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31/2, 217-222 (1985)

Comparison of Improved Precipitation Methods for Quantification of High-

Density Lipoprotein Cholesterol
G. Russell Warnicl#{231}
Thuy Nguyen, and Alegrla A. Albers

We compared the standard Lipid Research Clinics heparin- insoluble lipoproteins in the presence of high concentrations
Mn2 (46 mmol/L) method and five improved precipitation of triglyceride. However, in this combination the manganese
methods for quantification of high-density lipoprotein (HOL) can interfere with some of the common enzymic cholesterol
cholesterol. Three of these methods-a dextran sulfate- methods (14).
Mg2 procedure,reportedas a Selected Method, a modified In an alternative method (15) dextran sulfate and magne-
heparin-Mn2 (92 mmol/L) method, and a modified phos- sium are used. This method, published as a Selected Method
photungstate-Mg2 procedure-all gave similar results. (16) for HDL, was designed to optimize the conditions for
Three other methods-the standard heparin-Mn2’ (46 removing LDL and VLDL, the apoB-associated lipoproteins,
mmol/L) method and two polyethylene glycolmethods (75g/ without excessive HDL precipitation. Previous similar
L or pH 10 reagent at 100 g/L final concentrations)-gave methods (17, 18) had involved use of a dextran sulfate of Mr
500 000 and a magnesium concentration that reportedly
slightly highervaluesforHDL cholesterol. Additionof NaCI or
(19) precipitatedsome of the HDL fraction, thereby causing
glucose to specimens did not significantly change protein
underestimation of the HDL cholesterol.
precipitation. In terms of sedimentation effectiveness with
A third precipitation method, with sodium phosphotung-
hypertriglyceridemic specimens,themethods were rankedin
state and magnesium, was originally described by Burstein
the following order: polyethylene glycol (pH 10, 100 gIL) > et al. (20) and subsequently reported (21) for routine quanti-
dextran sulfate-Mg2 > heparin-Mn2 (92 mmol/L) = poly- fication of HDL cholesterol. However, the recommended
ethylene glycol (75 g/L) > phosphotungstate-Mg2 > hepa-
concentrations were later reported (19) to precipitate some
rin-Mn2 (46 mmol/L). H])L and underestimate the HDL concentration. A subse-
quent modification (22) with less phosphotungstate and
Additional Keyphrases: hypertriglyceridemia variation, source magnesium reportedly had improved accuracy. Polyethyl-
of ene glycol (Mr 6000) was used at 120 g/L to precipitate the
lipoproteins (23), but this concentration was reported to
The differential association of the lipoproteins with risk of
coronary artery disease necessitates quantification of the precipitate significant quantities of HDL (19). In subsequent
modifications 100 g per liter of polyethylene glycol was used
individual lipoprotein classes. Recent studies (1-3) have
(24) and then 75 g/L (25). In another modification (26), the
emphasized the importance of lipoprotein measurement,
final polyethylene glycol concentration was 100 g/L, with
especially that of the high-density lipoprotein (HDL) class.’
Low-density lipoprotein (LDL) concentrations are often esti- the pH of the reagent adjusted to 10.
mated in the routine laboratory (4). The many reports of Our evaluation of improved or “second-generation” HDL
methods, modifications, and evaluations in the recent litera- methods was undertaken to assess their agreement on
normolipidemic specimens and on specimens with high and
ture signal not only the high interest in HDL measurement
but also the difficulty of performing this measurement. low concentrations of cholesterol, triglyceride, and HDL.
Therefore, the methods were compared on specimens with a
Recent proficiency surveys demonstrate the magnitude of
range of total cholesterol, triglyceride, and HDL cholesterol
the problem (5-7).
HDL generally is quantified as the cholesterol remaining values. We also tested specimens to which we added either
in the supernate afterchemical precipitation and sedimen- sodium chloride, to increase the ionic strength, or glucose, to
tation of other lipoproteins. The earliest commonly used approximate a specimen from a diabetic patient-factors
that reportedly affect lipoprotein precipitation (27). Methods
method involved precipitation with heparin and manga-
were compared for effectiveness of lipoprotein sedimenta-
nese. The original method, described by Burstein and Sa-
maille (8) for serum specimens, was adapted for use with tion, by checking the proportion of specimens with turbid
supernates. Cholesterol was measured in all supernates. We
EDTA-treated plasma (9, 10) without adjusting the manga-
measured total protein in a subset of supernates and precipi-
nese concentration. Although the method was reasonably
tates to determine whether the effectiveness of sedimenta-
accurate, the manganese concentration was shown in sever-
tion of hypertriglyceridemic specimens might be due to
al studies (11-13) to be borderline for use with plasma,
coprecipitation of other plasma proteins.
perhaps because of manganese binding by EDTA, and an
increase in manganese concentration was recommended.
Doubling the manganese concentration reportedly (11) not Materials and Methods
only improved precipitation of the non-HDL apoB-associat- Specimens
ed lipoproteins but also improved sedimentation of the
Blood samples were collected between June 1981 and May
1983 at the Northwest Lipid Research Clinic from males
Northwest Lipid Research Clinic, Harborview Medical Center, and females, ages 6 to 75 years, according to established
Seattle, WA 98104; and the Department of Medicine, University of protocol (10). A combination of apparently healthy volun-
Washington, Seattle, WA.
‘Nonstandard abbreviations: HDL, LDL, VLDL, high-, low-,and teers and hyperlipidemic referral patients provided speci-
very-low-density lipoproteins, respectively; apo, apolipoprotein; mens with a broad range of values for cholesterol, triglycer-
EDTA, disodium ethylenediaminetetracetate. ide, and HDL cholesterol. Plasma total cholesterol ranged
Received May 8, 1984; accepted September 17, 1984. 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 mL with de-ionized water. The concentration of the MgCl2
morning alter a 12- to 14-h fast. Blood from the antecubi- solution was 2.0 mol/L. For the combined working reagent
tal vein was collected into 15-mL Vacutainer Tubes contain- we mixed four volumes of the sodium phosphotungstate
ing 22.5 mg of EDTA, mixed thoroughly, and cooled mime- solution with one volume of the MgCl2 solution.
diately at 4 #{176}C. Lipoproteins were precipitated from 2-mL aliquots of
Within 3 h we removed blood cells by centrifugation at specimens by adding, with thorough mixing, 200 L of the
1500 x g for 20 mm. Plasma specimens were then stored at combined phosphotungstate-Mg2 reagent. After incuba-
for lipoprotein separation by the various precipitation tion for 5 win at room temperature, the samples were
methods. This was done the day of sample collection or, at promptly centrifuged at 5000 x g for 10 win.
the latest, the following day. Polyethylene glycol 6000 (100 gIL final concentration, pH
10) (26): The reagent solution was prepared by dissolving 20
Separation of Lipoproteinsby Precipitation g of polyethylene glycol 6000 (lot no. 81260; Fluka AG
The indicated precipitation methods were applied concur- Chemische Fabrik, CH-9470 Buchs SG, F.R.G.) and 1.50 g
rently to aliquots from each specimen. In some cases, (20 mmol) of glycine (lot no. 003362; J. .T. Baker Co.) in
specimen volume was insufficient to perform all precipita- about 60 mL of de-ionized water. We then adjusted the pH of
tion methods and some methods were deleted. Precipitating the solution to pH 10 with 1.0 mol/L NaOH before diluting it
reagents at concentrations described below were added and to 100 mL with de-ionized water.
vortex-mixed to samples that were either equilibrated to To 1.0 mL of specimen we added an equal volume of the
room temperature (23-27 #{176}C)or kept at 4 #{176}Cas indicated. polyethylene glycol solution, mixed thoroughly for 8-10 s,
The temperature and duration of incubation varied as and then incubated the mixture at room temperature for 10
indicated below. All sedimentation of insoluble lipoprotein rein before centrifugation at 1500 x g for 30 mm.
aggregates was done by centrifugation at 4#{176}C,
but the g- Polyethylene glycol 6000(75 gIL final concentration) (25):
force and duration used were specific for each method. This reagent was prepared by dissolving 45 g of polyethyl-
Dextran sulfate (50)-Mg2 (15): We prepared a 20 g/L ene glycol 6000 in 100 mL of de-ionized water. We added 400
solution of dextran sulfate (Dextralip 50, 5 x iO D, lot no. iL of the reagent to a 2.0-mL aliquot of plasma. Because
251-118A; Sochibo S.A., 92100 Boulogne, France), to which this polyethylene glycol solution was viscous, we were very
was added a sodium azide-chloramphenicol-gentamicin so- careful to transfer the indicated volume. We mixed the
lution (15) as preservative, and a 1.0 mol/L solution of sample thoroughly, incubated it at room temperature for 15
MgCl2 61120 (lot no. 946-7510; J. T. Baker Co., Phillips-
. mm, and then centrifuged it at 1500 x g for 30 mm.
burg, NJ 08865), adjusted to pH 7.0. Both were stored in
glass-stoppered vials at 4 #{176}C. The working reagent was Lipid Analysis
prepared freshly each week by mixing equal volumes of the Cholesterol and triglycerides were quantified by Lipid
above two solutions. Research Clinic procedures (10) for continuous-flow analysis
To 2.0 mL of plasma we added 200 1zL of the dextran (AutoAnalyzer II). Supernates were mixed thoroughly to
sulfate-Mg2+ combined solution, mixed thoroughly for 8-10 resuspend any precipitates before extraction into isopro-
s, and incubated at room temperature for 15 mm before panol in the presence of a zeolite mixture. We determined
centrifuging at 1500 x g for 30 win. cholesterol by a method involving the Liebermann-Bur-
Heparin-Mn2: We evaluated two methods involving chard reagent (10) and triglyceride by a fluorometric proce-
one at a final concentration of 46 mmol/L and the dure involving 2,4-pentanedione (10). Standard solutions
second at 92 mmolIL. The final concentration of heparin was and quality-control samples were provided by the Clinical
1.3 g/L in both cases. Chemistry Standardization Laboratory, Centers for Disease
Heparin-Mn2 (46 mmol/L) (10): The heparin reagent, Control, Atlanta, GA 30333; moreover, our own analytical
stored at 4#{176}C, contained 5 x i03 USP units/L, prepared by procedures met their standardization requirements. Preci-
diluting one part of sodium heparin (Liquaemin sodium, 4 x sion was excellent, with CVs of <1.0% for cholesterol
io USP unitsfL; Organon Co., West Orange, NJ 07052) with measurement, including the HDL cholesterol range. For the
seven parts 0.15 mol/L NaCl solution. A 1.0 mol/L solution precipitation step and HDL cholesterol measurement, the
of MnCl2 4H0 (reagent grade, J. T. Baker Co.) was also
. CVs were approximately 3%.
prepared. Analysis conditions were also designed to maximize the
To 2.0 mL of each specimen we added sequentially (with measurement precision in the supernates that have low
thorough mixing after each addition) 80 pL of the above HDL concentrations. To eliminate between-run and be-
heparin reagent and 100 pL of 1.0 moWL MnCl2 solution. tween-tray variation, comparison samples from each subject
This mixture was incubated at 4#{176}Cfor 30 win, then were analyzed consecutively in one tray. Systentatic bias
centrifuged at 1500 x g for 30 win. effects of within-tray baseline drift and scaleexpansion were
Heparin-Mn2 (92 inolIL) (11): For this method, the minimized by randomizing the order of the comparison
working reagent was a heparin-Mn2 combined solution samples for each subject. The precipitating reagents did not
obtained by mixing 0.6 mL of the above heparin stock (4 x interfere with this cholesterol procedure.
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 heparin- Ultrafiltration
Mn2 combined reagent, mixed, and incubated the solution The incomplete sedimentation of lipoprotemn aggregates
for 10 mm at room temperature before centrifugation as in that may occur in hypertriglyceridemic specimens results in
the other heparin-Mn2 method. turbid or cloudy supernates and overestimation of HDL. In
Phosphotungstate-Mg2 (22): We prepared a 40 g/L sodi- this study we wiped tubes free of condensate and carefully
um phosphotungstate solution by dissolving 4 g of inspected for turbidity immediately after their removal from
phosphotungstate acid (lot no. 68C-0366; Sigma Chemical the centrifuge. Any turbid supernates were clarified by
Co., St. Louis, MO 63178) in approximately 50 mL of de- ultrafiltration (28) through 25-mm Millipore filters(0.22-
ionized water, adding 16 mL of 1.0 mol/L NaOH (reagent p.m pore size; Millipore Co., Bedford, MA 01730), protected
grade; Mallinckrodt, Inc., St. Louis, MO 63147) adjusting by two prefilters. Turbid supernates (volume must be 1.5
the pH of the solution to 7.4 with NaOH, and diluting to 100 mL or greater) were transferred by pipetting into a Luer-tip

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

syringe barrel attached to the Swinnex upper part and were In the earlier work (15, 16) dextran sulfate-Mg super-
forced through the filter with moderate pressure from the nates of EDTA-treated plasma contained virtually no apoB,
plunger. The clear ifitrates were collected for analysis. indicating specific separation of LDL. On the other hand,
the dextran sulfate-Mg precipitate fractions contained
Matrix Effects little apoA-I, suggesting that precipitation of HDL was not
We pipetted 9.0-mL aliquots of each plasma pool into each excessive.
of three 10-mL volumetric flasks, to compare the methods The agreement of each precipitation method with the
for specimens to which either sodium chloride or glucose dextran sulfate-Mg comparison method is illustrated in
was added to approximate extreme specimens that might be Figure 1. Results by the heparin-Mn2 (46 mmol/L) method
encountered. To one flask we added 8.77 mg of NaC1 were highly correlated (0.995) and in reasonably good
(approximately 10% increase in final concentration); to the agreement with those by the dextran sulfate method, as
second flask we added 40 mg of glucose. All three flasks shown by a slope of 1.05 and a y-intercept of -21 mgfL
were carefully adjusted to 10-mL final volume with 0.15 (Figure la). Although heparin-Mn2” (46 mmol/L) values
moIJL NaCl solution. We then precipitated duplicate ali- were generally somewhat higher at higher HDL, this was
quota from each flask, applying each, separation method. compensated by a slight tendency for underestimation at
low HDL levels, and the overall difference in this set of
Protein Measurement specimens was small and not statistically significant.
We prepared standards of bovine serum albumin (Cohn The modified heparin-Mn2 (92 mmol/L) method gave
Fraction V, fatty acid free; Sigma Chemical Co.) in saline slightly lower values. The agreement with the dextran
solution. Aliquots of the supernates obtained from the sulfate method was excellent, with a correlation coefficient
various I{DL precipitation methods were diluted with 0.15 of 0.997, slope of 1.01, andy-intercept of -10.4 mg/L (Figure
mol/L NaCl. The corresponding precipitates were resolubi- lb). Even though the heparin-Mn2 results averaged only 6
lized in 0.15 mo)JL NaCl solution, and the final volume mg/L lower (464 vs 470 mgfL) the small difference was
accurately measured. To aliquots (in triplicate) of the dilut- consistent throughout the range and statistically significant
ed supernate and precipitate solutions we added 25 p.L of (p sO.005).
sodium deoxycholate (Sigma) 20 g/L, and the appropriate These relationships are consistent with previous reports.
volume of saline to give a final volume of 1.5 mL. After The standard heparin-Mn2 (46 mmol/L) method has been
thorough vortex-mixing and standing for about 15 rein, 0.5 reported (11-13, 15, 16) to slightly overestimate HDL in
mL of trichloroacetic acid (24%) was added, and the mixture EDTA-treated plasma specimens, owing to incomplete pre-
was then centrifuged at 1500 x g for 30 mm. The trichloro- cipitation of the apoB-associated lipoproteins (VLDL and/or
acetic acid supernates were aspirated carefully, and the LDL). At 46 mmol/L the 2+ concentration reportedly was
protein in the remaining pellet was assayed by use of a marginal for complete precipitation of LDL, and a higher
modified Lowry procedure (29). Protein concentrations were 2+ concentration was recommended (11, 13, 30).
reported in terms of the original specimen volume. More than 10 years of experience with the standard
heparmn-Mn2 (46 mmolJL) method in this laboratory sup-
port these conclusions and suggest that in inexperienced
Results and Discussion hands the overestimation by this method might be larger
The dextran sulfate-Mg’ method was selected as the than that observed here. From 1976 to 1980 we quantified
comparison method for this report because previous studies apoB-associated lipoproteins by a radial immunodiffusion
(15, 16) demonstrated good specificity for HDL separation. assay in our routine heparin-Mn2 (46 mmolJL) supernates.

C ,1
I, /
/ ‘I
/‘ 2



// 5IRrE so -G2. COOI#{128}5tEROl GIL

Fig. 1. Relationship of each precipitationmethod (.) to the dextran sulfate-Mg2 method (x) by linear regression

CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985 219

With this feedback on the specificity of the separation, the been reported for enzymic cholesterol methods. The interfer-
amount of apoB-associated lipoprotein in supernates de- ence of Mn2’4’ with enzymic cholesterol analyses is well
creased, probably because of improvements in technique. known (14). Dextran sulfate of Mr 500000 reportedly (31)
Greater care was taken in preparing the reagents to ensure interferes negatively with enzymic assay. The 50 000-Da
that the critical Mn2 concentration was exact and not low. dextran sulfate used here was demonstrated not to interfere
In addition, technologists became increasingly aware that with some enzymic methods (15). Sodium phosphotungstate
even slightly turbid supernates contained significant apoB, reportedly(32) may interfere with some methods.
because of incomplete LDL/VLDL sedimentation, and more The ionic strength of the sample has been reported (27) to
care was given to detect turbid supernates that were subject- affect lipoprotein precipitation by some methods. In our
ed to dilution or ultrafiltration. Without this experience and study, addition of sodium chloride to plasma pools to in-
care, overestimation by the standard heparin-Mn2 proce- crease the ionic strength by about 10% did not significantly
dure could likely be greater. change the mean values for cholesterol in the supernate in
Results by the modified phosphotungstate method were any of the precipitation methods: for each of the various
also in excellent agreement with those by the dextran precipitation methods on four pools to which sodium chlo-
sulfate method (Figure ic). The correlation coefficient was ride was added these values were all within 10 mgIL of those
0.993, with slope of 0.998 andy-intercept of 2 mg/L. For 59 for the unaltered control pools by each precipitation method.
specimens the means for the phosphotungstate method and The differences between the various precipitation methods
for the dextran sulfate method were 470 mg/L and 469 mgI on the pools with and without added sodium chloride were
L, respectively, not statistically significantly different. Note similar to those illustrated in Figure 1. These results
that the final concentrations of phosphotungstate and Mg2 suggest that even though ionic strength has been reported
we used in this comparison were lower than in most to affect lipoprotein precipitation, variations within the
previous studies. Because the higher concentrations used expected physiological range are unlikely to have a signifi-
previously resultedin underestimated HDL cholesterol (19), cant effect on HDL cholesterol as measured by any of these
this modification appears to improve the accuracy of the methods. Similarly, the addition of approximately 500mg of
phosphotungstate-Mg method. glucose per deciliter to pools, to approximate a diabetic
On the other hand, the polyethylene glycol method with a specimen, also did not significantlychange values forsuper-
final reagent concentration of 75 g/L gave slightly higher natant cholesterol by any of the precipitation methods:
results(492 vs 484 mg/L), but again the difference was not mean values for glucose treated and control pools differed by
statistically significant in this set. Figure id illustrates that less than 10 mg/L for each of the precipitation methods.
the tendency for positive bias is especially apparent on The next problem to be considered is that of turbidity or
specimens with high HDL concentrations, as evidenced by cloudiness seen in supernates immediately after centrifuga-
the slope of 1.06 partly compensated by a negative y- tion, a problem usually associated with incomplete sedimen-
intercept. Clearly, the final concentration of 75 g/L used tation of the insoluble VLDLILDL aggregates. This often
here improved the accuracy of HDL measurement as com- occurs with hypertriglyceridemic specimens, because the
pared with previous versions in which higher polyethylene triglycerides make the overall density of the aggregate
glycol concentrations were used. However, a 75 g/L final about the same as that of the solution. In this instance, the
concentration of polyethylene glycol may be slightly low. A insoluble lipoproteins are not sedimented by low-speed
disadvantage of this particular polyethene glycol method is centrifugation. In our study, some precipitation methods
the viscosity of the concentrated polyethylene glycol solu- produced better sedimentation than others. The polyethyl-
tion, which necessitates considerable care to transfer the full ene glycol method (pH 10-100 g/L) had 4% (one of 28) turbid
volume of reagent. supernates (Table 1). Only one specimen, with triglycerides
The final method, in which we used polyethylene glycol of 77.6 g/L, did not give a clear supernate, and specimens
adjusted to pH 10 at a final concentration of 100 g/L, gave a with triglycerides up to 13.9 g/L separated well. The dextran
similar pattern (Figure le) with slope of 1.05, y-intercept of sulfate method had 10% (sixof 59) turbidsupernatesand
-13, but slightly more scatter (r = 0.985). Adjustment of the specimens with triglycerides up to 11.3 g/L gave clear
pH to 10 apparently inhibits lipoprotein precipitation and supernates, while specimens with triglyceride concentra-
compensates for the higher reagent concentration as com- tions as low as 5.4 g/L gave turbid supernates. The heparin-
pared with the previous methods. Mn2 (92 mmol/L) method and the polyethylene glycol (75 gI
This comparison demonstrates that these modifications of L) methods were similar, with 11% and 12% turbid super-
the precipitation methods improve their accuracy. The nates, respectively. The heparin-Mn2’4 method appeared to
agreement observed here is much betterthan that observed be slightly better overall, because the range of triglycerides
with earlier versions of the same methods (19). Further- in specimens with both clear and turbid supernates was
more, the small between-method differences observed here higher. This version of the phosphotungstate-Mg method
resultprimarily from specimens with high HDL concentra- had 17% turbid supernates. The range of triglyceride values
tions. Considering only the subset of specimens with high for this method was somewhat unusual. Some high-triglyc-
HDL (500 mgIL by dextran sulfate-Mg), we observed eride specimens gave clear supernates, but some specimens
the following positive or negative differences in comparison with low triglycerides gave turbid supernates. The heparin-
with dextran sulfate-Mg values: + 10 mg/L (for heparin- Mn2 (46 mmolIL) method gave the highest proportion of
Mg2, 46 mmol/L), -6 mg/L (heparin-Mn2, 92 mmolJL), turbid supernates, 37%.
-3 mg/L (phosphotungstate-Mg), + 13 mg/L (polyethyl- Turbidity of the supernate generally is the result of
ene glycol, 7.5%), and + 10 mg/L (polyethylene glycol, pH 10, incomplete sedimentation of the insoluble lipoproteins-in
10%). The differences for heparin-Mn2 (46 mrnol/L) and our experience one of the major sources of error in HDL
polyethylene glycol (7.5%) were statistically significant (p cholesterol quantitation. In proficiency surveys (5, 6) the
0.05). inter-laboratory variation was generally highest for hyper-
The Lipid Research Clinics’ cholesterol method used for triglyceridemic specimens. Measurement of cholesterol in a
this evaluation was not subject to interference by any of the turbid supernate overestimates HDL cholesterol, in some
precipitation reagents. Therefore, the comparison results cases substantially.
were not confounded by the interference effects that have To avoid this overestimation, further treatment is re-

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

Table 1. Effectiveness of Sedimentation by the Various Precipitation Methods
Triglyceride di strlbutlon, g/L
Turbid supernates Turbid Clear

Method Number Per cent i a SD Range i a SD Range

Polyethylene glycol, pH 10, 1/28 (4) (77.6) 2.4 ± 2.8 0.3-13.9
100 g/L
Dextran sulfate-Mg2 6/59 (10) 24.4 ± 2.71 5.4-77.6 2.0 ± 2.0 0.3-11.3
Heparin_Mn2*, 92 mmol/L 5/46 (11) 19.6 ± 33.6 3.8-77.6 2.1 ± 2.2 0.3-11.3
Polyethylene glycol, 75 g/L 5/40 (12) 12.1 ± 9.4 2.8-23.6 1.7 ± 1.7 0.4- 7.0
Phosphotungstate-Mg2 10/59 (17) 15.3 ± 22.5 3.6-77.6 2.0 ± 3.4 0.3-23.6
Heparin-Mn 22/60 (37) 9.3 ± 15.9 1.4-77.6 1.0 ± 0.6 0.3- 3.3
a No. turbid/total no. supernate S. by method.

quired. One approach (8) is to dilute the original specimen ranged in the following order: polyethylene glycol (100 gIL,
with an equal volume of 0.15 mol/L NaC1 solution before pH 10) > dextran sulfate-Mg2 > heparin-Mn2’4’ (92 mmoll
adding the precipitating reagents. A convenient alternative L) = polyethylene glycol (75 g/L) > phosphotungstate-Mg
is to dilute the turbid supernate and then add the propor- > heparin-Mn” (46 mmol/L).
tionate additional volume of precipitating reagent to
achieve the recommended final concentration for that rea- We are grateful to Chien Yu for the lipid analyses and to Carol
Lum and Carolyn Walden for assistance with data analysis. This
gent. This reduces the solution density by diluting the
work was supported in part by contract NOI-HV-12 157 and grant
protein and other constituents, thereby facilitating sedimen- HL 30086 from the National Heart, Lung, and Blood Institute, and
tation as well as decreasing the lipoprotein concentration, grant 1372 A from the Council for Tobacco Research.
which probably promotes precipitation. Another approach is
to clear turbid supernates by ultrafiltration (28). However,
ultrafiltration must be done carefully, because a plugged References
ifiter can remove HDL along with the insoluble material. In 1. Miller GJ, Miller NE. Plasma-high-density-lipoprotein concen-
our experience a 25-mm ifiter, protected by two prefilters, tration and developmentofischaemic heart-disease. Lancet i, 16-23
gives the best results. This configuration requires a mini-
2. Castelli WP, Doyle JT, Gordon T, et al. HDL cholesterol and
mum volume of 1.5-mL of supernate, owing to the void other lipids in coronaryheart disease: The cooperative lipoprotein
volume. This latter approach was used to clear any turbid phenotyping study. Circulation 55, 767-772 (1977).
supernates encountered in this study. 3. Gordon T, Castelli WP, Hjortland MC, et al. High density
Because incomplete sedimentation is a function of the lipoprotein as a protective factor against coronary heart disease:
precipitatedensity in relation to the solution density, an The Framinghain Study. Am J Med 62, 707-714 (1977).
increasein precipitatedensity or decrease in solution densi- 4. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the
ty would be expected to enhance sedimentation. Therefore, concentration of low-density lipoprotein cholesterol in plasma,
the effectiveness of precipitation by a particular method without use of the preparative ultracentrifuge. Clin Clam 18,499-
might be related to the amount of other serum proteins 502 (1972).
coprecipitating with the lipoproteins. To test this question, 5. Warnick GR, Albers JJ, Leary ET. HDL cholesterol: Results of
interlaboratory proficiency tests. Clin Clam 26, 169-170 (1980).
we measured total protein in supernatant and precipitate
6. Warnick GR, Benderson JM, Albers JJ. Interlaboratory profi-
fractions from some of the precipitation methods, using five
ciency survey of high-density lipoprotein cholesterol measurement.
separate plasma pools. Of the methods tested, only the Clin Clam 29, 516-519 (1983).
polyethylene glycol (75 g/L) method had on the average 7. Hainline A Jr,Cooper GR, Olansky AS, et al. CDC survey of
significantly more protein in the precipitates (74 ± 42 mg high density lipoprotein cholesterol measurement: A report.Cen-
per 2.0 mL) and less in the supernates (140 ± 50 mg). The ters for Disease Control, Atlanta, GA, 1980.
other precipitation methods were similar in the amount of 8. Burstein M, Samaille J. Sur un dosage rapide du cholesterol lie
protein in the precipitate and supernate fractions, respec- aux a- etaux /3-lipoproteines du serum. Clin Chim Acta 5,609-613
tively: dextran sulfate-Mg (26 ± 14 and 174 ± 70), (1960).
heparin-Mn24’ (46 mmolJL) (25 ± 17 and 188 ± 50), 9. Fredrickson DS, Levy RI, Lindgren yr. A comparison of herita-
heparin-Mn2 (92 mmol/L) (21 ± 12 and 195 ± 72), ble abnormal patterns as defined by two different
techniques. J Clin Invest 47, 2446-2457 (1968).
phosphotungstate-Mg2 (26 ± 12 and 190 ± 73). The
polyethylene glycol (pH 10, 100 g/L) method that gave the 10. Manual of Laboratoiy Operations, Lipid Research Clinics Pro-
gram, Lipid and Lipoprotein Analysis, Revised 1982, NIH, U.S.
lowest proportion of turbid supernates was not tested. Dept. of Health and Human Services, Washington, DC.
Nevertheless, no association between sedimentation effec- 11. Warnick GR, Albers JJ. A comprehensive evaluation of the
tiveness and protein precipitation was apparent. heparin-manganese precipitation procedure for estimating high
density lipoprotein cholesterol. J Lipid Res 19,65-76 (1978).
In summary, all of the modified precipitation methods 12. Albers JJ, Warnick GR, Wiebe D, et al. Multi-laboratory
tested here gave similar results for HDL cholesterol, sug- comparison of three heparin-Mn2’ precipitation procedures for
gesting that modifications of the methods have led to estimating cholesterol in high-density lipoprotein. Clin Clam 24,
improved accuracy. Results by the heparin-Mn2 (92 mmolJ 853-856 (1978).
L) and phosphotungstate-Mg2 methods agreed best with 13. Ishikawa TI’, Brazier JB, Steiner PM, et al. A study of the
those by the dextran sulfate-Mg procedure. The heparin- heparin-manganese chloride method fordeterminationofplasma a-
lipoprotein cholesterol concentration. Lipids 11, 628-633 (1976).
Mn2’ (46 mmol/L) and the two polyethylene glycol methods
14. Steele BW, Koehler DF, Azar MM, et al. Enzymatic determina-
gave slightly higher results. Observed differences were tions of cholesterol in high-density lipoprotein fractions prepared by
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222 CLINICAL CHEMISTRY, Vol. 31, No. 2, 1985