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

Martin M. Bednar, Cordell E. Gross, Maria K. Balazy, Yuri Belosludtsev, Danette T. Colella, J R Falck, Michael Balazy

Research output: Contribution to journalArticle

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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 P450ω- and ω-4 oxygenase(s). (C) 2000 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)447-455
Number of pages9
JournalBiochemical Pharmacology
Volume60
Issue number3
DOIs
StatePublished - Aug 1 2000

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Arachidonic Acids
Metabolites
Hydroxyeicosatetraenoic Acids
Neutrophils
Arachidonic Acid
Stereoisomerism
Calcium Ionophores
Cytochrome P-450 Enzyme System
4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine
Lipids
Arachidonate 5-Lipoxygenase
Oxygenases
Stereochemistry
Radioactivity
Chromophores
Metabolism
Hydroxyl Radical
Hydrolysis
Derivatives
16-hydroxy-5,8,11,14-eicosatetraenoic acid

Keywords

  • Arachidonic acid metabolism
  • Cytochrome P450
  • Hydroxyeicosatetraenoic acid (HETE)
  • Mass spectrometry
  • Polymorphonuclear leukocytes
  • Stereochemistry

ASJC Scopus subject areas

  • Pharmacology

Cite this

16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes. / Bednar, Martin M.; Gross, Cordell E.; Balazy, Maria K.; Belosludtsev, Yuri; Colella, Danette T.; Falck, J R; Balazy, Michael.

In: Biochemical Pharmacology, Vol. 60, No. 3, 01.08.2000, p. 447-455.

Research output: Contribution to journalArticle

Bednar, Martin M. ; Gross, Cordell E. ; Balazy, Maria K. ; Belosludtsev, Yuri ; Colella, Danette T. ; Falck, J R ; Balazy, Michael. / 16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes. In: Biochemical Pharmacology. 2000 ; Vol. 60, No. 3. pp. 447-455.
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AU - Balazy, Michael

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N2 - 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 P450ω- and ω-4 oxygenase(s). (C) 2000 Elsevier Science Inc.

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KW - Stereochemistry

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