Isocyanates such as methylisocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, or diisocyanates, used in the production of plastics, are highly reactive toxic compounds that spontaneously bind to biological macromolecules. In vivo formation of stable adducts with blood protein globin offers possibilities for biomonitoring of internal exposure to various reactive species. Thus, biomonitoring of the isocyanates through determination of their specific adducts with globin is a challenge. In this study, we characterized the adducts formed in human globin upon treatment with 100-fold molar excess of MIC. The globin was subject to enzymatic hydrolysis with pronase, and the hydrolysate was analysed by high performance liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometric detection (HPLC/APCI-MS). The two major MIC adducts were those with N-terminal Val and side-chain of Lys, as confirmed by comparison with the synthetic standards. About 20 other adducts were observed, and several of them were tentatively identified using their MS and MS/MS spectra. Whereas detection of the adducts with Tyr and His was expected, the adducts with Trp and Phe, and a Lys adduct containing two MIC moieties, were probably analytical artifacts resulting from the transcarbamoylation during globin hydrolysis rather than products of direct carbamoylation. The other detected products were MIC-Val-His, derived from the N-terminal dipeptide of globin beta-chain, and dipeptides consisting of MIC-Lys attached to Gly, Val, Leu, Thr, and Glu. Failure to detect the corresponding non-modified dipeptides suggests that the pronase action may be hampered by the amino acid modification. MIC is known as a metabolic intermediate of the industrial solvents N,N-dimethylformamide (DMF) and N-methylformamide (MF) in humans and rats. The HPLC/APCI-MS analysis of globin from rats injected with DMF or MF, 1000 mg/kg, revealed the presence of the MIC adducts with both Val and Lys. The level of the Val adduct in globin from the DMF-dosed rats, determined using Edman degradation and GC/MS, was ca. 40 nmol/g, which is a level common in workers occupationally exposed to DMF. This suggests that also the Lys adduct in such human globin samples can be feasible to analysis and is therefore considered for further studies as a potential biomarker of exposure to DMF.

• MeSH
• biologické markery analýza   MeSH
• dimethylformamid farmakokinetika   MeSH
• erytrocyty chemie   metabolismus   MeSH
• formamidy farmakokinetika   MeSH
• globiny chemie   metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• hydrolýza MeSH
• isokyanatany chemie   metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• lysin chemie   metabolismus   MeSH
• monitorování životního prostředí metody   MeSH
• pronasa chemie   MeSH
• valin chemie   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• dimethylformamid MeSH
• formamidy MeSH
• globiny MeSH
• isokyanatany MeSH
• lysin MeSH
• methyl isocyanate MeSH Prohlížeč
• methylformamide MeSH Prohlížeč
• pronasa MeSH
• valin MeSH

Diisocyanates, reactive compounds used in plastics industry and potent occupational allergens, readily bind to proteins both in vitro and in vivo, however, the pattern of adducts with individual amino acids has not been investigated systematically. In this study, potential of the proteinogenic amino acid residues for carbamoylation with 2,4-toluenediisocyanate (2,4-TDI) and hexamethylenediisocyanate (HDI) was evaluated. The diisocyanates were incubated in an in vitro system (buffer pH 7.4/dioxane 50:50) with: (a) a series of Nalpha-benzyloxycarbonyl amino acids (Z-amino acids) and N-acetylcysteine (Ac-Cys), model compounds for non-N-terminal amino acids of the protein chain; (b) dipeptides Val-Phe and Asp-Phe, model compounds for N-termini of globin and albumin, respectively. Reactivity of the compounds tested, evaluated from their depletion during incubation with the diisocyanates (measured by HPLC), was in the order: Ac-Cys = Asp-Phe > Val-Phe = Nalpha-Z-Lys >> Nalpha-Z-His for 2,4-TDI, and Ac-Cys > Asp-Phe > Val-Phe = Nalpha-Z-Lys > Nalpha-Z-His > N-Z-Tyr for HDI, however, the adducts with Ac-Cys were unstable. Reactions of other amino acid residues (e.g. Ser, Thr, Met, Trp, Arg, Asn, Gln) with 2,4-TDI and HDI were not observed. Thus, N-terminal amino acids and Lys residues are likely to produce most abundant adducts with diisocyanates in proteins. Further, three amino compounds with increasing pKa values (Val-Phe, Val and Nalpha-Z-Lys) were incubated with 2,4-TDI and N-acetyl-S-[4-(2-amino)tolylcarbamoyl]cysteine, a 2,4-TDI-derived thiocarbamate with carbamoylating activity, in media with 10% and no dioxane, respectively. Here, reactivity of the amino compounds was decreasing in the order: Val-Phe > Val > Nalpha-Z-Lys, which reflects the mechanism of the amine-isocyanate reaction. The experiments also demonstrate the effect of a solvent (organic phase content) on the yield of the carbamoylation reactions.

• MeSH
• albuminy chemie   MeSH
• aminokyseliny chemie   metabolismus   MeSH
• chromatografie na tenké vrstvě MeSH
• globiny chemie   MeSH
• isokyanatany MeSH
• krevní proteiny chemie   metabolismus   MeSH
• kyanatany metabolismus   MeSH
• techniky in vitro MeSH
• toluendiisokyanát chemie   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,6-hexamethylene diisocyanate MeSH Prohlížeč
• albuminy MeSH
• aminokyseliny MeSH
• globiny MeSH
• isokyanatany MeSH
• krevní proteiny MeSH
• kyanatany MeSH
• toluendiisokyanát MeSH

Metabolism of the solvents N,N-dimethylformamide (DMF) and N-methylformamide (MF) results in the formation of N-methylcarbamoyl adducts at the N-terminal valine and lysine in blood protein globin, of which the lysine adduct has so far only been reported in rats given high doses of both solvents [Mráz, J., Simek, P., Chvalová, D., Nohová, H., Smigolová, P., 2004. Studies on the methyl isocyanate adducts in globin. Chem. Biol. Interact. 148, 1-10]. Here we examined whether the lysine adduct is produced, and accessible to analysis, in humans occupationally or experimentally exposed to DMF. Globin from exposed subjects (n=35) and unexposed controls (n=5) was analyzed by two methods. Edman degradation was used as a sensitive reference method to measure the valine adduct by converting it to 3-methyl-5-isopropylhydantoin (MVH). The MVH levels in globin of the exposed subjects were in the range of 1-441 nmol/g, in controls <1 nmol/g. The principal method of globin analysis consisted of enzymatic hydrolysis with pronase to release free N(epsilon)-(N-methylcarbamoyl)lysine (MLU) and N-methylcarbamoylvaline (MVU), which were determined by HPLC/MS/MS, with no clean-up or preconcentration steps needed. For MLU, the parent and product ions were m/z 204-->173, and the limit of detection was approximately 5 nmol/g globin. MLU was found in most globins from the exposed subjects but not in the controls. A close correlation between the MLU and MVH levels (nmol/g) was observed: MLU=7+0.48 MVH (R(2)=0.84, n=32). In conclusion, MLU can be easily measured in globin of workers exposed to DMF. The findings also indicate a long-term persistence of MLU in the human body, and consequently, its potential as a biomarker of chronic exposure to DMF.

• MeSH
• biologické markery MeSH
• dimethylformamid metabolismus   MeSH
• globiny metabolismus   MeSH
• hydantoiny metabolismus   MeSH
• lidé MeSH
• lysin analogy a deriváty   metabolismus   MeSH
• pracovní expozice analýza   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-methyl-5-isopropylhydantoin MeSH Prohlížeč
• biologické markery MeSH
• dimethylformamid MeSH
• globiny MeSH
• hydantoiny MeSH
• lysin MeSH