title: Exogenous vs. endogenous gamma-glutamyltransferase activity: Implications for the specific determination of S-nitrosoglutathione in biological samples.
abstract: The determination of S-nitrosoglutathione (GSNO) levels in biological fluids is controversial, partly due to the laborious sample handling and multiple pretreatment steps required by current techniques. GSNO decomposition can be effected by the enzyme gamma-glutamyltransferase (GGT), whose involvement in GSNO metabolism has been suggested. We have set up a novel analytical method for the selective determination and speciation of GSNO and its metabolite S-nitrosocysteinylglycine, based on liquid chromatography separation coupled to on-line enzymatic hydrolysis of GSNO by commercial GGT. In a post-column reaction coil, GGT allows the specific hydrolysis of the gamma-glutamyl moiety of GSNO, and the S-nitrosocysteinylglycine (GCNO) thus formed is decomposed by copper ions originating oxidized cysteinylglycine and nitric oxide (NO). NO immediately reacts with 4,5-diaminofluorescein (DAF-2) forming a triazole derivative, which is detected fluorimetrically. The limit of quantitation (LOQc) for GSNO and GCNO in plasma ultrafiltrate was 5 nM, with a precision (CV) of 1-6% within the 5-1500 nM dynamic linear range. The method was applied to evaluate the recovery of exogenous GSNO after addition of aliquots to human plasma samples presenting with different total GGT activities. By inhibiting GGT activity in a time dependent manner, it was thus observed that the recovery of GSNO is inversely correlated with plasmatic levels of endogenous GGT, which indicates the need for adequate inhibition of endogenous GGT activity for the reliable determination of endogenous GSNO.
authors.affiliation: Italian National Research Council-Istituto per i Processi Chimico-Fisici, Laboratory of Instrumental & Analytical Chemistry, 56124 Pisa, Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Bramanti
authors.forename: Valeria
authors.initials: V
authors.lastname: Angeli
authors.forename: Maria
authors.initials: M
authors.lastname: Franzini
authors.forename: Cecilia
authors.initials: C
authors.lastname: Vecoli
authors.forename: Riccardo
authors.initials: R
authors.lastname: Baldassini
authors.forename: Aldo
authors.initials: A
authors.lastname: Paolicchi
authors.forename: Renata
authors.initials: R
authors.lastname: Barsacchi
authors.forename: Alfonso
authors.initials: A
authors.lastname: Pompella
mesh_terms: D053000:Analytic Sample Preparation Methods; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D001774:Blood Chemical Analysis / Q000379:methods*; D002417:Cattle; D002853:Chromatography, Liquid; D019793:Fluorescein / Q000378:metabolism; D005470:Fluorometry; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism; D009603:Nitroso Compounds / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D026422:S-Nitrosoglutathione / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014527:Uric Acid / Q000737:chemistry; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000378:metabolism*
chemical_list: C111205:4,5-diaminofluorescein; D009603:Nitroso Compounds; C417091:S-nitrosocysteinylglycine; D013438:Sulfhydryl Compounds; D014527:Uric Acid; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D005723:gamma-Glutamyltransferase; D001205:Ascorbic Acid; D019793:Fluorescein