Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver

A liquid chromatography/tandem mass spectrometry assay of glutathione (GSH), glutathione disulfide (GSSG) and of precursors (gamma-glutamyl-cysteine, cysteinyl-glycine, cysteine, cystine, homocysteine and homocystine) was developed to study glutathione synthesis in mice liver. After iodoacetic acid derivatization, the analytes were analyzed using reversed-phase gradient HPLC and detected using multiple reaction monitoring. Linear calibrations were performed over the concentrations range of 100-10,000 ng/mL for the thiol-containing precursors and extended up to 100,000 ng/mL for GSH and GSSG. The method was validated for each compound with inter-day accuracy below 11.9% and with precision below 15%. The method showed low limits of quantitation of 100 ng/mL for each thiol-containing compound and GSSG and of 200 ng/mL for other disulfides.     

Enzymatic determination of BPA by means of tyrosinase immobilized on different carbon carriers

Different tyrosinase carbon paste modified electrodes to determine bisphenol A (BPA) concentration in aqueous solutions have been constructed. Variables examined were in the carbon paste composition and in particular: (i) the immobilized enzyme amount; (ii) the carbon type (powder, single or multi-walled nanotubes); (iii) the nature of the pasting oil (mineral oil, hexadecane and dodecane). For each biosensor type the amperometric response was evaluated with reference to the linear range and sensitivity. Constant reference has been made to the amperometric signals obtained, under the same experimental conditions, towards the catechol, a specific phenolic substrate for tyrosinase. The most efficient biosensors were those constructed by using the following composition for the carbon paste: 10% of tyrosinase, 45% of single wall carbon nanotubes (SWCN) and 45% of mineral oil. This biosensor formulation displayed the following electrochemical characteristics: a sensitivity equal to 138 microA/mM, LOD of 0.02 microM (based on three times the S/N ratio), linear range of 0.1-12 microM and response time of 6 min. This experimental work represents a first attempt at construction of a new carbon nanotube-tyrosinase based biosensor able to determine the concentration of BPA, one of the most ubiquitous and hazardous endocrine disruptors which can pollute the drinking and surface water, as well as many products of the food chain.     

Kinetics and mechanism of reduction of ferricytochrome c by glutathione and L-cysteine: a comparative study

The kinetics and mechanism of the reduction of ferricytochrome c [Cyt c(III)] by substrates namely glutathione (GSH) and L-cysteine (L-cys) have been investigated spectrophotometrically employing [substrate]T > [Cyt c(III)]T. The reaction exhibits first order dependence in [substrate]T and [Cyt c(III)]T. The pseudo-first order rate constant increases with an increase in pH, indicating that the conjugate base form of the HCyt c(III) is a better oxidant than the parent HCyt c(III). The electron transfer rate constants between the oxidants and GSH for both the k1 and k2 paths are found to be greater than that with L-cysteine. Hence, GSH is a better reductant of Cyt c(III) as compared to L-cysteine. A suitable mechanism has been proposed on the basis of experimental findings. The deprotonation constant for HCyt c(III) and the second order rate constants of k1 and k2 paths for the present reaction at 25 degrees C have been determined.     

Oxidative stress and genotoxic effects in gill and kidney of Anguilla anguilla L. exposed to chromium with or without pre-exposure to beta-naphthoflavone

Fish in the aquatic environment can be subjected to a multipollution state and the occurrence of sequential exposures is an important aspect of eco-toxicological research. In this context, a preceding exposure can affect a toxic response to a subsequent exposure. Therefore, the current study was based on sequential exposures, viz. to a PAH-like compound (beta-naphthoflavone, BNF) followed by a heavy metal (chromium, Cr), focusing on the assessment of oxidative stress responses and their role in induction of genotoxicity. Oxidative stress responses in gill and kidney were investigated in European eel (Anguilla anguilla L.), and measured as lipid peroxidation (LPO), glutathione peroxidase (GPX), catalase (CAT) and glutathione S-transferase (GST) activity, and reduced glutathione (GSH) concentration, whereas genotoxicity was measured as DNA strand breakage. Fish were exposed for 24 h to two Cr concentrations (100 microM, 1 mM), with or without pre-exposure to BNF (2.7 microM, 24 h). In gill, a GSH decrease was observed along with loss of DNA integrity at all exposure conditions except at the lowest Cr concentration, showing a crucial role of GSH over genotoxicity. Moreover, sporadic induction of antioxidant enzymes was not effective in the protection against genotoxicity. However, a different mechanism seems to occur in kidney, since the loss of DNA integrity detected for all exposed groups was not accompanied by alterations in antioxidant levels. With regards to peroxidative damage, both organs showed an LPO increase after sequential exposure to BNF and 100 microM Cr. However, no association between LPO induction and antioxidant responses could be established, showing that LPO is not predictable solely on the basis of antioxidant depletion. The interference of BNF pre-exposure with the response of organs to Cr showed a marked dependence on the Cr concentration. Gill showed synergistic effects on LPO and GPX increase, as well as on CAT and GSH decrease for the lowest Cr concentration. However, for the highest concentration an additive effect on decrease of DNA integrity and an antagonistic effect on the increase of GPX were observed. In kidney, synergistic effects were evident on LPO increase and GSH decrease for the lowest Cr concentration, as well as on CAT and GST decrease for the highest concentration. In contrast, an antagonistic action was observed on DNA integrity loss for both Cr concentrations. The current results are relevant in assessing the interactions of PAHs and metals and contribute to a better knowledge about oxidative stress and mechanisms of genotoxicity in fish.     

Allosteric and non-allosteric forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: differential inhibition of activity by adenosine 2'-monophospho-5'-diphosphoribose

Adenosine 2'-monophospho-5'-diphosphoribose (P-ADP-Rib) is a structural analog of NADPH which was reported to competitively inhibit (Kiapp = 21.7 microM) solubilized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Tanazawa, K., and A. Endo. 1979. Eur. J. Biochem. 98: 195-201). However, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib (Kiapp = 550 microM at 4.5 mM GSH). Gradual shift of the microsomal reductase towards a non-allosteric form by increasing glutathione (GSH) concentrations resulted in a higher inhibition by P-ADP-Rib. Under these conditions, Ki values for P-ADP-Rib were 165 microM and 53 microM at 9 mM and 27 mM GSH, respectively. The largest change in the degree of inhibition by P-ADP-Rib was observed within the 10 mM range of GSH. By contrast, freeze-thaw solubilized HMG-CoA reductase, which does not display allosteric properties, is readily inhibited by P-ADP-Rib, even when assayed at a low concentration of GSH (Kiapp = 50 microM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP-Rib (22 microM at 27 mM GSH). Microsomal HMG-CoA reductase is allosterically activated by various nucleotides. When activated by NADH, the enzyme is effectively inhibited by P-ADP-Rib even at a 4.5-mM GSH concentration (Kiapp = 175 microM in the presence of 300 microM NADH).(ABSTRACT TRUNCATED AT 250 WORDS)     

L-Cysteine influx in type 2 diabetic erythrocytes

Erythrocyte oxidative stress has been implicated in the pathogenesis of diabetes mellitus, and the deficiency of antioxidant defense by the glutathione (GSH) pathway is thought to be one of the factors responsible for development of complications in diabetes. Erythrocytes require L-cysteine for the synthesis of GSH and the rate of synthesis is determined only by L-cysteine availability. In the present study we have found that the L-cysteine influx in erythrocytes from type 2 diabetic patients was significantly lower compared to age-matched controls. The decreased influx may be one of the factors leading to low GSH concentration observed in type 2 diabetes. Since L-cysteine is the limiting amino acid in GSH synthesis, any strategy aimed to increase L-cysteine influx in erythrocytes may be beneficial for type 2 diabetic patients.     

Inhibition of glutathione disulfide reductase by glutathione

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 microM, about 1000-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (Vmax) and decreased Km for GSSG.     

Kinetics of hydroperoxide degradation by NADP-glutathione system in mitochondria

Hydroperoxide decomposition by the NADP-glutathione system in rat liver mitochondria was analyzed. Mitochondria were found to contain high concentrations of the reduced form of glutathione (GSH) (4.32 +/- 0.50 nmol/mg) and NADPH (4.74 +/- 0.64 nmol/mg), and high activities of glutathione peroxidase and reductase. In the initial phase of the reaction, the rate of hydroperoxide decomposition was proportional to both the GSH level and the activity of GSH peroxidase. However, in the later steady state, the step of NADP reduction was rate-limiting, and the overall reaction rate was independent of the initial concentration of GSH, and activities of glutathione peroxidase and reductase. Some GSH was released from mitochondria during incubation, but the rate of the decomposition could be simply expressed as kappa [GSH]/2, where kappa is the first-order rate constant of the peroxidase and [GSH] is the intramitochondrial level of GSH in the steady state. The rate of the reaction in the steady state was also dependent on the NADPH level, its reciprocal being linearly correlated with [NADPH]-1. The rate of decomposition of hydroperoxide was influenced by the respiratory state. During state 3 respiration, the rate was greatly depressed, but was still considered to exceed by far the rate of physiological generation of hydroperoxide.     

Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding

Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase. It has the highest specific activity (11 micromol/min/mg) reported for any mammalian glutathione synthetase. The apparent K(m) values for ATP and glycine are 37 and 913 microM, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0. 576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which attacks this intermediate to form GSH, exhibit any cooperativity. The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration. Thus, mammalian glutathione synthetase is an allosteric enzyme.     

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.     

L-Cysteine influx and efflux: a possible role for red blood cells in regulation of redox status of the plasma

The objective of this study was to investigate if erythrocytes play a role in the maintenance of redox homeostasis of the plasma. Thus, we studied L-cysteine efflux and influx in vitro in human erythrocytes. In the present study, we exposed the erythrocytes to different concentrations of L-cysteine and then measured the intracellular free -SH concentrations. Erythrocytes treated in the same manner were later utilized for the cysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. Change in the free -SH content of the buffer was evaluated as a measure for the presence of an efflux process. The effects of free -SH depletion on L-cysteine transport is also investigated. We also determined the rate of L-cysteine efflux in the presence and absence of buthionine sulfoximine (BSO) in erythrocytes that are pretreated with 1-chloro-2,4-dinitro benzene, a glutathione (GSH) depletory. Our L-cysteine influx studies demonstrated that erythrocytes can respond to increases in L-cysteine concentration in the extracellular media and influx L-cysteine in a concentration-dependent manner. Free -SH concentrations in erythrocytes treated with 1 mM L-cysteine reached to 1.64 +/- 0.06 mM in 1 h whereas this concentration reached to 4.30 +/- 0.01 mM in 10 mM L-cysteine treated erythrocytes. The L-cysteine efflux is also determined to be time-and concentration-dependent. Erythrocytes that are pretreated with higher L-cysteine concentrations displayed a higher efflux process. Outside concentration of free -SH in 1 mM L-cysteine pretreated erythrocytes reached to 0.200 +/- 0.005 mM in 1 h whereas this concentration reached to 1.014 +/- 0.002 with 10 mM L-cysteine pretreated erythrocytes. Our results also indicate that the rate of inward and outward transport of L-cysteine is affected by the oxidative status of the erythrocytes. When GSH is depleted and GSH synthesis is blocked, the L-cysteine uptake and the efflux processes are significantly decreased. Depending on our results, it could be concluded that erythrocytes play a role in the regulation of the plasma redox status and intracellular level of GSH determines the rate of the L-cysteine efflux.     

The Synthesis of a Coumarin Carbohydrazide Dinuclear Copper Complex Based Fluorescence Probe and Its Detection of Thiols

Small-molecule thiols, such as cysteine (CYS) and glutathione (GSH), are essential for maintaining the cellular redox environment and play important roles in regulating various cellular physiological functions. A fluorescence probe (compound 1-Cu²⁺) for thiols based on coumarin carbohydrazide dinuclear copper complex was developed. Compound 1 was synthesized from the reaction of 7-(diethylamino)-2-oxo-2H-chromene-3-carbohydrazide with 4-tert-butyl-2,6- diformylphenol. Accordingly, the copper complex (compound 1-Cu²⁺) was prepared by mixing compound 1 with 2 equivalents copper ions. Compound 1 had strong fluorescence while compound 1-Cu²⁺ hardly possessed fluorescence owing to the quenching nature of paramagnetism Cu²⁺ to the fluorescence molecule excited state. However, the fluorescence intensity of compound 1-Cu²⁺ was increased dramatically after the addition of thiol-containing amino acids, but not the other non-sulfhydryl amino acids. UV-vis absorption and fluorescence spectra indicated that compound 1-Cu²⁺ had good selectivity and sensitivity for thiols such as glutathione in CH₃CN:H₂O (3:2, v/v) PBS solution. The fluorescence imaging experiments implied that compound 1-Cu²⁺ has potential application in thiol-containing amino acids detection in living cells.     

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%. The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer. The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol. The reduced flavin is instantaneously reoxidized by addition of GSSG. The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158). This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+. However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism. NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates. The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.     

Distribution of mercury 203 in pregnant rats and their fetuses following systemic infusions with thiol-containing amino acids and glutathione during late gestation

To investigate the effect of amino acids and the tripeptide glutathione (GSH) on tissue uptake of methylmercury (MeHg) in the developing rat fetus in utero, pregnant rats were continuously infused into the external jugular vein with 0.1 mM L-cysteine, 0.1 mM L-leucine, 0.1 mM GSH or saline commencing on day 17 of gestation. This was followed at 24, 48, and 72 hours by external jugular infusion of 50 microM [203Hg]-MeHgCl administered in 1 ml over 1 hour. Pups were surgically removed from the uterus on gestational day 21. Whole body, brain, kidney, liver, and placental 203Hg radioactivity was measured by means of gamma-spectrometry. Brain 203Hg concentration in pups exposed in utero to L-cysteine was significantly higher compared with pups exposed to saline (P less than 0.05). Brain 203Hg concentration in pups exposed in utero to L-leucine and GSH was significantly depressed compared with pups exposed to saline (P less than 0.05). Kidney 203Hg concentration was not significantly changed in all treatment groups compared with controls. Liver 203Hg concentration was significantly depressed in L-leucine- and GSH-treated pups compared with controls (P less than 0.05). Placental 203Hg concentration was not affected by any treatment compared with controls. These effects occurred despite no difference in total 203Hg body burden among pups, irrespective of the treatment. In addition, infusion with L-cysteine resulted in a significant increase in 203Hg brain concentration in dams compared with controls, and 203Hg brain concentration in L-leucine- and GSH-treated dams was significantly depressed compared with controls. Thus 203Hg distribution in both adult and developing animals is altered by chronic amino acid or GSH infusions and suggests that MeHg uptake may be mediated through the formation of a cysteine-MeHg complex which is transported across the blood-brain barrier by the neutral amino acid carrier transport system.     

Synthesis and evaluation of 2',4',6'-trihydroxychalcones as a new class of tyrosinase inhibitors

In this study, we synthesized a series of hydroxychalcones and examined their tyrosinase inhibitory activity. The results showed that 2',4',6'-trihydroxychalcone (1), 2,2',3,4',6'-pentahydroxychalcone (4), 2',3,4,4',5,6'-hexahydroxychalcone (5), 2',4',6'-trihydroxy- 3,4-dimethoxychalcone (9) and 2,2',4,4',6'-pentahydroxychalcone (15) exhibited high inhibitory effects on tyrosinase with respect to l-tyrosine as a substrate. By the structure-activity relationship study, it was suggested that the 2',4',6'-trihydroxyl substructure in the chalcone skeleton were efficacious for the inhibition of tyrosinase activity. And also, the catechol structure on B-ring of chalcones was not advantageous for the inhibitory potency. Furthermore, 15 (IC(50)=1microM) was found to show the highest activity out of a set of 15 hydroxychalcones, even better than both 2,2',4,4'-tetrahydroxychalcone (13, IC(50)=5microM) and kojic acid (16, IC(50)=12microM), which were known as potent tyrosinase inhibitors. Kinetic study revealed that 15 acts as a competitive inhibitor of tyrosinase with K(i) value of 3.1microM.     

Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K(m) values of the substrates PGH(2) and GSH were 14 microM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 micromol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V(max) but does not affect the K(m) values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE(2) formation.     

An amperometric inhibitor biosensor for the determination of reduced glutathione (GSH) without any derivatization in some plants

In this study, an amperometric biosensor based on cucumber tissue homogenate was developed for the determination of glutathione. Cucumber (Cucumis sativus L.) tissue homogenate was used as the biological material. The cucumber tissue homogenate was cross-linked with gelatine using glutaraldehyde and fixed on a pretreated teflon membrane. The principle of the measurements was based on the determination of the decrease in the differentiation of oxygen level which had been caused by the inhibition of ascorbate oxidase in the biological material by glutathione. Determinations were carried out by standard curves which were obtained by the measurement of the decrease in the consumed oxygen level related to glutathione concentration. Optimization and characterization studies of the biosensor were carried out and a linearity in the gamma-L-glutamyl-L-cysteinyl-glycine (GSH) concentration range 0.1-2 microM was obtained when 600 microM ascorbic acid was used as a substrate. The repeatability experiments (n = 7) revealed that for 1.5 microM GSH, the average value (x), standard deviation (S.D.) and variation coefficient (C.V.) were 1.517 microM, 4.72 x 10(-5) 3.11%, respectively. The biosensor useful lifetime was at least 2 months. The results of some plant samples analyzed with the presented biosensor agreed well with the spectrophotometric method (Ellman's reagent) used as a reference.     

Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor

The use of an amperometric graphite-Teflon composite tyrosinase biosensor for the rapid monitoring of alkaline phosphatase (ALP), with no need of an incubation step and using phenyl phosphate as the substrate, is reported. Phenol generated by the action of ALP is monitored at the tyrosinase composite electrode through the electrochemical reduction of the o-quinone produced to catechol, which produces a cycle between the tyrosinase substrate and the electroactive product, giving rise to the amplification of the biosensor response and to the sensitive detection of ALP. The current was measured at -0.10 V 5 min after the addition of ALP. As a compromise between high ALP activity and high sensitivity for the detection of phenol, a pH of 8.5 was chosen. The substrate concentration was also optimized. A linear calibration plot was obtained for ALP between 2.0 x 10(-13) and 2.5 x 10(-11), with a detection limit of 6.7 x 10(-14) M. Different types of milk were analyzed with good results, using an extremely simple and rapid procedure.