Quantitative assessment of primary cerium reaction product formed by glucose-6-phosphatase activity in a male rat liver: an image analysis study

Abstract

Glucose-6-phosphatase (G6Pase) activity has been determined in periportal and pericentral areas of the liver of normal male rats. Measurements were performed on unfixed cryostat sections mounted on semipermeable membranes. In the present study, the oxidized primary reaction product of a cerium-based histochemical method [Ce(IV)perhydroxyphosphate] instead of the final reaction product after a second-step incubation was measured. For quantification of the amount of Ce(IV)perhydroxyphosphate formed the digital image analyzing system Quantimet 500+ was used. Estimated values of optical densities of Ce(IV)perhydroxyphosphate over test areas were employed for calculation of kinetic parameters of (G6Pase). Highest activities of G6Pase (higher Km and Vmax levels) were found in periportal areas of the rat liver, indicating a higher amount of active enzyme molecules and a lower affinity for the substrate. Differences in values for both Km and Vmax between periportal and pericentral zones were highly significant and closely comparable to those for male fed rats. Correlations between Km and Vmax were significant for periportal as well for pericentral liver areas. The results of the present study thus allow the same biological implications as histochemical methods employing a final reaction for quantification of enzyme activities. The present method avoids the drawbacks of enhancement reactions and demonstrates the feasibility of in situ analysis of enzyme kinetic parameters by quantification of oxidized primary cerium reaction products.