Stereoselective carbonyl reductases from rat skin and leukocyte microsomes converting 12-ketoeicosatetraenoic acid to 12(S)-HETE

Cell-free preparations from rat polymorphonuclear leukocytes and skin were found to catalyze the reduction of 12-keto-5,8,10,14-eicosatetraenoic acid (12-KETE) to 12-hydroxyeicosatetraenoic acid (12-HETE). The reductase activity was associated with the microsomal fraction and showed a marked preference for NADH over NADPH as reducing cofactor. Characterization of the reaction product by chiral phase HPLC of the methyl ester derivative indicated that 12-KETE reduction generated almost exclusively 12(S)-HETE. The results demonstrate that rat skin and leukocyte microsomes possess an NADH-dependent 12-KETE reductase activity that forms 12(S)-HETE as a major product. The identification of stereoselective 12-KETE reductases provides a basis for further defining the role these enzymes may play in the regulation of 12-KETE levels and in the protection against degradation of 12-KETE to the pro-inflammatory 12(R)-HETE by selectively generating 12-HETE of the S configuration.     

12(R)-hydroxyeicosatetraenoic acid synthesis by 3-methylcholanthrene- and clofibrate-inducible cytochrome P450 in porcine ciliary epithelium.

Porcine ciliary epithelial microsomes synthesized 12[S]-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12[S]-HETE) from arachidonic acid by a membrane-bound lipoxygenase and 12[R]-isomer by the cytochrome P450-dependent monooxygenase system. The activity to form 12(R)-isomer was markedly enhanced by 3-methylcholanthrene and clofibrate. Both basal and induced levels of 12(R)-HETE synthesizing activity were considerably higher in nonpigmented epithelial cells than in pigmented cells of the ciliary processes. The induced activity was suppressed by polyclonal antibodies raised against purified cytochrome P450 IA1 and NADPH-P450 reductase but not by substrates for clofibrate-inducible omega/omega-1 hydroxylases (P450 IVA-mediated). These results suggest that 12(R)-HETE synthesis by porcine ciliary microsomes may be mediated by a cytochrome P450 of the IA family.     

Metabolism of 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE) in corneal tissues: formation of novel metabolites

12(R)-Hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], a cytochrome P450 arachidonate metabolite, is metabolized by corneal tissues via three distinct metabolic pathways: beta-oxidation, omega-hydroxylation, and keto-reduction. The major metabolite released from the intact rabbit corneal epithelium or cultured cells was identified by mass spectrometric analysis as 8-hydroxy-4,6,10-hexadecatrienoic acid, the tetranor metabolite derived following two steps of beta-oxidation from the carboxy terminus. The beta-oxidation pathway was expressed in both microsomes and mitochondria isolated from bovine corneal epithelium and was dependent on the addition of oxidizing equivalents. The major metabolite of 12(R)-HETE in subcellular fractions of bovine corneal epithelial cells was a dihydro compound, 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). This derivative is presumably formed by an oxidation of the hydroxyl group followed by two keto-reduction steps, since its formation was accompanied by the appearance of a keto metabolite identified as 12-oxo-5,8,14-eicosatrienoic acid. The omega-hydroxylation, in contrast to other cell types, was a minor route for 12(R)-HETE metabolism in these tissues. Since 12(R)-HETE has been implicated as a modulator of Na(+)-K(+)-ATPase activity and its related functions in ocular tissues, these findings raise the possibility that the newly described metabolites may be involved in regulating corneal functions. In addition, the presence of a keto reductase in the cornea may be of great importance following injury since 12(R)-HETrE resulting from 12(R)-HETE by this activity is a potent ocular proinflammatory compound.     

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid is a more potent neutrophil chemoattractant than the 12(R) epimer in the rat cornea

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE] is reported to be more potent than its epimer 12(S)-HETE as a chemoattractant for human neutrophils in vitro and following topical application to the skin. To assess the in vivo neutrophil chemoattractant potencies of 12(S)-HETE and 12(R)-HETE in the rat, we injected 1 microgram, 5 micrograms, or 10 micrograms of these eicosanoids into the corneal stroma. Rats were killed 12-15 hours after injection, and the number of neutrophils in the stroma was counted in a histological section of the cornea including the injection site. The number of neutrophils was significantly increased in corneas injected with 5 micrograms (+103% of control) or 10 micrograms (+456% of control) of 12(S)-HETE and in those injected with 10 micrograms of 12(R)-HETE (+111% of control). The neutrophilic infiltrate in corneas injected with 1 microgram or 5 micrograms of 12(S)-HETE was not significantly different from that in corneas injected with 1 microgram of leukotriene B4. The data for the 10 micrograms injections indicate that 12(S)-HETE is a more potent neutrophil chemoattractant than 12(R)-HETE in the rat cornea. Our results suggest that species or tissue specificity may determine the relative potencies of 12-HETE epimers as chemoattractants for neutrophils, and that 12(S)-HETE may be an important inflammatory mediator in the rat cornea.     

Biosynthesis of 18(RD)-hydroxyeicosatetraenoic acid from arachidonic acid by microsomes of monkey seminal vesicles. Some properties of a novel fatty acid omega 3-hydroxylase and omega 3-epoxygenase

Microsomes of seminal vesicles of the cynomolgus monkey were incubated with [14C]5,8,11,14-eicosatetraenoic (arachidonic) acid and NADPH for 40 min at 37 degrees C and the products were characterized. Prostaglandins F2 alpha and E2 were the two main metabolites (approximately 52% of radioactivity), while 18(R)-hydroxy-cis-5,8,11,14-eicosatetraenoic acid (18(R)-HETE) was identified as the main, less polar product (approximately 13%). Significant biosynthesis of the 19-hydroxy or 20-hydroxy metabolites of arachidonic acid could not be detected. The formation of 18(R)-HETE was further investigated in the presence of a prostaglandin synthesis inhibitor, diclofenac sodium. The omega 3-hydroxylation was only partly supported by substituting NADH for NADPH. The hydroxyl oxygen of 18(R)-HETE was derived from the atmosphere and the omega 3-hydroxylation was inhibited by proadifen and partly inhibited by carbon monoxide. These findings suggest that 18(R)-HETE is formed by a cytochrome P-450 (P-450 omega 3). Linoleic acid and 8,11,14-eicosatetraenoic acid were also substrates of the enzyme, but stearic acid was not metabolized. 5,8,11,14,17-Eicosatetraenoic acid was oxygenated under these conditions mainly to 17,18-dihydroxy-5,8,11,14-eicosatetraenoic acid, presumably formed from 17(18)-epoxy-5,8,11,14-eicosatetraenoic acid by hydrolysis. The seminal microsomes thus seem to possess both omega 3-hydroxylase and omega 3-epoxygenase activity. These seminal vesicles also contain prostaglandin E 19-hydroxylase (Oliw, E.H., Kinn, A.-C., and Kvist, U. (1988) J. Biol. Chem. 263, 7222-7227). The presence of arachidonate omega 3-hydroxylase and prostaglandin E 19-hydroxylase was assessed in microsomes of adult and juvenile monkey livers. Arachidonic acid was metabolized extensively to diols (via epoxides), but 18-HETE could not be detected. In contrast, prostaglandin E1 was slowly hydroxylated mainly to 19-hydroxyprostaglandin E1 by both adult male and female juvenile hepatic microsomes. The results indicate that P-450 omega 3 of seminal vesicles might be a tissue-specific enzyme.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant for human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B4. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

Distribution of tritium labeled 12(S) hydroxy-eicosatetraenoic acid (12-HETE) in the rat.

The in vivo metabolism of 12-(S)-Hydroxy-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets, has been investigated in the rat. Fifty microcuries of 5,6-[3H]-12-HETE (50 Ci/mmol) were injected to anesthetized rats and the radioactivity was followed in plasma. At the end of the experiment, various organs of the animal were removed and the radioactivity attached to them was determined. The label of the plasma plateaued to approximately one third of the initial radioactivity ten minutes after the injection. Among the various organs tested (brain, heart, intestine, kidney, liver, lungs, spleen, testis/uterus) the kidney was far the most active to accumulate 12-HETE and/or its labeled metabolites, and no radioactivity could be detected in urine during the course of the experiment. The analysis of lipid extracts from the various tissues revealed that 12-HETE was not accumulating in its unesterified form but was likely bound to phospholipids. We conclude that, although the label providing from the initial 12-HETE did not completely disappear from plasma, circulating 12-HETE cannot be considered as a circulating marker of cell activation.     

Microsomal carbonyl reductase responsible for reduction of 4-phenyl-3-buten-2-one in rats

trans-4-Phenyl-3-buten-2-one (PBO), a flavoring additive, was transformed to the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) by rat liver microsomes, but not by liver cytosol, in the presence of NADH or NADPH. PBOL formed was identified by comparison with an authentic sample. The reductase activity was not inhibited by quercitrin, an inhibitor of cytosolic carbonyl reductase. The carbonyl reduction product of PBO by liver microsomes was identified as the R-enantiomer of PBOL by HPLC analysis. Rat blood also exhibited the carbonyl reductase activity in the presence of NADH or NADPH, but to a lesser extent.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant fo human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy - 5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B₄. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Mechanism for the formation of dihydro metabolites of 12-hydroxyeicosanoids. Conversion of leukotriene B4 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid to 12-oxo intermediates.

Eicosanoids containing a 12-hydroxyl group preceded by at least two conjugated double bonds are metabolized to 10,11-dihydro and 10,11-dihydro-12-oxo products by porcine polymorphonuclear leukocytes (PMNL) (Wainwright, S. L., Falck, J. R., Yadagiri, P., and Powell, W. S. (1990) Biochemistry 29, 10126-10135). These 10,11-dihydro metabolites could either have been formed by the direct reduction of the 10,11-double bond of the substrate, as previous evidence suggested, or via an initially formed 12-oxo intermediate. To gain some insight into the mechanism for the formation of dihydro products by this pathway, we investigated the metabolism of leukotriene B4 (LTB4), 12(S)-hydroxy-5,8,10,14-eicosatetraenoicacid(12(S)-HETE), and 12(R)-HETE by subcellular fractions from porcine PMNL. In the presence of NAD+ and a microsomal fraction from PMNL, each of the above 12-hydroxyeicosanoids was converted to a single product with a lambda max approximately 40 nm higher than that of the substrate, indicating that the conjugated diene or triene chromophore had been extended by one double bond, presumably by oxidation of the 12-hydroxyl group to an oxo group. In the case of LTB4, this was confirmed by mass spectrometry, which indicated that the product was identical to 12-oxo-LTB4. LTB4 was not converted to any products by a cytosolic fraction from PMNL, but was converted to both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 by the 1500 x g supernatant in the presence of NAD+. Negligible amounts of dihydro products were formed in the presence of NADH or NADPH, suggesting that initial oxidation of the 12-hydroxyl group is a requirement for reduction of the 10,11-double bond. Consistent with this hypothesis, 12-oxo-LTB4 was rapidly metabolized to 10,11-dihydro-12-oxo-LTB4 by the cytosolic fraction in the presence of NADH. Only small amounts of this product, along with some LTB4, were formed by the microsomal fraction. These results indicate that the initial step in the formation of 10,11-dihydro products from 12-hydroxyeicosanoids is oxidation of the 12-hydroxyl group by a microsomal 12-hydroxyeicosanoid dehydrogenase in the presence of NAD+, which is followed by reduction of the olefinic double bond by a cytosolic delta 10-reductase in the presence of NADH.     

Properties and biochemical characterization of NADH 5 alpha-reductase from rat liver microsomes

NADH 5 alpha-reductase is present in microsomes of various rat organs: heart and skeletal muscle, liver, adrenal glands, kidney, testes and prostate. The enzyme from rat liver microsomes utilizes B-hydrogen from the coenzyme NADH for steroid reduction. After solubilization of the enzyme with the nonionic detergent lubrol, phosphatidylcholine is necessary to restore the activity. This reactivation of the enzyme activity is paralleled by a corresponding increase of Vmax for testosterone (17 beta-hydroxy-4-androsten-3-one). Km and Vmax for testosterone change, Km and Vmax for the coenzyme NADH remain constant with an alteration of phosphate concentration in the incubation medium. The NADH 5 alpha-reductase is inhibited by numerous substances: amytal, phenobarbital, mepacrin, thenoyltrifluoracetone, gallic acid propyl ester, dicoumarol, pentachlorophenol, NADP and antibodies against rat liver NADPH ferrihemoprotein reductase. Antibodies against rat liver cytochrome-b5 reductase cause an activation of NADH 5 alpha-reductase.     

A comparison of the proinflammatory effects of 12(R)- and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid in human skin

Topical application of racemic 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R,S)-HETE] produces erythema and leucocyte accumulation in human skin. Since 12(R)-HETE is more potent than its epimer 12(S)-HETE as a neutrophil chemoattractant in vitro, their proinflammatory effects have now been compared in vivo. 12(R)- and 12(S)-HETE (0.5 - 20 ug/site) were applied topically to the forearm skin of 5 healthy volunteers and the sites occluded for 6 h. Five ug each of the two enantiomers were also applied to the opposite forearm. At 6 and 24 h blood flow and the areas of erythematous responses were measured. The 5 ug application sites were biopsied at 24 h. Both enantiomers caused dose related erythema and increased blood flow at 6 and 24 h, which were not significantly different at either of the time points tested. In contrast, pronounced neutrophil infiltrates were seen in the epidermis (25.2 +/- 13 cells/hpf) and dermis (13.2 +/- 5.1 cells/hpf) 24 h after application of 12(R)-, but not 12(S)-HETE (0.02 +/- 0.02 and 1.02 +/- 0.7 cells/hpf in epidermis and dermis respectively). However, the numbers of dermal mononuclear cells accumulating in response to the two enantiomers were similar. 12(R)-HETE thus appears to be a more potent neutrophil chemoattractant than 12(S)-HETE in human skin in vivo and may be of potential importance as a mediator of inflammation in man.     

12(S)-hydroperoxyeicosatetraenoic acid (12-HETE) increases mitochondrial nitric oxide by increasing intramitochondrial calcium

12(S)-Hydroxyeicosatetraenoic acid (12-HETE) is one of the metabolites of arachidonic acid involved in pathological conditions associated with mitochondria and oxidative stress. The present study tested effects of 12-HETE on mitochondrial functions. In isolated rat heart mitochondria, 12-HETE increases intramitochondrial ionized calcium concentration that stimulates mitochondrial nitric oxide (NO) synthase (mtNOS) activity. mtNOS-derived NO causes mitochondrial dysfunctions by decreasing mitochondrial respiration and transmembrane potential. mtNOS-derived NO also produces peroxynitrite that induces release of cytochrome c and stimulates aggregation of mitochondria. Similarly, in HL-1 cardiac myocytes, 12-HETE increases intramitochondrial calcium and mitochondrial NO, and induces apoptosis. The present study suggests a novel mechanism for 12-HETE toxicity.     

12(R)-hydroxyeicosatetraenoic acid is a neutrophil chemoattractant in the cavine, lapine, murine and canine dermis

Psoriasis is a disease state characterized by epidermal proliferation, neutrophil infiltration, along with release of the proinflammatory mediators leukotriene-B4(LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE]. LTB4 and 12(R)-HETE are chemoattractant to the neutrophil, the latter approximately 1000x less potent. LTB4 and 12(R)-HETE are present in psoriatic scale, the latter in quantities so much greater than LTB4 that it is proposed as a primary mediator of neutrophil infiltration in psoriasis. 12(R)-HETE, synthesized in optically pure form by a new, shorter route, was injected into the dermis of the cavine, lapine, canine, mouse and rat. At doses up to 50 mu gm per intradermal site, 12(R)-HETE was chemoattractant to the neutrophil (as assessed by dermal myeloperoxidase levels) with response in the cavine greater than canine greater than lapine greater than mouse greater than rat.     

NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome C reductase and cytosolic aldehyde oxidase

NAD (P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.     

Reduction of 7-ketolithocholic acid to chenodeoxycholic acid by rat liver preparations in vitro

The formation of chenodeoxycholic acid via 7-ketolithocholic acid by rat liver preparations was examined in vitro. Results showed that a rat liver preparation reduced 7-ketolithocholic acid mainly to chenodeoxycholic acid and to ursodeoxycholic acid in a smaller amount, and that the reductase required NADPH but not NADH as coenzyme and was mainly localized in the microsomes.     

12-Hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineals

The role of arachidonic acid metabolites in norepinephrine (NE)-induced N-acetyltransferase (NAT) activity and melatonin release was examined from 6 h-incubations of rat pineal glands. A cyclooxygenase inhibitor, indomethacin (5 x 10(-8) - 5 x 10(-6) M) was ineffective on melatonin release, in the presence of absence of NE (5 x 10(-6) M) while a lipoxygenase inhibitor, nordihydroguaiaretic acid (5 x 10(-7) -5 x 10(-5) M) had an inhibitory effect. Among the lipoxygenase metabolites, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulated both NAT activity and melatonin release in a dose-dependent manner, with a maximal effect occurring at 10(-6) M, while 5-HPETE or hydroxy derivatives of these compounds (12-HETE, 15-HETE and 5-HETE) were ineffective. These results indicate that 12-HPETE and 15-HPETE can be involved in NE-induced melatonin release.     

Regulation of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on human epidermal cells by interferon-gamma

We recently detected specific high-affinity binding sites for 12(S)-HETE, the main arachidonic acid metabolite in skin, on epidermal cells. The putative receptor is involved in keratinocyte chemotaxis toward 12(S)-HETE, which points to its participation in wound healing. In an effort to further characterize the 12(S)-HETE receptor, we investigated its regulation by various cytokines. Of the tested cytokines, only interferon (IFN)-gamma led to a massive induction of the 12(S)-HETE receptors. The effect was dose and time dependent and blocked by cycloheximide. The up-regulation of 12(S)-HETE receptors by IFN-gamma may represent an amplification mechanism of the assumed role of 12(S)-HETE in skin wound repair.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.