16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes

doi:10.1016/S0006-2952(00)00345-2

Biochemical Pharmacology

Volume 60, Issue 3, 1 August 2000, Pages 447-455

https://doi.org/10.1016/S0006-2952(00)00345-2 Get rights and content

Abstract

Intact human polymorphonuclear leukocytes (PMNL) incubated with substimulatory amounts of arachidonic acid in the absence of a calcium ionophore formed four metabolites that were isolated by reverse-phase HPLC and characterized structurally by GC/MS. A major metabolite eluting as the most abundant peak of radioactivity lacked UV chromophores above 215 nm, and its formation was sensitive to 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF525A) but not 3-amino-1-[m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), suggesting that it was likely to be a product of cytochrome P450. The GC/MS analysis revealed the presence of two components: 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) in an approximate ratio of 4:1. The minor metabolites were identified as 15-HETE and 5-HETE. Although 20-HETE has been observed previously as a product of arachidonic acid metabolism in PMNL, the occurrence of 16-HETE was a novel finding. The stereochemistry of the hydroxyl group in PMNL-derived 16-HETE was established by analysis of 1-pentafluorobenzyl-16-naphthoyl derivatives on a chiral-phase chromatographic column and comparison with authentic synthetic stereoisomers. The PMNL-derived radioactive metabolite co-eluted with the synthetic 16(R)-HETE stereoisomer. Analysis of the total lipid extracts from intact PMNL followed by mild alkaline hydrolysis resulted in detectable amounts of 16-HETE (108 ± 26 pg/10⁸ cells) and 20-HETE (341 ± 69 pg/10⁸ cells), which suggested that these HETEs were formed from endogenous arachidonic acid and esterified within PMNL lipids. Thus, in contrast to calcium ionophore-stimulated neutrophils that generate large amounts of 5-lipoxygenase products, the intact PMNL generate 20-HETE and 16(R)-HETE via a cytochrome P450 ω- and ω-4 oxygenase(s).

Section snippets

Materials

HETE standards [16(R)-, 16(S)-, 17-, 18-, 19-, and 20-HETE] and 19-HETE-d₃ (99+ atom% deuterium) were synthesized as described previously 15, 18. The standards of lipoxygenase-derived HETEs 5, 8, 11, 12, 15 and octadeuterium-labeled arachidonic acid were obtained from Biomol. All solvents used were HPLC grade (Burdick & Jackson), and other reagents were of the highest grade commercially available. BSTFA and pentafluorobenzyl bromide were obtained from the Aldrich Chemical Co., and [1-¹⁴

Results

Incubations of arachidonic acid with human PMNL in the absence of a Ca²⁺ ionophore resulted in the formation of four radiolabeled products (Fig. 1A). The major peak of radioactivity (D) was unmodified arachidonic acid. The material in peak A contained 10–15% of the radioactivity, and it did not show UV absorbance above 205 nm. The material in peaks B and C, combined, accounted for 5–8% of the total radioactivity injected into the HPLC column, and displayed absorbance at 234 nm (Fig. 1B),

Discussion

Biosynthesis of leukotrienes in PMNL requires calcium influx [2]. In the absence of extracellular calcium, stimulation with PAF or fMLP fails to induce arachidonic acid release and LTB₄ synthesis. Addition of Ca²⁺ with either PAF or fMLP to Ca²⁺-depleted PMNLs results in a Ca²⁺ influx proportional to the extracellular concentration of Ca²⁺ and causes a substantial release of arachidonic acid and synthesis of LTB₄[3]. Our previous work has shown that in the absence of Ca²⁺ influx, PMNL

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Structural biology16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes

Abstract

Section snippets

Materials

Results

Discussion

J Biol Chem

Biochem Biophys Res Commun

FEBS Lett

J Biol Chem

Arch Biochem Biophys

J Biol Chem

Biochem Pharmacol

Biochem Biophys Res Commun

J Biol Chem

Arch Biochem Biophys

J Biol Chem

Tetrahedron Lett

J Biol Chem

Kidney Int

Arch Biochem Biophys

Arch Biochem Biophys

Prog Lipid Res

Structural biology
16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes