Metabolomic evaluation of NO production in human erythrocytes: an LC-MS/MS method to asses arginine and its metabolites

Experimental studies suggest that red blood cells are involved in nitric oxide (NO) synthesis and delivery. Erythrocytes acts as producers, scavengers and vehicles of NO affecting several physiological processes. NO bioavailability is linked not only to arginine and its metabolic products, ornithine and citrulline, but also to methylarginines which are inhibitors of NO sinthesis. Existing methodologies do not permit a systematic evaluation of the metabolic and biosynthetic pathway of NO in RBC. A metabolomic approach, considering a larger number of compounds involved in NO metabolic pathway, could be important to better understand the role of red blood cells in physiological and pathological conditions. We set up an HPLC-electrospray ionization-tandem mass spectrometry method, to simultaneously detect and quantify arginine metabolome, i.e. arginine, symmetric and asymmetric dimethylarginine, monomethyarginine, ornithine, citrulline. Stable isotope-labelled internal standards have been used to minimize analytical variations before organic protein precipitation of the isolated and lysated erythrocytes. The ion source was operating in positive ion mode and the analytes were detected using multiple reaction monitoring after hydrophilic-interaction chromatography. A 8 min run time allowed the simultaneous evaluation of all the analytes involved in the NO pathway. The chromatograms were free from interferences, the calibration functions were linear and the inter-day CV were<10% for all the analytes. LOQ values make this method suitable for low concentration samples. Our validated LC-MS/MS method might be useful to clarify the role of RBCs in the synthesis of NO and will be of help for studies assessing the involvement of these cells in the regulation of blood flow under physiologic or pathological conditions. Moreover, a simple sample processing without the derivatization step, allows to consider this method a valuable tool for diagnostic evaluation of arginine metabolome in red blood cells and it could be applied in clinical chemistry.