Role of human CYP4F2 in hepatic catabolism of the proinflammatory agent leukotriene B4.

Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its omega-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 +/- 0.3 nmol/min/nmol P450; 0.25 +/- 0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparent Km of 74.8 microM with a VMAX of 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp --> 12Gly) over the first 17 residues with the human CYP4F2 cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 omega-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 +/- 5%; n = 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1 but was unaffected by lauric acid, palmitic acid, and PGF2alpha. Finally, a strong correlation (r = 0.86; P < 0.002; n = 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 omega-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid.