Determination of the kinetics of rat UDP-glucuronosyltransferases (UGTs) in liver and intestine using HPLC

Abstract

Uridinediphosphoglucuronosyl transferases (UGTs) are a group of membrane bound proteins which catalyze the transfer of glucuronic acid from UDP-glucuronic acid to a wide variety of xenobiotics and drug molecules enabling them to be eliminated. The major UGT isoforms found in the rat are 1A1, 1A6, 2B1 and 2B12. Conventional methods for the assay of glucuronides (GLs) include TLC, extraction and colorimetry or quantification of the aglycone, liberated after hydrolyzing the GL with β-glucuronidase. However these techniques cannot distinguish between isomeric GLs or GLs of multiple acceptor site substrates. Therefore the purpose of this study was to develop simple and sensitive HPLC methods for the direct and simultaneous analysis of the GL(s) and their aglycones without the drawbacks of the conventional methods. The three classical substrates we chose were 4-methylumbelliferone (4MU), testosterone (TES) and 8-hydroxyquinoline (8HOQ) representing UGT isoforms 1A6, 2B1 and 2B12 of the rat family, respectively. Here we report the validated HPLC conditions, for the detection and separation of 4-methylumbelliferone glucuronide (4MUG), testosterone glucuronide (TESG) and 8-hydroxyquinoline glucuronide (8HOQG) and their aglycones in incubation media containing male Sprague–Dawley rat liver and intestinal microsomal preparations. The separations were achieved on a Zorbax SB-CN column (150×4.6 mm, 5 μ). The analysis time for the separation of TES, 8HOQ and 4MU and their glucuronides were 17, 12 and 30 min, respectively. The methods showed excellent linearity (r²>0.99) over the concentration ranges tested (0.25–5.0 nmoles of TESG; 0.125–18.75 nmoles of 8HOQG and 0.125–12.5 nmoles of 4MUG), good precision and accuracy (RSD<2.5%). Inter-day variability studies (n=3) showed no significant difference between the regression lines obtained on the three days. Recoveries were good (>90%) at all three points (low, mid-point, high) of the standard curve. The limits of detection were 0.125, 0.1 and 0.1 nmole for TESG, 8HOQG and 4MUG, respectively. The above methods were used to estimate kinetic parameters such as V_max and K_m for the GLs of the three substrates in both liver and intestinal tissue preparations and the values were comparable with previously reported results. UGT2B1 was found primarily in the liver while UGTs 1A6 and 2B12 were present in comparable amounts in both tissues.

Introduction

Uridinediphosphoglucuronosyl transferases (UGTs, EC 2.4.1.17) constitute a major class of Phase II enzymes that are involved in the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to a variety of acceptor groups like phenols, alcohols, aliphatic amines, carboxylic acids and acidic carbon atoms found in a wide range of drugs and xenobiotics [1]. This represents a major step in the detoxification and elimination of many potentially toxic compounds [1], [2]. The UGTs are primarily classified into families 1 and 2, in both rats and humans and their nomenclature has been assigned based on evolutionary divergence [3]. In rat family 1, UGTs 1A1 and 1A6 are two important isoforms involved in the glucuronidation of bilirubin and small planar phenols respectively [3]. In family 2, UGTs 2B1 and 2B2 are involved in the glucuronidation of steroids and UGT 2B12 glucuronidates bulky phenols and mono-terpenoid alcohols [3]. Characterization of these isoforms based on substrate specificity is essential to understand their role in drug metabolism. The model substrates of the isoforms 1A6, 2B1 and 2B12 whose glucuronidation we decided to study were testosterone (TES) for rat UGT2B1 [4], 8-hydroxyquinoline (8HOQ) for rat UGT2B12 [5] and 4-methylumbelliferone (4MU) for rat UGT1A6 [4]. It was therefore necessary to develop bio-assays to quantify the glucuronides. Looking into literature, the most commonly used assay was the universal TLC method which used the incorporation of (¹⁴C)-UDPGA into the acceptor site(s) of a substrate to form radio-labeled glucuronide(s) which can be quantified using a radio TLC scanner [6]. The problem with this method is the lack of specificity, sensitivity and the inability to distinguish between glucuronides of substrates with multiple acceptor sites or between isomeric glucuronides. Alternatively quantification of glucuronides, for e.g. 4MU glucuronide (4MUG) is commonly done through indirect means involving extraction of the resultant glucuronide, hydrolysis with acid or β-glucuronidase and then assaying for the liberated aglycone using fluorescence [7]. This method has problems of sensitivity, recovery, incomplete hydrolysis and instability of the aglycone in acid media. Other investigators have separated 4MU from 4MUG in various tissues using open column chromatography with Dowex AG-50W resins [8]. Methods not involving such complex extractions or non-specificity and those which quantify the glucuronide directly in the incubation mixture would be preferred. HPLC would then be an obvious choice. In the case of TES and 8HOQ, previously reported HPLC methods are radiometric assays utilizing either radiolabeled UDPGA or substrate and are gradient elutions [9], [10]. Gradient separations have problems with baseline drifting and reproducibilty. Radiometric HPLC assays have problems of availability of labeled substrate or co-factor and have high background noise due to the scintillant thereby requiring the use of high concentration of labeled material which makes routine analysis expensive. HPLC methods for the assay of 4MU and 4MUG have been reported and involve either the use of ion-pair reagents [11] or are gradient elutions with a run time of 40 min [12].

This paper describes simple isocratic methods for the direct quantification of the glucuronides of TES, 8HOQ and 4MU in incubation mixtures which contained their respective aglycones and all the separations were achieved on the same stationary phase. Using these methods enzyme kinetic parameters like V_max and K_m were calculated for these substrates in rat liver and intestinal microsomal preparations.