Simultaneous histochemical assay of two dehydrogenases in the same cell

A new approach has been developed for the simultaneous assay of the activities of two enzymes (lactate and succinate dehydrogenases) in the same cell in sections of unfixed liver. The sections, mounted on coverslips, were placed on top of 0.6-mm thick 0.8% low gelling-temperature agarose films containing the substrates of both enzymes (70 mM lactate and 50 mM succinate, respectively) plus 80 mM Tris-HCl buffer (pH 7.5), 5 mM EDTA, 10 mM NaN3, 1.5 mM NAD+, 1.2 mM Nitro BT and 0.26 mM phenazine methosulphate. The integrated absorbance (A) at 585 nm of the final reaction product formazans deposited by the two enzymes in a selected hepatocyte was measured continuously at 37 degrees C as a function of incubation time, using a Vickers M85 microdensitometer. The intercept A0 on the A-axis of the linear regression line of A on time was determined. After a known incubation time t, the absorbance A1, was noted and the section placed on another gel film lacking the substrates in order to estimate the final reaction product either formed in the gel film or lost from the cell. The absorbance A2 of the hepatocyte was remeasured. The reaction velocities (activities) vL and vS of lactate and succinate dehydrogenases, respectively, were calculated from the following equations: vL = [(A1-A2) - A0(1- alpha L)]/(1-alpha L)t and vS = (A2-alpha LA1)/(1-alpha L)t where alpha L = A2/A1 for hepatocytes incubated on gel films containing only lactate as the substrate. This parameter was found to be virtually constant (0.44) over a wide range of vL.(ABSTRACT TRUNCATED AT 250 WORDS)