Background: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 μmol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. Methods: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector. Results: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30–150 μmol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide. Conclusions: GSSG is present in healthy human blood at low concentrations (2–6 μmol/L), and most published data on GSSG may be affected by artifacts.

The effect of oxidants, electrophiles, and NO donors in rat or human erythrocytes was analyzed to investigate the influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein mixed disulfide) was induced by menadione only in human erythrocytes. The treatment of rat red blood cells with electrophiles produced glutathione S-conjugates to a much lower extent than in human red blood cells; GSH was only minimally depleted in rat red blood cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing high levels of S-nitrosohemoglobin; this reaction in human red blood cells was negligible. All drugs were cleared more rapidly in rat than in human erythrocytes. Unlike human Hb, rat hemoglobin contains three families of protein SH groups; one of these located at position ␤125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.Glutathione is the major low molecular weight thiol in mammalian cells where it constitutes the most important antioxidant defense. Its action is usually favored by ubiquitous enzymes (e.g. glutathione S-transferases, glutathione peroxidase). GSH also regenerates other important defensive resources (e.g. vitamins E and C) and directly participates in the destruction of reactive oxygen species (1). The alkylation of glutathione by electrophilic reagents and the reduction of chemically reactive oxidant species are biological functions associated with the protection of SH groups of critical cellular macromolecules (2).The cell concentration of protein SH groups, ranging from 10 to 30 mM, is larger by far than that of GSH (2-10 mM) (3); however, the metabolic role against electrophiles and oxidants is thought to be rather marginal. In fact, the modification of PSH 1 has been usually considered only a potential damaging reaction, even if some authors suggested that chemical alteration of some cysteine residues may have a protective or regulatory role (4, 5).The participation of PSH in quantitatively important reactions is proportional to their concentration and reactivity. The intrinsic reactivity of PSH is dependent on the pK a value (the thiolate anion is much more reactive than the undissociated form) and its accessibility (structural and conformational features). In fact, some proteins such as albumin (6) with relatively low pK a values have an apparent reactivity much lower than that of GSH. It follows from this that PSH may have a wide range of apparent reactivity that spans 4 orders of magnitude (7). This fact has abrogated any efforts to understand the effective defensiv...

No abstract

A bi-directional, saturable transport of glutathione (GSH) was found in rat liver microsomal vesicles. GSH transport could be inhibited by the anion transport blockers flufenamic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid. A part of GSH taken up by the vesicles was metabolized to glutathione disulfide (GSSG) in the lumen. Microsomal membrane was virtually nonpermeable toward GSSG; accordingly, GSSG generated in the microsomal lumen could hardly exit. Therefore, GSH transport, contrary to previous assumptions, is preferred in the endoplasmic reticulum, and GSSG entrapped and accumulated in the lumen creates the oxidized state of its redox buffer. The endoplasmic reticulum (ER)1 of the cell is the site of the synthesis, posttranslational modification, and folding of proteins transported along the secretory pathway. The oxidizing environment in the lumen of the ER is necessary for the formation of disulfide bonds and for the proper folding of these proteins (1). The oxidative effects are reflected in and supported by the GSH redox buffer; the ratio of GSH and GSSG is around 2:1 within the lumen of ER and along the secretory pathway, whereas the cytosolic ratio ranges from 30:1 to 100:1 (2). However, the primary source(s) of the oxidative effect has not been demonstrated. Recent observations suggest two possible mechanisms. First, the preferential uptake of the oxidized member of a redox couple through the ER membrane and/or the efflux (or exocytosis) of its reduced form could ensure the oxidative environment. Alternatively, enzymes resident in the membrane or lumen of the ER could produce oxidizing compounds (e.g. reactive oxygen species) toward the lumen. Experimental evidence supports both mechanisms. Favoring the transport-based hypothesis, the preferential transport of dehydroascorbate (the oxidized form of ascorbate) has been described in rat liver microsomal vesicles (3). Similarly, the selective microsomal transport of GSSG was also reported (2, 4). On the other hand, several microsomal enzymes (cytochrome P-450s, NADPH cytochrome P-450 reductase, gulonolactone oxidase, microsomal iron protein, NADPH-dependent oxidase, sulfydryl oxidase, etc.) can produce reactive oxygen species (5-10). The recent exploration of the ER oxidase protein (ERO1) and its role in the protein folding also support the latter mechanism (11, 12). Because of the conflicting opinions, the microsomal transport of GSH and GSSG has not been unequivocally established. The presently available data are based on the detection of microsome-associated radioactivity by applying a rapid filtration method and radiolabeled compounds (2, 4); however, intraluminal GSH or GSSG contents upon transport have not been directly demonstrated. Therefore, experiments were undertaken to reinvestigate the transport of GSH and GSSG through the ER membrane.The main difficulties in the investigation of microsomal transport processes are deriving from the very small intraluminal space, the presence of (intraluminal) reactions affecting the transported c...

No abstract

No abstract

The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mM), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t1 ⁄2 <100 ms; slow reacting (sHbSH), with t1 ⁄2 30 -50 s; and very slow reacting (vsHbSH), with t1 ⁄2 180 -270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125␤(H3)) conjugates with DTNB 100 times faster than glutathione and ϳ4000 times more rapidly than (v)sHbSH (Cys-13␣(A11) and Cys-93␤(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (ϳ6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123␤(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mM, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.Human but not rat erythrocytes are reported (1) to be able to restore the cellular pool of GSH, which is strongly decreased after treatment with diazenedicarboxylic acid bis(N,N-dimethylamide), a thiol-oxidizing agent known by the trivial name diamide (2). Such a difference in redox behavior was attributed to a lower enzymatic capacity in reducing disulfides of rat red cells relative to the human ones (3). However, recent work in this laboratory demonstrated that the reversibility of such process can be observed also in rat erythrocytes, depending on diamide dose (4). Additional evidence (3) may suggest that these differences in behavior between rat and human erythrocytes could be related to diversities in reactivity of sulfhydryl groups of the corresponding hemoglobins.

The present study reports the activities of quercetin and its main circulating conjugates in man (quercetin-3 0 -sulphate (Q3 0 S) and quercetin-3-glucuronide (Q3G)) on in vivo angiogenesis induced by vascular endothelial growth factor (VEGF) and examines the effects of these molecules on cultured endothelial cells. We found opposing effects of quercetin and its metabolites on angiogenesis. While quercetin and Q3G inhibited VEGFinduced endothelial cell functions and angiogenesis, Q3 0 S per se promoted endothelial cell proliferation and angiogenesis. The inhibitory effect elicited by Q3G was linked to inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation elicited by VEGF. The activation of endothelial cells by Q3 0 S was associated to stimulation of VEGF receptor-2 and to downstream signalling activation (phosphatidylinositol-3 kinase/Akt and nitric oxide synthase pathways), ultimately responsible for ERK1/2 phosphorylation. These data indicate that the effects of circulating quercetin conjugates on angiogenesis are different depending on the nature of the conjugate. Q3G and Q3 0 S are the two major conjugates in plasma, but their ratio is dependent on several factors, so that inhibition or activation of angiogenesis could be subtly shifted as a result of metabolism in vivo.