Characterizing Bile Acid and Lipid Metabolism in the Liver and Gastrointestinal Tract of Mice

Salvatore Modica1, Stefania Murzilli2, Antonio Moschetta3

1 Institute of Food, Nutrition, and Health, ETH Zurich, Schwerzenbach, Switzerland, 2 Laboratory of Lipid Metabolism and Cancer, Department of Translational Pharmacology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy, 3 Clinica Medica “A. Murri,” Department of Internal and Public Medicine, University Aldo Moro of Bari, Bari, Italy
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo100226
Online Posting Date:  June, 2011
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Abstract

Mouse models that mimic human diseases are invaluable tools to study and discover genetic and pharmacological therapies for human diseases. The protocols described in this article are intended to assess general clinical parameters in the context of the enterohepatic system under both normal and pathological conditions. Methods are presented for characterizing liver and intestinal function with a focus on bile acid and lipid metabolism in the gut‐liver axis. Curr. Protoc. Mouse Biol. 1:289‐321 © 2011 by John Wiley & Sons, Inc.

Keywords: nuclear receptors; bile acids; gut‐liver axis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Enzymatic Measurement of Bile Acids
  • Basic Protocol 2: Hepatic Bile Acid Extraction
  • Basic Protocol 3: Determination of Total Bile Acid Pool Size and Composition
  • Support Protocol 1: HPLC Measurement of Bile Acids
  • Basic Protocol 4: Determination of Fecal Bile Acid Pool Size and Composition
  • Basic Protocol 5: Bile Flow Measurement
  • Basic Protocol 6: Intestinal Bile Acid Absorption
  • Basic Protocol 7: Intestinal Bacterial Counts
  • Basic Protocol 8: Measurement of Serum and Hepatic Triglycerides
  • Basic Protocol 9: Measurement of Serum, Biliary, and Hepatic Cholesterol
  • Basic Protocol 10: Measurement of Biliary, Hepatic, and Serum Phospholipids
  • Basic Protocol 11: Hepatic Lipid Extraction
  • Basic Protocol 12: Intestinal Cholesterol Absorption
  • Basic Protocol 13: Measurement of In Vivo Fatty Acid Synthesis
  • Serum Transaminases (AST, ALT, ALP)
  • Basic Protocol 14: Determination of Serum Alanine Aminotransferase (ALT) Activity
  • Basic Protocol 15: Determination of Serum Aspartate Aminotransferase (AST) Activity
  • Basic Protocol 16: Determination of Serum Alkaline Phosphatase (ALP) Activity
  • Basic Protocol 17: Measurement of Serum and Biliary Total Bilirubin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enzymatic Measurement of Bile Acids

  Materials
  • Mice
  • Colorimetric Total Bile Acid Assay kit (containing Reagents R1 and R2, and a calibrator; http://www. diazyme.com; also see recipe for total bile acid reagents in Reagents and Solutions)
  • Ethanol
  • Refrigerated centrifuge
  • 1‐ml syringe
  • 30‐G needle
  • 1.5‐ml cuvettes or 96‐well plates
  • Spectrophotometer capable of measuring absorbance at 405 nm
  • Additional reagents and equipment for hepatic bile acid extraction ( protocol 2) and obtaining fecal bile acid pool ( protocol 5)

Basic Protocol 2: Hepatic Bile Acid Extraction

  Materials
  • Mice
  • 75% (v/v) ethanol
  • Surgical instruments including scissors and clamps
  • Dounce homogenizer with tight‐fitting glass pestle
  • 50°C heating block
  • 5‐ml glass tubes (5 ml)
  • Refrigerated centrifuge
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, )

Basic Protocol 3: Determination of Total Bile Acid Pool Size and Composition

  Materials
  • Mice
  • Ethanol (HPLC grade)
  • 1 mg/ml deuterated glycol‐cholic acid (glycol‐CA; Point‐Clare)
  • Ultrapure H 2O
  • Methanol (HPLC‐grade)
  • Formic acid (mass spectrometry grade; Fluka)
  • Animal balance
  • Surgical instruments including scissors and clamps
  • 250‐ml beakers with watch glasses
  • Hemostats
  • Multi‐position hot plate/stirrer
  • Glass funnels
  • No. 2 Whatman filter paper
  • 100‐ml volumetric flasks
  • 5‐ml glass tubes
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, ), enzymatic BA assay ( protocol 1), and HPLC‐MS/MS ( protocol 4)

Support Protocol 1: HPLC Measurement of Bile Acids

  Materials
  • Extracted BAs ( protocol 3)
  • Ultrapure water via Milli‐Q system (Millipore)
  • Methanol (HPLC grade)
  • Formic acid (mass spectrometry grade; Fluka)
  • BA standard working solutions (see recipe for BA standards and internal standards) in the range of 0.01 to 10 µg/ml for preparing calibration curves
  • Internal standard working solutions (1S1 and IS2; see recipe for BA standards and internal standards)
  • Gradient solution A: methanol (HPLC grade) containing 10 mM ammonium acetate (analytical grade) and 0.25% (v/v) formic acid (mass spectrometry grade; Fluka)
  • Gradient solution B: Ultrapure H 2O containing 10 mM ammonium acetate (analytical grade) and 0.25% (v/v) formic acid (mass spectrometry grade; Fluka)
  • Perkin Elmer 200 autosampler with 20‐µl sample loop
  • Perkin Elmer 200 quaternary HPLC pump
  • 250 × 3.0 mm i.d., 5‐µm Luna C18(2) column provided with a 4.0 × 2.0–mm i.d., 5‐µm Luna C18(2) SecurityGuard System (Phenomenex)
  • API 365 triple quadrupole mass spectrometer (PE Sciex, http://www.absciex.com/)
  • Turbo ion spray source (PE Sciex, http://www.absciex.com/)
  • TurboQuan 1.0 software (PE Sciex, http://www.absciex.com/)

Basic Protocol 4: Determination of Fecal Bile Acid Pool Size and Composition

  Materials
  • Mice
  • 1 µg/100 µl deuterated cholic acid (CA) (https://www.cdnisotopes.com/)
  • 2 mg/ml sodium borohydride in ethanol (prepare fresh)
  • 2 N HCl
  • 10 N NaOH
  • Nitrogen source
  • 20% (v/v) methanol and 100% methanol (HPLC grade)
  • Ultrapure H 2O
  • Ethanol (HPLC grade)
  • Formic acid (mass spectrometry grade)
  • BA standard working solutions (see recipe for BA standards and internal standards) in the range of 0.01 to 10 µg/ml for preparing calibration curves
  • 100‐ml glass beakers
  • 50‐ml plastic and glass tubes with caps
  • 60°C, 70°C, and 120°C heat blocks
  • No. 2 Whatman filter paper
  • C18 Bond Elute column (500 mg/6 ml; Varian)
  • Suction manifold (Varian)
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Additional reagents and equipment for HPLC‐MS/MS analysis of BA composition (see protocol 4)

Basic Protocol 5: Bile Flow Measurement

  Materials
  • Mice, fasted for 4 hr
  • Ketamine (Sigma‐Aldrich)
  • Xylazine (Bayer)
  • Animal balance
  • Syringe and 26‐G needle
  • Surgical instruments
  • PE‐10 catheter
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, )

Basic Protocol 6: Intestinal Bile Acid Absorption

  Materials
  • Mice
  • Ketamine (Sigma‐Aldrich)
  • Xylazine (Bayer)
  • 100 µM [3H(G)] taurocholic acid (sp. act. 1 µCi/100 µl; American Radiolabeled Chemicals)
  • Solvable sample solubilizer (Perkin‐Elmer)
  • 10% (v/v) H 2O 2
  • Opti‐Fluo (Perkin‐Elmer)
  • Surgical instruments including scissors and clamps
  • Syringes with 26‐G needles
  • Tared 20‐ml glass vials
  • Scintillation counter
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, )

Basic Protocol 7: Intestinal Bacterial Counts

  Materials
  • Mice
  • Trypticase soy broth (BD Difco; also see recipe in Reagents and Solutions)
  • Trypticase soy agar plates with 5% sheep blood (BD Difco)
  • Surgical instruments including scissors and clamps
  • Balance
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, ) and anaerobic culture of bacteria (Speers et al., )

Basic Protocol 8: Measurement of Serum and Hepatic Triglycerides

  Materials
  • Triglyceride‐containing samples
  • Colorimetric Triglycerides Liquid Kit (containing Reagent 1 and Standard; Sentinel, http://www.sentinel.it); see recipe for Triglyceride reagents in Reagents and Solutions for compositions of items
  • Methanol
  • Triton X‐100
  • 1.5‐ml cuvettes or 96‐well plates (300‐µl well volume)
  • Spectrophotometer at 546 nm

Basic Protocol 9: Measurement of Serum, Biliary, and Hepatic Cholesterol

  Materials
  • Cholesterol‐containing samples
  • Colorimetric Cholesterol Liquid Kit (containing Reagent 1 and Standard; Sentinel, http://www.sentinel.it); see recipe for Cholesterol reagents in Reagents and Solutions for compositions of items
  • Methanol
  • Triton X‐100
  • 1.5‐ml cuvettes or 96‐well plates (300‐µl well volume)
  • Spectrophotometer at 546 nm

Basic Protocol 10: Measurement of Biliary, Hepatic, and Serum Phospholipids

  Material
  • Phospholipid‐containing samples
  • Colorimetric Phospholipids Kit (containing Reagent 1a, Reagent 1b, and Standard; Sentinel, http://www.sentinel.it); see recipe for Phospholipid reagents in Reagents and Solutions for descriptions of items
  • Methanol
  • Triton X‐100
  • 96‐well plates
  • Spectrophotometer at 520 nm

Basic Protocol 11: Hepatic Lipid Extraction

  Materials
  • Mouse liver tissue
  • 2:1 (v/v) chloroform/methanol
  • 50 mM NaCl
  • 0.36 M CaCl 2 in methanol
  • Chloroform
  • Nitrogen source
  • 10% (v/v) Triton X‐100 in methanol
  • 5‐ml volumetric glass tubes
  • Polytron homogenizer
  • Centrifuge

Basic Protocol 12: Intestinal Cholesterol Absorption

  Materials
  • Mice
  • Dosing mixture of labeled cholesterol and stigmastanol (see recipe)
  • Soluene 350 (Perkin Elmer)
  • 2‐propanol
  • 30% (v/v) hydrogen peroxide
  • UltimaGold scintillation fluid (Perkin Elmer)
  • Animal balance
  • Gavage needle for mice
  • Mortar and pestle
  • 20‐ml scintillation vial
  • Heat block
  • β‐scintillation counter

Basic Protocol 13: Measurement of In Vivo Fatty Acid Synthesis

  Material
  • Mice
  • [3H] 2O (tritiated water; American Radiolabeled Chemicals)
  • 1 M KOH in 66% (v/v) ethanol
  • Petroleum ether
  • Concentrated HCl
  • Hexane
  • Methanol
  • Scintillation cocktail
  • Syringe and 26‐G needles
  • Animal balance
  • Surgical instruments including scissors and clamps
  • 50‐ml glass tubes
  • Heat block
  • 50‐ or 100‐ml volumetric flask
  • Scintillation vials
  • 80°C vacuum oven
  • β‐scintillation counter
  • Additional reagents and equipment for euthanasia of the mouse (Donovan and Brown, )

Basic Protocol 14: Determination of Serum Alanine Aminotransferase (ALT) Activity

  Materials
  • Serum sample
  • Colorimetric ALT (SGPT) kit (containing ALT substrate, ALT color reagent, ALT color developer, ALT calibrator; BioQuant, http://www.bio‐quant.com); see recipe for ALT reagents in Reagents and Solutions for compositions of items
  • 2‐ml test tubes with rack
  • Heat block
  • Spectrophotometer with detection at 505 nm

Basic Protocol 15: Determination of Serum Aspartate Aminotransferase (AST) Activity

  Materials
  • Serum sample
  • Colorimetric AST (SGOT) kit (containing AST substrate, AST color reagent, AST calibrator; BioQuant, http//www.bio‐quant.com); see recipe for AST reagents in Reagents and Solutions for compositions of items
  • 0.1 N HCl
  • 2 ml test tubes with rack
  • Spectrophotometer with detection at 530 nm

Basic Protocol 16: Determination of Serum Alkaline Phosphatase (ALP) Activity

  Materials
  • Serum sample
  • Colorimetric ALP kit (containing ALP substrate, ALP color developer, ALP standard; BioQuant, http://www.bio‐quant.com); see recipe for ALP reagents in Reagents and Solutions for compositions of items
  • 2‐ml test tubes with rack
  • Spectrophotometer detecting at 590 nm

Basic Protocol 17: Measurement of Serum and Biliary Total Bilirubin

  Materials
  • Serum or bile sample
  • Colorimetric Bilirubin Total assay kit (containing Reagent R1, Reagent R2, and calibrator; Diazyme, (http://www.diazyme.com); see recipe for total bilirubin reagents in Reagents and Solutions for compositions of items
  • Cuvettes or 96‐well plates
  • Spectrophotometer detecting at 450 nm
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Figures

Videos

Literature Cited

Literature Cited
   Allain, C.C., Poon, L.S., Chan, C.S., Richmond, W., and Fu, P.C. 1974. Enzymatic determination of total cholesterol. Clin. Chem. 20:470‐475.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Doumas, B. and Biggs, H.G. 1969. A colorimetric method for assaying serum aspartate aminotransferase activities. Clin. Chim. Acta 23:75‐82.
   Falany, C.N., Johnson, M.R., Barnes, S., and Diasio, R.B. 1994. Glycine and taurine conjugation of bile acids by a single enzyme: Molecular cloning and expression of human liver bile acid CoA:amino acid N‐acyltransferase. J. Biol. Chem. 269:19375‐19379.
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