Hydroxylation of prostaglandins A₁ and E₁ by liver microsomal monooxygenase. Characteristics of the enzyme system in the guinea pig

Guinea pig liver microsomes in the presence of NADPH catalyzed the hydroxylation of prostaglandin A₁ (PGA₁) and prostaglandin E₁ (PGE₁) primarily (>95%) at the ω-1 and to a minor extent (<5%) at the ω position, yielding 19-hydroxy and 20-hydroxy derivatives. The identity of the 19-hydroxy metabolites was established in the form of 19-OH-PGB₁ after isolation with the help of high pressure liquid chromatography (HPLC). The isolated product having characteristic UV absorption of PGB₁ (λ(max) = 278 nm) was compared with authentic 19-OH-PGB₁. The metabolites from PGA₁ and PGE₁ and authentic 19-OH-PGB₁ exhibited identical retention times in HPLC as free acids and as the corresponding methyl esters. The corresponding t-butyldimethylsilyl ethers-methyl esters also exhibited identical retention times in the gas chromatography (GC) and similar fragmentations in GC/mass spectrometry. Both NADPH and NADH supported the hydroxylation of PGA₁ and PGE₁, NADH being less effective. There was no synergism by NADH of the NADPH-supported hydroxylation. Inhibitors of microsomal monooxygenase such as SKF 525A, metyrapone, nicotinamide, and carbon monoxide inhibited the hydroxylation of PGA₁ and PGE₁; similarly cytochrome c and antibodies to NADPH-cytochrome c reductase inhibited these reactions, indicating that the hydroxylation of these prostaglandins is catalyzed by a typical monooxygenase system. The kinetic constants for the hydroxylation of PGA₁ and PGE₁ were determined; the K(m) values were 2.1 x 10^-4 M and 1.4 x 10^-4 M, respectively. The V(max) values for the two prostaglandins were also similar, being for PGA₁ and PGE₁, 21.8 and 16.7 nmol/h/mg of protein, respectively.