Targeted Precise Quantification of 12 Human Recombinant Uridine-Diphosphate Glucuronosyl Transferase 1A and 2B Isoforms Using Nano-Ultra-High-Performance Liquid Chromatography/Tandem Mass Spectrometry with Selected Reaction Monitoring

Abstract

Quantification methods employing stable isotope-labeled peptide standards and liquid chromatography-tandem mass spectrometry are increasingly being used to measure enzyme amounts in biologic samples. Isoform concentrations, combined with catalytic information, can be used in absorption, distribution, metabolism, and excretion studies to improve accuracy of in vitro/in vivo predictions. We quantified isoforms of uridine-diphosphate glucuronosyltransferase (UGT) 1A and 2B in 12 commercially available recombinant UGTs (recUGTs) (n = 49 samples) using nano-ultra-high-performance liquid chromatography-tandem mass spectrometry with selected reaction monitoring). Samples were trypsin-digested and analyzed using our previously published method. Two MRMs were collected per peptide and averaged. Where available, at least two peptides were measured per UGT isoform. The assay could detect UGTs in all recombinant preparations: recUGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17, with limit of detection below 1.0 pmol/mg protein for all isoforms. The assay had excellent linearity in the range observed (2-15.5 pmol/mg, after dilution). Examples of concentrations determined were 1465, 537, 538, 944, 865, 698, 604, 791, 382, 1149, 307, and 740 pmol/mg protein for the respective isoforms. There was a 6.9-fold difference between the maximum and minimum recUGT concentrations. The range of concentrations determined indicates that catalytic rates per mg total protein in vitro will not accurately reflect isoform inherent specific activity for a particular drug candidate. This is the first report of a targeted precise quantification of commercially available recUGTs. The assay has potential for use in comparing UGT amounts with catalytic activity determined using probe substrates, thus allowing representation of catalysis as per pmol of UGT isoform. IntroductionUridine-diphosphate glucuronosyltransferase (UGT) enzymes catalyze formation of the glucuronide conjugates of phase II metabolism and are important for the elimination of drugs, xenobiotics, and endogenous molecules (Tukey and Strassburg, 2000;Rowland et al., 2013). In drug development studies potential drug candidates are tested with a range of metabolic enzymes, including UGTs, to determine possible routes of disposition. Catalytic activity of enzymes in the studies is normally presented as amount of substrate converted per unit of time (e.g., mmol min 21 ) or, for specific activity, the amount converted per unit of time per amount of total protein in the enzyme preparation (e.g., mmol min 21 mg 21) (Court, 2005;Wen et al., 2007). These units fail to account for differences in the actual amount of enzyme in a preparation, which is generally only estimated or unknown. It is suggested, for example, that in recombinant UGT (recUGT) preparations the UGT content is approximately 5% to 15% of the total protein content (BD Biosciences, personal communication). Targeted isotope dilution techniques with tandem mass spectrometry have recently been used to ...

Key to Opening Kidney for In Vitro–In Vivo Extrapolation Entrance in Health and Disease: Part I: In Vitro Systems and Physiological Data

“…Expression and activity data suggest that UGT1A9 and UGT2B7 are the major UGT enzymes in human kidney, with UGT1A9 expressed at levels close to or higher than those measured in liver (91). These findings are in agreement with the majority of quantitative abundance data from commercially available pooled human kidney microsomes, acquired using targeted LC-MS/MS proteomic methods (14,92). Large variability in both mRNA (72-85% CV, n = 11) and protein abundance (76-159% CV, n = 10) has been noted for UGT1A6, 1A9 and 2B7 in kidney homogenates prepared from unspecified regions of healthy kidney (93).…”

“…High variability in abundance, not only between enzymes but also between batches for the same enzyme, has been reported for recombinantly expressed UGTs (e.g. 30.6% coefficient of variation for rUGT1A4), which may hinder IVIVE-based prediction of both hepatic and renal glucuronidation clearance (14). In addition, UGTs expressed in insect cells may have a substantial amount of inactive protein present that can be reduced by lowering the amount of baculovirus used to infect cells (21).…”

“…It is evident from Table I that human kidney subcellular fractions (microsomes, S9) and recombinant enzyme expression systems are the most commonly applied sources of kidney drug-metabolising enzymes in in vitro assays (7,(14)(15)(16). Subcellular fractions require supplementation with appropriate cofactors lost during the preparation procedure and these are highlighted in Table I.…”

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity

“…In-solution sample preparation was performed as reported previously (Fallon et al, 2008(Fallon et al, , 2013a. Briefly, sample protein content was assessed using bicinchoninic acid assay and samples (n = 60) were diluted 1:20 in 50 mM ammonium bicarbonate.…”

Key to Opening Kidney for In Vitro-In Vivo Extrapolation Entrance in Health and Disease: Part II: Mechanistic Models and In Vitro-In Vivo Extrapolation

“…Enzyme abundances were found to be between 7-and 11-fold and 19-and 43-fold higher in recombinant expression systems than in human kidney microsomes for uridine diphosphate glucuronosyltransferase (UGT) 1A9 and UGT2B7, respectively (17,27,28). These data suggest that for calculation of the appropriate REF values for IVIVE, enzyme abundance data should be collected for every batch of recombinantly expressed UGT.…”