ADDUCTS TO N-TERMINAL VALINES IN HEMOGLOBIN FROM BUTADIENE METABOLITES

In order to identify a hemoglobin adduct useful for monitoring of dose s of butadiene metabolites, particularly the strongly genotoxic, bifun ctional diepoxybutane (DEB), the reaction of DEB with valinamide, a re levant model of globin N-termini, was studied. A preliminary kinetic a nalysis showed that the primary reaction product of DEB with valine-N gives, as was expected, rise to a ring-closed pyrrolidine-structured c ompound, N,N-(2,3-dihydroxybuta-1,4-diyl)valine (PYRV), in a reaction which is fast when compared to hydrolysis of the second oxirane ring w ith formation of N-(2,3,4-trihydroxybutyl)valine (THBV). The ring clos ure is also fast when compared to the rate of formation of a cross-lin ked divaline product. PYRV can therefore be used as a specific marker of in vivo doses of DEB whereas THBV may be applied for the dosimetry of the metabolite (1,2-dihydroxyethyl)oxirane. The latter is formed by half-hydrolysis of DEB or oxygenation of 1,2-dihydroxy-3-butene. The N-alkyl Edman method, used for specific cleavage and gas chromatograph ic/mass spectrometric (GC/MS) determination of adducts to N-terminal v aline in hemoglobin, could be used for measurement of THBV, as shown i n alkylation experiments with blood. However, the adduct specific for DEB, PYRV, requires-due to its tertiary amine structure-other techniqu es. The reaction products were identified by GC/MS, PYRV by C-13 and H -1 NMR, and THBV because of its formation by reduction of the Schiff b ases of threose and erythrose with hemoglobin.