Semiautomated Measurement of Nitrate in Biological Fluids

Abstract

The nitric oxide radical (NO·) plays an important role as a physiological messenger (1). NO is formed from l-arginine (2) by NO synthase (NOS; EC 1.14.13.39), which exists in several isoforms (3). Constitutive calcium-dependent isoforms (cNOS) modulate the control of vascular tone in endothelial cells or the neurotransmission in neurons, whereas inducible calcium-independent isoforms (iNOS) are located in macrophages, chondrocytes, and hepatocytes and are induced by cytokines and endotoxin (4)(5). Pathological conditions associated with increased release of cytokines and endotoxin, e.g., inflammation or sepsis (6), can therefore increase NO production. NO is a very unstable, short half-life gas that breaks down rapidly into the stable products nitrate and nitrite (7). Upon coming into the bloodstream, nitrite reacts immediately with oxyhemoglobin to form methemoglobin. Consequently, most NO produced is detected in serum as the remaining product, nitrate (8). Recently, several reports focused on methods to measure nitrate concentrations in biological fluids (9)(10)(11). One of the most commonly used methods is based on the reduction of nitrate to nitrite by cadmium or nitrate reductase, the nitrite produced being determined by Griess reaction (9)(12)(13). Other methods for monitoring NO production are based on chemiluminescence (11)(14), enzymatic assay with an internal standard (10), or chromatographic procedures (8) for nitrate (15)—all of which are time-consuming for routine application in clinical chemistry laboratories. We describe a rapid semiautomated method based on the Griess reaction, involving a shortened incubation period of nitrate with cadmium. The method is applicable to several types of biological fluids. Serum or plasma samples from healthy individuals after a 12-h fast were obtained in accordance with the Medical Ethical Committee of our hospital. Samples were stored at −20 °C and were stable for at least 6 months. The method was …