CLIN.CHEM. 26/9,1275-1277(1980)
Determinationof High-DensityLipoproteinPhospholipidsin Serum
Yoshihisa Yamaguchi
I describe a method formeasuring high-density
lipoprotein
phospholipids. Magnesium chloride and dextran sulfate
are
used to precipitatealllow-densityand very-low-density
lipoproteins.
The supernatecontainsonlyhigh-density
Iipoproteins,the phospholipidconcentrationof which is
determined by an enzymic method. The precision of the
method (CV) is 2.35% (10 repeated assays), and the mean
value for HDL.phospholipids was 1006 (SD 248) mg/L for
30 apparently heaithy subjects. I used electrophoresis and
enzymic color development to confirm the presence of
HDL-phospholipids.
Results are compared
obtained by an ultracentrifugation
method.
with those
AdditIonal
Keyphrases: enzymic method
reference intervals
ultracentrifugat ion method compared
heart dis-
.
ease
.
lipoproteins
buffer
agents
solution was used to dissolve the same amount of refor color development
of phospholipids
mentioned
above.
Phosphatidyicholine
(Type III-L; Sigma Chemical Co., St.
Louis, MO 63178) was dissolved in water containing 5 g of
Triton X-100 per liter. A 5 mg/2.5 mL solution was prepared
just before
use.
For color development
of cholesterol
I used a Determiner
TC cholesterol
test kit (Kyowa Hakko Co., Machida-shi,
Tokyo, Japan); to stain for HDL-cholesterol
on agarose film,
the solution for color development
of cholesterol was prepared
in double concentration
as compared to the solution usually
used.
Precipitating reagent was prepared by dissolving of 780mg
of dextran sulfate (Mr 500 000; Sigma) in 1 L of magnesium
chloride solution (72 mmol/L).
Procedure
High-density
determined
lipoprotein
(HDL)
cholesterol
has been widely
in clinical tests by use of precipitation
such as heparin/manganese
and by electrophoretic
and magnesium/dextran
separation
with MgC12/dextran
lipoprotein
sulfate,
sulfate,
on cellulose acetate mem-
branes followed by enzymic color development
terol oxidase (1-3).
I describe
a method in which the low-density
and very-low-density
reagents
with choleslipoprotein
are precipitated
from
and the phospholipid
serum
content
of
the supernate,
the HDL-phospholipids,
is measured enzymically with phospholipase
D (phosphatidylcholine
phosphatidehydrolase;
EC 3.1.4.4). The efficiency of this separation is demonstrated
by electrophoresis
and enzymic color
development.
Materials and Methods
Equipment
Electrophoresis
was performed
with Pfizer product no. 618
E and pol-E-Film
(agarose film; Pfizer, Inc., New York NY
10017). I used an automatic
preparative ultracentrifuge
to
separate
lipoproteins
for comparison
of tests (Hitachi Ltd.,
Chiyoda-ku,
Tokyo. Japan).
Reagents
The color development of phospholipids
was as proposed
by Takayama et al. (4). The enzymic assay solution contained
Electrophoresis
of 2 L of serum on duplicate
was performed
for 40 mm with barbital
buffer
pH 8.6) at 90 V. After electrophoresis,
the enzyme
pipetted
onto the film, the enzyme solution
phospholipid
agarose films
(50 mmol/L,
solution was
for staining
on agarose film being used for one sample and
the enzyme solution for staining cholesterol
on agarose film
for the other. After incubation
for 30 mm at 37 #{176}C,
the films
were placed on a white paper and scanned densitometrically
at 500 nm with a dual-wavelength
TLC-scanner
CS-910
(Shimazu).
Ultracentrifugation
was used as the method for HDL isolation against which the results of polyanion precipitation
methods were compared. A 3-mL serum aliquot was adjusted
to background density 1.063 g/mL with solid KBr. The density-adjusted
sample was overlaid with KBr solution, d =
1.063, to fill the tube and then centrifuged at 105 000 X g for
20 hat 4#{176}C.
The d> 1.063 lipoprotein fraction was recovered
and reconstituted
to 6 mL (6).
Determinationof HDL-phospholipids
To 0.2 mL of sample, add 0.2 mL of precipitating
reagent
and immediately vortex-mix for 5g. Let the tubes set for 5 mm
at 25 #{176}C.
Centrifuge for 15 mm at 3000 rpm. Analyze 100 1zL
of the supernatant
enzyme solution.
fraction for phospholipids
with 2 mL of
Multiply the result by 2 to correct for dilu-
tion.
45 U of phospholipase
Results
from Toyo Jozo Co., Ltd., Japan),
Patterns
of HDL-phospholipids
were stained after electrophoresis to confirm the position of the HDL fraction in
serum and in the supernate from the precipitation.
As shown
in Figure 1, the color zone of phospholipids
at the position of
a- and -lipoproteins
was the same as for cholesterol, but the
pattern of phospholipids
was different from that of cholesterol.
Precision (CV) with normal serum samples was 2.35%, mean
509.0 (SD 11.9), and 1.47%, mean 856 (SD 12.6) mg/L for two
D (from Streptomyces
spp., obtained
100 U of choline oxidase
(from Art hrobacter spp., EC 1.1.3.17) (5), 220 U of peroxidase
(EC 1.11.1.7), 12 mg of 4-aminoantipyrine,
20 mg of phenol,
and 8 mg of calcium chloride dihydrate,
dissolved in 100 mL
of 50 mmol/L tris(hydroxymethyl)aminomethane
(Tris).HC1
buffer (pH 7.8) containing 2 g of Triton X-100 per liter. To
stain HDL-phospholipids
on agarose film, 30 mL of Tris-HC1
The Central
Laboratory
for Clinical
Investigation,
Hospital, Fukushima-ku,
Osaka, Japan 553.
Received Feb. 29, 1980; accepted May 6, 1980.
Osaka
University
series of repeated
assays.
Values for the HDL-cholesterol/HDL-phospholipid
ratio
CLINICALCHEMISTRY,Vol.26,No.9,1980 1275
Table 1. Serum HDL-Cholesterol and HDL-Phospholipid Concentrations in 21 Randomly Selected
Hospitalized Patients
Ag
HDLcholesterol
Totalcholesterol
Sx
Totalphosphollds
HOt.plIospholIpIds
HOt-Cl
HDL-P
Concn, mg/L
19
M
1500
420
1620
850
20
F
1740
580
2030
1200
24
M
1810
420
1740
820
0.51
26
M
2050
710
2240
1330
0.53
26
29
31
F
F
F
1380
2000
3230
610
1700
1150
0.53
590
1950
990
0.60
400
2970
860
0.47
44
F
1570
420
1680
850
45
47
M
M
1410
1760
440
480
1760
1990
910
1010
0.49
0.48
0.48
48
49
49
M
F
M
1760
1980
2530
570
680
530
1910
2140
2560
1030
1120
1140
0.55
0.61
0.47
51
55
F
M
1900
2010
400
420
1910
2280
860
1060
0.47
0.40
63
F
2280
740
2680
1490
0.50
65
M
2660
600
2600
1120
0.54
69
F
2200
690
2500
1270
0.54
70
F
2090
400
2010
850
102
107
M
M
2960
2610
570
830
2630
2680
1170
1360
Sample
a
1
0.49
0.48
0.47
0.49
0.61
2000
b
mg/I
1500
U,
a.
0
i.iooo
U,
0
a.
Sample
a
y.1.16X
L
HDL
LDL,HDL
C
I
+
466
n 30
500
2
b
0
500
1000
mg/I
H DL- Cholesterol
FIg. 2. Correlation between HDL-cholesterol and HDL-phospholipids
in Table 1 for some hospitalized
patients selected
at random. Another comparison between HDL-cholesterol
and phospholipids
values is shown in Figure 2.
Serum HDL-phospholipids,
measured for 30 apparently
healthy subjects, was 1006 (SD 248) mg/L.
I compared HDL-phospholipids
value obtained by the
present method (y) and by ultracentrifugation
(x); the coefficient of correlation was 0.902 and the equation for the regression line was y = 1.09x + 16 mg/L (n = 15).
Comparison
of the phospholipids
concentration
in the
are listed
LDL
HDL
HDL
Fig.1. Electrophoretlc pattern for HDL and LDL phospholipids
after color development with the enzymic method and densi-
tometry
- - - -phosphollpids,
cholesterol. (a) Sample before precipitation, (b) after
precipitation. Serum (2 iL) was applied to agarose film for sample a, and supernate (4 paL) of dextran sulfate/Mg
was applied to agarose film for sample
b. Electrophoresis,
enzymlc color development, and densitometric scanning
as described In Methods
-
1276
CLINICAL CHEMISTRY,
Vol. 26, No. 9, 1980
mgi
i
lesterol
and HDL-triglyceride
in the supernate
from precipare shown in Figure 3 and the correlation
between
HDL- phospholipids
and HDL-triglyceride
in Figure 4. In this
study, I used a test kit for triglycerides,
the fully enzymatic
UV-method
from Boehringer
Mannheim.
itation
1000
V
‘0
YzO,22X
+ 257
I-
V
U
.2’ 500
Discussion
I.J
C
I
500
0
1000
HDL-Cholesterol
mg/I
Fig. 3. CorrelatIon between HDL-cholesterol and HOL-triglycerides
mg/I
1000
Y.O.17X
500
‘S
+210
References
.
#{149}#{149}
____________
0
500
1000
HDI
Phospholiplds
Fig.4. CorrelatIon between HDL-phosphollplds
glycerides
1500
mg/I
and HDL-tri-
sulfate/Mg2
supernate
by the present method (y)
and by the chemical method of Hoeflmayr and Fried (x) (7)
gave a coefficient of correlation of 0.924 and the equation for
the regression line was y = 1.02x + 12 mg/L (n = 30).
Preliminary results for the correlation between HDL-chodextran
The concentration
of HDL-cholesterol
has been found to
correlate
with coronary heart disease. Apoprotein,
triglycerides, and phospholipids
of HDL may also have significance
for this disease.
The enzymic method for serum phospholipids
is specific
to choline-containing
phospholipids
such as phosphatidylcholine,
sphingomyelmn,
and lysophosphatidylcholine
in
serum; as reported
by Takayaina
et al. (4), these comprise
about 95% of the phospholipids
in serum.
As shown in Figure 2, HDL-phospholipids
correlated fairly
well with HDL-cholesterol,
and so the ratio of HDL-phospholipids
may have significance
for clinical tests.
1. Bachorik, P. S., Wood, P. D., Albers, J. J., et al., Plasma highdensity lipoprotein cholesterol concentration determined after removal of other lipoproteins by heparin/manganese
precipitation
or
by ultracentrifugation.
GUn. Chem. 22, 1828 (1976).
2. Finley, P. R., Schifman, R. B., Williams, R. J., and Lichti, D. A.,
Cholesterol in high-density lipoprotein: Use of Mg2/dextran
sulfate
in its enzymic measurement. Clin. Chem. 24,931 (1978).
3. Cobb, S. A., and Sanders, J. L., Enzymic determination
of cholesterol in serum lipoproteins separated by electrophoresis.
Clin.
Chem. 24, 1116 (1978).
4. Takayama, M., Itoh, S., Nagasaki, T., and Tanimizu, I., A new
enzymatic method for determination
of choline-containing
phospholipids. Clin. Chim. Acta 79,93 (1977).
5. Ikuta, S., Imamura, S., Misaki, H., and Horiuti, Y., Purification
and characterization
of choline oxidase from Art hroba cter globiformis. J. Biochem. 82, 1741 (1977).
6. Gt,fman, J. W., and Lindgren, F. T., Ultracentritugal
studies on
lipoproteins of human serum. J. Biol. Chem. 179,973 (1949).
7. Hoefimayr, J., and Fried, R., Eine Methode zur routinem#{225}ssigen
Bestimmung des Lipidphosphors
und der Phosphatide.
Med. Ernaehr. 7, 9 (1966).
CLINICAL CHEMISTRY, Vol. 26, No. 9, 1980
1277