Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile cysteine derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all cysteine derivatives, alkylation rates are faster than their dynamic interchange. However, when the interconversion of cysteine derivatives is not rate limiting, electrophilic labelling is under Curtin-Hammett control; hence, the final alkylated mixture may not represent the speciation that prevailed before alkylation. EXPERIMENTAL APPROACH: Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. KEY RESULTS: In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration- and time-dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. CONCLUSIONS AND IMPLICATIONS: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Metabolism of sulfur compounds in homocystinurias.

BACKGROUND AND PURPOSE: Homocystinurias are rare genetic defects characterized by altered fluxes of sulfur compounds including homocysteine and cysteine. We explored whether the severely perturbed sulfur amino acid metabolism in patients with homocystinurias affects the metabolism of hydrogen sulfide. EXPERIMENTAL APPROACH: We studied 10 treated patients with a block in the conversion of homocysteine to cysteine due to cystathionine ß-synthase deficiency (CBSD) and six treated patients with remethylation defects (RMD) and an enhanced flux of sulfur metabolites via transsulfuration. Control groups for CBSD and RMD patients consisted of 22 patients with phenylketonuria on a low-protein diet and of 12 healthy controls respectively. Plasma and urine concentrations of selected sulfur compounds were analysed by HPLC and LC-MS/MS. KEY RESULTS: Patients with CBSD exhibited plasma concentrations of monobromobimane-detected sulfide similar to appropriate controls. Urinary homolanthionine and thiosulfate in CBSD were increased significantly 1.9 and 3 times suggesting higher hydrogen sulfide synthesis by γ-cystathionase and detoxification respectively. Surprisingly, patients with RMD had significantly lower plasma sulfide levels (53 and 64% of controls) with lower sulfite concentrations, and higher taurine and thiosulfate levels suggesting enhanced cysteine oxidation and hydrogen sulfide catabolism respectively. CONCLUSION AND IMPLICATIONS: The results from this study suggest that severe inherited defects in sulfur amino acid metabolism may be accompanied by only moderately perturbed hydrogen sulfide metabolism and lends support to the hypothesis that enzymes in the transsulfuration pathway may not be the major contributors to the endogenous hydrogen sulfide pool. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Rapid analysis of sulfur mustard oxide in plasma using gas chromatography-chemical ionization-mass spectrometry for diagnosis of sulfur mustard exposure.

Sulfur mustard (SM) is the most utilized chemical warfare agent in modern history and has caused more casualties than all other chemical weapons combined. SM still poses a threat to civilians globally because of existing stockpiles and ease of production. Exposure to SM causes irritation to the eyes and blistering of skin and respiratory tract. These clinical signs of exposure to SM can take 6-24 h to appear. Therefore, analyzing biomarkers of SM from biological specimens collected from suspected victims is necessary for diagnosis during this latent period. Here, we report a rapid, simple, and direct quantitative analytical method for an important and early SM biomarker, sulfur mustard oxide (SMO). The method includes addition of a stable isotope labeled internal standard, SMO extraction directly into dichloromethane (DCM), rapid drying and reconstitution of the extract, and direct analysis of SMO using gas chromatography-chemical ionization-mass spectrometry. The limit of detection of the method was 0.1 µM, with a linear range from 0.5 to 100 µM. Method selectivity, matrix effect, recovery, and short-term stability were also evaluated. Furthermore, the applicability of the method was tested by analyzing samples from inhalation exposure studies performed in swine. The method was able to detect SMO from 100% of the exposed swine (N = 9), with no interferences present in the plasma of the same swine prior to exposure. The method presented here is the first of its kind to allow for easy and rapid diagnosis of SM poisoning (sample analysis <15 min), especially important during the asymptomatic latency period.

Simultaneous determination of sulfur compounds from the sulfur pathway in rat plasma by liquid chromatography tandem mass spectrometry: application to the study of the effect of Shao Fu Zhu Yu decoction.

A sensitive, accurate, and time-saving approach was developed for the simultaneous quantification of eight sulfur compounds in the sulfur pathway, which could reflect the status of an organism, including oxidative stress, signal transduction, enzyme reaction, and so on. In order to overcome the instability of highly reactive sulfhydryl compounds, N-ethylmaleimide derivatization was adopted to effectively protect sulfhydryl-containing samples. Using isotope-labeled glutathione (GSH-13C2, 15N), the validated method was demonstrated to offer satisfactory linearity, accuracy, and precision. Separation was done by UHPLC, using a BEH amide column. Accordingly, 0.1% formic acid acetonitrile was selected as the precipitant. A tandem mass spectrometer was coupled to the chromatographic system and afforded a detection limit of 0.2 ng/mL. Good linearity was maintained over a wide concentration range (r2 > 0.994), and the accuracy was in the range of 86.6-114% for all the studied compounds. The precision, expressed in RSD%, ranged from 1.1% to 9.4% as intraday variability and less than 13% as interday precision for all of the analytes. The approach was applied to study the potential therapeutic mechanism of a well-known traditional Chinese medicine, Shao Fu Zhu Yu decoction. The results suggested that Shao Fu Zhu Yu decoction might protect against oxidative damage by increasing the concentrations of sulfhydryl compounds. Graphical abstract An approach to quantitatively determining sulfur compounds in the sulfur pathway simultaneously wasestablished and applied to the study of the effect of Shao Fu Zhu Yu decoction.

Dietary Route of Exposure for Rabbit Developmental Toxicity Studies.

Dietary administration is a relevant route of oral exposure for regulatory toxicity studies of agrochemicals as it mimics potential human intake of the chemical via treated crops and commodities. Moreover, dietary administration of test compounds during a developmental toxicity study can deliver a prolonged and stable systemic exposure to the embryo or fetus at all stages of development. In this study, strategies were employed to optimize rabbit test material consumption via diet. Comparative toxicokinetic profiles of gavage versus dietary administration were evaluated in pregnant or non-pregnant New Zealand White rabbits for 2 novel agrochemicals with different plasma half-lives of elimination (sulfoxaflor, t½ = 13.5 h and halauxifen, t½ = 1 h). Dietary administration of sulfoxaflor resulted in stable 24-h plasma concentrations, whereas gavage administration resulted in a 3-fold fluctuation in plasma levels between Cmax and Cmin Dietary administration of sulfoxaflor resulted in a 2-fold higher nominal and diurnal systemic dose when compared with gavage dosing due to Cmax-related maternal toxicity following gavage. Results with the shorter half-life molecule, halauxifen, were more striking with a 6-fold diurnal fluctuation by the dietary route compared with a 368-fold fluctuation between Cmax and Cmin by gavage. Furthermore, plasma halauxifen was detectable only up to 12 h following gavage but up to 24 h following dietary administration. Finally, the presence of these compounds in fetal blood samples was demonstrated, confirming that dietary exposure is appropriate for achieving fetal exposure. Collectively, the results of these studies support the use of dietary exposure in rabbit developmental toxicity studies.

Expedition of in vitro dissolution and in vivo pharmacokinetic profiling of sulfur nanoparticles based antimicrobials.

A comprehensive pharmacokinetic profiling of novel drugs and therapeutics is a definite prerequisite of drug discovery and development. The present study expedites the in vivo and in vitro pharmacokinetic properties of colloidal sulfur nanoparticles (SNPs). In vitro dissolution properties of SNPs have been demonstrated and compared with the in vivo pharmacokinetic parameters of rabbit (Oryctolagus cuniculus) serum sample. The present study was also aimed at developing levels of correlation between in vitro and in vivo pharmacokinetic parameters. Cumulative results of the proposed study also suggest good in vitro-in vivo correlation of these novel nanocolloids and suggest their immediate profiling as an antimicrobial drug.

Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study.

A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid-liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLC™ BEH HILIC column (1.7 µm, 2.1 mm×50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6±0.1 and 2.1±0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5-4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from -6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats.

Measurement of sulfur-containing compounds involved in the metabolism and transport of cysteamine and cystamine. Regional differences in cerebral metabolism.

An HPLC method with coulometric detection is presented for the quantitation of cysteamine, cystamine, thialysine, glutathione, glutathione disulfide and an oxidized metabolite of thialysine [S-(2-aminoethyl)-L-cysteine ketimine decarboxylated dimer (AECK-DD)]. The advantage of coulometric detection is that derivatization is unnecessary if the analyte is redox sensitive. The method was used to quantitate several sulfur-containing compounds in plasma and brain following gavage feeding of cysteamine to rats. Cysteamine, cystamine, thialysine and AECK-DD were detected in the brains of these animals. Interestingly, cysteamine treatment resulted in greatly elevated levels of cerebral methionine, despite the fact that cysteamine is not a precursor of methionine.

High-throughput determination of ultra-low concentrations of LAG078, a lipid modulator, in human plasma.

A high throughput method with ultra-low level quantification limit (10 pg/ml) was developed and validated for the quantitative determination of LAG078, a lipid modulator, in human plasma to support clinical studies employing low doses of the compound. The method consisted of reverse phase chromatographic separation of the analyte from plasma extract followed by electrospray ionization (ESI) in the negative ion mode and tandem mass spectrometry in the multiple reaction monitoring mode (MRM). Extraction was performed using a combination of protein precipitation and liquid-liquid extraction in the 96-well plate format to increase the throughput of the method. Optimised chromatographic separation in a short and high-resolution column (50 mm x 2.0 mm i.d., 3 microm particle size) coupled with MRM mode of detection yielded clean chromatograms with minimal signal suppression. The standard curve was linear (r=0.996) within the concentration range of 0.01 (lower limit of quantification) to 50 ng/ml using 0.5 ml of human plasma. The accuracy of the method varied from 95-101% with a precision (CV) of 5.29-13.2% over the concentration range. The method was simple and rapid.

Identification and quantification of the conjugated metabolites derived from orally administered hesperidin in rat plasma.

Hesperidin is a biologically effective flavonoid. Several studies have reported that dietary hesperidin was converted to conjugated metabolites, such as hesperetin-glucuronides and sulfoglucuronides, during absorption and metabolism. However, the chemical structures of the conjugated metabolites, especially the sites of glucuronidation and sulfoglucuronidation in plasma, were unconfirmed. Therefore, the concentrations of the metabolites conjugated at various sites in plasma could not be individually quantified. In the present study, we identified the chemical structures and concentrations of the major conjugated metabolites in rat plasma after oral administration of hesperidin. Two hesperetin-glucuronides were prepared and identified as hesperetin-7-O-beta-D-glucuronide and hesperetin-3'-O-beta-D-glucuronide. Using these authentic compounds, the concentrations of hesperetin-7-O-beta-D-glucuronide and hesperetin-3'-O-beta-D-glucuronide in rat plasma were individually determined by liquid chromatography-mass spectrometry. In rat plasma, hesperetin-glucuronides were primarily comprised of hesperetin-7-O-beta-D-glucuronide and hesperetin-3'-O-beta-D-glucuronide. The concentration of hesperetin-7-O-beta-D-glucuronide was slightly higher than that of hesperetin-3'-O-beta-D-glucuronide. Furthermore, not only hesperetin conjugates but also homoeriodictyol conjugates were observed in rat plasma. The present study is the first report elucidating the chemical structures and changes in individual concentrations in rat plasma of glucuronides derived from orally administered hesperidin.

Determination of LAG078, a lipid-lowering compound, in dog plasma using liquid chromatography-tandem mass spectrometry: application in a toxicokinetic study.

A high throughput method was developed and validated for the quantitative determination of LAG078, a lipid-lowering compound, in dog plasma obtained during toxicokinetic studies. The method was based on reverse phase liquid chromatographic separation of the analyte from plasma extract followed by turbo-ionspray (TIS) in the negative ion mode and tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. Extraction was performed using a combination of protein precipitation and liquid-liquid extraction in the 96-well plate format to increase the throughput of the method. Optimized chromatographic separation under basic condition (pH approximately 10) in a short polymer based column (50 mm x 2.0 mm i.d.) coupled with MRM mode of detection yielded clean chromatograms with minimal signal suppression. The standard curve was linear (r = 0.997) within the concentration range of 0.05 (lower limit of quantification; LLOQ) to 50 ng/ml using only 0.1 ml of dog plasma. The accuracy of the method varied from 95 to 100% with a precision (CV) of 3.04-10.8% over the concentration range. The method was simple, rapid, and robust.

Antioxidative and antiglycative effects of six organosulfur compounds in low-density lipoprotein and plasma.

Low-density lipoprotein (LDL) and plasma were isolated from patients with non-insulin-dependent diabetes. The protective effects of six organosulfur compounds (DAS, diallyl sulfide; DADS, diallyl disulfide; SAC, S-allylcysteine; SEC, S-ethylcysteine; SMC, S-methylcysteine; SPC, S-propylcysteine) against further oxidation and glycation in these already partially oxidized and glycated samples were studied. DAS and DADS showed significantly greater oxidative-delaying effects than four cysteine-containing compounds in both partially oxidized LDL and plasma samples (P < 0.05). However, cysteine-containing agents were superior to DAS and DADS in delaying glycative deterioration in already partially glycated LDL (P < 0.05). The observed delays of oxidative and glycative effects from each agent were significantly concentration-dependent (P < 0.05). Furthermore, six organosulfur agents significantly decreased the loss of catalase and glutathione peroxidase activities in plasma and increased alpha-tocopherol retention in LDL and plasma (P < 0.05). These results suggested that the use of these organosulfur agents derived from garlic at these concentrations could protect partially oxidized and glycated LDL or plasma against further oxidative and glycative deterioration, which might benefit patients with diabetic-related vascular diseases.

Kinetic spectrophotometric determination of some sulfur containing compounds in pharmaceutical preparations and human serum.

A kinetic spectrophotometric method was developed for the determination of carbocisteine, ethionamide, thioctic acid and penicillamine based on the catalytic effect on the reaction between sodium azide and iodine in aqueous solution. Ten to 100 microg ml(-1) of carbocisteine and ethionamide, 0.1-1 microg ml(-1) of thioctic acid and 0.01-0.1 microg ml(-1) of penicillamine could be determined, respectively, by measuring the decrease in the absorbance of iodine at 348 nm by a fixed time method. The decrease in the absorbance in the first 5 min from the initiation of the reaction is related to the concentration of the drugs. The detection limits were 0.47, 0.71, 0.018 and 9.38 x 10(-4) microg ml(-1) for the four drugs, respectively. The proposed procedure was successfully applied in the determination of these drugs in pharmaceutical preparations and human serum.

Halitosis in medicine: a review.

This review deals with the different forms of halitosis. Halitosis can be subdivided according to its original location. At present, halitosis of oral origin is quite well understood and some excellent reviews have already appeared in the literature. Special attention is given here to extra-oral halitosis. Extra-oral halitosis can be subdivided into: halitosis from the upper respiratory tract including the nose; halitosis from the lower respiratory tract; blood-borne halitosis. In blood-borne halitosis, malodourant compounds in the bloodstream are carried to the lungs where they volatilise and enter the breath. Potential sources of blood-borne halitosis are some systemic diseases, metabolic disorders, medication and certain foods. The methods of analysis of halitosis are critically reviewed. Attention is also given to odour characterisation of various odourants.

Determination of a sulfur-containing drug in human plasma by an improved method for sulfur chemiluminescence detection in combination with capillary gas chromatography.

An improved method for sulfur chemiluminescence detection in combination with capillary gas chromatography was established. The method was applied to the analysis of a sulfur-containing antiasthma drug, S-1452, and its nine metabolities in human plasma. The high selectivity and sensitivity of the method allowed accurate quantitation of trace levels of these compounds in human plasma with no interferences from the biological components present. To enable stable measurements and maintain reproducibility over a long period, the performance characteristics of a commercially available instrument were investigated. The gas seal in the injection port was found to easily corrode through interaction of the sulfur analyte with the metal gas seal. To prevent this, a disk made from an alloy of platinum and gold (60:40) was mounted on the gas seal. The reproducibility of the measurement was improved remarkably by using the disk. The use of compressed air of high purity significantly lowered the signal-to-noise ratio. The optical filter was kept clean by using a nickel catalyst to trap ozone in place of copper manganese oxide (CuMn2O4). These improvements raised the sensitivity and selectivity with the lower quantitation limits of 0.5-1.0 ng/mL in human plasma.