Identification of an in vivo chrysene diol epoxide adduct in human hemoglobin

Human blood samples were analyzed by fluorescence and mass spectrometry for the presence of polycyclic aromatic hydrocarbon metabolites covalently bound to hemoglobin. Globin was prepared by HCl-acetone precipitation and enzymatically digested. The polycyclic aromatic alcohols generated from ester adducts during digestion were concentrated by monoclonal antibody immunoaffinity chromatography and separated by C-18 HPLC. Analysis of collected fractions by room temperature fluorescence spectroscopy revealed the presence of the anti-tetrahydrotetrols of benzo[a]pyrene and another component possessing a phenanthrene chromophore. Gas chromatographic-mass spectral analysis of the HPLC fractions revealed that the latter compound was r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene, arising from a hemoglobin-chrysene diol epoxide adduct.