8
398 BBA 52448 Biochwmca et Biophysics Acta 917 (1987) 398-405 Elsevier Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A 5 and B5, mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes Bing K. Lam a, Aizan Hirai b, Sho Yoshida b, Yasushi Tamura b and Patrick Y-K. Wong a Department of Pharmacology, New York Medical College, Valhalla, NY (U.S.A.) and h 2nd Department of Internal Medrcrne, Chiha University, School of Medicine, Chiha (Japan) (Received 27 May 1986) (Revised manuscript received 20 October 1986) Key words: 15-Hydroperoxyicosapentaenoic acid; Icosanoid metabolism; Lipoxin; (Porcine leukocyte) 15-Hydroperoxy[l-14C]eicosapentaenoic acid derived from eicosapentaenoic acid (EPA) was incubated with suspensions of porcine leukocytes. Incubation with porcine leukocytes resulted in the formation of seven dihydroxy compounds, one monohydroxy and one hydroxyepoxy compound. After separation by reverse-phase and straight-phase HPLC, GC/MS analysis revealed that these metabolites were four isomers of 8,15-di- HEPEs, two isomers of 14,15-diHEPEs, one isomer of 5,15-diHEPE, 19HEPE and an epoxyalcohol: 13-hydroxy-14,15-epoxyeicosatetraenoic acid. In addition to the above metabolites, two trihydroxytetraene derivatives were also isolated. GC/MS and utlraviolet spectroscopy identified the two trihydroxypentaene derivatives as 5,6,15-trihydroxy-7,9,11,13,17-eicosapentaenoic acid (lipoxin As) and 5,14,15+ihydroxy- 6,8,10,12,17-eicosapentaenoic acid (iipoxin B,). This study demonstrated that the 15-hydroperoxide of EPA can be actively converted to various hydroxylated products via the 5-, 12- and 19lipoxygenase as well as epoxyisomerase pathways in the porcine leukocytes. Introduction Abbreviations: EPA, 5,8,11,14,17-eicosapentaenoic acid; 15- HPEPE, 15-hydroperoxy-5,8,11,13,17-eicosapentaenoic acid; 15-HPETE, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; 15-HEPE, 15-hydroxy-5,8,11,13,17-eicosapentaenoic acid; 15- HETE, 15-hydroxy-5,8,11,13-eicosatetraenoic acid; 5-HPETE, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid; leukotriene C5, 5( S)-hydroxy-6( R)-S-glutathionyl-7,9-tracts-11,14,17-cis-eico- sapentaenoic acid; HPLC, high-performance liquid chromatog- raphy: lipoxin A,, 5,6,15-trihydroxy-7,9,11,13,17-eicosapen- taenoic acid; lipoxin B,, 5,14,15-trihydroxy-6,8,10,12,17- eicosapentaenoic acid; diHETE, dihydroxyeicosatetraenoic acid; diHEPE, dihydroxyeicosapentaenoic acid; fMet-Leu-Phe, N-formylmethionylleucylphenylalanine; DHPETE. dihydro- peroxyeicosatetraenoic acid; DHPEPE dihydroperoxyeicosa- pentaenoic acid. Correspondence: P.Y.-K. Wong, Department of Pharmacol- ogy, New York Medical College, Valhalla. NY 10595, U.S.A. Eicosapentaenoic acid (EPA) has been reported to be a substrate for cyclooxygenase in the bio- synthesis of prostaglandins and thromboxanes [1,2]. Recently, Ochi et al. [3,4] demonstrated that EPA was a better substrate than arachidonic acid for the 5-lipoxygenase in guinea-pig peritoneal polymorphonuclear leukocytes for the synthesis of 5-hydroperoxy-6,8,11,14,17-eicosapentaenoic acid (5-HPEPE), a precursor for leukotrienes A, and R, 13941. It has been shown that EPA, like arachidonic acid [5], can be incorporated into cell membrane phospholipids which, upon stimulation, can be released and utilized by 5-lipoxygenase to yield pentaene leukotrienes [6]. In the presence of exo- 0005-2760/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A5 and B5, mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes

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398

BBA 52448

Biochwmca et Biophysics Acta 917 (1987) 398-405

Elsevier

Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A 5 and B5,

mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes

Bing K. Lam a, Aizan Hirai b, Sho Yoshida b, Yasushi Tamura b and Patrick Y-K. Wong a

’ Department of Pharmacology, New York Medical College, Valhalla, NY (U.S.A.)

and h 2nd Department of Internal Medrcrne, Chiha University, School of Medicine, Chiha (Japan)

(Received 27 May 1986)

(Revised manuscript received 20 October 1986)

Key words: 15-Hydroperoxyicosapentaenoic acid; Icosanoid metabolism; Lipoxin; (Porcine leukocyte)

15-Hydroperoxy[l-14C]eicosapentaenoic acid derived from eicosapentaenoic acid (EPA) was incubated with suspensions of porcine leukocytes. Incubation with porcine leukocytes resulted in the formation of seven dihydroxy compounds, one monohydroxy and one hydroxyepoxy compound. After separation by reverse-phase and straight-phase HPLC, GC/MS analysis revealed that these metabolites were four isomers of 8,15-di- HEPEs, two isomers of 14,15-diHEPEs, one isomer of 5,15-diHEPE, 19HEPE and an epoxyalcohol: 13-hydroxy-14,15-epoxyeicosatetraenoic acid. In addition to the above metabolites, two trihydroxytetraene derivatives were also isolated. GC/MS and utlraviolet spectroscopy identified the two trihydroxypentaene derivatives as 5,6,15-trihydroxy-7,9,11,13,17-eicosapentaenoic acid (lipoxin As) and 5,14,15+ihydroxy- 6,8,10,12,17-eicosapentaenoic acid (iipoxin B,). This study demonstrated that the 15-hydroperoxide of EPA can be actively converted to various hydroxylated products via the 5-, 12- and 19lipoxygenase as well as epoxyisomerase pathways in the porcine leukocytes.

Introduction

Abbreviations: EPA, 5,8,11,14,17-eicosapentaenoic acid; 15- HPEPE, 15-hydroperoxy-5,8,11,13,17-eicosapentaenoic acid;

15-HPETE, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid;

15-HEPE, 15-hydroxy-5,8,11,13,17-eicosapentaenoic acid; 15-

HETE, 15-hydroxy-5,8,11,13-eicosatetraenoic acid; 5-HPETE,

5-hydroperoxy-6,8,11,14-eicosatetraenoic acid; leukotriene C5,

5( S)-hydroxy-6( R)-S-glutathionyl-7,9-tracts-11,14,17-cis-eico-

sapentaenoic acid; HPLC, high-performance liquid chromatog- raphy: lipoxin A,, 5,6,15-trihydroxy-7,9,11,13,17-eicosapen-

taenoic acid; lipoxin B,, 5,14,15-trihydroxy-6,8,10,12,17-

eicosapentaenoic acid; diHETE, dihydroxyeicosatetraenoic

acid; diHEPE, dihydroxyeicosapentaenoic acid; fMet-Leu-Phe,

N-formylmethionylleucylphenylalanine; DHPETE. dihydro-

peroxyeicosatetraenoic acid; DHPEPE dihydroperoxyeicosa-

pentaenoic acid.

Correspondence: P.Y.-K. Wong, Department of Pharmacol-

ogy, New York Medical College, Valhalla. NY 10595, U.S.A.

Eicosapentaenoic acid (EPA) has been reported to be a substrate for cyclooxygenase in the bio- synthesis of prostaglandins and thromboxanes [1,2]. Recently, Ochi et al. [3,4] demonstrated that EPA was a better substrate than arachidonic acid for the 5-lipoxygenase in guinea-pig peritoneal polymorphonuclear leukocytes for the synthesis of 5-hydroperoxy-6,8,11,14,17-eicosapentaenoic acid (5-HPEPE), a precursor for leukotrienes A, and

R, 13941. It has been shown that EPA, like arachidonic

acid [5], can be incorporated into cell membrane phospholipids which, upon stimulation, can be released and utilized by 5-lipoxygenase to yield pentaene leukotrienes [6]. In the presence of exo-

0005-2760/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

399

genous EPA, mouse mastocytoma cells challenged with ionophore A23187 generate LTB, and LTC, in addition to LTB, and LTC, [6,7], whereas LTC, is equipotent to LT%, in contracting guinea-pig pulmonary parenchymal strips and guinea-pig ileum [8]. LTB, is 30- to 60-fold less potent than LTTZ, in eliciting neutrophil chem- otaxis. In addition, it has been shown that EPA and its oxygenated products, i.e., LTA,, inhibit LTB, formation, the site of its inhibitory action being perhaps at the level of LTA, hydrolase [9,10]. Although there are extensive studies of metabolism of EPA via cyclooxygenase and 5- lipoxygenase pathways in neutrophils and tumor cell lines, relatively little is known of its metabo- lism via 15-lipoxygenase pathways. Recently, we have demonstrated that 15-HPEPE can be con- verted to a novel series of trihydroxypentaenes, lipoxene A (lipoxin A,) and lipoxene B (lipoxin B,) by porcine leukocytes [ll]. In this study, we describe the metabolic profile of 15-HPEPE in porcine leukocytes.

Materials and Methods

~ateriais [I-i4C]EPA (specific activity, 55 mCi/mmol)

was purchased from New England Nuclear, Bos- ton, MA. 5,8,11,14,17-Eicosapentaenoic acid was obtained by treatment of eicosapentaenoate ethyl ester (99.7% pure, Central Research Laboratory, Nippon Suisan Kaisha, Tokyo, Japan) with 40% methanolic KOH overnight at room temperature. Soybean lipoxygenase I (EC 1.13.11.12) was from Sigma Co. Di-HETEs and leukotriene standards of the 5 series (i.e., 5,15-diHEPE; 8,15-diHEPE; 14,15-diHEPE, LTB, and LTCs, etc.) were ob- tained from WW Diagnostic Products, West Haverstraw, NY 10993. I5-[l-i4C]HPEPE was synthesized by incubating [1-“4C]EPA with lipoxy- genase I similar to the preparation of 15-HPETE described by Hamberg and Samuelsson [12]. Porcine venous blood was obtained from a nearby slaughterhouse. Dextran T-500 was from Phar- macia, Sweden.

Preparation of porcine leukocytes Porcine leukocytes were prepared from mixed

venous blood containing 10 mM EDTA 1111.

Venous blood was mixed with half volume of 6% dextran solution and allowed to settle for 60 min at room temperature. The upper phase was then centrifuged at 250 x g for 20 min and the con- taminating red cells were removed by hypotonic lysis with distilled water for 20 s. The final cell suspension was reconstituted by the addition of an appropriate volume of 36% saline. After centrifu- gation at 250 X g for 20 min, the pellet was resus- pended in Dulbecco’s phosphate-buffered saline (pH 7.4) to a final concentration of 100.10” cells/ml. The viability of the cells as measured by Trypan blue exclusion test was found to be greater than 95%.

Incubation, extraction and purification

After prewarming the leukocyte suspension to 37*C for 5 min, 15-HPEPE and ionophore A23187 (in ethanol) were added to final concentrations of 100 PM and 5 PM, respectively, and incubated for 30 min at 37°C with constant shaking. The amount of ethanol needed to dissolve 15-HPEPE never exceeded 0.1% of the incubation volume. The in- cubation was terminated by the addition of 2 vol. ethanol. The incubation precipitate was filtered and the ethanolic filtrate was evaporated to near dryness. The residue was dissolved in 5 ml of distilled water and acidified to pH 3.5 with 1 M HCl. The solution was then extracted with 10 vol. of ethyl acetate. The ethyl acetate fraction was evaporated to dryness under N,. The residue was dissolved in 50 ~1 of methanol and separated by HPLC on a Water’s Associates Dual Pump system equipped with a reverse-phase ultrasphere ODS column (C,,-ODS, 5~., 10 mm x 25 cm, Beckman, Palo Alto, CA), a U-6K injector and a 481 varia- ble wavelength detector. The products were eluted with a linear gradient of methanol/ water/ acetic acid (50: 50: 0.05, v/v) (solvent A) to methanol (solvent B) for 40 min at a flow rate of 3 ml/mm [ll]. Column effluents were monitored with a Water’s Associates 481 X,, variable wavelength detector set at 302 nm (O-8 mm), 270 nm (9-20 min) and 237 nm (21-40 min) for porcine leuko- cytes. Fractions of 3 ml were simultaneously col- lected with an on-line fraction collector and a 50 ~1 portion of each fraction was removed for esti- mation of recovered radioactivity. PGB,, 15- HETE, LTB,, 8,15- and 14,15-diHEPEs standards

400

were monitored routinely by reverse-phase-HPLC before and after sample injection.

Ultraviolet spectroscopy Samples eluted from the HPLC were evaporated

to dryness under vacuum, dissolved in absolute ethanol and examined with a Hewlett-Packard

8450-A ultraviolet/visible spectrophotometer.

Gas chromatography-mass spectrometry The methylester of each sample (Fig. 1) (1-5 pg

each) was converted to trimethylsilyl ethers by addition of 25 ~1 of pyridine followed by 50 ~1 of trimethylchlorosilane and 50 ~1 of hexamethyldi-

silazane (Supelco). The mixtures were kept at room temperature for 20 min and evaporated to dryness with N,. Next, the samples were dissolved in hexane (25 ~1) and injected into the gas chromato- graph-mass spectrometer (Hewlett-Packard 5895- B) equipped with a glass column (1.5 cm x 4 m)

packed with 1% SE-30 on chromosorb W (HP). SO/l00 mesh. The helium flow was set at 40 ml/min, with the oven temperature, injection temperature and ion source temperature set at 200, 260 and 2OO”C, respectively. The electron energy was set at 70 eV.

Results

Reverse-phase HPLC analysis of the ethyl acetate extract from porcine leukocytes incubated

with 15-[1-‘4C]HPEPE revealed six major peaks in porcine leukocytes (Fig. 1A). The radioactivities of these fractions indicated that these metabolites were derived from 15-[1-‘4C]HPEPE. All of the fractions were subsequently isolated and dried under N,. The residue of each fraction was meth- ylated with excess diazomethane for 20 min at room temperature. The methyl esters were further purified by straight-phase HPLC using a step gradient of solvent I (hexane/isopropanol/acetic acid, 99 : 1 : 0.01, v/v) and II (hexane/ isopropanol/ acetic acid, 94 : 6 : 0.01, v/v) as

described [13]. Fraction LI displayed an ultraviolet spectrum

with a X,, of 302 nm and shoulders at 289 and 316 nm, suggesting the presence of a conjugated tetraene structure [ll]. The methyl ester of frac- tion LI was further separated into two major

2’ L

0 5 10 15 20 25 30 35 4iv

c 2

I j-y 0 5 10 15

T

Time (min)

. 6%

25 30 35 40 45 50 55 60 65

e (mln)

Fig. 1. (A) Reverse-phase HPLC chromatogram of 15-[l- 14C HPEPE metabolites isolated after incubation of porcine 1 leukocytes with 15-[1-‘4C]HPEPE. Solid line shows ultraviolet absorption at 302,270 and 235 nm. Broken line shows radioac- tivity in each fraction estimated by liquid scintillation count- ing. (B) Straight-phase HPLC chromatogram of peak LI from incubation of 1%HPEPE with porcine leukocytes using step gradient as described in Results.

compounds (Fig. 1B). The two major compounds, compound A and compound B, underwent tri- methylsilylation (TMSi) and were analyzed by GC/MS. The mass spectrum of compound A (C value = 24.3) showed ions of high intensity at m/z = 203 (base peak); Me,SiO=CH-(CH&CO-

OCH,; 377 (M - 203, loss of Me,SiO=CH- (CH,),-COOH,); 171, (MeJiO+=CH-CH2- [CH],CH,CH,). Ions of low intensity were ob- served at m/z = 580 (M+); 490 (M - 90); 217 and 69. This mass spectrum was identical to that of lipoxene A (lipoxin A,) as described [ll]. Simi- larly, the mass spectrum of compound B (C value = 24.1) showed ions of high intensity at m/z = 171 (base peak, Me,SiO+=CH-CH,(CH),CH,CH,); 409, (M - 171, loss of Me,SiO+=CHCH,(CH),

401

CH,CH,); 203, (Me3SiO+=CH(CH,),-CO- OCH,); ions of low intensity were observed at M/z = 580 (M); 490 (M - 90); 69. These mass spectra are identical to that of lipoxene B (lipoxin B,) [ll]. Taking together the ultraviolet and mass

spectra, compound A was identified as 5,6,15-tri- hydroxy-7,9,11,13,17-eicosapentaenoic acid or lipoxin A, (lipoxene A) [ll]. Similarly, compound

B was identified as 5,14,15_trihydroxy- 6,8,10,12,17-eicosapentaenoic acid or lipoxin B,

(lipoxene B) [ll]. The methyl ester of the substance in peak LII

contains one single peak in straight-phase-HPLC. The ultraviolet spectrum showed X,, at 269.5 nm and shoulders at 260,281. The mass spectrum of trimethylsilyl derivatives of the methyl ester showed ions of high intensity at m/z = 171

(Me,SiO+=CHCH,(CH),CH,CH,); 351 (M - 141, loss of CH,(CH),(CH,),COOCH& 261 (351 - 90); 243 (Me,SiO+=CH(CH,)(CH),- (CH,),COOCH3). Ions of low intensity were ob- served at m/z = 477 (M - 15); 461 (M - 31) and 69 (Fig. 2A). The mass spectrum was similar to that of 8,15-diHETE [14]. The mass spectrum and ultraviolet spectrum strongly suggested that the methyl ester of 8,15-diHEPE is derived from EPA.

After methylation, peak LIII was further sep- arated by straight-phase HPLC into four major components. The first component displayed ultra-

violet h,, at 243 nm, shoulders at 226, which is

similar to 5,15-diHETE [15]. The mass spectrum of the TMSi derivatives of the methylesters (C value = 23.7) showed prominent ions at m/z: 203, 171, 223, 477, 461, 492 (Fig. 2B). The spectrum is very similar to that of 5,15-diHETE, except that it showed two mass units less than 5,15-diHETE. It was identified as the methyl ester of 5,15-diHEPE. The ultraviolet spectrum of the remaining three components showed X,, of 269.5, 269.5 and 270 nm, respectively. The mass spectra of the trimeth- ylsilyl derivatives of these three components were identical to those of fraction LII. These data sug- gest that they are isomers of 8,15-diHEPE.

The methyl ester of fraction LIV was further separated into two peaks by straight-pahse HPLC. The first peak displayed ultraviolet h,, at 273.5 nm and the second peak showed ultraviolet X,, at 273 nm. After trimethylsilylation, both frac- tions from straight-phase HPLC were analyzed by

GC/MS. The mass spectrum (C value = 23.6) of the first straight-phase HPLC peak showed promi- nent ions at m/z = 171 (base peak, Me,SiO+= CHCH,(CH),CH,CH,); 321 (M - 171); 394 (re- arrangement of ion of C,_,, MeSiO+=CH(CH), CH,(CH),(CH,),COMeOSiMe,) [16]; 477 (M - 15); 461 (M - 31); 423 (M - 69). The position of the hydroxy groups at Cl4 and Cl5 were de-

termined by ion fragments at m/z 171 and 321, as well as the rearrangement ion at 394. a character- istic ion of 14,15-dihydroxy compounds [15,16] (Fig. 2C). The mass spectrum (C value = 24.5) of the second isomer was identical to that of first

isomer. Based on the ultraviolet absorption spec- tra and GC/MS data, it is concluded that peak LIV comprised two isomers of 14,15-diHEPE.

Fraction LV did not display any ultraviolet

absorbance. However, the presence of radioactiv- ity indicated that it was derived from 15-[l- 14C]HPEPE. After methylation and trimethylsily-

lation, it was analyzed by GC/MS. The mass spectrum (C value = 21.2) showed ions of high

intensity at m/z = 405 (M - 15); 391 (M - 31); 351 (M- 69); 309 (base peak, Me,SiO’=CH

(CH),CH,(CH),-CH,(CH),(CH,),COOCH,); 219; 142; 71 and 69 (Fig. 2D). Although the molecular ion was not observed in the mass spec- trum, the molecular weight was deduced to be 420 by the presence of ions at 405 (M - 15) and 389

(M - 31). Ions at 351 and 69 indicated the clea- vage at C15-16 with the loss of the fragment of

C 15_20 containing an n - 3 double bond [17]. According to the reverse- and straight-phase HPLC retention times, the C value and the mass spec- trum, this compound was identified as 13-hy-

droxy-14,15-epoxyeicosatetraenoic acid, which is analogous to 13-hydroxy-14,15-epoxyeicosatri- enoic acid as reported by Bryant et al. and Narumiya et al. [18,19].

Fraction LVI displayed an ultraviolet spectrum

of XInax 237 nm, suggesting the presence of a

conjugated double bond [13]. The methyl ester of this fraction was further purified by straight- phase-HPLC (retention time = 7.5 mm). The methyl ester was then reacted with trimethylsilane and the derivative was analyzed by GC/MS. The mass spectrum (C value = 20.9) (Fig. 2E) showed ions at m/z = 404 (M+); 389 (M - 15); 373 (M - 31); 335 (M- 69); 314 (M - 90); 233 (M -

402

20 111 lli 371

A 360 400 440 460 520 560 600

m/z

461 !I#

,I,

. L, .I, , 1; . . . , , 360 400 440 480 520 560 600

C m/z

171) and 171 (base peak) (Fig. 2E). This mass spectrum was similar to that of 15HETE except that there were two mass units less due to the presence of an n - 3 double bond. Furthermore, the mass spectrum of the hydrogenated derivatives was identical to the spectrum of hydrogenated derivatives of 15-HETE. This compound was therefore identified as 15HEPE.

322 119 239 280 , i

80 120 160 200 240 280 320

360 400 440 480 520 560 600

m/z

60 120 160 200 240 260 320

E 360 400 440 460 520 560 600

m/z

Fig. 2. Mass spectra of methyl ester and trimethylsilylated

derivatives of 8,15-diHEPE (A); 5,15-diHEPE (B); 14,15-di-

HEPE (C); 13-hydroxy-14,15-epoxyeicosatetraenoic acid (D)

and 15-HEPE (E).

Discussion

This report describes the formation of a new group of mono-, di- and trihydroxy compounds derived from 15-HPEPE in porcine leukocytes. Their structural elucidations were based on their chromatographic behavior on reverse- and straight-phase HPLC, on their ultraviolet spectra, C-values and finally by GC/MS (Table I). In- cubation with porcine leukocytes resulted in the

403

TABLE I

CHROMATOGRAPHIC, ULTRAVIOLET AND GC,‘MS DATA OF 15-HPEPE LMETABOLITES

Reverse-phase (RP) HPLC peak designations correspond to Fig. l(A). Some variation in straight-phase (SP) HPLC retention times

can occur as column activity changes. GC (CV) refers to the elution time of the Me-MesSi derivatives relative to a series of saturated

fatty acid Me standards. MS ions are representative fragment ions in the mass spectrum. UV,,,, denotes the wavelength of maximum

absorption. Amount is expressed as nanograms per ml of incubation media after straight-phase HPLC. The indicated quantities are

not corrected for losses incurred during isolation.

RP-HPLC SP-HPLC UVrnaX GC (0 Major mass ions

Peak retention vol. (nm) (m/z)

LI 55 302 24.3 203,377,171

LI 59 302 24.1 203,409,171

LII 35 269.5 23.9 171,351,492

LIII 35 269.5 23.9 171,351,492

LIKI 36 269.5 23.8 171,3X,492

LIII 37.5 270 23.9 171,351,492

LIII 33 243 23.7 171,203,492

LIV 33 273.5 23.6 171,321,492

LIV 33.6 273 24.5 171,321,492

LV 8 none 21.2 309,351,405

LVI 7.5 237 20.9 171,335,404

a 13-OH-14,15-epoxyETE = 13-hydroxy-I4,15-epoxyeicosate~aenoic acid.

Designation Amount

(ng)

lipoxin As 20

hpoxin Bs 30

8,15-diHEPE 686

f&15-diHEPE 174

8,15-diHEPE 50

815.diHEPE 500

5,15-diHEPE 560

14,15-diHEPE 480

14,15-diHEPE 142

13-OH-14,15-epoxyETE a 100

15-HEPE 3 320

formation of four isomeric 8,15-diHEPE, two iso- meric 14,15-diHETE, 5,lSdiHEPE and 15HEPE, as well as 13-hydroxy-14,15-epoxyeicosatetraenoic acid. The formation of the Cl5 series of leukotrienes of arachidonic acid by porcine leukocytes had previously been described by Maas et al. [20,21]. By incubating 15HPETE with porcine leukocytes and human platelet, Maas and co-workers had demonstrated that the formation of these Cl5 series of leukotrienes, i.e., 8,15-di- HETEs and 14,15-diHETEs, was via a lipo- xygenase mechanism similar to 12-lipoxygenase (201. Most recently, Brash et al. [22], using the purified porcine 12-lipoxygenase, have confirmed their hypothesis that conversion of 15-HPETE to Cl5 leukotrienes was indeed catalyzed by 12-lip- oxygenase in porcine leukocytes [22]. The mecha- nism was as follows: 12-lipoxygenase-like activity catalyzes the stereospecific removal of hydrogen at Cl0 from 15-HPETE, this being followed by radi- cal migration to Cl4 and resulting in the forma- tion of 14,15-LTA, and 14,15-diHPETE; hydroly- sis of 14,15-LTA, yields two 8,lSdiHETE and the reduction of 14,15-DHPETE yields 14,15-di-

HETEs [20,22]. Similar mechanisms are to be ex- pected for the formation of the Cl5 series leukotrienes of EPA, In this manner, the forma- tion of the two major 8,15-diHEPEs (peaks LII and LIII) was very likely to be via initial removal of the Cl0 hydrogen of 15-HPEPE by the action of a 12-lipoxygenase-like activity and the forma- tion of 14,15-LTA, followed by hydrolysis [20]. The formation of 14,15-diHEPE was also via a similar mechanism, i.e., radical migration to Cl4 after initial Cl0 hydrogen atom removal followed by intermolecular reaction of oxygen and reduc- tion [20]. In addition, hydrolysis of 14,15-LTA, can also contribute to certain percentages of 14,15-diHEPE formation [20].

The isolation and purification of 5-lipoxy- genase enzyme in porcine leukocytes has recently been reported by Yamamoto and co-workers [23]. This enzyme was also active with 12- and 15- HPETE, producing (5S,12S)- and (5S,15S)-di- HETEs, respectively. Therefore, the presence of Slipoxygenase in porcine leukocytes will also be expected to convert 15-HPEPE to 5,15-DHPEPE. 5,15-DHPEPE may subsequently be converted to

404

5,15-diHEPE. Alternatively, 5,lSDHPEPE may

be converted to 5,6-oxido-15-hydroperoxyeicosa- pentaenoic acid [24], an intermediate for the for- mation of lipoxin of the 5 series [LXA, and LXB,) [11,12]. The mechanism of lipoxin forma- tion may also be generated via the LTA, syn- thetase pathway associated with 5-lipoxygenase enzyme in a manner analogous to the formation of LTA, from 5-HPETE [25]. The possibility that LTA, synthetase catalyses the formation 5,6- oxido-15-hydroperoxyeicosapentaenoic acid from 5,15-DHPEPE is presently under investigation.

The nonenzymatic formation of 13-OH-14,15-

epoxyETE from 15-HPETE was first noticed by Bryant et al. and Narumiya et al. [18,19]. How- ever, recently, Hamberg et al. [26] had demon- strated that a two-step reaction may be involved in the formation of epqxide alcohol derivatives of arachidonic acid in the primitive fungus, Sapro-

legnia parasitica [26]. Hamberg’s mechanism in- volved an initial step with 15-lipoxygenase to gen- erate 15-HPETE from arachidonic acid followed by a second step in which 15-HPETE was con- verted to hydroxyepoxide derivatives by the action of hydroperoxide isomerase [26]. In addition, the

formation of hydroxyepoxide may also be cata- lysed by the possible contaminating hematin or hemoglobin, which was reported to catalyze the formation of hydroxyepoxide from 1ZHPETE

~271. In this study, we have demonstrated that 15-

HPEPE can be metabolized by porcine leukocytes to form monohydroxy and dihydroxy metabolites, as well as trihydroxypentaene derivatives. Unlike LTB,, these trihydroxypentaenes or lipoxins of the 5 series possess biological activities comparable to their arachidonic-acid-derived counterparts [28]. We had demonstrated that 15-HEPE was the major metabolite of 15-HPEPE in human platelets [29] and exhibited a more potent biological activity than 15HETE on neutrophil aggregation as pro- voked by the chemotactic peptide fMet-Leu-Phe [29]. Thus, it is possible that during nutritional manipulation, diets enriched in EPA or fish oil may result in the change of the biological spectra of the eicosanoids derived from platelets, leuko- cytes and other cell types. This new group of eicosanoids of EPA may alter cardiovascular homeostasis [30], and may function as im-

munoregulators, such as lipoxins, of certain auto-

immune disease [31]. The biological activities of these new groups of hydroxylated metabolites of EPA are now under investigation in our labora-

tory.

Acknowledgments

We wish to thank Miss Gail Price for the preparation of this manuscript. We thank Carl Ehmer Farm of Poughkeepsie, NY, for the gener- ous supply of porcine blood used in this study. This work was supported by NIH grant HL-25316. P.Y-K.W. is the recipient of an NIH Research

Career Development Award (HL-00811).

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