Hemoglobin is a blood protein that contains three unbound cysteine thiol moieties that are nucleophilic and that have been shown to covalently adduct to reactive electrophilic xenobiotics. In particular, the beta chain 93 cysteine thiol moiety is highly reactive as it resides on the surface of the protein. While measurement of adducted hemoglobin beta chain 93 cysteine thiols has been employed for environmental and occupational agents, its use for retrospective exposure assessment for drugs of abuse has not been extensively explored. Furthermore, identification and characterization of covalent hemoglobin thiol adducts is technically difficult, as the level of modified hemoglobin molecules is generally far lower than that of unmodified protein.
This study employed an in vitro enzymatic trapping assay combined with a newly developed enrichment method to selectively remove unadducted hemoglobin to allow for a greater sensitivity for identifying and characterizing covalent adducts at the beta chain 93 cysteine thiol as a potential biomarker of abused drug exposure. The assay utilized human liver microsomes, nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate, magnesium chloride, glucose-6-phosphate dehydrogenase, and human hemoglobin in ammonium bicarbonate buffer, pH 7.4. Test drugs (acetaminophen, clozapine, oxycodone, and diazepam) were added and incubated for 6-hours at 37°C. Treated hemoglobin was then subjected to an 18-hour incubation with Thiol Sepharose resin for removal of unmodified hemoglobin. Following centrifugation, supernatant containing enriched, drug-modified hemoglobin was collected and an aliquot subjected to tryptic digestion. An Agilent 1290/6530 Liquid Chromatography coupled Quadrupole-Time of Flight Mass Spectrometer in positive mode Electrospray Ionization was used for high resolution mass spectrometer peptide analysis. Agilent Qualitative and BioConfirm software, along with Protein Prospector software, were used to process full