Observations on the Heterogeneity of Malic and Lactic Dehydrogenase in Human Serum and Red Blood Cells1

Abstract

The enzyme malic dehydrogenase (MDH)2 catalyzes the conversion of oxalacetic acid to malic acid. It The method of zone electrophoresis, using a starch supporting medium in barbital buffer, pH 8.6, with an ionic strength of 0.1 or 0.05, was used to separate the serum proteins (4). After electrophoresis the starch block was cut into half inch segments, the protein eluted with 5.0 ml. buffer and its concentration determined by a modification of the Folin-Ciocalteau procedure (4). An aliquot of 2.5 ml. from each eluate was then assayed for MDH activity spectrophotometrically by the method of Wacker, Ulmer, and Vallee (5). Each eluate was incubated for 20 minutes at room temperature with 0.2 ml. of 0.003 molar DPNH. The mixture was transferred to a Beckman cuvette of 1 cm. path length; 0.1 ml. of 0.0076 molar oxalacetate was added, and the decrease in absorption at 340 mju was measured in the Beckman spectrophotometer. Readings were obtained every 30 seconds for 3 minutes. One unit of dehydrogenase activity was defined as a decrease in the optical density at 340 mu of 0.001 per minute. Under these conditions, the contribution to the observed MDH activity by the simultaneous measurement of LDH, caused by spontaneous decarboxylation of oxalacetate to form pyruvate, was found to be negligible. Lactic dehydrogenase obtained from rabbit muscle (Worthington laboratories) was found to have insignificant quantities of activity when oxalacetate was used as substrate instead of pyruvate.In several experiments, a 2.5 ml. sample of an hemolysate prepared from normal human red cells (6) was fractionated electrophoretically and an aliquot of the eluate was assayed for MDH activity. To determine the pH optima of the three peaks, 0.3 ml. was removed from the tube containing the maximum MDH activity, and added to tubes containing barbital buffers varying in pH from 7.4 to 9.2, all at 0.1 ionic strength. MDH activity was then reassayed.The effect of the inhibitors, ethylenediaminetetraacetic acid and p-chloromercuribenzoate, on each of the activity peaks was studied according to the method outlined below. Immediately after the serum fractions had been assayed for enzymatic activity the inhibitor was added, the solution thoroughly mixed and the activity redetermined. The decline in activity between the two measurements was attributed to the action of the inhibitor.The final concentrations of ethylenediaminetetraacetic 672

Isozymes of Lactic Dehydrogenase; Their Alterations in Arthritic Synovial Fluid and Sera*

“…Therefore, isozyme 5, if employed to localize pathology, could prove to be misleading. Although serum isozymes have been useful as an adjunct in diagnosis of certain conditions (3)(4)(5)(6)(7)(8)(9)(10)22), it is probably an oversimplification to expect, as some have (10), that specific localization of pathology to any organ can be inferred by an analysis of serum for LDH isozymes. Figures 1 and 2 illustrate the similarity among tissues in the number and the electrophoretic mobility of LDH isozymes and show that the main differences are in distribution of total LDH activity among the five isozymes.…”

“…Subsequently additional peaks of serum LDH activity were found to be raised early in the course of hepatitis (4). Multiple molecular forms of other enzymes such as malic dehydrogenase were demonstrated in normal human serum, and individual peaks of these were altered in various disease states (5). Enzymatic heterogeneity gained additional significance with the definition of an isozyme by Markert and Moller, who introduced the term to refer to multiple enzymes exhibiting similar substrate specificities (6).…”

“…Electrophoretic separation of homogenates of various mammalian tissues showed that each tissue contained several LDH isozymes. Differences among tissues, either in the number or mobility of these isozymes, were reported (3)(4)(5)(6)(7)(8)(9)(10). If each human tissue possessed a unique isozyme pattern, the possibility arose that diagnosis might be facilitated by electrophoretic separation of serum.…”

Identification of Lactate Dehydrogenase Isoenzymes by Rapid Kinetics

“…Vessell and Bearn noted that the isoenzyme pattern of serum changes after myocardial infarction (6). Changes in the isoenzyme pattern are observed as early as [6][7][8][9][10][11][12] hr after the initial attack of pain and have been shown, in dogs, to be a more sensitive indicator of myocardial necrosis than total serum activity (7). Changes in LDH isoenzyme patterns are a constant feature in diseases where liver-cell damage occurs (8).…”

Diagnostic dissection by isoenzymes

“…The apparent energies ofactivation (for the reaction: lactate-pyruvate) using LDHh LDH2, LDH 3 , LDH 4 and LDH 5 , as a function of the relative electrophoretic mobilities of these isoenzymes…”