ascorbic-acid and 4-5-diaminofluorescein

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.

Topics: Analytic Sample Preparation Methods; Animals; Ascorbic Acid; Blood Chemical Analysis; Cattle; Chromatography, Liquid; Fluorescein; Fluorometry; gamma-Glutamyltransferase; Humans; Hydrolysis; Kidney; Nitric Oxide; Nitroso Compounds; S-Nitrosoglutathione; Sensitivity and Specificity; Spectrophotometry; Sulfhydryl Compounds; Uric Acid

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N(2)O(3) from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.

Topics: Animals; Ascorbic Acid; Dehydroascorbic Acid; Fluorescein; Nitric Oxide; Spectrometry, Fluorescence; Spectrometry, Mass, Electrospray Ionization

Although in hypertension a defect in stimulated nitric oxide (NO) is well established, little is known about basal NO levels. Thus, we measured directly in vessels from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHR) both basal and stimulated NO production using a novel technique [4,5-diaminofluorescein (DAF-2) fluorescence].. Isolated vessels were exposed to the fluorescent probe DAF-2. After the technique was validated with increasing doses of acetylcholine in the presence and absence of NG-nitro-L-arginine methyl ester (l-NAME), we measured NO production in vessels from WKY rats and SHR in the same experimental setting. Finally, to explore the impact of reactive oxygen species (ROS) on NO release, we analysed the effect of an antioxidant, such as ascorbic acid, on basal and stimulated NO in aortic rings of WKY rats and SHR.. Aortic rings from SHR exhibited a higher basal NO production and a lower responsiveness to agonist-induced NO release as compared with those observed in WKY rats. Also in resistance vessels such as mesenteric arteries, basal NO production was higher in hypertension. In hypertensive rats, ascorbic acid was able to further increase basal NO release and recovered the impaired stimulated NO production, whereas no effect was detected in normotensive rats.. Our data reveal an increased basal NO availability in hypertension despite the increased production of ROS, suggesting a greater complexity in hypertensive endothelial dysfunction when the analysis is focused on direct NO measurement.

Topics: Acetylcholine; Animals; Antioxidants; Aorta; Ascorbic Acid; Enzyme Inhibitors; Fluorescein; Hypertension; In Vitro Techniques; Indicators and Reagents; Male; Mesenteric Arteries; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Reactive Oxygen Species

The quintessential nitrosating species produced during NO autoxidation is N(2)O(3). Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt(2)NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N(2)O(3) through the intermediacy of NO(2). This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO(2) was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.

Topics: Ascorbic Acid; Cyclic N-Oxides; Fluorescein; Glutathione; Humans; Imidazoles; Indicators and Reagents; Kinetics; Models, Chemical; Nitric Oxide; Nitrogen Oxides; Nitrosation; Oxygen; Protein Structure, Tertiary; Reactive Oxygen Species; Spectrometry, Fluorescence; Stress, Physiological; Time Factors; Tumor Cells, Cultured

4,5-Diaminofluorescein (DAF-2) is a newly developed indicator of nitric oxide (NO). Two amino groups of DAF-2 are oxidized by NO. We investigated the effects of reducers on the NO-induced oxidation of DAF-2. NOC-5 (0.1-10 microM), a NO-donor, concentration-dependently elicited fluorescence with 10 microM DAF-2. The rate of the fluorescence reaction was dependent on the width of the excitation band path. The presence of catecholamines (1 microM), but not tyrosine or phenylephrine, attenuated the fluorescence induced by NOC-5. Ascorbate and other reducers like dithiothreitol, 2-mercaptoethanol, or glutathione (all 1 mM) abolished the fluorescence. These results suggest that reducers attenuate the NO-induced fluorescence of DAF-2 mainly through an anti-oxidative action.

Topics: Antioxidants; Ascorbic Acid; Catecholamines; Fluorescein; Fluorescence; In Vitro Techniques; Indicators and Reagents; Nitric Oxide; Oxidation-Reduction