Comprehensive plasma thiol redox status determination for metabolomics

Thiol homeostasis plays an important role in human health and aging by regulation of cellular responses to oxidative stress. Due to major constraints that hamper reliable plasma thiol/disulfide redox status assessment in clinical research, we introduce an improved strategy for comprehensive thiol speciation using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that overcomes sensitivity, selectivity and dynamic range constraints of conventional techniques. This method integrates both specific and nonspecific approaches toward sensitivity enhancement for artifact-free quantification of labile plasma thiols without complicated sample handling. A multivariate model was developed to predict increases in ionization efficiency for reduced thiols when conjugated to various maleimide analogs based on their intrinsic physicochemical properties. Optimization of maleimide labeling in conjunction with online sample preconcentration allowed for simultaneous analysis of nanomolar levels of reduced thiols and free oxidized thiols as their intact symmetric or mixed disulfides. Identification of low-abundance thiols and various other polar metabolites detected in plasma was supported by prediction of their relative migration times using CE as a qualitative tool complementary to ESI-MS. Plasma thiol redox status determination together with untargeted metabolite profiling offers a systemic approach for elucidation of the causal role of dysregulated thiol metabolism in the etiology of human diseases.     

Nonequilibrium thermodynamics of thiol/disulfide redox systems: a perspective on redox systems biology

Understanding the dynamics of redox elements in biologic systems remains a major challenge for redox signaling and oxidative stress research. Central redox elements include evolutionarily conserved subsets of cysteines and methionines of proteins which function as sulfur switches and labile reactive oxygen species (ROS) and reactive nitrogen species (RNS) which function in redox signaling. The sulfur switches depend on redox environments in which rates of oxidation are balanced with rates of reduction through the thioredoxins, glutathione/glutathione disulfide, and cysteine/cystine redox couples. These central couples, which we term redox control nodes, are maintained at stable but nonequilibrium steady states, are largely independently regulated in different subcellular compartments, and are quasi-independent from each other within compartments. Disruption of the redox control nodes can differentially affect sulfur switches, thereby creating a diversity of oxidative stress responses. Systems biology provides approaches to address the complexity of these responses. In the present review, we summarize thiol/disulfide pathway, redox potential, and rate information as a basis for kinetic modeling of sulfur switches. The summary identifies gaps in knowledge especially related to redox communication between compartments, definition of redox pathways, and discrimination between types of sulfur switches. A formulation for kinetic modeling of GSH/GSSG redox control indicates that systems biology could encourage novel therapeutic approaches to protect against oxidative stress by identifying specific redox-sensitive sites which could be targeted for intervention.     

Mechanisms and dynamics in the thiol/disulfide redox regulatory network: transmitters, sensors and targets

Plant cells sense, weigh and integrate various endogenous and exogenous cues in order to optimize acclimation and resource allocation. The thiol/disulfide redox network appears to be in the core of this versatile integration process. In plant cells its complexity exceeds by far that of other organisms. Recent research has elucidated the multiplicity of the diversified input elements, transmitters (thioredoxin, glutaredoxins), targets and sensors (peroxiredoxins and other peroxidases), controlled processes and final acceptors (reactive oxygen species). An additional level of thiol/disulfide regulation is achieved by introducing dynamics in time and subcompartment and complex association.     

A novel and rapid apoptosis assay based on thiol redox status

We present here a novel probe, VitaBright-48, for the evaluation of the cellular level of reduced thiols. Using different cell lines and apoptogenic agents we show that a decrease in the level of reduced thiols correlates with well-known apoptotic markers such as phosphatidylserine translocation and caspase activity. The cell population to be investigated is added to the nonfluorescent stain VitaBright-48, which immediately permeates the cell membrane and reacts with intracellular thiols, forming a fluorescent compound. Quantification of the cell fluorescence directly after staining (without washing) can then be used to determine the population's cellular thiol level at the single cell level. Based on the results presented here, we suggest that measurement of changes in the level of free thiols should be added to the list of phenotypes which may be investigated in order to detect apoptosis.     

Thiol/disulfide redox states in signaling and sensing

Abstract

Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol–disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol–disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.     

Extracellular thiol/disulfide redox state affects proliferation rate in a human colon carcinoma (Caco2) cell line

Redox mechanisms function in regulation of cell growth, and variation in redox state of plasma thiol/disulfide couples occurs in various physiologic conditions, including diabetes, chemotherapy, and aging. The present study was designed to determine whether a systematic variation in extracellular thiol/disulfide redox state (E(h)) over a range (0 mV to -150 mV) that occurs in human plasma altered proliferation of cultured cells. Experiments were performed with a human colon carcinoma cell line (Caco2), which grows slowly in the absence of serum and responds to peptide growth factors with increased rate of cell division. The extracellular redox states were established by varying concentrations of cysteine and cystine, maintaining constant pool size in terms of cysteine equivalents. Incorporation of 5-bromo-2-deoxyuridine (BrdU) was used to measure DNA synthesis and was lowest at the most oxidized extracellular E(h) (0 mV). Incorporation increased as a function of redox state, attaining a 100% higher value at the most reduced condition (-150 mV). Addition of insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) increased the rate of BrdU incorporation at more oxidizing redox conditions (0 to -80 mV) but had no effect at -150 mV. Cellular GSH was not significantly affected by variation in extracellular E(h). In the absence of growth factors, extracellular E(h) values were largely maintained for 24 h. However, IGF-1 or EGF stimulated a change in extracellular redox to values similar to that for cysteine/cystine redox in plasma of young, healthy individuals. The results show that extracellular thiol/disulfide redox state modulates cell proliferation rate and that this control interacts with growth factor signaling apparently independently of cellular glutathione.     

Antioxidant enzymes and thiol/disulfide status in the digestive gland of the brown mussel Perna perna exposed to lead and paraquat

Lead (Pb) and paraquat (PQ) have different toxic mechanisms associated with cell damage. Pb may induce alterations in zinc containing proteins, including the known inhibitory effect on the enzyme delta-aminolevulinic acid dehydratase, disrupting the heme-synthesis pathway. During PQ biotransformation, redox cycle reactions enhance oxyradical production, which may lead to pro-oxidative conditions. In this study, we analyzed the effects of Pb and PQ on antioxidant enzymes and thiol status, using the digestive glands of the mussel Perna perna collected in a mussel farm on Santa Catarina Island. Mussels were exposed to Pb (1 ppm) and PQ (10 ppm), either separately or concomitantly, for 48 h. We were unable to detect an effect of Pb treatment on the enzymes, catalase, glucose 6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST) and glutathione reductase (GSSG-reductase), which contrasts to the effect of PQ, increasing GSSG-reductase and G6PDH, but decreasing GST activity. The thiol status showed a pro-oxidative trend, observed mainly through a decrease in the reduced/oxidized glutathione ratio, despite the total-glutathione increase. Protein-mixed disulfides and protein thiols did not change by the treatments. The observed effects of PQ and Pb were consistent with literature. Pb had a suppressive effect on the enzymatic changes elicited by PQ, while the changes in the thiol/disulfide parameters were retained.     

Physical activity and alpha-lipoic acid modulate inflammatory response through changes in thiol redox status

α-Lipoic acid (αLA), as an inductor of hydrogen peroxide (H₂O₂) and nitrogen oxide (NO) generation and modulator of thiol redox status, plays an important role in cell signalling pathways. The study was designed to observe the effect of αLA on inflammatory response through changes in H₂O₂ and NO levels as well as thiol redox status. Sixteen physically active males were randomly assigned to one of two groups: placebo or αLA (1,200 mg?d^?1 for 10 days prior to exercise). The exercise trial involved a 90-min run at 65 % VO₂max (0 % gradient) followed by 15-min eccentric phase at 65 % VO₂max (?10 % gradient). Blood samples were collected before the exercise trial and then again 20 min, 24, and 48 h after. αLA significantly elevated H₂O₂ but reduced NO generation before or after exercise. Thiol redox status (GSH_total-2GSSG/GSSG) increased by >50 % after αLA and exercise (ANOVA, P?<?0.05) and correlated with changes in cytokines interleukin-6 (IL-6) (r?=??0.478, P?<?0.05) and IL-10 (r?=??0.455, P?<?0.05). This was caused by strong effect of αLA on GSSG concentration. αLA elevated IL-6 and IL-10 levels at 20 min after exercise and decreased in interleukin-1β and tumor necrosis factor α before and after exercise. This enhanced the regeneration of injured muscles. Creatine kinase activity tended to lower values after αLA intake. The study suggests that the combination of intense exercise with α-lipoic acid intake might be useful to improve the skeletal muscle regeneration through changes in inflammatory response which are associated with H₂O₂ and NO generation as well as thiol redox status.     

Norethindrone plasma levels in human subjects

An analytical procedure for the estimation of norethindrone (17β-hydrox-19-nor-17α-pregn-4-en-20-yn-3-one) in human plasma has been developed. After extraction and purification on a silver ion exchange column, norethindrone is converted to a methoxime-trimethylsilyl ether derivative and measured by a gas chromatograph-mass spectrometer-accelerating voltage alternator system. Norethindrone-¹³C_20,21^?7?2H is used as a carrier and internal standard. Results correlated well with those obtained by radiochromatography. Maximum plasma levels of norethindrone in chronically treated and naive subjects ranged from 17–38 ng/ml.     

Evidence that the heme regulatory motifs in heme oxygenase-2 serve as a thiol/disulfide redox switch regulating heme binding

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-dependent conversion of heme to biliverdin, CO, and iron. The two forms of HO (HO-1 and HO-2) share similar physical properties but are differentially regulated and exhibit dissimilar physiological roles and tissue distributions. Unlike HO-1, HO-2 contains heme regulatory motifs (HRMs) (McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. D. (1997) J. Biol. Chem. 272, 12568-12574). Here we describe UV-visible, EPR, and differential scanning calorimetry experiments on human HO-2 variants containing single, double, and triple mutations in the HRMs. Oxidized HO-2, which contains an intramolecular disulfide bond linking Cys(265) of HRM1 and Cys(282) of HRM2, binds heme tightly. Reduction of the disulfide bond increases the K(d) for ferric heme from 0.03 to 0.3 microm, which is much higher than the concentration of the free heme pool in cells. Although the HRMs markedly affect the K(d) for heme, they do not alter the k(cat) for heme degradation and do not bind additional hemes. Because HO-2 plays a key role in CO generation and heme homeostasis, reduction of the disulfide bond would be expected to increase intracellular free heme and decrease CO concentrations. Thus, we propose that the HRMs in HO-2 constitute a thiol/disulfide redox switch that regulates the myriad physiological functions of HO-2, including its involvement in the hypoxic response in the carotid body, which involves interactions with a Ca(2+)-activated potassium channel.     

Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-cysteine synthetase) and, particularly, GLR1 (glutathione reductase) are required for survival on DPS. DPS is uniquely thiol-specific, and we found that the cellular mechanisms for DPS detoxification differ substantially from that of the commonly used thiol oxidant diamide. In contrast to this oxidant, the full antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS sensitivity. DPS seems to induce a specific disulfide stress, where an increase in the cytoplasmic/nuclear GSSG/GSH ratio results in putative DPS target(s) becoming sensitive to DPS.     

Role of thiol/disulfide exchange in newcastle disease virus entry

Newcastle disease virus (NDV) entry into host cells is mediated by the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. We previously showed that production of free thiols in F protein is required for membrane fusion directed by F protein (S. Jain et al., J. Virol. 81:2328-2339, 2007). In the present study we evaluated the oxidation state of F protein in virions and virus-like particles and its relationship to activation of F protein by HN protein, F protein conformational intermediates, and virus-cell fusion. F protein, in particles, does not have free thiols, but free thiols were produced upon binding of particles to target cells. Free thiols were produced at 16°C in F protein in virions bound to the target cells. They also appeared in different fusion defective mutant F proteins. Free thiols were produced in the presence of mutant HN proteins that are defective in F protein activation but are attachment competent. These results suggest that free thiols appear prior to any of the proposed major conformational changes in F protein which accompany fusion activation. These results also indicate that HN protein binding to its receptor likely facilitates the interaction between F protein and host cell isomerases, leading to reduction of disulfide bonds in F protein. Taken together, these results show that free thiols are produced in F protein at a very early stage during the onset of fusion and that the production of free thiols is required for fusion in addition to activation by HN protein.     

Baseline levels of elemental concentrations in whole blood,plasma, and erythrocytes of Nigerian subjects

Biological Trace Element Research - In this pioneering effort on Nigerians, the arithmetic means, ranges, and standard deviations for the concentrations of the 13 elements, Br, Ca, Cl, Cu, Fe, K,...     

The Bacillus subtilis YkuV is a thiol:disulfide oxidoreductase revealed by its redox structures and activity

The Bacillus subtilis YkuV responds to environmental oxidative stress and plays an important role for the bacteria to adapt to the environment. Bioinformatic analysis suggests that YkuV is a homolog of membrane-anchored proteins and belongs to the thioredoxin-like protein superfamily containing the typical Cys-Xaa-Xaa-Cys active motif. However, the biological function of this protein remains unknown thus far. In order to elucidate the biological function, we have determined the solution structures of both the oxidized and reduced forms of B. subtilis YkuV by NMR spectroscopy and performed biochemical studies. Our results demonstrated that the reduced YkuV has a low midpoint redox potential, allowing it to reduce a variety of protein substrates. The overall structures of both oxidized and reduced forms are similar, with a typical thioredoxin-like fold. However, significant conformational changes in the Cys-Xaa-Xaa-Cys active motif of the tertiary structures are observed between the two forms. In addition, the backbone dynamics provide further insights in understanding the strong redox potential of the reduced YkuV. Furthermore, we demonstrated that YkuV is able to reduce different protein substrates in vitro. Together, our results clearly established that YkuV may function as a general thiol:disulfide oxidoreductase, which acts as an alternative for thioredoxin or thioredoxin reductase to maintain the reducing environment in the cell cytoplasm.     

Reversible change in thiol redox status of the insulin receptor alpha-subunit in intact cells

In this study, we used maleimidobutyrylbiocytin to examine possible alteration that may occur in the redox state of the insulin receptor (IR) sulfhydryl groups in response to reduced glutathione (GSH) or N-acetyl-L-cysteine (NAC). Short-term treatment of intact cells expressing large numbers of IR with GSH or NAC led to a rapid and reversible reduction of IR alpha-subunit disulfides, without affecting the receptor beta-subunit thiol reactivity. The overall integrity of the oligomeric structure of IR was maintained, indicating that neither class I nor class II disulfides were targeted by these agents. Similar findings were obtained in cells transfected with IR mutants lacking cysteine524, one of the class I disulfides that link the two IR alpha-subunits. Membrane-associated thiols did not participate in GSH- or NAC-mediated reduction of IR alpha-subunit disulfides. No difference in insulin binding was observed in GSH-treated cells; however, ligand-mediated increases in IR autophosphorylation, tyrosine phosphorylation of cellular substrates, and dual phosphorylation of the downstream target mitogen-activated protein kinase were inhibited at concentrations of GSH (10 mM or greater) that yielded a significant increase in IR alpha-subunit thiol reactivity. GSH did not affect IR signaling in the absence of insulin. Our results provide the first evidence that the IR alpha-subunit contains a select group of disulfides whose redox status can be rapidly altered by the reducing agents GSH and NAC.     

Measurement of fasting serum apoB-48 levels in normolipidemic and hyperlipidemic subjects by ELISA

The present study was designed to evaluate the metabolism of chylomicron and chylomicron remnants by measuring serum apolipoprotein B-48 (apoB-48) levels in 335 normolipidemic and 253 hyperlipidemic subjects using a novel ELISA system. The distribution of fasting serum apoB-48 levels in normolipidemic subjects varied widely, ranging from <1 to >24 microgram/ml (mean, 5.2 +/- 3.8 microgram/ml; median, 3.9 microgram/ml). Serum apoB-48 levels correlated with serum triglyceride (TG) concentrations (r = 0.45, P < 0.001), but not with total cholesterol levels. Serum apoB-48 levels were 7 to 18 times higher in patients with Type I, Type V, and Type III hyperlipidemia, and only slightly higher in patients with Type IIa, Type IIb, and Type IV hyperlipidemia, compared with normolipidemic subjects. The calculated apoB-48/TG ratio was elevated only in patients with dysbetalipoproteinemia (apoE2/2 phenotype). In normolipidemic subjects, oral fat loading resulted in about 2-fold increase in serum apoB-48 levels, with a peak level recorded at 3-4 h postloading, and then returned to the baseline level within 6 h. On the other hand, in patients with dysbetalipoproteinemia, serum apoB-48 levels did not change considerably. Our results indicate that serum apoB-48 is a very useful parameter for evaluating lipoprotein metabolism in exogenous pathways.     

Protective effect of atorvastatin on circadian regulation of liver marker enzymes and redox status in hyperlipidemic rats

Hyperlipidemia was induced in rats by administering 2% cholesterol, 20% coconut oil, and 0.125% cholic acid for 10 weeks. Atorvastatin (0.8 mg/kg b.w.) was administered orally to rats together with high-fat diet for 10 weeks. At the end of the experimental period, the circadian characteristics (acrophase, amplitude, and mesor) of liver marker enzymes (aspartate aminotransferase and alanine transferase), lipid peroxidation products (thiobarbituric acid reactive substances (TBARS), and antioxidants (superoxide dismutase, catalase, reduced glutathione, and glutathione peroxidase) were analyzed. Circadian characteristics (mesor, amplitude, and acrophase) of liver marker enzymes, TBARS, and antioxidants were altered in high-fat diet-induced rats, and the diminished amplitude along with decreased mesor levels of antioxidants were observed in high-fat diet-induced rats. Further, oral administration of atorvastatin to high-fat diet-induced rats showed the normalized mesor, amplitude, and acrophase. These findings suggest that the antihyperlipidemic potential of atorvastatin could modulate the circadian patterns of liver marker enzymes and redox status in hyperlipidemic rats.