Lipid and Bile Acid Analysis

Carmen A. Argmann1, Sander M. Houten2, Marie‐France Champy3, Johan Auwerx4

1 Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, 2 Academic Medical Center, Amsterdam, 3 Institut Clinique de la Souris, Illkirch, 4 Institut de Genetique et de Biologie Moleculaire et Cellulaire and Institut Clinique de la Souris, Illkirch
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 29B.2
DOI:  10.1002/0471142727.mb29b02s75
Online Posting Date:  August, 2006
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Abstract

Lipids are important body constituents that are vital for cellular, tissue, and whole‐body homeostasis. Lipids serve as crucial membrane components, constitute the body's main energy reservoir, and are important signaling molecules. As a consequence of these pleiotropic functions, many common diseases, including atherosclerosis, chronic inflammatory disorders, and obesity, have been associated with altered lipid homeostasis. Lipid abnormalities are hence increasingly analyzed in mouse models. This unit describes commonly used methods to analyze mouse lipid metabolism, with techniques that evaluate lipids both in blood and in tissues. Despite the similarities between men and mice in many aspects of metabolism, important differences also exist in the area of lipid homeostasis. These differences are discussed and should be taken into account when extrapolating lipid data from mouse to men.

Keywords: atherosclerosis; bile acids; cholesterol; fatty acids; triglycerides

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Manual Measurement of Serum Lipids
  • Alternate Protocol 1: Blood Lipid Analysis Using Automated Laboratory Workstation
  • Basic Protocol 2: Lipoprotein Fractionation by Differential Ultracentrifugation
  • Alternate Protocol 2: Lipoprotein Analysis by Gel‐Exclusion Chromatography
  • Basic Protocol 3: Tissue Lipid Analysis
  • Basic Protocol 4: Enzymatic Serum Bile Acid Measurement
  • Alternate Protocol 3: Serum Bile Acid Measurement by HPLC Tandem Mass Spectrometry
  • Alternate Protocol 4: Bile Acid Pool Size and Composition
  • Support Protocol 1: Modified Lowry Assay for Lipoprotein Determination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Manual Measurement of Serum Lipids

  Materials
  • Blood serum, plasma, or isolated lipid fraction obtained via other protocols in this unit
  • Lipid analysis kits (Wako):
    • Total cholesterol kit
    • LDL cholesterol kit
    • HDL cholesterol kit
    • FFA kit
    • Free cholesterol kit
    • TG kit
  • 5‐ml plastic or glass tubes
  • Spectrophotometer

Alternate Protocol 1: Blood Lipid Analysis Using Automated Laboratory Workstation

  Materials
  • Calibrators for biochemistry on AU400:
    • Olympus OE 66300 for cholesterol and triglyceride (lyophilized human serum with chemical additives and appropriate enzymes of human, animal, and plant origin)
    • Olympus ODC0011 for HDL cholesterol and ODC0012 for LDL cholesterol (lyophilized human serum containing human HDL or LDL cholesterol)
    • Wako 994‐75409 for FFA
  • Intralaboratory controls for biochemistry on AU400:
    • ODC0003 (Level 1) and ODC0004 (Level 2) Olympus System Reagent Control Serum (lyophilized preparation of stabilized human serum with adjusted levels of defined analytes)
    • ODC0005 LDL/HDL Cholesterol Control Serum, Levels 1/2 (lyophilized preparation of stabilized human serum with adjusted levels of defined analytes)
  • Interlaboratory controls: Lyphochek unassayed chemistry control, human, levels 1 and 2 (Bio‐Rad C‐320‐10 and C‐325‐10)
  • Serum or plasma samples (80 µl minimum)
  • Olympus AU400 automated laboratory workstation and operating manual (http://www.olympus‐global.com) with appropriate sample tubes and software
  • Unity Plus quality control software (Bio‐Rad)
  • 4‐ml, 50 × 14–mm hemolysis tubes (Sarstedt) with bar‐coded labels added in‐house
NOTE: For general operation of the analyzer, refer to the operator manual of the AU400.

Basic Protocol 2: Lipoprotein Fractionation by Differential Ultracentrifugation

  Materials
  • Serum or plasma: preferably 400 µl of freshly collected sample (samples collected under the same conditions can be pooled)
  • Density gradient solutions (see recipe)
  • Potassium bromide, solid
  • Dialysis buffer (see recipe)
  • 1.0‐ml polycarbonate ultracentrifuge tubes (Beckman)
  • Optima TL‐100 ultracentrifuge with TLA‐100.2 rotor (Beckman) or equivalent ultracentrifuge and rotor
  • Digital Density Meter DMA3.5 (Anton Paar; http://www.anton‐paar.com)
  • Dialysis tubing (Spectra/Por membrane 3.5, Spectrum)
  • Beaker for dialysis
  • Additional reagents and equipment for dialysis ( appendix 3C), lipid assays ( protocol 1), and protein quantitation ( protocol 9)

Alternate Protocol 2: Lipoprotein Analysis by Gel‐Exclusion Chromatography

  Materials
  • Serum or plasma
  • Elution buffer (see recipe)
  • 20% (v/v) ethanol
  • FPLC system (Fig. ) consisting of:
    • HPLC pump (P580 A, Dionex)
    • Multi‐sample automatic injector (GINA 50, Dionex)
    • Column (Superose 6 PC 3.2/30, Amersham Biosciences)
    • UV/VIS detector (UV170S, Dionex)
    • Fraction collector (FRAC‐100, Amersham Biosciences)
    • Integrator software (Chromeleon 6.4, Dionex)
  • 0.22‐ or 0.45‐µm HPLC analytical filter for Biocompatible UPCA
  • Additional reagents and equipment for lipid assays ( protocol 1)

Basic Protocol 3: Tissue Lipid Analysis

  Materials
  • Tissue (∼0.5 g)
  • 2:1 (v/v) chloroform/methanol
  • Methanol
  • 1α,2α‐(n)‐[3H]Cholesteryl oleate (Amersham Biosciences)
  • Glycerol tri[1‐14C]oleate (Amersham Biosciences)
  • Chloroform
  • 0.73% (w/v) NaCl
  • Chloroform/methanol/NaCl (see recipe)
  • 95% ethanol or 1% (v/v) Triton X‐100 in chloroform
  • 200 µg/ml cholesterol/triglyceride standard solution: dilute cholesterol and triolein to a final concentration of 200 µg/ml in isopropanol
  • 14 × 100–mm Pyrex or borosilicate glass round‐bottom tubes with screw caps
  • Homogenizer (Polytron or equivalent)
  • 12 × 75–mm glass hemolysis tubes with corks
  • Whatman filter paper (lipid free)
  • Nitrogen evaporator with dry bath at ∼50°C (e.g., N‐EVAP from Organomation)
  • Scintillation vials, scintillation fluid, and β‐counter with dual‐channel program for 3H and 14C

Basic Protocol 4: Enzymatic Serum Bile Acid Measurement

  Materials
  • Colorimetric Total Bile Acids Assay Kit (containing reagents R1, R2, and R3; Bio‐Quant, http://www.bio‐quant.com)
  • 10 mM taurocholic acid
  • Bile acid control/calibrator (high and low level; Diagnostics Chemical Limited, http://www.dclchem.com)
  • 200‐µl cuvettes
  • Spectrophotometer

Alternate Protocol 3: Serum Bile Acid Measurement by HPLC Tandem Mass Spectrometry

  Materials
  • Plasma
  • Internal deuterium‐labeled standards:
    • Chenodeoxycholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐2772)
    • Cholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐2452)
    • Glycochenodeoxycholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐5673)
    • Glycocholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐3878)
    • Taurine conjugates (custom synthesized)
  • Acetonitrile (HPLC grade)
  • 1:3 (v/v) methanol/water
  • 1.5‐ml microcentrifuge tubes
  • 4‐ml glass vials
  • Nitrogen evaporator with dry bath at ∼40°C (e.g., N‐EVAP from Organomation)
  • HPLC‐MS/MS (e.g., Hewlett‐Packard HP1100 binary pump and a Waters Micromass Quatro II tandem mass spectrometer equipped with electrospray ionization)
  • Additional reagents and equipment for liquid chromatography–mass spectrometry (unit 10.21)

Alternate Protocol 4: Bile Acid Pool Size and Composition

  Materials
  • Test animal of interest
  • 100% ethanol (HPLC grade)
  • Recovery standard (see recipe)
  • Internal deuterium‐labeled standards:
    • Chenodeoxycholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐2772)
    • Cholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐2452)
    • Glycochenodeoxycholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐5673)
    • Glycocholic‐2,2,4,4‐d 4 acid (C/D/N Isotopes cat. no. D‐3878)
    • Taurine conjugates (custom synthesized)
  • Dissecting instruments
  • 50‐ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • 50‐ml glass beaker
  • Watch glass suitable as cover for 50‐ml beaker
  • Filter paper (Schleicher‐Schuell, type 597)
  • Glass funnels
  • 50‐ml volumetric flask
  • HPLC‐MS/MS (e.g., Hewlett‐Packard HP1100 binary pump and a Waters Micromass Quatro II tandem mass spectrometer equipped with electrospray ionization)
  • Additional reagents and equipment for liquid chromatography–mass spectrometry (unit 10.21)

Support Protocol 1: Modified Lowry Assay for Lipoprotein Determination

  Materials
  • Lowry reagents A and B (see recipe)
  • Folin & Ciocalteau's 2N phenol reagent (e.g., Sigma)
  • 1 mg/ml bovine serum albumin (BSA) standard solution (store at 4°C)
  • 2 N NaOH
  • Spectrophotometer
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Figures

Videos

Literature Cited

Literature Cited
   Alaupovic, P. 1991. Apolipoprotein composition as the basis for classifying plasma lipoproteins: Characterization of Apo A– and Apo B–containing lipoprotein families. Prog. Lipid Res. 30:105‐138.
   Assmann, G. and Gotto, A.M. Jr. 2004. HDL cholesterol and protective factors in atherosclerosis. Circulation 109:III8‐III14.
   Brousseau, T., Clavey, V., Bard, J.M., and Fruchart, J.C. 1993. Sequential ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins, and lipoprotein particles. Clin. Chem. 39:960‐964.
   Carr, T.P., Andresen, C.J., and Rudel, L.L. 1993. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts. Clin. Biochem. 26:39‐42.
   De Lalla, O. and Gofman, J.W. 1954. Ultracentrifugation as a technique for separation of lipoprotein fractions. In Methods in Biochemical Analysis (D. Glick, ed.) pp. 459‐478. Wiley Interscience, New York.
   Einarsson, K. and Grundy, S.M. 1980. Effects of feeding cholic acid and chenodeoxycholic acid on cholesterol absorption and hepatic secretion of biliary lipids in man. J. Lipid Res. 21:23‐34.
   Folch, J., Ascoli, I., Lees, M., Meath, J.A., and LeBaron, N. 1951. Preparation of lipid extracts from brain tissue. J. Biol. Chem. 191:833‐841.
   Grundy, S.M. 2004. Obesity, metabolic syndrome, and cardiovascular disease. J. Clin. Endocrinol. Metab. 89:2595‐2600.
   Houten, S.M. and Auwerx, J. 2004. The enterohepatic nuclear receptors are major regulators of the enterohepatic circulation of bile salts. Ann. Med. 36:482‐491.
   Houten, S.M., Watanabe, M., and Auwerx, J. 2006. Endocrine functions of bile acids. EMBO J. 25:1419‐1425.
   Lada, A.T. and Rudel, L.L. 2004. Associations of low density lipoprotein particle composition with atherogenicity. Curr. Opin. Lipidol. 15:19‐24.
   Lee, C., Olson, P., and Evans, R.M. 2003. Minireview: Lipid metabolism, metabolic diseases, and peroxisome proliferator‐activated receptors. Endocrinology 144:2201‐2207.
   Markwell, M.A.K., Haas, S.M., Bieber, L.L., and Tolbert, N.E. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206‐210.
   Mashige, F., Osuga, T., Tanaka, N., Imai, K., and Yamanaka, M. 1978. Continuous‐flow determination of bile acids in serum, and its clinical application. Clin. Chem. 24:1150‐1154.
   Schaefer, E.J., Eisenberg, S., and Levy, R.I. 1978. Lipoprotein apoprotein metabolism. J. Lipid Res. 19:667‐687.
   Setchell, K.D. and Matsui, A. 1983. Serum bile acid analysis. Clin. Chim. Acta 127:1‐17.
   Shepherd, J. and Packard, C.J. 1989. Lipoprotein metabolism. In Human Plasma Lipoproteins (J.C. Fruchart and J. Shepherd, eds.) pp. 55‐78. Walter de Gruyter, New York.
   Tall, A. 1995. Plasma lipid transfer proteins. Annu. Rev. Biochem. 64:235‐257.
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