An improved HPLC assay for the assessment of liver slice metabolic viability using 7-ethoxycoumarin

The use of precision-cut liver slices constitutes a new in vitro metabolism technique for the study of coupled phase I and phase II biotransformations. This technique has the advantage of being easily amenable to studies with human tissue. As a means of characterizing the metabolic viability of diverse liver samples, a standard substrate capable of undergoing oxidative and conjugative pathways of metabolism would be desirable. An assay based on 7-ethoxycoumarin was developed whereby the metabolites--7-hydroxycoumarin, 7-hydroxycoumarin sulfate, and 7-hydroxycoumarin glucuronide--could be quantitated using a single HPLC analytical method. This required the synthesis of metabolite standards. 7-Hydroxycoumarin glucuronide was prepared by coupling 7-hydroxycoumarin with methyl 2,3,4-tri-O-acetyl-1-bromo-1-alpha-D- glucopyranuronate, under phase transfer conditions, to give the protected conjugate that was then hydrolyzed to give the glucuronide as the sodium salt. Assignment of configuration at the anomeric center was based on analysis of the simulated 1H and gated 13C NMR spectra, in addition to enzymatic hydrolysis. The sulfate conjugate was prepared by treatment of 7-hydroxycoumarin with Bu4N+ HSO4-/dicyclohexylcarbodiimide/pyridine. 7-Ethoxycoumarin, 7-hydroxycoumarin, and the glucuronide and sulfate conjugates were resolved by HPLC on a C8 Hypersil BDS column using ion-pairing conditions. Incubation of 7-ethoxycoumarin with rat or human liver slices in Krebs-Henseleit buffer resulted in the formation of these metabolites that were readily quantitated in the media with UV detection at 320 nm, using external standard curves. Although the sulfate was seen as the major metabolite in rats, the glucuronide predominated in human tissue. Two different human livers were examined.(ABSTRACT TRUNCATED AT 250 WORDS)