Substrate-specific glucuronidation in rat hepatocytes.

Glucuronidation is an important detoxification pathway for many xenobiotics and endogenous compounds. This reaction is catalysed by microsomal UDP-glucuronosyltransferases (GT EC 2.4.1.17). which exhibit latency. Their functional heterogeneity has been demonstrated by substrate specificity, differential induction profiles, and differences in development patterns [ 1). Hepatocytes in suspension provide a valuable system for the study of integrated metabolism/toxicity in v i m . The rate of glucuronidation in whole cells depends o n several factors. including substrate lipophilicity, the intracellular content and rate o f synthesis of the cofactor UDP-glucuronic acid (UDPGA), the presence o f endogenous activators, and intrinsic enzyme activity. As a result, cell homogenates are often used to allow greater control of these factors. In this paper, we describe the comparison of the rates of glucuronidation of phenolphthalein. 1 -naphthol and bilirubin each specific for a different G T isoform in freshly-isolated rat hepatocytes and hepatocyte homogenates. Hepatocytes were isolated from male Sprague-Dawley rats (250-350 g) by collagenase perfusion 121. Cell viability was assessed by Trypan Blue exclusion. Hepatocyte assays were carried out at 37°C in Krebs-Hepes buffer, pH 7.4, gassed with 9 5 : s (v/v) O,:COz, with substrate as the only addition. Hepatocyte homogenates were prepared at 1 0 " viable cells per ml in 0. I M sodium phosphate buffer pH 7.6, stored at 80°C, and used within two weeks of preparation. Protein concentration was measured spectrophotometrically by Coomassie Brilliant Blue binding 13 I. Homogcnate assays wcre carried ou t at 37°C in Tris buffer, pH 7.4, containing