Hydroxylation of Prostaglandins A, and E, 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 A, (PGA,) and prostaglandin E, (PGE,) primarily (>95%) at them-1 and to a minor extent (~5%) at the w position, yielding 19-hydroxy and 20-hydroxy derivatives. The identity of the 19-hydroxy metabolites was established in the form of 19-OH-PGB, after isolation with the help of high pressure liquid chromatography (HPLC). The isolated product having characteristic UV absorption of PGB, (A,,, = 278 nm) was compared with authentic 19-OH-PGB,. The metabolites from PGA, and PGE, and authentic 19-OH-PGB, 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 PGA, and PGE,, 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 PGA, and PGE,; 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 PGA, and PGE, were determined; the K, values were 2.1 x 10e4 M and 1.4 x 10e4 M, respectively. The V,,,,, values for the two prostaglandins were also similar, being for PGA, and PGE,, 21.8 and 16.7 nmol/h/mg of protein, respectively. These findings are discussed in respect to the possibility that prostaglandins are natural substrates for the hepatic monooxygenase.