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Abstract
We report the appearance of a sixth isoenzyme of lactate dehydrogenase (LD, E.C. 1.1 1.27) migrating cathodic to LD-5 following agarose electrophoresis and visualization with iodonitrotetrazolium based reagents. We have shown that LD-6 is not an electrophoretic artifact nor a complex of LD with another protein. LD-6 activity is not inhibited by the addition of pyrazole as is alcohol dehydrogenase. We have shown that LD-6 is larger than other LD isoenzymes by Ferguson plots. Immunoprecipitation studies with antibodies to the LD subunits have shown LD-6 to consist of M-subunits. Isoelectric focusing has shown LD-6 to have two bands of activity at pH 9.0 to 9.6. Heat inactivation studies have shown LD-6 to be more heat stable than LD-5.
We have also demonstrated LD activity in the mitochondria of human hepatocytes. Digitonin fractionation showed the inner membrane/inner matrix and the outer membrane to contain LD activity. The mitochondrial isoenzymes were designed as LD-Mt$\sb1$ and LD-Mt$\sb2$ based on their chromatographic separation. Both migrate cathodically on agarose electrophoresis and are immunoprecipitated by antibodies to the LD M-subunit but not by antibodies to the H-subunit. LD-Mt$\sb2$ is similar to LD-5 with respect to subunit molecular weight (34 000 daltons) and pI (two bands of LD activity at 9.6 and 9.8). LD-Mt$\sb1$ is larger than the other isoenzymes as two bands, one at 34 000 daltons and one at 14 500 daltons, were seen following SDS electrophoresis. After electrofocusing of LD-Mt$\sb1$, a band at pH 4.8 was seen by protein staining in addition to the two bands at pH 9.6 and 9.8 observed with LD activity. The physical similarities between LD-6 and LD-Mt$\sb1$ suggest they may be the same.
The LD-6 electrophoretic pattern was observed in the sera of 17 patients, 15 of whom died during their hospitalization. Severe hypotension, metabolic acidosis, acute renal failure, and sepsis were clinical conditions preceding the appearance of the band. Enzyme studies revealed the release of the mitochondrial enzymes aspartate aminotransferase, mitochondrial creatine kinase, adenylate kinase, malate dehydrogenase, and $\beta$-hydroxybutyrate dehydrogenase. We propose that LD-6 is a mitochondrial enzyme which reflects profound, possibly irreversible cellular damage. Therefore, the detection of LD-6 indicated a poor prognosis.
