Adducts of N-terminal valines in hemoglobin with isoprene diepoxide, a metabolite of isoprene

Rapid Commun Mass Spectrom. 2004;18(18):2177-84. doi: 10.1002/rcm.1608.


Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • Animals
  • Butadienes / analysis*
  • Butadienes / chemistry
  • Cells, Cultured
  • Epoxy Compounds / analysis
  • Epoxy Compounds / chemistry
  • Erythrocytes / metabolism*
  • Gas Chromatography-Mass Spectrometry / methods*
  • Hemiterpenes / analysis*
  • Hemiterpenes / chemistry
  • Hemoglobins / analysis*
  • Hemoglobins / chemistry
  • Mice
  • Pentanes / analysis*
  • Pentanes / chemistry
  • Protein Binding
  • Rats
  • Species Specificity
  • Valine / analysis*
  • Valine / chemistry


  • Butadienes
  • Epoxy Compounds
  • Hemiterpenes
  • Hemoglobins
  • Pentanes
  • isoprene
  • Valine