NO detection in biological samples: Differentiation of 14NO and 15NO using infrared laser spectroscopy

Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between 14NO and 15NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples.With the cavity leak-out spectroscopy setup (CALOS) a differentiation between 14NO and 15NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of 14NO/N2 and 15NO/N2 were used to calibrate the system. 14NO2- and 15NO2- of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation.The correlation received for both methods for the detection of NO in the gas phase was r = 0.999, p < 0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for 14NO2- 0.3 pmol and 15NO2- 0.5 pmol; correlation 14NO: p < 0.0001, r = 0.975, 15NO: p < 0.0001, r = 0.969).The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.