Mass spectral analyses of hemoglobin adducts formed after in vitro exposure of erythrocytes to hydroxymethylvinyl ketone

Hydroxymethylvinyl ketone (HMVK) is a reactive oxidation product of 3-butene-1,2-diol, a metabolite of 1,3-butadiene. The potential for HMVK (0.1 and 1 mM) to form hemoglobin (Hb) adducts in erythrocytes from Sprague–Dawley rats was investigated at physiological conditions (pH 7.4, 37 °C) using electrospray ionization mass spectrometry (ESI/MS). With the 0.1 mM HMVK globin samples, the results indicate HMVK adduction on the α2, β2 and β3 chains. With the 1.0 mM HMVK globin samples, adducts were detected on the β2 and β3 chains. However, no correlation was observed between incubation time and the extent of adduct formation, and additional adducts were detected when globin samples were fractionated by HPLC before the ESI/MS analyses. For specific localizations of adducts on the globin chains, trypsin digested peptides from the 1 mM HMVK globin samples were subjected to liquid chromatography/mass spectrometry analyses. The results, which are consistent with formation of HMVK adducts on several specific peptides within the α- and β-chains, suggest selectivity in the interaction of HMVK with the different cysteine residues in Hb. Because adducts were also detected in peptides containing no cysteine residues and multiple HMVK moieties were detected on some of the cysteine-containing peptides, the results suggest other amino acids may be also reactive with HMVK. Adduct profiles and their relative intensities were consistent between the 1 and 2 h samples providing evidence for the HMVK reactions being fast and selective. The finding that fewer peptides were adducted in the 0.1 mM HMVK globin samples provides further evidence for selectivity of the HMVK reaction. Collectively, the results show HMVK readily and selectively forms adducts on Hb. Characterization of these adducts will facilitate development of useful biomarkers of exposure to HMVK and its precursor 1,3-butadiene.