Plasma Cysteine Levels Research Articles

Glutathione and N-acetylcysteine reduce gastric mucosal blood flow in rats.

Glutathione has been studied as a possible mediator in gastric mucosal protection and healing, but its extracellular function is not fully understood. This study evaluates blood flow changes in normal gastric mucosa secondary to glutathione modulation under stable central hemodynamic conditions. Thiol substances were quantified by reverse-phase ion-pair liquid chromatography and fluorescence detection. Central hemodynamics remained stable when glutathione and N-acetylcysteine were administered in a dose of 0.5 mmol/kg. Higher doses than 0.5 mmol/kg of glutathione and N-acetylcysteine caused unstable hemodynamics. Glutathione (0.5 mmol/kg intravenously) and N-acetylcysteine (0.5 mmol/kg intravenously) reduced corpus mucosal blood flow by 28% and 26% (P < 0.0005), respectively, and glutathione reduced antral mucosa blood flow by 22% (P < 0.01). L-Buthionine-[S,R]-sulfoximine (2 mmol/kg intravenously) did not effect gastric mucosal blood flow. Cysteine content in mucosa and plasma increased while mucosal glutathione levels were largely unchanged after administration of reduced glutathione and N-acetylcysteine. Plasma glutathione only increased after injection of glutathione. L-Buthionine-[S,R]-sulfoximine reduced the glutathione level in both plasma and mucosa. We conclude that glutathione and N-acetylcysteine reduce gastric mucosal blood flow and that the effect may be related to increased cysteine levels in plasma or mucosa.

Vitamin B-6 Normalizes the Altered Sulfur Amino Acid Status of Rats Fed Diets Containing Pharmacological Levels of Niacin without Reducing Niacin's Hypolipidemic Effects , ,

Niacin (nicotinic acid) in large doses (>2 g) has been increasingly the choice of lipid-lowering agent by clinicians. However, the potential risks of the use of high doses of the vitamin have not been critically considered in the same way as has the use of other lipid-lowering drugs. The present study provides evidence that pharmacological levels of niacin interfere with the metabolism of methionine, leading to hyperhomocysteinemia and hypocysteinemia. Male Sprague-Dawley rats were fed a semisynthetic diet supplemented with either 400 or 4000 mg niacin/kg (compared with 47 mg/kg diet in the control diet). In Experiment 1, feeding these diets for 3 wk resulted in a dose-related increase in the plasma and urine methionine concentrations while cysteine levels were decreased. This altered methionine metabolism was accompanied by a lower plasma vitamin B-6 concentration in niacin-supplemented rats compared with controls. In Experiment 2, the methionine and cysteine levels in plasma and urine were normalized when vitamin B-6 (10 mg/kg diet) was added to the diet containing 4000 mg niacin/kg and fed for 6 wk. This experiment also showed that plasma and urine homocystein concentrations were increased by niacin and normalized by vitamin B-6. The hypolipidemic action of niacin was unaffected by the presence of vitamin B-6. These results indicate that niacin at large dose levels interferes with methionine metabolism by affecting vitamin B-6 status. The treatment of dyslipidemia with simultaneous administration of niacin and vitamin B-6 could be a better therapy than the use of niacin alone.

Oxygen radical release by neutrophils of HIV-infected patients

Neutrophils from asymptomatic HIV-infected patients have an increased Nitroblue tetrazolium (NBT) reduction, that is an increased production of oxygen radicals. Plasma from these patients can activate normal neutrophils to an increased NBT-reduction and the neutrophil activating factor thus seems to be mainly plasma bound. Further, the patients also have increased levels of plasma malondialdehyde and thus an increased lipid peroxidation. Plasma cysteine levels are low, a sign of increased consumption of antioxidants. Treatment of the asymptomatic HIV-infected patients with N-acetylcysteine corrected the plasma cysteine levels and had some beneficial effects, but did not inhibit the increased radical production by the neutrophils.

Redox status and protein binding of plasma homocysteine and other aminothiols in patients with homocystinuria

Elevations of homocyst(e)ine levels in the blood of patients with homocystinuria may explain the high cardiovascular morbidity. We determined levels of reduced, oxidized, and protein-bound homocyst(e)ine, cyst(e)ine, and cyst(e)inylglycine in plasma from eight patients with homocystinuria. The technique used involved trapping of reduced thiols by collecting blood directly into tubes containing sulfhydryl-reactive reagents. All patients had high levels of homocysteine (range, 1.9 to 91.2 μmol/L), and among the aminothiols investigated, this species showed the most drastic elevation compared with trace levels (<0.4 μmol/L) found in healthy subjects. The ratio between free homocysteine and total homocyst(e)ine (reduced to total ratio) was above normal and positively correlated to the reduced to total ratio for cyst(e)ine, suggesting that an equilibrium exists between these species through sulfhydryl disulfide exchange. The other homocyst(e)ine species (oxidized and protein-bound) were also markedly increased in patients with homocystinuria. Plasma cysteine and cysteinylglycine levels were moderately increased, whereas plasma concentrations of protein-bound cyst(e)ine, protein-bound cyst(e)inylglycine, and free cystine were below normal. Homocysteine in particular and other homocyst(e)ine species are markedly increased in plasma of homocystinurics, and these changes are associated with pronounced alterations in the level and the redox status of other aminothiols. This should be taken into account when considering homocyst(e)ine as an atherogenic agent, and the role of various homocyst(e)ine species in the pathogenesis of homocystinuria.

Acute effects of nitroglycerin depend on both plasma and intracellular sulfhydryl compound levels in vivo. Effect of agents with different sulfhydryl-modulating properties.

Changes in sulfhydryl (SH) compound availability may alter the hemodynamic effect of nitroglycerin (NTG). Data on the relation between NTG effect and thiol levels are, however, limited to in vitro experiments. The present study investigates how intracellular and extracellular changes in SH group concentrations (cysteine and glutathione [GSH]) affect the responsiveness to NTG in vivo. GSH and cysteine levels in plasma, vena cava, and aorta were measured after administration of N-acetylserine (placebo, n = 6), N-acetylcysteine (NAC, extracellular and intracellular SH donor, n = 6), oxothiazolidine (OXO, intracellular SH donor, n = 6), buthionine sulfoximine (BSO, intracellular GSH-depleting agent, n = 6), BSO+NAC (n = 6), and BSO+OXO (n = 6) in chronically catheterized conscious rats. In addition, the effect of 2.5 mg NTG/kg i.v. on mean arterial pressure (MAP) was determined before and after the same treatment. NAC (5 mmol/kg i.v. for 2 hours) significantly (p < 0.05) increased extracellular cysteine and GSH levels and potentiated the hypotensive effect of NTG (from 26 +/- 3 to 31 +/- 4 mm Hg [mean +/- SEM], p < 0.05). OXO (5 mmol.kg-1 x hr-1 i.v. for 2 hours) significantly increased intracellular cysteine and GSH levels but had no effect on NTG responsiveness (p > 0.05). BSO (1 g i.p. three times within 24 hours) significantly decreased intracellular GSH levels (p < 0.05) and attenuated the effect of NTG (from 28 +/- 3 to 16 +/- 2 mm Hg). The results suggest that the acute hypotensive effect of NTG in vivo is: 1) increased by high extracellular GSH and/or cysteine levels (NAC), 2) decreased by low intracellular GSH levels (BSO), and 3) unaffected by high intracellular levels of cysteine and GSH (OXO).

Requirement for prooxidant and antioxidant states in T cell mediated immune responses. — Relevance for the pathogenetic mechanism of AIDS?

The discovery of decreased plasma cysteine and cystine levels and elevated plasma glutamate levels in HIV-infected patients has led to intense investigations into the role of cysteine in T cell-mediated immune responses. A large body of evidence indicates that certain aspects of the T cell response require the action of active oxygen derivatives while other aspects of the response require the action of antioxidants such as cysteine and glutathione (GSH). The prooxidant and antioxidant states may be required sequentially at different times during T cell activation. The extremely weak cystine transport activity of T cells together with oxidizing metabolites from inflammatory microenvironments appear to be important factors that support the prooxidant state. The relatively high cystine transport activity of the antigen-presenting macrophages, in contrast, provides these cells with a "cysteine pumping" function that allows the antigen binding T cells in their vicinity to shift to the antioxidant state. The difference between the membrane transport activities for cysteine of T cells and macrophages thus appears to be the key element of a mechanism that facilitates both, the prooxidant state of T cells and their regulated shift to the antioxidant state. When T cells do not receive sufficient amounts of cysteine, the intracellular GSH levels and rates of DNA synthesis activity decrease, and the cells may suffer from various manifestations of oxidative damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of N-acetylcysteine on the release of oxygen radicals from neutrophils and alveolar macrophages

N-acetylcysteine (NAC) is rapidly de-acetylated in vivo to cysteine (CYSH), a precursor of glutathione (GSH) which is an antioxidant in cells and body fluids. We investigated the effect of oral administration of N-acetyl cysteine for 5 days on the spontaneous and stimulated generation of hydrogen peroxide (H2O2) and superoxide anion (O2-) from human and rat phagocytic leucocytes. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage (BAL) in control rats and rats given NAC in their drinking water. Neutrophils (PMNL) were harvested from whole blood in normal nonsmoking volunteers before and after NAC was given by mouth. The stimulated release of H2O2 and O2 from both rat AM and human PMN was not changed by administration of NAC. However, a small but significant increase was observed in both the spontaneous generation of O2- from rat AM and the spontaneous generation of H2O2 from human PMNL. Administration of NAC significantly increased cysteine levels in human plasma and rat BAL, but the levels in human PMNL and rat AM after NAC did not differ from control levels. GSH levels were not altered significantly by NAC.

Inhibition of HIV-1 replication and NF-x B activity by cysteine and cysteine derivatives

HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor alpha (TNF alpha) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-kappa B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.

Integrity of the Blood-Brain Barrier System against Methylmercury Acute Toxicity.

To examine the possibility of methylmercury (MeHg)-induced damage at the bloodbrain barrier system, male C57BL/6N mice were orally administered MeHg chloride at doses from 40 to 200 μmol/kg of body weight. Although the Hg levels in plasma and blood at 24 h after the administration increased linearly with dosed amount, the increase in the brain levels exceeded that in the circulation levels. The ratio of the brain Hg to the dosed amount increased from 0.443±0.026% in the 40 μmol/kg dosed group to 0.667±0.062% in the 200 μmol/kg dosed group. The Hg concentration ratio of brain to plasma also increased from 1.26 to 1.90. Although some disturbance was expected in the brain uptake system of MeHg for the accelerated accumulation of the brain Hg, brain uptake of 125 I-labeled bovine serum albumin and 14C-L-methionine was not affected by MeHg treatment at all. This indicated that transport systems for albumin- and cysteine-conjugate of MeHg in the brain remained unchanged under the present circumstances. Ultrafiltration analysis revealed that Hg distribution rates in low molecular weight fraction of plasma markedly increased with dose. Glutathione and cysteine conjugates of MeHg were dominant constituents in this fraction. Although plasma cysteine levels increased after MeHg administration, glutathione levels remained unchanged. In contrast, albumin and total SH contents in plasma were markedly decreased in a dose-dependent manner by MeHg treatment. Furthermore, affinity of MeHg for albumin, a dominant MeHg carrier in mouse plasma, was suggested to be lowered by MeHg treatment. These results appeared to indicate that the mouse blood-brain barrier system was strongly protected from MeHg acute toxicity, as indicated by the unchanged protein and amino acid uptake rates and its γ-glutamyltranspeptidase activity. Plasma albumin, however, was shown to be liable to undergo quantitative and qualitative changes by MeHg treatment, thereby resulting in dose-dependent acceleration of the uptake of the low molecular weight Hg conjugate (s) in the brain.

Plasma cysteine and sulphate levels in patients with motor neurone, Parkinson's and Alzheimer's disease

Elevated plasma cysteine to sulphate ratios were found in patients with Motor neurone disease (MND), Parkinson's disease (PD) and Alzheimer's disease (AD). Cysteine and sulphate were measured by colourimetric methods. Following recent discovery of a defect in sulphoxidation and sulphation of xenobiotics in these diseases, this finding confirms that endogenous sulphur metabolism is disturbed. The mean cysteine:sulphate ratios (× 10 3) in fasting early morning plasma were 506, 521 and 477 for MND, PD and AD whereas it was 96 for normal controls ( P < 0.001). This excess of cysteine thiol groups may interfere with neural protein function. The deficiency of sulphate ions may lead to reduced xenobiotic detoxification.

Effects of ethanol feeding and withdrawal on plasma glutathione elimination in the rat.

Chronic ethanol feeding increases hepatic turnover and sinusoidal efflux of glutathione in rats. The present study was performed to determine whether the observed increase in glutathione efflux was due to increased extrahepatic requirements for glutathione. The concentration and disposition of plasma glutathione were determined in rats fed liquid diets containing 36% of calories as ethanol or pair-fed an isocaloric mixture with carbohydrate replacing ethanol calories for 5 to 8 weeks. The half-life and plasma clearance of [35S]glutathione were found to be similar in ethanol-fed and control rats and in rats withdrawn 24 hr from ethanol. Uptakes of the sulfur moiety of [35S]glutathione by kidney, jejunal mucosa, liver, lung, spleen, muscle and heart were also unchanged by ethanol feeding. The plasma glutathione concentration was significantly higher in ethanol-withdrawn rats 22.30 +/- 3.06 nmoles per ml (p less than 0.05) compared to pair-fed controls (13.51 +/- 2.04), while rats continuing to drink ethanol had intermediate levels (16.96 +/- 2.22). Plasma cysteine levels were slightly, but not significantly, higher in ethanol-fed rats. These findings suggest that increased sinusoidal efflux of glutathione in ethanol-fed rats is due to a direct effect of ethanol on hepatic glutathione transport and not due to an alteration in extrahepatic disposition of glutathione. In order to characterize further the effects of ethanol feeding on glutathione-dependent detoxification, activities of glutathione S-transferase, glutathione reductase and gamma-glutamyltransferase were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Homocystinuria due to cystathionine β-synthase deficiency—The effects of betaine treatment in pyridoxine-responsive patients

Homocystinuria due to cystathionine β-synthase deficiency may be responsive to pyridoxine, a precursor of the cofactor pyridoxal phosphate, and the amount of residual enzyme activity present is the probable determinant of this. In six treated pyridoxine-responsive patients whose biochemical control of fasting plasma amino acid levels appeared optimal, we assessed the effects on plasma amino acids of standard oral methionine loads (4g/m 2 of body area) before and after adding betaine (trimethylglycine) 6g/d, to the treatment regimen of pyridoxine and folic acid. Our aim was to define the capacity of these patients to metabolize methionine and to determine whether betaine would effect a reduction in postload homocysteine levels. During the 24 hours after the methionine challenge all patients had higher plasma methionine and homocysteine and lower cysteine than did 17 normal subjects. After betaine these homocysteine responses were reduced to near normal, and there was a trend toward increased methionine. There was a direct correlation between premethionine fasting homocysteine and mean homocysteine responses during the 24 hours following the methionine load, both before ( r = 0.79) and after betaine ( r = 0.71). Betaine also increased plasma cysteine levels in patients with the more severe biochemical abnormalities. After betaine there were modest increases in plasma serine (mean increase 25%; P < 0.025). Since the vascular complications of homocystinuria are related to increased plasma homocysteine, betaine therapy may reduce this risk in patients receiving a standard pyridoxine and folic acid regimen in whom there are abnormal homocysteine responses after a standard methionine load.

Effect of inhibition of gamma-glutamyltranspeptidase by AT-125 (acivicin) on glutathione and cysteine levels in rat brain and plasma.

AT-125 (Acivicin) is an inhibitor of gamma-glutamyltranspeptidase (gamma-GTP) which initiates glutathione catabolism to cysteine. We measured plasma and brain glutathione and cysteine in rats treated with AT-125. Six h after AT-125 treatment, plasma glutathione had increased 6-fold and plasma cysteine had fallen significantly. Brain cysteine fell after 24 h of AT-125 treatment, and brain glutathione had also decreased 18%. AT-125 pretreatment inhibited brain uptake of 35S when it was given as 35S-GSH but had no effect when it was given as 35S-cysteine. These results suggest that plasma glutathione is catabolized by gamma-GTP, and cysteine derived from it is taken up by the brain. N-acetylcysteine was administered to AT-125 treated rats in an attempt to supply cysteine to the brain in the face of gamma-GTP inhibition. N-acetylcysteine supported brain glutathione levels, suggesting that it can serve as a source of cysteine under these conditions.

Amino acid content of erythrocytes in uremia

The amino acid content of plasma and erythrocytes in patients with severe renal failure (serum creatinine < 8 mg/100 ml) treated with selective low-protein diets, in patients on regular hemodialysis, and in a control group of healthy subjects were studied. Most amino acids in erythrocytes of the patients showed the same changes as in plasma with the exception of histidine, serine, and alanine. In spite of low histidine plasma levels, the erythrocytes level is increased as compared with healthy controls. In uremic patients the plasma serine was constantly reduced whereas the serine content of the erythrocytes did not differ from healthy controls. Alanine concentrations in the erythrocytes of uremic patients were increased in spite of normal alanine plasma levels. This finding may be due to the increased glycolytic ratio of red cells from uremic subjects delivering more pyruvate for transamination to alanine. In the control group the cysteine content of erythrocytes was decreased with a gradient between plasma and erythrocytes of 3.5:1. The same gradient could be observed in uremic patients in spite of the elevation in their plasma cysteine levels by a factor of 2.7 compared with controls. The low cysteine levels in erythrocytes may be due to loss of cysteine for glutathione synthesis in red cells. High glutathione levels in the erythrocytes of uremic patients support this hypothesis.

The cleavage and adsorption of parathyroid hormone at high dilution Implications for receptor binding studies

Like other polypeptide hormones, purified intact parathyroid hormone (1–84)parathyroid hormone is notoriously unstable and is subject to large adsorptive losses in routine laboratory manipulation. The present studies were undertaken with 125I-labeled hormone to quantitate the problem and to develop preventative measures, particularly with concentrations of physiological interest, 1 · 10 −10 M. It was found that spontaneous cleavage of the hormone takes place upon its incubation in air or oxygen. This can be prevented by the presence of mercaptoethanol or by plasma levels of cysteine and ascorbate. Under non-cleavage conditions, adsorption was found to be extensive on all materials tested. This adsorption increased with time up to 2 h, was independent of ionic strength, increased with increasing temperature and was presumed to involve hydrophobic interactions. Under given conditions, adsorption was proportional to concentration (constant percentage). However, at very high concentrations, 1 · 10 −6 M, adsorption was markedly reduced. Adsorption was minimized at low pH (2). Bovine serum albumin reduced adsorption under all conditions when present at concentrations of 2 mg/ml or more. Coating laboratory ware with cetyl alcohol also was helpful. Using optimal conditions, cleavage is prevented and losses are less than 5% at neutral pH, and under 2% at pH 2.

Modulation of cysteine metabolism in mice—Effects of propargylglycine and l-cyst(e)ine-degrading enzymes

In an attempt at evaluating the therapeutic effects of cysteine depletion on the growth of cysteine-dependent L1210 leukemia, the effects in vivo of a cysteine- and cystine-degrading enzyme as well as the combined use of an inhibitor of cysteine biosynthesis with cystine-free diets, were measured in mice. The kinetic properties of rat liver γ-cystathionase (EC 4.2.1.13) and Enterobacter cloacae cysteine desulfhydrase (EC 4.4.1.1) toward the catabolism of cystine and cysteine, as well as the inhibitory properties of some possible physiological components, are described. A novel and sensitive assay for the detection of cysteine in biological fluids was used to measure the ability of these enzymes to deplete plasma cysteine levels when injected into normal mice. Cysteine desulfhydrase caused no alteration in mouse plasma cysteine concentrations, presumably due to rapid clearance ( T 1 2 less than 10 min) of the enzyme. γ-Cystathionase caused a 60 per cent drop in plasma cysteine concentrations which returned to normal with the clearance of the enzyme ( T 1 2 = 2 hr ). The properties and limitations of these enzymes are discussed within the context of their ability to deplete plasma cys(e)ine in vivo. When propargylglycine, an effective covalent inhibitor of γ-cystathionase, both in vitro ( K i = 0.1 mM, saturating T 1 2 = 0.5 min ) and in vivo, was combined with diets lacking cystine, no reduction in plasma cysteine or increase in survival of mice bearing L1210 leukemia could be observed. The compound would induce a cystathioninemia and a cystathioninuria when given to mice and was not toxic in cell culture or in animals when tested with an adequate source of cystine, but was highly toxic in the absence of cystine.