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Particle size measurement of lipoprotein fractions using diffusion-ordered NMR spectroscopy

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Abstract

The sizes of certain types of lipoprotein particles have been associated with an increased risk of cardiovascular disease. However, there is currently no gold standard technique for the determination of this parameter. Here, we propose an analytical procedure to measure lipoprotein particles sizes using diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY). The method was tested on six lipoprotein fractions, VLDL, IDL, LDL1, LDL2, HDL2, and HDL3, which were obtained by sequential ultracentrifugation from four patients. We performed a pulsed-field gradient experiment on each fraction to obtain a mean diffusion coefficient, and then determined the apparent hydrodynamic radius using the Stokes–Einstein equation. To validate the hydrodynamic radii obtained, the particle size distribution of these lipoprotein fractions was also measured using transmission electron microscopy (TEM). The standard errors of duplicate measurements of diffusion coefficient ranged from 0.5% to 1.3%, confirming the repeatability of the technique. The coefficient of determination between the hydrodynamic radii and the TEM-derived mean particle size was r 2 = 0.96, and the agreement between the two techniques was 85%. Thus, DOSY experiments have proved to be accurate and reliable for estimating lipoprotein particle sizes.

 

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Abbreviations

CVD:

Cardiovascular disease

DOSY:

Diffusion-ordered NMR spectroscopy

DSTE:

Double-stimulated echo

GGE:

Gradient gel electrophoresis

HDL:

High-density lipoprotein

IDL:

Intermediate density lipoprotein

LDL:

Low-density lipoprotein

LED:

Longitudinal eddy current delay

LS:

Light scattering

NMR:

Nuclear magnetic resonance

PFG:

Pulsed-field gradient

RMSPE:

Root mean squared percentage error

sdLDL:

Small, dense LDL

SE:

Standard error

SNR:

Signal-to-noise ratio

TEM:

Transmission electron microscopy

TSP:

3-Trimethylsilyl[2,2,3,3-d4]propionate

VLDL:

Very low-density lipoprotein

References

  1. Krauss RM (2010) Lipoprotein subfractions and cardiovascular disease risk. Curr Opin Lipidol 21:305–311

    Article  CAS  Google Scholar 

  2. Berneis KK, Krauss RM (2002) Metabolic origins and clinical significance of LDL heterogeneity. J Lipid Res 43:1363–1379

    Article  CAS  Google Scholar 

  3. Musunuru K, Orho-Melander M, Caulfield MP, Li SG, Salameh WA, Reitz RE, Berglund G, Hedblad B, Engstrom G, Williams PT, Kathiresan S, Melander O, Krauss RM (2009) Ion mobility analysis of lipoprotein subfractions identifies three independent axes of cardiovascular risk. Arterioscler Thromb Vasc Biol 29:1975–U1628

    Article  CAS  Google Scholar 

  4. Campos H, Genest JJ, Blijlevens E, McNamara JR, Jenner JL, Ordovas JM, Wilson PWF, Schaefer EJ (1992) Low-density-lipoprotein particle-size and coronary-artery disease. Arterioscler Thromb 12:187–195

    Article  CAS  Google Scholar 

  5. Coresh J, Kwiterovich PO, Smith HH, Bachorik PS (1993) Association of plasma triglyceride concentration and LDL particle diameter, density, and chemical-composition with premature coronary-artery disease in men and women. J Lipid Res 34:1687–1697

    CAS  Google Scholar 

  6. Roheim PS, Asztalos BF (1995) Clinical-significance of lipoprotein size and risk for coronary atherosclerosis. Clin Chem 41:147–152

    CAS  Google Scholar 

  7. Krauss RM (1995) Dense low-density lipoproteins and coronary-artery disease. Am J Cardiol 75:B53–B57

    Article  Google Scholar 

  8. Glomset JA (1968) Plasma lecithin—cholesterol acyltransferase reaction. J Lipid Res 9:155–167

    CAS  Google Scholar 

  9. Johnson WJ, Mahlberg FH, Rothblat GH, Phillips MC (1991) Cholesterol transport between cells and high-density-lipoproteins. Biochim Biophys Acta 1085:273–298

    CAS  Google Scholar 

  10. Stampfer MJ, Sacks FM, Salvini S, Willett WC, Hennekens CH (1991) A prospective-study of cholesterol, apolipoproteins, and the risk of myocardial-infarction. N Engl J Med 325:373–381

    Article  CAS  Google Scholar 

  11. Stampfer MJ, Krauss RM, Ma J, Blanche PJ, Holl LG, Sacks FM, Hennekens CH (1996) A prospective study of triglyceride level, low-density lipoprotein particle diameter, and risk of myocardial infarction. Jama-J Am Med Assoc 276:882–888

    Article  CAS  Google Scholar 

  12. Sacks FM, Campos H (2003) Clinical review 163—cardiovascular endocrinology 4—low-density lipoprotein size and cardiovascular disease: a reappraisal. J Clin Endocrinol Metab 88:4525–4532

    Article  CAS  Google Scholar 

  13. Mora S, Otvos JD, Rifai N, Rosenson RS, Buring JE, Ridker PM (2009) Lipoprotein particle profiles by nuclear magnetic resonance compared with standard lipids and apolipoproteins in predicting incident cardiovascular disease in women. Circulation 119:931–U944

    Article  CAS  Google Scholar 

  14. Kulkarni KR, Garber DW, Marcovina SM, Segrest JP (1994) Quantification of cholesterol in all lipoprotein classes by the VAP-II method. J Lipid Res 35:159–168

    CAS  Google Scholar 

  15. Jeyarajah EJ, Cromwell WC, Otvos JD (2006) Lipoprotein particle analysis by nuclear magnetic resonance spectroscopy. Clin Lab Med 26:847–870

    Article  Google Scholar 

  16. Krauss RM, Burke DJ (1982) Identification of multiple subclasses of plasma low-density lipoproteins in normal humans. J Lipid Res 23:97–104

    CAS  Google Scholar 

  17. Hoefner DM, Hodel SD, O’Brien JF, Branum EL, Sun D, Meissner I, McConnell JP (2001) Development of a rapid, quantitative method for LDL subfractionation with use of the Quantimetrix Lipoprint LDL System. Clin Chem 47:266–274

    CAS  Google Scholar 

  18. Witte DR, Taskinen MR, Perttunen-Nio H, van Tol A, Livingstone S, Colhoun HM (2004) Study of agreement between LDL size as measured by nuclear magnetic resonance and gradient gel electrophoresis. J Lipid Res 45:1069–1076

    Article  CAS  Google Scholar 

  19. Ensign W, Hill N, Heward CB (2006) Disparate LDL phenotypic classification among 4 different methods assessing LDL particle characteristics. Clin Chem 52:1722–1727

    Article  CAS  Google Scholar 

  20. McNamara JR, Warnick GR, Cooper GR (2006) A brief history of lipid and lipoprotein measurements and their contribution to clinical chemistry. Clin Chim Acta 369:158–167

    Article  CAS  Google Scholar 

  21. Chung M, Lichtenstein AH, Ip S, Lau J, Balk EM (2009) Comparability of methods for LDL subfraction determination: a systematic review. Atherosclerosis 205:342–348

    Article  CAS  Google Scholar 

  22. Mora S (2009) Advanced lipoprotein testing and subfractionation are not (yet) ready for routine clinical use. Circulation 119:2396–2404

    Article  Google Scholar 

  23. Rosenson RS, Brewer HB, Chapman MJ, Fazio S, Hussain MM, Kontush A, Krauss RM, Otvos JD, Remaley AT, Schaefer EJ (2011) HDL measures, particle heterogeneity, proposed nomenclature, and relation to atherosclerotic cardiovascular events. Clin Chem 57:392–410

    Article  CAS  Google Scholar 

  24. Johnson CS (1999) Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications. Prog Nucl Magn Reson Spectrosc 34:203–256

    Article  CAS  Google Scholar 

  25. Antalek B (2002) Using pulsed gradient spin echo NMR for chemical mixture analysis: how to obtain optimum results. Concepts Magn Reson 14:225–258

    Article  CAS  Google Scholar 

  26. Morris GA (2007) Diffusion-ordered spectroscopy (DOSY). Wiley, New York

    Google Scholar 

  27. Hinton DP, Johnson CS (1993) Diffusion ordered 2D-NMR spectroscopy of phospholipid-vesicles—determination of vesicle size distributions. J Phys Chem 97:9064–9072

    Article  CAS  Google Scholar 

  28. Canzi G, Mrse AA, Kubiak CP (2011) Diffusion-ordered NMR spectroscopy as a reliable alternative to TEM for determining the size of gold nanoparticles in organic solutions. J Phys Chem C 115:7972–7978

    Article  CAS  Google Scholar 

  29. Squires TM, Mason TG (2010) Fluid mechanics of microrheology. Annu Rev Fluid Mech 42:413–438

    Article  Google Scholar 

  30. Forte TM, Nordhausen RW (1986) Electron-microscopy of negatively stained lipoproteins. Methods Enzymol 128:442–457

    Article  CAS  Google Scholar 

  31. Schumaker VN, Puppione DL (1986) Sequential flotation ultracentrifugation. Methods Enzymol 128:155–170

    Article  CAS  Google Scholar 

  32. Rumsey SC, Galeano NF, Arad Y, Deckelbaum RJ (1992) Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low-density lipoproteins. J Lipid Res 33:1551–1561

    CAS  Google Scholar 

  33. Jerschow A, Muller N (1997) Suppression of convection artifacts in stimulated-echo diffusion experiments. Double-stimulated-echo experiments. J Magn Reson 125:372–375

    Article  CAS  Google Scholar 

  34. Mallol R, Rodríguez M, Heras M, Vinaixa M, Cañellas N, Brezmes J, Plana N, Masana L, Correig X (2011) Surface fitting of 2D diffusion-edited 1H NMR spectroscopy data for the characterisation of human plasma lipoproteins. Metabolomics 7:572–582

    Article  CAS  Google Scholar 

  35. Duell PB, Illingworth DR, Connor WE (2001) Endocrinology and metabolism, 4th edn. McGraw-Hill, New York

    Google Scholar 

  36. Chen A, Wu DH, Johnson CS (1995) Determination of molecular-weight distributions for polymers by diffusion-ordered NMR. J Am Chem Soc 117:7965–7970

    Article  CAS  Google Scholar 

  37. O’Neal D, Harrip P, Dragicevic G, Rae D, Best JD (1998) A comparison of LDL size determination using gradient gel electrophoresis and light-scattering methods. J Lipid Res 39:2086–2090

    Google Scholar 

  38. Sakurai T, Trirongjitmoah S, Nishibata Y, Namita T, Tsuji M, Hui SP, Jin S, Shimizu K, Chiba H (2010) Measurement of lipoprotein particle sizes using dynamic light scattering. Ann Clin Biochem 47:476–481

    Article  CAS  Google Scholar 

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Acknowledgments

We acknowledge CIBER de Diabetes y Enfermedades Metabólicas Asociadas (ISCIII, Ministerio de Ciencia e Innovación), for partially funding this work, as well as the FIS (project PI 081409). This work was partly supported by the Engineering and Physical Sciences Research Council (Grant Numbers EP/H024336/1 and EP/I007989/1). We also acknowledge Dr. M. Moncusí and Dr. R. Marimon for their assistance with the TEM analysis of the lipoprotein fractions as well as Dr. S. Pujol for her assistance with viscosity measurements.

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Authors and Affiliations

  1. Department of Electronic Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain

    Roger Mallol, Maria Vinaixa & Xavier Correig

  2. Metabolomics Platform, IISPV, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain

    Roger Mallol, Miguel A. Rodríguez, Maria Vinaixa & Xavier Correig

  3. CIBERDEM, Carrer Mallorca 183, 08036, Barcelona, Spain

    Roger Mallol, Miguel A. Rodríguez, Mercedes Heras, Maria Vinaixa, Núria Plana, Lluís Masana & Xavier Correig

  4. Lipids and Atherosclerosis Research Unit, Sant Joan University Hospital, IISPV, Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201, Reus, Spain

    Mercedes Heras, Núria Plana & Lluís Masana

  5. School of Chemistry, University of Manchester, Manchester, M13 9PL, UK

    Gareth A. Morris

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  1. Roger Mallol
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Correspondence to Roger Mallol.

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Mallol, R., Rodríguez, M.A., Heras, M. et al. Particle size measurement of lipoprotein fractions using diffusion-ordered NMR spectroscopy. Anal Bioanal Chem 402, 2407–2415 (2012). https://doi.org/10.1007/s00216-011-5705-9

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