Leukotriene B4 ω-side chain hydroxylation by CYP4F5 and CYP4F6

Bylund, Johan; Harder, Adam; Maier, Kristopher G.; Roman, Richard J.; Harder, David R.

doi:10.1016/s0003-9861(03)00030-4

Cited by 27 publications

(22 citation statements)

References 24 publications

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“…Minor activity was detected with CYP4F5, whereas CYP4F6 was devoid of LTB 4 -hydroxylase activity. These results are in conflict with the extensive side-chain hydroxylation of LTB 4 reported for yeast-expressed CYP4F5 and CYP4F6 (Bylund et al, 2003). Although CYP4F1 and CYP4F4 are clearly implicated in the metabolism of LTB 4 and regulation of inflammation, further studies will be necessary to clarify the role of CYP4F5 and CYP4F6 in this pathway.…”

Section: Discussionmentioning

confidence: 77%

“…In contrast, little is known about the ability of the CYP4F proteins to catalyze arachidonic acid metabolism. Although the rat CYP4F isoforms have been heterologously expressed in both yeast and E. coli (Kawashima et al, 1997;Kikuta et al, 1999;Bylund et al, 2003), this represents the first study in which the catalytic function of all four rat isoforms has been directly compared. Despite more than 70% amino acid sequence identity, the rat CYP4F isoforms show differences in substrate specificity and sensitivity to known P450 inhibitors.…”

Section: Discussionmentioning

confidence: 99%

“…Escherichia coli-expressed CYP4F5 had low -hydroxylation activity toward LTB 4 , whereas the solubilized membrane fraction of CYP4F4 recombinant protein catalyzed the -hydroxylation of prostaglandin A 1 , prostaglandin E 1 , 6-trans-LTB 4 , and LTB 4 (Kawashima and Strobel, 1995). More recently, yeast-expressed CYP4F5 was found to convert LTB 4 primarily into 18-hydroxy-LTB 4 , and a relatively small amount of 19-and 17-hydroxy-LTB 4 and yeast-expressed CYP4F6 was found to convert LTB 4 into 19-and 18-hydroxy-LTB 4 (Bylund et al, 2003). Neither CYP4F4 nor CYP4F1 was able to metabolize arachidonic acid or lauric acid, two typical substrates for CYP4A enzymes (Kawashima et al, 1997;Kikuta et al, 1999).…”

mentioning

confidence: 99%

“…However, the recent identification of CYP4F isoforms expressed in the rat kidney (Kalsotra et al, 2002;Bylund et al, 2003) and the finding that CYP4F2 is the major arachidonic acid -hydroxylase in human kidney (Lasker et al, 2000) led us to hypothesize that the CYP4F enzymes also contribute to renal 20-HETE formation in the rat. In the present study, the four rat CYP4F isoforms were heterologously expressed in E. coli, and their metabolism of fatty acids was characterized.…”

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confidence: 99%

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Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes

Xu¹,

Falck²,

Montellano³

et al. 2003

J Pharmacol Exp Ther

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Arachidonic acid is -hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome P450 (P450) 4A isoforms are generally considered the major arachidonic acid -hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B 4 (LTB 4 ) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K s of 25 to 59 M, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the -hydroxylation of LTB 4 with a K m of 24 and 31 M, respectively, and CYP4F5 had minor activity in LTB 4 metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the -hydroxylation of arachidonic acid with an apparent k cat of 9 and 11 min Ϫ1 , respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid -hydroxylase activity. The P450 -hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1-and CYP4F4-catalyzed -hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify P450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

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Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Catalytic Activity and Isoform-Specific Inhibition of Rat Cytochrome P450 4F Enzymes

Xu¹,

Falck²,

Montellano³

et al. 2003

J Pharmacol Exp Ther

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“…Additionally, rat liver microsomes were found to convert LTB 4 into 20-OH-LTB 4 along with small amounts of 19-OH-LTB 4 (31). Recently, recombinant CYP4F5 and CYP4F6 were found to convert LTB 4 into 17-OH-, 18-OH-, 19-OH-, and 20-OH-LTB 4 during in vitro incubation (37). These P450 isozymes were found expressed in hepatocytes of rats (37).…”

Section: Discussionmentioning

confidence: 99%

Urinary Metabolites of Leukotriene B4 in the Human Subject

Berry

Borgeat²,

Gosselin³

et al. 2003

Journal of Biological Chemistry

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Leukotriene B 4 (LTB 4 ) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans. The metabolism of LTB 4 in vitro is complex with several competing pathways of biotransformation, but metabolism in vivo, especially for normal human subjects, is poorly understood. As part of a Phase I Clinical Trial of human tolerance to LTB 4 , four human subjects were injected with 150 nmol/kg LTB 4 with one additional subject as placebo control. The urine of the subjects was collected in two separate pools (0 -6 and 7-24 h), and aliquots from these urine collections were analyzed using high performance liquid chromatography, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry for metabolites of LTB 4 . In the current investigation, 11 different metabolites of LTB 4 were identified in the urine from those subjects injected with LTB 4 , and none were present in the urine from the placebo-injected subject. The unconjugated LTB 4 metabolites found in urine were structurally characterized as 18-carboxy-LTB 4 , 10,11-dihydro-18-carboxy-LTB 4 , 20-carboxy-LTB 4 , and 10,11-dihydro-20-carboxy-LTB 4 . Several glucuronide-conjugated metabolites of LTB 4 were characterized including 17-, 18-, 19-, and 20-hydroxy-LTB 4 , 10-hydroxy-4,6,12-octadecatrienoic acid, LTB 4 , and 10,11-dihydro-LTB 4 . The amount of LTB 4 glucuronide (16.7-29.4 pmol/ml) and 20-carboxy-LTB 4 (18.9 -30.6 pmol/ml) present in the urine of subjects injected with LTB 4 was determined using an isotope dilution mass spectrometric assay before and after treatment of the urine samples with ␤-glucuronidase. The urinary metabolites of LTB 4 identified in this investigation were excreted in low amounts, yet it is possible that one or more of these metabolites could be used to assess LTB 4 biosynthesis following activation of the 5-lipoxygenase pathway in vivo.Leukotriene B 4 (LTB 4 , 1 (5S,12R)-dihydroxy-6,14Z-8,10E-eicosatetraenoic acid) is a biologically active metabolite of arachidonic acid that is chemotactic for the human neutrophil and a potent lipid mediator of inflammation (1). LTB 4 is not stored within a cell but synthesized following activation of certain cells through release of arachidonic acid by the cytosolic phospholipase A 2 (2) and activation of the enzyme 5-lipoxygenase (3). The immediate product of the 5-lipoxygenase pathway is the reactive epoxide intermediate leukotriene A 4 that is transformed by leukotriene A 4 hydrolase into LTB 4 (4). A specific G-protein-coupled receptor for LTB 4 has now been cloned and expressed (5) and is known to be expressed by many cell types including the neutrophil (6). Through this receptor, LTB 4 is a potent stimulus of many functional responses of cells that are involved in immune responses (7) such as neutrophils, monocytes, macrophages, and various lymphocytes. LTB 4 is therefore thought to be an important component of the host defense mechanisms and is currently under investigation as a potent drug for the prophylaxis and/...

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