Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs

Abstract

While ethanol is primarily metabolized to acetaldehyde and acetic acid via alcohol dehydrogenase, a minor but increasingly important pathway in the field of forensic science involves the conjugation of glucuronic acid to form an ethyl glucuronide (EtG) metabolite. The kinetics of ethyl glucuronide formation were examined in human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferases (UGTs). The metabolite exhibited a relatively slow rate of formation in a human liver microsome mix of 75.4 pmol/(min/mg). Further investigation identified multiple UGT isoforms to be responsible for catalyzing the addition of glucuronic acid to ethanol, with UGT1A1 and 2B7 being the two most prevalent isoforms. Co-incubation with bilirubin or 3′-azido-3′-deoxythymidine (UGT1A1 and 2B7 inhibitors, respectively) inhibited the greatest amount of ethyl glucuronide formation, though other UGT inhibitors also showed some effect. Enzyme kinetics were performed in human liver microsomes and recombinant UGT enzymes. The apparent K_m ($K_{{\text{m}}_{\text{app}}}$) and V_max values were determined to be 0.17 ± 0.08 mM and 75.98 ± 5.63 pmol/(min/mg) (human liver microsomes), 0.03 ± 0.01 mM and 25.22 ± 3.45 pmol/(min/mg) (UGT1A1), and 0.11 ± 0.04 mM and 52.03 ± 9.8 pmol/(min/mg) (UGT2B7). Thus, it appears that multiple UGTs are responsible for the formation of ethyl glucuronide and that any functional differences in the enzymology underlying ethyl glucuronide formation would most likely be masked by a combination of other enzymatic pathways.

Introduction

A significant amount of research has focused on finding a useful and reliable marker of alcohol consumption. Such a marker would potentially allow clinicians, researchers, and forensic experts to focus on groups with an increased risk of alcoholism, monitor current treatment programs more effectively, and determine the extent to which alcohol plays a role in the neurological impairment of drivers involved in accidents [1]. While hepatic clearance of ethanol is primarily catalyzed by alcohol dehydrogenase, the microsomal ethanol-oxidizing system and aldehyde dehydrogenase [2], [3], a minor, but forensically significant clearance pathway, involves ethanol conjugation with glucuronic acid [4], [5], [6], [7].

Formation of ethyl glucuronide (EtG) is characterized by the net addition of glucuronic acid to ethanol. This clearance pathway is catalyzed by the UDP-glucuronosyltransferase (UGT) superfamily of enzymes, which utilize UDP-glucuronic acid as a cofactor [8]. UGTs catalyze a very wide range of both xenobiotic and endogenous compounds, and as such, any noted polymorphisms in this family could potentially have toxicological, pharmacological, and forensic implications. UGT1A1, 1A6, 1A7, 2B4, 2B7, and 2B15 have all been shown to be polymorphic enzymes [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], though only the UGT1A1 polymorphism appears to translate into any functional significance [19], [20].

Approximately 0.02–0.06% of the total amount of ethanol consumed is eliminated as EtG [21], [22]. This minor pathway often results in urine EtG concentrations in the low ng/mL range [23]; however, the forensic importance of the pathway lies in the fact that clearance of EtG occurs at a much slower rate than that of ethanol [24], [25]. While serum and/or urine ethanol levels can normally only be detected for a few hours post-intake, urinary levels of EtG have been detected as long as 3–5 days following alcohol consumption [26], [27], [28]. Even though alcohol dehydrogenase, CYP2E1, and catalase mediated formation of acetaldehyde represent the major clearance pathways of ethanol, well-documented polymorphisms in the above-noted pathways coupled with rapid sequential metabolism to acetic acid would make analysis of this clearance mechanism much more complex [29]. Thus, forensic scientists have been using the advantageous properties of EtG in studying drunk driving cases, covert alcohol use among psychiatric inpatients, and multiple other situations in which alcohol consumption was thought to play a role [30], [31].

While the clearance of ethanol is a well-researched topic, the enzymology underlying the glucuronidation of ethanol has not received as much attention to date. Considering its use as a significant tool in forensic science, coupled with the polymorphic tendencies of the UGT family of enzymes, we felt it only prudent to explore the individual UGT isoforms responsible for catalyzing the formation of EtG. Metabolism by a single, polymorphic UGT could potentially lead to a skewed analysis of alcohol consumption. With this in mind, we have examined the formation of EtG in various in vitro systems. Using human liver microsomes (HLM) and recombinant UGTs, kinetic values were determined for the formation of EtG, and the effects of various inhibitors on the formation rate of the metabolite were studied. Finally, these data were scaled and compared to reported in vivo data for the clearance of ethanol in humans.