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

Federico M. Rubino, Marco Pitton, Daniela Di Fabio, Giuseppe Meroni, Enzo Santaniello, Enrico Caneva, Marco Pappini, Antonio Colombi

Research output: Contribution to journalArticlepeer-review

Abstract

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-[CD 3]-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. 500μg/L while SMC concentrations were in the 0.02-0.7μg/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 90-810μg/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes.

Original languageEnglish
Pages (from-to)330-343
Number of pages14
JournalBiomedical Chromatography
Volume25
Issue number3
DOIs
Publication statusPublished - Mar 2011

Keywords

  • Husek derivatization
  • Isotope dilution
  • S-methyl-cysteine
  • S-methyl-mercapturic acid
  • Urine

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Molecular Biology
  • Drug Discovery
  • Pharmacology

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