Fabrication of a "Selenium Signature" Chemical Probe-Modified Paper Substrate for Simultaneous and Efficient Determination of Biothiols by Paper Spray Mass Spectrometry.

Significant efforts have been made to develop robust and reliable methods for simultaneous biothiols determination in different matrices, but there still exist the problems such as easy oxidation, tedious derivatization, and difficulty in discrimination, which brings unsatisfactory results in their accuracy and fast quantification in biological samples. To overcome these problems, a simultaneous biothiols detection method combining a "selenium signature" chemical probe and paper spray mass spectrometry (PS-MS) was proposed. In the strategy, the modified-paper substrate is used to enhance the analytical performance. Chemical probe Ebselen-NH2 that has a specific response to biothiols was designed and covalently fixed on the surface of an oxidized paper substrate. By the identification of derivatized product with distinctive selenium isotope distribution and employment of the optimized PS-MS method, qualitative and quantitative analysis of five biothiols including glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), N-acetylcysteine (Nac), and homocysteine (Hcy) were realized. Biothiols in plasma and cell lysates were measured with satisfactory results. The established method not only provides a novel protocol for simultaneous determination of biothiols, but also is helpful for understanding the biological and clinical roles played by these bioactive small molecules.

Prospective randomized multicentre comparison on sibling oocytes comparing G-Series media system with antioxidants versus standard G-Series media system.

RESEARCH QUESTION: Does the inclusion of three antioxidants (A3), acetyl-l-carnitine (ALC), N-acetyl-l-cysteine (NAC) and alpha-lipoic acid (ALA) improve human embryo development and pregnancy potential? DESIGN: Prospective randomized multicentre comparison of sibling oocytes. A total of 1563 metaphase II oocytes from 133 patients in two IVF centres. Day 3 embryo and day 5/6 blastocyst quality were assessed. Good embryo quality on day 3 was defined as 8 to 10 cells with even cells and low fragmentation; good quality blastocysts as 3BB or greater. Clinical outcome was assessed on transfers of fresh or vitrified-warmed blastocyst on day 5. RESULTS: Of the two-pronuclei, 40.7% (G-Series) and 50.2% (G-Series with A3 group) resulted in good quality embryos on day 3 (P < 0.05). The implantation rate by fetal sac was 39.2% and 50.6%, and by fetal heartbeat was 37.8% and 47.1% for the G-Series and G-Series with A3 group, respectively. When stratified by female patient age, patients 35-40 years had an implantation rate by fetal sac and heart of 23.5% in the G-Series compared with 57.5% (P < 0.05) and 50.0% (P < 0.05) in the A3 group. The ongoing pregnancies in patients 35-40 years were significantly higher in the A3 group (50%) compared with the control (25.8%) (P < 0.05). CONCLUSIONS: The presence of antioxidants during IVF and embryo culture for patients 35-40 years resulted in a significant increase in implantation and pregnancy rate. Supplementation of antioxidants to IVF and culture media may therefore improve the viability of human embryos in assisted reproductive technologies, plausibly through the reduction of oxidative stress.

Effect of N-acetyl cysteine and glycine supplementation on growth performance, glutathione synthesis, anti-oxidative and immune ability of Nile tilapia, Oreochromis niloticus.

An 8-week feeding trial was conducted to evaluate the effect of N-acetyl cysteine (NAC) and glycine supplementation on growth performance, glutathione (GSH) synthesis, anti-oxidative and immune ability of Nile tilapia, Oreochromis niloticus. Four practical diets were formulated, control, control +0.2% NAC, control +0.5% glycine, control +0.2% NAC +0.5% glycine. Each diet was randomly assigned to quadruplicate groups of 30 fish (approximately 9.5 g). The weight gain and specific growth rate were significantly increased with the supplementation of NAC and glycine. While they had no effect on feed efficiency feed intake and survival. Glutathion peroxidase (GPx) was increased by NAC and γ-glutamine cysteine synthase (γ-GCS) in plasma were increased by glycine. After the feeding trail, fish were challenged by Streptococcus iniae, fish fed the diet supplemented with NAC obtained significantly higher survival rate after 72 h challenge test. NAC also decreased malonaldehyde (MDA) in liver, increased glutathione S-transferase (GST) activity in plasma, up-regulated mRNA expression of Superoxide dismutase (SOD) and GPx in liver and headkidney. Dietary supplementation of glycine increased the anti-oxidative ability of tilapia through increase anti-oxidative enzyme activity (SOD, glutathione reductase, myeloperoxidase) and up-regulate anti-oxidative gene expression (SOD). Immune ability only enhanced by the supplementation of NAC through increased interleukin-1ß (IL-1ß) mRNA expression. These results clearly indicated that the supplementation of NAC and glycine can significantly improve the growth performance of tilapia, and NAC also enhance the anti-oxidative and immune capacity of tilapia, glycine could only enhance the anti-oxidative ability.

Analysis of 18 urinary mercapturic acids by two high-throughput multiplex-LC-MS/MS methods.

Mercapturic acids (MAs) are metabolic end products formed from conjugates between glutathione and electrophilic compounds. MAs are, therefore, suitable biomarkers of exposure to toxicants, which are either electrophiles by themselves or metabolized to electrophilic intermediates. We developed and validated two LC-MS/MS methods which allow the complementary, rapid, and sensitive determination of MAs derived from acrolein, acrylamide, acrylonitrile, benzene, 1,3-butadiene, crotonaldehyde, N,N-dimethylformamide, ethylene, ethylene oxide, vinyl chloride, propylene oxide, styrene, toluene as well as methylating and ethylating agents. Since separate determinations of single or small groups of MAs are time-consuming and expensive, we multiplexed several individual methods into two LC-MS/MS methods covering 18 individual mercapturic acids. Method validation according to FDA guidelines showed excellent results in terms of robustness, accuracy, and sensitivity of the methods. Moreover, the use of a minimal, simple, and straightforward sample cleanup procedure further accelerated the analytical workflow, which allows a time- and cost-efficient analysis of up to 18 MAs derived from various toxicants in environmental levels. The methods were applied to urine samples derived from a strictly diet-controlled clinical study, including 25 smoking and 25 non-smoking subjects. Significant increase in the urine concentrations in smokers as compared to non-smokers (p < 0.01; Student t test) was observed for 13 individual MAs. Moreover, a dose dependence was obtained for the majority of the analytes. In conclusion, the newly developed assays represent a powerful tool for the fast and reliable quantification of 18 MAs in clinical studies. A first method application suggests several suitable biomarkers for nine relevant toxicants in tobacco smoke.

Dietary antioxidants prevent alcohol-induced ciliary dysfunction.

Previously we have shown that chronic alcohol intake causes alcohol-induced ciliary dysfunction (AICD), leading to non-responsive airway cilia. AICD likely occurs through the downregulation of nitric oxide (NO) and cyclic nucleotide-dependent kinases, protein kinase G (PKG) and protein kinase A (PKA). Studies by others have shown that dietary supplementation with the antioxidants N-acetylcysteine (NAC) and procysteine prevent other alcohol-induced lung complications. This led us to hypothesize that dietary supplementation with NAC or procysteine prevents AICD. To test this hypothesis, C57BL/6 mice drank an alcohol/water solution (20% w/v) ad libitum for 6 weeks and were concurrently fed dietary supplements of either NAC or procysteine. Ciliary beat frequency (CBF) was measured in mice tracheas, and PKG/PKA responsiveness to ß-agonists and NOx levels were measured from bronchoalveolar lavage (BAL) fluid. Long-term alcohol drinking reduced CBF, PKG and PKA responsiveness to ß-agonists, and lung NOx levels in BAL fluid. In contrast, alcohol-drinking mice fed NAC or procysteine sustained ciliary function and PKG and PKA responsiveness to ß-agonists. However, BAL NO levels remained low despite antioxidant supplementation. We also determined that removal of alcohol from the drinking water for as little as 1 week restored ciliary function, but not PKG and PKA responsiveness to ß-agonists. We conclude that dietary supplementation with NAC or procysteine protects against AICD. In addition, alcohol removal for 1 week restores cilia function independent of PKG and PKA activity. Our findings provide a rationale for the use of antioxidants to prevent damage to airway mucociliary functions in chronic alcohol-drinking individuals.

Correlative analysis of metabolite profiling of Danggui Buxue Tang in rat biological fluids by rapid resolution LC-TOF/MS.

In this work, the metabolite profiles of Danggui Buxue Tang (DBT) in rat bile and plasma were qualitatively described, and the possible metabolic pathways of DBT were subsequently proposed. Emphasis was put on correlative analysis of metabolite profiling in different biological fluids. After oral administration of DBT, bile and plasma samples were collected and pretreated by solid phase extraction. Rapid resolution liquid chromatography coupled to time-of-flight mass spectrometry (RRLC-TOFMS) was used for characterization of DBT-related compounds (parent compounds and metabolites) in biological matrices. A total of 142 metabolites were detected and tentatively identified from the drug-containing bile and plasma samples. Metabolite profiling shows that rat bile contained relatively more glutathione-derived conjugates, more saponins compounds and more diverse forms of metabolites than urine. The metabolite profile in plasma revealed that glucuronide conjugates of isoflavonoids, dimmers, acetylcysteine conjugates and parent form of phthalides, as well as saponin aglycones were the major circulating forms of DBT. Collectively, the metabolite profile analysis of DBT in different biological matrices provided a comprehensive understanding of the in vivo metabolic fates of constituents in DBT.

Fluorimetric determination of some sulfur containing compounds through complex formation with terbium (Tb+3) and uranium (U+3).

Two simple, sensitive and specific fluorimetric methods have been developed for the determination of some sulphur containing compounds namely, Acetylcysteine (Ac), Carbocisteine (Cc) and Thioctic acid (Th) using terbium Tb+3 and uranium U+3 ions as fluorescent probes. The proposed methods involve the formation of a ternary complex with Tb+3 in presence of Tris-buffer method (I) and a binary complex with aqueous uranyl acetate solution method (II). The fluorescence quenching of Tb+3 at 510, 488 and 540 nm (lambda(ex) 250, 241 and 268 nm) and of uranyl acetate at 512 nm (lambda(ex) 240 nm) due to the complex formation was quantitatively measured for Ac, Cc and Th, respectively. The reaction conditions and the fluorescence spectral properties of the complexes have been investigated. Under the described conditions, the proposed methods were applicable over the concentration range (0.2-2.5 microg ml(-1)), (1-4 microg ml(-1)) and (0.5-3.5 microg ml(-1)) with mean percentage recoveries 99.74+/-0.36, 99.70+/-0.52 and 99.43+/-0.23 for method (I) and (0.5-6 microg ml(-1)), (0.5-5 microg ml(-1)), and (1-6 microg ml(-1)) with mean percentage recoveries 99.38+/-0.20, 99.82+/-0.28 and 99.93+/-0.32 for method (II), for the three cited drugs, respectively. The proposed methods were successfully applied for the determination of the studied compounds in bulk powders and in pharmaceutical formulations, as well as in presence of their related substances. The results obtained were found to be in agree statistically with those obtained by official and reported ones. The two methods were validated according to USP guidelines and also assessed by applying the standard addition technique.

Nonenzymatic antioxidant activity of four organosulfur compounds derived from garlic.

The nonenzymatic antioxidant activity of diallyl sulfide (DAS), diallyl disulfide (DADS), S-ethyl cysteine (SEC), and N-acetyl cysteine (NAC) in the liposome system was examined. The antioxidant protection from these organosulfur agents was concentration dependent (p < 0.05). SEC and NAC showed significantly lower lipophilicity and greater reducing power than DAS and DADS (p < 0.05). Greater antioxidant protection was presented in the combinations of alpha-tocopherol with four organosulfur agents than alpha-tocopherol treatment alone (p < 0.05), and SEC and NAC showed greater sparing effects on alpha-tocopherol (p < 0.05). Four organosulfur agents lost antioxidant activity when the temperature was 65 degrees C (p < 0.05). At pH 2.5 and 10, DAS and DADS still showed antioxidant activity (p < 0.05). On the basis of the observed nonenzymatic antioxidant protection, these organosulfur compounds are potent agents for enhancing lipid stability.