Simultaneous assessment of endogenous thiol compounds by LC-MS/MS.

Biological thiol compounds are very important molecules that participate in various physiological events. Alteration in levels of endogenous thiols has been suggested as a biomarker of early stage of pathological changes. We reported a chemical derivatization- and LC-MS/MS-based approach to simultaneously determine thiol compounds including glutathione (GSH), cysteine (Cys), N-acetyl cysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples. Thiol-containing samples were derivatized with monobromobimane (mBrB) at room temperature, followed by LC-MS/MS analysis. Assessment of the analytes with baseline separation was completed within 10min, using a gradient elution on a C18 reversed-phase column. Excellent linearity was observed for all analytes over their concentration ranges of 1-400µM. The lowest limits of detection (S/N=3) in a range from 0.31fmol (for NAC) to 4.98fmol (for CysGly) were achieved. The results indicate that this approach was sensitive, selective, and well suited for high-throughput quantitative determination of the biological thiols.

Plasma-aminothiols status and inverse correlation of total homocysteine with B-vitamins in arsenic exposed population of West Bengal, India.

Chronic arsenic toxicity is a serious environmental health problem across the world. Bangladesh and India (particularly the state of West Bengal) are the worst affected countries with such problem. The present study reports plasma-aminothiols (p-aminothiols) like L-cysteine (L-Cys), cysteinyl glycine (Cys-gly), total homocysteine (t-Hcy) and glutathione (GSH) status, and the inverse relationship of t-Hcy with B-vitamins (B1, B6, B9 and B12) in arsenic exposed population of West Bengal, India. Reverse phase HPLC was used to measure p-aminothiols and serum B-vitamins in different arsenic exposed population. Arsenic in drinking water and urine were measured by flow injection analysis system - Atomic Absorption Spectrometry (FIAS-AAS) and Transversely heated graphite atomizer (THGA-AAS) techniques, respectively. Water arsenic exposure was >50 µg/L in 50% population, of which majority (33.58%) belong to the range of >50-500 µg/L and more than 8% were even >1000 µg/L. Urine arsenic (µg/g creatinine) levels increased with arsenic exposure. The variability among p-aminothiols was also observed with higher exposure to arsenic in drinking water. A significant difference between exposed and control population was noticed for plasma L-Cys. The difference of B-vitamins between the population exposed to <50 and >50 µg/L arsenic in drinking water was also found to be significant. B9 and B12 deficiency with increased consumption of arsenic in water corroborates the anemic conditions commonly observed among arsenic exposed population. The aminothiol status indicated oxidative stress in exposed population. This study demonstrated progressive increase in plasma t-Hcy as well as inverse relationships of serum B-vitamins with increased water arsenic concentration.

Serum thiols and cardiovascular risk scores: a combined assessment of transsulfuration pathway components and substrate/product ratios.

BACKGROUND: Serum thiols have shown associations with surrogate markers of cardiovascular disease. However, little information is available on their combined association with validated cardiovascular risk scores for primary prevention at population level. We sought to determine whether individual serum thiol concentrations and substrate/product ratios within the transsulfuration pathway are independently associated with such scores. METHODS: Data on clinical and demographic characteristics, serum thiols (homocysteine, cysteine, taurine, glutamylcysteine, total glutathione and cysteinylglycine) and high-sensitivity C-reactive protein (CRP) were collected from a sample of the Hunter Community Study without previous cardiovascular events [n=350, median age (IQR) = 62 (59-66) years]. Five-year absolute cardiovascular risk score for each subject was calculated using the Framingham Risk Equation. RESULTS: Median risk score was 7% (IQR 4-10). After adjusting for body mass index, estimated glomerular filtration rate and physical activity regression analysis showed independent associations between cardiovascular risk scores and a) higher serum homocysteine (B 0.066, 95% CI 0.040 to 0.091, P<0.001) and lower cysteine (B -0.003, 95% CI -0.005 to -0.001, P=0.003) and glutathione (B -0.029, 95% CI -0.056 to -0.003, P=0.03) concentrations; and b) higher homocysteine/cysteine (B 0.114, 95% CI 0.066 to 0.161, P<0.001) and lower glutathione/cysteinylglycine (B -1.145, 95% CI -2.030 to -0.260, P=0.011) ratios. No significant associations were observed between cardiovascular risk scores, taurine and CRP. CONCLUSIONS: Serum homocysteine, cysteine and glutathione are independently associated with cardiovascular risk scores at population level. Enzymatic pathways involved in reduced bioconversion of homocysteine into cysteine and increased glutathione degradation might play an important role in such associations.

Energy expenditure, satiety, and plasma ghrelin, glucagon-like peptide 1, and peptide tyrosine-tyrosine concentrations following a single high-protein lunch.

High-protein (HP) foods are more satiating and have a higher thermogenic effect than normal protein foods over the short-term as well as the long-term. We hypothesized that acute effects of higher protein intake on satiety may be related to acute metabolic and hormonal responses. The study was a single-blind, randomized, crossover design. Subjects underwent 2 indirect calorimetry tests for measurement of energy expenditure (EE) and substrate oxidation. After a standard subject-specific breakfast, subjects received 1 of 2 randomly assigned treatments: an appropriate protein (AP) lunch (10% energy (E) protein, 60%E carbohydrate, 30%E fat), or a HP lunch (25%E protein, 45%E carbohydrate, 30%E fat). The increase in postlunch EE tended to be greater after the HP lunch (0.85 +/- 0.32 kJ/min) than after the AP lunch (0.73 +/- 0.22 kJ/min) (P = 0.07). The respiratory quotient did not differ between the HP (0.84 +/- 0.04) and the AP (0.86 +/- 0.04) treatments. Satiety visual analogue scales (VAS) scores were significantly higher 30 and 120 min after the HP lunch than after the AP lunch. The area under the curve of the VAS score for satiety was higher after the HP lunch (263 +/- 61 mm/h) than after the AP lunch (AP 236 +/- 76 mm/h) (P < 0.02). Effects of the meals on satiety and diet-induced thermogenesis did not occur simultaneously with changes in plasma ghrelin, glucagon-like peptide 1, and peptide tyrosine-tyrosine concentrations. A single HP lunch, therefore, does not exert its acute effect on satiety through increased concentrations of satiety-related hormones. Other factors, which may explain the HP effect on satiety, may be metabolites or amino acids.