Date of Submission

5-2026

Document Type

Thesis

Degree Name

Master of Science in Forensic Science

Department

Criminal Justice

Advisor

Robert Powers, Ph.D.

Committee Member

Marisia Fikiet, Ph.D.

Committee Member

Christina Zito, Ph.D.

Committee Member

Na Liu, Ph.D.

Keywords

Morphine, Alcohol, Enzyme Kinetics

MeSH

Morphine, Alcohol, Enzymes

LCSH

Morphine, Alcohol, Enzyme Kinetics

Abstract

Glucuronidation is the primary route of morphine metabolism, and co-ingestion of ethanol is associated with increased opioid toxicity and a decreased rate of elimination of morphine from the body. However, the effect of ethanol on morphine glucuronidation has not been fully characterized. In this study, an LC-MS/MS method was developed and optimized for the detection of morphine metabolites, morphine-3- and morphine-6-glucuronide (M3G/M6G), and applied to investigate the impact of ethanol and redox balance on morphine metabolism. Initial evaluation with mouse microsomes showed no significant direct inhibition of morphine glucuronidation by ethanol, suggesting that inhibition of morphine UDP­ glucuronosyltransferase (UGT), the enzyme responsible for this pathway, alone does not account for the interaction. Because formation of the glucuronidation cofactor UDP-glucuronic acid (UDPGA) requires NAD+-dependent oxidation of UDP-glucose, we further investigated a potential cofactor-dependent mechanism. Morphine (0.5, 1, and 2 µM) was incubated with or without ethanol (0.1 and 0.2 g/dL) at 37°C for 30 minutes in mouse S9 fractions, and M3G/M6G formation was quantified by LC-MS/MS. Samples exposed to ethanol exhibited a significant, dose-dependent reduction in M3G/M6G formation compared to controls. In addition, modulation of the NAD+fNADH ratio demonstrated that increased NADH levels reduced glucuronidation capacity, supporting a redox-dependent effect. These findings indicate that ethanol impairs morphine glucuronidation through cofactor-dependent mechanisms rather than direct UGT inhibition. This interaction may contribute to increased toxicity during co­ ingestion and has important implications for forensic and clinical interpretation.

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