Skip to main content
SpringerLink
Log in

Development of a Method to Measure preβHDL and αHDL apoA-I Enrichment for Stable Isotopic Studies of HDL Kinetics

  • Methods
  • Published:
Lipids

Abstract

Our understanding of HDL metabolism would be enhanced by the measurement of the kinetics of preβHDL, the nascent form of HDL, since elevated levels have been reported in patients with coronary artery disease. Stable isotope methodology is an established technique that has enabled the determination of the kinetics (production and catabolism) of total HDL apoA-I in vivo. The development of separation procedures to obtain a preβHDL fraction, the isotopic enrichment of which could then be measured, would enable further understanding of the pathways in vivo for determining the fate of preβHDL and the formation of αHDL. A method was developed and optimised to separate and measure preβHDL and αHDL apoA-I enrichment. Agarose gel electrophoresis was first used to separate lipoprotein subclasses, and then a 4–10 % discontinuous SDS-PAGE used to isolate apoA-I. Measures of preβHDL enrichment in six healthy subjects were undertaken following an infusion of l-[1-13C-leucine]. After isolation of preβ and αHDL, the isotopic enrichment of apoA-I for each fraction was measured by gas chromatography–mass spectrometry. PreβHDL apoA-I enrichment was measured with a CV of 0.51 % and αHDL apoA-I with a CV of 0.34 %. The fractional catabolic rate (FCR) of preβHDL apoA-I was significantly higher than the FCR of αHDL apoA-I (p < 0.005). This methodology can be used to selectively isolate preβ and αHDL apoA-I for the measurement of apoA-I isotopic enrichment for kinetics studies of HDL subclass metabolism in a research setting.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

2-DE:

2-Dimensional electrophoresis

α-KIC:

α-Ketoisocaproate

αHDL:

Alpha High density lipoprotein

AP:

Alkaline phosphatase

APE:

Atom percent excess

ApoA-I:

Apolipoprotein A-I

CAD:

Coronary artery disease

CETP:

Cholesterol ester transfer protein

FCR:

Fractional catabolic rate

FPLC:

Gel filtration fast protein liquid chromatography

FSR:

Fractional secretion rate

GC–MS:

Gas chromatography–mass spectrometry

HDL:

High density lipoprotein

HDL-C:

High density lipoprotein cholesterol

LCAT:

Lecithin-cholesterol acyltransferase

m/z :

Mass to charge ratio

PR:

Production rate

preβHDL:

Prebeta high-density lipoprotein

SDS-PAGE:

Sodium dodecylsulfate polyacrylamide gel electrophoresis

TAG:

Triacylglycerol

References

  1. Barter PJ (2000) Hugh sinclair lecture: the regulation and remodelling of HDL by plasma factors. Atheroscler Suppl 4(3):39–47

    Google Scholar 

  2. Rye KA, Barter PJ (2004) Formation and metabolism of prebeta-migrating, lipid-poor apolipoprotein A-I. Arterioscler Thromb Vasc Biol 24:421–428

    Article  PubMed  CAS  Google Scholar 

  3. Huang Y, von Eckardstein A, Wu S, Maeda N, Assmann G (1994) A plasma lipoprotein containing only apolipoprotein E and with gamma mobility on electrophoresis releases cholesterol from cells. Proc Natl Acad Sci USA 91:1834–1838

    Article  PubMed  CAS  Google Scholar 

  4. Miida T, Sakai K, Ozaki K, Nakamura Y, Yamaguchi T, Tsuda T, Kashiwa T, Murakami T, Inano K, Okada M (2000) Bezafibrate increases prebeta 1-HDL at the expense of HDL2b in hypertriglyceridemia. Arterioscler Thromb Vasc Biol 20:2428–2433

    Article  PubMed  CAS  Google Scholar 

  5. Moyad MA, Merrick GS (2007) Cholesterol, cholesterol-lowering agents/statins, and urologic disease: part VI–the recent rise and fall of the HDL-boosting drug torceptrapib. Urol Nurs 27:169–173

    PubMed  Google Scholar 

  6. Asztalos BF, Roheim PS, Milani RL, Lefevre M, McNamara JR, Horvath KV, Schaefer EJ (2000) Distribution of ApoA-I-containing HDL subpopulations in patients with coronary heart disease. Arterioscler Thromb Vasc Biol 20:2670–2676

    Article  PubMed  CAS  Google Scholar 

  7. Cheung MC, Brown BG, Wolf AC, Albers JJ (1991) Altered particle size distribution of apolipoprotein A-I-containing lipoproteins in subjects with coronary artery disease. J Lipid Res 32:383–394

    PubMed  CAS  Google Scholar 

  8. Xu Y, Fu M (2003) Alterations of HDL subclasses in hyperlipidemia. Clin Chim Acta 332:95–102

    Article  PubMed  CAS  Google Scholar 

  9. Miida T, Yamaguchi T, Tsuda T, Okada M (1998) High prebeta1-HDL levels in hypercholesterolemia are maintained by probucol but reduced by a low-cholesterol diet. Atherosclerosis 138:129–134

    Article  PubMed  CAS  Google Scholar 

  10. Yang Y, Yan B, Fu M, Xu Y, Tian Y (2005) Relationship between plasma lipid concentrations and HDL subclasses. Clin Chim Acta 354:49–58

    Article  PubMed  CAS  Google Scholar 

  11. Cohn JS, Wagner DA, Cohn SD, Millar JS, Schaefer EJ (1990) Measurement of very low density and low density lipoprotein apolipoprotein (Apo) B-100 and high density lipoprotein Apo A-I production in human subjects using deuterated leucine. Effect of fasting and feeding. J Clin Invest 85(3):804–811

    Article  PubMed  CAS  Google Scholar 

  12. Ikewaki K, Rader DJ, Schaefer JR, Fairwell T, Zech LA, Brewer HB Jr (1993) Evaluation of apoA-I kinetics in humans using simultaneous endogenous stable isotope and exogenous radiotracer methods. J Lipid Res 34:2207–2215

    PubMed  CAS  Google Scholar 

  13. Kunitake ST, Kane JP (1982) Factors affecting the integrity of high density lipoproteins in the ultracentrifuge. J Lipid Res 23:936–940

    PubMed  CAS  Google Scholar 

  14. Cheung MC, Wolf AC (1988) Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations. J Lipid Res 29:15–25

    PubMed  CAS  Google Scholar 

  15. Cheung MC, Albers JJ (1979) Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios. J Lipid Res 20:200–207

    PubMed  CAS  Google Scholar 

  16. Dwyer KP, Barrett PH, Chan D, Foo JI, Watts GF, Croft KD (2002) Oxazolinone derivative of leucine for GC-MS: a sensitive and robust method for stable isotope kinetic studies of lipoproteins. J Lipid Res 43:344–349

    PubMed  CAS  Google Scholar 

  17. Ford GC, Cheng KN, Halliday D (1985) Analysis of (1–13C)leucine and (13C)KIC in plasma by capillary gas chromatography/mass spectrometry in protein turnover studies. Biomed Mass Spectrom 12:432–436

    Article  PubMed  CAS  Google Scholar 

  18. Ishida BY, Frolich J, Fielding CJ (1987) Prebeta-migrating high density lipoprotein: quantitation in normal and hyperlipidemic plasma by solid phase radioimmunoassay following electrophoretic transfer. J Lipid Res 28:778–786

    PubMed  CAS  Google Scholar 

  19. Ramakrishnan R (2006) Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments. J Lipid Res 47:2738–2753

    Article  PubMed  CAS  Google Scholar 

  20. Umpleby AM, Das S, Stolinski M, Shojaee-Moradie F, Jackson NC, Jefferson W, Crabtree N, Nightingale P, Shahmanesh M (2005) Low density lipoprotein apolipoprotein B metabolism in treatment-naive HIV patients and patients on antiretroviral therapy. Antivir Ther 10:663–670

    PubMed  CAS  Google Scholar 

  21. Pearson TC, Guthrie DL, Simpson J, Chinn S, Barosi G, Ferrant A, Lewis SM, Najean Y (1995) Interpretation of measured red cell mass and plasma volume in adults: Expert Panel on Radionuclides of the International Council for Standardization in Haematology. Br J Haematol 89:748–756

    Article  PubMed  CAS  Google Scholar 

  22. Asztalos BF, Sloop CH, Wong L, Roheim PS (1993) Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations. Biochim Biophys Acta 41(1169):291–300

    Google Scholar 

  23. Wieland H, Seidel D (1973) Improved techniques for assessment of serum lipoprotein patterns. II. Rapid method for diagnosis of type III hyperlipoproteinemia without ultracentrifugation. Clin Chem 19:1139–1141

    CAS  Google Scholar 

  24. Chétiveaux M, Ouguerram K, Zair Y, Maugère P, Falconi I, Nazih H, Krempf M (2004) New model for kinetic studies of HDL metabolism in humans. Eur J Clin Invest 34(4):262–267

    Article  PubMed  Google Scholar 

  25. Collins P (2008) HDL-C in post-menopausal women: an important therapeutic target. Int J Cardiol 124(3):275–282

    Article  PubMed  Google Scholar 

  26. Nakamura Y, Kotite L, Gan Y, Spencer TA, Fielding CJ, Fielding PE (2004) Molecular mechanism of reverse cholesterol transport: reaction of pre-beta-migrating high-density lipoprotein with plasma lecithin/cholesterol acyltransferase. Biochemistry 43(46):14811–14820

    Article  PubMed  CAS  Google Scholar 

  27. Castro GR, Fielding CJ (1988) Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein. Biochemistry 27(1):25–29

    Article  PubMed  CAS  Google Scholar 

  28. Schaefer EJ, Zech LA, Jenkins LL, Bronzert TJ, Rubalcaba EA, Lindgren FT, Aamodt RL, Brewer HB Jr (1982) Human apolipoprotein A-I and A-II metabolism. J Lipid Res 23:850–862

    PubMed  CAS  Google Scholar 

  29. Fisher WR, Venkatakrishnan V, Zech LA, Hall CM, Kilgore LL, Stacpoole PW, Diffenderfer MR, Friday KE, Sumner AE, Marsh JB (1995) Kinetic evidence for both a fast and a slow secretory pathway for apolipoprotein A-I in humans. J Lipid Res 36:1618–1628

    PubMed  CAS  Google Scholar 

  30. Chan DC, Barrett PH, Watts GF (2004) Lipoprotein transport in the metabolic syndrome: methodological aspects of stable isotope kinetic studies. Clin Sci (Lond) 107(3):221–232

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Xuefei Li was the recipient of a University of Surrey PhD scholarship. We are grateful to Mrs J. Batt for her technical assistance.

Author information

Authors and Affiliations

  1. Diabetes and Metabolic Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK

    Xuefei Li & A. Margot Umpleby

  2. School of Life Sciences, Faculty of Science, Engineering and Computing, Kingston University, London, UK

    Michael Stolinski

Authors
  1. Xuefei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Stolinski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Margot Umpleby
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xuefei Li.

About this article

Cite this article

Li, X., Stolinski, M. & Umpleby, A.M. Development of a Method to Measure preβHDL and αHDL apoA-I Enrichment for Stable Isotopic Studies of HDL Kinetics. Lipids 47, 1011–1018 (2012). https://doi.org/10.1007/s11745-012-3703-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-012-3703-0

Keywords

Search

Navigation