Investigation of the Optimum Conditions for Measurement of Human Lactate Dehydrogenase Activity

Abstract

Optimal reaction conditions for assaying human serum lactate dehydrogenase (LDH EC 1.1.1.27)(lactate-to-pyruvate) were established for purified isoenzymes 1, 3, 5 and whole sera at 30°C in diethanolamine (DEA) buffer.(1). The optimal reaction conditions with equal efficiency for each isoenzyme were pH 8.8, 60 mmol/L lactate, 6mmol/L NAD⁺, and 300 mmol/L DEA at 30°C.(2). The pH optima were within the range of 8.8-9.4 for LDH 3 and 5, >9.0 for LDH 1.(3). Optimal concentration of DEA buffer ranged from 200 to 400 mmol/L for all isoenzymes.(4). Optimal concentration of L-lactate were 20 mmol/L for LDH 1, 60 mmol/L for LDH 3, and>100 mmol/L for LDH 5, respectively.(5). Optimal activity was obtained at a NAD⁺ concentration of 6 mmol/L for all isoenzymes.(6). Initial velocities were most accurately determined within 75 sec after a lag time of 25 sec.(7). Higher LDH activity was obtained.when the reaction was initiated with the enzyme.(8). The standard curve for this assay is linear to 350 U/L of serum.(9). Coefficients of variation within a run and between days were 1.6% (379.6 U/L) and 2.1% (367.4 U/L), respectively.(10), Normal range of LDH activity in fresh serum was 60-120 U/L.(11). The order of storage stability of serum LDH was-60°C>-20°C>25°C>4°C.