Determination of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in the general population using gas chromatography-mass spectrometry.
Carbamoylation of glutathione, peptides and DNA is thought to be one of the most important reactions occurring in an organism after exposure to nitrosoureas, methylformamides or isocyanates. The carcinogenic effects of carbamoylation are not yet fully clarified. Although carbamoylation is known to occur after occupational exposure, it has never been reported in the general population. To clarify the situation, we investigated the levels of N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) in urine samples from persons without occupational exposure using a sensitive and specific method (gas chromatography-mass spectrometry, GC-MS). AMCC is the degradation product of N-methylcarbamoylated glutathione. The clean-up procedure of urine samples includes two liquid-liquid extraction steps and solid phase extraction using a cation-exchange resin to separate AMCC from other urinary components. N,N-Dimethylpropionic acid amide (DMPA) is used as internal standard. During the preparation of the samples, AMCC is converted to ethyl-N-methylcarbamate (EMC) in the presence of anhydrous potassium carbonate (K2CO3) and ethanol. The reliability and accuracy of this method have been proven in detail. The relative standard deviation for the within-series imprecision for three different concentrations was determined to be between 10.9% and 14.3%, while the relative standard deviation for the between-day imprecision was between 11.3% and 14.8%. The mean recovery for AMCC was determined to be between 79.2% and 85.6%. The limit of detection for the simultaneous measurement of two fragment masses was 30 micrograms L-1. Using this GC-MS method, we analysed urine samples from 42 individuals of the general population in order to determine their urinary excretion of AMCC. It was identified in 40 samples. The mean concentration was 40 micrograms L-1. AMCC can be formed in two ways. The first possibility is the dietary intake of isothiocyanates, especially methyl isothiocyanate, which is a component of wine and cruciferous vegetables (such as cabbage, turnips and cress). During the metabolism of isothiocyanates in humans, the sulfur is partly exchanged for oxygen resulting in the formation of the corresponding isocyanate derivatives. The other possibility is the physiological formation of AMCC. In humans, this may occur via a two step process: carbamoylation and methylation, or vice versa. However, as AMCC was identified in about 95% of urine samples, and the standard deviation for the level of AMCC excreted was low, physiological formation seems to be the more probable pathway.
Käfferlein
H U
HU
Institut und Poliklinik für Arbeits-, Sozial- und Umweltmedizin, Friedrich-Alexander Universität Erlangen-Nürnberg, Schillerstr. 25/29, D-91054 Erlangen, Germany.
Angerer
J
J
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000652:urine*; D000293:Adolescent; D000328:Adult; D000970:Antineoplastic Agents / Q000652:urine*; D015415:Biomarkers / Q000032:analysis; D004032:Diet; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D017879:Isothiocyanates / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D016273:Occupational Exposure
D000970:Antineoplastic Agents; D015415:Biomarkers; D017879:Isothiocyanates; C052469:N-acetyl-S-(N-methylcarbamoyl)cysteine; D000111:Acetylcysteine