Metabolism of 4,7,10,13,16-docosapentaenoic acid by human platelet cyclooxygenase and lipoxygenase

29

Biochimica et Biophysics Acta 835 (1985) 29-35

Elsevier BBA 51920

~eta~lism

of 4,7,10,~3,l~d~osa~n~enoic acid by human platelet cyclooxygenase and lipoxygenase Michael M. Milks and Howard Sprecher *

Department of Physiological Chemistry, Coiiege

ofMedicine,Ohio State University. 333

W. 10th Avenue, Columbus, OH

43210 (U.S.A.)

(Received December 6th. 1984)

Key words: 4.7,10,13,16-Docosapentaenoic acid; Thromboxane: Hydroxy fatty acid: (Human platelet)

Washed human platelets are shown to metabolize 4,7,10,13,16-docosapentaenoic acid into three major metabolites which were purified by reverse-phase HPLC. The mass spectra of the methyl ester-trimethylsilyl ether and ethyl ester-trfmethylsilyl ether of compound A established it as A4-dihomo-thromboxane Bz. Corn~~d B was shown to be 14-hy~oxy~,7,lO,l~nonad~~~enoic acid, which is analogous to 12-hydroxy-5,8,10-heptadecatrienoic acid from arachidonic acid. Compound C was produced via an indomethacininsensitive pathway and was identified as 14-hydroxy-4,7,10,12,16-docosapentaenoic acid. Time- and substrate-dependent studies showed that compounds A, B and C were produced approximately IO, 15 and 65% of the extent to which thromboxane &, 12-hy~oxy-5,8,lO-hep~d~a~ienoic acid and 12-hydroxy-5,8,10,14eicosatetraenoic acid were produced, respectively, from arachidonic acid.

Introduction

Materials and Methods

Dietary linoleic acid is metabolized via a series of alternating desaturation and chain-elongation reactions in the microsomal fraction of the cell as follows: 18:2~18:3-+20:3+20:4+22:4+ 22 : 5 [l]. Human platelet phospholipids contain large amounts of arachidonic acid and smaller amounts of other (n - 6) acids [2,3]. Arachidonic acid [4], 20 : 3(8,11,14) [5] and 22 : 4(7,10,13,16) [6] are all metabolized by platelets to yield thromboxanes which differ in chain length as well as the position of their first double bond. The studies reported here show that 22 : 5(4,7,10,13,16), like its three immediate precursors, is metabolized by both platelet cyclooxygenase and lipoxygenase.

Furry acids. [l-14C]22 : 5(4,7,10,13,16) and the corresponding nonradioactive acid were made by couphng the diGrignard complex of 3-butyn-l-01 with l-bromo-2,5,8,11-heptadecatetrayn. The resulting 3,6,9,12,15-heneicosapentayn-l-01 was converted to the desired acids by using established procedures [7]. [l-‘4C]Arachidonic acid (56.9 Ci/ mol) was purchased from New England Nuclear (Boston, MA) while arachidonic acid was from Nu-Chek Preparations (Elysian, MN). Platelet incubations. Blood was obtained from healthy volunteers who had not taken any medications within two weeks of collection. The blood was mixed with 7.5% (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 1000 X g, at 10°C for 15 min and the platelet pellet was resuspended in 0.15 M NaCl/O.lS M Tris HCl (pH 7.4)/77 mM dis-

* To whom correspondence should be addressed. Abbreviatjons: HHT, lZ-hydroxy-5,8,10-h~tad~at~enoic acid; 12-HETE, 12-hydroxy-5,8,10,14cosatetraenoic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; TMS, trimethylsilyl. ~5-27~/85/$03.30

0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

30

odium EDTA (90 : 8 : 2, v/v). The platelet suspension was centrifuged at 1000 x g, for 15 min at 10°C and again resuspended in the above medium. The final concentration of the platelet suspension was adjusted to 3.0. lo8 platelets/ml. Incubations of 0.5 ml (1.5. 10’ platelets) were performed in siliconized glass tubes placed in an orbital shaker (200 rpm; 37°C). Platelet suspensions were preincubated at 37°C for 2 min. Inhibitors, when used, were then added in 2 ~1 of ethanol or water. The platelets were incubated for an additional 3 min prior to the addition of the sodium salt of the fatty acid. Incubations were terminated by the addition of 0.2 vol. of 2 M formic acid and were extracted three times with 6 vol. of ethyl acetate. The ethyl acetate extracts were washed once with 1 ml of water and dried over anhydrous sodium sulfate. The ethyl acetate was removed under a stream of N, and the residue was dissolved in methanol. Chromatography. Thin-layer chromatography was carried out on Whatman LK6D plates (Whatman Inc., Clifton, NJ). The plates were developed with the upper phase obtained after mixing ethyl acetate/ 2,2,4-trimethyl pentane/ acetic acid/ water (90 : 50 : 20 : 100, v/v). Several pg of thromboxane B, and prostaglandins E,, D, and Fla (kindly supplied by Dr. John Pike, The Upjohn Co.) were applied as standards. The prostaglandins were visualized by exposure to iodine vapor, while radioactive compounds were detected by scanning with a Packard Model 7720 radiochromatogram scanner. HPLC was performed on a DuPont system consisting of an 870 pump, 8800 series gradient controller, a column oven set at 35°C and a variable wavelength detector. Radioactivity was quantitated with a radioactive flow detector (Model HP, Radiomatic Instruments and Chemical CO., Tampa, FL). A 25 X 0.46 cm Zorbax ODS 5 p particle column was used, preceded by a guard column (5 x 0.46 cm) packed with Permaphase ODS (DuPont, Wilmington, DE). Separations were carried out by elution (1.5 ml/mm) for 10 min with 34% acetonitrile in water adjusted to pH 2.2 with HPLC grade H,PO, (Fisher Chemical Co., Cincinnati, OH). After this time, the instrument was programmed to 75% acetonitrile over 30 min using the

-2 exponential gradient. At 40 min, the concentration of acetonitrile was increased linearly to 100% over 5 min to elute unreacted substrate. The radioactive detector was set so the ratio of scintillation fluid to column effluent was 3 : 1 (v/v). Counting efficiency was approx. 75% and was not significantly altered by changes in acetonitrile concentration. Normal-phase HPLC was carried out with a Beckman system consisting of two model 112 pumps, a model 420 controller and variable wavelength detector (Model 1305, Bio-Rad. Richmond, CA). Separations were carried out at room temperature using a 25 x 0.46 cm Zorbax-Sil column (DuPont, Wilmington, DE). Methyl esters of hydroxy acids were purified by eluting with hexane/isopropanol(99.5 : 0.5, v/v). Gas chromatography - mass spectrometq. Metabolites collected from reverse-phase chromatography were recovered by extracting with ethyl acetate which was then removed under a stream of N,. The metabolites were dissolved in either methanol or ethanol and converted to methyl or ethyl esters by reaction with ethereal diazomethane or diazoethane, respectively. Hydroxyl groups were converted to TMS ethers by reaction with 10 ~1 of N.O-bis(trimethylsilyl)trifluoroacetamide (Pierce Chemical Co., Rockford IL) in 10 ~1 of pyridine for 30 min at 60°C. Methyl esters of hydroxy acids were hydrogenated by bubbling hydrogen for 30 s into 0.5 ml of a methanol solution of the compounds with about 1 mg of PtO. Gas chromatography was carried out on a Varian 6000 Vista gas chromatograph equipped with a 2 m x 2 mm i.d. glass column packed with 1% SP-2100 on 100/120 mesh Supelcoport. The flow rate of helium was 30 ml/mm and the temperatures of the injector, oven, and detector were 230, 210, and 260°C respectively. Equivalent chain lengths were calculated by comparing the retention times of compounds with a saturated series of fatty acid methyl esters. Mass spectrometry was performed on a Hewlett-Packard 5970A mass selective detector and a 5790A gas chromatograph using a 15 m X 0.25 mm i.d. DB-1 J and W capillary column (obtained from Applied Science Laboratories Bellefonte, PA). Samples were injected in isooctane in the splitless mode at 70°C. After 1 min, the oven was pro-

31

grammed at 30°C/min to the final which is defined in individual figures. ature of the detector was 230°C. voltage was 70 eV. Spectrophotometry. Spectra were methanol with a Bausch and Lomb photometers.

temperature The temperThe ionizing

100

measured in 2000 spectro-

60

20

Results I

0

The thin-layer radiochromatogram (Fig. 1) shows that [l-14C]22 : 5( n - 6) is metabolized into two metabolites which comigrate with thromboxane I& and 12-HETE. When the metabolites produced from [ l-l4 Cl22 : 5(n - 6) were separated by reverse-phase HPLC, three major radioactive products were detected (Fig. 2). Both compounds B and C showed intense absorption at 234 nm. Indomethacin (25 PM) inhibited the synthesis of both A and B, indicating that both of these metabolites were produced via the cyclooxygenase pathway. OKY 1581 (1 PM), a specific inhibitor of thromboxane synthetase [8], blocked the production of compound A without affecting the synthesis of compounds B and C. ETYA (50 FM) totally inhibited the synthesis of all three metabolites. Fig. 3 shows that the mass spectra of the methyl ester-TMS ether of compound A (equivalent chain length = 26.4) had major ions at m/r 536 (M - 90, loss of Me,SiOH), 465 [M - (90 + 71) loss of Me,SiOH and .(CH,),CH,], 446 [M- (2 X 90)], 392 [M - 234, loss of ‘CH(OSiMeJ)-CH,CH-

22.5 (3

ORIGIN

(n-6)

MIN 1

0

5

DISTANCE

IO

FROM

OR!GIN

15 SOLVENT FRONT ( cm )

Fig. 1. Thin-layer radiochromatogram of the metabolites produced when 1.5.10’ platelets were incubated for 3 min with 20 yM 4,7,10,13,16-[1-‘4C]docosapentaenoic acid.

I

IO

20 ELUTION

30

40

50

60

TO

TIME (MIN)

Fig. 2. Reverse-phase HPLC radiochromatogram of the metabolites produced from 4,7,10,13,16-[1-‘4C]docosapentaenoic acid. Platelets (l.S~10s/0.5 ml) were incubated for 3 min and metabolites were extracted into ethyl acetate. The ethyl acetate was removed and metabolites were injected in 50 pl of methanol.

(OSiMe,)O‘], 375 [M - (2 x 90) + 711, 363, 349, 321 (392 - 71) 301 [Me,SiO=CH-CH=CH-CH(OSiMe,)-(CH,),-CH_T], 282 [Me,SiO-CH= CH-CH2-(CH=CH-CH,),CH,COOCH;], 225 [392 - 167, loss of ‘CHI-(CH= CH-CH,),CH,COOCHr], 217 (Me,SiO=CHCH=CH-OSiMel), 211 (301-90) 173 [Me,SiO+=CH(CH,),CH,] and 166. The ion at 166 shifted to 180 when the ethyl ester-TMS ether of compound A was analyzed. This ion thus results from cleavage between carbons 9 and 10 with additional loss of a proton from the C, chain. The spectrum of the ethyl ester-TMS ether also had ions at m/z 550, 479 and 460. In neither spectrum was it possible to observe the expected molecular ion. Simultaneous selective monitoring of ions at m/z 626 (M), 611 (M - 15) 555 (M - 71). 536 (M - 90) and 446 (M - 2 x 90) showed that these ions were produced from a compound with a retention time identical with that of the methyl ester TMS ether of compound A. These results show that compound A is A4-dihomo-thrombo xane B,. The methyl ester of compound B was purified by normal phase HPLC prior to conversion to the TMS ether. The equivalent chain length of the methyl ester-TMS ether was 21.1 and, as shown in Fig. 4, the mass spectrum had ions at m/r 392 (M+), 377 (M - 15), 321 [M - 71, loss of .(CH,),CH,], 302 (M- 90, loss of Me,SiOH), 225 (M- 167 loss of ‘CH,(CH=CH-CH,),CH2

200

300

250

450

400

500

550

6cx)

640

Fig. 3. Mass spectrum of the methyl ester-TMS ether of compound A. The sample was injected at an oven temperature of 70°C’ and after 1 min the oven was programmed to 260°C at 30’C/min.

377

392

0 100

150

400

350

300

250

200

ml2 Fig. 4. Mass spectrum of the methyl ester-TMS ether of compound B. After injection at 70°C the oven was programmed to 215’C at 30”C/min.

1

255 I@_;t.., 100

150

200

250

,

.,* , 300

/.

ir. ,

)

/

,

,

350

m72 Fig. 5.

Mass spectrum of the methyl ester-TMS ether of compound C. Conditions were as described in Fig.

4.

-7-w 400

417 440

33

COOCH,) and 199 [321 - (90 + 32)]. Compound B is thus 14-hydroxy-4,7,10,12-nonadecatetraenoic acid. The methyl ester of compound C was also purified by normal-phase HPLC prior to analysis. Compound C had X,,, 236 nm with f = 2’7500 in methanol, suggesting that it contained a pair of double bonds in the cis/trans configuration [9], The mass spectrum (Fig. 5) of the methyl esterTMS ether (equivalent chain length = 23.0) had ions at m/z 417 (M - 15, loss of .CH,) 321 (M - 111, loss of -CH,-CH=CH-(CH,)&H& 231 (321 - 90) and 199 1321 - (90 + 32)]. The mass spectrum of the hydrogenated methyl ester-TMS ether (equivalent chain length = 24.0) of compo~d C had ions at wr/z 427 ( M - 15; l-l%), 411 (M- 31; 2.5%), 395 [M- (32 + 15); 7.1%], 329 [(M - 113), loss of ‘(CH,),CH,; base peak], 300 [(M - 142), loss of CH,-(CH,),CHO followed by a rearrangement of the trimethylsilanol to the carbomethoxy group, 20.6% [IO]] and 215 (M - 227), loss of ‘CH2-(CH,),,COOCH,; 93.0%]. Compound C is thus 14-hydroxy4,7,10,12,16-docosapentaenoic acid. A comparison of the substrate dependent conversion of 20 : 4(n - 6) and 22 : 5( n - 6) to their respective metabolites is shown in Fig. 6. With

‘Ofi’

5 25

50

75

100

200

@JBSTRATE~~M

Fig. 6. Substrate-dependent metabolism of fl-‘qC]arachidonic acid and 4,7,~0,~3,1~-[l-‘4~]d~~~nlae~oic acid. Platelets (l.S-108/0.5 ml) were incubated for 3 min. Metabolites were separated by reverse-phase HPLC: 12-HETE (a), Whydroxy4,7,10,12,16-docosapentaenoic acid (O), HHT (A), thromboxane B, (m), 14-hydroxy-4,7,10,12-nonadecatetraenoic acid (A) and A4-dihomo-thromboxane h (Cl),

TIME

Fig. 7. Time-dependeat

I minf

metabolism of [l-‘4C]arachidonic acid

and 4,7,10,13,16-[1-‘4C]dccosapentaenoic acid. Platelets (1.5. 108/0.5 ml) were incubated with 60 PM substrate. Metabolites were separated by reverse-phase HPLC: 12-HETE (o), 14-hydroxy-4,7,10,12,16-docosapentaenoic acid (U), HHT (A), thromboxane B, (8). l~hydroxy~,?,lO,~2-nonad~atetraenoic acid (A) and A4-d~om~t~omboxane 3, (El).

both substrates, maximum amounts of cyclooxygenase-derived metabolites were produced when the substrate concentration was ibout 10 PM, Approx. 10% as much A4-d~homo-thromb~ xane B, and 15% as much 14-hydroxy-4,7,10,12nonadecatetraenoic acid was formed from 22 : 5( n - 6) as compared to the analogous metabolites derived from arachidonic acid. About 50 FM levels of substrate were required to achieve maximum lipoxygenase activity at which point the amount of 14-hydroxy-4,7,10,12,16docosapentaenoic acid produced was about 65% of that for 12-HETE synthesis. Time-dependent studies using 60 PM substrate (Fig. 7) showed that with both fatty acids, maximum cyclooxygenase activity had been achieved by 1 min. Conversely, there was almost a linear increase in lipoxygenase-derived products for about 6 min. lkxtssion

It is well known that platelets can metabolize 20: 4(5,8,11,14) to thromboxane B, [4]. Subsequent studies have demonstrated that

34

20: 3(8,11,14) [5] and 22 : 4(7,10,13,16) [6] are metabolized to thromboxane B, and dihomothromboxane B,, respectively. The studies reported here show that 22: 5(4,7,10,13,16) is also metabolized by platelets to a thromboxane. All four 20- and 22-carbon unsaturated acids derived from linoleate thus can serve as substrates for cyclooxygenase to give thromboxanes. Linolenate, like linoleate, is metabolized to a series of long-chain polyunsaturated fatty acids as follows: 18 : 3(9,12,15) -+ 18 : 4(6,9,12,15) 20:4(8,11,14,17) -+ 20: 5(5.8,11,14,17) + 22:5 (7,10,13,16,19) -+ 22 : 6(4,7,10,13,16.19). These (n - 3) acids differ from the corresponding linoleate metabolites in that they contain an additional double bond at the (n - 3) carbon atom. This double bond is not required for endoperoxide synthesis. The (n - 3) acids are however metabolized quite differently than are the analogous linoleate metabolites. Platelets metabolize 20 : 5(5,8,11,14,17) into thromboxane B, to about 20% of the extent to which arachidonic acid is converted to thromboxane B, and only a single 12-hydroxy acid is produced from both acids via the lipoxygenase pathway [l I]. Conversely 22 : 5(7,10,13,16,19) [12] and 22 : 6(4,7,10,13,16,19) 1131 are not metabolized to thromboxanes but are each metabolized by platelets via an indomethatin-insensitive pathway to a pair of isomeric lland 14-hydroxy acids. Platelet phospholipids contain small amounts of 22-carbon acids [2,3]. These acids are readily incorporated into platelet phospholipids, and they also are produced in platelets by chain elongation of their 20”carbon precursors [14]. Agonist-induced activation of phospholipases thus has the potential of liberating not only arachidonic acid but other unsaturated fatty acids from phospholipids. Platelet aggregation, therefore, may well be regulated by the collective action of the various metabolites produced from a number of polyunsaturated fatty acids. In addition, our previous studies showed that inhibition of thromboxane B, synthesis by exogenous 22:4(7,10,13,16) [6] and 22 : 5(7.10.13,16,19) [12] resulted in shunting of arachidonate to the lipoxygenase pathway and as a result increased the synthesis of 12-HETE. The 22-carbon acids were simultaneously metabolized to hydroxy fatty acids. IZHPETE not only stimu-

lates neutrophil 5-lipoxygenase [15] but it is also further metabolized by these cells [16--181. It remains to be determined whether hydroxy acids produced from 22-carbon acids can modulate the activity of 5-lipoxygenase or if they are substrates for further metabolism by neutrophils. 22 : 5(4,7,10,13,16)is the only acid in membrane lipids with a double bond at position 4 which is metabolized by cyclooxygenase. The model compound, 19 : 4(4,7,10,13) is metabolized to a thromboxane that does not induce platelet aggregation even though the corresponding endoperoxide is a partial agonist. In contrast, this cY-northromboxane A, contracted the thoracic aorta even though the endoperoxide was inactive 1191. Synthetic prostaglandins with their first double bond at position 4 have unusually long half-lives in the circulation 1201. Even though 22 : 5(4,7,10,13,26) is generally found in relatively small amounts in membrane phospholipids, our studies show that it is metabolized by platelet cyclooxygenase and lipoxygenase. thus su~esting that autocoids produced from this acid may have unique biological properties. Acknowledgements This study was supported 20387 and AM 18844.

by NIH

grants

AM

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17 Wong, P.U.-K., Westlund, P., Hamberg, M., Granstrom, E., Chao, P.W.-H. and Samuelsson, B. (1984) J. Biol. Chem. 259, 2683-2686 18 Marcus, A., Safier, L.B., Broekman, M.J., Islam, N., Oglesby, T.D. and Gorman, R.R. (1984) Proc. Natl. Acad. Sci. USA 81, 903-907 19 LeDuc, L.E., Wyche, A.A., Sprecher, H., Sankarappa, S.K. and Needleman, P. (1981) Mol. Pharmacol. 19, 242-247 20 Green, K., Samuelsson, B. and Magerlein, B.J. (1976) Eur. J. B&hem. 62, 527-537