Universita′ Di Pisa

Renata Barsacchi

Emilia Bramanti

Maria Franzini

Aldo Paolicchi

Alfonso Pompella

Michele Emdin

Cecilia Vecoli

A method is described for detecting the gamma-glutamyl transferase enzyme isoforms (GGT, EC 2.3.2.2) in a sample of biological fluid, such as for example plasma or serum. The method comprises an HPLC separation step of the sample proteins based on the molecular size and a second step for detecting the GGT isoforms by post-column reaction with a GGT enzyme substrate capable of generating a detectable final product, preferably by spectrophotometric or fluorimetric means. The GGT isoforms can be separated by ultra-centrifugation, thereby obtaining three enzymatic isoforms characterized by molecular weights of approximately 2000, 940, and 140 KDa, respectively.

Method of detecting serum gamma-glutamyl transferase isoforms in a biological fluid

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 &#x3b3;-glutamyltranspeptidase (GGT). This enzyme hydrolizes the residual &#x3b3;-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.

Process for determining S-nitrosothiols in biological fluids

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Localita S. Maria del Giudice, Italy

Renata Barsacchi