Characterization of Blood Protein Modifications by Reactive Drug of Abuse Metabolites

Authors

William J. Morrison IV, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Chemistry

First Advisor's Name

Anthony DeCaprio

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Jeremy Chambers

Second Advisor's Committee Title

Committee Member

Third Advisor's Name

Watson Lees

Third Advisor's Committee Title

Committee Member

Fourth Advisor's Name

Bruce McCord

Fourth Advisor's Committee Title

Committee Member

Fifth Advisor's Name

John Berry

Fifth Advisor's Committee Title

Committee Member

Keywords

Covalent Adducts, Drug of Abuse, Mass Spectrometry, Reactive Metabolites

Date of Defense

2-21-2023

Abstract

Hemoglobin (Hb) is an abundant blood protein that contains three cysteine amino acid residues at the α¹⁰⁴Cys, β⁹³Cys, and β¹¹²Cys positions. Each cysteine contains an unbound thiol moiety that is nucleophilic in nature. These nucleophilic sites have the potential to form covalent protein modifications, or protein ‘adducts’ with reactive electrophilic xenobiotics. A unique feature of a protein adduct is that the covalent bond is stable and will remain for the life cycle of the protein. While measurement of covalent protein modifications as a biomarker of exposure for occupational and environmental xenobiotics has been employed, its use for retrospective exposure assessment for drugs of abuse has not been extensively explored. This can primarily be attributed to the technical difficultly of identifying covalent thiol adducts, as the level of modified Hb molecules is far lower than that of unmodified protein. The first objective of this research was to develop a selective enrichment procedure for Hb adducted at β⁹³Cys to increase the sensitivity and selectivity for the detection of potential covalent thiol adducts. The developed enrichment assay was then used to assist in the identification and characterization of in vitro generated Hb β⁹³Cys-adducted species by reactive drug metabolites of acetaminophen (APAP), clozapine, cocaine, diazepam, oxycodone, and tetrahydrocannabinol. Major findings included MS identification of ten different β⁹³Cys-adducted drug metabolites. Identified adducts were reanalyzed using MS/MS peptide sequencing, where eight covalent Hb adducts were characterized, confirming the β⁹³Cys thiol was the site of adduction. The last portion of this work involved a proof-of-concept screen of authentic user blood for identification of Hb β⁹³Cys adducts using Hb isolated from whole blood, the developed enrichment procedure, and LC-HRMS analysis. Full scan MS and MS/MS characterization data was successfully collected for an in vivo characterization of a Hb β⁹³Cys covalent NAPQI adduct from blood with a positive confirmation for the presence of APAP. Overall, this research has positive implications for the field of forensic drug testing, as there is currently a lack of reliable long-term exposure assessment biomarkers. The analysis of covalent Hb protein modifications could be a potential alternative biomarker for long-term exposure assessment for illicit drugs.

Identifier

FIDC011057

Recommended Citation

Morrison, William J. IV, "Characterization of Blood Protein Modifications by Reactive Drug of Abuse Metabolites" (2023). FIU Electronic Theses and Dissertations. 5277.
https://digitalcommons.fiu.edu/etd/5277