Hemoglobin adducts of benzene oxide in neonatal and adult dried blood spots

Adducts of reactive chemicals with hemoglobin (Hb) or human serum albumin can be used as biomarkers of internal doses of carcinogens. Because dried blood spots are easier to collect and store than conventional venous blood samples, they encourage applications of biomarkers of exposure in large epidemiologic studies. In addition, neonatal dried blood spot can be used to investigate chemical exposures in utero. Here, we report a simple method to isolate Hb from dried blood spot with high recovery and purity using the addition of ethanol to aqueous dried blood spot extracts. To prove the concept that dried blood spot-derived proteins can be used to assay for adducts, we measured Hb adductsof benzene oxide, a reactive metabolite of the ubiquitousair pollutant benzene in nine neonatal and nine adult dried blood spots (from volunteer subjects), using a gas chromatography-mass spectrometry method that we had previously developed. For comparison, benzene oxide-Hb adducts were measured in the same nine adult subjects using Hb that had been isolated and purified using our conventional method for venous blood. The geometric mean of benzene oxide-Hb levelsin all dried blood spot samples ranged from 27.7 to 33.1 pmol/g globin. Neither of the comparisons of mean (logged) benzene oxide-Hb levels between sources (adult conventional versus adult dried blood spot and adult dried blood spot versus newborn dried blood spot) showed a significant difference. Based upon the estimated variance of the benzene oxide-Hb levels, we had 80% power to detect a 1.7-fold difference in geometric mean levels of benzene oxide-Hb in our sample of nine subjects.