GSSG Concentrations Research Articles

Heritability of glutathione and related metabolites in stored red blood cells

Red blood cells (RBCs) collected for transfusion deteriorate during storage. This deterioration is termed the “RBC storage lesion.” There is increasing concern over the safety, therapeutic efficacy, and toxicity of transfusing longer-stored units of blood. The severity of the RBC storage lesion is dependent on storage time and varies markedly between individuals. Oxidative damage is considered a significant factor in the development of the RBC storage lesion. In this study, the variability during storage and heritability of antioxidants and metabolites central to RBC integrity and function were investigated. In a classic twin study, we determined the heritability of glutathione (GSH), glutathione disulfide (GSSG), the status of the GSSG,2H+/2GSH couple (Ehc), and total glutathione (tGSH) in donated RBCs over 56 days of storage. Intracellular GSH and GSSG concentrations both decrease during storage (median net loss of 0.52±0.63mM (median ± SD) and 0.032±0.107mM, respectively, over 42 days). Taking into account the decline in pH, Ehc became more positive (oxidized) during storage (median net increase of 35±16mV). In our study population heritability estimates for GSH, GSSG, tGSH, and Ehc measured over 56 days of storage are 79, 60, 67, and, 75%, respectively. We conclude that susceptibility of stored RBCs to oxidative injury due to variations in the GSH redox buffer is highly variable among individual donors and strongly heritable. Identifying the genes that regulate the storage-related changes in this redox buffer could lead to the development of new methods to minimize the RBC storage lesion.

Oxidative stress markers after a race in professional cyclists.

The aim was to determine the levels and activities of the oxidative stress markers in erythrocytes, plasma, and urine after a flat cyclist stage. Eight voluntary male professional trained-cyclists participated in the study. Exercise significantly increased erythrocyte, leukocyte, platelet, and reticulocyte counts. The exercise induced significant increases in the erythrocyte activities of catalase (19.8%) and glutathione reductase (19.2%), while glutathione peroxidase activity decreased significantly (29.3%). Erythrocyte GSSG concentration was significantly increased after exercise (21.4%), whereas GSH was significantly diminished (20.4%). Erythrocyte malondialdehyde levels evidenced a significant decrease 3 h after finishing the stage (44.3%). Plasma malondialdehyde, GSH and GSSG levels significantly decreased after 3 hr recovery (26.8%, 48.6%, and 31.1%, respectively). The exercise significantly increased the F2-isoprostane concentration in urine from 359 ± 71 pg/mg creatinine to 686 ± 139 pg/mg creatinine. In conclusion, a flat cycling stage induced changes in oxidative stress markers in erythrocytes, plasma, and urine of professional cyclists. Urine F2-isoprostane is a more useful biomarker for assessing the effects of acute exercise than the traditional malondialdehyde measurement.

Anti-inflammatory effects of betaine on AOM/DSS‑induced colon tumorigenesis in ICR male mice.

Betaine is an important human nutrient obtained from various foods and studies in animals and humans have provided results suggesting their pathogenesis of various chronic diseases and points to a role in risk assessment and disease prevention. However, the molecular mechanisms of its activity remain poorly understood and warrant further investigation. This study was performed to investigate the anti-inflammation and tumor preventing capacity of betaine on colitis-associated cancer in mice. In in vivo experiments, we induced colon tumors in mice by azoxymethane (AOM) and dextran sulfate sodium (DSS) and evaluated the effects of betaine on tumor growth. Administration with betaine significantly decreased the incidence of tumor formation with downregulation of inflammation. Treatment with betaine inhibited ROS generation and GSSG concentration in colonic mucosa. Based on the qPCR data, administration of betaine inhibited inflammatory cytokines such TNF-α, IL-6, iNOS and COX-2. In in vitro experiments, LPS-induced NF-κB and inflammatory-related cytokines were inhibited by betaine treatment in RAW 264.7 murine macrophage cells. Our findings suggest that betaine is one of the candidates for the prevention of inflammation-associated colon carcinogenesis.

Iron toxicity in field-cultivated rice: contrasting tolerance mechanisms in distinct cultivars

Iron toxicity is a major nutritional disorder in irrigated and rainfed waterlogged rice. To elucidate mechanisms involved in tolerance to iron toxicity, plants from one cultivar susceptible to iron toxicity (BR-IRGA 409) and two tolerant cultivars (EPAGRI 108 and EPAGRI 109) were grown in the field, at an iron-toxic site and at a control site in Southern Brazil. We evaluated chlorophyll concentrations, carbonyl concentrations, iron concentrations in leaves and roots, antioxidative enzyme activities (SOD, APX, CAT, GR and DHR), concentrations of reduced and oxidized forms of ascorbate and glutathione, and gene expression profile of four SOD genes in rice leaves. Only plants from the susceptible cultivar showed symptoms of iron toxicity when grown at the iron-toxic site, accumulating high levels of iron in leaves. EPAGRI 108 plants had the lowest iron concentration in leaves and reached the highest iron concentration in the root symplast, suggesting that the capacity to safely store iron in root cells and to limit iron translocation to shoots could be a tolerance mechanism in this cultivar. Plants from the susceptible cultivar showed higher APX activity as well as higher DHA and GSSG concentrations. Plants from the EPAGRI 109 cultivar accumulated high iron levels in leaves, and showed the highest SOD, GR and DHR activities when grown in the iron-toxic site. The same cultivar also showed the highest expression of three out of four SOD genes tested. Therefore, the two tolerant cultivars seem to rely on different mechanisms to deal with iron toxicity in field conditions: limiting iron translocation to the shoot or inducing enzymes-dependent leaf tolerance.

Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization

Summary A high-performance liquid chromatography (HPLC) method was used to assess the concentration of reduced and oxidized glutathione (GSH and GSSG) in rat striatum. Following decapitation, striatum was isolated from the male Wistar rat brain and immediately homogenized with double distilled water. GSH level was determined after pre-column derivatization with o-phthalaldehyde (OPA). The optimal incubation time with OPA was tested. The concentration of GSSG was determined after blockage the thiol groups of GSH by N-ethylmaleimide (NEM). The useful time for incubation with NEM was optimized. Next, disulfide bounds of GSSG were reduced by dithiothreitol (DTT), and released GSH is derivatized with OPA. The total glutathione, tGSH (sum of free and bound GSH, GSSG, and other low-molecular-mass aminothiols), was determined after reduction with DTT and then derivatization with OPA. The level of GSSG was calculated of the difference in concentrations of tGSH and GSH, but we showed that the calculated concentrati...

89: Early Postnatal Exposure to Oxidants Induces Prolonged Oxidative Stress and Increases the Incidence of Oxidative Stress Related Diseases in Preterm Infants les Less than 29 Weeks Gestational Age

In preterm infants, the antioxidant defenses are still poorly developed. The two major sources of oxidants during intensive care stay are oxygen and parenteral nutrition (PN) which is contaminated with peroxides. We hypothesize that early O2 supplementation and longer PN duration increase the risk for oxidative stress associated diseases such as bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP) and necrotizing enterocolitis (NEC). To assess the effect of early oxygen (on day 7 and 28) and the PN duration on oxidative stress markers at 36 weeks corrected age and on oxidative stress related diseases. A prospective observational study including all infants <29 weeks GA without major congenital anomalies admitted to our NICU during one year period (116 infants). During the first week of life, in fifty-one out of 116 infants, consent for blood sample at 36 corrected age (CA) was obtained. GSH and GSSG (nmol/mg protein) were measured by capillary electrophoresis and were used for redox potential (mV) calculation using Nernst equation -expressed as mean (sem). BPD was defined as the need of O2 supplement at 36 weeks CA. Significant ROP was defined as any ROP stage 3 or higher or that required laser or anti-VEGF treatment. Cases of NEC grade 2 or higher according to Bell's criteria were included. Student's t test or χ2 were used, as appropriate, *=P<0.05, **=P<0.01. Basline chracteristics was similar in the consent group and the whole cohort. FiO2 ≥25 on day 7 and 28 of life and PN duration >14 days resulted in higher GSSG concentration and more oxidized redox potential at 36 weeks CA indicating prolonged oxidative stress. FiO2 ≥25 early on life and PN duration >14 days increased the incidence of BPD, significant ROP and NEC (Table 1). Early life exposure to oxidants is associated with prolonged oxidative stress and higher incidence of oxidative stress-related diseases. These results suggest that strategies targeting judicious O2 use and either decreasing the duration or using safer formulation PN will help decreasing the incidence of BPD, ROP and NEC.

Ontogeny of redox regulation in Atlantic cod (Gadus morhua) larvae

The reduction potential of a cell is related to its fate. Proliferating cells are more reduced than those that are differentiating, whereas apoptotic cells are generally the most oxidized. Glutathione is considered the most important cellular redox buffer and the average reduction potential (Eh) of a cell or organism can be calculated from the concentrations of glutathione (GSH) and glutathione disulfide (GSSG). In this study, triplicate groups of cod larvae at various stages of development (3 to 63 days post-hatch; dph) were sampled for analyses of GSSG/2GSH concentrations, together with activities of antioxidant enzymes and expression of genes encoding proteins involved in redox metabolism. The concentration of total GSH (GSH+GSSG) increased from 610±100 to 1260±150 μmol/kg between 7 and 14dph and was then constant until 49dph, after which it decreased to 810±100μmol/kg by 63dph. The 14- to 49-dph period, when total GSH concentrations were stable, coincides with the proposed period of metamorphosis in cod larvae. The concentration of GSSG comprised approximately 1% of the total GSH concentration and was stable throughout the sampling series. This resulted in a decreasing Eh from −239±1 to −262±7mV between 7 and 14dph, after which it remained constant until 63dph. The changes in GSH and Eh were accompanied by changes in the expression of several genes involved in redox balance and signaling, as well as changes in activities of antioxidant enzymes, with the most dynamic responses occurring in the early phase of cod larval development. It is hypothesized that metamorphosis in cod larvae starts with the onset of mosaic hyperplasia in the skeletal muscle at approximately 20dph (6.8mm standard length (SL)) and ends with differentiation of the stomach and disappearance of the larval finfold at 40 to 50dph (10–15mm SL). Thus, metamorphosis in cod larvae seems to coincide with high and stable total concentrations of GSH.

Roles of sedentary aging and lifelong physical activity in exchange of glutathione across exercising human skeletal muscle

Reactive oxygen species (ROS) are important signaling molecules with regulatory functions, and in young and adult organisms, the formation of ROS is increased during skeletal muscle contractions. However, ROS can be deleterious to cells when not sufficiently counterbalanced by the antioxidant system. Aging is associated with accumulation of oxidative damage to lipids, DNA, and proteins. Given the pro-oxidant effect of skeletal muscle contractions, this effect of age could be a result of excessive ROS formation. We evaluated the effect of acute exercise on changes in blood redox state across the leg of young (23±1 years) and older (66±2 years) sedentary humans by measuring the whole blood concentration of the reduced (GSH) and oxidized (GSSG) forms of the antioxidant glutathione. To assess the role of physical activity, lifelong physically active older subjects (62±2 years) were included. Exercise increased the venous concentration of GSSG in an intensity-dependent manner in young sedentary subjects, suggesting an exercise-induced increase in ROS formation. In contrast, venous GSSG levels remained unaltered during exercise in the older sedentary and active groups despite a higher skeletal muscle expression of the superoxide-generating enzyme NADPH oxidase. Arterial concentration of GSH and expression of antioxidant enzymes in skeletal muscle of older active subjects were increased. The potential impairment in exercise-induced ROS formation may be an important mechanism underlying skeletal muscle and vascular dysfunction with sedentary aging. Lifelong physical activity upregulates antioxidant systems, which may be one of the mechanisms underlying the lack of exercise-induced increase in GSSG.

Short term feeding of a high fat diet exerts an additive effect on hepatocellular damage and steatosis in liver-specific PTEN knockout mice.

BackgroundHepatospecific deletion of PTEN results in constitutive activation of Akt and increased lipogenesis. In mice, the addition of a high fat diet (HFD) downregulates lipogenesis. The aim of this study was to determine the effects of a HFD on hepatocellular damage induced by deletion of PTEN.Methods12 Week old male flox/flox hepatospecific PTEN mice (PTENf/f) or Alb-Cre controls were fed a HFD composed of 45% fat-derived calories (from corn oil) or a normal chow. Animals were then analyzed for hepatocellular damage, oxidative stress and expression of enzymes involved in fatty acid metabolism.ResultsIn the Alb-Cre animals, the addition of a HFD resulted in a significant increase in liver triglycerides and altered REDOX capacity as evidenced by increased GPX activity, decreased GST activity and decreased hepatic concentrations of GSSG. In addition, SCD2, ACLY and FASN were all downregulated by the addition of HFD. Furthermore, expression of PPARα and PPARα-dependent proteins Cyp4a and ACSL1 were upregulated. In the PTENf/f mice, HFD resulted in significant increased in ALT, serum triglycerides and decreased REDOX capacity. Although expression of fatty acid synthetic enzymes was elevated in the chow fed PTENf/f group, the addition of HFD resulted in SCD2, ACLY and FASN downregulation. Compared to the Alb-Cre HFD group, expression of PGC1α, PPARα and its downstream targets ACSL and Cyp4a were upregulated in PTENf/f mice.ConclusionsThese data suggest that during conditions of constitutive Akt activation and increased steatosis, the addition of a HFD enhances hepatocellular damage due to increased CD36 expression and altered REDOX status. In addition, this work indicates HFD-induced hepatocellular damage occurs in part, independently of Akt signaling.

Interaction of plasma glutathione redox and folate deficiency on arsenic methylation capacity in Bangladeshi adults

Inorganic arsenic(As) is metabolized through a series of methylation reactions catalyzed by arsenic(III)-methyltransferase (AS3MT), resulting in the generation of monomethylarsonic (MMAs) and dimethylarsinic acids (DMAs). AS3MT activity requires the presence of the methyl donor S-adenosylmethionine, a product of folate-dependent one-carbon metabolism, and a reductant. Although glutathione (GSH), the primary endogenous antioxidant, is not required for As methylation, GSH stimulates As methylation rates in vitro. However, the relationship between GSH redox and As methylation capacity in humans is unknown. We wished to test the hypothesis that a more oxidized plasma GSH redox status is associated with decreased As methylation capacity and examine whether these associations are modified by folate nutritional status. Concentrations of plasma GSH and GSSG, plasma folate, total blood As (bAs), total urinary As (uAs), and uAs metabolites were assessed in a cross-sectional study of n=376 Bangladeshi adults who were chronically exposed to As in drinking water. We observed that a decreased plasma GSH/GSSG ratio (reflecting a more oxidized redox state) was significantly associated with increased urinary %MMA, decreased urinary %DMA, and increased total bAs in folate-deficient individuals (plasma folate ≤9.0nmol/L). Concentrations of plasma GSH and GSSG were independently associated with increased and decreased As methylation capacity, respectively. No significant associations were observed in folate-sufficient individuals, and interactions by folate status were statistically significant. Our findings suggest that GSH/GSSG redox regulation might contribute to the large interindividual variation in As methylation capacity observed in human populations.

Genetic regulation of glutathione concentrations and redox balance in kidney (1134.5)

Glutathione is an essential component of the endogenous antioxidant defense system. A decline in glutathione redox balance ― the ratio of reduced (GSH) to oxidized (GSSG) glutathione ― suggests oxidative stress and is associated with kidney disease. Despite the importance of GSH/GSSG, the genetic regulation of this trait is not well defined. In this study, we measured kidney concentrations of GSH and GSSG in mice from 30 genetically‐diverse inbred mouse strains. Genetic background caused an approximately three‐fold difference in kidney GSH/GSSG among the most disparate strains. Haplotype association mapping revealed single nucleotide polymorphisms associated with glutathione concentrations and redox balance. We narrowed the number of significant loci by placing emphasis on those located within protein‐coding genes, and we compared these candidate genes to the results of our previous genetic analysis of hepatic GSH/GSSG. No candidate genes were associated with both hepatic and renal GSH/GSSG, suggesting that genetic regulation of GSH/GSSG occurs predominantly in a tissue‐specific manner. To our knowledge, this is the first quantitative trait loci study to examine the genetic regulation of glutathione concentrations and redox balance in mammals. We identified novel candidate genes that have the potential to redefine our knowledge of renal redox biochemistry and possibly inform future therapies for kidney disease.Grant Funding Source: Supported by the following NIH grants: GM101723 (R.P.) and AG032333 (D.E.H.).

The heart as an endocrine source of metabolic vasodilators (1079.26)

During heightened cardiac work, O2 consumption by the heart increases. This increase benefits energy production via mitochondrial e¯ transport, but the process of e¯ transport is not perfect and some e¯ “leak” from respiratory chain and reduce O2. This reduction yields O2˙¯, which reacts with thiol groups, iron, and is rapidly metabolized into H2O2 by mitochondrial superoxide dismutase (SOD2). To understand the potential systemic effects of the metabolic dilator, H2O2, we studied mice with cardiac specific SOD2 overexpression, which would increase the amount of H2O2 produced by the heart. To determine if this enhanced production of H2O2 results in a humoral action, i.e., systemic vasodilation, we first evaluated the effectiveness of SOD2 overexpression to effectively remove O2˙¯ from the mitochondria (EPR). SOD2 transgenic mice produced less O2, and had lower redox activity. Mitochondria of SOD2‐tg (vs WT) had lower GSSG concentration (decreased by 37.2±4.5%, GSH/GSSG ratio was increased by 66.7±6.8%). EPR analysis of the isolated mitochondria showed significant decrease of semiquinone in the SOD2‐tg vs WT at the Qi site (by 69.4±9.9%), and a diminished complex I protein thiyl radical(s) in the mitochondria of SOD2‐tg. These results support a more reducing milieu in the mitochondria of the SOD2‐tg heart. Cardiac mitochondria isolated from SOD2‐tg exhibited no significant differences in the respiratory control index between WT and SOD2‐tg (6.30.3 vs. 5.70.5, p&gt;0.05). These results support the concept that the SOD2 overexpression is effective and results in increased production of H2O2. Moreover, arterial pressure was lower in the SOD2‐tg, but was normalized by iv injection of catalase, implying that the metabolic dilator, H2O2, “spilled” over inducing systemic vasodilation. In conclusion, SOD2 overexpression in myocytes triggers a more reductive milieu in murine heart enhancing the production of H2O2, which then acts as an endocrine dilator causing reductions in arterial pressure.Grant Funding Source: NIH RO1 HL083237

Exhaled breath condensate in intubated neonates- a window into the lung’s glutathione status

BackgroundAnalysis of exhaled breath condensates (EBC) is a non-invasive technique to evaluate biomarkers such as antioxidants in the pediatric population, but limited data exists of its use in intubated patients, particularly newborns. Currently, tracheal aspirate (TA) serves as the gold standard collection modality in critically ill newborns, but this method remains invasive. We tested the hypothesis that glutathione status would positively correlate between EBC and TA collections in intubated newborns in the Newborn Intensive Care Unit (NICU). We also hypothesized that these measurements would be associated with alveolar macrophage (AM) glutathione status in the newborn lung.MethodsReduced glutathione (rGSH), glutathione disulfide (GSSG), and total GSH (rGSH + (2 X GSSG)) were measured in sequential EBC and TA samples from 26 intubated newborns via high performance liquid chromatography (HPLC). Additionally, AM glutathione was evaluated via immunofluorescence. Pearson’s correlation coefficient and associated 95% confidence intervals were used to quantify the associations between raw and urea-corrected concentrations in EBC and TA samples and AM staining. Statistical significance was defined as p ≤ 0.05 using two-tailed tests. The sample size was projected to allow for a correlation coefficient of 0.5, with 0.8 power and alpha of 0.05.ResultsEBC was obtainable from intubated newborns without adverse clinical events. EBC samples demonstrated moderate to strong positive correlations with TA samples in terms of rGSH, GSSG and total GSH. Positive correlations between the two sampling sites were observed in both raw and urea-corrected concentrations of rGSH, GSSG and total GSH. AM glutathione staining moderately correlated with GSSG and total GSH status in both the TA and EBC.ConclusionsGSH status in EBC samples of intubated newborns significantly correlated with the GSH status of the TA sample and was reflective of cellular GSH status in this cohort of neonatal patients. Non-invasive EBC sampling of intubated newborns holds promise for monitoring antioxidant status such as GSH in the premature lung. Further studies are necessary to evaluate the potential relationships between EBC biomarkers in the intubated premature newborn and respiratory morbidities.

Glutathione Preservation during Storage of Rat Lenses in Optisol-GS and Castor Oil

BackgroundGlutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations.Methodology/Principal FindingsRat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree.Conclusions/SignificanceIt was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours.

Assay of total glutathione and glutathione disulphide in seminal plasma of male partners of couples presenting for a fertility evaluation

A method is described here for the determination of total glutathione (TGSH) and glutathione disulphide (GSSG) in the seminal plasma of the male partners of couples requesting a fertility evaluation. A suitable sample preparation procedure prior to high-performance liquid chromatography analysis is discussed. After adequate sample preparation, the samples were derivatised with ortho-phthaldialdehyde to form a stable, highly fluorescent tricyclic derivative. Reversed-phase column chromatography was used for the separation, and the effluent was monitored with a fluorescence detector at an excitation wavelength of 350nm and an emission wavelength of 420nm. The analytical performance of this method was satisfactory. The intra-assay and inter-assay coefficients of variation were below 10%. The recoveries were as follows: 94.1% (CV 2.3%) for TGSH and 93.2% (CV 4.0%) for GSSG. No significant differences were found in either TGSH or GSSG concentration between the smokers and nonsmokers (2.07±1.28μm versus 1.56±1.20μm, P=0.431 and 95±56nm versus 112±138nm, P=0.825).

A biophysically based mathematical model for the catalytic mechanism of glutathione reductase

Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as the reducing cofactor, and thereby maintains a constant GSH level in the system. GSH scavenges superoxide (O2−) and hydroxyl radicals (OH) nonenzymatically or by serving as an electron donor to several enzymes involved in reactive oxygen species (ROS) detoxification. In either case, GSH oxidizes to GSSG and is subsequently regenerated by the catalytic action of GR. Although the GR kinetic mechanism has been extensively studied under various experimental conditions with variable substrates and products, the catalytic mechanism has not been studied in terms of a mechanistic model that accounts for the effects of the substrates and products on the reaction kinetics. The aim of this study is therefore to develop a comprehensive mathematical model for the catalytic mechanism of GR. We use available experimental data on GR kinetics from various species/sources to develop the mathematical model and estimate the associated model parameters. The model simulations are consistent with the experimental observation that GR operates via both ping–pong and sequential branching mechanisms based on relevant concentrations of its reaction substrate GSSG. Furthermore, we show the observed pH-dependent substrate inhibition of GR activity by GSSG and bimodal behavior of GR activity with pH. The model presents a unique opportunity to understand the effects of products on the kinetics of GR. The model simulations show that under physiological conditions, where both substrates and products are present, the flux distribution depends on the concentrations of both GSSG and NADP+, with ping–pong flux operating at low levels and sequential flux dominating at higher levels. The kinetic model of GR may serve as a key module for the development of integrated models for ROS-scavenging systems to understand protection of cells under normal and oxidative stress conditions.

Glutathione controls the redox state of the mitochondrial carnitine/acylcarnitine carrier Cys residues by glutathionylation

BackgroundThe mitochondrial carnitine/acylcarnitine carrier (CAC) is essential for cell metabolism since it catalyzes the transport of acylcarnitines into mitochondria allowing the β-oxidation of fatty acids. CAC functional and structural properties have been characterized. Cys residues which could form disulfides suggest the involvement of CAC in redox switches. MethodsThe effect of GSH and GSSG on the [3H]-carnitine/carnitine antiport catalyzed by the CAC in proteoliposomes has been studied. The Cys residues involved in the redox switch have been identified by site-directed mutagenesis. Glutathionylated CAC has been assessed by glutathionyl-protein specific antibody. ResultsGSH led to increase of transport activity of the CAC extracted from liver mitochondria. A similar effect was observed on the recombinant CAC. The presence of glutaredoxin-1 (Grx1) accelerated the GSH activation of the recombinant CAC. The effect was more evident at 37°C. GSSG led to transport inhibition which was reversed by dithioerythritol (DTE). The effects of GSH and GSSG were studied on CAC Cys-mutants. CAC lacking C136 and C155 was insensitive to both reagents. Mutants containing these two Cys responded as the wild-type. Anti-glutathionyl antibody revealed the formation of glutathionylated CAC. ConclusionsCAC is redox-sensitive and it is regulated by the GSH/GSSG couple. C136 and C155 are responsible for the regulation which occurs through glutathionylation. General significanceCAC is sensitive to the redox state of the cell switching between oxidized and reduced forms in response to variation of GSSG and GSH concentrations.

The state of reduced glutathione in patients with impaired glycemic states and coronary heart disease

Antioxidative mechanisms prevent human body from the damaging action of free radicals and reactive oxygen species. Glutathione and related to it enzyme systems detoxicate H2O2 and hyperoxide and from there, reduced glutathione is a potential marker of oxidative stress level. Purpose: To investigete glutathione of erythrocytes in patients with prediabetes (IGT & IFG) and type 2 diabetes and concomitant coronary heart disease (CHD). Matherial and methods: We examined 25 patients with T2D and concomitant CHD and atherosclerosis (group 1), 20 patients with prediabetes (IGT & IFG) newly diagnosed and the same co-morbidity (group 2) and 47 almost healthy person (group 3). Examinations included clinical laboratory evaluation, fasting glicose, HbA1c. Total glutathione (GSHt) and oxidized glutathione (GSSG) were measered by use of glutathione reductase reaction. Redox potential (Eh) of glutathione was evaluated by use of Nernst equation. Results: Concentrations of GSHt and GSH were greater in group 2 to compare with groups 1 and 3 (by 4,2 times, p=0,012 and by 3,1 times, p=0,005 respectively). Increase of concentration of GSSG in group 1 may change extracellular redox condition. Calculation of Eh revealed its change to oxidized condition in group 1 to compare with group 2 (19,0 mV greater) and group 3 (15,4 mV greater). Conclusion: Antioxidant defense in red-cells is overactivated during prediabetes (IGT & IFG) with concomitant CHD and atherosclerosis while in patients with T2D was noticed depletion of compensatory mechanisms with increased GSSG and Eh. Reduction in 2GSH/GSSG suggests that intracellular antioxidant is lost; herewith cells become sensitive to oxidative stress.

Analysis of GSH and GSSG after derivatization with N-ethylmaleimide

This protocol describes a procedure for determining glutathione (GSH) and glutathione disulfide (GSSG) concentrations in blood and other tissues. Artifactual oxidation to GSSG of 5-15% of the GSH found in a sample can occur during deproteination of biological samples with any of the commonly used acids, with consequent marked overestimation of GSSG. This can be prevented by derivatizing GSH with the alkylating agent N-ethylmaleimide (NEM) to form GS-NEM before acid deproteination, followed by back-extraction of excess NEM from the deproteinized samples with dichloromethane. GSSG concentration is then measured by spectrophotometry with the GSH recycling method, on the basis of conversion of GSSG to GSH by glutathione reductase and NADPH and reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). GSH concentration is instead measured by either of two methods: by analysis of GS-NEM conjugates by HPLC in the same sample that is used to measure GSSG or, alternatively, by analysis of GSH by spectrophotometry (GSH recycling method) on one additional sample aliquot that has not been derivatized with NEM. The procedure can assay GSH and GSSG in blood and other tissues in 30 min or less.

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.