ABSTRACT
A convenient method for the enzymic conversion of multimilligram quantities of 3-hydroxybenzo[a]pyrene to 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid in 90% yield is described. Commercially available freeze-dried rabbit liver microsomes were incubated in the presence of UDPGA, 3-hydroxybenzo[a]pyrene, and Triton X-100 detergent (Figure 1). The course of the biosynthetic reaction was followed by fluorimetry. The glucuronide product was extracted from the acidified incubation supernate with ethyl acetate and the acid function of the glucuronide was utilized in an acid-base extraction procedure to purify the glucuronide from biological and unreacted starting material. The glucuronide precipitated from ethyl acetate and was collected by centrifugation. High pressure liquid chromatography and spectroscopic techniques were used to verify the structure and purity of 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid.
Subject(s)
Benzopyrenes , Glucuronates , Glucuronosyltransferase , Microsomes, Liver/enzymology , Animals , Glucuronates/isolation & purification , Glucuronidase , Microchemistry , RabbitsABSTRACT
The aglycone, 3-hydroxybenzo[a]pyrene, was metabolized to 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid in the presence of uridine 5'-diphosphoglucuronic acid and rabbit liver microsomes. The course of the biosynthetic reaction was followed by fluorimetry and reverse-phase, paired-ion high pressure liquid chromatography (HPLC). Also, the HPLC system was used to analyze for glucuronide and 3-hydroxybenzo[a]pyrene during the isolation procedure. The existence of a glucuronide of 3-hydroxybenzo[a]pyrene was determined by radiotracer and enzymic techniques, utilizing the HPLC system. Field desorption and direct inlet mass spectral techniques were used to characterize the 3-hydroxybenzo[a]pyrene glucuronide.
