Measurements of UDP‐ Glucuronosyltransferase (UGT) Activities

Birgit L. Coffman1, Gladys R. Rios1, Thomas R. Tephly1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.3
DOI:  10.1002/0471140856.tx0403s13
Online Posting Date:  November, 2002
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Abstract

Mammalian UDP‐glucuronosyltransferases are a family of isoenzymes that catalyze the reaction of endobiotics and xenobiotics with glucuronic acid resulting in the formation of hydrophilic glucuronides. This pathway is an important step in the metabolism and subsequent excretion of many compounds that would otherwise have toxic effects. This unit describes three methods for measuring UGT activity. Thin layer chromatography is a powerful screening method and may be used to analyze multiple substrates simultaneously. The Sep‐Pak C18 cartridge extraction method has been developed to specifically separate opioid glucuronides from UDP‐glucuronic acid. Finally, the ethyl acetate extraction method is used to separate the glucuronides of bilirubin, sterols, and vile acids from UDP‐glucuronic acid. These methods may be applied to a microsomal fraction or to cultured cells transformed with cDNA for UGT.

     
 
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Table of Contents

  • Basic Protocol 1: Thin‐Layer Chromatography Assay for UGT Activity
  • Basic Protocol 2: Sep‐Pak Assay for UGT Activity
  • Basic Protocol 3: Ethyl Acetate Extraction Assay for UGT Activity
  • Support Protocol 1: Preparation of Microsomes from Tissue
  • Support Protocol 2: Preparation of Microsomes from Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Thin‐Layer Chromatography Assay for UGT Activity

  Materials
  • Microsomal pellet (see protocol 4Support Protocol 1 or protocol 52)
  • Enzyme buffer (see recipe), ice cold
  • Bio‐Rad protein assay kit
  • Reaction buffer (see recipe and Table 4.3.1)
  • Phosphatidylcholine (see recipe)
  • Saccharolactone (see recipe)
  • Substrate solutions (see recipe and Table 4.3.1)
  • 20 mM [14C]UDPGlcUA solution (1 µCi/µmol; see recipe)
  • 100% ethanol
  • 1‐Butanol
  • Acetone
  • Glacial acetic acid
  • 28% (w/w) ammonium hydroxide
  • 48% (w/w) hydrofluoric acid
  • 3a70B counting cocktail (Research Products International) or equivalent
  • TLC plates: PSC plates (silica gel 60F 254, 20 × 20 cm, concentration zone 4 × 20 cm, 0.5 mm thickness; Merck)
  • TLC tanks for 20 × 20–cm plates
  • EN3HANCE (NEN Life Science)
  • Kodak XAR 5 or Kodak BioMax MR film
  • Electronic flash (Vivitar or equivalent), dimmed by covering the emission surface with Whatman no. 3 filter paper
    Table 4.3.1   MaterialsOptimal Assay Conditions for Glucuronidation of Common Substrates

    Substrate Species pH Substrate concentration (mM) Reaction time (min) Basic Protocol(s)
    Androsterone Rat 8.0 0.1 10‐15 protocol 11 and protocol 33
    Human 8.4 0.1 15‐30 protocol 11 and protocol 33
    Bilirubin Rat 7.7 0.345 30‐60 protocol 33
    Human 7.8 0.345 30‐60 protocol 33
    Codeine Human 8.4 5 30‐60 protocol 22
    Morphine Rat 8.4 5 15‐30 protocol 22
    Human 8.4 5 15‐30 protocol 22
    Sapogenins Human 7.5 0.1 30‐120 protocol 11 and protocol 33
    Testosterone Human 7.5 0.1 15‐30 protocol 11 and protocol 33

Basic Protocol 2: Sep‐Pak Assay for UGT Activity

  Materials
  • 20 mM [14C]UDPGlcUA solution (0.5 µCi/µmol; see recipe)
  • 100% methanol
  • 10 mM ammonium acetate (see recipe)
  • 1 M ammonium acetate, pH 9.2 (see recipe), ice cold
  • 13 × 100–mm glass or 75 × 12–mm polypropylene tubes
  • Sep‐Pak C18 cartridges (Millipore)
  • Budget‐Solve counting cocktail (Research Products International) or similar
  • Additional reagents and equipment for UGT reaction (see protocol 1)

Basic Protocol 3: Ethyl Acetate Extraction Assay for UGT Activity

  Materials
  • 20 mM [14C]UDPGlcUA solution (0.5 µCi/µmol; see recipe)
  • Glycine/Triton buffer (see recipe)
  • Water‐saturated ethyl acetate
  • Budget‐Solve counting cocktail (Research Products International) or similar
  • Additional reagents and equipment for UGT reaction (see protocol 1)

Support Protocol 1: Preparation of Microsomes from Tissue

  Materials
  • Organ tissue, freshly recovered or frozen tissue from a commercial source
  • 1.15% (w/v) KCl
NOTE: Perform all steps at 4°C.

Support Protocol 2: Preparation of Microsomes from Cultured Cells

  Materials
  • Cultured cells, such as HK293, V79, or COS cells, expressing UGT after transfection with vector containing UGT cDNA
  • PBS ( appendix 2A)
  • Cell suspension buffer (see recipe)
  • Cell homogenate dilution buffer (see recipe)
  • Tissue tearer: 985‐370 (Biospec Products) or equivalent
  • Sonifier cell disrupter: Hat system (Ultrasonic) or equivalent
NOTE: Perform all procedures at 4°C.
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Figures

Videos

Literature Cited

Literature Cited
   Bansal, S.K. and Gessner, T. 1980. A unified method for the assay of uridine diphosphoglucuronyltransferase activities towards various aglycones using uridine diphospho {U‐14C} glucuronic acid. Anal. Biochem. 109:321‐329.
   Bock, K.W. 1991. Role of UDP‐glucuronosyltransferases in chemical carcinogenesis. Crit. Rev. Biochem. Mol. Biol. 26:129‐150.
   Burchell, B., Brierley, C.H. and Rance, D. 1995. Minireview. Specificity of human UDP‐glucuronosyltransferases and xenobiotic glucuronidation. Life Sci. 57:1819‐1831.
   Christrup, L.L. 1997. Morphine metabolites. Acta Anasthesiol. Scan. 41:116‐122.
   Clarke, D.J. and Burchell, B. 1994. The uridine diphosphate glucuronosyltransferase multigene family: Function and regulation. In Handbook of Experimental Pharmacology, Vol. 112. Conjugation‐Deconjugation Reactions in Drug Metabolism and Toxicity (F.C. Kauffman ed.) pp.3‐43. Springer‐Verlag Berlin
   Del Villar, E., Sanchez, E. and Tephly, T.R. 1974. Morphine metabolism. II Studies on morphine glucuronosyltransferase activity in intestinal microsomes of rats. Drug. Metab. Dispos. 2:370‐374.
   Frei, I., Schmid, E. and Bischmeyer, H. 1974. UDP‐glucuronosyltransferase. In Methoden der Enzymatischen Analyze (H.W. Beregmeyser ed.) pp.763‐768. Verlag Chemie, Weinheim.
   Herweigh, K.P.M.., Van De Vijer, M. and Fevery, J. 1972. Assay and properties of digitonin‐activated bilirubin uridine diphosphate glucuronyltransferase from rat liver. Biochem. J. 129:605‐618.
   King, C.D., Rios, G.R., Green, M.D., Mackenzie, P.I. and Tephly, T.R. 1997. Comparison of stably expressed rat UGT1.1 and UGT2B1 in the glucuronidation of opioid compounds. Drug. Met. Dispos. 25:251‐255.
   Lillienblum, W. and Bock, K.W. 1984. N ‐Glucuronide formation of carcinogenic aromatic amines in rat and human liver microsomes. Biochem. Pharmacol. 33:2041‐2046.
   Mackenzie, P.I., Owens, I.S., Burchell, B., Bock, K.W., Bairoch, A., B´langer, A., Fournel‐Gigleux, S., Green, M., Hum, D.W., Iyanagi, T., Lancet, D., Louisot, P., Magdalou, J., Roy Chowdhury, J., Ritter, J.K., Schachter, H., Tephly, T.R., Tipton, K.F. and Nebert, D.W. 1997. The UDP‐glycosyltransferase gene superfamily: Recommended nomenclature update based on evolutionary divergence. Pharmacogenetics. 7:255‐269.
   Matern, H., Heinemann, H. and Matern, S. 1994. Radioassay of UDP‐glucuronosyltransferase activities toward endogenous substrates using labeled UDP‐glucuronic acid and an organic solvent extraction procedure. Anal. Biochem. 219:182‐188.
   Nebert, D.W. 1991. Proposed role of drug‐metabolizing enzymes regulation of steady state levels of the ligands that effect growth, homeostasis, differentiation, and neuroendocrine function. Mol. Endocrinol. 5:1203‐1214.
   Puig, J.P. and Tephly, T.R. 1986. Isolation and purification of rat liver morphine UDP‐glucuronosyltransferase. Mol. Pharmacol. 30:558‐565.
   Spivac, W. and Yuey, W. 1986. Application of a rapid and efficient H.P.L.C. method to measure bilirubin and its conjugates from native bile and in model bile systems. Biochem. J. 234:101‐109.
   Svensson, J.‐O. 1986. Determination of morphine, morphine‐6‐glucuronide and normorphine in plasma and urine with high‐performance liquid chromatography and electrochemical detection. 1986. J. Chromatogr. 375:174‐178.
   Tukey, R.H. and Tephly, T.R. 1981. Purification and properties of rabbit liver estrone and p‐nitrophenol UDP‐glucuronosyltransferase. Arch. Biochem. Biophys. 209:565‐578.
   Yue, Q., Von Bahr, C., Odar‐Cederlöf, I. and Säwe, J. 1990. Glucuronidation of codeine and morphine in human liver and kidney microsomes: Effect of inhibition. Pharmacol. Toxicol. 66:221‐226.
Key References
   Bansal and Gessner 1980. See
  Describes in detail the TLC method, which is applicable for analyzing the glucuronidation formation of many substrate samples simultaneously. A modification of this method is described in .
   Matern et al., 1994. See .
  Describes use of the extraction method to determine UGT activity toward bilirubin, steroid, and hyodeoxycholic acid. A modification of this method is described in .
   Puig and Tephly, 1986. See .
  Describes the Sep‐Pak C18 method () for determining UGT activity toward opioids and amines.
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