Assessment of S-nitrosothiols in biological fluids: content of S-nitrosothiols in plasma.
Tyurin-VA; Zhao-Q; Jiang-J; Borisenko-GG; Gandley-RE; Tyurina-YY; Bair-H; Kapralov-AA; Shvedova-AA; Komarov-AM; Hubel-CA; Taylor-RN; Stoyanovsky-DA; Kagan-VE
Toxicologist 2004 Mar; 78(S-1):411-412
In plasma, nitrosylated thiols, mainly S-nitrosoalbumin, (Alb-S-NO), are the major reservoirs of releasable NO. Estimates of plasma concentrations of RSNO have been reported to range from 5 nM up to 15 uM. Both underestimates and overestimates in the assessment of RSNO are implicated as potential sources of high variability. We utilized four independent protocols to assess Alb-S-NO in plasma. The samples were pretreated with NEM and separated by affinity-chromatography to obtain an albumin-enriched fraction. Decomposition of RSNO results in the stoichiometric production of NO and a reduced thiol as the two primary relatively stable products. We employed two assays to detect released NO (an EPR-based trapping using an NO chelator, MGD2-Fe, as well as a fluorogenic scavenger of NO, DAF2) as well as two protocols to evaluate Alb-SH (biotin-switch assay using NitroGlo kit, and ThioGlo-1-based assay with a maleimide fluorogenic SH-reagent, and subsequent fluorescence assay of Alb-SH after reduction of Alb-S-NO by high concentrations of ascorbate). Procedures utilizing sulfanilamide gave false negative results due to decomposition of RSNO by the reagent. This may explain very low concentrations of RSNO reported by workers who utilized sulfanilamide treatments. All the protocols used give reasonably concordant results: low icromolar concentrations (20-100 pmol/mg protein). These levels are physiologically ustified as they provides for its physiologically-relevant vasodilatory functions s assessed in our experiments with isolated mouse mesenteric arteries.
Biological-factors; Fluids; Sampling; Physiological-factors; Physiological-effects; Laboratory-animals; Animals; Animal-studies
Disease and Injury: Allergic and Irritant Dermatitis
The Toxicologist. Society of Toxicology 43nd Annual Meeting and ToxExpo, March 21-25, 2004, Baltimore, Maryland