-eicosatetraenoic acid by human platelet lipoxygenase and cyclooxygenase

94

BBA 52547

Metabolism of 8,11,14,17-eicosatetraenoic

acid by human platelet tipcvxygenase

and cyclooxygenase

Maria Mbnica Careaga and Howard Sprecher The Department of Physiological Chemistry, College of Medicine, The Ohio Stare University, Columbus, OH (U.S.A.)

(Received 1 December 1986)

Key words: Lipoxygenase; Cyclooxygenase; Hydroxy fatty acid; 8,11,14.17-Icosatetraenuic acid; (Human platelet)

Human plateIets metabolize 8,11,14,17-ei~osate~aenoi~ acid prim~ily into 12-hydro~y-~,l~~l4,17-eicosatetraenoic acid. Several other hydroxy acids were also produced in small amounts via an indometbacin insensitive pathway. Platelet cyclooxygenase metabolized this acid only into 12-hydroxy-8,1CJY14-heptadecatrienoic acid. It was not possible to detect any cyclic products even though vesicular gland cyclooxygenase metabolizes this (n - 3) acid to 17,18-dehydroprostaglandin E, (Oliw, E.H., Sprecher, H. and Hamberg, M. (19%) J. Biol. Chem. 261, 26752683).

Platetet p~osph~l~p~ds are characterized by a high level of arachidonic acid [lf, These cells also contain smaller amounts of other (n - 6) acids as well as long chain 22-carbon (n -- 3) acids. When (n - 3) acids are added to the diet some of the arachidonic acid is replaced by 20 : S(n - 3) and other (n - 3) acids [2]. Dietary linolenate, 18 : 3(n - 3), is metabolized to long-chain (n - 3) fatty acids via a pathway in which 6,9,12,35-18:4 and 8,11,14,17-20 : 4 are obligatory intermediates f3]. Platelets metabolize (n - 6) and (n - 3) acids quite differently. Arachidonic acid ]4], 8,11,14-20 : 3 [S]. 7,t0,13,IS-22 : 4 [6] and 4,7,10,13,l6-22 : 5 [7j are

Abbreviations: SHETE, S-hydroxy-6,8,11,14-eicosatetraenoic acid; I2-HETE, 12-hydroxy-5,8,10,14-eicasatetraenoic acid; IS-NETE, 15-hydroxy-5,8,11,13-eicosatetraenoic acid; 12” HPETE, 12-bydsoperoxy-5,8,10,14-eicosatetraenoic acid; 15HPETE, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. Correspondence: H. Sprecher, Department of PhysioXogical Chemistry, The Ohio State University, 333 W. l&h Avenue. Columbus, OH 43224 U.S.A. ~~-276~/‘%7/$03.50

6 1987 E&tier

all (n - 6) acids and they are all metabolized into t~omboxa~es. In addition, each of these acids is metabolized primarily to a single hydroxy fatty acid by the lipoxygenase pathway. Platelets also metabolize arachidonic acid into small amounts of 1%HETE 181. 5,8,11,14,17-Eicosapentaenoic acid is an (n ^- 3) acid and it is metabolized into thromboxane A3 and 12-hydroxy-5,8,10,14,17eicosapentaenoic acid [9]. Conversely, the Xongchain (n - 3) acids, 7,10,13,16,19-22 : 5 [lo] and 4,7,10,13,16,19-22 : 6 [fl] are metabolized only into an isomeric pair of II- and f4-hydroxy acid isomers. This later finding suggested that platelets may contain a second fipoxygenase which only utilizes long-chain (n - 3) acids as a substrate, Hamberg [12] however showed that 6,9,12-18 : 3, an (n - 6) acid, was metabohzed by platelets into lo-hydroxyd,8,12-octadecatrienoic acid and 13hydroxy-6,9,11=octadecatrienoic acid via a Iipoxygenase pathway and to lo-hydroxy-6,&pentadecadienoic acid by cyclooxygenase. This I&carbon (n - 6) acid, like f&9,12,15-18 : 4 and 8,11,14,17-20 : 4, is a minor membrane component but it is an obligatory ~nterm~iate in the conversion of dietary linoleate to arachidonate 133. We

Science Publishers B.S. ~~~orncd~ca~Division)

95

thus prepared 6,9,12,15-[l-14C]18 : 4 and 8,11,14,17-]1-14C]20 : 4 to further define what differences exist in the metabolism of essential (n - 3) and (n - 6) fatty acids by platelets. Materials and Methods Fatty acids. 6,9,12,15-[1-‘4C]Octadecatetraenoic acid (34 Ci/ mol) and 8,11,14,17-[1-14C]eicosatetraenoic acid (39 Ci/mol) as well as the corresponding noma~oactive acids were made by total synthesis using estab~shed procedures ]13]. In~bfftion of platelets. Blood was drawn from healthy volunteers who had not taken any medication for two weeks prior to collection. Blood was collected in 7S%(v/v) 77 mM disodium EDTA and centrifuged at 200 x g for 15 min. Platelets were recovered by centrifuging the platelet-rich plasma at 2000 x g for 20 mm at 4’ C. The platelets were suspended in 0.15 M NaC1/0.15 M Tris-HCI (pH 7.4)/77 mM disodium EDTA (90 : 8 : 2, v/v). After centrifugation at 2000 X g for 15 min the platelets were resuspended at a concentration of 3 . 108fml for analytical studies and at 6 1lOafm1 for large scale incubations in order to isolate metabolites. Platelets (0.5 ml) were preincubated at 37 ’ C in a shaking-water bath for 2 min prior to the addition of the sodium salt of the fatty acid (15 Ci/mol). Reactions were terminated by addition of 2 vol. of 2 M formic acid. The products were recovered by extracting three times with 3 vol. of ethyl acetate. The pooled ethyl acetate extracts were washed with 1 ml of water. The ethyl acetate was removed under a stream of N, and the residue was dissolved in methanol. High-performance liquid chromatography. HPLC was carried out with a DuPont system equipped with an 870 pump, 8800 series gradient controller and a variable wavelength detector. Radioactivity was quantitated with a Model HP radioactive flow detector (Radiomatic Instruments and Chemical Co., Inc. Tampa, FL). Reverse phase chromatography was carried out using a 0.46 X 25 cm Zorbax ODS column which was preceded by a 5 x 0.46 cm guard column packed with Permaphase ODS (DuPont, Wilmington, DE). Separations were carried out at 35” C using a gradient of acetonitrile in water which was

adjusted to pH 2.4 with H,PO, (Fisher Chemical Co., Cmcinnati, OH). The initial solvent was 38% acetonitrile in water. After 20 min, the concentration of acetomtrile was linearly increased to 42% over 5 min. The instrument was then programmed to 55% acetonitrile over 30 min, using the -2 exponent. Finally, remaining substrate was removed by increasing the acetonitrile concentration to 100% over 10 min. The flow rate through the column was 1.5 ml/mm, while the flow rate of Scinti Verse LC (Fisher Chemical Co.) was 4.5 ml/min. In order to isolate metabolites 6 *10’ platelets/ ml were incubated for 10 min, with 80 PM 1-14Clabeled 8,11,14,17-20:4 (3 Ci/mol). The HPLC effluent was collected in siliconized tubes containing a small amount of sodium bicarbonate. Acetonitrile was removed at room temperature with a stream of N,. The pH was adjusted to about 3 by addition of formic acid. Metabolites were recovered by extraction with ethyl acetate. The ethyl acetate was removed under N, and metabolites were esterified by reaction with an ethereal solution of diazomethane. Normal phase HPLC was carried out at 35 *C with a Zorbax Sil column (0.46 X 25 cm). The methyl esters of metabolites were injected in 0.3% isopropyl alcohol in hexane and they were separated by isocratic elution with this solvent. The flow through the column was 1.5 ml/mm, while the flow rate of Scinti Verse LC was 3 ml/mm. Gas-chromatography-mass spectrometry. The methyl esters of metabolites isolated from normal phase HPLC were converted to t~ethylsilyl ethers by reaction with 10 ~1 of ~,~-bis(t~methylsilyl)trifluoroacet~ide (Pierce Chemical Co., Rockford, IL) in an equal volume of pyridine for 1 h at 50°C. Methyl esters were hydrogenated by bubbling H, for 30 s into a solution of methyl esters in 0.5 ml of methanol containing about 1 mg of platinum oxide. The reaction mixture was transferred to a siliconized Pasteur pipette packed with silicic acid and the products were recovered by eluting with methanol. Gas chromato~aphy was carried out on a Varian Vista 6000 gas chromatograph equipped with a glass column (6 ft x 2 mm internal diameter) packed with 1% SP-2100 on lOO/ZOO mesh Supelcoport (Supelco, Bellefonte, PA). Helium, 30

96

ml/mm, was the carrier gas and the temperature of the injector, oven and detector were 220, 190 and 250 ’ C, respectively. Equivalent chain lengths (ECL) were determined by comparing retention times with a saturated series of methyl esters. Mass spectrometry was carried out with a Hewlett-Packard model 5970A mass selective detector and a 5790 gas chromatograph. The oven contained a 30 m x 0.25 mm internal diameter DB-1 J and W capillary column (Ranch0 Cordova, CA). All injections were done in the splitless mode with an oven temperature of 70 o C. The temperature of the injector was 220” C and the transfer line was 280 o C. 1 min after injection the oven was programmed at 30 Cdeg/min to 21O“C and 230 o C, respectively, for analysis of unsaturated and saturated compounds.

Fig. 1 shows a reverse-phase HPLC radiochromatogram when 8,11,14,17-[1-‘4C]20 : 4 was the substrate. The synthesis of the minor metabolites eluting at 32-36 min (fraction A), were blocked when incubations were carried out with 10 PM indomethacin. When these metabolites were collected and reanalyzed by normal phase HPLC it was also possible to detect several compounds (Fig. 2). The mass spectrum of compound I (ECL = 19.4) as shown in Fig. 3 had ions at m/z 366 (M+; 0.2%), 351 (M15; 0.8%) 335 (M - 31; 0.8%) and 297 (M - 69, loss of CH,CH,CH= CHCH;; base peak). The mass spectrum of the hydrogenated compound had ions at m/z 357 (M - 15; 2.1%), 341 (M - 31; 5.7%) 325 (M - 47; 15.4%), 301 (M- 71, loss of CH,(CH,),, base peak), 173 (CH,(CH,),CH=OSi(Me):; 78.2%) and 272 (M - 100; 19.6%, loss of CH,(CH,),CHO followed by a rearrangement of the trimethylsilyl group to the carbomethoxy carbon). This compound is thus 12-hydroxy-8,10,14-heptadecatrienoic acid. It is produced in very small

Results When 6,9,12,15-[1-‘4C]18 : 4 was incubated with platelets no radioactive metabolites were detected.

100 -

[I-k]

8.11.14.17-20:4

B

60

-

/

0

8

16

24

32

40

46

36

64

72

MINUTES

Fig. 1. Reverse-phase HPLC radiochromatogram of the metabolites produced from 8,11,14,17-[1-‘4C]eicosatetraenoic acid. Platelets (1.5. 108/0.5 ml) were incubated for 3 min with 20 gM substrate. The metabolites were removed by extracting into ethyl acetate. The ethyl acetate was removed and metabolites were dissolved in methanol for analysis.

97

phase HPLC, the radiochromatogram shown in Fig. 4 was obtained. The mass spectrum of compound I (ECL = 21.5) had ions at m/z 391 (M 15; 1.8%), 375 (A4 - 31; 1.4%), 337 (M - 69, loss of CH,CH,CH=CHCH;; base peak), 271 (12.3%) and 247 (337-90; 8.9%). The spectrum of the hydrogenated compound had ions at m/z 399 (M 15; 1.6%), 383 (M - 31; 4.0%), 367 (A4 - 47; 12.7%), 343 (M- 71, loss of CH,(CH,),; 95%), 314 (rearrangement ion involving loss of HC,(CH,),CHO; 25.5%) and 173 (CH,(CH,),CH=OSi(CH,)l, base peak. Compound I is thus 15-hydroxy-8,11,13,17-eicosatetraenoic acid. Only very small amounts of compound II were produced. The mass spectrum of this metabolite (ECL = 21.5) had a base peak at m/z = 183. This is consistent with an ion of composition CH,CH,(CH=CH),CHO=Si(CH,)l. The spectrum of the hydrogenated compound had ions at m/z 399 (1.5%), 383 (4.4%), 367 (11.6%) as well as ions at 329 (base peak) and 187 (84.2%). These later two ions correspond to +(CHj)sSiO= CH(CH,),,COOCH, and CH,(CH,),CH=OSi (CH,):. The presence of an ion at m/z = 300 (20.2%) formed by loss of CH,(CH2),CH0 further supports the conclusion that compound II is 14-hydroxy-8,11,15,17-eicosatetraenoic acid. The methyl ester of compound III had A,, 235 in methanol with E = 25 000. The mass spectrum (Fig. 5) of the methyl ester-trimethylsilyl

IO

6

FRACTION A

6 : 0 i4

2

0 c

II

n 4

h 12

20

26

36

44

MINUTES

Fig. 2. Normal phase HPLC radiochromatogram of the methyl esters of metabolites eluting between 32 and 37 min in Fig. 1.

amounts so it was not possible to obtain an ultraviolet spectrum. Since its synthesis was blocked by indomethacin it is most likely formed in a manner analogous to the way in which arachidonic acid is metabolized to 12-hydroxy-5,8,10-heptadecatrienoic acid. When the compounds eluting between 40 and 45 min (fraction B, Fig. 1) were collected, converted to methyl esters and separated by normal

297

~coocn” OSi(CH.&

r-

xl0

I 4 100

150

200

250

300

m/z Fig. 3. Mass spectrum of the methyl ester-trimethylsilyl ether of compound I in Fig. 2.

’ “i”’ MT(536t3 350

98 IO

FRACTION

B

6 N 4 X E

0. ”

4

2

=h

0

t 4

I2

20

28

36

44

52

MINUTES Fig. 4. Normal

ether (ECL 0.6%), 375 and 297 CH),CH;;

phase HPLC

radiochromatogram

of the methyl

= 21.5) had ions at m/z 391 (M - 15; (M- 31; l.l%), 316 (M - 90; 1.1%) (M - 109, loss of CH,(CH,CH= base peak). The spectrum of the hy-

esters of compounds

of the methyl

bteween

40 and 45 min of Fig. 1

drogenated compound had ions at m/z 399 (M 15; 1.2%), 383 (M - 31; 2.5%), 367 (M - 47; 8.9%), 301 (M - 113, loss of CH,(CH,);, base peak), 272 (M - 142, loss of CH,(CH,),CHO; 15.7%)

29

Fig. 5. Mass spectrum

eluting

ester-trimethylsilyl

‘I

ether of compound

III in Fig. 4

99

and 215 (CH,(CH,),CH=OSi(CH,):; 82.4%). Compound III is thus 12-hydroxy-8,10,14,17eicosatetraenoic acid in which the double bond at position 10 is most likely trapls. The base peak in the mass spectrum of compound IV (ECL = 21.6) was at m/z 223 which is consistent with an ion of the composition CH,CH,CH=CHCH,(CH=CH)2CE?o=;si(CH,>:. The spectrum of the saturated compound had ions at 399 (M - 15; 1.3%), 383 (N - 31; 2.3%), 367 (M - 47; 7.2%), 287 (M - 127, loss of CH,(CH,)& base peak), 258 (M- 156, loss of CH J(CH,),CHO; 16.1%) and 229 ~CH~(CH~~sCH~i(CH~~~, 76%). Compound IV is thus ~l-hy~o~-~,l2,I4,17-ei&osatetraenoi~ acid, Compound V had ECL= 21.7 and the base peak in the mass spectrum was at m/z = 297. The spectrum of the hydrogenated compound was identical with compound III. This compound is thus also a 12-hydroxyeicosatetraenoic acid in which the double bonds are most likely at positions 8,10,14,17. The radioactive pattern (Fig. 4) as well as the absorption at 235 nm with the HPLC detector (not shown)? suggested that pedk VI contained more than one compound. It was not possible to obtain a mass spectrum of the unsaturated compounds. When this fraction was hydrogenated and analyzed by mass sp~tromet~ it was possible to identify two hydroxy acids which separated on the 30 m capillary column. The first component was the trimethylsilyl ether methyl ester of 12-hydroxyeicosanoate. The mass spectrum of the second component had ions at m/z 399 (M - 15; 1.9%), 385 (M- 29 loss of *CH,CH,; 45.3%) 367 fM - 47; 14.3%), 356 (M - loss of CH,CH,CHO; 16.5%:) and 131 (CH~CH~CH~Si(CH~~~, base peak). This compound thus has its hydroxyl group at carbon 18 and suggests that the original unsaturated metabolite was probably 18-hydroxy8,11,14,16-20 : 4. The equivalent chain lengths of compounds VII and VIII were 21.6 and 22.2, resp&ctively. The spectra of both compounds had ions at m/z 297 and 263. By comparing these spectra with that reported for 5-HETE [14] it is apparent that these two ions would be produced if compounds 8 and 9 had their hydroxyl at carbon-g. The ion at m/z

297 would correspond to M - 109, loss of CH,CH,(CH=KHCH,);, while the ion at m/z = 263 would result from cleavage between carbons 7 and 8 to yield CH,CH,(CH=CHCH2), (CH~H)~CH~Si(CH~)~. The spectra of both hydrogenated compounds had ions at m/z 399 (M - 151, 383 (M - 31), 367 (M - 47), 271 (CH,(CH,),,CHO=Si(CfI,):), 245 (M - 169, loss of CH,(CH,);,) and 216 (M - 198, loss of CH,(CH,),,CHO). These results suggest that compounds VII and VIII are both &hydroxy9,11,14,17-eicosatetraenoic acids. Table I shows the percentage composition of the hydroxy fatty acids produced from 8,11,14,1720 : 4. In contrast to the results obtained with the 22-carbon (n - 3) acids [lO,llf this (n - 3) acid was metabolized primarily into a single hydroxy fatty acid via an indomethac~-insensitive pathway. Fig. 6 shows the time-dependent metabolism of 8,11,14,17-20 : 4 at three different substrate concentrations. At ail three substrate levels there was rapid metabolism to hydroxy acids until the substrate was depleted. Conversely, the amount of 12-hydroxy-~,lO,l~~7 : 3 produced was relatively independent of the substrate level. Substrate dependent studies (not shown) established that the synthesis of hydroxy acids via the indometha~in insensitive pathway had not reached saturation levels even when 100 FIG 8,11,14,17-20:4 were incubated with platelets for 1 min.

TABLE I CUM~~ITI~N OF HYDRUXY FATTY ACIDS PRODUCED FROM 8,11,14,17-EICOSATET~E~OIC ACID Peak number from Fig. 4

Component(s)

I

15-OH-8,11,13,17-20:4 14-OH-8,11,15,17-20 : 4 12-OH-8,10,14,17-20 : 4 11-OH-8,12,14,17-20: 4 12-Ok-8,10,14,17-20: 4 12-OH-8,10,14,17-20: 4 and X3-OH-8,11,14,16-20 : 4 8-OH-9,11,14,17-20: 4 S-UH-9~11,14~17-20: 4

II III IV V VI VII VIII

% of total

2.3 0.5 90.2 2.4 0.9 1.0 1.1 1.6

36

32

28

24 s 2

20

E

f

I6 12

8

4

0

Fig. 6. Time-dependent metaboIism of various concentrations of 8~l~,14,~7-[l-‘4C]ei~satetraeno~c acid. Platelets (1.5. lOs/ 0.5 ml) were incubated with 20 PM (*I, 40 FM (or) or 80 gM (m) substrate. Metabolites were separated by reverse phase HPLC. Fraction A and fraction B denote nmols of product eluting between 32-37 and 40-45 min, respectively.

Discussion 8,11,14,17-Eicosatetraenoic acid is not a major component of membrane phospholipids. This acid is however an obligatory intermediate in the metabolism of linolenic acid to 5,8,11,14,17eicosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoic acid [3]. Ram seminal vesicle microsomes metabolize this acid primarily to 17,18-dehydroprostaglandin E,, [15]. In addition, this prosta~~d~ was isolated from ram seminal fluid [16] but it was not possible to detect it in human seminal fluid [15]. 8,11,14-Eicosatrienoic acid is metabolized by human platelets into thromboxane B,, 12-hydroxy-8,10-heptadecadienoic acid as well as into two trihydroxy acids [5]. 8,11,14,17-Eicosa-

tetraenoic acid is the analogous metabolite from the (8 - 3) fatty acid family of polyunsaturated fatty acids. This acid, like 5,8,11,14,17-20: 5 [f’i’], is a poor substrate for platelet cyclooxygenase. It was not metabolized to any polar metabolite and only small amounts of 12-hydroxy-8,10,14heptadecatrienoic acid were produced. Conversely, 8,11,14,17-20: 4 was an excellent substrate for a platelet lipoxygenase. The primary product produced via this pathway was 12-hydroxy8,10,14,17-eicosatetraenoic acid. The initial and rate-limiting step in the metabolism of arachidonic acid to 12-HETE involves abstraction of a proton from carbon-10 1181. Presumably an identical pathway is utilized for the synthesis of the isomerit hydroxy acid from 8,11,14,17-20 : 4. We previously reported that platelets metabolized 7,10,13,16-22 : 4 primarily into 14-hydroxy7,10,12,16-22 : 4, although small amounts of several isomeric hydroxy acids were also partially characterized [B]. In this study we also found small amounts of other hydroxy acids made via an indomethacin-insensitive pathway. These compounds had their hydroxyl groups at carbons 8, 11, 12, 14, I5 and 18. Presumably, the initial step in their synthesis would involve the abstraction of a proton from carbons 10, 13, 10, 16, 13 and 16, respectively. In addition, it was possible to characterize two isomeric 8-hydroxy acids and three isomeric 12-hydroxy acids. The nonenzymatic peroxidation of pol~~saturated fatty acids results in the synthesis of two pairs of racemic hydroxy acids for each methylene carbon in the substrate 1191. The stereochemistry of the hydroxyl groups and the configuration of the double bond of the hydroxy acids produced from 8,11,14,17-20 : 4 was not established. The possibility thus exists that the eight minor metabolites were produced nonenzymatically. We feel that this is unlikely, since no products were detected in zero time incubations and, in addition, these metabolites were produced in about the same ratio with several different preparatiuns of platelets. Our previuus studies with 7,10,13,16,19-X? : 5 [10] and 4,7,10,13,16,19-22 : 6 [ll], as well as those by Hamberg [12] with 6,9,12-18 : 3, showed that these three acids were metabolized into two lipoxygenase products by human platelets. These results could be explained if lipoxygenase had a

101

dual specificity for initial proton abstraction. For example, a purified reticulocyte lipoxygenase metabolizes arachidonic acid to 15-HPETE and 12-HPETE [20]. Alternatively, platelets may contain more than one Iipoxygenase. Our previous studies showed that 0.05 PM 5,8,11,14-heneicosatetraynoic acid resulted in a 50% inhibition in the synthesis of both the ll- and 1Chydroxy acids from 7,10,13,16,19-22 : 5, while 0.5 PM concentrations were required for 50% inhibition of 12-HETE synthesis from arachidonic acid [lo]. Those findings suggested that a Iipoxygenase with dual specificity metabolized 7,10,13,16,19-22 : 5 into two isomeric hydroxy acids but that this enzyme was different than the enzyme which metabolized arachidonic acid to 12-HETE. The present study shows that 8,11,14,17-20:4 is metabolized primarily to 12-hydroxy-8,10,14,17-20 : 4. It remains to be established whether the Iipoxygenase which metabolizes this acid is the same enzyme which converts arachidonic acid to the isomeric 12HETE. In addition, it is not clear why some polyunsaturated acids are metabolized to more than one hydroxy fatty acid. Although inhibition studies suggest the presence of more than one lipoxygenase, a definite answer may depend on the purification of the platelet enzymes which metabolize polyunsaturated acids. Acknowledgements

This study was supported by NIH grants AM18844 and AM-20387.

References 1 Marcus, A.J., Uflman, H.L. and Safier, L.B. (1969) J. Lipid Res. 10, 108-114 2 Siess, W., Roth, P., Scherer, B., Kurzrnann, I., B&Jig, B. and Weber, P.C. (1980) Lancet i, 441-444 3 Sprecher, H. and Lee, C.-J. (1975) Biochim. Biophys. Acta 388,113-125 4 Hamberg, M. and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. USA 71,3400-3404 5 Falardeau, P., Hamberg, M. and Samuelsson, B. (1976) B&him. Biophys. Acta 491,193-200 6 VanRollins, M., Horrocks, L. and Sprecher, H. (1985) B&him. Biophys. Acta 833, 272-280 7 Milks, M. and Sprecher, H. (1985) B&him. Biophys. Acta 835, 29-35 8 Wong, Y.-K., Westhmd, P., Hamberg, M., Granstrom, E., Chao, PH-W. and Samuelsson, B. (1985) J. Biol. Chem. 260, 9162-9165 9 Hamberg, M. (1980) Biochim. Biophys. Acta 618, 389-398 10 Careaga, M.M. and Sprecher, H. (1984) J. Biol. Chem. 259, 14413-14417 11 Aveldano, ML and Sprecher, H. (1983) J. Biol. Chem. 258, 9339-9343 12 Hamberg, M. (1983) B&hem. Biophys. Res. Commun. 117, 593-600 13 Sprecher, H. and Sankarappa, S.K. (1982) Methods Enzymol. 86, 357-366 14 Borgeat, P., Hamberg, M. and Samuelsson, B. (1976) J. Biol. Chem. 251, 7816-7820 15 Oliw, E.H., Sprecher, H. and Hamberg, M. (1986) J. Biol. Chem. 261, 2675-2683 16 Oliw, E.H., Sprecher, H. and Hamberg, M. (1986) Acta Physiol. Stand. 127, 45-49 17 Needleman, P., Raz, A., Minks, M., Ferrendelli, J.A. and Sprecher, H. (1979) Proc. Natl. Acad. Sci. USA 70,944-948 18 Hamberg, M. and Hamberg, G. (1980) B&hem. Biophys. Res. Commun. 95,1090-1097 19 Brash, A.R., Porter, A.T. and Maas, R.L. (1985) J. Biol. Chem. 260, 4210-4216 20 Bryant, R.W., Schewe, T., Rappoport, SM. and Bailey, J.M. (1985) J. Biol. Chem. 260, 3548-3555.