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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 H₂ O
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 H₂ O 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 H₂ O
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 [³ H(G)] taurocholic acid (sp. act. 1 µCi/100 µl; American Radiolabeled Chemicals)
Solvable sample solubilizer (Perkin‐Elmer)
10% (v/v) H₂ O₂
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 CaCl₂ 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
[³ H]₂ O (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

Figure 1. Common bile duct ligation.

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Figure 2. Removal of various small intestinal segments from the mouse.

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

Abstract

Table of Contents

Materials

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

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

Figures

Videos

Literature Cited