Characterization of Whole Body Cholesterol Fluxes in the Mouse

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 [¹⁴ C]cholesterol, 5 µCi [³ H]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 ¹⁴ C and ³ H 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 [¹⁴ C]cholesterol and 2 µCi [³ H]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 [³ H]cholesterol (NEN Life Science Products) dissolved in Intralipid (20%, w/v) (Pharmacia)
Solution of 1 µCi [¹⁴ H]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

D₇ ‐cholesterol (purity >90%) (Cambridge Isotope Laboratories Inc.)
Absolute ethanol (Merk, Darmstadt, Germany)
D₅ ‐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

Figure 1. Simulated data for lymphatic transport of cholesterol. Approximately 35% of the administered [¹⁴ C] cholesterol and 93% [³ H]palmitic acid is recovered in the lymph.

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Literature Cited

Literature Cited
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	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.
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	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.