Isocyanate-specific hemoglobin adduct in rats exposed to 4,4 '-methylenediphenyl diisocyanate

4,4'-Methylenediphenyl diisocyanate (MDI) is the most important of the isoc yanates used as intermediates in the chemical industry. Among the main type s of damage after exposure to low levels of MDI are lung sensitization and asthma. Protein adducts of MDI might be involved in the etiology of sensiti zation reactions. It is therefore necessary to have sensitive and specific methods for monitoring the isocyanate exposure of workers. To date, urine m etabolites or protein adducts have been used as biomarkers in workers expos ed to MDI. However, with these methods it is not possible to determine if t he biomarkers result from exposure to MDI or to the parent aromatic amine 4 ,4'-methylenedianiline (MDA). This work presents a procedure for quantitati ng isocyanate-specific hemoglobin adducts. Blood proteins are used as marke rs of exposure and possibly as markers of dose size for the modifications o f macromolecules in the target organs where the disease develops. For the q uantitation of hemoglobin adducts, N-1- [4-(4-isocyanatobenzyl)phenyl] acet amide (AcMDI) was reacted with the tripeptide valyl-glycyl-glycine and with valine yielding N-[4-(4-acetylaminobenzyl)phenyl] carbamoyl]valyl-glycyl-g lycine and N-[4-[4-(acetylaminobenzyl)phenyl] carbamoyl] valine, respective ly. N- [4- [4(Acetylamino-3,5-dideuteriobenzyl)-2,6-dideuteriophenyl]carbam oyl]valine was synthesized from valine, as was N-1-[4-(4-isocyanato-3, 5-di deuteriobenzyl)-2,6-dideuteriophenyl] acetamide, for use as an internal sta ndard. These adducts were cleaved in 2 M HCl to yield the corresponding hyd antoins, 3-[4-(4-aminobenzyl)phenyl]-5-isopropyl-1,3-imidazoline-2,4-dione (MDA-Val-Hyd) and 3-[4-(4-amino-3,5-dideuteriobenzyl)-2,6dideuteriophenyl]- 5-isopropyl-1,3-imidazoline-2,4-dione, respectively. In globin of rats expo sed to MDI, MDA-Val-Hyd could be found in a dose-dependent manner. The addu ct was identified by HPLC/MS/MS and quantified by GC/MS after derivatizatio n with heptafluorobutyric anhydride. The amount of MDA-Val-Hyd found after acid hydrolysis of globin at 100 degrees C is about 12 times larger than th e sum of N-acetyl-4,4'-methylenedianiline (AcMDA) and MDA obtained from mil d base hydrolysis of hemoglobin. The MDA-Val-Hyd is an isocyanate-specific adduct. MDA and AcMDA released after mild base hydrolyses result most likel y from a sulfinamide adduct which is a typical adduct of arylamines. Accord ing to these results, higher amounts of isocyanate adducts than arylamine a dducts should be expected in workers exposed to isocyanates.