Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes.

A specific high performance liquid chromatographic assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol glucuronidation kinetics in human livers microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction. Mean apparent Km and Vmax values were 7.37 +/- 0.99 mM and 4.76 +/- 1.35 nmol/min/mg, respectively. Addition of the non-ionic detergent Brij 58 to microsomal incubations resulted in approximately 50% activation of microsomal paracetamol UDPGT-activity. This contrasts to the approximately three-fold activation of 4-methylumbelliferone, morphine and 4-nitrophenol glucuronidation observed following Brij 58 treatment of human liver microsomes. The glucuronidated xenobiotics chloramphenicol, digitoxigenin monodigitoxoside, 4-hydroxybiphenyl, 4-methylumbelliferone, morphine, 1-naphthol and 4-nitrophenol were screened for inhibitory effects on paracetamol glucuronidation. Of these compounds, only digitoxigenin monodigitoxoside and 1-naphthol were found to cause significant inhibition of paracetamol UDPGT activity. Along with the results of previous studies of the kinetics and inhibitor profile of human liver glucuronidation reactions (Miners et al., Biochem Pharmacol 37: 665-671, 1988 and 37: 2839-2845, 1988), these data indicate that the model glucuronidated substrates paracetamol, morphine and 4-methyllumbelliferone may be used to differentiate at least four human liver UDPGT isozyme activities.