Glucuronidation of 7-hydroxycoumarin in periportal and pericentral regions of the liver lobule

Abstract

Rates of glucuronidation were measured at high substrate concentrations in specific zones of the liver lobule using micro-light guides placed on periportal and pericentral regions on the surface of livers from phenobarbital-treated rats. Livers were perfused with sulfate-free buffer under normoxic conditions, and fluorescence of free 7-hydroxycoumarin was monitored in the tissue. The formation of nonfluorescent 7-hydroxycoumarin glucuronide was then inhibited completely by perfusion with N2-saturated perfusate containing 20 mM ethanol. Under these conditions, fluorescence recorded from the surface of the liver was directly proportional to the concentration of substrate infused. The difference in 7-hydroxycoumarin fluorescence between N2 plus ethanol and normoxic perfusion was due to glucuronidation. Maximal rates of glucuronidation in periportal and pericentral regions of the liver lobule calculated with this new method were 9.6 and 35 mumoles/g/hr, respectively. Glucuronidation was half-maximal with 25-50 microM 7-hydroxycoumarin in both regions. Glucuronosyltransferase activity assayed in microdissected, freeze-dried tissue samples in vitro was 3-fold greater in pericentral areas than in periportal areas. This activity was half-maximal with 0.2 mM UDP-glucuronic acid and 54 microM 7-hydroxycoumarin in both regions of the liver lobule. Thus, the maximal capacity of the glucuronidation system determined in vitro is about 3-fold greater in pericentral than in periportal regions of the liver lobule, a difference which correlates well with measured rates of glucuronidation of 7-hydroxycoumarin in the two zones of the lobule in the intact, perfused liver.