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.

Metabolic signatures of extreme longevity in northern Italian centenarians reveal a complex remodeling of lipids, amino acids, and gut microbiota metabolism.

The aging phenotype in humans has been thoroughly studied but a detailed metabolic profiling capable of shading light on the underpinning biological processes of longevity is still missing. Here using a combined metabonomics approach compromising holistic (1)H-NMR profiling and targeted MS approaches, we report for the first time the metabolic phenotype of longevity in a well characterized human aging cohort compromising mostly female centenarians, elderly, and young individuals. With increasing age, targeted MS profiling of blood serum displayed a marked decrease in tryptophan concentration, while an unique alteration of specific glycerophospholipids and sphingolipids are seen in the longevity phenotype. We hypothesized that the overall lipidome changes specific to longevity putatively reflect centenarians' unique capacity to adapt/respond to the accumulating oxidative and chronic inflammatory conditions characteristic of their extreme aging phenotype. Our data in centenarians support promotion of cellular detoxification mechanisms through specific modulation of the arachidonic acid metabolic cascade as we underpinned increased concentration of 8,9-EpETrE, suggesting enhanced cytochrome P450 (CYP) enzyme activity. Such effective mechanism might result in the activation of an anti-oxidative response, as displayed by decreased circulating levels of 9-HODE and 9-oxoODE, markers of lipid peroxidation and oxidative products of linoleic acid. Lastly, we also revealed that the longevity process deeply affects the structure and composition of the human gut microbiota as shown by the increased extrection of phenylacetylglutamine (PAG) and p-cresol sulfate (PCS) in urine of centenarians. Together, our novel approach in this representative Italian longevity cohort support the hypothesis that a complex remodeling of lipid, amino acid metabolism, and of gut microbiota functionality are key regulatory processes marking exceptional longevity in humans.

The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p.

The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta-king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings.