Plasma Redox Status Research Articles

Dietary Chlorophyll Metabolites Catalyze the Photoreduction of Plasma Ubiquinone

Ubiquinol is a plasma antioxidant. The mechanisms responsible for maintenance of plasma ubiquinol are poorly understood. Here, we show that metabolites of chlorophyll can be found in blood plasma of animals that are given a chlorophyll-rich diet. We also show that these metabolites catalyze the reduction of plasma ubiquinone to ubiquinol in the presence of ambient light, in vitro. We propose that dietary chlorophyll or its metabolites, together with light exposure, regulate plasma redox status through maintaining the ubiquinol pool.

Treatment of chronic hemodialysis patients with low-dose fenofibrate effectively reduces plasma lipids and affects plasma redox status

Dyslipidemia is common in chronic hemodialysis patients and its underlying mechanism is complex. Hemodialysis causes an imbalance between antioxidants and production of reactive oxygen species, which induces the oxidative stress and thereby may lead to accelerated atherosclerosis. Statins have been found to be little effective in end-stage kidney disease and other lipid-lowering therapies have been only scarcely studied. The study aimed to assess the effect of low-dose fenofibrate therapy on plasma lipids and redox status in long-term hemodialysis patients with mild hypertriglyceridemia.Twenty seven chronic hemodialysis patients without any lipid-lowering therapy were included in a double-blind crossover, placebo-controlled study. The patients were randomized into two groups and were given a sequence of either 100 mg of fenofibrate per each hemodialysis day for 4 weeks or placebo with a week-long wash-out period between treatment periods. Plasma lipids, high sensitive C-reactive protein (CRP), urea, creatinine, electrolytes, phosphocreatine kinase (CK), GOT, GPT and plasma thiols (total and free glutathione, homocysteine, cysteine and cysteinylglycine) were measured at baseline and after each of the study periods. Plasma aminothiols were measured by reversed phase HPLC with thiol derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate.Fenofibrate therapy caused a significant decrease of total serum cholesterol, LDL cholesterol and triglycerides and an increase of HDL cholesterol. The treatment was well tolerated with no side-effects but there was a small but significant increase of CK not exceeding the upper limit of normal range. There were no changes of serum CRP, potassium, urea, and creatinine and liver enzymes during the treatment. Neither total nor total free cysteinylglycine and cysteine changed during the study but both total and free glutathione increased during the therapy with fenofibrate and the same was observed in case of plasma homocysteine.The study shows that a treatment with reduced fenofibrate dose is safe and effective in reducing serum triglycerides and cholesterol in chronic dialysis patients and may shift plasma aminothiol balance towards a more antioxidative pattern.

Formation of micronuclei and other nuclear anomalies in exfoliated nasal and oral cells: Results of a human study with workers in a power plant processing poultry litter

Handling of chicken litter leads to exposure to toxic gases, endotoxins and airborne microorganisms. Aim of this study was to investigate if this results in acute cytotoxicity and to damage of the genetic material which is involved in the etiology of various diseases including cancer. Nuclear anomalies which reflect genotoxic and cytotoxic effects were monitored in exfoliated buccal and nasal cells which were collected from workers (n = 25) of a power plant which processes chicken manure and from controls (n = 21). Furthermore, biochemical parameters of the redox status (malondialdehyde, oxLDL and TEAC) and C-reactive protein (CRP) in plasma and the concentrations of toxic gases and endotoxins in the air were determined. No increase of anomalies which reflect chromosomal damage (micronuclei, binucleates, nuclear buds) but significantly higher rates of nuclear aberrations which are indicative for cytotoxicity (karyolysis, karyorrhexis, condensed chromatin) were found in the workers. These effects were in nasal cells more pronounced as in buccal cells. MDA, oxLDL and CRC levels were in both study groups similar. Chemical analyses show that the workers are exposed to high concentrations of NO and endotoxins, while the levels of NO2, NH3 and H2S were below the MAK levels. Taken together, the results show that anomalies that are due to cytotoxicity are increased in the workers and suggest that the exposure may lead to inflammations in the respiratory tract. However, the lack of induction of anomalies that reflect chromosomal damage indicate that no health effects will take place which are due to instability of the genetic material.

Olive oil supplemented with Coenzyme Q10: Effect on plasma and lipoprotein oxidative status

Olive oil consumption is associated with protective cardiovascular properties, including some beneficial modifications in lipoprotein profile and composition. Coenzyme Q(10) (CoQ(10)) exerts a protective effect on plasma lipoproteins. Aim of the study was to investigate whether extra virgin (EV) olive oil enriched with CoQ(10) affects CoQ(10) levels and oxidative status in plasma and in isolated lipoproteins. Twelve subjects were administered 20 mL olive oil per day for 2 weeks, followed by 2 weeks of olive oil enriched with 20 mg and 2 more weeks with 40 mg of CoQ(10). Plasma and isolated lipoproteins were collected in each phase of the study and subsequently analyzed to assess lipid profile, CoQ10 levels, ORAC assay, resistance of lipoproteins to peroxidation and paroxonase 1 activity. Plasma CoQ(10) levels significantly increased with the 20 mg (+73%) and 40 mg dose (+170%), while the percentage of oxidized CoQ(10) decreased. A significant inverse correlation was found in plasma between percentage of oxidized CoQ(10) and total antioxidant capacity. A lower susceptibility of LDL to peroxidation was also found. Finally, a positive correlation was observed between concentration of CoQ(10) in HDL and paraoxonase-1 activity. EV olive oil enriched with both doses of CoQ(10) significantly affects its bioavailability and plasma redox status. These changes are associated with a decreased susceptibility of plasma lipoproteins to peroxidation associated with a chain-breaking antioxidant activity of the formulation.

Clinical parameters and biomarkers of oxidative stress in agricultural workers who applied copper-based pesticides

Copper based-pesticides are widely used in agricultural practice throughout the world. We studied the (i) concentration of Cu and proteins involved in Cu homeostasis, (ii) plasma redox status, and (iii) biomarkers of exposure in Cu-based pesticide applicators in order to compare them with clinical biochemical tests. Thirty-one professional applicators and 32 control subjects were recruited. Oxidative stress biomarkers, ceruloplasmin (CRP), metallothioneins (MTs), copper, hematological parameters, and biochemical markers for pancreatic, hepatic and renal function were measured in plasma. Copper was increased in the exposed group compared to the control group concomitantly with TBARS, protein carbonyls, and nitrate+nitrite levels. In the exposed group, α-tocopherol and the FRAP assay were lower and LDH, transaminases, GGT, ALP, urea, creatinine, CRP and MTs were higher than in the control group. The relative leukocyte subclasses were also different between the two groups. Clinical chemistry tests did not surpass the upper reference limit. Our results suggest that the incorporation of oxidative stress biomarkers to biochemical/clinical tests should be considered for validation and included in the human health surveillance protocols.

Astaxanthin limits fish oil-related oxidative insult in the anterior forebrain of Wistar rats: Putative anxiolytic effects?

The habitual consumption of marine fish is largely associated to human mental health. Fish oil is particularly rich in n-3 polyunsaturated fatty acids that are known to play a role in several neuronal and cognitive functions. In parallel, the orange-pinkish carotenoid astaxanthin (ASTA) is found in salmon and displays important antioxidant and anti-inflammatory properties. Many neuronal dysfunctions and anomalous psychotic behavior (such as anxiety, depression, etc.) have been strongly related to the higher sensitivity of cathecolaminergic brain regions to oxidative stress. Thus, the aim of this work was to study the combined effect of ASTA and fish oil on the redox status in plasma and in the monoaminergic-rich anterior forebrain region of Wistar rats with possible correlations with the anxiolytic behavior. Upon fish oil supplementation, the downregulation of superoxide dismutase and catalase activities combined to increased “free” iron content resulted in higher levels of lipid and protein oxidation in the anterior forebrain of animals. Such harmful oxidative modifications were hindered by concomitant supplementation with ASTA despite ASTA-related antioxidant protection was mainly observed in plasma. Although it is clear that ASTA properly crosses the brain-blood barrier, our data also address a possible indirect role of ASTA in restoring basal oxidative conditions in anterior forebrain of animals: by improving GSH-based antioxidant capacity of plasma. Preliminary anxiolytic tests performed in the elevated plus maze are in alignment with our biochemical observations.

Role of endothelial dysfunction in modulating the plasma redox homeostasis in visceral leishmaniasis

BackgroundEvidence in the literature suggests that down-regulation of nitric oxide (NO) is associated with the pathophysiological conditions during visceral leishmaniasis (VL). Here we have investigated the mechanism that leads to the down regulation of systemic NO in the infected condition. Moreover, we have determined whether down regulation of NO is associated with increased generation of reactive oxygen species (ROS) during this disease. Therapeutic strategy targeting signaling molecules of these events was evaluated. MethodsPlasma protein-nitrotyrosine was examined by ELISA kit. Generation of superoxides and peroxynitrites was investigated by flow cytometry. NO bioavailability in endothelial cells was evaluated using DAF-2DA fluorescence. Ceramide contents were evaluated using FACS analysis, HPTLC and HPLC. ResultsL. donovani infected reticulo-endothelial cells regulated the activity of eNOS and NAD(P)H oxidase in the endothelial cells through the generation of intercellular messenger, ceramide. Activation of SMases played an important role in the generation of ceramide in animals during chronic infection. These events led to generation of ROS within endothelial cells. Modulation of redox status of plasma and accumulation of ROS in endothelial cells were critically involved in the regulation of NO bioavailability in plasma of the infected animal. Endothelial dysfunction and decline of NO were resulted from an increased production of superoxide where upregulation of eNOS expression appeared as an ineffective compensatory event. Inhibition of ceramide generation increased NO bioavailability, prevented endothelial dysfunction and concomitant oxidative stress. Conclusion and general significanceDecreased NO bioavailability and endothelial dysfunction were the downstream of ceramide signaling cascade. ROS accumulation promoted peroxynitrite generation and reduced NO bioavailability. Inhibition of ceramide generation may be a potential therapeutic option in preventing the co-morbidity associated with VL.

Effect of dioxin exposure on several indices of blood redox status in lactating buffalo cows

Dioxins are lipophilic compounds with a small molecular weight and are highly persistent, bioaccumulative and toxic. Dioxin detoxification is associated with an increased production of reactive oxygen species (ROS). In physiological conditions the body is protected against ROS and their toxic products by a wide range of antioxidant systems. We hypothesize that the imbalance between ROS production, associated with dioxin exposure, and the antioxidant defence capacity, may lead to oxidative stress, with consequent increased consumption of antioxidants and accumulation of toxic compounds in blood and tissues. The objective of this study was to evaluate the effect of exposure to dioxins on the plasma redox status of lactating buffalo cows. To this aim, the major liposoluble (retinol and α-tocopherol) and water-soluble (ascorbate) antioxidants, the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, the total antioxidant capacity (TAC), as well as specific protein oxidation markers (protein bound carbonyls and nitro-tyrosine) and lipid oxidation markers (hydroperoxides), were chosen as indices of blood redox status. The concentration of antioxidants, protein-bound carbonyls (PC), nitro-tyrosine (N-Tyr), and hydroperoxides (LPO), the SOD and GPx activity, and the TAC were measured in plasma samples obtained from buffalo cows exposed to environmental levels of dioxins higher (n=21, group A) or lower (n=29; group B) than those permitted. Plasma titres of antioxidants, as measured by HPLC, and the total antioxidant capacity, as measured by trolox equivalents capacity, were higher in group B than in A. Similarly, SOD and GPx activities were higher in group B than in A. Conversely, plasma levels of PC, N-Tyr and LPO, as measured by ELISA, were higher in group A than in B. Our results suggest that exposure to dioxins impairs the plasma antioxidant defence system of lactating buffalo cows, and that metabolic processes associated with dioxin detoxification might induce or enhance oxidation of protein and lipids. This adverse effect on blood redox status might have negative implications for animal health and reproduction, and might compromise animal welfare.

An Oxidized Extracellular Oxidation-Reduction State Increases Nox1 Expression and Proliferation in Vascular Smooth Muscle Cells Via Epidermal Growth Factor Receptor Activation

To examine the effect of an oxidized extracellular oxidation-reduction (redox) state (E(h)) on the expression of NADPH oxidases in vascular cells. The generation of reactive oxygen species by NADPH oxidase (Nox)-based NADPH oxidases activates redox-dependent signaling pathways and contributes to the development of "oxidative stress" in vascular disease. An oxidized plasma redox state is associated with cardiovascular disease in humans; however, the cellular mechanisms by which the extracellular redox state may cause disease are not known. Aortic segments and cultured aortic smooth muscle cells were exposed to E(h) between -150 mV (reduced) and 0 mV (oxidized) by altering the concentration of cysteine and its disulfide, cystine, the predominant redox couple in plasma. A more oxidized E(h) increased the expression of Nox1 and resulted in Nox1-dependent proliferation of smooth muscle cells. Oxidized E(h) rapidly induced epidermal growth factor receptor phosphorylation via shedding of epidermal growth factor-like ligands from the plasma membrane and caused extracellular signal-regulated kinase 1/2-dependent phosphorylation of the transcription factors activating transcription factor-1 and cAMP-response element-binding protein. Inhibition of epidermal growth factor receptor or extracellular signal-regulated kinase 1/2 activation, or addition of small interference RNA to activating transcription factor-1, prevented the increase in Nox1 expression. Our results identify a novel mechanism by which extracellular oxidative stress increases expression and activity of Nox1 NADPH oxidase and contributes to vascular disease.

Redox status of equine seminal plasma reflects the pattern and magnitude of DNA damage in sperm cells

Antioxidant status of seminal plasma from 23 stallions was evaluated. We found a negative correlation between total antioxidant capacity (ABTS •+ decolorization assay) and thiol content of seminal plasma, and sperm DNA damage (8-oxoG immunostaining, TUNEL reaction, comet assay). Low seminal redox status was the strongest correlated with 8-oxoG level which may indicate that seminal total antioxidant capacity influences mainly the formation of single strand DNA breaks in sperm cells. Since inter-individual differences in seminal antioxidant status were reported, we postulated that the redox status of seminal plasma may be an additional important parameter, both with sperm quantitative and morphological analysis, for evaluation of equine semen quality.

Comparison between Glucose-6-Phosphate Dehydrogenase-Deficient and Normal Individuals after Eccentric Exercise

Theoretically, glucose-6-phosphate dehydrogenase (G6PD)-deficient compared with nondeficient individuals may be less capable of performing physical activities and/or may be more vulnerable to muscle damage and oxidative stress. Therefore, the purpose of the present study was to examine the effects of a resistance muscle-damaging exercise bout on muscle function and damage, redox status in plasma, and erythrocytes and hemolysis. Nine males with established G6PD deficiency and nine males with normal G6PD activity performed an eccentric muscle-damaging exercise protocol. Isometric torque, range of motion, delayed onset muscle soreness, and creatine kinase were measured as indices of muscle function and damage. Reduced glutathione, oxidized glutathione, thiobarbituric acid-reactive substances, protein carbonyls, catalase, uric acid, and total antioxidant capacity were measured as indices of blood redox status. Plasma hemoglobin and bilirubin were measured as indices of hemolysis. All measurements conducted before, immediately after, and 1, 2, 3, 4, and 5 d after exercise. All indices measured confirmed that eccentric exercise induced severe muscle damage, oxidative stress, and hemolysis, peaking at 2 and 3 d postexercise. Lower resting levels of reduced glutathione were detected in the G6PD-deficient group compared with the control group. Nevertheless, both the time course and the magnitude of the changes of the selected muscle performance, redox status (both in plasma and in erythrocytes), and hemolysis indices measured were similar between the two groups. The present study indicates that G6PD-deficient individuals may participate in high-intensity muscle-damaging activities, without a negative impact on muscle function, blood redox status, and hemolysis.

B Chronic oral tetrahydrobiopterin treatment in patients with coronary artery disease elevates total biopterin levels but does not improve biopterin redox status or vascular function: a randomised placebo-controlled trial

Background Tetrahydrobiopterin (BH4), a critical endothelial nitric oxide synthase (eNOS) cofactor, is an important determinant of endothelial function. Oral BH4 therapy has been proposed as a treatment for vascular disease. We investigated the pharmacokinetics and pharmacodynamics of oral BH4 in both plasma and vascular tissue to determine whether oral BH4 therapy would improve vascular function in patients with established atherosclerosis and oxidative stress. Methods In a double-blind, randomised trial, 49 patients with coronary artery disease (CAD) awaiting coronary artery bypass graft surgery received oral BH4 700 mg/d (n=16) or 400 mg/d (n=14), or placebo (n=19), for a mean duration of 30 days prior to surgery. Biopterin levels were measured in plasma at baseline and after the treatment period, and in saphenous vein (SV) and internal mammary artery (IMA) tissue collected at the time of surgery. Vascular superoxide and endothelial function were measured ex vivo. Cardiovascular MRI was used to determine brachial artery flow-mediated dilatation and indices of arterial stiffness before and after treatment. Results Plasma BH4 levels were elevated threefold by both low- and high-dose BH4 treatment compared to placebo (p ex vivo , or in MRI indices of vascular function. Conclusion Although absolute BH4 levels in plasma and SV are increased, chronic oral BH4 therapy does not alter biopterin redox status (BH4:BH2 ratio) in either plasma or vascular tissue. Accordingly, oral BH4 does not improve vascular function or oxidative stress in patients with established CAD.

Effects of Sulphur-Containing Compounds on Plasma Redox Status in Muscle-Damaging Exercise

The aim of the study was to compare effects of three-day N-acetylcysteine, alpha-lipoic acid or taurine administration on plasma antioxidant status and oxidative damage markers in healthy men after performing muscle-damaging exercise. Fifty-five healthy and trained men were randomly assigned to N-acetylcysteine (NAC, 1.8 g/day, 3 days), alpha-lipoic acid (ALA, 1.2 g/day, 3 days), taurine (TAU, 3 g/day, 3 days) and control group (CON), and exposed to intense resistance exercise. The resistance exercise induced the muscle damage which was observed by significant increase in total creatine kinase (CK) activity at 24 h rest. The administration of NAC and ALA significantly elevated the resting or/and postexercise plasma total antioxidant status (TAS) and total thiols (TT). Uric acid (UA) concentration was decreased by NAC, ALA and TAU at 24 h rest compared with CON. The plasma lipid peroxidation (TBARS) and protein carbonylation (PC) were considerably reduced by NAC and ALA administration at rest and after exercise. TAU did not have any influence on TAS, TT, TBARS and PC levels. Our study has shown that three-day oral N-acetylcysteine and alpha-lipoic acid administration enhanced plasma total antioxidant status and attenuated oxidative damage whereas taurine did not demonstrate any antioxidant action in healthy men after performing a single muscle-damaging exercise.

Antioxidant level and redox status of coenzyme Q10in the plasma and blood cells of children with diabetes mellitus type 1

Hyperglycaemia has been reported to cause increased production of oxygen free radicals. Oxidative stress may contribute to the pathogenesis of diabetic complications. Coenzyme Q(10) (CoQ(10)) is known for its key role in mitochondrial bioenergetics and is considered as a potent antioxidant and free radical scavenger. This study was conducted to evaluate plasma and blood cell concentrations of CoQ(10) in accordance to its redox capacity in children with diabetes mellitus type 1. CoQ(10) plasma and blood cell concentrations and redox status were measured using high-performance liquid chromatography with electrochemical detection in 43 children with diabetes mellitus type 1 and compared with 39 healthy children. In addition, the diabetic patients were subdivided according to their haemoglobin A1c (HbA1c) values into two groups, that is, those with good control (<8%) and those with poor control (>8%), and the CoQ(10) status was compared between the two groups. Children with type 1 diabetes showed increased plasma levels of CoQ(10) in comparison to healthy children. While CoQ(10) erythrocyte and platelet concentrations did not differ, in the diabetes group, the platelet redox status differed with a significantly increased part of reduced CoQ(10). This difference in concentration and redox status in comparison to healthy controls may be attributed to the subgroup of patients with poor control, as the subdivision of diabetic patients according to their HbA1c values shows. In diabetic children, especially in those with poor control, an increase in plasma concentration and intracellular redox capacity of the antioxidant CoQ(10) may contribute to the body's self-protection during a state of enhanced oxidative stress.

Plasma redox status is impaired in the portacaval shunted rat – the risk of the reduced antioxidant ability

BackgroundPortacaval shunting in rats produces a reduction of hepatic oxidant scavenging ability. Since this imbalance in hepatic oxidant/antioxidant homeostasis could coexist with systemic changes of oxidant stress/antioxidant status, plasma oxidants and antioxidant redox status in plasma of portacaval shunted-rats were determined.ResultsMale Wistar male: Control (n = 11) and with portacaval shunt (PCS; n = 11) were used. Plasma levels of the oxidant serum advanced oxidation protein products (AOPP), lipid hydroperoxides (LOOH), the antioxidant total thiol (GSH) and total antioxidant status (TAX) were measured. Albumin, ammonia, Aspartate-aminotransferase (AST), Alanine-aminotransferase (ALT), thiostatin and alpha-1-acid glycoprotein (α1-AGP) were also assayed 4 weeks after the operation. AOPPs were significantly higher (50.51 ± 17.87 vs. 36.25 ± 7.21 μM; p = 0.02) and TAX was significantly lower (0.65 ± 0.03 vs. 0.73 ± 0.06 mM; p = 0.007) in PCS compared to control rats. Also, there was hypoalbuminemia (2.54 ± 0.08 vs. 2.89 ± 0.18 g/dl; p = 0.0001) and hyperammonemia (274.00 ± 92.25 vs. 104.00 ± 48.05 μM; p = 0.0001) and an increase of thiostatin (0.23 ± 0.04 vs. 0.09 ± 0.01 mg/ml; p = 0.001) in rats with a portacaval shunt. The serum concentration of ammonia is correlated with albumin levels (r = 0.624; p = 0.04) and TAX correlates with liver weight (r = 0.729; p = 0.017) and albumin levels (r = 0.79; p = 0.007)ConclusionThese findings suggest that in rats with a portacaval shunt a systemic reduction of oxidant scavenging ability, correlated with hyperammonemia, is principally produced. It could be hypothesized, therefore, that the reduced antioxidant defences would mediate a systemic inflammation.

Influence of Dietary Quercetin on Glutathione Redox Status in Mice

Flavonoids such as quercetin have been shown to serve as a protective defense against oxidative damage in vivo. However, the bioavailability of quercetin depends on the food source and type of glycosidic moiety linked to the molecule. In this study, mice were fed 1 mg/day quercetin in the form of quercetin aglycone, rutin, apple, or onion, and reduced glutathione (GSH), oxidized glutathione (GSSG), and protein-GSH mixed disulfides were determined to investigate the influence of dietary quercetin on the GSH redox status in metabolically active tissues, mitochondria, and plasma of mice. All quercetin treatment groups produced increases in the GSH:GSSG ratio and decreases in mixed disulfide levels in hepatic tissue. Cardiac tissue did not change in response to dietary quercetin; however, cardiac mitochondria demonstrated a reduction in the GSH:GSSG ratio and an increase in protein mixed disulfide levels. No significant changes were observed in the plasma GSH:GSSG ratio, but mixed disulfide levels were decreased for all of the diets. The changes in plasma redox status did not parallel the changes in the tissues. Onion fed mice demonstrated the greatest increases in GSH:GSSG ratios and the greatest decreases in protein mixed disulfide levels of all diets compared. For all treatment groups, increases in the GSH:GSSG ratios corresponded with decreases in protein mixed disulfide levels. The results of this study indicate that quercetin influences GSH:GSSG ratios and protein thiolation in a tissue-specific manner and that these effects are dependent on food source and bioavailability.

Hyperosmolarity Induced Cystine Transport and Cystine-cysteine Cycle between Erythrocytes and the Plasma

The objective of the present study was to determine if a cystine-cysteine cycle operates between the erythrocytes and the plasma. In the present study we incubated the erythrocytes in krebs ringer phosphate buffer with different osmolarity containing different amounts of cystine. Our results show that erythrocytes do not uptake cystine from the environment when the osmolarity of the buffer is 310 mOsmol/l. Erythrocytes also do not operate a cystinecysteine cycle in this isoosmolar buffer. However, when exposed to hyperosmolar buffer in the ranges that occur in th ek idne ym edulla which is in between 1200–1400 mOsmol/l erythrocytes start to uptake cystine from the environment and induce a cystine-cysteine cycle. The cystine uptake and cystine-cysteine cycle were characterized by measurement of changes in the free -SH concentrations in erythrocytes and in the buffer. Following incubation of erythrocytes in 1 mM cystine containing 1250 and 1300 mOsmol/l buffer, the free -SH concentrations in the buffer reached to 0.102 ± 0.002 and 0.241 ± 0.013µmol/ml erythrocyte respectively. Our results demonstrate that erythrocytes display a cystine-cysteine cycle in hyperosmolar environment which is prevailed mainly in the kidney medulla. Our results also display that this process is biologically active and energy dependent. The observed cystine-cysteine cycle is inhibited when the erythrocytes are incubated at lower temperatures and in the absence of glucose. Our results suggest that erythrocytes uptake cystine, intracellulary reduce it to cysteine and release it back to the environment whe ne xposed to hyperosmolar conditions. Erythrocytes may have a role in the regulation of plasma cystine and cysteine concentrations and may contribute to the regulation of plasma redox status.

Anti-Oxidant Treatment with α-Lipoic Acid and Acetyl L-Carnitine in Hemoglobinopathies.

Oxidative stress is integral to pathophysiology of sickle cell disease and thalassemia. Precipitated globin chains in thalassemia generate free radicals, while increased superoxide, vaso-occlusive crises (VOC) and depletion of NO contribute to pro-oxidant state in SCD. To combat oxidative stress in hemoglobinopathies, potent antioxidant compounds able to neutralize free radicals in both aqueous and lipophilic environments are required. A combination of α-lipoic acid (LA) and acetyl L-carnitine (ALCAR) was given to 14 subjects (SCD = 11, HbH-Constant Spring = 3) who were not on regular red cell transfusions. All subjects except 1 with SCD completed the study. SCD subjects (SS=9, SC=1) had history of frequent VOC with 7/10 receiving hydroxyurea therapy. All subjects were at baseline clinical status with no acute complications from the disease. LA 400 mg and ALCAR 1,000 mg per day were administered for 3 weeks. Some subjects received a higher dose (LA 800/ALCAR 2000 per day) for an extra 3 weeks to test tolerability. Paired t-test was performed to compare variables at baseline and after 3 weeks. Plasma redox sulfur-amino acids were measured by liquid chromatography/tandem mass spectrometry. The redox state of plasma free glutathione (reduced:oxidized GSH) was significantly better at 3 weeks (14.3±4.9 vs. 18.6±3.9, p=.004). Plasma cysteine, methionine and homocysteine redox ratios were unchanged. Intracellular amino acid profile of RBC was significantly altered by LA/ALCAR. LA is a known inducer of GSH synthesis. There was 4-fold increase in cysteine (p=.027), 1.4-fold increase in glycine (p=.003), and 1.7-fold increase in serine (p=.026), all of which are precursors of GSH. The increase in glutamate (1.3-fold) was not significant. Total red cell GSH content was unchanged, though there was a trend towards better GSH redox (1.5-fold, p=.109). RBC arginine content was significantly higher following therapy (1.7-fold, p=.026). Ornithine decreased slightly (0.87-fold, p=n.s.), and arginine:ornithine ratio increased by 2.1-fold (p=.099). The content of other amino acids did not change significantly. The effect of antioxidant therapy on serum markers of inflammation was examined. CRP was elevated (>8 mg/L) at baseline in 5/10 subjects with SCD (range 9–27 mg/L). CRP returned to normal in 3/5 subjects at the end of 3 weeks. The other two subjects with elevated CRP at baseline developed VOC during the study, which was associated with further rise in CRP. Plasma IL-6, elevated at baseline in one subject (16.2 pg/mL), also returned to normal (0.3 pg/mL). Only 1/5 subjects with a normal CRP at baseline (2.7 mg/L) had a higher level (9.1 mg/L) at the end of study. No significant change in the plasma sVCAM level was noted. IL-1β level was below the limit of detection in all cases. Treatment was well tolerated, except for 1 subject (Hb SC) who dropped out after 1 week following a localized neck rash. Three SCD subjects experienced vaso-occlusive (VOC) pain episodes while on study (2 in week 3, 1 in week 6, including 1 death). No adverse events were recorded in subjects with thalassemia. The data demonstrate that treatment with LA/ALCAR is capable of improving plasma redox status in hemoglobinopathies. There is a beneficial impact on RBC amino acid profile, with increase in GSH precursors and a significant improvement in arginine content. Systemic inflammation appeared to improve in subjects with SCD who did not develop VOC while on study.

Glutamate-cysteine ligase polymorphism, hypertension, and male sex are associated with cardiovascular events. Biochemical and genetic characterization of Italian subpopulation

Glutathione (GSH) is an important intravascular scavenger that protects endothelial cells from atherosclerosis. However, it is still unknown whether cardiovascular (CV) events are associated with metabolic and genetic factors, linked to GSH synthesis in an Italian subpopulation, and if a glutamate-cysteine ligase polymorphism within the catalytic subunit (GCLC) could affect blood and plasma GSH concentrations. One hundred subjects, with or without CV risk factors, were enrolled to evaluate plasma and erythrocyte redox status (GSH, homocysteine, cysteine, cysteinylglycine), antioxidant vitamins (alpha-tocopherol and ascorbate), malondialdehyde, a lipid peroxidation product, and the presence of the GCLC-129 C/T polymorphism; an experimental hyperhomocysteinemia after methionine-induced stimulation of transsulfuration pathway was performed in 91% of enrolled subjects. Clinical, biochemical, and genetic variables were correlated with the presence of CV events (myocardial infarction, transient ischemic attacks, and stroke). By multiple logistic regression analysis, male sex (P = .027), hypertension (P = .001), and GCLC C/T genotype (P = .009) were the only variables associated with events. Plasma alpha-tocopherol content decreased postmethionine in the T allele subjects compared with wild type (P for time x group interaction = .001). Plasma-reduced GSH level was higher in C/T than in C/C genotype subjects at both time points (P for group = .03), whereas intracellular GSH concentration did not differ between the 2 genotype groups either at baseline or postmethionine. GCLC T allele, together with hypertension and male sex, is associated with CV events in our study population. Moreover, after stimulation of transsulfuration, intracellular GSH content is preserved in T allele subjects, probably by increases in GSH turnover and export, and consumption of alpha-tocopherol.

Obesity induced insulin resistance and oxidative stress in mice fed a high fat diet

Insulin resistance (IR) is a common feature in obesity. There is a growing body of evidence linking the induction of obesity-related IR to an increase in oxidative stress. Objective: To determine if a high fat diet can induce IR and oxidative stress in a mouse model. To determine if there is an increase in oxidative stress related to adipocyte differentiation. Methods: Male mice (C57B6) were fed a high fat diet (HF) (45% kcal) or a control fed diet (CF) (10% kcal) ad libidum for 6 wk. IR was measured using an intraperitoneal glucose tolerance test. Oxidative stress was measured using thiol redox status in mouse plasma (Cys/CySS) and in differentiated and undifferentiated murine 3T3L1 cells (GSH/GSSG). Results: HF mice were significantly heavier than CF (p<.05). HF mice exhibited hyperglycemia (162±15) vs CF (82±7 mg/dL), as well as glucose intolerance (p<.05). HF mice had a 10 mv increase in plasma oxidation as compared to CF (p<.05). Cellular thiol redox in differentiated 3T3L1 cells was significantly higher (20 mv) than in undifferentiated cells. Conclusion: In this study we demonstrated that HF mice have increased IR and oxidative stress. We also showed that differentiation induces an increase in oxidative stress. This data suggests that oxidative stress and IR occur with increased adiposity. Further studies should determine mechanisms by which oxidative stress plays a role in obesity-induced IR.