Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans, trans-Muconic acid (1,3-butadiene-1, 4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C18 column (5 x 0.46 cm I.D., 3 microns particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50-500 microgram/l range; the quantification limit was 6 micrograms/l; day-to-day precision, at 300 micrograms/l, was C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean +/- S.D. = 77 +/- 54 micrograms/l, n = 82) were statistically different from those of smokers (169 +/- 85 micrograms/l, n = 30) (P < 0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits

A study of the glutathione metaboloma peptides by energy-resolved mass spectrometry as a tool to investigate into reference of toxic heavy metals with their metabolic processes

To better understand the fragmentation processes of the metal-biothiol conjugates and their possible significance in biological terms, an energy-resolved mass spectrometric study of the glutathione conjugates of heavy metals, of several thiols and disulfides of the glutathione metaboloma has been carried out. The main fragmentation process of gamma-glutamyl compounds, whether in the thiol, disulfide, thioether or metal-bis-thiolate form, is the loss of the gamma-glutamyl residue, a process which ERMS data showed to be hardly influenced by the sulfur substitution. However, loss of the gamma-glutamyl residue from the mono-S-glutathionyl-mercury (II) cation is a much more energetic process, possibly pointing at a strong coordination of the carboxylic group to the metal. Moreover, loss of neutral mercury from ions containing the gamma-glutamyl residue to yield a sulfenium cation was a much more energetic process than those not containing them, suggesting that the redox potential of the thiol/disulfide system plays a role in the formal reduction of the mercury dication in the gas phase. Occurrence of complementary sulfenium and protonated thiol fragments in the spectra of protonated disulfides of the glutathione metaboloma mirrors the thiol/disulfide redox process of biological importance. The intensity ratio of the fragments is proportional to the reduction potential in solution of the corresponding redox pairs. This finding has allowed the calculation of the previously unreported reduction potentials for the disulfide/thiol pair of cysteinylglycine, thereby confirming the decomposition scheme of bis- and mono-S-glutathionyl-mercury (II) ions. Finally, on the sole basis of the mass spectrometric fragmentation of the glutathione-mercury conjugates, and supported by independent literature evidence, an unprecedented mechanism for mercury ion-induced cellular oxidative stress could be proposed, based on the depletion of the glutathione pool by a catalytic mechanism acting on the metal (II)-thiol conjugates and involving as a necessary step the enzymatic removal of the glutamic acid residue to yield a mercury (II)-cysteinyl-glycine conjugate capable of regenerating neutral mercury through the oxidation of glutathione thiols to the corresponding disulfides

Comparison between blood and urinary toluene as biomarkers of exposure to toluene

Objectives: To compare blood toluene (TOL-B) and urinary toluene (TOL-U) as biomarkers of occupational exposure to toluene, and to set a suitable procedure for collection and handling of specimens. Method: An assay based on headspace solid-phase microextraction (SPME) was used both for the determination of toluene urine/air partition coefficient (lambda(urine/air)) and for the biological monitoring of exposure to toluene in 31 workers (group A) and in 116 non-occupationally exposed subjects (group B). Environmental toluene (TOL-A) was sampled during the work shift (group A) or during the 24 h before specimen collection (group B. Blood and urine specimens were collected at the end of the shift (group A) or in the morning (group B) and toluene was measured. Results: Toluene lambda(urine/air) was 3.3 +/- 0.9. Based on the specimen/air partition coefficient, it was calculated that the vial in which the sample is collected had to be filled up to 85% of its volume with urine and 50% with blood in order to limit the loss of toluene in the air above the specimen to less than 5%. Environmental and biological monitoring of workers showed that the median personal exposure to toluene (TOL-A) during the work-shift was 80 mg/m(3), the corresponding TOL-B was 82 mu g/l and TOL-U was 13 mu g/l Personal exposure to toluene in environmentally exposed subjects was 0.05 mg/m(3), TOL-B was 0.36 mu g/l and TOL-U was 0.20 mu g/l. A significant correlation (P < 0.05) was observed between TOL-B or TOL-U and TOL-A (Pearson's r = 0.782 and 0.754) in workers, but not in controls. A significant correlation was found between TOL-U and TOL-B both in workers and in controls (r = 0.845 and 0.681). Conclusion: The comparative evaluation of TOL-B and TOL-U showed that they can be considered to be equivalent biomarkers as regards their capacity to distinguish workers and controls and to correlate with exposure. However, considering that TOL-U does not require an invasive specimen collection, it appears to be a more convenient tool for the biological monitoring of exposure to toluene

Measurement of S-methylcysteine and S-methyl-mercapturic acid in human urine by alkyl-chloroformate extractive derivatization and isotope-dilution gas chromatography\u2013mass spectrometry

S-methylcysteine (SMC) is a minor amino acid naturally excreted in human urine, a protective agent against oxidative stress and a biotransformation product of the fumigant biocide methyl bromide and of nicotine. A metabolic source of SMC is catabolism of the repair catalytic protein MGMT (EC 2.1.1.37), which specifically removes the methyl group from the modified DNA nucleotide O-6-methyl-guanine to revert the normal GC base pairing. To assess the value of SMC and of S-methylmercapturic acid (SMMA) as candidate biomarkers of proliferative phenomena, a sensitive analytical method by GC-MS was applied in a pilot study of healthy subjects to assess their urinary elimination and the intra- and inter-individual variability. Extractive alkylation with butylchloroformate-n-butanol-pyridine (Husek technique) was employed for sample derivatization and isotope dilution GC-MS with S-[CD3]-SMC and -SMMA was applied for specific and sensitive detection. To resolve the target analytes from the main coeluting interferents in the derivatized urine extract a medium-polarity stationary phase was employed. SMMA was not detected in the morning urine of three healthy fertile-age women followed for one month above the minimum detectable level of approx. 500\u2009\ub5g/L while SMC concentrations were in the 0.02\u20130.7\u2009\ub5g/mL range (n = 61) with large inter-day and inter-individual variations. In a young healthy male urine samples taken throughout a few days yielded concentrations in the same 90\u2013810\u2009\ub5g/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes