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Serum high density lipoprotein cholesterol determination: A simple modification
307
Clinica Chimica Acta, 92 (1979) 307-310 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION -__-
~_
._~_
CCA 10012
SERUM HIGH DENSITY LIPOPROTEIN CHOLESTEROL DETERMINATION: A SIMPLE MODIFICATION
ALAN MADDISON
*, RAMESH MOTWANI and ANN B. SPEAIGHT
Clinical Services Ltd., 6 Harley Street,
London
WIN IAA
(U.K.)
(Received September 8th, 1978)
Summary A simple modification to the method of Burnstein et al. (Burnstein, M., Scholnick, H.R. and Morfin, R. (1970) J. Lip. Res. 11, 583) [l] for the measurement of serum high-density lipoprotein (HDL) cholesterol concentrations is reported. The modification obviates the need for a refrigerated centrifuge and makes the method suitable for routine use in almost any laboratory. Good correlation was obtained for values in 80 healthy adults using the method as originally described by Burnstein et al. [l] and then with the modification. The presented method was linear and precise and the study showed that it was not necessary to fast before giving a blood sample for the assay. It was found that HDL cholesterols were higher in women than in men, but did not change significantly with age in either group.
Introduction Miller et al. [ 21 reported an inverse relationship between serum high density lipoprotein (HDL) cholesterol and the risk of coronary heart disease (CHD), in a prospective study involving 6595 men from Tromsq5, Norway. Castelli et al. [ 31 have published similar findings for middle aged North Americans. In the U.K. about 50% of male deaths are due to CHD [4]. A report [ 51 made by a joint working party of the Royal College of Physicians (London) and the British Cardiac Society examined the feasibility of primary and secondary prevention of CHD and commented upon the high cost of preventive trials involving the detection and monitoring of CHD. It seems that if the findings of Miller et al. [ 21 and Castelli et al. [3] are accepted then serum HDL cholesterol may prove to be a very useful and * To whom correspondence should be addressed.
308
inexpensive screening test for use in the identification of those at risk from CHD. Lopes-Virella et al. [6] had recognised the need for an assay system for the determination of HDL cholesterol which was suitable for routine use. In their paper they showed that the relatively simple method of Burnstein et al. [ 11, using a phosphotungstate-magnesium chloride precipitation technique, correlated well with methods using heparin-manganese precipitation and ultracentrifugation techniques. The method of Burnstein et al. [l] does, however, involve using a refrigerated centrifuge. As many laboratories do not possess this instrument, adoption of the Burnstein et al. [l] method would involve some capital expenditure. This study has shown that the Burnstein et al. method can be successfully modified to use centrifugation at room temperature instead of at 4’C. Also investigated were the effects of fasting upon serum HDL cholesterol. Finally, a sex and age population distribution study for serum HDL cholesterol has been performed. Materials and methods Samples Serum samples were obtained from overtly healthy adults attending the B.U.P.A. Medical Centre (London) for the purpose of health screening examinations. Venous blood was collected into plain tubes and the serum separated after centrifugation at room temperature. Assays were performed within 24 h of specimen collection. HDL cholesterol determination The method of Burnstein et al. [ 11 was used as originally described, and also with the modification that the centrifugation step was performed at room temperature and not +4”C. To 2 ml of serum was added 200 ~1 of sodium phosphotungstate solution (40 g/l phosphotungstic acid in 0.1 M sodium hydroxide) and 50 ~1 of 2 M magnesium chloride solution. Each sample was mixed well and then centrifuged at either +4”C or room temperature for 30 min at 1500 X g. Aliquots of the supernate and serum specimens were then assayed for cholesterol concentration using the method of Hiinteler and Van der Slik [7] on a Technicon AA1 system (Technicon Instruments). Results Fig. 1 shows the results for serum HDL cholesterol determined in 80 healthy adults using the Burnstein et al. method and the modified procedure. The regression equation was y = 0.85x + 0.082, with a correlation coefficient (r) of 0.97 (p < 0.001). Fig. 2 illustrates the linearity of the modified method and Table I presents the results of replicates for within- and between-batch analyses. Fifteen healthy adults had serum and HDL cholesterols determined on samples taken after an overnight fast, and on samples taken a few hours after their
309
Y= 0.65X r =o
+ 0082
/
HDL our modified
Fig.
0.2
03
HDL
0 5 0.6 03
1 g Ilitrel (x).
0.9
0
FIGURES
”
healthy
measured
by
original
method
of Burnstein
et al. (Y),
and
adults.
of our modified
method
FOR
THE
MODIFIED
for
serum
HDL
cholesterol
determinations.
METHOD
Within-batch
Between-batch
10
50
(g/l)
0.574
0.585
S.D.
(g/I)
0.0069
0.0093
C.V.
(%)
1.1
1.6
TABLE
II
SERUM
CHOLESTEROL
AND
HDL
CHOLESTEROLS
Fasting
IN
FASTING
AND
NON-FASTING
SUBJECTS
Non-fastmg
number Serum (g/I)
cholesterol
HDL (g/I)
cholesterol
Serum
cholesterol
HDL
(g/I)
(g/I)
1
2.00
0.40
2.05
0.40
2
2.15
0.58
2.10
0.60
3
2.90
0.55
2.70
0.57
4
2.40
0.52
2.55
0.52
5
2.30
0.46
2.30
0.45
6
2.20
0.51
2.30
0.53
7
1.55
0.35
1.80
0.35
8
2.30
0.62
2.20
0.57
9
1.80
0.56
1.80
0.53
10
2.80
0.65
2.95
0.64
11
1.95
0.58
1.95
0.58
12
1.65
0.56
1.70
0.54
13
1.85
0.76
1.80
0.71
14
2.25
0.67
2.25
0.63
15
1.85
0.65
1.80
0.65
15
15
15
”
0.9 wlue
I
PRECISION
Subject
0.1 02 03 04 05 0.6 07 08 HDL cholesterol [gllitrcl-estimated
method
concentrations
in 80
the linearity
08
-modified
cholesterol
method
2. Assessing
Mean
0 4
cholesterol
1. Serum
TABLE
line
.
0.1
Fig.
Ideal
97(P<.OOl)
15
MfXUl
2.13
0.561
2.15
0.551
S.D.
0.38
0.10
0.37
0.095
cholesterol
by
310 TABLE
III
DISTRIBUTION
OF
SERUM
HDL
CHOLESTEROL
BY
AGE
Female
Age
AND
SEX
Male ___._
group
n Mean S.D.
25-34
3 5-44
45-54
5544
25-34
35-44
16
16
15
9
41
36
(g/l) (g/l)
45-54 ~_~._ 72
55-64 36
0.635
0.656
0.60
0.62
0.524
0.519
0.505
0 539
0.127
0.186
0.134
0.24
0.129
0.152
0.156 ____
0.148
-__
_
normal midday meal. These results are shown in Table II. Results for fasting and non-fasting specimens, upon statistical analysis, show no significant difference. Finally, serum HDL cholesterol concentrations were measured in 241 healthy adults, and the results were divided into groups by age and sex. The mean and standard deviation for each group is presented in Table III. There was no significant change with age in serum HDL cholesterol for either men or women. The results for women, however, were significantly higher than those for men (p < 0.05). Discussion It has been shown that the modification to the Burnstein method for the precipitation of low density and very low density lipoprotein is valid and gives results that correlate extremely well with those of the original method. The modified method is linear over the population range of the healthy subjects and gives good reproducibility. Although relatively simple, this modification does mean that the method can be more easily performed in a large number of laboratories. Surprisingly, diet seems to exert very little effect upon serum concentrations of HDL cholesterol. This information would obviously prove useful in the organisation of any screening procedure. The finding that there appears to be no change in serum HDL cholesterol concentrations with increasing age perhaps supports the concept that the assays could be used to detect risk for CHD in the young, and that preventative measures could be applied at an early age. The population studies also showed that women had higher serum HDL cholesterols than men. Higher values are purported to indicated reduced risk for CHD and this would explain the lower incidence of CHD in women. References Burnstein, Miller, CasteUi. Zukel.
M.,
N.E.. W.P., W.J.
Registrar
of
Physicians
O.H.,
Doyle.
(1975)
J.L.A.
and Morfin,
The&.
J.T..
M.F., and
and Stone. Van
D.S.
Gordon.
Statistical
Coronary
of London
Lopes-Virella,
H.R.
Circulation
General’s
Prevention
Hiinteler,
Scholnick,
FOrde,
52,
T.,
the
Hames,
of
British
P., Ellis.
O.D. C..
England
Disease.
der Slik.
(1970)
Mi$s,
J. Lip.
Res.
(1977)
Lancet
Hulley,
S.B.,
11,
583 i, 965
Kagen.
A..
Mctiee,
D..
Vicic.
W.J.
and
II-97
Review
Heart
R.
and
Report
Cardiac S. and
W.
(1972)
and Wales, of
Society
Colwell. CIin.
a
1973.
Joint
(1976) J.A.
(1977)
Chim.
Acta
H.M.S.O.
Working
Party
J. R. Coll. Clin. 42.
449
(1975) of
the
Physicians
Chem.
23,
10, 882
Royal 3
College
of
Clinica Chimica Acta, 92 (1979) 307-310 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION -__-
~_
._~_
CCA 10012
SERUM HIGH DENSITY LIPOPROTEIN CHOLESTEROL DETERMINATION: A SIMPLE MODIFICATION
ALAN MADDISON
*, RAMESH MOTWANI and ANN B. SPEAIGHT
Clinical Services Ltd., 6 Harley Street,
London
WIN IAA
(U.K.)
(Received September 8th, 1978)
Summary A simple modification to the method of Burnstein et al. (Burnstein, M., Scholnick, H.R. and Morfin, R. (1970) J. Lip. Res. 11, 583) [l] for the measurement of serum high-density lipoprotein (HDL) cholesterol concentrations is reported. The modification obviates the need for a refrigerated centrifuge and makes the method suitable for routine use in almost any laboratory. Good correlation was obtained for values in 80 healthy adults using the method as originally described by Burnstein et al. [l] and then with the modification. The presented method was linear and precise and the study showed that it was not necessary to fast before giving a blood sample for the assay. It was found that HDL cholesterols were higher in women than in men, but did not change significantly with age in either group.
Introduction Miller et al. [ 21 reported an inverse relationship between serum high density lipoprotein (HDL) cholesterol and the risk of coronary heart disease (CHD), in a prospective study involving 6595 men from Tromsq5, Norway. Castelli et al. [ 31 have published similar findings for middle aged North Americans. In the U.K. about 50% of male deaths are due to CHD [4]. A report [ 51 made by a joint working party of the Royal College of Physicians (London) and the British Cardiac Society examined the feasibility of primary and secondary prevention of CHD and commented upon the high cost of preventive trials involving the detection and monitoring of CHD. It seems that if the findings of Miller et al. [ 21 and Castelli et al. [3] are accepted then serum HDL cholesterol may prove to be a very useful and * To whom correspondence should be addressed.
308
inexpensive screening test for use in the identification of those at risk from CHD. Lopes-Virella et al. [6] had recognised the need for an assay system for the determination of HDL cholesterol which was suitable for routine use. In their paper they showed that the relatively simple method of Burnstein et al. [ 11, using a phosphotungstate-magnesium chloride precipitation technique, correlated well with methods using heparin-manganese precipitation and ultracentrifugation techniques. The method of Burnstein et al. [l] does, however, involve using a refrigerated centrifuge. As many laboratories do not possess this instrument, adoption of the Burnstein et al. [l] method would involve some capital expenditure. This study has shown that the Burnstein et al. method can be successfully modified to use centrifugation at room temperature instead of at 4’C. Also investigated were the effects of fasting upon serum HDL cholesterol. Finally, a sex and age population distribution study for serum HDL cholesterol has been performed. Materials and methods Samples Serum samples were obtained from overtly healthy adults attending the B.U.P.A. Medical Centre (London) for the purpose of health screening examinations. Venous blood was collected into plain tubes and the serum separated after centrifugation at room temperature. Assays were performed within 24 h of specimen collection. HDL cholesterol determination The method of Burnstein et al. [ 11 was used as originally described, and also with the modification that the centrifugation step was performed at room temperature and not +4”C. To 2 ml of serum was added 200 ~1 of sodium phosphotungstate solution (40 g/l phosphotungstic acid in 0.1 M sodium hydroxide) and 50 ~1 of 2 M magnesium chloride solution. Each sample was mixed well and then centrifuged at either +4”C or room temperature for 30 min at 1500 X g. Aliquots of the supernate and serum specimens were then assayed for cholesterol concentration using the method of Hiinteler and Van der Slik [7] on a Technicon AA1 system (Technicon Instruments). Results Fig. 1 shows the results for serum HDL cholesterol determined in 80 healthy adults using the Burnstein et al. method and the modified procedure. The regression equation was y = 0.85x + 0.082, with a correlation coefficient (r) of 0.97 (p < 0.001). Fig. 2 illustrates the linearity of the modified method and Table I presents the results of replicates for within- and between-batch analyses. Fifteen healthy adults had serum and HDL cholesterols determined on samples taken after an overnight fast, and on samples taken a few hours after their
309
Y= 0.65X r =o
+ 0082
/
HDL our modified
Fig.
0.2
03
HDL
0 5 0.6 03
1 g Ilitrel (x).
0.9
0
FIGURES
”
healthy
measured
by
original
method
of Burnstein
et al. (Y),
and
adults.
of our modified
method
FOR
THE
MODIFIED
for
serum
HDL
cholesterol
determinations.
METHOD
Within-batch
Between-batch
10
50
(g/l)
0.574
0.585
S.D.
(g/I)
0.0069
0.0093
C.V.
(%)
1.1
1.6
TABLE
II
SERUM
CHOLESTEROL
AND
HDL
CHOLESTEROLS
Fasting
IN
FASTING
AND
NON-FASTING
SUBJECTS
Non-fastmg
number Serum (g/I)
cholesterol
HDL (g/I)
cholesterol
Serum
cholesterol
HDL
(g/I)
(g/I)
1
2.00
0.40
2.05
0.40
2
2.15
0.58
2.10
0.60
3
2.90
0.55
2.70
0.57
4
2.40
0.52
2.55
0.52
5
2.30
0.46
2.30
0.45
6
2.20
0.51
2.30
0.53
7
1.55
0.35
1.80
0.35
8
2.30
0.62
2.20
0.57
9
1.80
0.56
1.80
0.53
10
2.80
0.65
2.95
0.64
11
1.95
0.58
1.95
0.58
12
1.65
0.56
1.70
0.54
13
1.85
0.76
1.80
0.71
14
2.25
0.67
2.25
0.63
15
1.85
0.65
1.80
0.65
15
15
15
”
0.9 wlue
I
PRECISION
Subject
0.1 02 03 04 05 0.6 07 08 HDL cholesterol [gllitrcl-estimated
method
concentrations
in 80
the linearity
08
-modified
cholesterol
method
2. Assessing
Mean
0 4
cholesterol
1. Serum
TABLE
line
.
0.1
Fig.
Ideal
97(P<.OOl)
15
MfXUl
2.13
0.561
2.15
0.551
S.D.
0.38
0.10
0.37
0.095
cholesterol
by
310 TABLE
III
DISTRIBUTION
OF
SERUM
HDL
CHOLESTEROL
BY
AGE
Female
Age
AND
SEX
Male ___._
group
n Mean S.D.
25-34
3 5-44
45-54
5544
25-34
35-44
16
16
15
9
41
36
(g/l) (g/l)
45-54 ~_~._ 72
55-64 36
0.635
0.656
0.60
0.62
0.524
0.519
0.505
0 539
0.127
0.186
0.134
0.24
0.129
0.152
0.156 ____
0.148
-__
_
normal midday meal. These results are shown in Table II. Results for fasting and non-fasting specimens, upon statistical analysis, show no significant difference. Finally, serum HDL cholesterol concentrations were measured in 241 healthy adults, and the results were divided into groups by age and sex. The mean and standard deviation for each group is presented in Table III. There was no significant change with age in serum HDL cholesterol for either men or women. The results for women, however, were significantly higher than those for men (p < 0.05). Discussion It has been shown that the modification to the Burnstein method for the precipitation of low density and very low density lipoprotein is valid and gives results that correlate extremely well with those of the original method. The modified method is linear over the population range of the healthy subjects and gives good reproducibility. Although relatively simple, this modification does mean that the method can be more easily performed in a large number of laboratories. Surprisingly, diet seems to exert very little effect upon serum concentrations of HDL cholesterol. This information would obviously prove useful in the organisation of any screening procedure. The finding that there appears to be no change in serum HDL cholesterol concentrations with increasing age perhaps supports the concept that the assays could be used to detect risk for CHD in the young, and that preventative measures could be applied at an early age. The population studies also showed that women had higher serum HDL cholesterols than men. Higher values are purported to indicated reduced risk for CHD and this would explain the lower incidence of CHD in women. References Burnstein, Miller, CasteUi. Zukel.
M.,
N.E.. W.P., W.J.
Registrar
of
Physicians
O.H.,
Doyle.
(1975)
J.L.A.
and Morfin,
The&.
J.T..
M.F., and
and Stone. Van
D.S.
Gordon.
Statistical
Coronary
of London
Lopes-Virella,
H.R.
Circulation
General’s
Prevention
Hiinteler,
Scholnick,
FOrde,
52,
T.,
the
Hames,
of
British
P., Ellis.
O.D. C..
England
Disease.
der Slik.
(1970)
Mi$s,
J. Lip.
Res.
(1977)
Lancet
Hulley,
S.B.,
11,
583 i, 965
Kagen.
A..
Mctiee,
D..
Vicic.
W.J.
and
II-97
Review
Heart
R.
and
Report
Cardiac S. and
W.
(1972)
and Wales, of
Society
Colwell. CIin.
a
1973.
Joint
(1976) J.A.
(1977)
Chim.
Acta
H.M.S.O.
Working
Party
J. R. Coll. Clin. 42.
449
(1975) of
the
Physicians
Chem.
23,
10, 882
Royal 3
College
of