Separation and Enrichment of Alkylated Globin Chains as a Means of Improving the Sensitivity of Hemoglobin Adduct Measurements.

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Abstract
Measurement of adducts of hemoglobin is a reliable and quantitative method for monitoring exposure to genotoxic chemicals. To make the approach applicable to the low levels of adducts originating from exposure to chemicals in the environment, increased sensitivity of the analytical procedures is required. The method presented here is based on quantitative determination of low levels of adducts after purification and enrichment of chemically modified (adducted) globin chains on CM-Sepharose CL-6B. In the development work, human globin was used after alkylation by radiolabelled ethylene oxide, styrene oxide or N-ethyl-N-nitrosourea. Ethylene oxide reacts mainly with the amino terminal valine and nitrogens in the imidazole ring of histidine, while N-ethyl-N-nitrosourea has a particularly high reactivity towards free carboxy group of acidic amino acids. Globin chains with adducts to the carboxy groups were especially easy to separate from the non-modified chains. Ethyl adducts to carboxy groups in hemoglobin were shown to be sufficiently stable in vivo to be used for dose monitoring.