DETERMINATION OF NITRITE IN HUMAN BLOOD BY COMBINATION OF A SPECIFIC SAMPLE PREPARATION WITH HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHYAND ELECTROCHEMICAL DETECTION

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human bl ood because of its rapid metabolism within the erythrocytes. We now el aborate on method to prevent nitrite degradation during sample prepara tion which in combination with high-performance anion-exchange chromat ography and electrochemical detection allows a sensitive measurement o f nitrite. A linear current response in the concentration range of 10- 1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In-addition, the combination of the electrochemical with a UV d etector allowed us to simultaneously quantify nitrate within one analy tical run, which is the end product of NO/nitrite metabolism. Basal le vels for nitrate and nitrite in human blood were determined with 25+/- 4 mu mol/l and 578+/-116 nmol/l (n=8), respectively and thus were in t he same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial consti tutive NO synthase in humans under physiological and pathophysiologica l conditions.