EFFECTS OF TISSUE PROTECTANTS ON THE KINETICS OF LACTATE-DEHYDROGENASE IN CELLS

We studied the effects of two tissue protectants, polyvinyl alcohol (P VA) and agarose gel, on a kinetic parameter of lactate dehydrogenase L DH that is assumed to be related to the extent of diffusion of the enz yme out of tissue sections during its histochemical assay. The kinetic s of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and pe riportal hepatocytes were determined in unfixed sections incubated eit her on substrate (L-lactate)-containing agarose gel films or in aqueou s assay media in the presence or absence of 18% PVA. The absorbances o f the formazan final reaction products at their isobestic point were m easured continuously in the cytoplasm of individual cells as a functio n of incubation time, using a real-time image analysis system. Whichev er incubation medium was used, the absorbances in the two cell types i ncreased nonlinearly during the first minute of incubation but linearl y for incubation times between 1 and 3 min. The nonlinearity of the LD H reaction was analyzed using the equation v(i) - v = a degrees A, whe re v(i) is the observed initial velocity determined from the absorbanc e changes during the first 10 sec of incubation and v and degrees A ar e respectively the gradient and intercept on the absorbance axis of th e linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed (v(i)-v) against degrees A wer e linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12-43 % lower than those for hepatocytes. The a value for hepatocytes obtain ed with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by t he two methods were almost the same. Addition of excess pyruvate to th e aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide g els containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to t he extent to which the enzyme diffuses out of sections during incubati on but not to product inhibition of LDH by pyruvate. PVA was more effe ctive for protecting diffusion of LDH from hepatocytes than from skele tal muscle fibers, possibly because hepatocytes contain a greater prop ortion of diffusable (unbound) LDH than skeletal muscle fibers.