1 INSERM U507 and 2 Laboratory of Biochemistry A, Necker Hospital, Paris and 3 Laboratory of Biochemistry A, Cochin Hospital, Paris, France
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Abstract |
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Methods. Plasma levels of S-nitrosothiols and nitrotyrosine were determined in 22 non-smoking HD patients and 12 healthy control subjects, together with albumin, homocysteine, haemoglobin, highly sensitive C-reactive protein (hsCRP) and various components of the oxidantantioxidant system at the plasma and erythrocyte levels.
Results. While plasma nitrosothiol levels were significantly higher in HD patients than in controls (2.25±1.17 vs 0.45±0.45 µmol/l, respectively, P<0.0001), nitrotyrosine levels were not different. HD patients also exhibited a marked deficit of ascorbate and low plasma glutathione peroxidase activity. An inverse relationship was found between plasma S-nitrosothiol and blood haemoglobin in HD patients (P<0.005). No direct relationship was observed between plasma S-nitrosothiol levels and any of the oxidative stress markers, or hsCRP levels.
Conclusion. This study demonstrates high plasma S-nitrosothiol levels in HD patients, which are partially related to low blood haemoglobin concentrations. The pathophysiological significance of this elevation remains to be elucidated. A possible protective role against nitrosative stress is suggested in presence of normal plasma nitrotyrosine levels in such patients.
Keywords: atherosclerosis; haemodialysis; nitric oxide; nitrotyrosine; oxidantantioxidant system; S-nitrosothiol
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Introduction |
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First, NO can form adducts with molecules containing functional sulph-hydryl groups to yield S-nitrosothiols (Figure 1) [7]. Circulating NO is primarily complexed in S-nitrosothiol species [13]. S-nitrosothiols are considered as a NO pool, buffering NO, which is important for its storage and transport. Notably, S-nitrosothiols are potent vasodilators whose action is commonly associated with the ability to release NO at physiologically relevant sites [14]. This release may occur via a reduction by transition metal ions, ascorbate, thiol compounds, and several enzymes including plasma glutathione peroxidase (GSH-Px) [7,15].
Secondly, NO can also react with superoxide anion to form peroxynitrite (Figure 1) [7]. The latter can react with protein tyrosyl residues to form nitrotyrosine, and with thiols in proteins or in glutathione to form S-nitrosothiols [7]. The reaction with thiols may prevent the accumulation of peroxynitrite towards toxic levels.
To the best of our knowledge, no information is available at present concerning plasma levels of S-nitrosothiols and nitrotyrosine in HD patients. Therefore, the aim of our study was to determine whether plasma S-nitrosothiols and nitrotyrosine concentrations are elevated in chronic HD patients, compared with those of healthy controls, and if so, to evaluate factors potentially involved, including oxidative stress and inflammatory status.
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Subjects and methods |
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Biochemical determinations
Iron and transferrin were determined using routine methods on a Hitachi 917 analyser (Roche, Meylan, France). Plasma levels of albumin and highly sensitive CRP (hsCRP) were determined using immunonephelometric procedures (Dade Behring, Paris, France). Plasma total homocysteine concentrations were measured using a radio-enzymatic method [16], and blood haemoglobin by Coulter analyser (STKS; Coultronics, Margency, France).
Plasma S-nitrosothiols levels were determined by a fluorimetric method [17]. Briefly, ammonium-sulphamate solution (50 µl of 0.1 mmol/l) was added to 100 µl of a 2-fold diluted plasma to trap nitric dioxide (NO2). After a 10-min incubation, 50 µl of the reaction mixture (one part of 1.1 mmol/l mercuric chloride and four parts of 0.05 g/l diaminonaphthalene in 0.62 M HCl) were added. Following a 10-min incubation at room temperature and in darkness, the reaction was stopped with 20 µl of 2.8 M NaOH. Fluorescence intensity was measured in a 96-well microtitre plate (Pharmacia Biotech, Saint-Quentin en Yvelines, France) at excitation and emission wavelengths of 360 and 450 nm, respectively, and compared to a S-nitrosothiol standard curve prepared using various concentrations of reduced glutathione (GSH) (50 µl of 0.15 to 10.0 µmol/l GSH in 1 M HCl), incubated with 50 µl NaNO2 (10 µmol/l) at room temperature in darkness for 2 h, and then with 50 µl of 100 µM ammonium-sulphamate.
Plasma levels of 3-nitrotyrosine-modified proteins were measured by a commercial ELISA method (TCS Cell Works Ltd, Buckinghamshire, UK) [18]. Briefly, in a precoated microtitre plate with 4 µg/ml nitro-BSA, standards or samples were incubated in the wells with immunoaffinity-purified polyclonal anti-nitrotyrosine rabbit IgG for 2 h at 37°C, followed by washing with wash buffer. Sequential incubations were then performed with biotinylated donkey anti-rabbit IgG HRP-conjugated antibody for 1 h at 37°C. After further washing, colour development was initiated by the addition of substrate, was allowed to develop for up to 30 min at room temperature, was stopped by the addition of 0.18 M sulphuric acid and was read at 450 nm on a microplate reader (MR 5000; Dynatech, France). A standard curve was constructed by incubating a serial dilution of nitro-BSA in PBS pH 7.4 (5.691.10 ng/ml). The concentration of nitrated proteins was estimated from the standard curve and expressed as nitro-BSA equivalent.
To eliminate the influence of plasma storage on nitrosothiol and nitrotyrosine levels, fresh plasma from healthy subjects and HD patients was evaluated and compared with that observed after storage. Mean plasma levels of nitrosothiols and nitrotyrosine were of the same magnitude in fresh and stored samples (data not shown).
Plasma levels of lipoperoxidation products, e.g. thiobarbituric acid-reactive substances (TBARS) were measured by fluorimetry, using a commercial kit from Sobioda (Grenoble, France) and those of protein oxidation, e.g. carbonyls and advanced oxidation protein products (AOPP), by spectrophotometry, as described previously [19]. Plasma ascorbate, the reduced form of vitamin C, was measured by spectrophotometry using ascorbate oxidase [20]. GSH-Px and glutathione reductase (GSSG-Red) activities were measured both in plasma and in erythrocytes, as described previously [21]. Red blood cell (RBC) enzyme activities were expressed as micromoles of NADPH oxidized per minute and per gram of haemoglobin (IU/g Hb). Erythrocyte glutathione content was simultaneously determined as reduced (GSH) and oxidized (GSSG) form by HPLC method, as reported previously [20]. The intracellular redox potential was evaluated by GSSG/GSH ratio, GSSG being expressed as GSH equivalent (one mole of GSSG corresponding to two equivalents GSH).
Statistical analysis
Data have been expressed as mean±SD. The MannWhitney U test was used to compare means between HD patients and controls. Simple regression analysis and Spearman correlation coefficient were used to determine relationships between parameters. Independent associations between one dependent variable and more than two independent variables were assessed by multiple regression analysis.
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Results |
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Relationship with levels of thiol substances, markers of oxidative stress and markers of inflammation
Using regression analysis, no relationship was observed between plasma S-nitrosothiol levels and serum albumin (35.9±4.0 g/l, mean±SD) or plasma homocysteine (30.3±13.8 µmol/l) levels in HD patients. An inverse relationship was found between plasma S-nitrosothiol levels and blood haemoglobin (10.1±1.2 g/dl) concentrations in HD patients (r=-0.58, P<0.005).
Table 3 shows oxidative stress markers and antioxidant parameters in the study population. Although HD patients exhibited a combination between an increase in oxidative stress markers and a profound deficiency in antioxidant systems, we did not observe a direct relationship between plasma S-nitrosothiol levels and any of these markers. However, when HD patients were subdivided into two groups based on the upper limit of normal plasma S-nitrosothiol levels (i.e. 1.57 µmol/l), those patients with elevated plasma S-nitrosothiol levels (n=16) had lower red blood cell reduced glutathione levels than those with normal plasma S-nitrosothiol levels (2.66±1.31 vs 4.16±1.46 µmol/g Hb, P<0.05, respectively). Finally, we found no relationship between plasma S-nitrosothiol and hsCRP (17.4±38.0 mg/l) levels or Kt/V urea index in HD patients.
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Discussion |
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The observed elevation of plasma S-nitrosothiol concentrations clearly points to the presence of circulating NO metabolites presenting a potentially biological activity. Previous reports have suggested the presence of high levels of unreactive nitrate and nitrite ions, but these findings have been criticized because of limitations of the ion-detection methods used [9,12]. Collectively, these data argue against a quantitative NO deficiency in HD patients.
The cause of the elevation of plasma S-nitrosothiols cannot be fully delineated in the present study. We found a negative correlation between S-nitrosothiols and haemoglobin. Data obtained in vitro suggest that haemoglobin can scavenge the NO generated from S-nitrosothiols [22], and S-nitrosohaemoglobin may also act as a reservoir of NO [23]. Indeed, a slight elevation of predialysis S-nitrosohaemoglobin concentration has been observed in HD patients [23]. However, in the present study HD patients, who had normal Hb levels (i.e. 11 g/dl) also had high plasma S-nitrosothiol levels (1.81±0.94 µmol/l). Notably, plasma S-nitrosothiol levels were slightly, but not significantly, different from those of HD patients who had Hb levels <11 g/dl (2.51±1.23 µmol/l). Moreover, in another, non-uraemic control group with low haemoglobin levels (9.5±0.9 g/dl, n=8 subjects) due to different conditions not related to ESRD, we found only a slight elevation of S-nitrosothiol concentrations (0.75±0.1 µmol/l) (our unpublished results). Therefore modification of the haemoglobin levels appears to exert only a partial influence on the level of S-nitrosothiols, and other factors could be responsible in HD patients as well.
Increased production of NO under the influence of inflammatory stimuli is a possible mechanism, since uraemia can be considered as an inflammatory state [20,24]. However, we observed no relationship between plasma S-nitrosothiol and hsCRP levels. An accumulation of sulph-hydryl amino acids, such as homocysteine and cysteine, has been demonstrated in ESRD patients [25,26]. However, S-nitrosothiol compounds in control plasma are present mainly as S-nitrosoalbumin [13]. Therefore it is not surprising that there was no correlation between S-nitrosothiols and homocysteine in the present study. The elevation of plasma S-nitrosothiol levels may also be related to abnormal breakdown, since HD patients have a deficit in ascorbate and low levels of plasma glutathione peroxidase activity, which is responsible for the release of NO from S-nitrosothiols [7,15]. However, we did not observe a correlation between plasma ascorbate or glutathione peroxidase activity and plasma S-nitrosothiols.
Under the oxidative stress condition encountered in HD patients, NO can react rapidly with superoxide anion to form peroxynitrite, which can attack protein, leading to nitrotyrosine. Moreover, inducible NO synthase can act as NO, as well as a peroxynitrite-producing enzyme, depending on the antioxidant capacity of the microenvironment [27]. Paradoxically, plasma nitrotyrosine levels in HD patients were not increased, compared with controls. Peroxynitrite detoxification via its reaction with thiols (e.g. glutathione) to form S-nitrosothiols could explain the high S-nitrosothiol levels observed in the present study. The observation of normal plasma nitrotyrosine levels, and of a difference of reduced glutathione red-cell levels between patients with normal and those with high plasma S-nitrosothiol levels are in favour of this explanation. Nossuli et al. [28] have demonstrated an increase in S-nitrosoglutathione when peroxynitrite was co-incubated with reduced glutathione.
S-nitrosothiols are considered as a NO reservoir and they are potent vasodilators [14]. However, in view of the marked deficit of ascorbate and the low levels of plasma glutathione peroxidase activity in HD patients, an enhanced vasodilatory function of S-nitrosothiols is questionable. A deficiency of bioactive NO has been found to be associated with arterial thrombosis in animal models, in individuals with endothelial dysfunction, and in patients with a low extracellular glutathione peroxidase activity [7]. The lack of S-nitrosothiol bioavailability might participate in the generation of hypertension and thrombotic events in HD patients, and consequently favour the increased frequency of cardiovascular events. Further studies will be necessary to test whether supplementation with vitamin C or exogenous glutathione peroxidase may correct such a deficiency of bioactive NO, by enhancing the release of NO from S-nitrosothiols, and therefore decrease the frequency of cardiovascular events in these patients. Finally, it remains possible, as shown above, that elevated circulating S-nitrosothiols reflect a protective mechanism against nitrosative stress in HD patients. Again, studies with antioxidants are necessary to verify this hypothesis.
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Notes |
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References |
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