Sequential injection analysis of nitrites and nitrates in human serum using nitrate reductase.

BACKGROUND

Nitrates and nitrites, as products of nitric oxide in human serum, are currently used as markers in many diseases. A sequential injection analysis (SIA) flow system, for their determination in serum, is presented. The exact timing of fluidic manipulations and the small volumes used in the automated SIA systems provide exquisite control of the reaction conditions and an economy in the biological fluid and enzymes used.

METHODS

For nitrite determinations, 150 microl of sample and 50 microl of Griess reagent were sequentially aspirated to the system and sent to the detector. Nitrates were determined as nitrites after reduction through 0.09 U of nitrate reductase and 75 microl of NADPH.

RESULTS

Nitrates and nitrites were determined at concentrations up to 4 and 3 mg/l, respectively, with relative standard deviation (rsd) <5%. Detection limits of 0.14 and 0.03 mg/l, respectively, were calculated. Statistical evaluation showed good agreement between the results obtained, for 15 deproteinized serum samples, with both the SIA system and the comparison batch procedure.

CONCLUSIONS

The automatic developed method seems to be a good alternative for routine implementation since it is four times faster, and it requires one third of sample and one half of nitrate reductase than the comparison batch procedure.