Comparative study of methods for quantitation of lactate dehydrogenase isozymes

Abstract

The amounts of the lactate dehydrogenase (LDH) isozymes in homogenates of rat heart, skeletal muscle, and liver were measured by means of ( 1) the standard spectrophotometric assay at 340 mμ of eluates from DEAE Sephadex columns and ( 2) densitometric determination of formazan bands on acrylamide gel electropherograms. Reproducible quantitation was achieved by the chromatographic method. All the activity applied to the columns could be recovered, and the purity of isozyme fractions could be verified electrophoretically on cellulose acetate strips. Spectrophotometric determination of formazan deposition on acrylamide gels was found to be less satisfactory in quantitating isozymes because of the necessity of applying very small amounts (i.e., less than 0.002 International Unit per isozyme to each gel). Application of larger amounts of enzyme to the 5 mm diameter gel columns resulted in a nonlinear staining response. LDH-5 and LDH-4, known to be present in small amounts in rat cardiac muscle, appeared in disproportionately large amounts when too much enzyme was used for electrophoresis. The percentages of A and B subunits determined by the chromatographic and electrophoretic methods were compared to the percentages of A and B subunits determined by the low/high pyruvate ratio method. There was only moderate agreement between the methods.