Sample Pretreatment with Nitrate Reductase and Glucose-6-Phosphate Dehydrogenase Quantitatively Reduces Nitrate While Avoiding Interference by NADP+ When the Griess Reaction Is Used to Assay for Nitrite

An assay for the simultaneous measurement of nitrite and nitrate, products of nitric oxide metabolism, is described. Others have reported pretreating sample by using nitrate reductase (NR) and NADPH to reduce endogenous NO−3 before assaying the resultant NO−2 using the Griess reaction. However, we found that the NADP+ formed during pretreatment interfered with the Griess reaction when NADPH was used at concentrations necessary to drive the NR reaction. For instance, 500 μM NADP+ in 100 μM NaNO−3 (without NR) causes a 90% interference with the formation of Griess reaction product. To limit interference, we modified the method by decreasing the NADPH concentration to 1 μM. NADPH was regenerated by coupling the NR reaction with that catalyzed by glucose-6-phosphate dehydrogenase (GD). Using this method, NaNO−3 standard curves were linear up to 100 μM and coincided with control curves obtained using NaNO−2 incubated in parallel. Addition of urine up to a strength of 20% did not interfere with the assay. Comparison with an alternative assay based on cadmium reduction resulted in the following linear regression: [Cd method] = 0.915∗[NR-GD method] + 0.37, r2 = 0.997. Coupling GD to NR to recycle NADPH allows this cofactor to be used at a low concentration so that interference with the Griess reaction is negligible.