METABOLISM OF N-HYDROXYGUANIDINES (N-HYDROXYDEBRISOQUINE) IN HUMAN AND PORCINE HEPATOCYTES: REDUCTION AND FORMATION OF GLUCURONIDES DMD#4572 -page 2 Running Title Metabolism of N-hydroxydebrisoquine in hepatocytes Corresponding author

Abstract

Abstract The biotransformation of N-hydroxydebrisoquine, a model substrate for Nhydroxyguanidines, was studied in vitro with cultured and characterized porcine and human hepatocytes. The objective of the present work was to compare the N-oxidative and N-reductive metabolism of this compound using a monolayer culture system with previously described microsomal studies and to investigate the phase 2 metabolism, in particular glucuronidation of this class of compounds. At the same time the suitability of pig hepatocytes as model system for the human metabolism could be investigated. Two glucuronides of the parent compound N-hydroxydebrisoquine were analyzed. For the first time, one of these phase 2 metabolites could be identified as O-glucuronide of an N-hydroxyguanidine by comparing to a synthesized authentic compound. The involvement of certain human UDP-glucuronosyltransferases (UGTs) was evaluated by incubating the substrate with eight human hepatic recombinant UGT enzymes. Metabolites were determined by a newly developed LC-MS (liquid chromatography/mass spectrometry) analyses using electrospray ionisation (ESI). The known microsomal reduction of the N-hydroxylated compound was also demonstrated with hepatocytes. The N-hydroxylation of the corresponding reduced compound (debrisoquine), which was previously described with microsomes, could not be detected in hepatocytes. There was no qualitative difference in the formation of the described derivatives by human and porcine hepatocytes. All phase 2 metabolites identified in hepatocyte culture were also formed by glucuronosyltransferases. In culture, the N-reduction of the N-hydroxylated substrate is the dominating reaction, indicating a predominance of N-reduction in vivo