Quantitative Enzyme Histochemistry of Normal and Injured Livers

Abstract

G6PD and ICD activity within the hepatic lobule was determined quantitatively by the ultramicrochemical method of Lowry in CCl4-injured liver of rats and their controls.The activity of G6PD, ICD and NADPH diaphorase was evaluated simultaneously by two histochemical staining methods : the Meijer method using phenadine methosulfate (PMS) and menadione as intermediate electron acceptors and the Pearse method without using PMS or menadione.By the ultramicrochemical method, the G6PD activity increased remarkably and ICD activity decreased in the central area which was most severely affected in CCl4-injured rats.By the Pearse method, the activities of these three enzyme decreased or diminished in the central area. The Meijer method, however, demonstrated considerable G6PD and ICD activity in the central area. Further examination confirmed that such discrepancies were related to NADPH diaphorase which is generally considered the rate-limiting factor in the staining of NADP-linked dehydrogenases. Increased G6PD activity and considerable ICD activity were detected even in necrotic cells by the ultramicrochemical method and the Meijer method.Previous studies in man and experimental animals have shown significant changes in hepatic enzyme activity of the injured liver. Glucose-6-phosphate dehydrogenase (G6PD) performs a vital function in the pentose phosphate pathway which produces NADPH. NADPH is required for many synthetic processes, such as fatty acid and steroid synthesis. In various kinds of liver injury, the activity of G6PD increases (11,14), although the activity of most enzymes of the glycolytic and tricarboxylic acid cycles decrease. Isocitric acid dehydrogenase (ICD) is one of the important dehydrogenase enzymes which produces NADPH in the TCA cycle. This enzyme probably decreases in activity after liver injury. Previous investigation (8) has shown some discrepancies between the staining and quantitative data in the lobular distribution of NAD or NADP-linked dehydrogenases in normal rats. It was pointed out that these discrepancies must be related to NADP or NADPH diaphorase activity in the tetrazolium staining method and to the diffusion of enzymes into the incubation medium (8).In the present paper such discrepancies were examined in fine detail. G6PD193