CLIN.

CHEM.

27/6,

838-841

(1981)

Separationof Serum High-DensityLipoproteinfor Cholesterol

Determination:Ultracentrifugationvs Precipitationwith Sodium
Phosphotungstate
and MagnesiumChloride
Larry Seigler and Wei T. Wu1
We compared two methods for separation of serum
high-density lipoprotein: selective precipitation with sodium
phosphotungstate
centrifugation

and magnesium

in sodium

chloride

chloride, and ultra-

solution

(relative

density

1.063). When the cholesterol content (determined enzymically with a centrifugal analyzer) of fractions obtained
by each method was compared (ultracentrifugation = x),
the correlation coefficient was 0.97; y = 1.Olx
11.2
mg/L; p <0.05; n = 54. The within-day and between-day
coefficients of variation for this method were 1.1 and
4.0%, respectively. Reference intervals for high-density
lipoprotein cholesterol in subpopulations categorized by
-

age and sex were based on data obtained from volunteer

blood donors.
AdditionalKeyphrases: reference interval

corona,y heart

disease
The concentration
of high-density lipoprotein
cholesterol
(HDLC)2 in serum is reported to be inversely related to the
incidence of coronary heart disease (1-8). Consequently,
the
measurement
of HDLC has become popular as an adjunct in
assessing

individual

risk to this disease.

In the modified

pro-

cedure recommended
by the Lipid Research Clinic Program
(9-12) heparin
and manganese
are used to precipitate
verylow-density lipoprotein (VLDL) and low-density lipoprotein
(LDL) selectively, the extent of precipitation
being in some
dispute (10, 13). The manganese ion interferes with enzymic
determination
of cholesterol (14, 15), so ethylenediaminetetraacetate
(EDTA), a chelator, must be added (15, 16).
Moreover, because the precipitating ability of heparin varies
with the supplier and the biological source, each new lot of
heparin must be standardized
to assure maximum precipitation of the apolipoprotein
B-containing
lipoproteins
(14,
17).
Sodium phosphotungstate
(NaPT)
and magnesium
ion
have been used as an alternative to heparin-manganese
in the
selective precipitation
of VLDL and LDL (12, 13, 17, 18). The
pH of the NaPT solution, however, must be lower than 7.6 for

the precipitation
of VLDL and LDL to be complete (18).
We have compared the NaPT_Mg2+ procedure with ultracentrifugation

in the isolation

of HDL, and have found

the

former to be suitable for routine use by the clinical laboratory

in the determination

of serum

HDLC

concentration.

Department
of Pathology, Louisiana State University Medical
Center, 1542 Tulane Ave., New Orleans, LA 70112.
1 Also at the Clinical Chemistry
Laboratory, Chemical Pathology
Section, Department of Pathology, Charity Hospital of Louisiana at
New Orleans, New Orleans, LA 70140.
2 Nonstandard
abbreviations: high-density lipoprotein cholesterol
(HDLC); very-low-density
lipoprotein
(VLDL); low-density lipoprotein (LDL); high-density lipoprotein (HDL); and sodium phosphotungstate
(NaPT).
Received Oct. 17, 1980; accepted March 4, 1981.
838

CLINICAL CHEMISTRY,

Vol. 27, No. 6, 1981

Materials and Methods

Specimens
Blood specimens
were collected
at Charity
Hospital
of
Louisiana
at New Orleans
from laboratory
personnel
and
students
who had fasted overnight.
For serum specimens,
blood was collected from an arm vein into 7-mL Vacutainer
Tubes (Becton-Dickinson,
East Rutherford,
NJ 07073) containing
no anticoagulant,
and was allowed to clot at room
temperature.
For plasma specimens,
blood was collected into
7-mL Vacutainer Tubes containing
10.5mg of KSEDTA or 143
USP units of sodium heparmnate and mixed thoroughly.
Cells
were separated
by centrifugation
(1500 X g, 15 mm) and the
plasma or serum was stored at 4#{176}C.
The assay was done within
48 h.
Specimens
for the reference
interval study were obtained
from the Blood Bank of Charity Hospital of Louisiana
at New
Orleans.

Reagents
Sodium phosphotungst
ate, 40 gIL, pH 7.4: Eight grams of
phosphotungstate
acid (reagent grade; Sigma Chemical
Co.,
St. Louis, MO 63178) was added to 160 mL of water. After
addition of 32 mL of 1 mol/L NaOH, the pH of the solution
was adjusted to 7.4 with 1 mol/L HC1, and it was diluted to 200

mL with water.
Magnesium
chloride,
0,5 moliL: Freshly
grade magnesium
chloride hexahydrate,

Chemical Co.), was dissolved

in deionized

opened reagent10.17 g (Sigma

water in a volu-

metric flask and diluted to 100 mL.

Sodium
chloride,
relative
density
1.063 or 1.177: These
solutions
were prepared
by the method of Havel et a!. (19)
from reagent-grade
sodium chloride
(Mallinckrodt,
Inc., St.
Louis, MO 63147).
Cholesterol
reagent: Worthington
Cholesterol
Reagent (cat.
no. 27059; Worthington
Diagnostics,
Freehold, NJ 07728) was
reconstituted
in 14.0 mL of the Tris buffer supplied
with the

reagent.
Standards:

Miami,
FL 33152) and
Sera (Data Medical Associates, Arlington, TX 76011) were used as reference cholesterol
standards. After reconstitution
according to the suppliers
directions, these solutions were diluted fourfold and sixfold,
respectively, with isotonic (8.5 g/L) saline. The cholesterol
concentrations
in these diluted solutions, as calculated from
information
supplied by the manufacturers,
were 612.5 and
617 mgfL, respectively.
Preciset#{174}Cholesterol
Standard
(Boehringer-Mannheim
Biochemicals,
Inc., Indianapolis,
IN
43250), 500, 1000, 1500, 2000, 3000, and 4000 mg/L solutions
of cholesterol in methanol/water
(10/90 by vol), were used as
supplied.

Cholesterol

Monitrol

Calibrated

II (Dade,

Reference

Procedures
Precipitation

of VLDL

and

LDL:

The LDL and VLDL

Table 1. Operational Parameters for Quantitatlon of

High-Density Lipoprotein Cholesterol on the GEMSAEC Analyzer
Vol, zL

Sample

Flush
Reagent

iiL

Pump,

24

50

42

200
1000

420

%
48

w.n

21

42

1, water; 2, cholesterol

standard;

3,

control; 4-16, unknown samples

Reaction temperature: 30 #{176}C
Wavelength:

500 nm

Filter position: 430-560 nm

Reaction mode: Auto/Rate
IR

(initial reading)

RI

NR

(read interval)
(number of

SC

readings)
(standardconcn)

Results and Discussion

Modified
enzymic cholesterol
assay: The ratio of specimen
volume to total reaction mixture volume for the cholesterol
assay was increased from 1:100, as recommended
by the reagent manufacturer, to 1:20, (a) to keep the absorbance
change of most specimens (those containing 300 to 700mg of
HDLC per liter) within an absorbance range where the most
nearly accurate
photometric
measurements
could be made

Sample tip: Polypropylene

Blank switch: Reagent
Cuvet no. and contents:

C8dj
is the concentration of standard used, in mg/L, and
1.2 is the dilution factor.

60

12
#{149}
(enter concn
of standard)

and (b) to reduce the effect of absolute volume errors in pipetting.

Precipitation:
Unlike precipitation
procedures
in which
heparin-Mn2
is used (14-16), this NaPT-Mg2
procedure
does not produce pseudocholesterol
turbidity
in the presence of enzymic cholesterol
reagents. Addition of as much as
fourfold excess NaPT and MgC12(compared with the concentrations normally used in this precipitation
procedure)
to
the Preciset 1500 mg/L cholesterol standard
caused no more
than 0.6%higher cholesterol values than was expected. This
implies that NaPT and MgC12 neither inhibit nor enhance
these enzyme
reactions.
Sample
blanks: Absorbancevalues of sample blanks observed at 500 nm in 66 serum supernates ranged from 0.0002

0.0430, with a mean of 0.0122. Because this absorbance

corresponds to an HDLC concentration
of 18 mgfL, we included specimen
blanks in the procedure. The absorbance

to

data obtained
26s after reaction initiation
(the earliest possible reading on our GEMSAEC)
could not be used for the
sample blank abeorbances
because a significant portion of the
reaction had occurred by that time. The separate run we found
necessary for obtaining appropriate
blank absorbances would
not be needed if the GEMSAEC were equipped
with early
reading capability.
Linearity:
To assess the linearity of the modified cholesterol
analysis.
assay procedure,
we used volumetric
dilutions of a 1150 mg/L
Lipoprotein
fractionation
by ultracentrifugation:
Adjust
cholesterol standard. The results of the assay are linear with
to relative density 1.063 by adding 2.0 mL of NaCl solution
concentration
up to 1150 mg/L (y = 1.02x - 13.4 mgfL; r =
(relative density 1.177) to a cellulose nitrate tube containing
0.999; p <0.01).
4.0 mL of serum; then centrifuge the tubes (105 000 X g, 5#{176}C,
Precision:
The coefficients of variation obtained on using
72 h). Quantitatively
recover the infranatant
fractions conpooled sera as the reference with this HDLC procedure were:
taining the HDL by a tube-slicing technique, adjust the volwithin-run, 0.9% ( = 392 mg/L; SD = 3 mgfL; n = 14); beume of each fraction to 5.0 mL with isotonic saline, and store
tween-run, 1.1% ( = 359 mg/L; SD = 4 mg/L; n = 12); and
at 5 #{176}C
until analysis for cholesterol.
Observed concentrations
between-day, 4.0% (1=364 mgfL; SD = 14 mg/L; n = 31). The
of cholesterol should be multiplied by 1.25 to compensate for

were precipitated

with NaPT-Mg2
by a modification of the
method of Burstein
(20,21), as follows:
Add 50 iL of 40 g/L NaPT and 50 tL of 0.5 mol/L MgCl2
sequentially,
with thorough vortex-mixing
after addition of
each reagent, to 500 sL of serum or plasma. Then centrifuge
the mixture (1500 X g, 15 min) and promptly
remove the supernatant
fraction
with a Pasteur pipette for cholesterol

dilution.

Qua ntitation

of HDLC:

We set up the centrifugal

(GEMSAEC, Electro-Nucleonics,
shown in Table
each

run recorded

Fairfield,

NJ

analyzer
07006) as

1. A multiple-data
printout obtained after
the final absorbance (at 12 mm) of each

cuvet.

A blank absorbance value for each sample was obtained by

using a separate
distribution disc. The procedure for loading
this disc was identical to that for loading the assay disc except
that the reagent wells were filled with water instead of cholesterol reagent. The control and analyzer module
settings
were the same as those for the assay except: RI = 5 and NR
1.
The blank absorbance for each cuvet was obtained from a
multiple-data
printout after each blank run.
(Note: We used the Auto/Rate
mode to obtain absorbance
information at 60-s intervals, which can be useful in evaluating
the overall performance
of the reactions. If this information
is not desired, the Auto/End Point reaction mode may be used
with the additional alteration:
IR = 720 and NR = 1.)
Calculation:
The concentration
of HDLC in the original
serum or plasma specimen was calculated as follows:

pooled sera used for determination

of the within-run coefficient of variation differed from those used for the other determinations.
Preconditioning
of the polypropylene
loader
sample tip was found to be unnecessary,
in contrast to results
obtained with a stainless steel tip (22). HDLC concentrations
observed for 14 replicate specimens in each of two successive
runs were randomly distributed about the mean concentration. No consistent trend was noted from the first to the 28th
specimen.
Correlation
with ultracentrifugation:
The HDLC concentration data obtained after lipoprotein fractionation
by
ultracentrifugation

compared

HDLC (mg/L)

(Aunknown/Astandard)

X Cstandard

X 1.2

of

and

by NaPT-Mg2

precipitation
are
and coefficient
with 54 split serum samples

in Figure 1. The slope y-intercept,

correlation

obtained

in a study

1.01, -11.2 mg/L, and 0.97, respectively.

Premixing the NaPT and MgCl2: Incomplete
precipitation
of the VLDL and LDL fractions resulted when NaPT
and
MgCls were mixed before addition to serum or plasma. The
order in which the NaPT and MgCl2 were added, however, had
no effect on the precipitation
of VLDL and LDL, an observation in agreement with that of Grove (18). We believe that
the divalent cation, Mg2, forms a bridge between LDLphospholipids
and the polyanion, phosphotungstate.
Premixing the polyanion and the divalent cation leads to the

were

CLINICAL CHEMISTRY,

Vol. 27, No. 6, 1981

839

457, and 456 mg/L. The differences between the mean observed in serum and those observed in heparinized
plasma and
EDTA-treated plasma (8 and 9 mg/L, respectively)
were
within the limits of analytical
error
associated
with the
assay.
Linear regression
analysis of HDLC values obtained
from
serum (x) and heparmnized
plasma (y) yielded a slope, y-intercept, and coefficient of correlation
of 1.01, -11.8 mg/L, and
0.99, respectively.
The corresponding
values for serum and
EDTA-treated
plasma were similar, being 0.96, -2.3 mg/L,
and 0.98, respectively.
Specimen
stability:
Aliquots of pooled serum stored at -20
were thawed at intervals
and analyzed for HDLC. The
mean concentration
obtained
on eight aliquots
stored two
months
was 0.9% lower than the initial mean, a difference

y. 101*- II.?
0.97

en

en

en

HD1-Choleiterol.rrg/L
DITRACENTRIFUGATION d

Fig. 1. Fractionation

of high-density

1.063

lipoprotein:

ultracentrifu-

gatlon (relative density = 1.063) vs precipitation with sodium

phosphotungstate
and magnesium chloride

formation
of a stable matrix that will not react with the
LDL-phospholipids.
Formation
of such matrices significantly
reduces the concentration of polyanions
and divalent cations

available for interaction

with the lipoproteins. Support for

such a mechanism is provided by Bernfield and Kelly (23),
who observed that LDL treated with proteolytic enzymes
retained its ability to interact with polyanions in the presence
of divalent cations. They concluded that the protein moiety
of LDL is not essential for the formation of insoluble complexes. Srinivasan
et al. (24) observed that LDL precipitation
with heparin

and Ca2

is inversely

proportional

to the degree

of LDL-phospholipid
hydrolysis by phospholipase
C. They
concluded that insoluble LDL-divalent
cation-polyanion
complexes form through
the phospholipid
moiety of LDL.
Presumably,
polyanions
and divalent cations interact with
VLDL and HDL through a similar mechanism.
Effects
of anticoagulants:
In an experiment designed to

determine
nations,

the effects of anticoagulants

specimens

were collected

on HDLC determi-

from 38 volunteers.

During

a single venipuncture,
separate blood samples were drawn
from each volunteer into Vacutainer Tubes containing either
heparin, EDTA, or no anticoagulant.
The mean HDLC concentrations
observed in the respective specimens were 465,

Table 2. Reference Ranges for Serum High-Density

Lipoprotein Cholesterol
Ags, yr

HOL chofs.t.rol,
90% rang.

mg/L
95% rang.

Men

18-25

85

300-600

270-630

26-35

49

280-690

260-760

36-45

47

260-660

240-720

46-55

29

240-650

220-720

28
33
20

290-590
350-690
280-740

260-620
340-740
250-830

Women

18-25
26-35
36-45

The 90% range includes 90% of the reference population; the 95% range
includes 95% of the reference population.

840

CLINICAL CHEMISTRY,

Vol.

27, No. 6, 1981

within the limits of analytical error of the assay. Aliquots assayed after storage for four, six, and eight months, however,
yielded mean values that were less than the initial mean
HDLC value by 8.3% (n = 10), 15.3% (n = 11), and 27.1% (n
=
8), respectively.
Reference
intervals:
Serum HDLC concentrations
with
respect to age and sex were studied on a reference population
comprising 210 men and 81 women blood donors at the Blood
Bank at Charity Hospital of Louisiana at New Orleans. About
two-thirds of the donor population was black. Although the
medical histories of the donors were not available, each donor
met the health criteria established by the Blood Bank for
donation. The distribution
of HDLC values was approximately log normal among all subpopulations
except among
men and women aged 18-25 years, which had an approximately gaussian distribution.
Reference ranges for HDLC
based on the subpopulations
are shown in Table 2. The 95%
range is bounded by the HDLC concentrations
corresponding
to the 2.5th and 97.5th percentiles of the HDLC concentration
distribution
for that subpopulation.
The HDLC concentrations corresponding to the 5th and the 95th percentiles bound
each 90% range. The lower limit of each reference range is of
greater clinical interest than the upper limit because the incidence of coronary heart disease is inversely proportional to
the serum HDLC concentration.
Acceptance of the reference
range that includes 90% rather than 95% of the reference
population reduces the risk of accepting as normal a patient
whose serum HDLC concentration
value is actually abnormal (Type II error). The mean serum HDLC concentration
observed among men of this population
(451 mg/L) was
comparable with values reported in studies of other reference
populations
(18, 22, 25-28). Nevertheless,
the mean serum
HDLC concentration
observed among New Orleans women
(468 mg/L) was considerably
less than those reported
in
similar studies,
including
one in which a NaPT-Mg2
precipitation
procedure
was used (18). This observation
may
reflect the higher incidence
of mortality
from coronary heart
disease among New Orleans
residents
compared
with the
national average (29).

This research was supported in part by USPHS

grant no. AH-

00898.

References
1. Barr, D. P., Russ, E. M., and Eder, H. A., Protein-lipid relationships
plasma. Am. J. Med. 11,480-493 (1951).
2. Berg, K., Borrenson,
A.-L., and Dahlen, G., Serum-high-densitylipoprotein and atherosclerotic
heart disease. Lancet i, 499-501
(1976).
3. Miller, G. J., Miller, N. E., and Ashcroft, M. T., Inverse relationship
in Jamaica between plasma high-density
lipoprotein cholesterol
concentration
and coronary-disease
risk as predicted
by multiple
risk-factor states. Clin. Sci. Mol. Med. 51,475-482 (1976).
4. Miller, N. E., Thille, D. S., Forde, 0. H., and Mjos, 0. D., The
Troms#{216}
Heart Study: High-density lipoprotein and coronary heart
in human

disease:
(1977).

prospective

case-control

Lancet

study.

i, 965-968

J. P. D., Betteridge,
P. J., Wu, P., et al., High density and
low density lipoproteins and prevalence of vascular disease in diabetes
mellitus. Lancet i, 1242 (1978).
6. Rhoads, G. T., Galbrandsen,
C. L., and Kagan, A., Serum lipoproteins and coronary heart disease in a population study of Hawaiian
Japanese men. N. Engi. J. Med. 294, 293-298 (1976).
7. Stanhope, J. M., Sampson, V. M., and Clarkson, P. M., Highdensity lipoprotein cholesterol and other serum lipids in a New Zealand biracial adolescent sample: The Wairoa College Survey. Lancet
i, 968-970 (1977).
8. Gordon, T., Castelli, W. P., Hjortland, M. C., et a!., High density
lipoprotein as a protective factor against coronary heart disease: The
Framingham Study. Am. J. Med. 62,707-714 (1977).
9. Manual of Laboratory Operations,
Lipid Research
Clinics Program, 1, Lipid and Lipoprotein Analysis. DHEW Publication (NIH)
75,628 (1974).
10. Warnick, G. R., and Albers, J. J., A comprehensive evaluation of
the heparin-manganese precipitation
procedure for estimating high
density lipoprotein
cholesterol.
J. Lipid Res. 19,65 (1978).
11. Ishikawa, T. T., Brazier, J. B., Steiner, P. M., et al., A study of the
heparin-manganese
alpha-lipoprotein
(1976).

chloride
cholesterol

method
for determination
concentration.
Lipids

of plasma

11, 628-633

12. Warnick, G. R., Cheung, M. C., and Albers,

methods for high-density
Clin. Chem. 25, 596-604 (1979).
13. Kostner,
high-density
25, 939-942

precipitation method. Clin. Chem. 24, 161-165

(1978).

5. Reckless,

current

by the heparin/Mn2

lipoprotein

J. J., Comparison
of
cholesterol
quantitation.

G, M., Avogaro, P., Bon, G. B., et al., Determination

of
lipoproteins:
Screening methods compared. Clin. Chem.
(1979).

14. Bachorik,
P. S., Wood, P. D., Albers, J. J., et al., Plasma highdensity lipoprotein
cholesterol
concentrations
determined
after removal of other lipoproteins
by heparin/manganese
precipitation
or
by ultracentrifugation.
Clin. Chem. 22, 1828-1834 (1976).
15. Steele, B. W., Koehler,
D. F., Azar, M. M., et al., Enzymatic
determinations
of cholesterol
in high-density
lipoprotein
fractions
prepared
by a precipitation
technique.
Clin. Chem. 22, 98-101
(1976).
16. Liedtke, R. J., Busby, B., and Batzer, J. D., Use of the Dupont aca
to measure cholesterol in high-density lipoprotein
fractions prepared

17. Lopes-Virella,

M. F., Stone, P., Ellis, S., and Caldwell,

Cholesterol
determination
three different
methods.

in high-density

Clin. Chem.

lipoproteins
separated
23, 882-885 (1977).

J. A.,
by

18. Grove, T. H., Effect of reagent pH on determination

of highdensity lipoprotein
cholesterol
by precipitation
with sodium phosphotungstate-magnesium.
Clin. Chem. 25, 560-564 (1979).
19. Have!, R. J., Eder, H. A., and Bragdon, J. H., The distribution
and
chemical composition
of ultracentrifugally
separated lipoproteins in
human serum. J. Clin. Invest. 34, 1345-1353 (1955).

M., and Satnaille, J., On a rapid determination

of the
cholesterol bound to the serum alpha- and beta-lipoproteins.
Clin.
Chim. Acta 5,609 (1960).
21. Burstein, M., Rapid method for the isolation of lipoproteins from
human serum by precipitation with polyanions. J. Lipid Res. 11,
583-595 (1970).
22. Ash, K. 0., and Hentschel, W. M., High-density lipoproteins estimated by an enzymatic
cholesterol procedure, with a centrifugal
analyzer. Clin. Chem. 24, 2180 (1978).
23. Bernfeld, P., and Kelly, T. P., Proteolysis of human serum betalipoproteins. J. Biol. Chem. 239, 3341-3346 (1964).
24. Srinivasan, S. R., Lopez, A., Radhakrishnamurthy,
B., and Berenson, G. S., A simplified technique for semi-quantitative, clinical
estimation of serum beta- and pre-beta-lipoproteins. Clin. Chem. 17,
20. Burstein,

994-997

(1970).

25. Ononogbu, I. C., and Lewis, B., Lipoprotein fractionation by a

precipitation method. A simple quantitative procedure. Clin. Chim.
Acta 71, 397-402 (1976).
26. Frederickson, D. S., Levy, R. I., and Lindgren, F. T., A comparison
of heritable abnormal lipoprotein patterns as obtained by two different techniques. J. Clin. Invest. 47, 2446-2456 (1968).
27. Castelli, W. P., Doyle, J. T., Gordon, T., et al., HDL-cholesterol
and other lipids in coronary heart disease: The cooperative lipoprotein
phenotyping
study. Circulation
55, 767-772 (1977).
28. Allen, J. K., Hensley, W. J., Nichols, A. V., and Whitfield, J. B.,
An enzymatic and centrifugal method for estimating high-density
lipoprotein cholesterol. Clin. Chem. 25, 325-327 (1979).
29. Sauer, H. I., Epidemiology
of cardiovascular
mortality-geographic and ethnic. Am. J. Pub. Health 52, 94-101 (1962).

CLINICAL CHEMISTRY, Vol. 27, No. 6, 1981

841