Process for determining s-nitrosothiols in biological fluids

Abstract

A process for determining S-nitrosothiols, in particular S-nitrosoglutathione, in biological fluids that is easy, selective, cheap with respect to the prior art, which requires the use of equipment commonly available in laboratories, at low cost, which can be used by not qualified operators. The process is based on the hydrolysis of S-nitrosoglutathione (GSNO) by an enzyme, in particular γ-glutamyltranspeptidase (GGT). This enzyme hydrolizes the residual γ-glutamyl of GSNO for giving glutamate (GIu) and S-nitroso-cysteinylglycine (GIyCySNO). In the presence of ions of transition metals GGT speeds up the release of NO since the intermediate that is formed, the GIyCySNO, is much more sensitive to a metal-dependent decomposition. Advantageously, the amount of nitric oxide present in the sample is measured through a reaction thereof with 4,5 diaminof luorescein (DAF-2), said reaction creating a fluorescent compound in an amount proportional to the S-nitrosothiol amount present in the sample. Alternatively, the amount of released NO can be measured by a chemiluminescence analyser, commercially available. In the presence of biological fluids having complex matrix, the introduction of the enzyme is done after separation of the S-nitrosothiol from the other components of the fluid.