Comparisonof Improved PrecipitationMethodsfor Quantificationof High Density LipoproteinCholesterol

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CLIN. CHEM. 31/2, 217-222 (1985)

Comparison of Improved Precipitation Methods for Quantification of HighDensity Lipoprotein Cholesterol G.

Russell Warnicl#{231} Thuy Nguyen, and Alegrla A. Albers

We compared the standard Lipid Research Clinics heparinMn2 (46 mmol/L) method and five improved precipitation methods for quantification of high-density lipoprotein (HOL) cholesterol. Three of these methods-a dextran sulfateMg2 procedure,reportedas a Selected Method, a modified heparin-Mn2 (92 mmol/L) method, and a modified phosphotungstate-Mg2 procedure-all gave similar results. Three other methods-the standard heparin-Mn2’ (46 mmol/L) method and two polyethylene glycolmethods (75g/ L or pH 10 reagent at 100 g/L final concentrations)-gave slightly highervaluesforHDL cholesterol. Additionof NaCI or glucose to specimens did not significantly change protein precipitation. In terms of sedimentation effectiveness with hypertriglyceridemic specimens,themethods were rankedin the following order: polyethylene glycol (pH 10, 100 gIL) > dextran sulfate-Mg2 > heparin-Mn2 (92 mmol/L) = polyethylene glycol (75 g/L) > phosphotungstate-Mg2 > heparin-Mn2 (46 mmol/L). Additional Keyphrases: hypertriglyceridemia of

variation, source

The differential association of the lipoproteins with risk of coronary artery disease necessitates quantification of the individual lipoprotein classes. Recent studies (1-3) have emphasized the importance of lipoprotein measurement, especially that of the high-density lipoprotein (HDL) class.’ Low-density lipoprotein (LDL) concentrations are often estimated in the routine laboratory (4). The many reports of methods, modifications, and evaluations in the recent literature signal not only the high interest in HDL measurement but also the difficulty of performing this measurement. Recent proficiency surveys demonstrate the magnitude of the problem (5-7). HDL generally is quantified as the cholesterol remaining in the supernate afterchemical precipitation and sedimentation of other lipoproteins. The earliest commonly used method involved precipitation with heparin and manganese. The original method, described by Burstein and Samaille (8) for serum specimens, was adapted for use with EDTA-treated plasma (9, 10) without adjusting the manganese concentration. Although the method was reasonably accurate, the manganese concentration was shown in several studies (11-13) to be borderline for use with plasma, perhaps because of manganese binding by EDTA, and an increase in manganese concentration was recommended. Doubling the manganese concentration reportedly (11) not only improved precipitation of the non-HDL apoB-associated lipoproteins but also improved sedimentation of the Northwest Lipid Research Clinic, Harborview Medical Center, Seattle, WA 98104; and the Department of Medicine, University of

Washington, Seattle, WA. ‘Nonstandard abbreviations: HDL, LDL, VLDL, high-, low-,and very-low-density lipoproteins, respectively; apo, apolipoprotein; EDTA, disodium ethylenediaminetetracetate. Received May 8, 1984; accepted September 17, 1984.

insoluble lipoproteins in the presence of high concentrations of triglyceride. However, in this combination the manganese can interfere with some of the common enzymic cholesterol methods (14). In an alternative method (15) dextran sulfate and magnesium are used. This method, published as a Selected Method (16) for HDL, was designed to optimize the conditions for removing LDL and VLDL, the apoB-associated lipoproteins, without excessive HDL precipitation. Previous similar methods (17, 18) had involved use of a dextran sulfate of Mr 500 000 and a magnesium concentration that reportedly (19) precipitatedsome of the HDL fraction, thereby causing underestimation of the HDL cholesterol. A third precipitation method, with sodium phosphotungstate and magnesium, was originally described by Burstein et al. (20) and subsequently reported (21) for routine quantification of HDL cholesterol. However, the recommended concentrations were later reported (19) to precipitate some H])L and underestimate the HDL concentration. A subsequent modification (22) with less phosphotungstate and magnesium reportedly had improved accuracy. Polyethylene glycol (Mr 6000) was used at 120 g/L to precipitate the lipoproteins (23), but this concentration was reported to precipitate significant quantities of HDL (19). In subsequent modifications 100 g per liter of polyethylene glycol was used (24) and then 75 g/L (25). In another modification (26), the final polyethylene glycol concentration was 100 g/L, with the pH of the reagent adjusted to 10. Our evaluation of improved or “second-generation” HDL methods was undertaken to assess their agreement on normolipidemic specimens and on specimens with high and low concentrations of cholesterol, triglyceride, and HDL. Therefore, the methods were compared on specimens with a range of total cholesterol, triglyceride, and HDL cholesterol values. We also tested specimens to which we added either sodium chloride, to increase the ionic strength, or glucose, to approximate a specimen from a diabetic patient-factors that reportedly affect lipoprotein precipitation (27). Methods were compared for effectiveness of lipoprotein sedimentation, by checking the proportion of specimens with turbid supernates. Cholesterol was measured in all supernates. We measured total protein in a subset of supernates and precipitates to determine whether the effectiveness of sedimentation of hypertriglyceridemic specimens might be due to coprecipitation of other plasma proteins.

Materials and Methods Specimens Blood samples were collected between June 1981 and May 1983 at the Northwest Lipid Research Clinic from males and females, ages 6 to 75 years, according to established protocol (10). A combination of apparently healthy volunteers and hyperlipidemic referral patients provided specimens with a broad range of values for cholesterol, triglyceride, and HDL cholesterol. Plasma total cholesterol ranged from 1.42 to 10.35 g/L and total triglyceride from a low of CLINICALCHEMISTRY, Vol. 31, No. 2, 1985 217

0.33 g/L to a high of 77.60 g/L. All subjects reported in the morning alter a 12- to 14-h fast. Blood from the antecubital vein was collected into 15-mL Vacutainer Tubes containing 22.5 mg of EDTA, mixed thoroughly, and cooled mimediately at 4 #{176}C. Within 3 h we removed blood cells by centrifugation at 1500 x g for 20 mm. Plasma specimens were then stored at 4#{176}C for lipoprotein separation by the various precipitation methods. This was done the day of sample collection or, at the latest, the following day. Separation of Lipoproteinsby

Precipitation

The indicated precipitation methods were applied concurrently to aliquots from each specimen. In some cases, specimen volume was insufficient to perform all precipitation methods and some methods were deleted. Precipitating reagents at concentrations described below were added and vortex-mixed to samples that were either equilibrated to room temperature (23-27 #{176}C) or kept at 4 #{176}C as indicated. The temperature and duration of incubation varied as indicated below. All sedimentation of insoluble lipoprotein aggregates was done by centrifugation at 4#{176}C, but the gforce and duration used were specific for each method. Dextran sulfate (50)-Mg2 (15): We prepared a 20 g/L solution of dextran sulfate (Dextralip 50, 5 x iO D, lot no. 251-118A; Sochibo S.A., 92100 Boulogne, France), to which was added a sodium azide-chloramphenicol-gentamicin solution (15) as preservative, and a 1.0 mol/L solution of MgCl2 61120 (lot no. 946-7510; J. T. Baker Co., Phillipsburg, NJ 08865), adjusted to pH 7.0. Both were stored in glass-stoppered vials at 4 #{176}C. The working reagent was prepared freshly each week by mixing equal volumes of the above two solutions. To 2.0 mL of plasma we added 200 1zL of the dextran sulfate-Mg2+ combined solution, mixed thoroughly for 8-10 s, and incubated at room temperature for 15 mm before centrifuging at 1500 x g for 30 win. Heparin-Mn2: We evaluated two methods involving one at a final concentration of 46 mmol/L and the second at 92 mmolIL. The final concentration of heparin was 1.3 g/L in both cases. Heparin-Mn2 (46 mmol/L) (10): The heparin reagent, stored at 4#{176}C, contained 5 x i03 USP units/L, prepared by diluting one part of sodium heparin (Liquaemin sodium, 4 x io USP unitsfL; Organon Co., West Orange, NJ 07052) with seven parts 0.15 mol/L NaCl solution. A 1.0 mol/L solution of MnCl2 4H0 (reagent grade, J. T. Baker Co.) was also prepared. To 2.0 mL of each specimen we added sequentially (with thorough mixing after each addition) 80 pL of the above heparin reagent and 100 pL of 1.0 moWL MnCl2 solution. This mixture was incubated at 4#{176}C for 30 win, then centrifuged at 1500 x g for 30 win. Heparin-Mn2 (92 inolIL) (11): For this method, the working reagent was a heparin-Mn2 combined solution obtained by mixing 0.6 mL of the above heparin stock (4 x i07 USP units/L) with 10 mL of 1.06 mol/L MnC12 solution. To 2.0 mL of plasma we added 200 L of the heparinMn2 combined reagent, mixed, and incubated the solution for 10 mm at room temperature before centrifugation as in the other heparin-Mn2 method. Phosphotungstate-Mg2 (22): We prepared a 40 g/L sodium phosphotungstate solution by dissolving 4 g of phosphotungstate acid (lot no. 68C-0366; Sigma Chemical Co., St. Louis, MO 63178) in approximately 50 mL of deionized water, adding 16 mL of 1.0 mol/L NaOH (reagent grade; Mallinckrodt, Inc., St. Louis, MO 63147) adjusting the pH of the solution to 7.4 with NaOH, and diluting to 100 .

.

218 CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985

mL with de-ionized water. The concentration of the MgCl2 solution was 2.0 mol/L. For the combined working reagent we mixed four volumes of the sodium phosphotungstate solution with one volume of the MgCl2 solution. Lipoproteins were precipitated from 2-mL aliquots of specimens by adding, with thorough mixing, 200 L of the combined phosphotungstate-Mg2 reagent. After incubation for 5 win at room temperature, the samples were promptly centrifuged at 5000 x g for 10 win. Polyethylene glycol 6000 (100 gIL final concentration, pH 10) (26): The reagent solution was prepared by dissolving 20 g of polyethylene glycol 6000 (lot no. 81260; Fluka AG

Chemische Fabrik, CH-9470 Buchs SG, F.R.G.) and 1.50 g (20 mmol) of glycine (lot no. 003362; J. .T. Baker Co.) in about 60 mL of de-ionized water. We then adjusted the pH of the solution to pH 10 with 1.0 mol/L NaOH before diluting it to 100 mL with de-ionized water. To 1.0 mL of specimen we added an equal volume of the polyethylene glycol solution, mixed thoroughly for 8-10 s, and then incubated the mixture at room temperature for 10 rein before centrifugation at 1500 x g for 30 mm. Polyethylene glycol 6000(75 gIL final concentration) (25): This reagent was prepared by dissolving 45 g of polyethylene glycol 6000 in 100 mL of de-ionized water. We added 400 iL of the reagent to a 2.0-mL aliquot of plasma. Because this polyethylene glycol solution was viscous, we were very careful to transfer the indicated volume. We mixed the sample thoroughly, incubated it at room temperature for 15 mm, and then centrifuged it at 1500 x g for 30 mm.

Lipid Analysis Cholesterol and triglycerides were quantified by Lipid Research Clinic procedures (10) for continuous-flow analysis (AutoAnalyzer II). Supernates were mixed thoroughly to resuspend any precipitates before extraction into isopropanol in the presence of a zeolite mixture. We determined cholesterol by a method involving the Liebermann-Burchard reagent (10) and triglyceride by a fluorometric procedure involving 2,4-pentanedione (10). Standard solutions and quality-control samples were provided by the Clinical Chemistry Standardization Laboratory, Centers for Disease Control, Atlanta, GA 30333; moreover, our own analytical procedures met their standardization requirements. Precision was excellent, with CVs of dextran

L)

polyethylene

=

> heparin-Mn”

glycol (100 gIL,

> heparin-Mn2’4’ (92 mmoll glycol (75 g/L) > phosphotungstate-Mg (46 mmol/L). sulfate-Mg2

We are grateful to Chien Yu for the lipid analyses and to Carol Lum and Carolyn Walden for assistance with data analysis. This work was supported in part by contract NOI-HV-12 157 and grant HL 30086 from the National Heart, Lung, and Blood Institute, and grant 1372 A from the Council for Tobacco Research.

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