Quantitative Measurement of Low-and High-Km Aldehyde Dehydrogenase Activity in Rat Liver

Abstract

Quantitative measurement of low- and high-Km aldehyde dehydrogenase (ALDH) activity was performed in the liver acinus by combination of light microscopic enzyme histochemistry with simple video microscopy and routine spectrophotometry. After ALDH activity staining, integrated staining intensity of reference wide area and small area of interest were measured by image analysing program. The resulting relative values of the small areas were converted into absolute ALDH activity using the results of spectrophotometeric enzyme assay and staining intensity obtained from the reference wide areas. The validity of the present method was confirmed by the results showing the linear relationships between the activity and either incubation time (5∼20 min) or section thickness (6∼20㎛). In liver acinus, both total and high-Km ALDH activity were gradually decreased from zone 3 to 1. Total ALDH activity in zone 3, 2 and 1 was 19.6, 17.2 and 16.4 mU/㎟, respectively. However, the intra-acinar distribution of low-Km ALDH activity was nearly flat: 8.6, 7.9 and 8.2 mU/㎟ in zone 3, 2 and 1, respectively. Low-Km values of ALDH estimated by this histochemical method did not exhibit a significant difference between zone 1 and 3; 55 and 62 uM, respectively. In view of predominant localization of alcohol dehydrogenase activity in the zone 3, even distribution pattern and nearly equal Km values of low-Km ALDH in liver acinus may provide a metabolic background for alcohol-induced liver damage which occurs predominantly in the zone 3.