Loss of NADPH during assays of HMG-CoA reductase: implications and approaches to minimize errors.

In assays of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, preincubation of isolated washed microsomes with NADPH led to a time- and protein concentration-dependent loss of enzyme activity. This occurred despite the presence of an NADPH regenerating system. Addition of fresh NADP, glucose 6-phosphate and glucose 6-phosphate dehydrogenase restored activity. Of the individual components, only NADP was effective. Errors due to loss of NADPH are most pronounced in assays using high microsomal protein, low NADPH levels and preincubation with NADPH and when glutathione rather than dithiothreitol is present. To minimize the effects of NADPH depletion, it is recommended that (i) NADP and NADPH not be present during the preincubation period; (ii) incubation periods be relatively short; (iii) microsomal protein concentrations be less than 1 mg; and (iv) NADPH concentrations be 1 to 2 mM.