Micromethod for determination of lactate dehydrogenase isoenzyme C4 activity in human seminal plasma.

We have developed an automated enzymatic assay for LDH-C4, a lactate dehydrogenase (EC 1.1.1.27) isoenzyme found exclusively in spermatozoa, as a marker of human spermatogenesis. LDH-C4 activity is assayed bichromatically in the Abbott ABA-100 spectrophotometer, with 2-oxohexanoate as substrate. As many as 27 samples (2.5 microL) of human seminal plasma can be analyzed sequentially, with apparent zero-order kinetic conditions and with a precision (CV) of 5%. The apparent Km for the 2-oxohexanoate reaction is 11.2 mmol/L. Electrophoretic and enzymatic studies indicate that LDH-C4 acts on this substrate to produce hydroxyhexanoate, in contrast to lactate dehydrogenase isoenzymes 1 and 5--further evidence for the uniqueness of LDH-C4. The sensitivity, specificity, and speed of this assay system makes it practicable for studies on, and evaluation of, male fertility.