Jun 11, 1974 - but not the lipoxygenase, whereas 5,8,11,14-eicosatetray- noic acid ... The observed transformations of arachidonic acid in platelets also ...
Proc. Nat. Acad. Sci. USA Vol. 71, No. 9, pp. 3400-3404, September 1974
Prostagland Endoperoxides. Novel Transformations of Arachidonic Acid in Human Platelets* (platelet Uipoxygenase/fatty acid cyclo-oxygenase/endoperoxide metabolites/platelet aggregation)
MATS HAMBERG AND BENGT SAMUELSSON Department of Chemistry, Karolinska Institutet, 5-104 01 Stockholm, Sweden
Communicated by Hugo TheoreU, June 11, 1974 Arachidonic acid incubated with human ABSTRACT platelets was converted into three compounds, 12Lhydroxy-5,8,10,14-eicosatetraenoic acid, 12L-hydroxy-5,8,10-beptadecatrienoic acid, and the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid. The formation of the two latter compounds from arachidonic acid proceeded by pathways involving the enzyme, fatty acid cyclo-oxygenase, in the initial step and with the prostaglandin endoperoxide, PGG2, as an intermediate. The first mentioned compound was formed from 12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid, which in turn was formed from arachidonic acid by the action of a novel lipoxygenase. Aspirin and indomethacin inhibited the fatty acid cyclo-oxygenase but not the lipoxygenase, whereas 5,8,11,14-eicosatetraynoic acid inhibited both enzymes. The almost exclusive transformation of the endoperoxide structure into nonprostaglandin derivatives supports the hypothesis that the endoperoxides can participate directly and not by way of the classical prostaglandins in regulation of cell functions. The observed transformations of arachidonic acid in platelets also explain the aggregating effect of this acid.
Prostaglandins (PG) E2 and F2a are formed and released by human platelets during aggregation induced by various agents (3, 4). This biosynthetic capacity has also been demonstrated with labeled precursors (5, 6). Recent work in our laboratory led to the isolation (1) of an earlier postulated (7) endoperoxide intermediate in prostaglandin biosynthesis. This finding was confirmed and extended in subsequent studies in which an additional endoperoxide derivative, carrying a hydroperoxy group at C-15, was isolated (2, 8). Studies of the aggregation of human platelets also demonstrated that the biosynthetic process can stop at the endoperoxide stage resulting in release of the intermediates (PGG2 and/or PGH2) (2). Since the endoperoxides were found to be potent aggregating agents and since blockade of their formation is accompanied by inhibition of the second wave of aggregation, it was suggested that they play a physiological role in this process (2). In connection with these and other studies it became of particular interest to further investigate the transformation of arachidonic acid by human platelets. The present work demonstrates that arachidonic acid is oxygenated in this system both by the cyclo-oxygenase involved in prostaglandin Abbreviations: PGE2, prostaglandin E2; PGF2,, prostaglandin F2,,; PGG2, prostaglandin G2; PGH2, prostaglandin H2; Me3Si, trimethylsilyl. * This is paper no. III in this series. Papers no. I and II are refs. 1 and 2, respectively.
biosynthesis and by a novel dioxygenase. It was also found that the endoperoxide of the former reaction is almost exclusively metabolized to nonprostaglandin derivatives. These results provide additional evidence for a direct role of endoperoxides in the regulation of cell functions. MATERIAL AND METHODS
[1_-4C]Arachidonic acid, specific radioactivity 0.91 Ci/mole, prepared from 1-chloro-4,7,10,13-nonadecatetraene (kindly donated by Dr. W. Stoffel, Cologne, Germany; see ref. 9). [5,6,8,9,11,12,14,15-2HS]Arachidonic acid was prepared as described (10). 12-Hydroxyeicosanoic acid was prepared by anodic coupling of 10-acetoxyoctadecanoic acid [80 mg, prepared by acetylation of lO-hydroxyoctadecanoic acid (11)1 and methyl hydrogen succinate (300 mg). The product was hydrolyzed and subjected to silicic acid chromatography and preparative thin-layer chromatography, giving 30 mg (39%) of pure 12hydroxyeicosanoic acid. Thin-layer chromatography of the methyl ester showed a single spot with RF = 0.57. Gasliquid chromatographic analysis of the methyl ester showed a single peak with equivalent chain length C-22.0 (column, 1% SE 30). The mass spectrum showed ions of high intensity at m/e 292 [M-(18 + 32); loss of H20 and CHOH], 229 [M-113; loss of (CH2)7CH31, 200 [M-142; loss of CH(OH)-(CH2)7CH, minus 1 H], and 197 (229 - 32). This fragmentation pattern was in complete accordance with that expected for methyl 12-hydroxyeicosanoate on the basis of published spectra of the isomeric methyl hydroxyoctadecanoates (12). 12-Hydroxy-5cis,8trans,10trans-heptadecatrienoic acid was obtained in 40-50% yield by treatment of PGH2 (2) (2-5 mg) with 100 ml of H20 containing 1 mM FeCl2 at room temperature for 1 hr. The methyl ester of the purified material gave a single spot on thin-layer chromatography (R1 = 0.45), and the ultraviolet spectrum showed an absorption band with XEtOH = 232 nm (e = 33.400) [earlier reported for 12-hydroxy-8,10-heptadecadienoic acid, XF5 = 231 nm (13) ]. The equivalent chain length of the trimethylsilyl (Me;Si) derivative of the methyl ester was C-19.3 (1% SE 30), and the mass spectrum showed ions of high intensity at m/e 366 (M), 351 (M-15; loss of OCH), 335 (M-31; loss of OCH3), 295 [M-71; loss of . (CH2)4CH3], 276 (M-90; loss of Me3SiOH), and 225 [M-141; loss of CH2-CH==CH(CH2),-COOCH3] (see also ref. 2). Sodium borodeuteride (98 atom % deuterium) and [2Hg]trimethylchlorosilane (99 atom % deuterium) were purchased from Merck, Sharp & Dohme, Quebec, Canada. was
-
-
-
3400
Transformation of Arachidonic Acid
(1974)
Proc. Nat. Acad. Sci. USA 71 1800a. 0 t!
_ Methyl arachidonate
Compound E-Me
jO ~|Compound I-Me 900-
Compound il-Me
a
05a
0 5 10 Distance from origin (cm ) FIa. 1. Thin-layer radiochromatogram of product (methyl esters) obtained after incubation of [1-14C]arachidonic acid (38 pg) with 2 ml of platelet suspension at 370 for 2 min. Solvent system: organic layer of ethyl acetate-2,2,4-trimethylpentanewater (50:100:100, v/v/v).
15
Thin-layer chromatography was carried out with plates coated with Silica gel G and the organic layer of ethyl acetate2,2,4-trimethylpentane-water (50: 100: 100, v/v/v) as solvent when not otherwise indicated. Washed human blood platelets were prepared from blood collected with 7.5% (v/v) of 77 mM sodium EDTA as described (2). RESULTS [1-"4C]Arachidonic acid, 38 pg, was incubated with 2-ml suspensions of washed platelets (10/,sl) at 370 for 2 min. During this period, aggregation took place (see ref. 14). The reaction was stopped by the addition of 10 ml of ethanol, and the mixture was diluted with water, acidified, and extracted twice with diethyl ether. The residue obtained after evaporation of the ether (70-80% of the added radioactivity) was treated with diazomethane and subjected to thin-layer chromatography. Four peaks of radioactivity appeared (Fig. 1): methyl arachidonate (RF = 0.88; 32%0 of the recovered radioactivity), methyl ester of compound I (RF = 0.52; 29%O), methyl ester of compound II (RF = 0.45; 20%0), and the methyl ester of- compound III (RF = about 0.02; 19%). In order to obtain larger amounts of these compounds, 1 mg of arachidonic acid was incubated with 30 ml of platelet suspension at 370 for 5 min. The product was subjected to silicic
acid column chromatography. Compound I (0.4 mg) was eluted with diethyl ether-light petroleum (25:75, v/v) and compound II (0.1 mg) with diethyl ether-light petroleum (40:60, v/v). Compound III (0.05-0.1 mg), together with various amounts of a mixture of less polar compounds, probably formed by degradation of compound III, were obtained by elution with ethyl acetate. The acids were converted into the methyl esters and purified by preparative thin-layer chromatography. Structure of Compound I. The ultraviolet spectrum of the methyl ester showed an absorption band with XEtAOm = 237 nm (e = 30.500) [reported for 15-hydroperoxy-8cis,llcis,13trans-eicosatrienoic acid, X"1H = 235 nm (e = 30.000) (15)], indicating the presence of one pair of conjugated double bonds. The absorption band of the methyl ester of compound I shifted to 281 nm on oxidation into the corresponding keto ester with MnO2 in chloroform, demonstrating that the two conjugated double bonds were located a to the alcohol group. The configuration of the conjugated double bonds was cis! tranm, as shown by the infrared spectrum, which showed bands inter alia at 10.10 and 10.47 pm (16). Gas-liquid chromatographic analysis of the Me3Si ether derivative of the methyl ester of compound I showed a single peak with equivalent chain length C-21.3 (column, 1% SE 30). The mass spectrum is given in Fig. 2. Ions were present at m/e 406 (M), 391 (M-15), 375 (M-31), 295 [M-111; loss of CH2-CH=CH-(CH2)4CH3], 229, 205 (295 - 90), and 173 [295-(90 + 32)], suggesting a tetraunsaturated C20 acid carrying a hydroxyl group at C-12. This hypothesis was supported by the mass spectrum of the hydrogenated derivative, which was identical with that of authentic methyl 12-hydroxyeicosanoate (see above). About 0.4 mg of the methyl ester of compound I was converted into the menthoxycarbonyl derivative and subjected to oxidative ozonolysis (17). The product was treated with diazomethane and analyzed by gas-liquid chromatography on a column of 5% QF-1. Two major peaks were observed, one chromatographing with dimethyl glutarate and one with the menthoxycarbonyl derivative of dimethyl imalate. Under the conditions used, there was separation between the menthoxycarbonyl derivatives of dimethyl D- and -malates (separation factor, 1.06) (see ref. 17). This experiment, together with the ultraviolet spectrometry and mass spec-
100
~~~~i34
X OOCH3
60-
i
40-
130
170
2 10
250 m/e
3401
290
330
370
FIG. 2. Mass spectrum of Me3Si derivative of the methyl ester of compound I.
Biochemistry: Hamberg and Samuelsson
3402
Proc. Nat. A cad. Sci. USA 71
(1974) 15
301
M.3519
SY
M*3
,, 80-
60219
,r
a: 40212
103
173
20L[
t11
L
L
L,
L--
,.
1 10
150
190
230
LaIL
*iL
L-
...
270
310
350
3900
430
..
470
510
550
590
630
670
mil
FIG 3. Mass spectrum of Me3Si derivative of compound obtained by sodium borohydride reduction of the methyl ester of compound III. trometry data, showed that compound I contained double bonds at A5, A8, A10, and A14 and had the iLconfiguration at C-12. The structure of compound I established as described above was thus 12ihydroxy-5,8,10,14-eicosatetraenoic acid. Structure of Compound II. The methyl ester of compound II chromatographed with methyl 12-hydroxy-5cis,8trans,10trans-heptadecatrienoate on thin-layer chromatography. The ultraviolet spectra of the two compounds were identical (see above). The MeaSi derivative of the methyl esters of the two compounds cochromatographed on gas-liquid chromatography (C-19.3) and gave identical mass spectra (see above). Finally, oxidative ozonolysis performed on the menthoxycarbonyl derivative of the methyl ester of compound II (17) yielded methyl hydrogen glutarate and the menthoxycarbonyl derivative of 2L-hydroxyheptanoic acid. On the basis of these experiments, compound II was identified as 12ihydroxy-5,8,10-heptadecatrienoic acid.
Structure of Compound III. The methyl ester of compound III migrated close to the methyl ester of PGE2 on TLC (RF 0.73 and 0.71, respectively; solvent: water-saturated ethyl acetate). Compound III, in contrast to PGE2, was not converted into PGB2 on treatment with sodium hydroxide. The equivalent chain length of the MeSi derivative of the methyl ester of compound III was C-24.6 (column, 1% OV-1), and the mass spectrum showed a molecular ion at m/e 600. This ion shifted to m/e 627 in the mass spectrum of the [2H9]Mer. Si derivative and to m/e 608 in the mass spectrum of compound III prepared by incubation of [6,6,8,9,11,12,14,162H8]arachidonic acid. Compound III thus had three hydroxyl groups and retained the eight hydrogens at carbons 5, 6, 8, 9, 11, 12, 14, and 15 of the precursor acid. A clue to the structure of compound III was provided by ions at m/e 366, 295, and 225. These were the major ions of the MesSi derivative of methyl 12-hydroxy-5,8,10-heptadecatrienoate (see above) and, therefore, suggested that compound III was a substituted derivative of compound II. Further ions of high intensities were seen at m/e 585 (M-15), 529 [M-71; loss of -(CH2)4CH3], 510 (M-90), 439 [M-(71 + 90)], 420 (M-2 X 90), 323, 301 [probably MegSiO+-CH-CH=0CH-CH(OSiMe,)-(CH2)4CH3 formed by a rearrangement], 256 (base peak, [MeaSiO-CH=CH-CHs-CH=-CH-(CH2)r COOCH3]+, 217 (Me3SiO+-CH-CH=CH-OSiMe3), and 173 [Me3SiO+=CH-(CH2)4CH:]. The structures of the fragments above were supported in each case by the mass spectra of the [2H9]Me3Si derivative and of the Me3Si derivative of the methyl ester of [2H8]compound III. From the mass =
spectrum it was likely that compound III differed from compound II by the presence of the group CH(OH)-CH2CH(OH)-O. attached to carbons C-8 and 0-9. Support for the presence of a 3-dihydroxy structure was afforded by the ion at m/e 217 (see above), which is typical for Me3Si derivatives of PGF compounds (18). Treatment of the methyl ester of compound III with sodium borohydride in methanol yielded a more polar derivative (Rp 0.38; solvent: water-saturated ethyl acetate). The mass spectrum (Fig. 3) recorded on the Me3Si derivative (equivalent chain length, C-25.1) showed a molecular ion at m/e 674 as well as ions of high intensity at m/e 411 [M-(90 + 173); loss of MevSiOH plus CH(OSiMea)-(CH2)4CH3], 301 -
-
[MeaSiO+=CH-CH=CH-CH(OSiMes)-(CH2)4CIH], 219 (Me3SiO+=CH-CH2-CH2-OSiMes), 211 (301 90), 173 [Me3SiO+==CH-(CH2)4CH3J, and 103 (MeaSiO+=CH2). -
The ions at m/e 301 and 211 demonstrated that the reduced derivative of compound III contained a hydroxyl group at C-9. Therefore, the ether oxygen of the six-membered ring of compound III must be attached to C-9. One atom of deuterium was incorporated upon reduction of compound III with sodium borodeuteride. This atom was localized to the -CH20H group by the shift of the ion at m/e 103 to m/e 104. The hemiacetal structure of compound III should be in equilibrium with the corresponding acyclic aldehyde-alcohol, which in turn should react with an aldehyde reagent. Accordingly, treatment of the methyl ester of compound III with methoxyamine hydrochloride in pyridine at room temperature for 18 hours afforded a slightly less polar compound that was converted into the Me3Si derivative and analyzed by mass spectrometry. Ions were present at m/e 629 (M), 614 (M-15), 598 (M-31), 558 [M-71; loss of (CH2)4CH3], 508 [M-(31 + 90)], 301 [MeSiO+-CH-CH=CHCH(OSiMe,)-(CH2)4CH3, 211 (301 90), 191 (Me3SiO+CH-OSiMe3), 174 (MeSiO+-CH-CH2-CH-NOCH3), and 173 [Me3SiO+=CH-(CH2)4CH3J. This mass spectrum was in agreement with that expected for the Me3Si-O-methyloxime derivative of the aldehyde form of the methyl ester of compound III (see ref. 19). Finally, the menthoxycarbonyl derivative of the methyl ester of compound III was subjected to oxidative ozonolysis. Methyl hydrogen glutarate, the menthoxycarbonyl derivative of 2i,-hydroxyheptanoic acid, as well as two unidentified compounds corresponding to the central part of the molecule, were present in the product. This result showed that the double bonds of compound III were located at A5 and A10 and that the alcohol group at 0-12 had the iconfiguration. -
-
Proc. Nat. Acad. Sci. USA 71
(1974)
Transformation of Arachidonic Acid COOH
TABLE 1. Effect of inhibitors Inhibitor Not present Aspirin, 1 ug/ml Aspirin, 10,pg/ml Aspirin, 100l g/ml Indomethacin, 10 pg/ml Indomethacin, 100 pg/ml ETA, 0.3 pg/ml ETA, 3 pg/ml
Percentage composition of product II AA I III 32 28 20 20 32 22 28 18 36 32 20 12 43 51 3 3 47 2 49 2 45 0 53 2 41 26 16 17 66 7 8 8
Arachidonic acid Lipoxygenr s
Fa tty acid
Effect of Inhibitors. [1-14C]Arachidoriic acid, 38 sg, was incubated with 2-ml suspensions of washed platelets for 2 min at 37°. As seen in Table 1, aspirin and indomethacin inhibited formation of compounds II and III but not compound I. On the other hand, 5,8,11,14-eicosatetraynoic acid inhibited formation of all three compounds. DISCUSSION In the present study three oxygenated derivatives were isolated after incubation of [1-"4C]arachidonic acid with human platelets. The least polar compound was found to be 12i, hydroxy-5,8,10,14-eicosatetraenoic acid, a compound not earlier recognized. This acid was apparently formed by reduction of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid, which could be isolated when a preparation of homogenized platelets was used as an enzyme source. The presence of one pair of conjugated double bonds a to the hydroperoxy group indicated that it was formed from arachidonic acid by action of a lipoxygenase. This was also supported by the fact that the oxygen in the hydroxyl group of 12L-hydroxy-5,8,10,14eicosatetraenoic acid was derived from molecular oxygent. t Hamberg, M.
&
Samuelsson, B., to be published.
cycio-oxygenase
OOH
0---
Prostaglandin 62
ETA, 5,8,11,14-eicosatetraynoic acid; AA, arachidonic acid; I, compound I; II, compound II; III, compound III.
From this work it could be concluded that compound III was identical with the hemiacetal derivative of 8-(1-hydroxy3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid. Isolation of 12-Hydroperoxy-6,8,1O,14-eioatetaenoic Acid (Methyl Ester). Suspensions of 40 ml of washed platelets in Krebs' medium not containing Ca2+ were homogenized and subsequently centrifuged at 650 X g. The sediment was suspended in 20 ml of medium and incubated with 2 mg of [I- 4C~arachidonic acid at 370 for 2-10 min. Analysis of the product (methyl esters) by thin-layer chromatography [solvent: diethyl ether-petroleum ether (30: 70, v/v) I showed the presence of methyl arachidonate, as well as variable amounts of methyl 12-hydroxy-5,8,10,14-eicosatetraenoate (RF = 0.45; 10-30% of the radioactivity applied) and methyl 12-hydroperoxy-5-8,10,14-eicosatetrenoate (RF =- 0.68; 515%). The structure of the last mentioned compound was based on the following data: (i) positive reaction with a peroxide reagent, (ii) conversion into methyl 12-hydroxy-5,8,10,14-eicosatetraenoate by treatment with 10 mg of SnCl2 in 2 ml of methanol at room temperature for 2 min (17), and (iii) ultraviolet spectrometry, which showed XEtOH = 237 nm, e= 31.000.
3403
0 .%C
tH
Compound r
0
-f
COOH
'H Compound 11
00
HON
OHCompound m
FIG. 4. Transformations of arachidonic acid in human platelets.
The positional specificity (C-12, or A9) of this novel lipoxygenase differs from that of previously recognized lipoxygenases (co6 and/or 40) (20). A second monohydroxy acid (compound II) formed from arachidonic acid in platelets was found to be identical with 12i.hydroxy-5,8,10-heptadecatrienoic acid. This compound had been isolated earlier after incubation of arachidonic acid with microsomes of the sheep vesicular gland (21) and as a byproduct in chemical reduction of PGG2 and PGH2 (2). Platelets contain the enzyme fatty acid cyclo-oxygenase (1), since they can produce prostaglandins (3, 4) as well -as prostaglandin endoperoxides (2). It seems likely that formation of 12L-hydroxy-5,8,10-heptadecatrienoic acid in platelets occurs by the same mechanism as that postulated for formation of the analogous 12-hydroxy-8,10-heptadecadienoic acid in preparations of the vesicular gland of sheep, i.e., fragmentation of the endoperoxide intermediate with expulsion of malonaldehyde (22, 28, 13). The most polar compound formed from arachidonic acid (compound III) was found to be the hemiacetal derivative of
8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid. This compound was probably formed from PGG2 (directly or by way of PGII2) by rearrangement and incorporation of one molecule of H20. Short incubations of [1-'4C]arachidonic acid revealed that the time courses of the formation of the hemiacetal derivative (compound III) and 12Lhydroxy-5,8,10>heptadecatrienoic acid (compound II) were identical and differed from that of 12L-hydro'x-5,8,10,14cicosatetraenoic acid (compound I)t. This result supported the view that PGG2 serves as a common precursor for the two
3404
Biochemistry: Hamberg and Samuelsson
former compounds (Fig. 4). The mechanism of formation of the hemiacetal derivative has not been established. However, experiments with 1802 have shown that both oxygens of the peroxide bridge of the endoperOxide precursor are retained in the six-membered ring moiety of the hemiacetal derivativet. The finding that aspirin and indomethacin, inhibitors of prostaglandin biosynthesis (24)i blocked formation of the hemiacetal derivative as well as 12-hydroxy-5,8,10-heptadecatrienoic acid but not 12Lhydroxy-5,8,10,14-eicosatetraenoic acid, gave independent support for formation of the two former compounds by pathways involving the fatty acid cyclo-oxygenase and of the last mentioned compound by action of a Iipoxygenase. With respect to inhibitors, the platelet lipoxygenase resembles soybean lipoxygenase, which is inhibited by 5,8,11,14-eicosatetraynoic acid (25) but not by indomethacin (26). Our earlier results on the release and biological activity of the endoperoxides indicated that they might play a role not only as precursors of the classical prostaglandins, but also as regulators of cell functions (2, 27). The present work with human platelets reinforces this hypothesis since arachidonic acid, except for transformation by the novel lipoxygenase, was almost exclusively converted into non-prostaglandin metabolites derived from the endoperoxide structure. The extensive transformation of added arachidonic acid into the potent aggregating agent PGG2 (2) and its metabolites provides a biochemical explanation for earlier observations on platelet aggregation induced by arachidonic acid (14, 28) and appearance of an unidentified factor with aggregating properties (29, 30). This work was supported by grants from the Swedish Medical Research Council (Project no. 03X-217). 1. Hamberg, M. & Samuelsson, B. (1973) Proc. Nat. Acad. Sci. USA 70, 899-903. 2. Hamberg, M., Svenssoh, J., Wakabayashi, T. & Samuelsson, B. (1974) Proc. Nat. Acad. Sci. USA 71, 345-349. 3. Smith, J. B. & Willis, A. L. (1970) Brit. J. Pharmacol. 40, 545P.
4. Smith, J. B., Ingerrman, C., Kocsis, J. J. & Silver, M. J. (1973) J. Clin. Invest. 52, 965-969. 5. Clausen, J. & Srivastava, K. C. (1971) Lipids 7, 246-250.
Proc. Nat. Acad. Sci. USA 71 (1974) 6. Schoene, N. W. & Iacono, J. M. (1974) Prostaglandins 5, 387-395. 7. Samuelsson, B. (1972) Fed. Proc. 31, 1442-1450. 8. Nugteren, D. H. & Hazelhof, E. (1973) Biochim. Biophys. Acta 326, 448-461. 9. Stoffel, W. (1964) Hoppe-Seyler's Z. Physiol. Chem. 673, 26-36. 10. Hamberg, M., Niehaus, W. G. & Samuelsson, B. (1968) Anal. Biochem. 22, 145-153. 11. Bergstrom, S., Aulin-Erdtman, G., Rolander, B., Stenhagen, E. & Ostling, S. (1952) Acta Chem. Scand. 6, 11571174. 12. Ryhage, R. & Stenhagen, E. (1960) Arkiv Kemi 15, 545-574. 13. Hamberg, M. & Samuelsson, B. (1967) J. Biol. Chem. 242, 5344-5354. 14. Ingerman, C., Smith, J. B., Kocsis, J. J. & Silver, M. J. (1973) Fed. Proc. 32, 219Abstr. 15. Hamberg, M. & Samuelsson, B. (1967) J. Biol. Chem. 242, 5329-5335. 16. Chipault, J. R. & Hawkins, J. M. (1959) J. Amer. Oil Chem. Soc. 36, 535-539. 17. Hamberg, M. (1971) Anal. Biochem. 43, 515-526. 18. Hamberg, AI. & Israelsson, U. (1970) J. Biol. Chem. 245, 5107-5114. 19. Laine, R. A. & Sweeley, C. C. (1973) Carbohyd. Res. 27, 199-213. 20. Hamberg, M., Samuelsson, B., Bjorkhem, I. & Danielsson, H. (1974) in Molecular Mechanisms of Oxygen Activation, ed. 0. Hayaishi (Academic Press, New York), pp. 29-85. 21. Wiodawer, P. & Samuelsson, B. (1973) J. Biol. Chem. 248, 5673-5678. 22. Hamberg, M. & SamueLsson, B. (1966) J. Amer. Chem. Soc. 88, 2349-2350. 23. Nugteren, D. H., Beerthuis, R. K. & Van Dorp, D. A. (1966) Rec. Trav. Chim. Pays-Bas 85, 405-419. 24. Vane, J. R. (1971) Nature New Biol. 231, 232-235. 25. Downing, D. T., Ahern, D. G. & Bachta, M. (1970) Biochem. Biophys. Res. Commun. 40, 218-223. 26. Downing, D. T. (1972) Prostaglandins 1, 437-441. 27. Samuelsson, B. & Hamberg, M. (1974) in Proceedings of an
International Symposium on Prostaglandin Synthetase Inhibitors, New York (1973), ed. J. R. Vane & H. J. Robinson, in press. 28. Silver, M. J., Hoch, W., Kocsis, J. J., Ingerman, C. & Smith, J. B. (1974) Science 183, 1085-1087. 29. Vargaftig, B. B. & Zirinis, P. (1973) Nature New Biol. 244, 114-116. 30. Willis, A. L. (1974) Science 183, 325-327.
