Blood Plasma's Protective Ability against the Degradation of S-Nitrosoglutathione under the Influence of Air-Pollution-Derived Metal Ions in Patients with Exacerbation of Heart Failure and Coronary Artery Disease.

One of the consequences of long-term exposure to air pollutants is increased mortality and deterioration of life parameters, especially among people diagnosed with cardiovascular diseases (CVD) or impaired respiratory system. Aqueous soluble inorganic components of airborne particulate matter containing redox-active transition metal ions affect the stability of S-nitrosothiols and disrupt the balance in the homeostasis of nitric oxide. Blood plasma's protective ability against the decomposition of S-nitrosoglutathione (GSNO) under the influence of aqueous PM extract among patients with exacerbation of heart failure and coronary artery disease was studied and compared with a group of healthy volunteers. In the environment of CVD patients' plasma, NO release from GSNO was facilitated compared to the plasma of healthy controls, and the addition of ascorbic acid boosted this process. Model studies with albumin revealed that the amount of free thiol groups is one of the crucial factors in GSNO decomposition. The correlation between the concentration of NO released and -SH level in blood plasma supports this conclusion. Complementary studies on gamma-glutamyltranspeptidase activity and ICP-MS multielement analysis of CVD patients' plasma samples in comparison to a healthy control group provide broader insights into the mechanism of cardiovascular risk development induced by air pollution.

Cardiovascular "Patterns" of H2S and SSNO--Mix Evaluated from 35 Rat Hemodynamic Parameters.

This work is based on the hypothesis that it is possible to characterize the cardiovascular system just from the detailed shape of the arterial pulse waveform (APW). Since H2S, NO donor S-nitrosoglutathione (GSNO) and their H2S/GSNO products (SSNO--mix) have numerous biological actions, we aimed to compare their effects on APW and to find characteristic "patterns" of their actions. The right jugular vein of anesthetized rats was cannulated for i.v. administration of the compounds. The left carotid artery was cannulated to detect APW. From APW, 35 hemodynamic parameters (HPs) were evaluated. H2S transiently influenced all 35 HPs and from their cross-relationships to systolic blood pressure "patterns" and direct/indirect signaling pathways of the H2S effect were proposed. The observed "patterns" were mostly different from the published ones for GSNO. Effect of SSNO--mix (≤32 nmol kg-1) on blood pressure in the presence or absence of a nitric oxide synthase inhibitor (L-NAME) was minor in comparison to GSNO, suggesting that the formation of SSNO--mix in blood diminished the hemodynamic effect of NO. The observed time-dependent changes of 35 HPs, their cross-relationships and non-hysteresis/hysteresis profiles may serve as "patterns" for the conditions of a transient decrease/increase of blood pressure caused by H2S.

Measurement of S -Nitrosoglutathione in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

This chapter describes an ultraperformance liquid chromatographic-tandem mass spectrometric (UPLC-MS/MS) method for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. S-[15N]Nitrosoglutathione (GS15NO) serves as the internal standard. The protocol involves inactivation of plasma γ-glutamyltransferase activity by serine-borate, stabilization of GSNO with EDTA, and avoidance of S-transnitrosylation reactions by blocking SH groups with N-ethylmaleimidide (NEM). Fresh blood is treated with NEM/serine-borate/EDTA, plasma is spiked with GS15NO (50 nM), ultrafiltered (cutoff 10 kDa) and 10-µL aliquots of ultrafiltrate are analyzed by UPLC-MS/MS in the positive electrospray ionization (ESI+) mode. LC is performed on a Nucleoshell column using isocratic (0.5 mL/min) elution with acetonitrile-20 mM ammonium formate (70:30, v/v), pH 7. Quantification is performed by selected-reaction monitoring the mass transition m/z 337 ([M+H]+) â†’ m/z 307 ([M+H-14NO]+●) for GSNO and m/z 338 ([M+H]+) â†’ m/z 307 ([M+H-15NO]+●) for GS15NO. Matrix effects are outweighed by the internal standard GS15NO. The lower limit of quantitation (LOQ) is 2.8 nM.

Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

UPLC-MS/MS measurement of S-nitrosoglutathione (GSNO) in human plasma solves the S-nitrosothiol concentration enigma.

We developed and validated a fast UPLC-MS/MS method with positive electrospray ionization (ESI+) for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. We used a published protocol for the inactivation of plasma γ-glutamyltransferase (γGT) activity by using the γGT transition inhibitor serine/borate and the chelator EDTA for the stabilization of GSNO, and N-ethylmaleimide (NEM) to block SH groups and to avoid S-transnitrosylation reactions which may diminish GSNO concentration. S-[(15)N]Nitrosoglutathione (GS(15)NO) served as internal standard. Fresh blood was treated with NEM/serine/borate/EDTA, plasma spiked with GS(15)NO (50nM) was ultrafiltered (cut-off 10kDa) and 10µL aliquots of the ultrafiltrate were analyzed by UPLC-MS/MS. Five HILIC columns and an Acquity UPLC BH amide column were tested. The mobile phase was acetonitrile-water (70:30, v/v), contained 20mM ammonium formate, had a pH value of 7, and was pumped isocratically (0.5mL/min). The Nucleoshell column allowed better LC performance and higher MS sensitivity. The retention time of GSNO was about 1.1min. Quantification was performed by selected-reaction monitoring the mass transition m/z 337 ([M+H](+))→m/z 307 ([M+H(14)NO](+)) for GSNO (i.e., GS(14)NO) and m/z 338 ([M+H](+))→m/z 307 ([M+H(15)NO](+)) for GS(15)NO. NEM/serine/borate/EDTA was found to stabilize GSNO in human plasma. The method was validated in human plasma (range, 0-300nM) using 50nM GS(15)NO. Accuracy and precision were in generally acceptable ranges. A considerable matrix effect was observed, which was however outweighed by the internal standard GS(15)NO. In freshly prepared plasma from heparinized blood donated by 10 healthy subjects, no endogenous GSNO was determined above 2.8nM, the limit of quantitation (LOQ) of the method. This study challenges previously reported GSNO plasma concentrations being far above the present method LOQ value and predicts that the concentration of low-molecular-mass and high-molecular-mass S-nitrosothiols are in the upper pM- and lower nM-range, respectively.

Visible photolysis and amperometric detection of S-nitrosothiols.

The concentration of S-nitrosothiols (RSNOs), endogenous transporters of the signaling molecule nitric oxide (NO), fluctuate greatly in physiology often as a function of disease state. RSNOs may be measured indirectly by cleaving the S-N bond and monitoring the liberated NO. While ultraviolet photolysis and reductive-based cleavage both decompose RSNOs to NO, poor selectivity and the need for additional reagents preclude their utility clinically. Herein, we report the coupling of visible photolysis (i.e., 500-550 nm) and amperometric NO detection to quantify RSNOs with greater selectivity and sensitivity. Enhanced sensitivity (up to 1.56 nA µM(-1)) and lowered theoretical detection limits (down to 30 nM) were achieved for low molecular weight RSNOs (i.e., S-nitrosoglutathione, S-nitrosocysteine) by tuning the irradiation exposure. Detection of nitrosated proteins (i.e., S-nitrosoalbumin) was also possible, albeit at a decreased sensitivity (0.11 nA µM(-1)). This detection scheme was used to measure RSNOs in plasma and illustrate the potential of this method for future physiological studies.

Determination of S-nitrosoglutathione in plasma: comparison of two methods.

In this work we compared the results of the GSNO determination in human plasma by two independent methods. The first method is based on the pre-column derivatization of GSNO thiolic part by p-hydroxymercury benzoate (PHMB) and followed by the determination of GS-PHMB product by reversed phase chromatography coupled to chemical vapour generation atomic fluorescence spectrometry (RPC-CVGAFS). The second method is based on RPC separation of GSNO from interfering compounds and the post-column, on-line enzymatic hydrolysis of GSNO by commercial gamma-glutamyl transferase (GGT) and fluorescence detection. Endogenous GSNO was determined only in plasma from blood sampled by syringe (not by Vacutainers) and ranged between 157 and 257nM on the basis of RPC-CVGAFS method, and between 90 and 225nM by RPC-FD method. There was a good correlation between the two methods (slope=1.06+/-0.09, R(2)=0.9543). RPC-CVGAFS method based on PHMB derivatization determined a GSNO concentration 60+/-20nM in excess with respect to RPC-FD method. Sampling issues connected with common blood sampling procedures like venipuncture and sampling in syringe or Vacutainers still introduce in GSNO analysis unknown factors, which require further investigations.

Transfer of nitric oxide by blood from upstream to downstream resistance vessels causes microvascular dilation.

The discovery that hemoglobin, albumin, and glutathione carry and release nitric oxide (NO) may have consequences for movement of NO by blood within microvessels. We hypothesize that NO in plasma or bound to proteins likely survives to downstream locations. To confirm this hypothesis, there must be a finite NO concentration ([NO]) in arteriolar blood, and upstream resistance vessels must be able to increase the vessel wall [NO] of downstream arterioles. Arteriolar blood NO was measured with NO-sensitive microelectrodes, and vessel wall [NO] was consistently 25-40% higher than blood [NO]. Localized suppression of NO production in large arterioles over 500-1,000 microm with L-nitroarginine reduced the [NO] approximately 40%, indicating as much as 60% of the wall NO was from blood transfer. Flow in mesenteric arteries was elevated by occlusion of adjacent arteries to induce a flow-mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor-mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, [NO] increased and both sets of vessels dilated. When isoproterenol was applied to the upstream vessels, they dilated, but neither the [NO] or diameter downstream arterioles increased. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This mechanism allows larger vessels that generate large [NO] to influence vascular tone in downstream vessels in response to both flow and receptor stimuli.

Exogenous vs. endogenous gamma-glutamyltransferase activity: Implications for the specific determination of S-nitrosoglutathione in biological samples.

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.

Potential oxidative stress of gold nanoparticles by induced-NO releasing in serum.

Nitric oxide (NO)-release in blood serum initiated by gold nanoparticles has been prove to be a reaction between RSNO and the gold nanoparitcles. In this reaction the NO production was catalyzed on the surface of the nanoparticles, and a new bond of Au-thiolate was simultaneously formed.

Glutathione supplementation to University of Wisconsin solution causes endothelial dysfunction.

INTRODUCTION: Glutathione (GSH) is added to University of Wisconsin (UW) organ preservation solution to protect against oxidative stress. This study assesses the effect of GSH-supplementation on endothelial function in tissues subjected to cold ischaemia and compares its effects to a mono-ethyl ester equivalent (GSH-MEE) and S-nitrosated GSH (GSNO). METHODS: Rat aortic rings were stored for 1 h or 48 h in cold, hypoxic UW solution with or without GSH (3 mM), GSH-MEE (3 mM) or GSNO (100 mciroM) supplementation. Aortic rings were reoxygenated in warm Krebs solution; smooth muscle function was assessed by responses to phenylephrine (PE), and endothelial function by vasodilatation to the endothelium-dependent dilator, acetylcholine (ACh). The protective effects against oxidant-induced endothelial cell death were assessed in cultured human umbilical vein endothelial cells (HUVEC). RESULTS: Supplementation of UW with either GSH or GSH-MEE had no effect on vascular responses to PE, but smooth muscle contraction was significantly attenuated in rings incubated for 48 h with GSNO. Endothelium-dependent relaxation was significantly impaired in tissues stored under hypoxic conditions in GSH, GSH-MEE and GSNO supplemented UW solution for 1 h. However, impairment at 48 h was significantly more pronounced in GSH-treated vessels. Cultured HUVEC death was exacerbated by GSH and GSH-MEE in unstressed cells and in those stressed with a superoxide anion generator. CONCLUSIONS: GSH supplementation of UW solution exacerbates cold-ischaemia induced endothelial dysfunction. GSNO did not share the detrimental effects of GSH and promoted NO-mediated vasodilatation.

Effects of low habitual cocoa intake on blood pressure and bioactive nitric oxide: a randomized controlled trial.

CONTEXT: Regular intake of cocoa-containing foods is linked to lower cardiovascular mortality in observational studies. Short-term interventions of at most 2 weeks indicate that high doses of cocoa can improve endothelial function and reduce blood pressure (BP) due to the action of the cocoa polyphenols, but the clinical effect of low habitual cocoa intake on BP and the underlying BP-lowering mechanisms are unclear. OBJECTIVE: To determine effects of low doses of polyphenol-rich dark chocolate on BP. DESIGN, SETTING, AND PARTICIPANTS: Randomized, controlled, investigator-blinded, parallel-group trial involving 44 adults aged 56 through 73 years (24 women, 20 men) with untreated upper-range prehypertension or stage 1 hypertension without concomitant risk factors. The trial was conducted at a primary care clinic in Germany between January 2005 and December 2006. INTERVENTION: Participants were randomly assigned to receive for 18 weeks either 6.3 g (30 kcal) per day of dark chocolate containing 30 mg of polyphenols or matching polyphenol-free white chocolate. MAIN OUTCOME MEASURES: Primary outcome measure was the change in BP after 18 weeks. Secondary outcome measures were changes in plasma markers of vasodilative nitric oxide (S-nitrosoglutathione) and oxidative stress (8-isoprostane), and bioavailability of cocoa polyphenols. RESULTS: From baseline to 18 weeks, dark chocolate intake reduced mean (SD) systolic BP by -2.9 (1.6) mm Hg (P < .001) and diastolic BP by -1.9 (1.0) mm Hg (P < .001) without changes in body weight, plasma levels of lipids, glucose, and 8-isoprostane. Hypertension prevalence declined from 86% to 68%. The BP decrease was accompanied by a sustained increase of S-nitrosoglutathione by 0.23 (0.12) nmol/L (P < .001), and a dark chocolate dose resulted in the appearance of cocoa phenols in plasma. White chocolate intake caused no changes in BP or plasma biomarkers. CONCLUSIONS: Data in this relatively small sample of otherwise healthy individuals with above-optimal BP indicate that inclusion of small amounts of polyphenol-rich dark chocolate as part of a usual diet efficiently reduced BP and improved formation of vasodilative nitric oxide. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00421499.

Speciation and quantification of thiols by reversed-phase chromatography coupled with on-line chemical vapor generation and atomic fluorescence spectrometric detection: method validation and preliminary application for glutathione measurements in human whole blood.

BACKGROUND: We developed a sensitive, specific method for the low-molecular-mass thiols cysteine, cysteinylglycine, glutathione, and homocysteine and validated the method for measurement of glutathione in blood. METHODS: The technique was based on reversed-phase chromatography (RPC) coupled on line with cold vapor generation atomic fluorescence spectrometry (CVGAFS). Thiols were derivatized before introduction on the column by use of a p-hydroxymercuribenzoate (PHMB) mercurial probe and separated as thiol-PHMB complexes on a Vydac C4 column. Postcolumn on-line reaction of derivatized thiols with bromine allowed rapid conversion of the thiol-PHMB complexes to inorganic mercury with recovery of 100 (2)% of the sample. HgII was selectively detected by atomic fluorescence spectrometry in an Ar/H2 miniaturized flame after sodium borohydride reduction to Hg0. RESULTS: The relationship between thiol-PHMB complex concentration and peak area (CVGAFS signal) was linear over the concentration range 0.01-1400 micromol/L (injected). The detection limits were 1, 1, 0.6, and 0.8 nmol/L for cysteine, cysteinylglycine, homocysteine, and glutathione in the injected sample, respectively. The CVs for thiols were 1.5%-2.2% for calibrator solutions and 2.1% and 3.0% for real samples. The RPC-CVGAFS method allowed speciation of glutathione (reduced and oxidized) in human whole blood from healthy donors and from the coronary sinus of patients with idiopathic dilated cardiomyopathy during and after chronotropic stress. CONCLUSION: The RPC-CVGAFS method could be used to measure reduced and oxidized glutathione in human whole blood as disease biomarkers.

Effects of melatonin on plasma S-nitrosoglutathione and glutathione in streptozotocin-treated rats.

The aim of this study was to determine the effects of streptozotocin-induced diabetes on plasma reduced glutathione (GSH) and S-nitrosoglutathione (GSNO) levels. Further, the study investigated whether an antioxidant, pineal hormone melatonin, could protect against STZ-induced effects. STZ significantly decreased plasma GSH but increased the levels of plasma GSNO. Daily supplementation with melatonin restored plasma thiol to control values. Data suggest that STZ-induced hyperglycemia and compounds that act as scavengers of free radicals and peroxynitrite like melatonin may exert protection against STZ-induced toxicity.

Red blood cell nitric oxide as an endocrine vasoregulator: a potential role in congestive heart failure.

BACKGROUND: A respiratory cycle for nitric oxide (NO) would involve the formation of vasoactive metabolites between NO and hemoglobin during pulmonary oxygenation. We investigated the role of these metabolites in hypoxic tissue in vitro and in vivo in healthy subjects and patients with congestive heart failure (CHF). METHODS AND RESULTS: We investigated the capacity for red blood cells (RBCs) to dilate preconstricted aortic rings under various O2 tensions. RBCs induced cyclic guanylyl monophosphate-dependent vasorelaxation during hypoxia (35+/-4% at 1% O2, 4.7+/-1.6% at 95% O2; P<0.05). RBC-induced relaxations during hypoxia correlated with S-nitrosohemoglobin (SNO-Hb) (R2=0.88) but not iron nitrosylhemoglobin (HbFeNO) content. Relaxation responses for RBCs were compared with S-nitrosoglutathione across a range of O2 tensions. The fold increases in relaxation evoked by RBCs were significantly greater at 1% and 2% O2 compared with relaxations induced at 95% (P<0.05), consistent with an allosteric mechanism of hypoxic vasodilation. We also measured transpulmonary gradients of NO metabolites in healthy control subjects and in patients with CHF. In CHF patients but not control subjects, levels of SNO-Hb increase from 0.00293+/-0.00089 to 0.00585+/-0.00137 mol NO/mol hemoglobin tetramer (P=0.005), whereas HbFeNO decreases from 0.00361+/-0.00109 to 0.00081+/-0.00040 mol NO/mol hemoglobin tetramer (P=0.03) as hemoglobin is oxygenated in the pulmonary circulation. These metabolite gradients correlated with the hemoglobin O2 saturation gradient (P<0.05) and inversely with cardiac index (P<0.05) for both CHF patients and control subjects. CONCLUSIONS: We confirm that RBC-bound NO mediates hypoxic vasodilation in vitro. Transpulmonary gradients of hemoglobin-bound NO are evident in CHF patients and are inversely dependent on cardiac index. Hemoglobin may transport and release NO bioactivity to areas of tissue hypoxia or during increased peripheral oxygen extraction via an allosteric mechanism.

Optimization of the principal parameters for the ultrarapid electrophoretic separation of reduced and oxidized glutathione by capillary electrophoresis.

Several factors can influence the analytical efficiency and rapidity of the quantitative determination of erythrocyte glutathione by capillary zone electrophoresis (CZE). We optimized the time, efficiency and resolution of the electrophoretic separation of reduced (GSH) and oxidized (GSSG) glutathione by studying the influence of the most important factors affecting the separation, i.e. the pH and ionic strength of the electrolyte solution, the capillary length and temperature. Best results in the shortest time are obtained at 25 degrees C, using an uncoated 37 cm x 75 microm i.d. capillary and a 300 mmol/l borate buffer pH 7.8. These conditions give a good reproducibility of the corrected peak areas (R.S.D. 1.41 and 1.31%) and of the migration time (R.S.D. 0.22 and 0.26%) for GSH and GSSG, respectively. The high concentration buffer, besides permitting a good resolution of standard GSH and GSSG mix, allows also N-nitrosoglutathione detection. By shortening the capillary length to 27 cm, the separation time of GSH and GSSG can be further decreased to less than 60s. This shortened method, the most rapid described in literature, can detect and quantify GSH in red blood cells despite a loss of sensitivity. To compare the new method here described with the Beutler colorimetric method, the data relative to the GSH content of red blood cells from young normal subjects were analyzed by the Passing and Bablok regression and the Bland-Altman test.

Interference with Saville's method in determination of low-molecular weight S-nitrosothiols by ultrafiltration.

To detect low-molecular weight S-nitrosothiols in human plasma, we used a system combining HPLC for separation and Saville's method for colorimetric detection of S-nitrosothiols. The sensitivity and detection limit was 1-2 nM for both S-nitrosocysteine and S-nitrosoglutathione. When plasma was analyzed after ultrafiltration (with units requiring higher g force [5000 g], irrespective to the material of the membrane) to eliminate high molecular substances, a signal corresponding to S-nitrosoglutahione was recognized. This signal behaved as real S-nitrosoglutathione as it was partially Hg(2+)-sensitive and gradually decayed with time. However, the use of pre-washed units or another ultrafiltration unit that required lower g force (1800 g) or direct application of plasma to the HPLC-Saville's method system did not result in such signal. Based on these observations, it is important to be aware of the interference originating from the ultrafiltration unit and its potential effect on the precise quantification of low molecular weight S-nitrosothiols using Saville's method.

Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo.

There is growing evidence for the involvement of nitric oxide (NO) -mediated nitrosation in cell signaling and pathology. Although S-nitrosothiols (RSNOs) have been frequently implicated in these processes, it is unclear whether NO forms nitrosyl adducts with moieties other than thiols. A major obstacle in assessing the significance of formation of nitrosated species is the limited reliability of available analytical techniques for measurements in complex biological matrices. Here we report on the presence of nitrosated compounds in plasma and erythrocytes of rats, mice, guinea pigs, and monkeys under basal conditions, in immunologically challenged murine macrophages in vitro and laboratory animals in vivo. Besides RSNOs, all biological samples also contained mercury-stable nitroso species, indicating the additional involvement of amine and heme nitros(yl)ation reactions. Significant differences in the amounts and ratios of RSNOs over N- and heme-nitros(yl)ated compounds were found between species and organs. These observations were made possible by the development of a novel gas-phase chemiluminescence-based technique that allows detection of nitroso species in tissues and biological fluids without prior extraction or deproteinization. The method can quantify as little as 100 fmol bound NO and has been validated extensively for use in different biological matrices. Discrimination between nitrite, RSNOs, and N-nitroso or nitrosylheme compounds is accomplished by use of group-specific reagents. Our findings suggest that NO generation in vivo leads to concomitant formation of RSNOs, nitrosamines, and nitrosylhemes with considerable variation between rodents and primates, highlighting the difficulty in comparing data between different animal models and extrapolating results from experimental animals to human physiology.