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.
Similar content being viewed by others
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
Krauss RM (2010) Lipoprotein subfractions and cardiovascular disease risk. Curr Opin Lipidol 21:305–311
Berneis KK, Krauss RM (2002) Metabolic origins and clinical significance of LDL heterogeneity. J Lipid Res 43:1363–1379
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
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
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
Roheim PS, Asztalos BF (1995) Clinical-significance of lipoprotein size and risk for coronary atherosclerosis. Clin Chem 41:147–152
Krauss RM (1995) Dense low-density lipoproteins and coronary-artery disease. Am J Cardiol 75:B53–B57
Glomset JA (1968) Plasma lecithin—cholesterol acyltransferase reaction. J Lipid Res 9:155–167
Johnson WJ, Mahlberg FH, Rothblat GH, Phillips MC (1991) Cholesterol transport between cells and high-density-lipoproteins. Biochim Biophys Acta 1085:273–298
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
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
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
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
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
Jeyarajah EJ, Cromwell WC, Otvos JD (2006) Lipoprotein particle analysis by nuclear magnetic resonance spectroscopy. Clin Lab Med 26:847–870
Krauss RM, Burke DJ (1982) Identification of multiple subclasses of plasma low-density lipoproteins in normal humans. J Lipid Res 23:97–104
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
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
Ensign W, Hill N, Heward CB (2006) Disparate LDL phenotypic classification among 4 different methods assessing LDL particle characteristics. Clin Chem 52:1722–1727
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
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
Mora S (2009) Advanced lipoprotein testing and subfractionation are not (yet) ready for routine clinical use. Circulation 119:2396–2404
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
Johnson CS (1999) Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications. Prog Nucl Magn Reson Spectrosc 34:203–256
Antalek B (2002) Using pulsed gradient spin echo NMR for chemical mixture analysis: how to obtain optimum results. Concepts Magn Reson 14:225–258
Morris GA (2007) Diffusion-ordered spectroscopy (DOSY). Wiley, New York
Hinton DP, Johnson CS (1993) Diffusion ordered 2D-NMR spectroscopy of phospholipid-vesicles—determination of vesicle size distributions. J Phys Chem 97:9064–9072
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
Squires TM, Mason TG (2010) Fluid mechanics of microrheology. Annu Rev Fluid Mech 42:413–438
Forte TM, Nordhausen RW (1986) Electron-microscopy of negatively stained lipoproteins. Methods Enzymol 128:442–457
Schumaker VN, Puppione DL (1986) Sequential flotation ultracentrifugation. Methods Enzymol 128:155–170
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
Jerschow A, Muller N (1997) Suppression of convection artifacts in stimulated-echo diffusion experiments. Double-stimulated-echo experiments. J Magn Reson 125:372–375
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
Duell PB, Illingworth DR, Connor WE (2001) Endocrinology and metabolism, 4th edn. McGraw-Hill, New York
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
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
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
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.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 878 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-011-5705-9