16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes.
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/10(8) cells) and 20-HETE (341+/-69 pg/10(8) 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 omega- and omega-4 oxygenase(s).
Bednar
M M
MM
Division of Neurosurgery, Vermont Center for Vascular Research, The University of Vermont, Burlington, VT 05405, USA.
Gross
C E
CE
Balazy
M K
MK
Belosludtsev
Y
Y
Colella
D T
DT
Falck
J R
JR
Balazy
M
M
D001095:Arachidonic Acids / Q000378:metabolism*; D002449:Cell Aggregation; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*
C410424:16-hydroxy-5,8,11,14-eicosatetraenoic acid; D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System