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Journal of Lipid Research, Vol. 19, 65-76, January 1978
Copyright © 1978 by Lipid Research, Inc.

A comprehensive evaluation of the heparin - manganese precipitation procedure for estimating high density lipoprotein cholesterol

G. Russell Warnick and John J. Albers

Northwest Lipid Research Clinic, Harborview Medical Center, University of Washington School of Medicine, Seattle, WA 98104

The accurate quantitation of high density lipoproteins has recently assumed greater importance in view of studies suggesting their negative correlation with coronary heart disease. High density lipoproteins may be estimated by measuring cholesterol in the plasma fraction of d > 1.063 g/ml. A more practical approach is the specific precipitation of apolipoprotein B (apoB)-containing lipoproteins by sulfated polysaccharides and divalent cations, heparin-Mn²⁺ being the most commonly used combination. The present heparin-Mn²⁺ procedure was found to be reasonably specific and not often subject to large errors; however, 9% (primarily hypertriglyceridemic samples) of the 966 plasma samples treated with heparin-Mn²⁺ had obvious supernatant turbidity, indicating incomplete sedimentation of apoB-associated lipoproteins. Furthermore, 48% of the nonturbid supernates contained more than 1 mg/dl (mean 2.5 mg/dl) of apoB-associated cholesterol when measured by a radial immunodiffusion procedure, indicating slight overestimation of HDL cholesterol. Determination of the extent of the unprecipitated apoB-associated lipoproteins by sensitive radioimmunoassay and of the amount of precipitated high density lipoprotein by radial immunodiffusion assay of apolipoproteins A-I and A-II at various heparin and Mn²⁺ concentrations indicated that the usual heparin level (approximately 1.3 mg/ml) was adequate. However, a twofold increase in Mn²⁺ concentration to 0.092 M improved precipitation of the apoB-associated lipoproteins without excessive precipitation of high density lipoprotein from plasma. This increased Mn²⁺ level also provided improved sedimentation of the apoB-associated lipoproteins from hypertriglyceridemic plasma. Additional observations suggested that, for convenience, the heparin and Mn²⁺ can be added simultaneously as a combined reagent, that samples can be incubated for 10 minutes at room temperature before centrifugation, and that turbid supernates from hypertriglyceridemic samples can usually be made free of apoB-associated lipoproteins by centrifugation at 12,000 g for 10 minutes.

Supplementary key words coronary heart disease • immunoassay • lipoprotein quantitation

Submitted on February 22, 1977
Accepted on July 5, 1977

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				S. F. Crouse, B. C. O'Brien, P. W. Grandjean, R. C. Lowe, J. J. Rohack, and J. S. Green Effects of training and a single session of exercise on lipids and apolipoproteins in hypercholesterolemic men J Appl Physiol, December 1, 1997; 83(6): 2019 - 2028. [Abstract] [Full Text] [PDF]

				T. A. B. Sanders, F. R. Oakley, G. J. Miller, K. A. Mitropoulos, D. Crook, and M. F. Oliver Influence of n-6 versus n-3 Polyunsaturated Fatty Acids in Diets Low in Saturated Fatty Acids on Plasma Lipoproteins and Hemostatic Factors Arterioscler. Thromb. Vasc. Biol., December 1, 1997; 17(12): 3449 - 3460. [Abstract] [Full Text]

				T. Dwyer, H. Iwane, K. Dean, Y. Odagiri, T. Shimomitsu, L. Blizzard, S. Srinivasan, T. Nicklas, W. Wattigney, M. Riley, et al. Differences in HDL Cholesterol Concentrations in Japanese, American, and Australian Children Circulation, November 4, 1997; 96(9): 2830 - 2836. [Abstract] [Full Text]

				R Munro, E Morrison, A G McDonald, J A Hunter, R Madhok, and H A Capell Effect of disease modifying agents on the lipid profiles of patients with rheumatoid arthritis Ann Rheum Dis, June 1, 1997; 56(6): 374 - 377. [Abstract] [Full Text]

				N. Harris, V. Galpchian, J. Thomas, E. Iannotti, T. Law, and N. Rifai Three generations of high-density lipoprotein cholesterol assays compared with ultracentrifugation/dextran sulfate–Mg2+ method Clin. Chem., May 1, 1997; 43(5): 816 - 823. [Abstract] [Full Text] [PDF]

				H. C. Lassila, K. S. Tyrrell, K. A. Matthews, S. K. Wolfson, and L. H. Kuller Prevalence and Determinants of Carotid Atherosclerosis in Healthy Postmenopausal Women Stroke, March 1, 1997; 28(3): 513 - 517. [Abstract] [Full Text]

				S. F. Crouse, B. C. O'Brien, P. W. Grandjean, R. C. Lowe, J. J. Rohack, J. S. Green, and H. Tolson Training intensity, blood lipids, and apolipoproteins in men with high cholesterol J Appl Physiol, January 1, 1997; 82(1): 270 - 277. [Abstract] [Full Text] [PDF]

				J. L. Isaacsohn, R. G. Bakker-Arkema, R. Fayyad, R. Whitcomb, and D. M. Black Atorvastatin, a New HMG-CoA Reductase Inhibitor, Does Not Affect Glucocorticoid Hormones in Patients with Hypercholesterolemia Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 1997; 2(4): 243 - 249. [Abstract] [PDF]

				S. T. Davidge, A. P. Signorella, C. A. Hubel, D. L. Lykins, and J. M. Roberts Distinct Factors in Plasma of Preeclamptic Women Increase Endothelial Nitric Oxide or Prostacyclin Hypertension, November 1, 1996; 28(5): 758 - 764. [Abstract] [Full Text]

				M. Sugano and N. Makino Changes in Plasma Lipoprotein Cholesterol Levels by Antisense Oligodeoxynucleotides against Cholesteryl Ester Transfer Protein in Cholesterol-fed Rabbits J. Biol. Chem., August 9, 1996; 271(32): 19080 - 19083. [Abstract] [Full Text] [PDF]

				K. Handa, M. Takao, J. Nomoto, K. Oku, K. Shirai, K. Saku, and K. Arakawa Evaluation of the Coagulation and Fibrinolytic Systems in Men with Intermittent Claudication Angiology, June 1, 1996; 47(6): 543 - 548. [Abstract] [PDF]

				V. Hachinski, C. Graffagnino, M. Beaudry, G. Bernier, C. Buck, A. Donner, J. D. Spence, G. Doig, and B. M. J. Wolfe Lipids and Stroke: A Paradox Resolved Arch Neurol, April 1, 1996; 53(4): 303 - 308. [Abstract] [PDF]

				D.R. Illingworth, E.A. Stein, R.H. Knopp, D.B. Hunninghake, M.H. Davidson, C.A. Dujovne, V.T. Miller, J.A. Tobert, P.M. Laskarzewski, J.L. Isaacsohn, et al. A Randomized Multicenter Trial Comparing the Efficacy of Simvastatin and Fluvastatin Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 1996; 1(1): 23 - 29. [Abstract] [PDF]

				R. R. Wing, R. W. Jeffery, and W. L. Hellerstedt A Prospective Study of Effects of Weight Cycling on Cardiovascular Risk Factors Arch Intern Med, July 10, 1995; 155(13): 1416 - 1422. [Abstract] [PDF]

				E. Talbott, D. Guzick, A. Clerici, S. Berga, K. Detre, K. Weimer, and L. Kuller Coronary Heart Disease Risk Factors in Women With Polycystic Ovary Syndrome Arterioscler. Thromb. Vasc. Biol., July 1, 1995; 15(7): 821 - 826. [Abstract] [Full Text]

				E. T. Stevenson, K. P. Davy, and D. R. Seals Hemostatic, Metabolic, and Androgenic Risk Factors for Coronary Heart Disease in Physically Active and Less Active Postmenopausal Women Arterioscler. Thromb. Vasc. Biol., May 1, 1995; 15(5): 669 - 677. [Abstract] [Full Text]

				F. Tato, G. L. Vega, and S. M. Grundy Bimodal Distribution of Cholesteryl Ester Transfer Protein Activities in Normotriglyceridemic Men With Low HDL Cholesterol Concentrations Arterioscler. Thromb. Vasc. Biol., April 1, 1995; 15(4): 446 - 451. [Abstract] [Full Text]

				M. F. Muldoon, T. B. Herbert, S. M. Patterson, M. Kameneva, R. Raible, and S. B. Manuck Effects of Acute Psychological Stress on Serum Lipid Levels, Hemoconcentration, and Blood Viscosity Arch Intern Med, March 27, 1995; 155(6): 615 - 620. [Abstract] [PDF]

				F. Tato, G. L. Vega, A. R. Tall, and S. M. Grundy Relation Between Cholesterol Ester Transfer Protein Activities and Lipoprotein Cholesterol in Patients With Hypercholesterolemia and Combined Hyperlipidemia Arterioscler. Thromb. Vasc. Biol., January 1, 1995; 15(1): 112 - 120. [Abstract] [Full Text]

				W. Insull Jr, D. Black, C. Dujovne, J. D. Hosking, D. Hunninghake, L. Keilson, R. Knopp, J. McKenney, E. Stein, A. J. Troendle, et al. Efficacy and Safety of Once-Daily vs Twice-Daily Dosing With Fluvastatin, a Synthetic Reductase Inhibitor, in Primary Hypercholesterolemia Arch Intern Med, November 14, 1994; 154(21): 2449 - 2455. [Abstract] [PDF]

				K. A. Matthews, R. R. Wing, L. H. Kuller, E. N. Meilahn, and P. Plantinga Influence of the Perimenopause on Cardiovascular Risk Factors and Symptoms of Middle-aged Healthy Women Arch Intern Med, October 24, 1994; 154(20): 2349 - 2355. [Abstract] [PDF]

				D. R. Illingworth, E. A. Stein, Y. B. Mitchel, C. A. Dujovne, P. H. Frost, R. H. Knopp, P. Tun, R. V. Zupkis, and R. A. Greguski Comparative Effects of Lovastatin and Niacin in Primary Hypercholesterolemia: A Prospective Trial Arch Intern Med, July 25, 1994; 154(14): 1586 - 1595. [Abstract] [PDF]

				Pravastatin Multicenter Study Group II Comparative Efficacy and Safety of Pravastatin and Cholestyramine Alone and Combined in Patients With Hypercholesterolemia Arch Intern Med, June 14, 1993; 153(11): 1321 - 1329. [Abstract] [PDF]

				D. B. Hunninghake, E. A. Stein, C. A. Dujovne, W. S. Harris, E. B. Feldman, V. T. Miller, J. A. Tobert, P. M. Laskarzewski, E. Quiter, J. Held, et al. The Efficacy of Intensive Dietary Therapy Alone or Combined with Lovastatin in Outpatients with Hypercholesterolemia N. Engl. J. Med., April 29, 1993; 328(17): 1213 - 1219. [Abstract] [Full Text]

				R. R. Wing, K. A. Matthews, L. H. Kuller, D. Smith, D. Becker, P. L. Plantinga, and E. N. Meilahn Environmental and Familial Contributions to Insulin Levels and Change in Insulin Levels in Middle-aged Women JAMA, October 14, 1992; 268(14): 1890 - 1895. [Abstract] [PDF]

				P. S. Bachorik, T. A. Cloey, C. A. Finney, D. R. Lowry, and D. M. Becker Lipoprotein-Cholesterol Analysis during Screening: Accuracy and Reliability Ann Intern Med, May 1, 1991; 114(9): 741 - 747. [Abstract] [PDF]

				R. R. Wing, K. A. Matthews, L. H. Kuller, E. N. Meilahn, and P. L. Plantinga Weight Gain at the Time of Menopause Arch Intern Med, January 1, 1991; 151(1): 97 - 102. [Abstract] [PDF]

				G. O. Perez, A. J. Mendez, R. B. Goldberg, R. Duncan, A. Palomo, E. DeMarchena, and S.L. Hsia Correlates of Atherosclerosis in Coronary Arteries of Patients Undergoing Angiographic Evaluation Angiology, July 1, 1990; 41(7): 525 - 532. [Abstract] [PDF]