USE OF PYRUVATE OXIDASE TO OVERCOME PYRUVATE INHIBITION DURING THE LACTATE TO PYRUVATE REACTION FOR ASSAYING LACTATE-DEHYDROGENASE IN SERUM

Automated assays of lactate dehydrogenase (LD) in serum are based on m easuring the rate of NADH produced in a reverse LD reaction using lact ate and NAD. The observed nonlinearity of LD reaction used in earlier assays performed in phosphate buffers has generally been attributed to the formation of a ternary complex of NAD, pyruvate, and phosphate. T his is not satisfactory to explain the course of assay reaction carrie d out in organic buffers. Investigation of the possible causes of nonl inearity during the course of the reverse LD reaction during LD assays performed in Tris or other organic buffers indicated that inhibition of LD activity by pyruvate may be chiefly responsible for the observed effects, especially in serum exhibiting abnormally high LD enzyme act ivity. Most of the LD activity in serum was inhibited by 5 mMoles/L py ruvate. By contrast, the LD isoenzyme activities were inhibited partia lly at 0.5 mMole/L pyruvate, LD1 being the most and LD4 the least susc eptible. In assays of serum samples with abnormally high LD and PYR co ncentration using LD reagent containing Tris buffer, pH 9.3, the inclu sion of a bacterial pyruvate oxidase (PO) enabled the removal of pyruv ate accumulating in situ, making it possible to assay LD activity in t he absence of inhibitory concentration of pyruvate. The inclusion of 1 0 U/L of PO in our routine LD reagent was sufficient to overcome pyruv ate inhibition, thus permitting the assay of serum exhibiting high LD activity, hence the extension of the upper limits of linearity of LD a ssay without compromising assay performance. (C) 1995 Wiley-Liss, Inc.