A STUDY OF DETERMINATION OF LACTIC DEHYDROGENASE ISOENZYMES BY MEANS OF AGAR GEL AND STARCH-AGAR GEL ZYMOGRAMS

Agar gel electrophoresis using veronal as buffer is very often used for the determination of LDH isoenzymes. Differences in optimal conditions for LDH isoenzymes, especially between LDH1 and LDH5 cause certain difficulties in their determination. When samples with the same activity of LDH1 and LDH5 were separated and estimated by the above mentioned technique, intensity of LDH5 was found to be less than 50% of that of LDH1 The steps in this procedure were investigated to explain the discrepancy of LDH1 and LDH5 intensities, but also aiming to improve the sensitivity of this method. Colloids, such as agar, agarose and albumin were shown to stimulate the formation of formazan in the colour reaction, while they stimulate LDH activity only slightly. Furthermore it was found that different buffer types combined wih cyanide, may interfere with colour formation and the determination of LDH activity. The possible effect of cyanide is discussed and the replacement of cyanide in the staining solution by pyrophosphate is suggested. The influence of veronal on the LDH isoenzyme determination was investigated also. Results showed that veronal selectively inhibits LDH isoenzymes. By adding citric acid, which is known to be a specific activator of LDH5, to veronal this discrepancy was removed. Results obtained with the original and the modified methods are presented. Finally, a method is described which allows the separation of LDH isoenzyrnes in starch gel followed by an estimation as described in the modified agar gel electrophoresis technique. Results obtained with these two methods were comparable. The reproducibility and simplicity of this method should make it a useful tool not only for biochemical studies but also in clinical diagnostic work. © 1968.