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Increased formation of S-nitrothiols and nitrotyrosine in cirrhotic rats during endotoxemia
Free Radical Biology & Medicine, Vol. 31, No. 6, pp. 790 –798, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
PII S0891-5849(01)00647-5
Original Contribution INCREASED FORMATION OF S-NITROTHIOLS AND NITROTYROSINE IN CIRRHOTIC RATS DURING ENDOTOXEMIA LONE H. OTTESEN,*† DAVID HARRY,* MATTHEW FROST,*‡ SUSAN DAVIES,* KORSA KHAN,* BARRY HALLIWELL,§ and KEVIN MOORE* *Centre for Hepatology, Royal Free and University College Medical School, Royal Free Campus, University College London, London, UK; †Centre for Clinical Pharmacology, Institute of Pharmacology, Aarhus University and University Hospital, Aarhus, Denmark; ‡International Antioxidant Research Centre, Guy’s, King’s and St. Thomas’s School of Biomedical Sciences (M.T.F.), London, UK; and §Department of Biochemistry, National University of Singapore, Kent Ridge Crescent, Singapore (Received 1 March 2001; Accepted 21 June 2001)
Abstract—Plasma S-nitrosothiols are believed to function as a circulating form of nitric oxide that affects both vascular function and platelet aggregation. However, the formation of circulating S-nitrosothiols in relation to acute and chronic disease is largely unknown. Plasma S-nitrosothiols were measured by chemiluminescence in rats with biliary cirrhosis or controls, and the effect of lipopolysaccharide (LPS) on their formation was determined. Plasma S-nitrosothiols were increased in rats with cirrhosis (206 ⫾ 59 nM) compared to controls (51 ⫾ 6 nM, p ⬍ .001). Two hours following injection of LPS (0.5 mg/kg) plasma S-nitrosothiols increased to 108 ⫾ 23 nM in controls (p ⬍ .01) and to 1335 ⫾ 423 nM in cirrhotic rats (p ⬍ .001). The plasma clearance and half-life of S-nitrosoalbumin, the predominant circulating S-nitrosothiol, were similar in control and cirrhotic rats, confirming that the increased plasma concentrations were due to increased synthesis. Because reactive nitrogen species, such as peroxynitrite, may cause the formation of Snitrosothiols in vivo, we determined the levels of nitrotyrosine by gas chromatography/mass spectrometry as an index for these nitrating and nitrosating radicals. Hepatic nitrotyrosine levels were increased at 7.0 ⫾ 1.2 ng/mg in cirrhotic rats compared to controls (2.0 ⫾ 0.2 ng/mg, p ⬍ .01). Hepatic nitrotyrosine levels increased by 2.3-fold and 1.5-fold in control and cirrhotic rats, respectively, at 2 h following injection of LPS (p ⬍ .01). Strong positive staining for nitrotyrosine was shown by immunohistochemistry in all the livers of the rats with cirrhosis. We conclude that there is increased formation of S-nitrosothiols and nitrotyrosine in biliary cirrhosis, and this is markedly upregulated during endotoxemia. © 2001 Elsevier Science Inc. Keywords—S-nitrosothiols, Nitrotyrosine, S-nitrosoalbumin, Half-life, Clearance, Cirrhosis, Bile duct ligation, Experimental study, Endotoxemia, LPS, Free radicals
INTRODUCTION
Lately, increased iNOS expression in the heart of rats with biliary cirrhosis has been shown to play a significant role in cirrhotic cardiomyopathy [5]. We and others have recently shown that there is an increased sensitivity and mortality following the injection of lipopolysaccharide (LPS), a component of the outer membrane of gramnegative bacteria, with a 40-fold decrease in the LD50 in rats with biliary cirrhosis [6,7]. Mechanisms that have been proposed to enhance the sensitivity and mortality to LPS in cirrhosis include an upregulated cytokine response, increased lipid peroxidation, induction of pulmonary intravascular phagocytosis, and increased synthesis of nitric oxide [6,7]. NO reacts in biological systems with oxygen, superoxide, and transition metals to form reactive nitrogen
Nitric oxide (NO) is a ubiquitous cell-signaling molecule that has a central role in the regulation of vascular tone, apoptosis, as well as platelet and enzyme functions [1]. There is considerable evidence that there is an upregulation of NO synthesis in chronic liver disease. For example, plasma nitrite/nitrate concentrations are increased, there is increased expression of inducible NO synthase (iNOS) in the arteries, and increased iNOS activity in the liver of rats with biliary cirrhosis [2– 4]. Address correspondence to: Dr. Kevin Moore, Centre for Hepatology, Royal Free & University College Medical School, Royal Free Campus, Rowland Hill St., London NW 3 2PF, UK; Tel: ⫹44 (207) 433-2876; Fax: ⫹44 (207) 433-2877; E-Mail: [email protected]. 790
S-nitrosothiols and nitrotyrosine in cirrhosis and endotoxemia
species that can support nitrosation reactions with nucleophiles such as thiol groups to form S-nitrosothiols, or can lead to the nitration of amino acids such as tyrosine. The S-nitrosation of extracellular proteins, such as albumin, gives rise to a circulating pool of NO, which may release NO directly, or transfer NO into the intracellular millieu via transnitrosation reactions with cell surface thiols, thus affecting vascular and platelet function [8 –11]. The S-nitrosation of intracellular proteins is thought to be critically important in the regulation of oxygen transport by hemoglobin, apoptosis by caspase-3, and proteins involved in cell signaling such as phosphatases [12–14]. Increased formation of nitrotyrosine has been demonstrated in plasma, kidney, and lung during endotoxemia, as well as in chronic inflammatory states such as chronic hepatitis C virus infection and rheumatoid arthritis [15–18]. Whereas the upregulation of NO synthesis and nitration of tyrosine residues in proteins have been demonstrated in a variety of disease processes, very little is known about the formation of S-nitrosothiols in disease. It is well recognized that cirrhosis, as well as sepsis, is causally linked to the development of decreased vascular tone and abnormal platelet function. Because S-nitrosothiols are thought to be important in the regulation of each of these functions we hypothesized that the formation of plasma S-nitrosothiols would be increased in cirrhosis and during endotoxemia. However, until recently, the study of S-nitrosothiols in health and disease has been hampered by an inability to measure these compounds with sufficient sensitivity in biological fluids. We have recently developed a chemiluminescence-based method to measure the concentration of S-nitrosothiols in human plasma [19]. In the present study we report for the first time that plasma S-nitrosothiols, as well as hepatic and plasma levels of nitrotyrosine, are increased in a rat model of biliary cirrhosis, and that the formation of S-nitrosothiols is markedly upregulated during endotoxemia. MATERIALS AND METHODS
Animals Male Sprague Dawley rats (body weight 230 –260 g) were obtained from the Comparative Biology Unit at the Royal Free and University College Medical School (Royal Free Campus, London, UK). Animals were given free access to normal rodent chow and water up until the time of bile duct ligation or subsequent study. A midline abdominal incision was made under general anesthesia. For those undergoing bile duct ligation, the common bile duct was isolated, triply ligated, and cut between the ligatures. Sham procedure involved a similar operation,
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but without ligation or cutting of the bile duct. The overall mortality in both the bile duct ligated and the sham group was less than 10% and usually occurred within 36 h of the operation. All subsequent studies were performed at 22 to 25 d, when biliary cirrhosis had developed in those undergoing bile duct ligation. Histological examination was performed on formalin-fixed liver stained with hematoxylin and eosin. Protocol To investigate the effects of LPS on the formation of S-nitrosothiols in normal and cirrhotic rats, animals were administered LPS (0.5 mg/kg) (Salmonella typhimurium, Sigma Chemical Co., Poole, Dorset, UK) by intraperitoneal injection, and sacrificed 2 h later. This time point was chosen because we have previously observed a significant mortality at 6 h in cirrhotic rats injected with LPS at this dose, but all of the animals survived 2 h [6]. Blood was then withdrawn from the inferior vena cava until full exsanguination into prechilled tubes containing EDTA, and an aliquot was immediately transferred to a tube containing N-ethylmaleimide (final concentration 5 mM) to stabilize the S-nitrosothiols. Following centrifugation at 10,000 ⫻ g for 2 min, plasma S-nitrosothiols were measured as described below. All assays were carried out within 5–10 min of collection. Measurement of plasma S-nitrosothiols S-Nitrosothiols were determined by a copper (II)/ iodine/iodine-mediated cleavage of S-nitrosothiols to NO, which was then quantified by its gas phase chemiluminescent reaction with ozone in a NO analyzer (NOA; Sievers, Boulder, CO, USA) by a method developed in our laboratory [19]. In short, plasma in volumes of 0.3 to 1 ml was rapidly injected into the reaction chamber of the NO analyzer after removal of background nitrite by addition of 0.5% sulphanilamide in 0.1 M hydrochloric acid. The reaction chamber contained glacial acetic acid, potassium iodide, and copper (II) sulphate, and was kept at 68°C via a water jacket. The solution was constantly purged with nitrogen and used only once per sample. A standard curve was obtained using S-nitrosoalbumin in phosphate-buffered saline with N-ethylmaleimide and sulphanilamide as above. Data collection and analysis were performed using the NOAnalysis software (Sievers). To determine the concentration of low molecular weight S-nitrosothiols such as S-nitrosoglutathione, 100 l of trichloroacetic acid (TCA) (100% w/v) was added to 0.9 ml of N-ethylmaleimide-treated plasma to precipitate proteins, centrifuged at 10,000 ⫻ g for 2 min, and low molecular weight
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S-nitrosothiols measured in the supernatant as described above. Pharmacokinetic studies For these studies a 22 G catheter (PVB Medizin Technik GMBH, Kirchseeon, Germany) was placed in the carotid artery to facilitate rapid and repeated blood sampling. An 18 G cannula was placed in the femoral vein for infusions. Saline was infused at 3 ml/h and the animals were heparinized with 25 U of heparin iv. Pharmacokinetic studies were performed in normal rats and rats with biliary cirrhosis. S-Nitrosoalbumin was synthesized as previously described [19] using human albumin and injected as an intravenous bolus at a dose of 200 nmol/kg. The albumin used to synthesize S-nitrosoalbumin has a single reduced cysteine residue, and although it varies from preparation to preparation, between 55– 75% of the available reduced cysteine residues are Snitrosylated. Blood samples for the measurements of plasma S-nitrosothiol concentrations were taken at times 0, 2, 5, 10, 15, 30, and 45 min, and continued up to 70 min. Following each blood sample, an equivalent volume of saline was injected to maintain euvolemia. Pharmacokinetic parameters were calculated using the Graph Pad Prism software. The data were fitted by iterative nonlinear regression analysis to a two compartment model by the equation C(t) ⫽ A e⫺␣t⫹ B e⫺t, where C(t) is the quantitated SNO-albumin concentration at a given time, and A and B represent the postinjection intercepts on the y-axis. The terms ␣ and  are composite rate constants and t is the time after the bolus injection. The plasma clearance, the volume of distribution (Vd), area under curve (AUC), and the half-life (t1/2) were calculated from: Clearance ⫽ Dose/AUC AUC ⫽ A/␣ ⫹ B/ Vd ⫽ Dose/(AUC 䡠 ) t1/2 ⫽ ln2/ Measurement of plasma and hepatic protein nitrotyrosine levels Nitrotyrosine levels were measured by a stable isotope dilution gas chromatography mass spectrometry method following the extraction of tissue or plasma proteins and alkaline hydrolysis [20]. In brief, liver proteins were precipitated from liver tissue that was initially homogenized in 13 ml of chloroform:methanol (2:1) with a blade homogenizer on ice, followed by the addition of 2 ml of saline and further homogenization. Plasma proteins were precipitated in 2 volumes of chloroform:methanol (2:1) [20]. Following centrifugation at
3000 ⫻ g for 30 min, the aqueous and organic layers were removed, and the protein precipitate lyophilized under vacuum overnight. A known amount of the lyophilized protein (⬃ 2 mg) was then hydrolyzed in 1 ml 4 M sodium hydroxide at 120°C overnight, following the addition of 10 ng [C139]-nitrotyrosine and 10 g [D4]tyrosine as internal standards. Alkaline hydrolysis of the protein is preferable to acid hydrolysis, because this avoids artifactual formation of nitrotyrosine by acid and nitrite/nitrate. Samples were then extracted and analyzed by gas chromatography mass spectrometry as previously described [20], following purification by LC18 and ENV⫹ cartridge extraction and derivatization to the heptafluorobutyric amide and tertbutyldimethylsilyl derivatives. This method has an intra-assay and inter-assay variation of less than 5%. All results are expressed in relation to dry weight of protein as measured by direct weighing prior to hydrolysis.
Immunohistochemistry To identify the region of nitration within the liver and kidneys from the bile duct, ligated rat immunohistochemistry was performed. After formalin fixation for 24 h the tissue was routinely processed by the Histopathology Department, Royal Free Hospital in a tissue processor (Miles Scientific, Slough, UK), embedded in paraffin (Bayer Diagnostics, Berkshire, UK) and cut in sections at 4 –5 m to dry overnight. Immunostaining for 3-nitrotyrosine was then achieved by the alkaline phosphatase development method. Optimization of the antibody dilution was achieved after staining of sections using a range of dilutions, 1:10 –1:2000. After sequential de-waxing through graded alcohols the sections were immersed in 15% acetic acid (20 min), washed with distilled water, immersed in 0.1 M sodium citrate buffer (pH 6.0) and, finally, microwaved (Panosonic, 600 W) for 10 min to unmask the antigen. Following incubation of the sections with 0.1% normal goat serum (Dako, Cambridgeshire, UK) for 20 min at room temperature to block nonspecific binding, the sections were incubated with the primary polyclonal antibody (TCS, Buckinghamshire, UK) (diluted 1:500) for 1 h at room temperature. After washing with Tris buffered saline (0.1 M, pH 7.6) the sections were incubated for a further hour at room temperature with the goat anti-rabbit conjugate containing 4% rat serum (Dako). Binding was visualized after washing and incubation with Fast Red for 30 min in distilled water. The Fast Red complex was prepared fresh and filtered immediately before use. The sections nuclei were then counter-stained with Mayers hematoxylin followed by bluing solution (0.5% sodium tetraborate).
S-nitrosothiols and nitrotyrosine in cirrhosis and endotoxemia
Fig. 1. Plasma S-nitrosothiol concentrations before (shaded bars) and after (solid bars) injection of lipopolysaccharide (0.5 mg/kg) (solid bars) in sham operated and rats with biliary cirrhotic (BDL) rats. Results are given as mean ⫾ SEM. *p ⬍ .01 compared to sham. **p ⬍ .001 compared to sham. #p ⬍ .001 compared to BDL.
Statistics The results are presented as mean ⫾ SEM apart from the pharmacokinetic data, which are given as medians with interquartile spans. Statistical analysis was carried out after logarithmic transformation of data. One-way analysis of variance was applied with post hoc paired comparisons done by the Bonferroni procedure using the SPSS 10.0 program. The pharmacokinetic data were analyzed by the Mann-Whitney U test; p values less than .05 were considered statistically significant. RESULTS
Plasma S-nitrosothiol concentrations and the effects of lipopolysaccharide Plasma S-nitrosothiols were present in sham control rats at a concentration of 51 ⫾ 6 nM (n ⫽ 20), and increased significantly following the development of biliary cirrhosis to 206 ⫾ 59 nM (n ⫽ 18, p ⬍ .001). To determine the effect of endotoxemia on the formation of S-nitrosothiols, blood was sampled at 2 h following the injection of LPS. Plasma concentrations of S-nitrosothiols increased from 51 ⫾ 6 nM to 108 ⫾ 23 nM, n ⫽ 11 in controls (p ⬍ .01). However, there was a marked increased in the concentration of plasma S-nitrosothiols in rats with biliary cirrhosis following injection of LPS from 206 ⫾ 59 nM to 1335 ⫾ 423 nM (n ⫽ 17, p ⬍ .001) (Fig. 1). The method employed for the measurement of Snitrosothiols does not distinguish between low and high
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molecular weight compounds such as S-nitrosoglutathione and S-nitrosoalbumin. Like others, we have previously observed that the concentration of low molecular weight S-nitrosothiols such as S-nitrosoglutathione is less than 5 nM in humans (limit of our assay) [19,21]. To determine whether low molecular weight S-nitrosothiols were detectable in the plasma of cirrhotic rats, plasma proteins were precipitated with TCA, and the supernatant analyzed in six rats with biliary cirrhosis. The concentration of low molecular weight S-nitrosothiols following TCA precipitation was less than 5 nM, whereas the concentration of total S-nitrosothiols was 100 ⫾ 26 nM. To determine whether this pool increased following LPS, five rats with biliary cirrhosis were studied 2 h following injection of LPS. Again, the concentration of low molecular weight S-nitrosothiols was less than 5 nM in the supernatant, and the mean concentration of total S-nitrosothiols was 590 ⫾ 220 nM.
The distribution and elimination of S-nitroso-albumin in normal and cirrhotic rats The observation that there is a 4-fold increase in plasma S-nitrosothiols in rats with biliary cirrhosis could be due to either increased synthesis or decreased elimination of endogenously formed S-nitrosothiols. We have previously shown that reaction of plasma with the NO donor papaNONOate forms a high molecular weight protein that has a molecular weight ⬎ 35,000, and binds to sepharoseBlue, which is strongly suggestive that the major species formed in vitro is S-nitrosoalbumin [19]. The observation that all of the measured S-nitrosothiols were precipitable by TCA suggests that the predominant species formed is a high molecular weight compound such as S-nitrosoalbumin. Therefore, the elimination and distribution of S-nitrosoalbumin was studied in normal and cirrhotic rats. Following the bolus injection of 200 nmol/kg S-nitrosoalbumin the plasma concentrations of S-nitrosothiols at 2 min were 2650 ⫾ 370 nM and 2970 ⫾ 1040 nM in the normal and cirrhotic rats, respectively (Fig. 2). As is evident in Fig. 2, the measured concentrations of plasma S-nitrosothiols are described by a two-compartment model of distribution and elimination. The median plasma-clearance of S-nitrosoalbumin was 2.1 ml/min (0.9 –3.7 ml/min) in cirrhotic rats compared to 1.4 ml/min (1.1–1.8 ml/min) in normal animals (ns) These equate to half-lives of 27 min (19 –36 min) in cirrhotics and 29 min (28 –52 min) in normal animals (ns). The volume of distribution was similar in rats with biliary cirrhosis at 85 ml (51–120 ml) compared with normal controls at 84 ml (60 –114 ml) (ns).
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Fig. 2. Elimination of S-nitrosoalbumin in a normal (Œ) and a cirrhotic rat (■) following bolus intravenous injection of 200 nmol/kg S-nitrosoalbumin. There is a decay of plasma concentrations of S-nitrosothiols indicative of a two-compartment model of distribution and elimination.
Plasma and hepatic nitrotyrosine concentrations Nitrotyrosine was present in plasma in normal rats at a concentration of 3.7 ⫾ 0.2 ng/mg dry protein, and increased in rats with biliary cirrhosis (8.2 ⫾ 1.1 ng/mg protein, p ⬍ .001), consistent with increased formation of reactive nitrogen species in cirrhosis. There was no significant change in the plasma levels of protein-bound nitrotyrosine following the injection of LPS (Fig. 3A). Injection of LPS normally causes a rapid influx of inflammatory cells into the liver, therefore nitrotyrosine was measured in the liver of normal rats (2.0 ⫾ 0.2 ng/mg dry weight of liver, n ⫽ 10). There was a marked increase of hepatic nitrotyrosine (7.0 ⫾ 1.2 ng/mg) in rats developing biliary cirrhosis (n ⫽ 10, p ⬍ .01) (Fig. 3B). Following the injection of LPS there was a significant increase of hepatic nitrotyrosine levels in the liver of normal controls (4.6 ⫾ 0.7 vs 2.0 ⫾ 0.2 ng/mg) (n ⫽ 9, p ⬍ .01), and in rats with biliary cirrhosis (10.4 ⫾ 1.8 vs 7.0 ⫾ 1.2 ng/mg) (n ⫽ 11, p ⬍ .01) (Fig. 3B). Liver pathology All bile duct ligated animals had macroscopic evidence of cirrhosis at the time of study. Histology confirmed the diagnosis of cirrhosis or severe fibrosis in all rats with chronic bile duct ligation; cirrhosis being defined as the presence of porto-portal septa with the formation of complete nodules. The presence of necrosis and inflammation was recorded and assessed semi-quantitatively based on individual apoptotic cells and neutrophil infiltrate, to foci of necro-inflammation to areas of coagulative necrosis. All rats with biliary cirrhosis showed evidence of hepatocyte degeneration ranging from vacuolation to individual cell necrosis and mitosis.
Fig. 3. (A) Plasma protein nitrotyrosine concentrations in sham-operated and biliary cirrhotic (BDL) rats before (shaded bars) or 2 h after injection of lipopolysaccharide (0.5 mg/kg) (solid bars). Results are given as mean ⫾ SEM. *p ⬍ .001 compared to sham. (B) Hepatic nitrotyrosine concentrations in sham-operated and biliary cirrhotic (BDL) rats before (shaded bars) or 2 h after injection of lipopolysaccharide (solid bars). Results are given as mean ⫾ SEM. *p ⬍ .01 compared to sham. #p ⬍ .01 compared to BDL.
In the bile duct ligated rats occasional neutrophils were seen in the sinusoids within cirrhotic nodules. Following the injection of LPS there was an increase in the number of neutrophils, which were focally aggregated within sinusoids and occasionally associated with hepatocyte necrosis in the liver of both control and cirrhotic rats. A small proportion of cirrhotic rats showed focal areas of coagulative necrosis with oval cell (stem cell) proliferation peripherally. Immunohistochemistry All of the BDL livers showed strong and diffuse positivity for nitrotyrosine antibody staining. In the ma-
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Fig. 4. The upper panels show liver sections from sham rats before (left upper) and after (right upper) injection of LPS. There is faint cytoplasmic staining of zone 3 hepatocytes (located around *). Following LPS there was a slight increase in zone 3 staining with increased granularity (see arrows right upper panel). In rats with biliary cirrhosis there is strong cytoplasmic positive staining in the hepatocytes (arrows) with relative sparing of the bile ductules (see arrows left lower panel, *indicates no staining in bile ductules). Injection of LPS caused an increase in staining positivity for nitrotyrosine in the neo-ductules (right lower panel arrows) as well as cytoplasmic staining of hepatocytes.
jority this was present within the hepatocyte cytoplasm and in three of eight livers additional cytoplasmaic positivity was found in neo-ductules. In some of the BDL rats occasional large blood vessels showed peri-nuclear positivity within the smooth muscle layer. Specificity was confirmed by the absence of staining by preincubation of the primary antibody with nitrotyrosine as the epitope. In comparison, faint but distinct granular cytoplasmic positivity for NT staining was observed in the hepatocytes in acinar zones 3 in control liver. Following the injection of LPS there was a modest increase in the intensity, but no increase in the distribution of staining in sham controls. By contrast, the BDL rat livers showed persistent strong positivity for NT staining in the hepatocytes, but with an increased staining for NT in the neo-ductules (Fig. 4). DISCUSSION
There have been few studies measuring the concentration of plasma S-nitrosothiols in vivo. In the first study
to report plasma concentrations of S-nitrosothiols, it was estimated that the concentration of S-nitrosoalbumin in human plasma was ⬃ 7 M [21]. However, recently a few groups have independently developed assays for plasma S-nitrosothiols, and reported a range of plasma S-nitrosothiol concentrations in healthy human volunteers from 28 –320 nM [9,19,22–25], with a general consensus that plasma concentrations of S-nitrosothiols are ⬍100 nM, with S-nitrosoglutathione being present at ⬍ 5 nM. The development of assays to measure such low levels will considerably accelerate our understanding of the role of S-nitrosothiols in disease states. Previous studies by Gaston et al. have shown that levels of S-nitrosothiols are decreased in the bronchoalveolar lavage fluid in asthmatic patients [26]. In a recent study, Jourd’heuil et al. observed that plasma S-nitrosothiols increased by ⬎ 3-fold in normal rats 5 h following injection of LPS [27]. This was associated with a 24-fold increase in S-nitrosohemoglobin concentrations, and an 8-fold increase in plasma nitrite/nitrate. The current study extends these observations to include an animal
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model of cirrhosis, in which there is upregulation of NO synthesis and increased formation of superoxide anion by neutrophils, thus providing a setting for the increased formation of reactive nitrogen species, and thereby the S-nitrosation of thiols and the nitration of proteins. Our observation that there is a 2– 4-fold increase in basal concentrations of plasma S-nitrosothiols, as well as increased levels of plasma and hepatic nitrotyrosine in cirrhosis, is consistent with these observations. The mechanisms for the observed increased formation of S-nitrosothiols in this model of biliary cirrhosis, and the marked effect of endotoxemia on the formation of S-nitrosothiols, are unknown. We have previously shown that the activity of nitric oxide synthase (NOS) is increased in the liver of rats with biliary cirrhosis, and this is presumably upregulated further following the injection of LPS [6]. Basal nitrite/nitrate concentrations are increased 2-fold in rats with cirrhosis and increase to a similar extent in both normal and cirrhotic rats at 2 h following the injection of LPS [6]. However, the induction and expression of iNOS normally takes 3– 4 h, whereas the rapid upregulation of plasma S-nitrosothiol concentrations (⬍ 2 h) suggest that NO is derived from other isoforms of NOS. Whilst it has long been recognized that S-nitrosation involves the direct nucleophilic attack of thiol groups by a nitrosonium group (NO⫹), it is still not known what pathway to generate NO⫹ is dominant in vivo. Thus, NO itself may cause S-nitrosation of plasma proteins in the presence of oxygen, presumably by reacting with oxygen to form nitrogen trioxide (N2O3). In this regard, we have recently shown that physiological rates of generation of NO in whole blood can generate physiological concentrations of S-nitrosothiols, and that the rate of S-nitrosothiol formation will increase 4-fold for every doubling of the rate of NO synthesis [28]. There has been much speculation on the relative contribution of peroxynitrite, formed by the reaction of NO with superoxide. Peroxynitrite is known to cause the oxidation or S-nitrosation of thiol groups [28 – 31], as well as nitration of tyrosine-containing proteins. However, Wink and colleagues have shown that cogeneration of superoxide with NO inhibits the S-nitrosation of glutathione in buffer and enhances the capacity to oxididize and presumably nitrate tyrosine residues [29], and we have independently observed that superoxide markedly attenuates the S-nitrosating capacity of NO in human plasma (unpublished observations). With respect to the nitration of tyrosine, endotoxemia would be expected to cause increased formation of both NO and superoxide in the liver. LPS causes upregulation of NO synthesis as well as a rapid infiltration of the liver by activated neutrophils that form superoxide. Superoxide anion reacts with NO to form peroxynitrite, thus nitrating hepatic proteins, although its contribution to the
formation of plasma S-nitrosothiols is difficult to predict [30,31]. Most studies that have tried to semi-quantitate the levels of nitrotyrosine in tissue have relied on immunostaining. This study has measured nitrotyrosine by both a quantitative method (mass spectrometry) as well as by an immunohistochemical method, and similar observations were demonstrated by both techniques. Our observation that the level of nitrotyrosine in the liver is increased in biliary cirrhosis supports the notion that there is increased formation of peroxynitrite or other reactive nitrogen species. Although it is generally assumed that increased formation of nitrotyrosine equates to increased peroxynitrite formation, it is now well recognized that nitrotyrosine can be formed by a variety of pathways. These include the nitration of tyrosine residues by nitryl chloride formed by reaction of nitrite and hypochlorous acid in activated neutrophils, or nitration by nitrite anion formed by oxidation of nitrite by peroxidases in activated neutrophils [32,33]. In this regard it would have been interesting to have carried out a histochemical study for the co-localization of myeloperoxidase and nitrotyrosine immunostaining. However, the data is also consistent with different NO-related pathways being involved in each process (i.e., S-nitrosation or nitration). The rapid increase of hepatic nitrotyrosine is probably secondary to a rapid accumulation of neutrophils in the liver following endotoxin challenge, but whether the nitration of tyrosine occurs through a peroxidase- or peroxynitrite-dependent pathway is unknown. Likewise, it is not known whether the circulating S-nitrosothiols are formed in the liver, and nothing is known about the concentrations of S-nitrosothiols in liver tissue. This is the first study in which the half-life of Snitrosoalbumin has been measured in vivo, and the relatively long terminal half-life of nearly 30 min suggests that S-nitrosoalbumin releases NO slowly. The description of plasma concentration data by a two-compartment model may indicate an initial phase of redistribution of S-nitrosoalbumin within the vasculature and albumin accessible tissue space, followed by a slower phase of metabolism. However, the model describes a sum of those two processes, and the actual contribution of each process at a given time point cannot be deduced from plasma data alone. In addition, the interpretation of these data should be tempered by the fact that we have not shown conclusively that S-nitrosoalbumin is the major circulating S-nitrosothiol in this model. There have been two reports on the metabolism and breakdown of Snitrosothiols in plasma, with low molecular weight thiols being responsible for the breakdown of S-nitrosoalbumin [9,34]. Recently, it was suggested that S-nitrosation of oxyhemoglobin in the lung is followed by release of NO when exposed to low oxygen tension in the microcircu-
S-nitrosothiols and nitrotyrosine in cirrhosis and endotoxemia
lation by further interaction with thiols to dilate blood vessels [35]. The increased formation of S-nitrosothiols seen in cirrhosis and endotoxemia may then be important in the pathogenesis of systemic vasodilatation observed in these disease processes. However, the role of endogenous S-nitrosothiols in the pathogenesis of vascular dysfunction in cirrhosis and endotoxemia remains to be determined. One of the major functions of S-nitrosothiols to emerge recently is their role as potent inhibitors of platelet aggregation. It is well recognized that platelet function is abnormal in cirrhosis, and several studies have shown that S-nitrosoalbumin and other S-nitrosothiols inhibit platelet aggregation. A bolus intravenous injection of S-nitrosoalbumin causes a marked prolongation of the bleeding time in dogs [11]. The anti-platelet effects of S-nitrosoalbumin are concentration dependent, with a dose as low as 10 nmol/kg causing a 50% reduction of platelet aggregation and 50 nmol/kg causing an 85% reduction of platelet aggregation in dogs [11]. The observation that plasma S-nitrosothiols increase markedly in cirrhotic rats with endotoxemia is likely to cause a prolongation of bleeding time, and may explain why there is an increased risk of variceal hemorrhage during infections in cirrhotic patients [36]. In summary, we have shown that experimental cirrhosis is associated with an increased formation of S-nitrosothiols and nitrotyrosine, indicative of increased nitrosative stress in this model of biliary cirrhosis. Endotoxemia causes a modest increased formation of nitrotyrosine, and a marked upregulation of S-nitrososthiol formation. Whether the increased formation of Snitrosothiols observed in rats with biliary cirrhosis following LPS injection contributes to the increased mortality during endotoxemia is unknown. However, further studies on the role of S-nitrosothiols on vascular function and platelet function in cirrhosis are underway. Acknowledgements — This research was supported by The Danish Medical Research Council (#9900809), and The Medical Research Council, UK (Component grant G000290); and MRC Cooperative Group grant (G0002) to the UCL Sepsis Research Group. We are grateful to Dr. Preben Jakobsen, Institute of Pharmacology, Aarhus University, Aarhus, Denmark for pharmacokinetic advice. REFERENCES [1] Grisham, M. B.; Jourd’Heuil, D.; Wink, D. A. Nitric oxide. I. Physiological chemistry of nitric oxide and its metabolites: implications in inflammation. Am. J. Physiol. 276:G315–G321; 1999. [2] Morales-Ruiz, M.; Jimenez, W.; Perez-Sala, D.; Ros, J.; Leivas, A.; Lamas, S.; Rivera, F.; Arroyo, V. Increased nitric oxide synthase expression in arterial vessels of cirrhotic rats with ascites. Hepatology 24:1481–1486; 1996. [3] Marley, R.; Holt, S.; Fernando, B.; Harry, D.; Anand, R.; Goodier, D.; Davies, S.; Moore, K. Lipoic acid prevents development of the hyperdynamic circulation in anesthetized rats with biliary cirrhosis. Hepatology 29:1358 –1363; 1999.
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[4] Heller, J.; Sogni, P.; Barriere, E.; Tazi, K. A.; Chauvelot-Moachon, L.; Guimont, M. C.; Bories, P. N.; Poirel, O.; Moreau, R.; Lebrec, D. Effects of lipopolysaccharide on TNF-alpha production, hepatic NOS2 activity, and hepatic toxicity in rats with cirrhosis. J. Hepatol. 33:376 –381; 2000. [5] Liu, H.; Ma, Z.; Lee, S. S. Contribution of nitric oxide to the pathogenesis of cirrhotic cardiomyopathy in bile duct-ligated rats. Gastroenterology 118:937–944; 2000. [6] Harry, D.; Anand, R.; Holt, S.; Davies, S.; Marley, R.; Fernando, B.; Goodier, D.; Moore, K. Increased sensitivity to endotoxemia in the bile duct-ligated cirrhotic rat. Hepatology 30:1198 –1205; 1999. [7] Chang, S. W.; Ohara, N. Chronic biliary obstruction induces pulmonary intravascular phagocytosis and endotoxin sensitivity in rats. J. Clin. Invest. 94:2009 –2019; 1994. [8] Kelm, M. Nitric oxide metabolism and breakdown. Biochim. Biophys. Acta 1411:273–289; 1999. [9] Jourd’Heuil, D.; Hallen, K.; Feelisch, M.; Grisham, M. B. Dynamic state of S-nitrosothiols in human plasma and whole blood. Free Radic. Biol. Med. 28:409 – 417; 2000. [10] Keaney, J. F. Jr.; Simon, D. I.; Stamler, J. S.; Jaraki, O.; Scharfstein, J.; Vita, J. A.; Loscalzo, J. NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties. J. Clin. Invest. 91:1582–1589; 1993. [11] Simon, D. I.; Stamler, J. S.; Jaraki, O.; Keaney, J. F.; Osborne, J. A.; Francis, S. A.; Singel, D. J.; Loscalzo, J. Antiplatelet properties of protein S-nitrosothiols derived from nitric oxide and endothelium-derived relaxing factor. Arterioscler. Thromb. 13: 791–799; 1993. [12] Bonaventura, C.; Ferruzzi, G.; Tesh, S.; Stevens, R. D. Effects of S-nitrosation on oxygen binding by normal and sickle cell hemoglobin. J. Biol. Chem. 274:24742–24748; 1999. [13] Mohr, S.; Zech, B.; Lapetina, E. G.; Brune, B. Inhibition of caspase-3 by S-nitrosation and oxidation caused by nitric oxide. Biochem. Biophys. Res. Commun. 238:387–391; 1997. [14] Caselli, A.; Chiarugi, P.; Camici, G.; Manao, G.; Ramponi, G. In vivo inactivation of phosphotyrosine protein phosphatases by nitric oxide. FEBS Lett. 374:249 –252; 1995. [15] Wizemann, T. M.; Gardner, C. R.; Laskin, J. D.; Quinones, S.; Durham, S. K.; Goller, N. L.; Ohnishi, S. T.; Laskin, D. L. Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia. J. Leukoc. Biol. 56:759 –768; 1994. [16] Bian, K.; Davis, K.; Kuret, J.; Binder, L.; Murad, F. Nitrotyrosine formation with endotoxin-induced kidney injury detected by immunohistochemistry. Am. J. Physiol. 277:F33–F40; 1999. [17] Garcia-Monzon, C.; Majano, P. L.; Zubia, I.; Sanz, P.; Apolinario, A.; Moreno-Otero, R. Intrahepatic accumulation of nitrotyrosine in chronic viral hepatitis is associated with histological severity of liver disease. J. Hepatol. 32:331–338; 2000. [18] Kaur, H.; Halliwell, B. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett. 350:9 – 12; 1994. [19] Marley, R.; Feelisch, M.; Holt, S.; Moore, K. A chemiluminescense-based assay for S-nitrosoalbumin and other plasma Snitrosothiols. Free Radic. Res. 32:1–9; 2000. [20] Frost, M. T.; Halliwell, B.; Moore, K. P. Analysis of free and protein-bound nitrotyrosine in human plasma by a gas chromatography/mass spectrometry method that avoids nitration artifacts. Biochem. J. 345:453– 458; 2000. [21] Stamler, J.; Jaraki, O.; Osborne, J.; Simon, D. I.; Keaney, J.; Vita, J.; Singel, D.; Valeri, C. R.; Loscalzo, J. Nitric oxide circulates in mammalian plasma as an S-nitroso-adduct of serum albumin. Proc. Natl. Acad. Sci. USA 89:7674 –7677; 1992. [22] Tsikas, D.; Sandmann, J.; Gutzki, F. M.; Stichtenoth, D. O.; Frohlich, J. C. 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. J. Chromatogr. B. Biomed. Sci. Appl. 726: 13–24; 1999. [23] Gladwin, M. T.; Shelhamer, J. H.; Schecter, A. N.; Pease-Fye, M.;
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L. H. OTTESEN et al. Waclawiw, M. A.; Panza, J. A.; Ognibene, F. P.; Cannon, R. O. Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans. Proc. Natl. Acad. Sci. USA 97:11482–11487; 2000. Ewing, J. F.; Janero, D. R. Specific S-nitrosothiol qunatification as solution nitrite after vanadium (III) reduction and ozonechemiluminescence detection. Free Radic. Biol. Med. 25:621– 628; 1998. Goldman, R. K.; Vlessis, A. A.; Trunkey, D. D. Nitrosothiol quantification in human plasma. Anal. Biochem. 259:98 –103; 1998. Gaston, B.; Sears, S.; Woods, J.; Hunt, J.; Ponaman, M.; McMahon, T.; Stamler, J. S. Bronchodilator S-nitrosothiol deficiency in asthmatic respiratory failure. Lancet 351:1317–1319; 1998. Jourd’heuil, D.; Gray, L.; Grisham, M. B. S-nitrosothiol formation in blood of lipopolysaccharide-treated rats. Biochem. Biophys. Res. Commun. 273:22–26; 2000. Marley, R.; Patel, R.; Orie, N.; Ceaser, E.; Darley-Usmar, V.; Moore, K. Formation of nanomolar concentrations of S-nitrosoalbumin in human plasma by nitric oxide. Free Radic. Biol. Med. 31:688 – 696; 2001. Wink, D. A.; Cook, J. A.; Kim, S. Y.; Vodovotz, Y.; Pacelli, R.; Krishna, M. C.; Russo, A.; Mitchell, J. B.; Jourd’heuil, D.; Miles, A. M.; Grisham, M. B. Superoxide modulates the oxidation and nitrosation of thiols by NO derived reactive intermediates. J. Biol. Chem. 272:11147–11151; 1997. Koppenol, W. H.; Moreno, J. J.; Pryor, W. A.; Ischiropoulos, H.; Beckman, J. S. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol. 5:834 – 842; 1992.
[31] Scorza, G.; Minetti, M. One-electron oxidation pathway of thiols by peroxynitrite in biological fluids: bicarbonate and ascorbate promote the formation of albumin disulphide dimers in human blood plasma. Biochem. J. 329:405– 413; 1998. [32] Eiserich, J. P.; Hristova, M.; Cross, C. E.; Jones, A. D.; Freeman, B. A.; Halliwell, B.; van der Vliet, A. Formation of nitric oxidederived inflammatory oxidants by myeloperoxidase neutrophils. Nature 391:393–397; 1998. [33] van der Vliet, A.; Eiserich, J. P.; Halliwell, B.; Cross, C. E. Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity. J. Biol. Chem. 272:7617–7625; 1997. [34] Scorza, G.; Pietraforte, D.; Minetti, M. Role of ascorbate and protein thiols in the release of nitric oxide from S-nitroso-albumin and S-nitroso-glutathione in human plasma. Free Radic. Biol. Med. 22:633– 642; 1997. [35] Gow, A. J.; Stamler, J. S. Reactions between nitric oxide and haemoglobin under physiological conditions. Nature 391:169 – 173; 1998. [36] Goulis, J.; Patch, D.; Burroughs, A. K. Bacterial infection in the pathogenesis of variceal bleeding. Lancet 353:139 –142; 1999. ABBREVIATIONS
LPS—lipopolysaccharide NO—nitric oxide TCA—trichloroacetic acid
PII S0891-5849(01)00647-5
Original Contribution INCREASED FORMATION OF S-NITROTHIOLS AND NITROTYROSINE IN CIRRHOTIC RATS DURING ENDOTOXEMIA LONE H. OTTESEN,*† DAVID HARRY,* MATTHEW FROST,*‡ SUSAN DAVIES,* KORSA KHAN,* BARRY HALLIWELL,§ and KEVIN MOORE* *Centre for Hepatology, Royal Free and University College Medical School, Royal Free Campus, University College London, London, UK; †Centre for Clinical Pharmacology, Institute of Pharmacology, Aarhus University and University Hospital, Aarhus, Denmark; ‡International Antioxidant Research Centre, Guy’s, King’s and St. Thomas’s School of Biomedical Sciences (M.T.F.), London, UK; and §Department of Biochemistry, National University of Singapore, Kent Ridge Crescent, Singapore (Received 1 March 2001; Accepted 21 June 2001)
Abstract—Plasma S-nitrosothiols are believed to function as a circulating form of nitric oxide that affects both vascular function and platelet aggregation. However, the formation of circulating S-nitrosothiols in relation to acute and chronic disease is largely unknown. Plasma S-nitrosothiols were measured by chemiluminescence in rats with biliary cirrhosis or controls, and the effect of lipopolysaccharide (LPS) on their formation was determined. Plasma S-nitrosothiols were increased in rats with cirrhosis (206 ⫾ 59 nM) compared to controls (51 ⫾ 6 nM, p ⬍ .001). Two hours following injection of LPS (0.5 mg/kg) plasma S-nitrosothiols increased to 108 ⫾ 23 nM in controls (p ⬍ .01) and to 1335 ⫾ 423 nM in cirrhotic rats (p ⬍ .001). The plasma clearance and half-life of S-nitrosoalbumin, the predominant circulating S-nitrosothiol, were similar in control and cirrhotic rats, confirming that the increased plasma concentrations were due to increased synthesis. Because reactive nitrogen species, such as peroxynitrite, may cause the formation of Snitrosothiols in vivo, we determined the levels of nitrotyrosine by gas chromatography/mass spectrometry as an index for these nitrating and nitrosating radicals. Hepatic nitrotyrosine levels were increased at 7.0 ⫾ 1.2 ng/mg in cirrhotic rats compared to controls (2.0 ⫾ 0.2 ng/mg, p ⬍ .01). Hepatic nitrotyrosine levels increased by 2.3-fold and 1.5-fold in control and cirrhotic rats, respectively, at 2 h following injection of LPS (p ⬍ .01). Strong positive staining for nitrotyrosine was shown by immunohistochemistry in all the livers of the rats with cirrhosis. We conclude that there is increased formation of S-nitrosothiols and nitrotyrosine in biliary cirrhosis, and this is markedly upregulated during endotoxemia. © 2001 Elsevier Science Inc. Keywords—S-nitrosothiols, Nitrotyrosine, S-nitrosoalbumin, Half-life, Clearance, Cirrhosis, Bile duct ligation, Experimental study, Endotoxemia, LPS, Free radicals
INTRODUCTION
Lately, increased iNOS expression in the heart of rats with biliary cirrhosis has been shown to play a significant role in cirrhotic cardiomyopathy [5]. We and others have recently shown that there is an increased sensitivity and mortality following the injection of lipopolysaccharide (LPS), a component of the outer membrane of gramnegative bacteria, with a 40-fold decrease in the LD50 in rats with biliary cirrhosis [6,7]. Mechanisms that have been proposed to enhance the sensitivity and mortality to LPS in cirrhosis include an upregulated cytokine response, increased lipid peroxidation, induction of pulmonary intravascular phagocytosis, and increased synthesis of nitric oxide [6,7]. NO reacts in biological systems with oxygen, superoxide, and transition metals to form reactive nitrogen
Nitric oxide (NO) is a ubiquitous cell-signaling molecule that has a central role in the regulation of vascular tone, apoptosis, as well as platelet and enzyme functions [1]. There is considerable evidence that there is an upregulation of NO synthesis in chronic liver disease. For example, plasma nitrite/nitrate concentrations are increased, there is increased expression of inducible NO synthase (iNOS) in the arteries, and increased iNOS activity in the liver of rats with biliary cirrhosis [2– 4]. Address correspondence to: Dr. Kevin Moore, Centre for Hepatology, Royal Free & University College Medical School, Royal Free Campus, Rowland Hill St., London NW 3 2PF, UK; Tel: ⫹44 (207) 433-2876; Fax: ⫹44 (207) 433-2877; E-Mail: [email protected]. 790
S-nitrosothiols and nitrotyrosine in cirrhosis and endotoxemia
species that can support nitrosation reactions with nucleophiles such as thiol groups to form S-nitrosothiols, or can lead to the nitration of amino acids such as tyrosine. The S-nitrosation of extracellular proteins, such as albumin, gives rise to a circulating pool of NO, which may release NO directly, or transfer NO into the intracellular millieu via transnitrosation reactions with cell surface thiols, thus affecting vascular and platelet function [8 –11]. The S-nitrosation of intracellular proteins is thought to be critically important in the regulation of oxygen transport by hemoglobin, apoptosis by caspase-3, and proteins involved in cell signaling such as phosphatases [12–14]. Increased formation of nitrotyrosine has been demonstrated in plasma, kidney, and lung during endotoxemia, as well as in chronic inflammatory states such as chronic hepatitis C virus infection and rheumatoid arthritis [15–18]. Whereas the upregulation of NO synthesis and nitration of tyrosine residues in proteins have been demonstrated in a variety of disease processes, very little is known about the formation of S-nitrosothiols in disease. It is well recognized that cirrhosis, as well as sepsis, is causally linked to the development of decreased vascular tone and abnormal platelet function. Because S-nitrosothiols are thought to be important in the regulation of each of these functions we hypothesized that the formation of plasma S-nitrosothiols would be increased in cirrhosis and during endotoxemia. However, until recently, the study of S-nitrosothiols in health and disease has been hampered by an inability to measure these compounds with sufficient sensitivity in biological fluids. We have recently developed a chemiluminescence-based method to measure the concentration of S-nitrosothiols in human plasma [19]. In the present study we report for the first time that plasma S-nitrosothiols, as well as hepatic and plasma levels of nitrotyrosine, are increased in a rat model of biliary cirrhosis, and that the formation of S-nitrosothiols is markedly upregulated during endotoxemia. MATERIALS AND METHODS
Animals Male Sprague Dawley rats (body weight 230 –260 g) were obtained from the Comparative Biology Unit at the Royal Free and University College Medical School (Royal Free Campus, London, UK). Animals were given free access to normal rodent chow and water up until the time of bile duct ligation or subsequent study. A midline abdominal incision was made under general anesthesia. For those undergoing bile duct ligation, the common bile duct was isolated, triply ligated, and cut between the ligatures. Sham procedure involved a similar operation,
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but without ligation or cutting of the bile duct. The overall mortality in both the bile duct ligated and the sham group was less than 10% and usually occurred within 36 h of the operation. All subsequent studies were performed at 22 to 25 d, when biliary cirrhosis had developed in those undergoing bile duct ligation. Histological examination was performed on formalin-fixed liver stained with hematoxylin and eosin. Protocol To investigate the effects of LPS on the formation of S-nitrosothiols in normal and cirrhotic rats, animals were administered LPS (0.5 mg/kg) (Salmonella typhimurium, Sigma Chemical Co., Poole, Dorset, UK) by intraperitoneal injection, and sacrificed 2 h later. This time point was chosen because we have previously observed a significant mortality at 6 h in cirrhotic rats injected with LPS at this dose, but all of the animals survived 2 h [6]. Blood was then withdrawn from the inferior vena cava until full exsanguination into prechilled tubes containing EDTA, and an aliquot was immediately transferred to a tube containing N-ethylmaleimide (final concentration 5 mM) to stabilize the S-nitrosothiols. Following centrifugation at 10,000 ⫻ g for 2 min, plasma S-nitrosothiols were measured as described below. All assays were carried out within 5–10 min of collection. Measurement of plasma S-nitrosothiols S-Nitrosothiols were determined by a copper (II)/ iodine/iodine-mediated cleavage of S-nitrosothiols to NO, which was then quantified by its gas phase chemiluminescent reaction with ozone in a NO analyzer (NOA; Sievers, Boulder, CO, USA) by a method developed in our laboratory [19]. In short, plasma in volumes of 0.3 to 1 ml was rapidly injected into the reaction chamber of the NO analyzer after removal of background nitrite by addition of 0.5% sulphanilamide in 0.1 M hydrochloric acid. The reaction chamber contained glacial acetic acid, potassium iodide, and copper (II) sulphate, and was kept at 68°C via a water jacket. The solution was constantly purged with nitrogen and used only once per sample. A standard curve was obtained using S-nitrosoalbumin in phosphate-buffered saline with N-ethylmaleimide and sulphanilamide as above. Data collection and analysis were performed using the NOAnalysis software (Sievers). To determine the concentration of low molecular weight S-nitrosothiols such as S-nitrosoglutathione, 100 l of trichloroacetic acid (TCA) (100% w/v) was added to 0.9 ml of N-ethylmaleimide-treated plasma to precipitate proteins, centrifuged at 10,000 ⫻ g for 2 min, and low molecular weight
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S-nitrosothiols measured in the supernatant as described above. Pharmacokinetic studies For these studies a 22 G catheter (PVB Medizin Technik GMBH, Kirchseeon, Germany) was placed in the carotid artery to facilitate rapid and repeated blood sampling. An 18 G cannula was placed in the femoral vein for infusions. Saline was infused at 3 ml/h and the animals were heparinized with 25 U of heparin iv. Pharmacokinetic studies were performed in normal rats and rats with biliary cirrhosis. S-Nitrosoalbumin was synthesized as previously described [19] using human albumin and injected as an intravenous bolus at a dose of 200 nmol/kg. The albumin used to synthesize S-nitrosoalbumin has a single reduced cysteine residue, and although it varies from preparation to preparation, between 55– 75% of the available reduced cysteine residues are Snitrosylated. Blood samples for the measurements of plasma S-nitrosothiol concentrations were taken at times 0, 2, 5, 10, 15, 30, and 45 min, and continued up to 70 min. Following each blood sample, an equivalent volume of saline was injected to maintain euvolemia. Pharmacokinetic parameters were calculated using the Graph Pad Prism software. The data were fitted by iterative nonlinear regression analysis to a two compartment model by the equation C(t) ⫽ A e⫺␣t⫹ B e⫺t, where C(t) is the quantitated SNO-albumin concentration at a given time, and A and B represent the postinjection intercepts on the y-axis. The terms ␣ and  are composite rate constants and t is the time after the bolus injection. The plasma clearance, the volume of distribution (Vd), area under curve (AUC), and the half-life (t1/2) were calculated from: Clearance ⫽ Dose/AUC AUC ⫽ A/␣ ⫹ B/ Vd ⫽ Dose/(AUC 䡠 ) t1/2 ⫽ ln2/ Measurement of plasma and hepatic protein nitrotyrosine levels Nitrotyrosine levels were measured by a stable isotope dilution gas chromatography mass spectrometry method following the extraction of tissue or plasma proteins and alkaline hydrolysis [20]. In brief, liver proteins were precipitated from liver tissue that was initially homogenized in 13 ml of chloroform:methanol (2:1) with a blade homogenizer on ice, followed by the addition of 2 ml of saline and further homogenization. Plasma proteins were precipitated in 2 volumes of chloroform:methanol (2:1) [20]. Following centrifugation at
3000 ⫻ g for 30 min, the aqueous and organic layers were removed, and the protein precipitate lyophilized under vacuum overnight. A known amount of the lyophilized protein (⬃ 2 mg) was then hydrolyzed in 1 ml 4 M sodium hydroxide at 120°C overnight, following the addition of 10 ng [C139]-nitrotyrosine and 10 g [D4]tyrosine as internal standards. Alkaline hydrolysis of the protein is preferable to acid hydrolysis, because this avoids artifactual formation of nitrotyrosine by acid and nitrite/nitrate. Samples were then extracted and analyzed by gas chromatography mass spectrometry as previously described [20], following purification by LC18 and ENV⫹ cartridge extraction and derivatization to the heptafluorobutyric amide and tertbutyldimethylsilyl derivatives. This method has an intra-assay and inter-assay variation of less than 5%. All results are expressed in relation to dry weight of protein as measured by direct weighing prior to hydrolysis.
Immunohistochemistry To identify the region of nitration within the liver and kidneys from the bile duct, ligated rat immunohistochemistry was performed. After formalin fixation for 24 h the tissue was routinely processed by the Histopathology Department, Royal Free Hospital in a tissue processor (Miles Scientific, Slough, UK), embedded in paraffin (Bayer Diagnostics, Berkshire, UK) and cut in sections at 4 –5 m to dry overnight. Immunostaining for 3-nitrotyrosine was then achieved by the alkaline phosphatase development method. Optimization of the antibody dilution was achieved after staining of sections using a range of dilutions, 1:10 –1:2000. After sequential de-waxing through graded alcohols the sections were immersed in 15% acetic acid (20 min), washed with distilled water, immersed in 0.1 M sodium citrate buffer (pH 6.0) and, finally, microwaved (Panosonic, 600 W) for 10 min to unmask the antigen. Following incubation of the sections with 0.1% normal goat serum (Dako, Cambridgeshire, UK) for 20 min at room temperature to block nonspecific binding, the sections were incubated with the primary polyclonal antibody (TCS, Buckinghamshire, UK) (diluted 1:500) for 1 h at room temperature. After washing with Tris buffered saline (0.1 M, pH 7.6) the sections were incubated for a further hour at room temperature with the goat anti-rabbit conjugate containing 4% rat serum (Dako). Binding was visualized after washing and incubation with Fast Red for 30 min in distilled water. The Fast Red complex was prepared fresh and filtered immediately before use. The sections nuclei were then counter-stained with Mayers hematoxylin followed by bluing solution (0.5% sodium tetraborate).
S-nitrosothiols and nitrotyrosine in cirrhosis and endotoxemia
Fig. 1. Plasma S-nitrosothiol concentrations before (shaded bars) and after (solid bars) injection of lipopolysaccharide (0.5 mg/kg) (solid bars) in sham operated and rats with biliary cirrhotic (BDL) rats. Results are given as mean ⫾ SEM. *p ⬍ .01 compared to sham. **p ⬍ .001 compared to sham. #p ⬍ .001 compared to BDL.
Statistics The results are presented as mean ⫾ SEM apart from the pharmacokinetic data, which are given as medians with interquartile spans. Statistical analysis was carried out after logarithmic transformation of data. One-way analysis of variance was applied with post hoc paired comparisons done by the Bonferroni procedure using the SPSS 10.0 program. The pharmacokinetic data were analyzed by the Mann-Whitney U test; p values less than .05 were considered statistically significant. RESULTS
Plasma S-nitrosothiol concentrations and the effects of lipopolysaccharide Plasma S-nitrosothiols were present in sham control rats at a concentration of 51 ⫾ 6 nM (n ⫽ 20), and increased significantly following the development of biliary cirrhosis to 206 ⫾ 59 nM (n ⫽ 18, p ⬍ .001). To determine the effect of endotoxemia on the formation of S-nitrosothiols, blood was sampled at 2 h following the injection of LPS. Plasma concentrations of S-nitrosothiols increased from 51 ⫾ 6 nM to 108 ⫾ 23 nM, n ⫽ 11 in controls (p ⬍ .01). However, there was a marked increased in the concentration of plasma S-nitrosothiols in rats with biliary cirrhosis following injection of LPS from 206 ⫾ 59 nM to 1335 ⫾ 423 nM (n ⫽ 17, p ⬍ .001) (Fig. 1). The method employed for the measurement of Snitrosothiols does not distinguish between low and high
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molecular weight compounds such as S-nitrosoglutathione and S-nitrosoalbumin. Like others, we have previously observed that the concentration of low molecular weight S-nitrosothiols such as S-nitrosoglutathione is less than 5 nM in humans (limit of our assay) [19,21]. To determine whether low molecular weight S-nitrosothiols were detectable in the plasma of cirrhotic rats, plasma proteins were precipitated with TCA, and the supernatant analyzed in six rats with biliary cirrhosis. The concentration of low molecular weight S-nitrosothiols following TCA precipitation was less than 5 nM, whereas the concentration of total S-nitrosothiols was 100 ⫾ 26 nM. To determine whether this pool increased following LPS, five rats with biliary cirrhosis were studied 2 h following injection of LPS. Again, the concentration of low molecular weight S-nitrosothiols was less than 5 nM in the supernatant, and the mean concentration of total S-nitrosothiols was 590 ⫾ 220 nM.
The distribution and elimination of S-nitroso-albumin in normal and cirrhotic rats The observation that there is a 4-fold increase in plasma S-nitrosothiols in rats with biliary cirrhosis could be due to either increased synthesis or decreased elimination of endogenously formed S-nitrosothiols. We have previously shown that reaction of plasma with the NO donor papaNONOate forms a high molecular weight protein that has a molecular weight ⬎ 35,000, and binds to sepharoseBlue, which is strongly suggestive that the major species formed in vitro is S-nitrosoalbumin [19]. The observation that all of the measured S-nitrosothiols were precipitable by TCA suggests that the predominant species formed is a high molecular weight compound such as S-nitrosoalbumin. Therefore, the elimination and distribution of S-nitrosoalbumin was studied in normal and cirrhotic rats. Following the bolus injection of 200 nmol/kg S-nitrosoalbumin the plasma concentrations of S-nitrosothiols at 2 min were 2650 ⫾ 370 nM and 2970 ⫾ 1040 nM in the normal and cirrhotic rats, respectively (Fig. 2). As is evident in Fig. 2, the measured concentrations of plasma S-nitrosothiols are described by a two-compartment model of distribution and elimination. The median plasma-clearance of S-nitrosoalbumin was 2.1 ml/min (0.9 –3.7 ml/min) in cirrhotic rats compared to 1.4 ml/min (1.1–1.8 ml/min) in normal animals (ns) These equate to half-lives of 27 min (19 –36 min) in cirrhotics and 29 min (28 –52 min) in normal animals (ns). The volume of distribution was similar in rats with biliary cirrhosis at 85 ml (51–120 ml) compared with normal controls at 84 ml (60 –114 ml) (ns).
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Fig. 2. Elimination of S-nitrosoalbumin in a normal (Œ) and a cirrhotic rat (■) following bolus intravenous injection of 200 nmol/kg S-nitrosoalbumin. There is a decay of plasma concentrations of S-nitrosothiols indicative of a two-compartment model of distribution and elimination.
Plasma and hepatic nitrotyrosine concentrations Nitrotyrosine was present in plasma in normal rats at a concentration of 3.7 ⫾ 0.2 ng/mg dry protein, and increased in rats with biliary cirrhosis (8.2 ⫾ 1.1 ng/mg protein, p ⬍ .001), consistent with increased formation of reactive nitrogen species in cirrhosis. There was no significant change in the plasma levels of protein-bound nitrotyrosine following the injection of LPS (Fig. 3A). Injection of LPS normally causes a rapid influx of inflammatory cells into the liver, therefore nitrotyrosine was measured in the liver of normal rats (2.0 ⫾ 0.2 ng/mg dry weight of liver, n ⫽ 10). There was a marked increase of hepatic nitrotyrosine (7.0 ⫾ 1.2 ng/mg) in rats developing biliary cirrhosis (n ⫽ 10, p ⬍ .01) (Fig. 3B). Following the injection of LPS there was a significant increase of hepatic nitrotyrosine levels in the liver of normal controls (4.6 ⫾ 0.7 vs 2.0 ⫾ 0.2 ng/mg) (n ⫽ 9, p ⬍ .01), and in rats with biliary cirrhosis (10.4 ⫾ 1.8 vs 7.0 ⫾ 1.2 ng/mg) (n ⫽ 11, p ⬍ .01) (Fig. 3B). Liver pathology All bile duct ligated animals had macroscopic evidence of cirrhosis at the time of study. Histology confirmed the diagnosis of cirrhosis or severe fibrosis in all rats with chronic bile duct ligation; cirrhosis being defined as the presence of porto-portal septa with the formation of complete nodules. The presence of necrosis and inflammation was recorded and assessed semi-quantitatively based on individual apoptotic cells and neutrophil infiltrate, to foci of necro-inflammation to areas of coagulative necrosis. All rats with biliary cirrhosis showed evidence of hepatocyte degeneration ranging from vacuolation to individual cell necrosis and mitosis.
Fig. 3. (A) Plasma protein nitrotyrosine concentrations in sham-operated and biliary cirrhotic (BDL) rats before (shaded bars) or 2 h after injection of lipopolysaccharide (0.5 mg/kg) (solid bars). Results are given as mean ⫾ SEM. *p ⬍ .001 compared to sham. (B) Hepatic nitrotyrosine concentrations in sham-operated and biliary cirrhotic (BDL) rats before (shaded bars) or 2 h after injection of lipopolysaccharide (solid bars). Results are given as mean ⫾ SEM. *p ⬍ .01 compared to sham. #p ⬍ .01 compared to BDL.
In the bile duct ligated rats occasional neutrophils were seen in the sinusoids within cirrhotic nodules. Following the injection of LPS there was an increase in the number of neutrophils, which were focally aggregated within sinusoids and occasionally associated with hepatocyte necrosis in the liver of both control and cirrhotic rats. A small proportion of cirrhotic rats showed focal areas of coagulative necrosis with oval cell (stem cell) proliferation peripherally. Immunohistochemistry All of the BDL livers showed strong and diffuse positivity for nitrotyrosine antibody staining. In the ma-
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Fig. 4. The upper panels show liver sections from sham rats before (left upper) and after (right upper) injection of LPS. There is faint cytoplasmic staining of zone 3 hepatocytes (located around *). Following LPS there was a slight increase in zone 3 staining with increased granularity (see arrows right upper panel). In rats with biliary cirrhosis there is strong cytoplasmic positive staining in the hepatocytes (arrows) with relative sparing of the bile ductules (see arrows left lower panel, *indicates no staining in bile ductules). Injection of LPS caused an increase in staining positivity for nitrotyrosine in the neo-ductules (right lower panel arrows) as well as cytoplasmic staining of hepatocytes.
jority this was present within the hepatocyte cytoplasm and in three of eight livers additional cytoplasmaic positivity was found in neo-ductules. In some of the BDL rats occasional large blood vessels showed peri-nuclear positivity within the smooth muscle layer. Specificity was confirmed by the absence of staining by preincubation of the primary antibody with nitrotyrosine as the epitope. In comparison, faint but distinct granular cytoplasmic positivity for NT staining was observed in the hepatocytes in acinar zones 3 in control liver. Following the injection of LPS there was a modest increase in the intensity, but no increase in the distribution of staining in sham controls. By contrast, the BDL rat livers showed persistent strong positivity for NT staining in the hepatocytes, but with an increased staining for NT in the neo-ductules (Fig. 4). DISCUSSION
There have been few studies measuring the concentration of plasma S-nitrosothiols in vivo. In the first study
to report plasma concentrations of S-nitrosothiols, it was estimated that the concentration of S-nitrosoalbumin in human plasma was ⬃ 7 M [21]. However, recently a few groups have independently developed assays for plasma S-nitrosothiols, and reported a range of plasma S-nitrosothiol concentrations in healthy human volunteers from 28 –320 nM [9,19,22–25], with a general consensus that plasma concentrations of S-nitrosothiols are ⬍100 nM, with S-nitrosoglutathione being present at ⬍ 5 nM. The development of assays to measure such low levels will considerably accelerate our understanding of the role of S-nitrosothiols in disease states. Previous studies by Gaston et al. have shown that levels of S-nitrosothiols are decreased in the bronchoalveolar lavage fluid in asthmatic patients [26]. In a recent study, Jourd’heuil et al. observed that plasma S-nitrosothiols increased by ⬎ 3-fold in normal rats 5 h following injection of LPS [27]. This was associated with a 24-fold increase in S-nitrosohemoglobin concentrations, and an 8-fold increase in plasma nitrite/nitrate. The current study extends these observations to include an animal
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model of cirrhosis, in which there is upregulation of NO synthesis and increased formation of superoxide anion by neutrophils, thus providing a setting for the increased formation of reactive nitrogen species, and thereby the S-nitrosation of thiols and the nitration of proteins. Our observation that there is a 2– 4-fold increase in basal concentrations of plasma S-nitrosothiols, as well as increased levels of plasma and hepatic nitrotyrosine in cirrhosis, is consistent with these observations. The mechanisms for the observed increased formation of S-nitrosothiols in this model of biliary cirrhosis, and the marked effect of endotoxemia on the formation of S-nitrosothiols, are unknown. We have previously shown that the activity of nitric oxide synthase (NOS) is increased in the liver of rats with biliary cirrhosis, and this is presumably upregulated further following the injection of LPS [6]. Basal nitrite/nitrate concentrations are increased 2-fold in rats with cirrhosis and increase to a similar extent in both normal and cirrhotic rats at 2 h following the injection of LPS [6]. However, the induction and expression of iNOS normally takes 3– 4 h, whereas the rapid upregulation of plasma S-nitrosothiol concentrations (⬍ 2 h) suggest that NO is derived from other isoforms of NOS. Whilst it has long been recognized that S-nitrosation involves the direct nucleophilic attack of thiol groups by a nitrosonium group (NO⫹), it is still not known what pathway to generate NO⫹ is dominant in vivo. Thus, NO itself may cause S-nitrosation of plasma proteins in the presence of oxygen, presumably by reacting with oxygen to form nitrogen trioxide (N2O3). In this regard, we have recently shown that physiological rates of generation of NO in whole blood can generate physiological concentrations of S-nitrosothiols, and that the rate of S-nitrosothiol formation will increase 4-fold for every doubling of the rate of NO synthesis [28]. There has been much speculation on the relative contribution of peroxynitrite, formed by the reaction of NO with superoxide. Peroxynitrite is known to cause the oxidation or S-nitrosation of thiol groups [28 – 31], as well as nitration of tyrosine-containing proteins. However, Wink and colleagues have shown that cogeneration of superoxide with NO inhibits the S-nitrosation of glutathione in buffer and enhances the capacity to oxididize and presumably nitrate tyrosine residues [29], and we have independently observed that superoxide markedly attenuates the S-nitrosating capacity of NO in human plasma (unpublished observations). With respect to the nitration of tyrosine, endotoxemia would be expected to cause increased formation of both NO and superoxide in the liver. LPS causes upregulation of NO synthesis as well as a rapid infiltration of the liver by activated neutrophils that form superoxide. Superoxide anion reacts with NO to form peroxynitrite, thus nitrating hepatic proteins, although its contribution to the
formation of plasma S-nitrosothiols is difficult to predict [30,31]. Most studies that have tried to semi-quantitate the levels of nitrotyrosine in tissue have relied on immunostaining. This study has measured nitrotyrosine by both a quantitative method (mass spectrometry) as well as by an immunohistochemical method, and similar observations were demonstrated by both techniques. Our observation that the level of nitrotyrosine in the liver is increased in biliary cirrhosis supports the notion that there is increased formation of peroxynitrite or other reactive nitrogen species. Although it is generally assumed that increased formation of nitrotyrosine equates to increased peroxynitrite formation, it is now well recognized that nitrotyrosine can be formed by a variety of pathways. These include the nitration of tyrosine residues by nitryl chloride formed by reaction of nitrite and hypochlorous acid in activated neutrophils, or nitration by nitrite anion formed by oxidation of nitrite by peroxidases in activated neutrophils [32,33]. In this regard it would have been interesting to have carried out a histochemical study for the co-localization of myeloperoxidase and nitrotyrosine immunostaining. However, the data is also consistent with different NO-related pathways being involved in each process (i.e., S-nitrosation or nitration). The rapid increase of hepatic nitrotyrosine is probably secondary to a rapid accumulation of neutrophils in the liver following endotoxin challenge, but whether the nitration of tyrosine occurs through a peroxidase- or peroxynitrite-dependent pathway is unknown. Likewise, it is not known whether the circulating S-nitrosothiols are formed in the liver, and nothing is known about the concentrations of S-nitrosothiols in liver tissue. This is the first study in which the half-life of Snitrosoalbumin has been measured in vivo, and the relatively long terminal half-life of nearly 30 min suggests that S-nitrosoalbumin releases NO slowly. The description of plasma concentration data by a two-compartment model may indicate an initial phase of redistribution of S-nitrosoalbumin within the vasculature and albumin accessible tissue space, followed by a slower phase of metabolism. However, the model describes a sum of those two processes, and the actual contribution of each process at a given time point cannot be deduced from plasma data alone. In addition, the interpretation of these data should be tempered by the fact that we have not shown conclusively that S-nitrosoalbumin is the major circulating S-nitrosothiol in this model. There have been two reports on the metabolism and breakdown of Snitrosothiols in plasma, with low molecular weight thiols being responsible for the breakdown of S-nitrosoalbumin [9,34]. Recently, it was suggested that S-nitrosation of oxyhemoglobin in the lung is followed by release of NO when exposed to low oxygen tension in the microcircu-
S-nitrosothiols and nitrotyrosine in cirrhosis and endotoxemia
lation by further interaction with thiols to dilate blood vessels [35]. The increased formation of S-nitrosothiols seen in cirrhosis and endotoxemia may then be important in the pathogenesis of systemic vasodilatation observed in these disease processes. However, the role of endogenous S-nitrosothiols in the pathogenesis of vascular dysfunction in cirrhosis and endotoxemia remains to be determined. One of the major functions of S-nitrosothiols to emerge recently is their role as potent inhibitors of platelet aggregation. It is well recognized that platelet function is abnormal in cirrhosis, and several studies have shown that S-nitrosoalbumin and other S-nitrosothiols inhibit platelet aggregation. A bolus intravenous injection of S-nitrosoalbumin causes a marked prolongation of the bleeding time in dogs [11]. The anti-platelet effects of S-nitrosoalbumin are concentration dependent, with a dose as low as 10 nmol/kg causing a 50% reduction of platelet aggregation and 50 nmol/kg causing an 85% reduction of platelet aggregation in dogs [11]. The observation that plasma S-nitrosothiols increase markedly in cirrhotic rats with endotoxemia is likely to cause a prolongation of bleeding time, and may explain why there is an increased risk of variceal hemorrhage during infections in cirrhotic patients [36]. In summary, we have shown that experimental cirrhosis is associated with an increased formation of S-nitrosothiols and nitrotyrosine, indicative of increased nitrosative stress in this model of biliary cirrhosis. Endotoxemia causes a modest increased formation of nitrotyrosine, and a marked upregulation of S-nitrososthiol formation. Whether the increased formation of Snitrosothiols observed in rats with biliary cirrhosis following LPS injection contributes to the increased mortality during endotoxemia is unknown. However, further studies on the role of S-nitrosothiols on vascular function and platelet function in cirrhosis are underway. Acknowledgements — This research was supported by The Danish Medical Research Council (#9900809), and The Medical Research Council, UK (Component grant G000290); and MRC Cooperative Group grant (G0002) to the UCL Sepsis Research Group. We are grateful to Dr. Preben Jakobsen, Institute of Pharmacology, Aarhus University, Aarhus, Denmark for pharmacokinetic advice. REFERENCES [1] Grisham, M. B.; Jourd’Heuil, D.; Wink, D. A. Nitric oxide. I. Physiological chemistry of nitric oxide and its metabolites: implications in inflammation. Am. J. Physiol. 276:G315–G321; 1999. [2] Morales-Ruiz, M.; Jimenez, W.; Perez-Sala, D.; Ros, J.; Leivas, A.; Lamas, S.; Rivera, F.; Arroyo, V. Increased nitric oxide synthase expression in arterial vessels of cirrhotic rats with ascites. Hepatology 24:1481–1486; 1996. [3] Marley, R.; Holt, S.; Fernando, B.; Harry, D.; Anand, R.; Goodier, D.; Davies, S.; Moore, K. Lipoic acid prevents development of the hyperdynamic circulation in anesthetized rats with biliary cirrhosis. Hepatology 29:1358 –1363; 1999.
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[31] Scorza, G.; Minetti, M. One-electron oxidation pathway of thiols by peroxynitrite in biological fluids: bicarbonate and ascorbate promote the formation of albumin disulphide dimers in human blood plasma. Biochem. J. 329:405– 413; 1998. [32] Eiserich, J. P.; Hristova, M.; Cross, C. E.; Jones, A. D.; Freeman, B. A.; Halliwell, B.; van der Vliet, A. Formation of nitric oxidederived inflammatory oxidants by myeloperoxidase neutrophils. Nature 391:393–397; 1998. [33] van der Vliet, A.; Eiserich, J. P.; Halliwell, B.; Cross, C. E. Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity. J. Biol. Chem. 272:7617–7625; 1997. [34] Scorza, G.; Pietraforte, D.; Minetti, M. Role of ascorbate and protein thiols in the release of nitric oxide from S-nitroso-albumin and S-nitroso-glutathione in human plasma. Free Radic. Biol. Med. 22:633– 642; 1997. [35] Gow, A. J.; Stamler, J. S. Reactions between nitric oxide and haemoglobin under physiological conditions. Nature 391:169 – 173; 1998. [36] Goulis, J.; Patch, D.; Burroughs, A. K. Bacterial infection in the pathogenesis of variceal bleeding. Lancet 353:139 –142; 1999. ABBREVIATIONS
LPS—lipopolysaccharide NO—nitric oxide TCA—trichloroacetic acid