Synthesis of glucuronides of propafenone and 5-hydroxypropafenone by Sepharose-bound uridine 5'-diphosphoglucuronyltransferase.

Propafenone (Rytmonorm) and its in vivo metabolite 5-hydroxypropafenone were glucuronidated by agarose-bound uridine 5'-diphospho (UDP)-glucuronyltransferase from bovine liver in the presence of UDP-glucuronic acid. The identification of the synthesized glucuronides was based on the specific enzymatic hydrolysis, by mass spectrometry and by chromatographic comparison with isolated metabolites from dog and man. The glucuronidation of propafenone and 5-hydroxypropafenone followed Michaelis-Menten kinetics up to substrate concentration of 0.4 mmol/l (propafenone) and 0.3 mmol/l (5-hydroxypropafenone). The pH optima were 7.5 and 8.0, respectively. The reaction was linear up to 30 min and for a protein concentration between 1 and 5 mg per assay. 5-Hydroxypropafenone showed the best affinity with a Km value of 1.4 mmol/l, in contrast to 11.6 mmol/l for propafenone. The use of agarose-bound UDP-glucuronyltransferase offers the possibility to synthesize propafenone glucuronide and 5-hydroxypropafenone glucuronide which are needed for kinetic and pharmacological studies in man and animal.