Measurement of Intestinal and Peripheral Cholesterol Fluxes by a Dual‐Tracer Balance Method

Onne A.H.O. Ronda1, Theo H. van Dijk2, H.J. Verkade1, Albert K. Groen3

1 Center for Liver, Digestive and Metabolic Diseases, Departments of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, 2 Department of Laboratory Medicine, Center for Liver Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, Groningen, 3 Amsterdam Diabetes Center, Department of Vascular Medicine, Academic Medical Center, Amsterdam
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.16
Online Posting Date:  December, 2016
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Long‐term elevated plasma cholesterol levels put individuals at risk for developing atherosclerosis. Plasma cholesterol levels are determined by the balance between cholesterol input and output fluxes. Here we describe in detail the methodology to determine the different cholesterol fluxes in mice. The percentage of absorbed cholesterol is calculated from a stable isotope–based double‐label method. Cholesterol synthesis is calculated from MIDA after 13C‐acetate enrichment. Cholesterol is removed from the body via the feces. The fecal excretion route is either biliary or non‐biliary. The non‐biliary route is dominated by trans‐intestinal cholesterol efflux, or TICE. Biliary excretion of cholesterol is measured by collecting bile. Non‐biliary excretion is calculated by computational modeling. In this article, we describe methods and procedures to measure and calculate dietary intake of cholesterol, fractional cholesterol absorption, fecal neutral sterol output, biliary cholesterol excretion, TICE, cholesterol synthesis, peripheral fluxes, and whole‐body cholesterol balance. © 2016 by John Wiley & Sons, Inc.

Keywords: cholesterol absorption; cholesterol synthesis; biliary excretion; fecal cholesterol excretion; mouse; trans‐intestinal cholesterol excretion

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Table of Contents

  • Introduction
  • Basic Protocol 1: Dietary Cholesterol Intake and Neutral Sterol and Bile Acid Excretion
  • Basic Protocol 2: Fractional Cholesterol Absorption Using Dual Tracers
  • Basic Protocol 3: Cholesterol Synthesis Measurement Using Labeled Acetate
  • Basic Protocol 4: Biliary and Non‐Biliary Cholesterol Excretion
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Dietary Cholesterol Intake and Neutral Sterol and Bile Acid Excretion

  • Appropriate animal model (mouse, rat)
  • Rodent diet (pellets preferred over powder)
  • 5α‐cholestane (Sigma, cat. no. C8003, or equivalent)
  • 1 M NaOH
  • Methanol
  • Petroleum ether, 60º to 80ºC boiling point
  • N 2 source
  • Coprostanol (Sigma, cat. no. C8003, or equivalent)
  • Epicoprostanol (Steraloids, cat. no. C5050, or equivalent)
  • Cholesterol (Sigma, cat. no. C8667, or equivalent)
  • Dehydrocholesterol (Sigma, cat. no. 30800)
  • Sitostanol (Stigmastanol, Sigma, cat. no. S4297)
  • N,O‐Bis(trimethylsilyl)trifluoroacetamide (BSTFA; Supelco, cat. no. 33027)
  • Pyridine
  • Trimethylchlorosilane (TMCS; Thermo, cat. no. TS‐88530)
  • Hexane (heptane has similar properties and can be used interchangeably)
  • C18 Sep‐Pak column (Waters)
  • 10% (v/v) acetone
  • Isolithocholic acid (Steraloids, cat. no. C1475)
  • Lithocholic acid (Steraloids, cat. no. C1420)
  • α‐Muricholic acid (Steraloids, cat. no. C1890)
  • β‐Muricholic acid (Steraloids, cat. no. C1895)
  • ω‐Muricholic acid (Steraloids, cat. no. C1888)
  • Deoxycholic acid (Sigma, cat. no. D2510)
  • Cholic acid (Sigma, cat. no. C1129)
  • Chenodeoxycholic acid (Sigma, cat. no. C9377)
  • Hyodeoxycholic acid (Steraloids, cat. no. C0860)
  • Ursodeoxycholic acid (Sigma, cat. no. U5127)
  • Hyocholic acid (Avanti Polar Lipids, cat. no. 700159)
  • 5β‐Cholanic acid‐7α, 12α‐diol (Steraloids, cat. no. C1170)
  • Acetyl chloride
  • Container to accommodate the animal during weighing
  • Individual housing (collection of feces)
  • Appropriate bedding [coarse wood chips, sparse paper‐based nesting material; not preferred are cotton‐based nestlets, (fine) sawdust, and (fine) wood shavings]
  • Scale with 0.1 g accuracy to weigh mice/rats
  • Scale with 1 mg accuracy to weigh food and feces
  • Mortar and pestle for grinding the diet and the feces
  • 10‐ml glass tubes that can be closed air‐tight
  • 50º, 55º, 65º, and 80ºC heat blocks or water baths
  • 2‐ml gas chromatography (GC) vials
  • Gas chromatograph
NOTE: The following steps can be applied to either mice or rats.

Basic Protocol 2: Fractional Cholesterol Absorption Using Dual Tracers

  • D 5‐cholesterol (2,2,4,4,6‐deuterium‐cholesterol; Medical Isotopes, Inc., cat. no. D201)
  • Absolute ethanol
  • D 7‐cholesterol (25,26,26,26,27,27,27‐deuterium‐cholesterol; Medical Isotopes Inc, cat. No. D897)
  • 20% Intralipid (Fresenius Kabi, or equivalent)
  • Medium‐chain triglyceride (MCT) oil (SHS International, Nutricia, or equivalent)
  • Appropriate animal model (mouse or rat)Acetone
  • N 2 source
  • N,O‐Bis(trimethylsilyl)trifluoroacetamide (BSTFA; Supelco, cat. no. 33027)
  • Trimethylchlorosilane (TMCS; Thermo, cat. no. TS‐88530)
  • Heptane
  • 40º and 50ºC heat blocks or water baths
  • Rollerbank (Stuart Roller mixer, or equivalent)
  • Sample carrier filter paper (TFN grade, 182 g/m2; Sartorius Stedim or equivalent)
  • Single‐hole puncher (diameter ∼6 mm)
  • 2‐ml microcentrifuge tubes
  • 10‐ml glass tube
  • GC/MS vials containing glass inserts (vials: Aluglas, cat. no. 1013679; inserts: Aluglas, cat. no. 1013586; caps: VWR, cat. no. 548‐0085)
  • GC/MS system
  • SAAM II software (version 1.2.1 SAAM Institute, University of Washington, or version 2.3 The Epsilon Group)
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Basic Protocol 3: Cholesterol Synthesis Measurement Using Labeled Acetate

  • Appropriate animal model (mouse, rat)
  • Sodium [1‐13C]acetate‐ (Sigma‐Aldrich, cat. No. 668656, or equivalent)
  • Sterilization filter (0.2‐µm, for sterilizing acetate solutions)
  • SAAM II software (version 1.2.1 SAAM Institute, University of Washington, or or version 2.3 The Epsilon Group)
  • Additional reagents and equipment for preparing blood spots ( protocol 2)

Basic Protocol 4: Biliary and Non‐Biliary Cholesterol Excretion

  • Appropriate animal model (mouse)
  • Chloroform
  • Methanol
  • Triton X‐100
  • Nitrogen source
  • Absolute ethanol
  • 0.2 M potassium phosphate buffer, pH 7.4
  • Sodium cholate
  • 4‐hydroxyphenylacetic acid (Sigma, cat. no. H50004)
  • Cholesterol oxidase enzyme (Sigma, cat. no. 26746 or C8868, or equivalent)
  • Cholesterol peroxidase enzyme (Roche, cat. no. 10108090001 or Sigma, cat. no. P8250 or equivalent)
  • Cholesterol esterase enzyme (Sigma, cat. no. C9281)
  • 5β‐Cholanic acid‐7α, 12α‐diol (Steraloids, cat. no. C1170)
  • Bile
  • 0.1 M sodium acetate buffer, pH 5.6
  • Choloyl glycine hydrolase (Sigma, cat. no. C4018)
  • 0.5‐ml tubes, labeled
  • Scale with 0.1 mg accuracy (to weigh tubes)
  • Gallbladder cannula
  • 37ºC incubator, or heat mat
  • Scale with 0.1 g accuracy (to weigh mice)
  • Incubator or heat pad to maintain body temperature
  • Container to accommodate the animal during weighing
  • ELISA Plate Reader capable of 340 nm excitation and 420 nm emission
  • Black non‐transparent 96‐wells ELISA plates
Additional reagents and equipment for determining bile acid profile by GC ( protocol 1)
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Literature Cited

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