Measurement of hemoglobin and albumin adducts of benzo(a)pyrenediolepoxide and their rate of elimination in the female Sprague-Dawley rat

Abstract

A high performance liquid chromatographic technique coupled with fluorometric detection was used to study the disappearance rate of the hemoglobin and albumin adducts of benzo(a)pyrene (BaP) in female Sprague-Dawley rats. The technique was considered adequate based on good recovery (> 80%) of the analyte and satisfactory intra-assay coefficients of variations of 7.8% and 4.2% for hemoglobin and albumin adducts, respectively. The persistence of the adducts was determined in rats injected intravenously with 550 nmol BaP diolepoxide, the electrophilic metabolite of BaP. In a first experiment the isolated protein fractions were directly subjected to mild acid hydrolysis to yield the ultimate analyte, BaP tetrol. In a second experiment these fractions were first digested by proteases in order to liberate any unbound lipophilic BaP tetrol possibly "hidden" in the hydrophobic portions of the protein matrices. It was found that 70-100% of the measured tetrols originated from true adducts. The half-life of the free tetrol was 2.8 and 1.6 days in the hemoglobin and albumin fractions, respectively, compared to 10.7 and 3.6 days for the true adducts. Based on the total amount of tetrols, the mean half-life of the latter in the hemoglobin and albumin fractions determined from the two experiments was 9.0 and 2.7 days, respectively, assuming first order kinetics. The greater persistence of the hemoglobin adduct compared to that of the albumin adduct is coherent with the biological half-life of the intact proteins. However, the observed disappearance profile obtained does not fit with current models of adduct formation and removal.