Determination of nitrite in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and 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 blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of 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 detector allowed us to simultaneously quantify nitrate within one analytical run, which is the end product of NO/nitrite metabolism. Basal levels 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 the 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 constitutive NO synthase in humans under physiological and pathophysiological conditions.