Physiological Thiols Research Articles

A novel concept for cleavable linkers applicable to conjugation chemistry - design, synthesis and characterization.

Linkers with disulfide bonds are the only cleavable linkers that utilize physiological thiol gradients as a trigger to initiate the intracellular drug release cascade. Herein, we present a novel concept exploiting the thiol gradient phenomena to design a new class of cleavable linker with no disulfide bond. To support the concept, an electron-deficient sulfonamide-based cleavable linker amenable to conjugation of drug molecules with targeting agents, was developed. Modulating the electron-withdrawing nature of the aryl sulfonamide was critical to the balance between the stability and drug release. Favorable stability and payload release in human serum under physiologically relevant thiol concentrations was demonstrated with two potent cytotoxics. Intracellular payload release was further validated in cell-based assay in context of antibody-drug conjugate generated from monoclonal antibody and sulfonamide containing linker. To support the proposed release mechanism, possible downstream by-products formed from the drug-linker adduct were characterized.

The Aging Kidney-As Influenced by Heavy Metal Exposure and Selenium Supplementation.

The aging process in the kidneys has been well studied. It is known that the glomerular filtration rate (GFR) declines with age in subjects older than 50–60 years. However, there is still insufficient knowledge regarding the response of the aged kidney to environmental toxicants such as mercury, cadmium, and lead. Here, we present a review on the functional decline and proposed mechanisms in the aging kidney as influenced by metal pollutants. Due to the prevalence of these toxicants in the environment, human exposure is nearly unavoidable. Further, it is well known that acute and chronic exposures to toxic metals may be detrimental to kidneys of normal adults, thus it may be hypothesized that exposure of individuals with reduced GFR will result in additional reductions in renal function. Individuals with compromised renal function, either from aging or from a combination of aging and disease, may be particularly susceptible to environmental toxicants. The available data appear to show an association between exposure to mercury, cadmium and/or lead and an increase in incidence and severity of renal disease in elderly individuals. Furthermore, some physiological thiols, as well as adequate selenium status, appear to exert a protective action. Further studies providing improved insight into the mechanisms by which nephrotoxic metals are handled by aging kidneys, as well as possibilities of therapeutic protection, are of utmost importance.

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria.

Yeast (Saccharomyces cerevisiae) essential for respiration and viability 1 (Erv1; EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html), a member of the flavin adenine dinucleotide‐dependent Erv1/ALR disulphide bond generating enzyme family, works together with Mia40 to catalyse protein import and oxidative folding in the mitochondrial intermembrane space. Erv1/ALR functions either as an oxidase or cytochrome c reductase by passing electrons from a thiol substrate to molecular oxygen (O2) or cytochrome c, respectively. However, the substrate specificity for oxygen and cytochrome c is not fully understood. In this study, the oxidase and cytochrome c reductase kinetics of yeast Erv1 were investigated in detail, under aerobic and anaerobic conditions, using stopped‐flow absorption spectroscopy and oxygen consumption analysis. Using DTT as an electron donor, our results show that cytochrome c is ~ 7‐ to 15‐fold more efficient than O2 as electron acceptors for yeast Erv1, and that O2 is a competitive inhibitor of Erv1 cytochrome c reductase activity. In addition, Mia40, the physiological thiol substrate of Erv1, was used as an electron donor for Erv1 in a detailed enzyme kinetic study. Different enzyme kinetic k cat and K m values were obtained with Mia40 compared to DTT, suggesting that Mia40 modulates Erv1 enzyme kinetics. Taken together, this study shows that Erv1 is a moderately active enzyme with the ability to use both O2 and cytochrome c as the electron acceptors, indicating that Erv1 contributes to mitochondrial hydrogen peroxide production. Our results also suggest that Mia40‐Erv1 system may involve in regulation of the redox state of glutathione in the mitochondrial intermembrane space.Erv1EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html.

Mechanistic and Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs.

Copper (Cu) is required for maturation of cuproenzymes, cell proliferation, and angiogenesis, and its transport entails highly specific protein-protein interactions. In humans, the Cu chaperone Atox1 mediates Cu(I) delivery to P-type ATPases Atp7a and Atp7b (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway and excess Cu efflux. Cu(I) handover is believed to occur through the formation of three-coordinate intermediates where the metal ion is simultaneously linked to Atox1 and to a soluble domain of Cu-ATPases, both sharing a CxxC dithiol motif. The ultrahigh thermodynamic stability of chelating S-donor ligands secures the redox-active and potentially toxic Cu(I) ion, while their kinetic lability allows facile metal transfer. The same CxxC motifs can interact with and mediate the biological response to antitumor platinum drugs, which are among the most used chemotherapeutics. We show that cisplatin and an oxaliplatin analogue can specifically bind to the heterodimeric complex Atox1-Cu(I)-Mnk1 (Mnk1 is the first soluble domain of Atp7a), thus leading to a kinetically stable adduct that has been structurally characterized by solution NMR and X-ray crystallography. Of the two possible binding configurations of the Cu(I) ion in the cage made by the CxxC motifs of the two proteins, one (bidentate Atox1 and monodentate Mnk1) is less stable and more reactive toward cis-Pt(II) compounds, as shown by using mutated proteins. A Cu(I) ion can be retained at the Pt(II) coordination site but can be released to glutathione (a physiological thiol) or to other complexing agents. The Pt(II)-supported heterodimeric complex does not form if Zn(II) is used in place of Cu(I) and transplatin instead of cisplatin. The results indicate that Pt(II) drugs can specifically affect Cu(I) homeostasis by interfering with the rapid exchange of Cu(I) between Atox1 and Cu-ATPases with consequences on cancer cell viability and migration.

Melatonin, glutathione and thiourea attenuates lead and acid rain-induced deleterious responses by regulating gene expression of antioxidants in Trigonella foenum graecum L.

Lead and acid rain are important abiotic stress factors that limit the growth, development, metabolic activity and yield of the crops. Melatonin (MT; an indoleamine molecule), glutathione (GSH; free thiol tripeptide) and thiourea (TU; non physiological thiol based ROS scavenger) have been known to mediate several physiological, biochemical and molecular processes in plants under different kinds of environmental threats. However, the roles of MT, GSH and TU in stress tolerance against combined effect of lead and simulated acid rain (SAR) remains inexpressible. In this study, we investigated the response of Trigonella foenum graecum L. (Fenugreek) to combined application of lead (1200 ppm) and SAR (pH 3.5), and the potential roles of MT (50 μM), GSH (1 mM) and TU (3 mM) in enhancing lead and SAR stress tolerance of Fenugreek. The results showed that co-application of each MT, GSH and TU along with lead and SAR improved the growth and development of seedlings. Moreover, MT, GSH and TU treatments stabilized the cell membrane integrity, reduced ROS accumulation [superoxide radical (O2ˉ) and hydrogen peroxide (H2O2)], malondialdehyde (MDA) content, lipoxygenase (LOX) activity and, enhanced protein accumulation and up-regulated the gene expressions of catalase (CAT) and superoxide dismutase (SOD) significantly. Furthermore, the present work provides strong evidence regarding protective roles of MT, GSH and TU against oxidative stress resulted from lead and SAR stress in Fenugreek. Considering these observations, MT, GSH and TU can be utilized as efficient ROS scavengers, for improving growth and increasing antioxidant capacity in lead and SAR stressed seedlings.

Thiol oxidase ability of copper ion is specifically retained upon chelation by aldose reductase.

Bovine lens aldose reductase is susceptible to a copper-mediated oxidation, leading to the generation of a disulfide bridge with the concomitant incorporation of two equivalents of the metal and inactivation of the enzyme. The metal complexed by the protein remains redox active, being able to catalyse the oxidation of different physiological thiol compounds. The thiol oxidase activity displayed by the enzymatic form carrying one equivalent of copper ion (Cu1-AR) has been characterized. The efficacy of Cu1-AR in catalysing thiol oxidation is essentially comparable to the free copper in terms of both thiol concentration and pH effect. On the contrary, the two catalysts are differently affected by temperature. The specificity of the AR-bound copper towards thiols is highlighted with Cu1-AR being completely ineffective in promoting the oxidation of both low-density lipoprotein and ascorbic acid.

Thiol-Based Redox Modulation of Soluble Guanylyl Cyclase, the Nitric Oxide Receptor.

Soluble guanylyl cyclase (sGC), which produces the second messenger cyclic guanosine 3', 5'-monophosphate (cGMP), is at the crossroads of nitric oxide (NO) signaling: sGC catalytic activity is both stimulated by NO binding to the heme and inhibited by NO modification of its cysteine (Cys) thiols (S-nitrosation). Modulation of sGC activity by thiol oxidation makes sGC a therapeutic target for pathologies originating from oxidative or nitrosative stress. sGC has an unusually high percentage of Cys for a cytosolic protein, the majority solvent exposed and therefore accessible modulatory targets for biological and pathophysiological signaling. Recent Advances: Thiol oxidation of sGC contributes to the development of cardiovascular diseases by decreasing NO-dependent cGMP production and thereby vascular reactivity. This thiol-based resistance to NO (e.g., increase in peripheral resistance) is observed in hypertension and hyperaldosteronism. Some roles of specific Cys thiols have been identified in vitro. So far, it has not been possible to pinpoint the roles of specific Cys of sGC in vivo and to investigate the molecular mechanisms in an animal model. The role of Cys as redox sensors, intermediates of activation, and mediators of change in sGC conformation, activity, and dimerization remains largely unexplored. To understand modulation of sGC activity, it is critical to investigate the roles of specific oxidative thiol modifications that are formed during these processes. Where the redox state of sGC thiols contribute to pathologies (vascular resistance and sGC desensitization by NO donors), it becomes crucial to design therapeutic strategies to restore sGC to its normal, physiological thiol redox state. Antioxid. Redox Signal. 26, 137-149.

Enhanced chemiluminescence detection of glutathione based on isoluminol-PSM nanoparticles probe

In this paper, a new chemiluminescence (CL) strategy was constructed for the determination of physiological thiols by using an isoluminol labeled nano-probe. The amino group on the surface of the magnetic beads (MBs) were converted into pyridyl disulfide groups by treatment with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), in the meantime, isoluminol and thiolated signal DNA were labeled on the surface of the polystyrene microspheres (PSMs). By treating the SPDP activated MBs with the modified PSMs, isoluminol molecules on the surface of the PSMs, along with the thiolated signal DNA, were attached to the surface of the MBs through disulfide bonds to form a CL probe. In the presence of glutathione (GSH), the disulfide bonds could be cleaved readily. The isoluminol molecules modified on the surface of the PSMs released from the CL probes were detached by magnetic separation and transferred to the dark closet for CL detection of isoluminol-H2O2-HRP system. Using GSH detection as a model, we prove a linear dose response in the range from 5×10−10 to 8×10−8M. The detection limit of this trial for GSH determined is 5×10−10M. The proposed design was successfully applied to the extracts of K562 cell for intracellular thiols detection, the average amount of thiols was about 4.114×10−13M per K562 cell.

Anethole dithiolethione lowers the homocysteine and raises the glutathone levels in solid tissues and plasma of rats: A novel non-vitamin homocysteine-lowering agent

High homocysteine (Hcys) levels are suspected to contribute to the pathogenesis of cardiovascular disease and of other chronic conditions. Failure of B vitamins to reduce the incidence of cardiovascular events while lowering the Hcys levels, has prompted the search for alternative treatments. We tested the ability of anethole dithiolethione (ADT) to lower the Hcys levels in rats and we explored possible underlying mechanisms. Parenteral administration of 10mg/kg ADT to normal rats for 3 days lowered the Hcys levels between 51.4% and 31.5% in kidneys, liver, testis and plasma. Concomitantly, glutathione (GSH) increased between 112% and 28% in kidneys, brain, liver and plasma whereas protein thiolation index decreased 30%. In hyperhomocysteinemic rats, the plasma Hcys levels dropped 70% following a single ip injection of 10mg/kg ADT, while they decreased 55% following oral administration of 2mg/kg/day ADT for one week. Significant additive effects occurred when sub-therapeutic doses of ADT and folic acid were used in combination. To test the possible mechanism(s) of these actions, we perfused isolated rat livers and kidneys with albumin-bound Hcys, the prevalent form of plasma Hcys, and physiological thiols and disulfides at different ratios. In both organ preparations, the elimination rate of albumin-bound Hcys was progressively faster as the amount of reduced thiols was increased in the perfusate. These findings indicate that ADT shifts the redox ratio of GSH and other thiols with their oxidized forms toward the reduced forms, thus favoring the dissociation of albumin-bound Hcys and its transfer to renal and hepatic cells for further processing.

A highly sensitive TTF-functionalised probe for the determination of physiological thiols and its application in tumor cells

A tetrathiafulvalene (TTF)-fused piazselenole as a novel redox-active and fluorescent probe for highly sensitive determination of physiological thiols is presented.

Thiourea, a ROS Scavenger, Regulates Source-to-Sink Relationship to Enhance Crop Yield and Oil Content in Brassica juncea (L.)

In the present agricultural scenario, the major thrust is to increase crop productivity so as to ensure sustainability. In an earlier study, foliar application of thiourea (TU; a non physiological thiol based ROS scavenger) has been demonstrated to enhance the stress tolerance and yield of different crops under field condition. Towards this endeavor, present work deals with the effect of TU on photosynthetic efficiency and source-to-sink relationship of Indian mustard (Brassica juncea) for understanding its mode of action. The application of TU increased the efficiency of both PSI and PSII photosystems and vegetative growth of plant. The comparative analysis of sucrose to starch ratio and expression level of sugar transporters confirmed the higher source and sink strength in response to TU treatment. The biochemical evidence in support of this was derived from higher activities of sucrose phosphate synthase and fructose-1,6-bis-phosphatase at source; and sucrose synthase and different classes of invertases at both source and sink. This indicated an overall increase in photoassimilate level at sink. An additional contribution through pod photosynthesis was confirmed through the analysis of phosphoenol pyruvate carboxylase enzyme activity and level of organic acids. The increased photoassimilate level was also co-ordinated with acetyl coA carboxylase mediated oil biosynthesis. All these changes were ultimately reflected in the form of 10 and 20% increase in total yield and oil content, respectively under TU treatment as compared to control. Additionally, no change was observed in oil composition of seeds derived from TU treated plants. The study thus signifies the co-ordinated regulation of key steps of photosynthesis and source-to-sink relationship through the external application of TU resulting in increased crop yield and oil content.

P39 Protoporphyrin modification by biological thiols and hydrogen sulfide

Sulfur metabolism furnishes cells with important metabolic compounds, among which glutathione (GSH), cysteine (Cys) and hydrogen sulfide (H2S) [1] , [2] . GSH is important in protecting cells against oxidative injuries and its intracellular redox couple varies within cells depending on their biological status. Cys is a precursor of several important macromolecules and participates in redox-based signaling via post-translational modifications (PTMs). H2S is a small “gaseous” molecule, which exerts important signaling functions in the vascular and neuronal systems and might signal through S-sulfhydration and HSNO formation [3] . As a result of their crucial role in cellular physiology and the reactivity of their SH group, sulfur metabolism is tightly controlled in response to environmental changes and defects affecting sulfur metabolic compounds and redox-based signaling are often associated with severe pathologies [e.g. [4] , [5] , [6] , [7] ]. In here we pursue the efforts in understanding the biology of sulfur compounds at the interface of chemistry and biology. We thus seek to explore in vitro the various effects elicited by RSH/H2S under normal versus pathological conditions. To “mimic” pathological conditions, we employed trace elements whose acute toxicity is well-known and are often used as tumor biomarkers, e.g. iron or copper. As model we chose a heme-containing constituent of the innate immune system, catalase (Cat) that has been reported to be inactivated due to sulfur compounds homeostasis deregulation, e.g. in animals bearing tumors due to aberrancy in cystine metabolism or in animal models of chronic hyperhomocysteinemia [8] , [9] , [10] , [11] . Our results indicate for the first time the peculiar formation of sulfcatalase in the presence of RSH alone. Furthermore, the observed IC50 greatly decreased in the presence of trace elements and felt within the physiological thiol concentrations, elucidating the aforementioned inactivation of Cat due to sulfur compounds homeostasis deregulation. In contrast, in the presence of H2S, we observed a new PTM of Cat. This latter is responsible for Cat activation in the presence of trace elements and physiological H2S concentrations.

Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus

FosB is a divalent-metal-dependent thiol-S-transferase implicated in fosfomycin resistance among many pathogenic Gram-positive bacteria. In the present paper, we describe detailed kinetic studies of FosB from Staphylococcus aureus (SaFosB) that confirm that bacillithiol (BSH) is its preferred physiological thiol substrate. SaFosB is the first to be characterized among a new class of enzyme (bacillithiol-S-transferases), which, unlike glutathione transferases, are distributed among many low-G+C Gram-positive bacteria that use BSH instead of glutathione as their major low-molecular-mass thiol. The K(m) values for BSH and fosfomycin are 4.2 and 17.8 mM respectively. Substrate specificity assays revealed that the thiol and amino groups of BSH are essential for activity, whereas malate is important for SaFosB recognition and catalytic efficiency. Metal activity assays indicated that Mn(2+) and Mg(2+) are likely to be the relevant cofactors under physiological conditions. The serine analogue of BSH (BOH) is an effective competitive inhibitor of SaFosB with respect to BSH, but uncompetitive with respect to fosfomycin. Coupled with NMR characterization of the reaction product (BS-fosfomycin), this demonstrates that the SaFosB-catalysed reaction pathway involves a compulsory ordered binding mechanism with fosfomycin binding first followed by BSH which then attacks the more sterically hindered C-1 carbon of the fosfomycin epoxide. Disruption of BSH biosynthesis in S. aureus increases sensitivity to fosfomycin. Together, these results indicate that SaFosB is a divalent-metal-dependent bacillithiol-S-transferase that confers fosfomycin resistance on S. aureus.

Generation of DNA-Damaging Reactive Oxygen Species via the Autoxidation of Hydrogen Sulfide under Physiologically Relevant Conditions: Chemistry Relevant to Both the Genotoxic and Cell Signaling Properties of H2S

Hydrogen sulfide (H(2)S) has long been known for its toxic properties; however, in recent years, evidence has emerged that this small, gaseous molecule may serve as an endogenous cell-signaling agent. Though perhaps surprising in light of its potential role as an endogenous signaling agent, a number of studies have provided evidence that H(2)S is a DNA-damaging mutagen. In the work reported here, the chemical mechanisms of DNA damage by H(2)S were examined. Using a plasmid-based DNA strand cleavage assay, we found that micromolar concentrations of H(2)S generated single-strand DNA cleavage. Mechanistic studies indicate that this process involved autoxidation of H(2)S to generate superoxide, hydrogen peroxide, and, ultimately, the well-known DNA-damaging agent hydroxyl radical via a trace metal-mediated Fenton-type reaction. Strand cleavage by H(2)S proceeded in the presence of physiological thiol concentrations, and the known byproducts of H(2)S oxidation such as thiosulfate, sulfite, and sulfate do not contribute to the strand cleavage process. However, initially generated oxidation products such as persulfide (S(2)(2-)) likely undergo rapid autoxidation reactions that contribute to the generation of superoxide. The potential relevance of autoxidation processes to the genotoxic and cell signaling properties of H(2)S is discussed.

Mechanism and biological implications of the NO release of cis-[Ru(bpy)2L(NO)]n+ complexes: A key role of physiological thiols

Nitric oxide (NO) has a critical role in several physiological and pathophysiological processes. In this paper, the reactions of the nitrosyl complexes of [Ru(bpy)2L(NO)]n+ type, where L = SO32− and imidazole and bpy = 2,2′-bipiridine, with cysteine and glutathione were studied. The reactions with cysteine and glutathione occurred through the formation of two sequential intermediates, previously described elsewhere, [Ru(bpy)2L(NOSR)]n+ and [Ru(bpy)2L(NOSR)2] (SR = thiol) leading to the final products [Ru(bpy)2L(H2O)]n+ and free NO. The second order rate constant for the second step of this reaction was calculated for cysteine k2(SR−)=(2.20±0.12)×109M−1s−1 and k2(RSH)=(154±2) M−1s−1 for L = SO32− and k2(SR−)=(1.30±0.23)×109M−1s−1 and k2(RSH)=(0.84±0.02) M−1s−1 for L = imidazole; while for glutathione they were k2(SR−)=(6.70±0.32)×108M−1s−1 and k2(RSH)=11.8±0.3M−1s−1 for L = SO32− and k2(SR−)=(2.50±0.36)×108M−1s−1 and k2(RSH)=0.32±0.01M−1s−1 for L = imidazole. In all reactions it was possible to detect the release of NO from the complexes, which it is remarkably distinct from other ruthenium metallocompounds described elsewhere with just N2O production. These results shine light on the possible key role of NO release mediated by physiological thiols in reaction with these metallonitrosyl ruthenium complexes.

Thiol-disulfide exchanges modulate aldo–keto reductase family 1 member B10 activity and sensitivity to inhibitors

The reversible thiol/disulfide exchange is an important regulatory mechanism of protein enzymatic activity. Many protein enzymes are susceptible to S-thiolation induced by reactive oxygen species (ROS); and the glutathione (GSH) and free amino acid cysteine (Cys) are critical cellular thiol anti-oxidants, protecting proteins from irreversible oxidative damage. In this study, we found that aldo–keto reductase family 1 member B10 (AKR1B10) contains 4 Cys residues, i.e., Cys45, Cys187, Cys200, and Cys299. Exposing AKR1B10 to ROS mixtures resulted in significant decrease of its free sulfhydryl groups, up to 40–50% in the presence of physiological thiol cysteine at 0.5 or 1.0 mM; and accordingly, AKR1B10 enzymatic activity was reversibly decreased, in parallel with the oxidation of the sulfhydryl groups. ROS-induced thiolation also affected the sensitivity of AKR1B10 to inhibitors EBPC, epalrestat, and statil. Together our results showed for the first time that AKR1B10's enzymatic activity and inhibitor sensitivity are modulated by thiol/disulfide exchanges.

A new strategy of photoelectrochemical analysis without an external light source based on isoluminol chemiluminescence probe

A new strategy of photoelectrochemical (PEC) analysis with chemiluminescence (CL) of the isoluminol-H(2)O(2)-Co(2+) system as a light source for the determination of physiological thiols in cancer cells was reported for the first time.

Detoxification of mercury species—an in vitro study with antidotes in human whole blood

To investigate the effects of mercury species intoxication and to test the efficiency of different commonly applied antidotes, human whole blood and plasma surrogate samples were spiked with inorganic mercury (Hg2+) and methylmercury (MeHg+, CH3Hg+) prior to treatment with the antidotes 2,3-dimercaptopropan-1-ol (British Anti Lewisite), 2,3-dimercaptosuccinic acid (DMSA), and N-acetylcysteine (NAC). For mercury speciation analysis in these samples, liquid chromatography was coupled to either inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionisation time-of-flight mass spectrometry (ESI-TOF-MS). Adduct formation between mercury species and physiological thiols (cysteine and glutathione) was observed as well as the release of glutathione under treatment with the antidotes DMSA and NAC.

Analysis of BNP7787 thiol-disulfide exchange reactions in phosphate buffer and human plasma using microscale electrochemical high performance liquid chromatography

BNP7787 (disodium 2,2′-dithio-bis ethane sulfonate; Tavocept™) is a novel water-soluble investigational agent that is undergoing clinical development for prevention and mitigation of cisplatin-induced nephrotoxicity. BNP7787 is a disulfide that undergoes thiol-disulfide exchange reactions in vivo with physiological thiols. Mesna-disulfide heteroconjugates that form as a result of these exchange reactions may play a key role in the protection against cisplatin-induced nephrotoxicity. Although several analytical methods have been used to detect thiols and disulfides, they have notable limitations including (i) low sensitivity, (ii) interference by chemical modification by derivatization reagents, and (iii) cumbersome sample preparation. In this paper, a sensitive micro-HPLC-EC method is described that identifies BNP7787 and mesna in plasma and phosphate buffer across a broad concentration range from 500 nM to 100 μM. This method utilizes a dual electrochemical detector equipped with a wall-jet gold electrode. The approach described here facilitates the identification of BNP7787 and mesna down to nanomolar levels. Although we did not focus on optimizing the approach for other thiol and disulfide compounds, we believe this approach could be optimized and used in the identification of other thiols and disulfides in plasma. The assay requires significantly less sample preparation and does not involve the use of derivatizing agents (i.e., the thiol and disulfide species can be detected directly) and represents an important advance over previous methods. This method was used to detect and quantitate BNP7787 and to monitor and kinetically characterize the interactions of BNP7787 with glutathione, cysteine, cysteinyl-glycine, cysteinyl-glutamate and homocysteine.

Simultaneous detection of N-acetyl-l-cysteine and physiological low molecular mass thiols in plasma by capillary electrophoresis

N-acetyl-L-cysteine (NAC) is a therapeutic drug widely used as mucolytic agent in the treatment of respiratory diseases. Recently it has been proposed that NAC administration may modify the plasma levels of low molecular weight thiols (LMW) like cysteine, homocysteine and glutathione, though it has been still debated if their plasma concentration increases or decreases during the therapy. Therefore research calls for methods able to analyze simultaneously NAC and the other plasma LMW thiols in order to evaluate if NAC is able to modify plasma thiols concentration and in particular to reduce homocysteine levels in hyperhomocysteinemia. In this paper we present a new capillary electrophoresis method that allows a baseline separation of plasma NAC from the physiological thiols. The proposed method has been utilized to measure the drug and the physiological LMW thiols in NAC administered chronic obstructive broncho-pneumopathy (COPB) disease patients.