Characterization ofp-hydroxyphenobarbital glucuronide generated from immobilized rat hepatic UDP-glucuronosyltransferase (original) (raw)

Immobilization of hepatic uridine-5'-diphosphoglucuronosyltransferase (UDPGT) is an accepted technique for the in vitro generation of glucuronides of interest. Parikh et al. first described the immobilization of rabbit hepatic UDPGT on agarose by the modified cyanogen bromide method and demonstrated the enzymatic activity by the subsequent generation of p-nitrophenol glucuronide. Other reports have demonstrated the nonspecificity of immobilized hepatic UD-PGT against a variety of s~b s t r a t e s~~~ and the simultaneous immobilization of hepatic cytochrome P-450 and UDPGT from solubilized microsomal protein preparation^.^ The method of Parikh et al.' has been modified by the immobilization and stabilization of rat hepatic UDPGT, instead of rabbit UDPGT. The applicability of this modification was demonstrated by generation of 5-ethyl-5-(p-hydroxyphenyl)barbiturate glucuronide (p-D-ghcopyranosiduronic acid, p-( 5-ethylhexahydro-2,4,6-trioxo-5-pyrimidinyl)phenyl[ 18743-41-41], the predominate metabolite of phenobarbital in huma1-1~~9~ and rats.7,* 5-ethyl-5-phenylbarbituric acid, 5-ethyl-5-(p-hydroxyphenyllbarbituric acid, uridine-5'-diphosphoglucuronic acid sodium salt (UDPGA), D-saccharic acid 1,4-lactone, and p-glucuronidase (Type H-2; 107 200 units/mL P-glucuronidase, 4500 units/mL sulfatase) were purchased from Sigma Chemical Co. (St. Louis, MO). CNBr-activated Sepharose 4B was obtained from Pharmacia LKB (Uppsala, Sweden). All other chemicals were of analytical reagent grade. Rat hepatic UDPGT was isolated according to a modification of the methods of Parikh et a1.I and Lehman et aL4 Five Sprague Dawley rats (300-400 g; Hilltop Laboratory Animals, Scottdale, PA, pretreated with phenobarbital, 80 mgkg ip x 5 days) were decapitated and the livers were removed. All subsequent steps were performed at 4 "C. The livers were minced and homogenized in 0.25 M sucrose buffer (25% w/v, Figure 3-(a) COSY ('H--'H-NMR, 499.87 MHz) and (b) HMQC (1H-13C-NMR, 499.87 MHz) spectra of the putative glucuronide conjugate of phydroxyphenobarbital.