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Research ArticleArticle

A Novel Method for the Immunoquantification of UDP-Glucuronosyltransferases in Human Tissue

Alison M. Milne, Brian Burchell and Michael W. H. Coughtrie
Drug Metabolism and Disposition December 2011, 39 (12) 2258-2263; DOI: https://doi.org/10.1124/dmd.111.041699

Abstract

Glucuronidation is a major pathway of drug and xenobiotic metabolism that is catalyzed by members of the UDP-glucuronosyltransferase (UGT) family. Predicting the contribution of individual UGTs to drug metabolism would be of considerable value in drug development and would be greatly aided by the availability of detailed absolute expression levels of these proteins; this is hampered by the lack of purified protein standards because of the hydrophobic membrane-associated nature of UGTs and the consequential difficulties in expression and purification. Here we describe a novel solution to this problem by expressing UGTs in Escherichia coli as fusion proteins with ribonuclease S-peptide, targeted to the periplasm with the pelB leader sequence. After addition of ribonuclease S-protein to membrane extracts, a functional ribonuclease is reconstituted that provides a direct and absolute quantification of the amount of UGT fusion protein; this is subsequently used to generate standard curves for immunoquantification by immunoblotting. To illustrate the value of the method, we have quantified the expression of UGT1A1 and UGT1A6 in human liver and kidney microsomes using new isoform-specific antibodies developed against peptides from these proteins. Expression levels of both proteins in liver were highly variable (28- and 20-fold, respectively) and correlated strongly with UGT enzyme activity toward the probe substrates bilirubin and 1-naphthol, respectively. The method is broadly applicable and provides a straightforward means of determining the absolute, as opposed to relative, quantities of UGT proteins present in human tissues.

Introduction

Glucuronidation is a key pathway for the metabolism, detoxification, and elimination of numerous xenobiotics as well as endogenous compounds such as bilirubin and steroid hormones (Burchell and Coughtrie, 1989; Tukey and Strassburg, 2000; Wells et al., 2004). The glucuronidation reaction, which involves the transfer of the glucuronic acid moiety from the cosubstrate UDP-glucuronic acid, is catalyzed by members of the UDP-glucuronosyltransferase (UGT) enzyme family. In humans, there are at least 20 members of the UGT enzyme family belonging to four different subfamilies: UGT1A (in which nine enzymes are produced from a single gene), UGT2A, UGT2B, and UGT3A. This diversity of UGTs facilitates glucuronidation of a vast array of chemical structures at several different functional groups, including phenols, alcohols, carboxylic acids, and amines as well as rare examples of glucuronidation at carbon and sulfur atoms (Burchell and Coughtrie, 1989; Tukey and Strassburg, 2000).

Adverse drug reactions (ADRs) are a major cause of morbidity and mortality. A survey (Pirmohamed et al., 2004) estimated that ADRs accounted for 6.5% of hospital admissions, with a financial burden to the U.K. health service of £0.5 billion per annum. It is increasingly clear that glucuronidation plays a significant role in individual response to drugs and therefore in susceptibility to ADRs. For instance, treatment-limiting toxicity of the anticancer drug irinotecan is associated with mutations in UGT1A1 (Nagar and Remmel, 2006; Hoskins et al., 2007), and a UGT1A haplotype involving mutations in UGT1A1, UGT1A3, and UGT1A7 predisposes to jaundice associated with atazanavir treatment (Lankisch et al., 2006). There is therefore considerable interindividual and intertissue variation in the expression of many UGTs [e.g., Strassburg et al. (2000) and Izukawa et al. (2009)] that have significant functional sequelae (Wells et al., 2004). Most studies aimed at determining expression levels of UGTs have relied upon mRNA measurements (often as relative rather than absolute values, e.g., Aueviriyavit et al., 2007), which have been shown to correlate poorly with protein expression levels (Izukawa et al., 2009). Therefore, there remains a need for quantitative data on functionally active UGT protein expression levels in human tissues. Such expression profiles have been generated for the cytochromes P450 in liver and intestine (Shimada et al., 1994; Paine et al., 2006) and also recently for the sulfotransferases in liver, kidney, intestine, and lung (Riches et al., 2009b). Such detailed information contributes importantly to software tools (e.g., SimCyp; http://www.simcyp.com/) that aim to predict the in vivo fate of drugs. However, absolute quantification of UGT protein expression in human tissues using immunochemical approaches poses a significant technical problem because the technique requires isoform-specific antibodies and authentic quantified standard protein. These standards would normally be in the form of the full-length purified protein (e.g., Shimada et al., 1994; Riches et al., 2009b), but because UGTs are integral membrane proteins, their purification from recombinant expression systems is challenging. Here, we describe a novel approach to this problem using UGTs expressed as fusion proteins coupled to S-peptide as standards for immunoquantification and validate this system using the important hepatic UGTs UGT1A1 and UGT1A6. The S-peptide (S-tag) is a 15-amino acid N-terminal peptide derived from ribonuclease that allows for direct quantification of the fusion protein without purification because high-affinity reconstitution with ribonuclease S-protein forms a functional ribonuclease enzyme for which the readily quantified activity yields a direct measurement of the concentration of the UGT fusion protein (Kim and Raines, 1993).

Materials and Methods

Materials.

pET-15b, pET-20b, pET-32b, BL21(λDE3), and all S-tag reagents were obtained from Merck (Nottingham, UK), and Pfu polymerase and pGEM-T Easy were from Promega (Southampton, UK). UDP glucuronic acid [glucuronosyl-14C] (>6.7 GBq/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Cambridge, UK). Multiple antigenic peptides were synthesized by AltaBioscience (Birmingham, UK). Anti-goat IgG conjugated to horseradish peroxidase was from Sigma-Aldrich (Poole, Dorset, UK). All other reagents were from commonly used local suppliers.

Tissue Samples and Preparation of Microsomal Fractions.

Human liver samples used in this study have been described previously (Thomas and Coughtrie, 2003; Riches et al., 2007, 2009b). Human kidney samples were obtained from the U.K. Human Tissue Bank (Leicester, UK). Ethical approval for local use of samples was obtained from the Tayside Research Ethics Committee. Frozen tissue was weighed and kept on ice, and 5 ml of SHM buffer (250 mM sucrose, 10 mM HEPES, and 3 mM 2-mercaptoethanol, pH 7.4) were added for every gram of tissue. Homogenates were prepared in a Teflon-glass homogenizer and subjected to differential centrifugation at 10,000g for 15 min, with the resulting supernatant centrifuged at 100,000g for 1 h. The pellet, representing the microsomal fraction, was resuspended in 1 ml/g of original tissue used and homogenized by hand, then aliquoted, snap-frozen in liquid nitrogen, and stored at −80°C until use (usually within 6 months).

Production of Antibodies against UGT1A1 and UGT1A6.

For UGT1A1, we selected a unique 19-amino-acid sequence (ENDSFLQRVIKTYKKIKKD) from the variable N-terminal region for synthesis as a multiple antigenic peptide (MAP). For UGT1A6, a 100-amino-acid peptide (from Pro61 to Ala160) was selected from within the N-terminal variable region on the basis of predicted antigenicity and sequence divergence from its nearest relatives. Antigenicity was assessed using a web-based application (http://www.bioinformatics.org/JaMBW/3/1/7/), and amino acid sequence similarity was determined by alignment of all of the UGT1A family members using ClustalW (http://www.ebi.ac.uk/clustalw/) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches. The sequence coding for this peptide was cloned into pET-15b with a C-terminal 6-histidine tag to facilitate purification and expression in Escherichia coli BL21 (λDE3) cells after induction with isopropyl β-d-thiogalactoside. In brief, the selected region for UGT1A6 was amplified using the following primers: 1A6VF PCR, 5′-CATATGCCATGGCCTGAAGTTAATTTGCTTTTGAAA-3′; and 1A6VR PCR, 5′-CTCGAGTCAAGCCAGGATCACCCCACAGGGTAA-3′. The PCR was performed using the pET-20b/pelB-UGT1A6 vector (see Expression of UGT-S-Tag Fusion Protein) as a template and Pfu polymerase (Promega). The PCR product was cloned into pGEM-T Easy (Promega) and sequenced, then it was excised with NdeI and XhoI and ligated with similarly digested pET-15b to produce an N-terminal 6-histidine-tagged peptide of UAG1A6 corresponding to the region Pro61 to Ala160. The expressed peptide was found in the pellet after centrifugation at 12,000g, suggesting that it was localized in inclusion bodies. Cells were harvested, and the pellet was resuspended in denaturing extraction buffer (50 mM sodium phosphate pH 7, 300 mM NaCl, and 6 M guanidine-HCl) and incubated on ice for 4 h with gentle shaking. The extract was centrifuged at 12,000g for 20 min at 4°C to pellet insoluble material. One milliliter of the supernatant was applied to a TALON cobalt affinity spin column (Clontech, Mountain View, CA) as described by the manufacturer. A total of 3 ml of extract was passed through spin columns, and the eluant was pooled and dialyzed against 500 volumes of sodium phosphate (45 mM, pH 7), NaCl (150 mM), and 20% (v/v) glycerol. This procedure produced a total of 9 mg of purified fusion peptide.

The immunization procedure was performed by Alba Bioscience (Edinburgh, UK), and 0.5 mg of peptide or MAP was used per injection in sheep. Four immunizations were administered 4 weeks apart with blood collected for analysis 1 week after the second, third, and fourth immunizations.

Expression of UGT-S-Tag Fusion Protein.

A PCR product representing part of the conserved C-terminal domain of the UGT1A family plus the S-tag sequence was generated using Pfu polymerase and cloned into pGEM-T Easy. After sequence confirmation, the PCR product was subcloned into the existing E. coli expression constructs pET-20b/pelB-UGT1A1 and pET-20b/pelB-UGT1A6, which contain the pelB leader sequence [which targets the protein to the bacterial periplasm (Better et al., 1988; Skerra and Pluckthun, 1988)] substituted for the natural N-terminal UGT signal sequence (Ouzzine et al., 1994). In brief, three primers were designed to bind in the constant region of the UGT1A [UGT1A(S)F], to link the UGT1A sequence to the S-tag sequence [UGT1A(S) Linker], and as an S-tag reverse (S-tag R). The sequences of these primers were UGT1A(S)F, 5′-ACCTGGTACCAGTACCATTCCTTG-3′, which binds across a unique internal restriction endonuclease site (KpnI) noted in italics; UGT1A(S) Linker, 5′-AAATCCAAGACCCATAAAGAAACCGCTGCTGCT-3′, which comprises the last 15 nucleotides of the UGT1A constant region before the stop codon (noted in italics) and the first 18 nucleotides of the S-tag sequence; S-tagR, 5′-CTCGAGGCGGCCGCCTAGCTGTCCATGTGCTG-3′, which comprises the final 15 nucleotides of the S-tag sequence, a stop codon, and restriction endonuclease sites NotI and XhoI. The cloning strategy included a two-stage PCR. In the first stage, a small PCR product of 74 base pairs is produced using the UGT1A(S) Linker and S-tag R primers and pET-32b as a template. In the second stage, the purified product from the first PCR is used alongside pET-20b/pelB-UGT1A6 as the template using the primers UGT1A(S) F and S-tag R. In the second PCR, the product was expected to be 217 base pairs. The PCR product was purified and sequenced. It was digested with KpnI and XhoI and ligated with similarly digested pET-20B/pelB-UGT1A1 or UGT1A6 to produce an S-tag version of these UGT isoforms. The modified expression constructs were transformed into the E. coli expression strain BL21 (λDE3). For expression of UGT1A1(S), a 500-ml culture of modified terrific broth was inoculated from a small starter culture. The cells were incubated for 16 h at 37°C with no induction. Subcellular fractionation was performed, and the membrane pellet was resuspended in TSE buffer (50 mM Tris-acetate pH 7.6, 250 mM sucrose, and 0.25 mM EDTA). Expression of UGT1A1(S) was confirmed using the S-tag rapid assay kit as described by the manufacturer (Merck) and by immunoblotting with peroxidase-conjugated S-protein and anti-UGT1A1 antibody. For expression of UGT1A6(S), a starter culture was used to inoculate 500 ml of Luria broth with ampicillin selection. The culture was incubated until the optical density was 0.57. After adding isopropyl β-d-thiogalactoside (1 mM) to induce expression, the culture was incubated at 27°C for 2 h. The membrane pellet was prepared and expression was confirmed as for UGT1A1(S).

Quantitative Immunoblot Analysis.

Crude IgG fractions of antisera were prepared by 50% ammonium sulfate precipitation followed by extensive dialysis against phosphate-buffered saline. For anti-UGT1A1, the best immunoblotting results were obtained by using affinity-purified IgG, which was prepared using the same peptide as for immunization, as described by the manufacturer (AltaBioscience). IgG prepared from both antisera was used at a concentration of 10 μg/ml for immunoblotting.

Proteins were resolved on denaturing SDS-polyacrylamide gel electrophoresis (PAGE) gels (10% acrylamide monomer) and transferred electrophoretically for 16 h at 27 V to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore Corporation, Billerica, MA) as described by Towbin et al. (1979). After transfer, the membranes were incubated in blocking buffer [5% (w/v) dried milk powder in 20 ml TBS-T (10 mM Tris-HCl, pH 9, 150 mM NaCl, and 0.1% (v/v) Tween 20)] for 1 h and then with the IgG (diluted to 10 μg/ml in 20 ml blocking buffer) for 3 h. Gels were stained with Coomassie Blue to ensure complete transfer of proteins; gels were routinely seen to be clear, suggesting efficient transfer of standards and test samples. Membranes were washed in TBS-T and then exposed to the anti-goat IgG-peroxidase conjugate (diluted at 1:20,000 in blocking buffer) for 2 h. After extensive washing in TBS-T, blots were developed using Western Lightning (PerkinElmer Life and Analytical Sciences) and ECL Hyperfilm (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Standard curves for quantification were generated using samples of UGT(S) loaded at a range between 0.25 and 5 ng.

Scans of the developed X-ray films were made using a desktop scanner attached to a personal computer and analyzed using Quantiscan 3.1 (Biosoft, Cambridge, UK) as described previously (Riches et al., 2009a). After pilot experiments, the quantity of microsomal protein loaded onto the gels was adjusted such that the densities of resulting bands were within the linear region of the standard curves on each blot. Each sample was analyzed on at least three separate blots with each antibody, and thus individual data points quoted are the means of these determinations.

Assay of UGT Activity.

UGT1A1 activity was assayed with bilirubin as substrate using a method described previously (Heirwegh et al., 1972), and UGT1A6 activity was assessed using 1-naphthol (500 μM) by the universal high-performance liquid chromatography assay method described by Ethell et al. (1998) (Taskinen et al., 2003).

Total Protein Concentration Analysis.

Quantification of total protein levels in microsomal samples and recombinant protein preparations was performed using the method originally described by Lowry et al. (1951) using bovine serum albumin as standard.

Results

We sought to develop a method that would overcome the problems posed by the lack of availability of purified UGT standards and facilitate the absolute immunoquantification of UGT expression in human tissue microsomes. The S-peptide/S-protein system provides a convenient mechanism for producing quantified protein standards and is readily applicable to membrane proteins such as UGTs because it does not rely upon purifying the protein from the expression system. The functional ribonuclease activity was reconstituted when incubating the recombinant E. coli membrane fraction containing the UGT/S-tag fusion protein with purified S-protein (the binding is of very high affinity, Kd = 10−9 M), and the enzymatic breakdown of the substrate polycytidylic acid was readily followed by measuring the absorbance at 280 nm. By comparison with results obtained with a known S-peptide standard, the molar concentration of UGT in the extract was determined. We used expression constructs generated with the pelB leader sequence to target the UGT expression to the periplasmic membrane because full-length UGTs are normally expressed in inclusion bodies in E. coli and require harsh conditions to solubilize them.

Antibodies against UGT1A1 and UGT1A6.

We designed two peptides that were used to generate isoform-specific antibodies against human UGT1A1 and UGT1A6 in sheep. For UGT1A1, we chose a short region in the N-terminal variable region that was synthesized as a MAP, and for UGT1A6, a 100-amino acid peptide was expressed in E. coli as a fusion protein with a 6-histidine tag and purified by affinity chromatography. We tested the specificity of the antibodies produced using immunoblot analysis against recombinant human UGTs as shown in Fig. 1.

Fig. 1.
Fig. 1.

Specificities of anti-UGT antibodies. Recombinant UGT isoforms were expressed in V79 cells or in FT293 cells as indicated, and cell lysates were prepared from frozen cell pellets resuspended in 200 μl SHM buffer (see Materials and Methods) and sonicated three times for 5 s. Lysates along with samples of human liver and kidney microsomes (30 μg of each) were resolved on SDS-PAGE gels and transferred to PVDF membranes, which were probed with each antibody as described under Materials and Methods. HLM, human liver microsomes; HKM, human kidney microsomes.

To validate the immunoquantification method, we chose to quantify the expression of UGT1A1 and UGT1A6 in microsomes prepared from 29 individual human liver samples. For each liver sample and antibody, conditions were optimized so that the bands were within the linear range of the standard curves generated using the S-tag-labeled UGTs. A representative immunoblot for each antibody is shown in Fig. 2.

Fig. 2.
Fig. 2.

Representative quantitative immunoblots with anti-UGT antibodies. Microsomes prepared from several liver samples were resolved on SDS-PAGE gels along with the appropriate recombinant UGT/S-tag fusion protein (values in nanograms). After transfer to PVDF membranes, blots were immunostained with either anti-UGT1A1 (A) or anti-UGT1A6 (B).

UGT1A1 and UGT1A6 Expression in Human Tissues.

There was considerable variation in the expression of UGT1A1 and UGT1A6 in the various liver samples (Table 1; Fig. 3), although expression could be detected in each sample. The quantity of UGT1A1 found in these liver samples varied from 0.04 to 1.14 μg/g tissue, a 28-fold variation, with a mean of 0.34 μg/g tissue. There is a common variable number tandem repeat polymorphism in the TATA box of the UGT1A1 gene (UGT1A1*28) that is associated with the reduced expression of UGT1A1, reduced glucuronidation of bilirubin, and the mild unconjugated hyperbilirubinemia seen in Gilbert's syndrome (Bosma et al., 1995; Monaghan et al., 1996) that presumably accounts for a significant proportion of the variability seen here. UGT1A1 expression was not detected in microsomes prepared from the human kidney samples.

View this table:
TABLE 1

Quantification of the expression and enzyme activity of UGT1A1 and UGT1A6 in human liver and kidney microsomes

Data are means ± S.D. for quantification in n tissue samples. Measurements were performed on between three and seven replicates.

Fig. 3.
Fig. 3.

Expression of UGT1A1 and UGT1A6 in human liver microsomes. Microsomes prepared from 29 human liver samples were subjected to immunoquantification for the expression of UGT1A1 (A) and UGT1A6 (B). Data are shown as mean ± S.D. for between three and seven separate determinations.

With the expression of UGT1A6 we again observed extensive variation in levels within liver microsomal samples, ranging from 0.07 to 1.42 μg/g tissue with a mean of 1.18 μg/g tissue, a 20-fold variation in the amount of UGT1A6. Expression of UGT1A6 was observed in microsomal samples prepared from human kidney, although this was approximately 3-fold lower than in the liver.

To confirm that expression levels measured with this assay were representative of enzyme function, we performed assays for UGT activity using hepatic microsomal samples. We used bilirubin as a probe substrate for UGT1A1 and 1-naphthol (at a concentration of 500 μM) as a probe substrate for UGT1A6 and performed correlation analysis between the protein expression and enzyme activity datasets. The results are presented in Table 1 and Fig. 4. There was a strong correlation between UGT1A1 expression and bilirubin UGT activity in the human liver microsomes (Pearson's r = 0.82 and p < 0.0001) and a somewhat lower, but still significant, degree of correlation between UGT1A6 protein expression and the glucuronidation of 500 μM 1-naphthol (Pearson's r = 0.61 and p < 0.005).

Fig. 4.
Fig. 4.

Correlation between UGT protein expression and enzyme activity. Microsomal fractions were subjected to immunoquantification as described and enzyme activity measurements with probe substrates bilirubin (A; for UGT1A1) and 1-naphthol (B; for UGT1A6). Correlation (linear regression) analysis was performed using Prism 4 (GraphPad Software Inc., San Diego, CA).

Discussion

The ability to reliably predict the contribution of individual drug-metabolizing enzymes to the in vivo fate of xenobiotics would be of considerable value and benefit to chemical and pharmaceutical industries as well as the regulatory bodies that oversee them. For many conjugating enzyme families, including the UGTs, we still do not have a good idea of the relative contributions of the major isoforms in this regard, not least because of the lack of reliable methods for quantifying protein expression. This is mainly because UGTs are integral membrane proteins that are difficult to express and purify from, e.g., E. coli, which means it is extremely challenging to produce accurately and absolutely quantified protein for the generation of standard curves for immunoquantification approaches. Another drawback is the lack of high-quality, isoform-specific antibodies for immunoquantification. Here we have described a novel approach to the immunoquantification of UGT protein expression in human tissues. The method, which uses expressed and readily quantified UGT/S-tag fusion proteins to generate standard curves, was validated using two important human UGTs, UGT1A1 and UGT1A6, against which we also produced new isoform-specific antibodies.

The UGT/S-tag fusion proteins with the pelB leader sequence were expressed in E. coli and were found in the membrane fraction upon ultracentrifugation, indicating that they had been correctly targeted to the periplasm. When combined with an excess of S-protein, they formed an active ribonuclease activity that was readily quantified spectrophotometrically using the S-tag rapid assay. These proteins were then successfully used to develop standard curves for quantification of UGT protein levels by immunoblotting.

Because of its role in the detoxification and elimination of bilirubin, UGT1A1 is considered to be one of the most important (if not the most important) UGTs and is expressed primarily in the liver and to a lesser extent in the gastrointestinal tract (Ohno and Nakajin, 2009). A proportion of the variability of UGT1A1 expression in the population is due to the common variable number tandem repeat polymorphism in the UGT1A1 gene promoter associated with Gilbert's syndrome (Bosma et al., 1995; Monaghan et al., 1996) [or other mutations in different populations, e.g., Koiwai et al. (1995))]; however, measurements of mRNA levels show very wide interindividual variation for all UGTs [e.g., Izukawa et al. (2009))], suggesting that other environmental, genetic, and/or epigenetic factors significantly affect expression levels. UGT1A6 is responsible for the glucuronidation of many xenobiotics, and several drugs are known to be substrates for this enzyme, including paracetamol (Court et al., 2001), valproate (Ethell et al., 2003), and deferiprone (Benoit-Biancamano et al., 2009). UGT1A6 also metabolizes several important endogenous substrates such as serotonin (Krishnaswamy et al., 2004). As with UGT1A1, previously reported mRNA-based quantification and other studies have demonstrated very widespread variability in the expression of UGT1A6 in liver (Krishnaswamy et al., 2005; Izukawa et al., 2009). One study failed to find an association between various polymorphisms in UGT1A6 and expression levels of the protein, suggesting instead that environmental factors such as alcohol consumption may play an important role in interindividual variability (Krishnaswamy et al., 2005); however, other studies have suggested an impact of the variant UGT1A6*2 on salicylic acid pharmacokinetics (Chen et al., 2007). We noticed that there is a reasonable similarity in the expression profiles of UGT1A1 and UGT1A6 in this set of liver samples. We know from Fig. 1 that there is no evidence for any significant cross-reactivity of these two new antibodies, so the possibility remains that there is a degree of coordinate regulation of these two UGTs.

The method reported here for producing recombinant protein standards as S-tag fusion proteins is readily applicable to potentially any recombinant protein expression system, particularly where proteins such as membrane proteins are difficult to purify, and we have recently successfully applied this method to the quantification of the ABC efflux transporters breast cancer resistance protein, multidrug-resistance protein-2, and P-glycoprotein in human tissues (T. Tucker, A. Milne, S. Fournel-Gigleux, K. Fenner, and M. Coughtrie, manuscript submitted for publication). The main drawbacks associated with this method include the requirement for the generation and characterization of high-quality, isoform-specific antibodies (which are often not available commercially) and the somewhat labor-intensive processes of gel electrophoresis and immunoblotting. However, unlike quantitative proteomic approaches such as AQUA, it does not rely on highly specialized and expensive mass spectrometry equipment or expensive labeled peptides (Gerber et al., 2003; Kettenbach et al., 2011); such a method has been described for the quantification of UGT1A1 and UGT1A6 (Fallon et al., 2008). Another problem with proteomic approaches is the potential for incomplete digestion of target proteins, particularly in complex samples such as microsomal membrane preparations where UGTs are located. A very recent publication (Sakamoto et al., 2011) details a mass spectrometry method for the quantification of several transporters and drug-metabolizing enzymes (including UGT1A1 and UGT2b7); however, only data on the reliability and reproducibility of the method are presented, not the absolute expression values for the proteins concerned.

In summary, we have devised a novel approach to the immunoquantification of UGT expression in human tissue samples. The method is straightforward to establish, is broadly applicable, and it provides the opportunity to determine the relative contributions of different UGT isoforms to the metabolism of drugs and other xenobiotics.

Authorship Contributions

Participated in research design: Milne, Burchell, and Coughtrie.

Conducted experiments: Milne.

Contributed new reagents or analytic tools: Milne.

Performed data analysis: Milne and Coughtrie.

Wrote or contributed to the writing of the manuscript: Milne, Burchell, and Coughtrie.

Acknowledgments

We thank Michelle Fettes for technical assistance and Dr. Sheila Sharp for helpful advice and discussion.

Footnotes

  • This work was funded by the Human Drug Conjugation Consortium (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, F. Hoffman-La Roche, Lilly, Novartis, Pfizer, and Wyeth-Ayerst); and an equipment grant from Tenovus Tayside.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.111.041699.

  • ABBREVIATIONS:

    UGT
    UDP-glucuronosyltransferase
    ADRs
    adverse drug reactions
    MAP
    multiple antigenic peptide
    S-tag
    S-peptide
    UGT1A(S)F
    primer designed to bind in the constant region of the UGT1A
    UGT1A(S) Linker
    primer to link the UGT1A sequence to the S-tag sequence
    S-tag R
    S-tag reverse primer
    PCR
    polymerase chain reaction
    PAGE
    polyacrylamide gel electrophoresis
    PVDF
    polyvinylidene difluoride.

  • Received July 11, 2011.
  • Accepted August 31, 2011.

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QUANTIFICATION OF UGT EXPRESSION IN HUMAN TISSUE

Alison M. Milne, Brian Burchell and Michael W. H. Coughtrie
Drug Metabolism and Disposition December 1, 2011, 39 (12) 2258-2263; DOI: https://doi.org/10.1124/dmd.111.041699
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