Rates of sulfation and glucuronidation of 7-hydroxycoumarin in periportal and pericentral regions of the liver lobule

Micro-light guides (tip diameter 170 μm) were placed on periportal (light) and pericentral (dark) regions of perfused liver to monitor the fluorescence (366 → 450 nm) of infused 7-hydroxycoumarin. During infusion of up to 30 μM 7-hydroxycoumarin, fluorescence of free 7-hydroxycoumarin could be detected only in periportal regions during anterograde persusion and in pericentral areas during retrograde perfusion. Over 95% of 7-hydroxycoumarin infused was converted into nonfluorescent sulfate and glucuronide conjugates. Thus, under these conditions, rates of sulfation and glucuronidation can be studied in the periportal and pericentral regions of the lobule by measuring conjugates of 7-hydroxycoumarin in the effluent perfusate via anterograde or retrograde perfusions. Sulfation predominated over glucuronidation when 2-10 μM 7-hydroxycoumarin was infused in the anterograde direction; however, with 20-30 μM 7-hydroxycoumarin, glucuronidation predominated. In contrast, rates of glucuronidation and sulfation were similar when 2-5 μM 7-hydroxycoumarin was infused in the retrograde direction; however, glucuronidation predominated at higher substrate concentrations. Thus, at low concentrations of 7-hydroxycoumarin, sulfation predominated over glucuronidation in periportal hepatocytes but not in pericentral hepatocytes. When livers were perfused with sulfate-free media, the glucuronide was the predominant conjugate formed in both periportal and pericentral hepatocytes. Taken together, these data indicate that sulfation successfully competes with glucuronidation for 7-hydroxycoumarin at low substrate concentrations. When 7-hydroxycoumarin was generated via mixed-function oxidation following the infusion of 7-ethoxycoumarin (5-20 μM), fluorescence from 7-hydroxycoumarin was detected predominantly in periportal areas during anterograde perfusions and in pericentral regions during retrograde perfusions. The predominant conjugate formed in both regions of the liver lobule during mixed-function oxidation was sulfate. Thus, rates of sulfation were significantly greater in periportal regions than in pericentral regions when 7-hydroxycoumarin was infused directly or generated indirectly via mixed-function oxidation.