Characterization of Whole Body Cholesterol Fluxes in the Mouse

Gemma Brufau1, Albert K. Groen2

1 Department of Pediatrics, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, 2 Department of Laboratory Medicine, Center for Liver, Digestive, and Metabolic Diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110118
Online Posting Date:  December, 2011
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Abstract

Atherosclerosis is characterized by excessive cholesterol accumulation in the vessel wall. Current therapies mainly aim at decreasing influx through lowering plasma LDL‐cholesterol levels. The challenge is to develop therapeutic interventions to increase efflux of excess cholesterol from the vessel wall. The pathway that mediates this efflux from vessel wall to final excretion in the feces is called reverse cholesterol transport. Recently, it has become apparent that the intestine plays an important regulatory role in this pathway. This article describes in detail a variety of experimental approaches to measure cholesterol fluxes in the hepatobiliary system as well as in the intestinal pathway. Curr. Protoc. Mouse Biol. 1:413‐427 © 2011 by John Wiley & Sons, Inc.

Keywords: mouse; fecal cholesterol excretion; biliary excretion; transintestinal cholesterol; excretion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Dietary Cholesterol Intake and Neutral Sterol Excretion
  • Basic Protocol 2: Measurement of Biliary Cholesterol Excretion
  • Basic Protocol 3: Direct Measurement of Fractional Cholesterol Absorption by Determination of Lymphatic Cholesterol Transport
  • Alternate Protocol 1: Indirect Measurement of Fractional Cholesterol Transport via Fecal Dual‐Isotope Ratio Method
  • Alternate Protocol 2: Indirect Measurement of Fractional Cholesterol Transport by the Plasma Dual‐Isotope Ratio Method
  • Basic Protocol 4: Estimation of Transintestinal Cholesterol Excretion by the Sterol Balance Method
  • Alternate Protocol 3: Measurement of Transintestinal Cholesterol Excretion by Modeling of Cholesterol Fluxes Determined via Stable Isotope Enrichment Estimation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Dietary Cholesterol Intake and Neutral Sterol Excretion

  Materials
  • Appropriate mouse strain
  • Mouse diet
  • Control feces (sample that has been repeatedly analyzed and has given consistent results)
  • Standards: 5α‐cholestane, coprostanol, epi‐coprostanol, cholesterol, dihydro‐cholesterol, coprostan‐3‐one and 3‐keto‐cholesterol (Sigma)
  • Absolute ethanol
  • 1 M sodium hydroxide (NaOH; Sigma)
  • Methanol (Merck)
  • Petroleum ether, boiling point range 60° to 80°C (Merck)
  • Pyridine (Thermo Scientific)
  • N,O‐Bis(trimethylsilyl)trifluoroacetamide (BSTFA; Sigma)
  • Trimethylchlorosilane (TMCS; Thermo Scientific)
  • Nitrogen source
  • Hexane (Merck)
  • Single‐housing cages for mice
  • Scale with 0.1 g accuracy (to weigh food and mice)
  • Container to accommodate mice during weighing
  • Separate mortar and pestle for grinding the feces and the food
  • Analytical balance with 0.1 mg accuracy
  • 10‐ml glass screw‐top vials
  • 80° and 40°C heat blocks
  • GC vials
  • Gas chromatographic (GC) system (van der Veen et al., )
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Basic Protocol 2: Measurement of Biliary Cholesterol Excretion

  Materials
  • Appropriate mouse strain
  • 0.315 mg/ml fentanyl citrate
  • 10 mg/ml fluanisone
  • 5 mg/ml diazepam
  • Phosphate‐buffered saline (PBS)
  • 70% (v/v) ethanol
  • Standards: 5α‐cholestane, coprostanol, epi‐coprostanol, cholesterol, dihydro‐cholesterol, coprostan‐3‐one, and 3‐keto‐cholesterol (Sigma)
  • Control bile (sample that has been repeatedly analyzed and has given consistent results)
  • Chloroform (Merck)
  • Methanol (Merck)
  • Nitrogen source
  • Pyridine (Thermo Scientific)
  • N,O‐Bis(trimethylsilyl)trifluoroacetamide (BSTFA; Sigma)
  • Trimethylchlorosilane (TMCS; Thermo Scientific)
  • Heptane
  • Single‐housing cages for mice
  • 0.5‐ml microcentrifuge tubes
  • Analytical balance with 0.1 mg accuracy
  • Scale with 0.1 g accuracy (to weigh food and mice)
  • Animal clippers
  • Surgical instruments including Backhaus towel clamps
  • Zoom stereomicroscope
  • Cotton swabs
  • 6–0 silk suture
  • 20‐G needle
  • PE‐10 cannula
  • Incubator to keep the animals at a constant temperature of 37°C
  • 10‐ml glass screw‐top tubes
  • 50°C heat block
  • Glass syringe
  • GC vials
  • Gas chromatographic (GC) system (van der Veen et al., )
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Basic Protocol 3: Direct Measurement of Fractional Cholesterol Absorption by Determination of Lymphatic Cholesterol Transport

  Materials
  • Appropriate mouse strain
  • Sodium pentobarbital
  • Solution of 2.5 µCi of [14C]cholesterol, 5 µCi [3H]palmitic acid, and 0.5% (w/v) taurocholate (NEN Life Science Products) dissolved in 100 µl medium‐chain triglyceride oil (Mead Johnson)
  • Solution of 0.5% taurocholate in medium‐chain triglyceride oil (Mead Johnson)
  • Scintillation fluid
  • 0.5‐ml heparinized microcentrifuge tubes
  • 3‐ml syringe
  • Zoom stereomicroscope
  • PE‐10 polyethylene catheter
  • 6–0 silk suture
  • Adhesive tape (13 mm × 30 m)
  • Infusion pump (Harvard Apparatus, cat. no. 70‐2209)
  • Incubator to keep the animals at a constant temperature of 37°C
  • Scintillation counter capable of measuring 14C and 3H in same measurement
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Alternate Protocol 1: Indirect Measurement of Fractional Cholesterol Transport via Fecal Dual‐Isotope Ratio Method

  • Appropriate mouse strain
  • Solution of 1 µCi of [14C]cholesterol and 2 µCi [3H]sitostanol (NEN Life Science Products) dissolved in 150 µl medium‐chain triglyceride oil (Mead Johnson)
  • Gavage needle

Alternate Protocol 2: Indirect Measurement of Fractional Cholesterol Transport by the Plasma Dual‐Isotope Ratio Method

  • Appropriate mouse strain
  • Solution of 2.5 µCi of [3H]cholesterol (NEN Life Science Products) dissolved in Intralipid (20%, w/v) (Pharmacia)
  • Solution of 1 µCi [14H]cholesterol (NEN Life Science Products, Boston, MA) dissolved in 150 µL medium‐chain triglyceride oil (Mead Johnson)
  • Heparinized microcentrifuge tubes
  • Additional reagents and equipment for obtaining blood from the mouse by cardiac puncture (Donovan and Brown, )

Basic Protocol 4: Estimation of Transintestinal Cholesterol Excretion by the Sterol Balance Method

  • D 7‐cholesterol (purity >90%) (Cambridge Isotope Laboratories Inc.)
  • Absolute ethanol (Merk, Darmstadt, Germany)
  • D 5‐cholesterol (purity >90%) (Medical Isotopes Inc.)
  • Intralipid solution (20%; v/v) (Pharmacia)
  • Medium chain triglyceride oil (Mead Johnson)
  • Small sharp scissors
  • Filter paper (Munktell & Filtrak, http://www.munktell.com)
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Figures

Videos

Literature Cited

Literature Cited
   Bligh, E.G. and Dyer, W.J. 1959. A rapid method of total lipid extraction and purification. Can. J Biochem. Physiol. 37:911–917.
   Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Donovan, J. and Brown, P. 2006b. Blood collection. Curr. Protoc. Immunol. 73:1.7.1‐1.7.9.
   Keys, A., Aravanis, C., Van Buchem, F.S.P., Blackburn, H., Buzina, R., Djordjevic, B.S., Dontas, A.S., Fidanza, F., Karvonen, M.J., Kimura, N., Menotti, A., Nedeljkovic, S., Puddu, V., Punsar, S., and Taylor, H.L. 1981. The diet and all‐causes death rate in the Seven Countries Study. Lancet 2:58–61.
   Ostlund, R.E. Jr. 2002. Phytosterols in human nutrition. Ann. Rev. Nutr. 22:533‐549.
   van der Veen, J.N., van Dijk, T.H., Vrins, C.L., van Meer, H., Havinga, R., Bijsterveld, K., Tietge, U.J., Groen, A.K., and Kuipers, F. 2009. Activation of the liver X receptor stimulates trans‐intestinal excretion of plasma cholesterol. J. Biol. Chem. 284:19211‐19219.
   van der Velde, A.E., Vrins, C.L., van den Oever, K., Seemann, I., Oude Elferink, R.P., van Eck, M., Kuipers, F., and Groen, A.K. 2008. Regulation of direct transintestinal cholesterol excretion in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 295:G203‐G208.
   Wang, D.Q.H. and Carey, M.C. 2003. Measurement of intestinal cholesterol absorption by plasma and fecal dual‐isotope ratio, mass balance, and lymph fistula methods in the mouse. J. Lipid Res. 44:1042–1059.
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