S-nitroso Albumin Research Articles

Cytoprotective effects of albumin, nitrosated or reduced, in cultured rat pulmonary vascular cells

S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H(2)S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to N(G)-nitro-L-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H(2)S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H(2)S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an N(G)-nitro-L-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H(2)S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS.

Application of a stable-isotope dilution technique to study the pharmacokinetics of human 15N-labelled S-nitrosoalbumin in the rat: Possible mechanistic and biological implications

In the year 1992, S-nitrosoalbumin (SNALB) has been proposed to be the most abundant physiological carrier and pool of nitric oxide (NO) activity in human circulation, by which NO-dependent biological functions are regulated. The concentration, the metabolism and the mechanisms of the biological actions of SNALB are controversial and still incompletely understood. Moreover, the suitability of SNALB as a biomarker of diseases associated with altered NO bioactivity in human circulation has not been demonstrated convincingly so far. In the present study, we report on the development and application of a stable-isotope technique to study the pharmacokinetics of 15N-labelled SNALB (S 15NALB) in anesthetized rats. S 15NALB was synthesized from albumin isolated by affinity chromatography from freshly prepared human plasma. This technique was also applied to study and quantify the formation of S 15NALB from endogenous rat plasma albumin and intravenously applied S-[ 15N]nitrosoglutathione (GS 15NO) or S-[ 15N]nitrosocysteine (S 15NC) in anesthetized rats. In these investigations the mean arterial pressure (MAP) was monitored continuously. The elimination half-life ( t 1/2) of S 15NALB from rat plasma was determined to be 4.1 min ( t 1/2 α) and 9.4 min ( t 1/2 β). S 15NALB (125 nmol) produced long-lasting decreases in MAP (by 49% for 18 min). Thirty minutes after intravenous (i.v.) injection of S 15NALB (125 nmol), repeated i.v. injection of l-cysteine or d-cysteine (10 μmol each) produced repeatedly potent (by 44–55%) but short-lasting (about 4 min) MAP falls. Intravenously administered GS 15NO and S 15NC (each 500 nmol) could not be isolated from rat blood. 15N-Labelled nitrite and nitrate were identified as the major metabolites of all investigated S-nitrosothiols in rat plasma. The results of this study suggest that in the rat S 15NALB is a potent S-transnitrosylating agent and that the blood pressure-lowering effect of S 15NALB and other S-nitrosothiols are mediated largely by l-cysteine via S-transnitrosylation to form S 15NC that subsequently releases 15NO. Our results also suggest that S-transnitrosylation of the single reduced cysteine moiety of albumin by endogenous GSNO or SNC in blood is possible but does not represent an effective mechanism to produce SNALB in vivo. This stable-isotope dilution GC–MS technique is suitable to perform in vivo studies on SNALB using physiologically and pharmacologically relevant doses.

Measurement of Nitric Oxide Levels in the Red Cell: VALIDATION OF TRI-IODIDE-BASED CHEMILUMINESCENCE WITH ACID-SULFANILAMIDE PRETREATMENT

The tri-iodide-based chemiluminescence assay is the most widely used methodology for the detection of S-nitrosothiols (RSNOs) in biological samples. Because of the low RSNO levels detected in a number of biological compartments using this assay, criticism has been raised that this method underestimates the true values in biological samples. This claim is based on the beliefs that (i) acidified sulfanilamide pretreatment, required to remove nitrite, leads to RSNO degradation and (ii) that there is auto-capture of released NO by heme in the reaction vessel. Because our laboratories have used this assay extensively without ever encountering evidence that corroborated these claims, we sought to experimentally address these issues using several independent techniques. We find that RSNOs of glutathione, cysteine, albumin, and hemoglobin are stable in acidified sulfanilamide as determined by the tri-iodide method, copper/cysteine assay, Griess-Saville assay and spectrophotometric analysis. Quantitatively there was no difference in S-nitroso-hemoglobin (SNOHb) or S-nitroso-albumin (SNOAlb) using the tri-iodide method and a recently described modified assay using a ferricyanide-enhanced reaction mix at biologically relevant NO:heme ratios. Levels of SNOHb detected in human blood ranged from 20-100 nM with no arterial-venous gradient. We further find that 90% of the total NO-related signal in blood is caused by erythrocytic nitrite, which may partly be bound to hemoglobin. We conclude that all claims made thus far that the tri-iodide assay underestimates RSNO levels are unsubstantiated and that this assay remains the "gold standard" for sensitive and specific measurement of RSNOs in biological matrices.

S -Nitrosoalbumin–Mediated Relaxation Is Enhanced by Ascorbate and Copper

S-nitrosoalbumin (SNO-Alb) is a major reservoir of releasable nitric oxide (NO) in plasma. In preeclampsia, a pregnancy-specific disorder associated with endothelial dysfunction, we previously found significant elevations in plasma SNO-Alb concentrations and decreased plasma ascorbate (Asc) levels. This increased SNO-Alb may result from low-plasma Asc if Asc, along with transition metals (eg, copper [Cu]) are necessary for release of NO from S-nitrosothiols. We propose that vasodilator effects of SNO-Alb, mediated by release of NO, are fully realized only when Asc/Cu availability is sufficient. Relaxation responses to SNO-Alb or the control reduced human serum albumin (SH-Alb), and responses to pooled plasma from normal or preeclamptic pregnancies were examined in isolated mouse arteries. Arteries preconstricted with phenylephrine were exposed to SNO-Alb or SH-Alb at physiologically relevant concentrations. When free Cu was added in excess (10 mumol/L), NO release was not dependent on Asc. However, when Cu was added at lower (physiological) levels, NO release was dependent on Asc. The addition of Asc and Cu to SNO-Alb stimulated vasodilatory responses in isolated arteries >90%, whereas no change in the SH-Alb (5%) response was observed. Preeclampsia plasma with higher levels of SNO-Alb caused arteries to relax 44.1+/-4.7%, whereas normal pregnancy plasma caused 11.9+/-4.2% relaxation (P=0.007). These data indicate that SNO-Alb alone or in plasma can act as a potent vasodilator, and that sufficient Asc/Cu promotes this action. We suggest that the higher circulating levels of SNO-Alb, in women with preeclampsia, reflect a deficiency in Asc/Cu-mediated release of NO from SNO-Alb.

Measurement of S-nitrosoalbumin by gas chromatography–mass spectrometry : III. Quantitative determination in human plasma after specific conversion of the S-nitroso group to nitrite by cysteine and Cu 2+ via intermediate formation of S-nitrosocysteine and nitric oxide

Highly contradictory data exist on the normal plasma basal levels in humans of S-nitrosoproteins, in particular of S-nitrosoalbumin (SNALB), the most abundant nitric oxide (·NO) transport form in the human circulation with a range of three orders of magnitude (i.e., 10 n M–10 μ M). In previous work we reported on a GC–MS method for the quantitative determination of SNALB in human plasma. This method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S 15NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, HgCl 2-catalysed conversion of the S-nitroso groups to nitrite and [ 15N]nitrite, respectively, their derivatization to the pentafluorobenzyl derivatives and quantification by GC–MS. By this method we had measured SNALB basal plasma levels of 181 n M in healthy humans. It is generally accepted that HgCl 2-catalysed conversion of S-nitroso groups into nitrite is specific. In consideration of the highly divergent SNALB plasma levels in humans reported so far, we were interested in an additional method that would allow specific conversion of S-nitroso groups into nitrite. We found that treatment with cysteine plus CuSO 4 is as effective and specific as treatment with HgCl 2. The principle of the cysteine/CuSO 4 procedure is based on the transfer of the S-nitroso group from SNALB to cysteine yielding S-nitrosocysteine, and its subsequent highly Cu 2+-sensitive conversion into nitrite via intermediate ·NO formation. Similar SNALB concentrations in the plasma of 10 healthy humans were measured by GC–MS using HgCl 2 (156±64 n M) and cysteine/CuSO 4 (205±96 n M). Our results strongly suggest that SNALB is an endogenous constituent in human plasma and that its concentration is of the order of 150–200 n M under physiological conditions.

S-Transnitrosylation of albumin in human plasma and blood in vitro and in vivo in the rat

S-Nitrosoalbumin (SNOALB) is the most abundant physiological circulating nitric oxide (NO) carrier regulating NO-dependent biological actions in humans. The mechanisms of its formation and biological actions are still incompletely understood. Nitrosation by authentic NO and S-transnitrosylation of the single sulfhydryl group located at Cys-34 of human albumin by the physiological S-nitroso compounds S-nitrosocysteine (SNOC) and S-nitrosoglutathione (GSNO) are two possible mechanisms. On a quantitative basis, we investigated by gas chromatography-mass spectrometry the contribution of these two mechanisms to SNOALB formation in human plasma and blood in vitro. GSNO and SNOC (0–100 μM) rapidly and efficiently (recovery=35%) S-transnitrosylated albumin to form SNOALB. NO (100 μM) S-nitrosated albumin to SNOALB at a considerably lower extent (recovery=5%). The putative NO-donating drugs glyceryl trinitrate and sodium nitroprusside (each 100 μM) failed completely in S-nitrosating albumin. Bubbling NO into human plasma and blood resulted in formation of SNOALB that inhibited ADP-induced platelet aggregation. Infusion of GS 15NO in the rat resulted in formation of S 15NOALB, [ 15N]nitrate and [ 15N]nitrite. Our results suggest that S-transnitrosylation of albumin by SNOC and GSNO could be a more favored mechanism for the formation of SNOALB in the circulation in vivo than S-nitrosation of albumin by NO itself.

Measurement of S-nitrosoalbumin by gas chromatography–mass spectrometry : II. Quantitative determination of S-nitrosoalbumin in human plasma using S-[ 15N]nitrosoalbumin as internal standard

A gas chromatographic–mass spectrometric method for the quantitative determination of S-nitrosoalbumin (SNALB) in human plasma is described. The method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S 15NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, Hg 2+-catalysed conversion of the S-nitroso groups to nitrite and [ 15N]nitrite, respectively, followed by their derivatization to the pentafluorobenzyl derivatives and quantification by GC–MS. Mean recovery of SNALB and S 15NALB from plasma was 45%. Mean precision and accuracy within the range 0–10 μ M was 95% and 99%, respectively. The limit of quantitation was determined as 100 n M at a precision of 93.8% and an accuracy of 94.8%. Considerable improvement of method sensitivity is possible by eliminating nitrite present in the elution buffer. The limit of detection was 0.2 n M corresponding to 67 amol of S 15NALB. In 0.4-ml aliquots of plasma samples from healthy humans, endogenous SNALB was determined at concentrations of 181±150 n M (mean±SD, n=23). External addition of SNALB to these plasma samples at 2 μ M and 5 μ M serving as quality control samples resulted in quantitative recovery of SNALB. Our results show that SNALB occurs in human plasma at concentrations at least one-order of magnitude smaller than those reported in the literature from measurements using chemiluminescence.

Measurement of S-nitrosoalbumin by gas chromatography–mass spectrometry : I. Preparation, purification, isolation, characterization and metabolism of S-[ 15N]nitrosoalbumin in human blood in vitro

S-Nitrosoalbumin (SNALB) and S-[ 15N]nitrosoalbumin (S[ 15N]ALB) were prepared by various methods, purified and isolated by a novel selective extraction procedure using HiTrapBlue Sepharose affinity columns and characterized by various techniques including SDS-PAGE electrophoresis, UV–Vis spectroscopy and gas chromatography–mass spectrometry (GC–MS). S-Nitrosylation of albumin in freshly obtained human plasma by unlabeled and 15N-labeled butylnitrite at neutral pH revealed the purest preparations. For GC–MS analysis, SNALB and S[ 15N]ALB were treated with HgCl 2 to obtain nitrite and [ 15N]nitrite, respectively, which were then analysed as their pentafluorobenzyl derivatives. S[ 15N]ALB preparations were standardized by GC–MS using nitrite as internal standard. S[ 15N]ALB was prepared and isolated at concentrations of 188±43 μ M (mean±SD, n=8) at a final yield of about 45%, an isotopic purity of 98%, and SDS-PAGE electrophoretic purity of 90%. 15N-Labeled SNALB was used to study its metabolism in human blood. The half-life of S[ 15N]ALB (25 μ M) in human heparinized blood in vitro was determined by GC–MS as 5.5 h. The GC–MS method described here could be useful for the quantitative determination of SNALB in human plasma using S[ 15N]ALB as an internal standard.

Kinetics of peroxynitrite reaction with amino acids and human serum albumin

S-Nitrosoalbumin (SNOALB) is the most abundant physiological circulating nitric oxide (NO) carrier regulating NO-dependent biological actions in humans. The mechanisms of its formation and biological actions are still incompletely understood. Nitrosation by authentic NO and S-transnitrosylation of the single sulfhydryl group located at Cys-34 of human albumin by the physiological S-nitroso compounds S-nitrosocysteine (SNOC) and S-nitrosoglutathione (GSNO) are two possible mechanisms. On a quantitative basis, we investigated by gas chromatography-mass spectrometry the contribution of these two mechanisms to SNOALB formation in human plasma and blood in vitro. GSNO and SNOC (0–100 μM) rapidly and efficiently (recovery=35%) S-transnitrosylated albumin to form SNOALB. NO (100 μM) S-nitrosated albumin to SNOALB at a considerably lower extent (recovery=5%). The putative NO-donating drugs glyceryl trinitrate and sodium nitroprusside (each 100 μM) failed completely in S-nitrosating albumin. Bubbling NO into human plasma and blood resulted in formation of SNOALB that inhibited ADP-induced platelet aggregation. Infusion of GS15NO in the rat resulted in formation of S15NOALB, [15N]nitrate and [15N]nitrite. Our results suggest that S-transnitrosylation of albumin by SNOC and GSNO could be a more favored mechanism for the formation of SNOALB in the circulation in vivo than S-nitrosation of albumin by NO itself.

Protein thiyl free radicals produced by nitric oxide and nitric oxide donors

The spin-trapping technique was used to study the radical intermediates produced by reaction of nitric oxide (*NO) and peroxynitrite with serum albumin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Our results show that the major radical product induced by *NO and by peroxynitrite with serum albumin and GAPDH was a thiyl radical. The same radical can be detected in the *NO-transfer from S-nitroso albumin to low molecular weight thiols. Moreover, *NO or peroxynitrite treatment of GAPDH was able to induce NAD-dependent covalent modification of the enzyme in erythrocyte ghosts.