Measurement of Xenobiotic Carbonyl Reduction in Human Liver Fractions

M. Jane Cox Rosemond1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.17
DOI:  10.1002/0471140856.tx0417s25
Online Posting Date:  September, 2005
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Abstract

Carbonyl reducing enzymes are involved in the metabolism of endogenous as well as xenobiotic molecules. Enzymes that catalyze the reversible oxidoreduction of aldehyde and ketone moieties include alcohol dehydrogenases, aldo‐keto reductases, quinone reductases, and short‐chain dehydrogenases/reductases. These enzymes differ with respect to subcellular location, cofactor dependence, and susceptibility to chemical inhibitors. Thus, it is possible to assess the relative contributions of these enzyme systems in the hepatic metabolism of a particular xenobiotic through simple in vitro experiments with commercially available reagents. The approaches described in this unit assume the availability of analytical procedures for measuring the parent compound and metabolites, such as HPLC with radiochemical, UV, or MS detection. Thus, the purpose of this unit is to outline methods for the study of the enzymatic carbonyl reduction of a drug development candidate or other xenobiotic molecule of interest.

Keywords: carbonyl reduction; human; liver; subcellular fraction; non‐P450 metabolism; xenobiotic; microsomes; cytosol

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Subcellular Location and Cofactor Dependence of a Carbonyl Reducing Enzyme
  • Basic Protocol 2: Use of Chemical Inhibitors in the Study of Carbonyl Reducing Enzymes
  • Basic Protocol 3: Determining pH Dependence of Carbonyl Reducing Enzymes
  • Basic Protocol 4: Discriminating Between Cytochrome P450 and Carbonyl Reducing Enzymes with Recombinant P450 Preparations
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Subcellular Location and Cofactor Dependence of a Carbonyl Reducing Enzyme

  Materials
  • Pooled human liver subcellular fractions (cytosol, microsomes; BD Biosciences, In Vitro Technologies, Xenotech), −80°C
  • 1 M potassium phosphate buffer, pH 7.4 (see recipe)
  • Cofactors:
    • 2 mM NADH (see recipe)
    • 2 mM NADPH (see recipe)
    • For NADPH‐regenerating system:
    • Glucose 6‐phosphate dehydrogenase (see recipe)
    • Regenerating system cofactor mixture (see recipe)
  • 1000× xenobiotic substrate solutions (see recipe)
  • Reagents for stopping reactions (Table 4.17.7)
  • Chromatography buffers and solutions
  • Thermomixer or shaking water bath, 37°C
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for HPLC and analysis (unit 4.4)

Basic Protocol 2: Use of Chemical Inhibitors in the Study of Carbonyl Reducing Enzymes

  Materials
  • Pooled human liver subcellular fractions (cytosol, microsomes; BD Biosciences, In Vitro Technologies, Xenotech)
  • 1 M potassium phosphate buffer, pH 7.4 (see recipe)
  • 1000× inhibitor solution (see Table 4.17.3 and recipes):
    • For cytosol:
    • Phenolphthalein, flufenamic acid (AKR probes)
    • Menadione, quercetrin (CR probes)
    • For microsomes:
    • Glycyrrhetinic acid, chenodeoxycholate (11β‐HSD probes)
    • Aminobenzotriazole (P450 probe)
    • Vehicle solutions (solvent for dissolving inhibitors, to be tested as controls):
  • 1000× xenobiotic substrate solution (see recipe)
  • Reagents for stopping reactions (see Table 4.17.7)
  • Chromatography buffers and solutions
  • Cofactors: 2 mM NADPH (see recipe)
  • Thermomixer or shaking water bath, 37°C
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for HPLC and analysis (unit 4.4)

Basic Protocol 3: Determining pH Dependence of Carbonyl Reducing Enzymes

  Materials
  • Pooled human liver cytosol subcellular fraction (BD Biosciences, In Vitro Technologies, Xenotech)
  • 1M potassium phosphate buffer, pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.4 (see reciperecipes)
  • 1000× xenobiotic substrate solution (see recipe)
  • Reagents for stopping reactions (Table 4.17.7)
  • Chromatography buffers and solutions
  • Cofactor: 2 mM NADPH (see recipe)
  • Thermomixer or shaking water bath, 37°C
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for HPLC and analysis (unit 4.4)

Basic Protocol 4: Discriminating Between Cytochrome P450 and Carbonyl Reducing Enzymes with Recombinant P450 Preparations

  Materials
  • Pooled human liver microsomes (BD Biosciences, In Vitro Technologies, Xenotech), gently thawed and placed on ice
  • 1 M potassium phosphate buffer, pH 7.4 (see recipe)
  • 1000× xenobiotic substrate solution (see recipe)
  • Recombinant P450 mixture (see Table 4.17.6), gently thawed and placed on ice
  • Recombinant control preparation, gently thawed and placed on ice
  • Cofactor:
    • 2 mM NADPH (see recipe)
    • For NADPH‐regenerating system:
    • Glucose 6‐phosphate dehydrogenase (see recipe)
    • Regenerating system cofactor mixture (see recipe)
  • Reagents for stopping reactions (Table 4.17.7)
  • Chromatography buffers and solutions
  • Thermomixer or shaking water bath, 37°C
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for HPLC and analysis (unit 4.4)
    Table 4.7.6   Materials   Sample Recipe for a Recombinant P450 Mixture   Sample Recipe for a Recombinant P450 Mixture

    Initial product concentrations (from vendor) Recombinant mixture preparation Final concentrations in incubations
    P450 Initial protein concentration (mg/ml) Initial P450 content (pmol/ml) Volume per P450 (ml) Total volume of protein Protein (mg/ml) P450 (pmol/ml) Amount recombinant mix per 1 ml incubation (ml) Protein (mg/ml) P450 (pmol/ml)
    1A2 6 1000 0.5 3.75 ml of 5.83 mg/ml 0.80 133 0.254 0.20 30
    2A6 10 1000 0.5 1.3 133 0.33 30
    2B6 7 1000 0.5 0.93 133 0.24 30
    2C9 3 1000 0.5 0.40 133 0.11 30
    2C19 3 1000 0.5 0.40 133 0.11 30
    2D6 6 1000 0.5 0.80 133 0.20 30
    2E1 4 2000 0.25 0.27 133 0.07 30
    3A4 7 1000 0.5 0.93 133 0.24 30
    Final total protein concentration in incubations = 1.5 mg/ml

     The recombinant P450 mixture helps to discriminate between P450‐ and non‐P450‐mediated metabolism. Aliquots of each recombinant cytochrome P450 isoform are combined to create an artificial human microsomal preparation with P450 and no other human microsomal enzymes. The quantities of each isoform will vary depending on the concentration of protein and P450 in each product. These characterizations are typically performed by the supplier (e.g., BD Biosciences, Invitrogen). P450 isoforms are mixed so that the final concentration of each P450 in the incubation mixture is 30 pmol/ml.
     The use of recombinant P450 mixtures is discussed in Crespi and Miller ( ).
     A typical protein concentration for control recombinant microsomal preparations is 5 mg/ml.
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Figures

Videos

Literature Cited

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Key References
   Snyder, L.R., Kirkland, J.J., and Glajch, J.L. 1997. Practical HPLC Method Development, 2nd ed. John Wiley and Sons, New York.
  References describing HPLC procedures.
   McClure, T.D. 2003. Detection of Metabolites Using High‐Performance Liquid Chromatography and Mass Spectrometry. In Current Protocols in Toxicology (M.D. Maines, L.G. Costa, E. Hodgson, D.J. Reed, and I.G. Sipes, eds.) pp. 4.4.1‐4.4.18. John Wiley & Sons, Hoboken, N.J.
  References describing stereochemistry procedures.
   Gross, A.S., Somogyi, A., and Eichelbaum, M. 2003. Stereoselective drug metabolism and drug interactions. In Handbook of Experimental Pharmacology 153 (Stereochemical Aspects of Drug Action and Disposition) pp. 313‐339. (K. Starke, ed.) Springer‐Verlag, New York.
  References describing enzyme kinetics.
   Testa, B. 1986. Chiral aspects of drug metabolism. Trends Pharmacol. Sci. 7:60‐64.
   Reddy, I.K. and Mehvar, R., ed. 2004. Chirality in Drug Design and Development. Marcel Dekker, N.Y.
   Allison, R.D. 1997. Kinetic assay methods. In Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., eds.), pp. A.3H.1‐A.3H.11. John Wiley & Sons, New York.
   Copeland, R.A. 2000. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis, 2nd ed., John Wiley & Sons, New York.
   Cornish‐Bowden, A. 2004. Fundamentals of Enzyme Kinetics, 3rd ed. Portland Press, Ltd. Colchester, UK.
   Cornish‐Bowden, A. 1995. Analysis of Enzyme Kinetic Data. Oxford Univ. Press. Oxford, UK.
Internet Resources
   http://www.med.upenn.edu/akr/
  A very useful Website for the aldo‐keto reductase superfamily.
   http://www.gentest.com/
  For fluorescence assays to confirm enzymatic activity of recombinant P450 mixture (BD Biosciences).
   http://www.abnova.com.tw
   http://www.biocatalytics.com
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