Comparing deproteinization methods for serum nitric oxide assay by Greiss reaction

Nitric oxide (NO) is involved in numerous physiologic and phathophysiologic processes. Recently, further investigators have focused on serum NO determination. In our previous study, we validated a simple, cheap and rapid method for serum NO determination based on the Greiss reaction. Deproteinization is a necessary step for this reaction, thus, the present study was designed to assess different deproteinization methods for serum NO determination.

Ten common protein precipitating chemicals including methanol, ethanol, zinc sulfate, methanol/diethyl ether, acetonitrile, TCA, PCA, sodium tangstate, ammonium sulfate and filter were used for deproteinization of 42 human sera, while results were compared to filter separation as a reference. Serum NO levels were determined in 60 sera of adult human.

Data showed that correlation coefficient of precipitating agents: methanol, ethanol, zinc sulfate, methanol-diethylether, acetonitrile, TCA, PCA, sodium tangstate, ammonium sulfate against filter separation method were 0.84, 0.92, 0.91, 0.79, 0.88, 0.85, 0.93, 0.53, and 0.78, respectively (p<0.001). Methanol, ethanol and methanol/diethylether caused overestimation, while TCA, PCA, sodium tangstate, and ammonium sulfate caused underestimation of serum NO results. Serum NO level had normal distribution with mean ±SE of 33±1.3 μmol/L.

Although different chemical protein precipitants are used for serum NO determination, our study revealed that zinc sulfate is the best choice for this purpose.