Histochemical demonstration of sinusoidal γ-glutamyltransferase activity by substrate protection fixation: Comparative studies in rat and guinea pig liver

Abstract

Most histochemical methods for the detection of an enzymatic activity are preceded by tissue fixation with chemical agents that partially inactivate the enzymes. It is well known that substrates exert a marked protection against fixative‐induced inactivation. The conventional histochemical methods for the demonstration of hepatic γ‐glutamyltransferase activity have not been successful in detecting the activity of the enzyme on the sinusoidal side of the hepatocytes despite mounting biochemical evidence for its presence on that pole of the hepatocyte. Under conventional fixation the enzymatic activity in hepatocytes is only seen on the bile canalicular side. This may be the result of a preferential protective effect of γ‐glutamyltransferase by its normal substrate, glutathione, present in the bile canaliculus at concentrations 500 times higher than in the sinusoidal lumen (8 mmol/L vs. 10 to 20 m̈mol/L). To test this hypothesis and to reduce the degree of fixative‐induced inhibition of the enzyme activity, glutathione was either incorporated in the fixative solution or the livers were perfused with high concentrations of glutathione (10 mmol/L) before fixation. Our results histochemically demonstrate, in the normal adult rat liver, the existence of γ‐glutamyl‐transferase activity not only on the bile canalicular pole but also on the sinusoidal pole of the hepatocytes. Visualization of the enzyme activity on the sinusoidal pole is dependent on glutathione protection. Guinea pig livers, which present a 10‐fold higher γ‐glutamyltransferase activity than rat livers (similar to that in human beings), showed marked sinusoidal γ‐glutamyltransferase activity even in the absence of glutathione protection. Glutathione protection further increased this sinusoidal activity. Histochemical data in the guinea pig paralleled the biochemical findings indicating that in a single pass guinea pig liver can remove considerably higher amounts (more than two thirds) of circulating glutathione than rat liver. The histochemical demonstration of γ‐glutamyl‐transferase activity in the sinusoidal pole of the hepatocyte in normal adult rat and guinea pig liver adds further evidence to the biochemical data supporting the existence of a sinusoidal γ‐glutamyltransferase ectoactivity capable of removing glutathione from the circulation. (HEPATOLOGY 1991;14:857–863).