Clinical Science (1999) 97, 183–192 (Printed in Great Britain)
Impaired postprandial clearance of squalene and
apolipoprotein B-48 in post-menopausal women
with coronary artery disease
Radhakrishnan A. RAJARATNAM, Helena GYLLING and Tatu A. MIETTINEN
Department of Medicine, Division of Internal Medicine, University of Helsinki, P.O. Box 340, FIN-00029 HYKS, Helsinki, Finland
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It is not known in detail whether postprandial lipaemia is associated with coronary artery disease
(CAD) in women. To investigate this, we administered an oral vitamin A/squalene/fat meal to
24 post-menopausal women with angiographically proven CAD who were not taking hormone
replacement therapy, and to 30 healthy controls (18 without and 12 with hormone replacement
therapy) to evaluate the effects of CAD on postprandial lipoprotein metabolism. This was done
by assessing squalene, triacylglycerols, retinyl palmitate and apolipoprotein B-48 (apoB-48)
during the subsequent 24 h. The subjects with CAD had significantly higher fasting concentrations of squalene and apoB-48 in triacylglycerol-rich lipoproteins (TGRL) compared with the
controls. The postprandial areas under the incremental curve of TGRL apoB-48, chylomicrons,
very-low-density lipoprotein (VLDL) and TGRL squalene, and of retinyl palmitate in VLDL only,
were significantly higher in women with CAD than in controls. Adjustment for fasting values did
not eliminate the differences in postprandial squalene and apoB-48 between CAD and controls.
The postprandial responses of control subjects were not influenced by hormone replacement
therapy. The peaks of squalene and retinyl palmitate of the controls, but not of the women with
CAD, occurred significantly earlier (P 0.01 for both) in chylomicrons than in VLDL. The
findings suggest that lipoproteins that accumulate postprandially are labelled by dietary
squalene, and that these lipoproteins may be atherogenic in post-menopausal women.
INTRODUCTION
Since humans spend a considerable portion of time in a
postprandial state, postprandial lipoproteins could have
an impact on atherogenesis [1,2]. The temporary accumulation of chylomicrons and chylomicron remnants
in the postprandial state may lead to lipid deposition into
the arterial wall and macrophages [3,4]. It has been
documented in several studies that men with coronary
artery disease (CAD) have pronounced postprandial
lipaemia in comparison with healthy controls [5–13].
Although men and women are postulated to have
different postprandial lipoprotein metabolism [14], there
is only one previous study that focused on women with
CAD in a postprandial state [15]. The women with CAD
had higher postprandial concentrations of intermediatedensity lipoprotein (IDL) than women with valvular
heart disease serving as controls. On the other hand, in
another study, women with angina only, without a
history of myocardial infarction, showed no accumulation of postprandial lipoproteins [12]. Accordingly, the
question of whether pronounced postprandial lipaemia is
associated with coronary atherosclerosis in women, as it
is in men, has not been answered in detail.
The vitamin A fat-loading test has been used frequently
to label postprandial lipoproteins. Since retinyl palmitate
Key words : atherosclerosis, chylomicron remnants, dietary fat, postprandial lipaemia, triacylglycerol-rich lipoproteins, very-lowdensity lipoprotein.
Abbreviations : apoB-48 (etc.), apolipoprotein B-48 (etc.) ; AUIC, area under the incremental curve ; CAD, coronary artery disease ;
IDL, intermediate-density lipoprotein ; LDL, low-density lipoprotein ; PIC, incremental peak concentration ; TGRL, triacylglycerol-rich lipoproteins ; VLDL, very-low-density lipoprotein.
Correspondence : Dr Tatu A. Miettinen.
# 1999 The Biochemical Society and the Medical Research Society
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R. A. Rajaratnam, H. Gylling and T. A. Miettinen
is also found in lipoproteins other than chylomicrons and
chylomicron remnants during the later stages of the
postprandial phase, apolipoprotein B-48 (apoB-48) was
suggested as a more specific indicator of lipoproteins that
originate intestinally [16]. The quantification of apoB-48
is laborious, and therefore searches have been made for
other postprandial markers. Squalene is a triterpenoid
hydrocarbon intermediate of cholesterol biosynthesis,
which in the fasting state is mainly associated with lowdensity lipoprotein (LDL) in the circulation [17]. Dietary
squalene, similarly to vitamin A, is incorporated into
chylomicrons and chylomicron remnants [18]. Elevation
of squalene levels in chylomicrons and chylomicron
remnants following a squalene\vitamin A mixed-fat meal
suggested that squalene is also a marker of intestinal
lipoproteins [19]. The postprandial metabolism of
squalene in women with CAD has not been elucidated.
Accordingly, the aim of the present study was to
investigate the postprandial metabolism of lipoproteins
and squalene in post-menopausal women with angiographically proven CAD, and in controls matched for age
and body mass index, by assessing postprandial responses
of triacylglycerols, squalene, retinyl palmitate and apoB48.
Part of this work was presented at the 11th International Symposium on Atherosclerosis, Paris, France,
October 5–9, 1997.
METHODS
Subjects
A total of 24 post-menopausal women aged 50–55 years
and with a positive coronary angiography were recruited
from the University Central Hospital of Helsinki. Twelve
of them had suffered from a myocardial infarction, nine
had undergone coronary artery bypass surgery, and
13 had undergone percutaneous transluminal angioplasty, all at least 6 months before entry into the study.
None of the patients had congestive cardiac failure,
renal, thyroid or hepatic diseases, or diabetes mellitus,
or suffered from extreme obesity (body mass index
30 kg\m#), or were taking hormone replacement therapy or hypolipidaemic medication. Beta-blocking agents
were used by 15 patients, and angiotensin-converting
enzyme inhibitor by one.
The healthy control group was selected randomly from
the population register of 50- and 55-year-old women
living in Helsinki. A total of 30 women that were not
taking hypolipidaemic treatment, without a history of
chest pain, with a normal electrocardiogram, and with a
body mass index of 30 kg\m# participated in the study.
Seven of them were hypertensive ; three were treated with
beta-blockers, two with angiotensin-converting enzyme
inhibitors and two with thiazide. Of the control women,
18 were not taking hormone replacement therapy,
# 1999 The Biochemical Society and the Medical Research Society
whereas two used transdermal oestrogen, eight used
peroral combined oestrogen and progestin, and two used
progestin alone.
The post-menopausal state was demonstrated by
amenorrhoea and elevated serum follicle-stimulating
hormone levels ( 30 units\litre). All subjects volunteered for the study, and gave informed consent. The
study protocol followed the ethical principles of the
Declaration of Helsinki, and was approved by the Ethics
Committee of our institution.
Oral vitamin A/squalene/fat tolerance test
After a 12 h fast, a blood sample was drawn to obtain
baseline values. The participants were then given a test
meal containing 200 ml of 38 % cream, an egg yolk,
345 000 i.u. of vitamin A and 0.5 g of squalene. This meal
consisted of 90 g of fat, 432 mg of cholesterol and
50 20 kJ (1200 kcal) of energy. During the 24 h period
following the meal, the subjects fasted except for a
routine hospital meal after 9 h. Blood samples were taken
at 3, 4, 6, 9, 12 and 24 h. The subjects were then advised
to continue with their normal diets, and dietary fat and
cholesterol were calculated from 7-day dietary recalls
[20].
Lipoprotein fractionation
Lipoproteins from fasting sera were separated by ultracentrifugation in a fixed-angle Ti 50.4 rotor (Beckman
Instruments) as follows : very-low-density lipoprotein
(VLDL), d 1.006 ; IDL, d 1.006–1.019 ; LDL, d
1.019–1.063 ; high-density lipoprotein, d 1.063–1.210.
Chylomicrons and VLDL were separated from postprandial samples. For this purpose, 7.2 ml of plasma was
overlayered with an NaCl solution of density 1.006 g\ml
and centrifuged at 34 873 g (18 000 rev.\min) for 30 min.
Chylomicrons were isolated by aspirating the top 3.6 ml.
The infranatant was then mixed with the 1.006 g\ml
NaCl solution and centrifuged at 131 849 g (35 000 rev.\
min) for 18 h to separate VLDL. Another aliquot of
3.0–3.5 ml of plasma was overlayered with NaCl solution
of density 1.006 g\ml and ultracentrifuged at 131 849 g
for 18 h. A combination of chylomicrons and VLDL,
defined as the triacylglycerol-rich lipoprotein (TGRL)
fraction, was recovered in the top 0.5 ml.
Assay of apoB-48
The method, described previously [21], was modified to
assess apoB-48 in the TGRL fraction at baseline and at 4,
6, 9 and 24 h postprandially. Isolated LDL apoB-100 was
used as a standard for apoB-48. Lipoprotein preparations
were dialysed overnight at 4 mC against 0.15 mol\l NaCl
in 1 mmol\l EDTA, pH 7.4. The apolipoprotein mass of
the LDL fraction was determined by the Lowry procedure, with BSA (Sigma) as standard [22]. The dialysed
LDL and TGRL fractions, each containing 60–200 µg of
protein, were delipidated with ice-cold ethanol\diethyl
Postprandial lipaemia in female coronary heart disease
ether (3 : 1, v\v) and centrifuged to precipitate apolipoproteins [23]. The protein sediment was then resolubilized in sample buffer containing 0.5 mol\l Tris,
0.15 mol\l sodium phosphate, 10 % (w\v) SDS, 87 %
(v\v) glycerol, 1.12 g\ml 2-mercaptoethanol and 0.05 %
Bromophenol Blue, and denatured at 80 mC for 10 min.
The apoB-100 standard (0.1–1.0 µg of protein) and TGRL
fractions were applied on to two 2–14 % (w\v) polyacrylamide gradient gels in the presence of SDS in a MiniProtean II vertical gel apparatus (Bio-Rad), and run
simultaneously at 90 V for 10 min and then at 200 V for
60 min. The gels were stained with 0.04 % Coomassie
Blue G-250 and destained with 5 % acetic acid. They
were scanned using a CliniScan 2 densitometer (Helena
Laboratories), and the area under each peak was integrated to obtain the absorbance. The linear regression
line between the absorbance and the known LDL apoB100 mass was used to calculate the apoB-48 concentration
in the TGRL fraction. ApoB-100 was not measured in
the TGRL fraction, as it needs extra gel application of
10–20-fold diluted samples.
were calculated. A P value of
significant.
RESULTS
Baseline characteristics
The mean age, body mass index, blood pressure, apoE
phenotype distribution, frequency of smokers, and dietary fat and cholesterol intakes were comparable in the
two groups (Table 1). The patients with CAD used betablockers more frequently than the control subjects. The
only differences between baseline lipids and lipoproteins
were the higher concentrations of LDL cholesterol,
squalene in serum, TGRL and d 1.006 lipoproteins,
and TGRL apoB-48 in the women with CAD compared
with the controls.
Table 1
Lipid and apolipoprotein analyses
Cholesterol and triacylglycerols were quantified using
commercial kits (Boehringer Diagnostica). ApoB was
assayed immunoturbidimetrically with anti-(human
apoB) antiserum using a commercial kit (Orion Diagnostica, Espoo, Finland). ApoE phenotypes were determined by isoelectric focusing [24]. Retinyl palmitate was
quantified by HPLC using a normal-phase LC-Si column
(Supelcosil ; 250 mmi4.6 mm) at 326 nm [25]. Squalene
was quantified by GLC using a Hewlett Packard Ultra I
column (50 m long) [26].
Data analysis
Continuous values are expressed as meanspS.E.M.
Responses to the fat meal were characterized in terms of
area under the incremental curve (AUIC) and incremental peak concentration (PIC). AUIC was calculated by
the trapezoidal method for 24 h curves. All postprandial
triacylglycerol and apoB-48 concentrations were below
baseline levels at 9 h, so that 9 h AUICs were calculated
for these parameters. PIC is defined as the difference
between the baseline concentration and the highest
postprandial concentration observed.
Statistical analysis was performed using the BMDP
statistical computer software package. Differences between continuous variables were tested using Student’s
two-tailed t-test and the Mann–Whitney rank sums test ;
those between discrete variables with the Chi-square test
and Fisher’s exact probability test ; and those between
postprandial concentrations and 24 h curves by analysis
of variance for repeated measures. Analysis of covariance
was used to calculate AUICs adjusted for baseline
parameters. Spearman’s rank correlation coefficients
0.05 was considered
Baseline characteristics
Values are meanspS.E.M. Significance of differences : *P 0.05, **P 0.01,
***P 0.001 compared with controls. For apoE, E2 l phenotypes 2/2 and
2/3 ; E3 l phenotype 3/3 ; E4 l phenotypes 2/4, 3/4 and 4/4.
Variable
CAD group (n l 24)
Controls (n l 30)
Age (years)
Body mass index (kg/m2)
Apolipoprotein E2/E3/E4 (n)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Smokers (n)
Beta-blocker users (n)
Dietary fat (g/day)
Dietary cholesterol (mg/day)
Serum cholesterol (mmol/l)
Total
VLDL
IDL
LDL
High-density lipoprotein
Serum triacylglycerol (mmol/l)
Total
VLDL
IDL
LDL
High-density lipoprotein
Serum apoB (mg/dl)
TGRL apoB-48 (mg/l)
Squalene (µg/dl)
Serum
TGRL
d 1.006 lipoproteins
Plasma retinyl palmitate (µg/l)
52.3p0.5
25.4p0.8
1/10/13
134.3p4.2
83.6p1.6
10
15***
75.8p4.4
280.5p21.1
52.9p0.4
27.1p1.0
1/19/10
135.1p3.7
86.4p1.8
10
3
78.1p6.0
281.4p18.7
6.08p0.21
0.38p0.05
0.24p0.02
3.75p0.17*
1.29p0.05
5.72p0.16
0.37p0.04
0.19p0.02
3.24p0.13
1.31p0.05
1.26p0.09
0.70p0.08
0.09p0.01
0.26p0.01
0.15p0.01
115.3p4.6
3.10p0.39**
1.27p0.08
0.67p0.07
0.09p0.01
0.26p0.01
0.17p0.01
103.9p4.6
1.67p0.22
89.3p3.5***
19.4p1.5*
71.0p3.0***
19.6p2.3
61.1p2.9
14.9p1.3
54.5p2.5
19.4p1.8
# 1999 The Biochemical Society and the Medical Research Society
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R. A. Rajaratnam, H. Gylling and T. A. Miettinen
Figure 1 Postprandial 24 h curves for triacylglycerols (triglycerides), squalene, retinyl palmitate and apoB-48 in TGRL and
in d 1.006 lipoproteins in women with CAD ($) and controls (#)
Curves and concentrations were significantly different for TGRL squalene (F l 2.50, P l 0.022 and F l 8.33, P l 0.006 respectively) and TGRL apoB-48 (F l
8.25, P 0.000 and F l 16.8, P 0.000 respectively) in the women with CAD compared with controls (as calculated by analysis of variance for repeated measures).
Postprandial responses
Triacylglycerols
The triacylglycerol concentrations, 9 h AUICs and PIC
for TGRL, chylomicrons, VLDL and d 1.006 lipoproteins were similar in the CAD and control subjects
(Figures 1 and 2 ; Table 2).
Squalene
The average concentrations and the 24 h curves for
squalene were higher in the TGRL (F l 8.33, P l 0.006
and F l 2.50, P l 0.022 respectively ; Figure 1), chylomicron (F l 6.01, P l 0.017 and F l 2.07, P l 0.056
# 1999 The Biochemical Society and the Medical Research Society
respectively) and VLDL (F l 8.22, P l 0.006 and F l
2.37, P l 0.029 respectively) fractions of the women with
CAD compared with the control group (Figure 2). In
addition, the AUICs [including AUICs adjusted
for fasting TGRL squalene (5.75p0.48 and 4.28p
0.43 mg:h−":dl−" respectively ; P 0.05) and for LDL
cholesterol (6.02p0.54 and 4.11p0.47 mg:h−":dl−" respectively ; P 0.01)] and PIC values in TGRL, chylomicrons and VLDL were clearly raised in the women
with CAD (Table 2). The CAD patients with and without
beta-blocker medication had similar AUICs for TGRL
squalene (5.90p0.90 and 6.64p1.05 mg:h−":dl−" respectively). Postprandial squalene concentrations in d Postprandial lipaemia in female coronary heart disease
Figure 2 Postprandial 24 h curves for triacylglycerols (triglycerides), squalene and retinyl palmitate in chylomicrons and
VLDL in women with CAD ($) and controls (#)
Concentrations and curves were different for chylomicron squalene (F l 6.01, P l 0.017 and F l 2.07, P l 0.056 respectively) and VLDL squalene (F l 8.22,
P l 0.006 and F l 2.37, P l 0.029 respectively) in the women with CAD compared with controls (as calculated by analysis of variance for repeated measures).
1.006 lipoproteins peaked at about 9 h, and subsequently
returned to basal values at 24 h (Figure 1), with similar
AUICs in the two groups.
The time to peak squalene concentration was significantly shorter in chylomicrons (6.97p0.36 h) than in
VLDL (8.63p0.46 h) in the healthy controls only (P
0.01). The squalene\triacylglycerol ratio was clearly
higher in chylomicrons than in VLDL throughout the
24 h period in both groups (Table 3). The ratios in
chylomicrons and VLDL at 24 h were significantly
higher in the women with CAD than in controls.
Retinyl palmitate
Postprandial retinyl palmitate concentrations, AUICs
and PIC in the TGRL, chylomicron and d 1.006
lipoprotein fractions were similar, but the AUICs and
PIC for the VLDL fraction were significantly higher in
the CAD patients compared with the controls (Figures 1
and 2 ; Table 2). The concentration peaked significantly
earlier in chylomicrons than in VLDL (6.10p0.28 and
8.06p0.50 h respectively ; P 0.01) in the control subjects only, and peaked at about 12 h in d 1.006
lipoproteins in both groups (Figure 1). Furthermore,
incremental 24 h retinyl palmitate concentrations in d 1.006 lipoproteins were increased in both the CAD
(j24.7p2.8 µg\l ; P 0.0001) and control (j30.4
p2.1 µg\l ; P 0.0001) groups, despite normalization of
the squalene concentration at this time. The retinyl
palmitate\triacylglycerol ratio was consistently higher in
chylomicrons than in VLDL throughout the postprandial
period, and at 3 h was higher in the controls than in
women with CAD (Table 3).
TGRL apoB-48
The TGRL apoB-48 concentration (F l 16.8, P 0.000),
especially at 6 h (2.63p0.63 and 1.21p0.25 mg\l re# 1999 The Biochemical Society and the Medical Research Society
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R. A. Rajaratnam, H. Gylling and T. A. Miettinen
Table 2
Postprandial AUIC and PIC values
Values are meanspS.E.M. Significance of differences compared with controls : *P 0.05 ; **P
incremental curves were used. Other AUICs were calculated using 24 h incremental curves.
0.01. For triacylglcerol and apoB-48 AUICs, the areas under 9 h
AUIC
Postprandial parameter
CAD subjects
Triacylglycerols
TGRL
Chylomicrons
VLDL
d 1.006 lipoproteins
Squalene
TGRL
Chylomicrons
VLDL
d 1.006 lipoproteins
Retinyl palmitate
TGRL
Chylomicrons
VLDL
d 1.006 lipoproteins
ApoB-48
TGRL
(mmol:h−1:l−1)
6.02p0.57
3.93p0.37
2.29p0.25
0.41p0.06
(mg:h−1:dl−1)
6.07p0.66**
3.27p0.39*
2.81p0.33*
1.23p0.12
(mg:h−1:l−1)
3.44p0.29
1.88p0.16
1.56p0.19*
0.54p0.05
(mg:h−1:l−1)
20.6p2.8**
Table 3
PIC
Controls
4.02p0.32
2.13p0.92
1.89p0.19
1.04p0.08
3.04p0.19
1.88p0.14
1.17p0.08
0.61p0.03
11.0p1.2
4
6
9
12
24
Controls
1.16p0.09
0.83p0.08
0.36p0.03
0.15p0.03
519.1p43.3
333.8p33.0
204.9p19.1
106.2p5.9
394.5p33.7
286.7p27.8
119.8p8.6
40.3p2.3
2.17p0.23
Squalene/triacylglycerol (SQ/TG) and retinyl palmitate/triacylglycerol (RP/TG) ratios in chylomicrons and VLDL
0.05, **P
SQ/TG ( µg/mmol)
3
(mmol/l)
1.32p0.13
0.93p0.09
0.42p0.04
0.13p0.03
(µg/dl)
761.1p78.1*
456.0p50.4*
315.3p33.5**
123.2p10.2
(µg/l)
452.9p40.2
294.9p28.6
169.6p19.2*
39.2p3.1
(mg/l)
3.61p0.53*
5.16p0.41
3.51p0.31
1.94p0.17
0.32p0.05
Values are meanspS.E.M. Significance of differences compared with controls : *P
Time (h)
CAD subjects
0.01, ***P
0.001 (Mann–Whitney rank sums test).
RP/TG ( µg/mmol)
Group
Chylomicrons
VLDL
Chylomicrons
VLDL
Cases
Controls
Cases
Controls
Cases
Controls
Cases
Controls
Cases
Controls
Cases
Controls
11.2p2.3
10.9p2.2
24.4p4.6
18.4p3.5
91.3p12.1
69.6p7.9
233.5p28.9
163.0p13.2
80.8p14.1
54.9p6.7
18.1p1.7**
14.5p2.1
4.82p0.81
4.53p0.84
10.1p1.4
7.56p1.05
34.8p5.3
27.3p3.4
163.0p13.2
62.7p7.4
44.7p7.1
35.9p3.6
4.94p0.44***
3.29p0.14
104.5p22.1**
160.3p19.3
205.4p37.8
222.8p33.3
594.5p88.4
643.9p73.2
1184.5p171.7
1131.8p81.4
360.6p55.1
347.3p38.4
54.7p7.4
60.9p8.4
33.1p7.96*
48.2p8.4
66.4p10.0
70.1p9.4
173.1p22.7
185.7p24.4
339.5p73.4
330.4p34.7
219.5p40.1
202.9p18.1
25.8p2.5
30.2p1.7
spectively ; P 0.01) and the curve (F l 8.25, P 0.000)
for the CAD patients differed significantly from those of
the controls (Figure 1, bottom panel). The women with
CAD had also higher 9 h AUICs [including AUICs
adjusted for baseline concentrations of TGRL apoB-48
(19.9p2.3 and 11.5p1.9 mg:h−":l−" respectively, P l
0.01) and for LDL cholesterol (19.2p2.2 and 11.7p
1.9 mg:h−":l−" respectively, P 0.05)], PIC and time
to peak concentration (4.87p0.21 and 4.33p0.14 h respectively, P 0.05) compared with controls (Table 2).
# 1999 The Biochemical Society and the Medical Research Society
The AUICs were similar in the subjects with and without beta-blocker medication (20.7p2.8 and 22.3p
6.3 mg:h−":l−" respectively).
Effects of hormone replacement therapy
The controls with and without post-menopausal hormone replacement therapy had similar postprandial 9 h
AUICs for triacylglycerols (5.17p0.63 and 5.15p
0.55 mmol:h−":l−" respectively) and apoB-48 (10.8p
Postprandial lipaemia in female coronary heart disease
Correlations between postprandial AUIC values for the TGRL fraction and baseline parameters in all subjects (n l 54)
AUICs were calculated under 24 h incremental curves, except for triacylglycerols and apoB-48, for which 9 h-incremental curves were used. Significance of correlations :
*P 0.05 ; **P 0.01 ; ***P 0.001.
Table 4
Correlation
Baseline variable
Serum triacylglycerol
Serum cholesterol
LDL cholesterol
HDL cholesterol
VLDL cholesterol
TGRL squalene
TGRL apoB-48
AUIC …
Squalene
Triacylglycerol
Retinyl palmitate
0.42**
0.29*
0.28*
k0.17
0.49***
0.61***
0.42**
0.52***
0.18
0.16
k0.20
0.58***
0.58***
0.41**
0.30*
0.27*
0.16
0.03
0.36**
0.64***
0.25
1.9 and 11.4p1.5 mg:h−":l−") and 24 h AUICs for
squalene (4.17p0.52 and 3.93p0.41 mg:h−":dl−" respectively) and retinyl palmitate (3.11p0.32 and 2.98p
0.26 mg:h−":l−" respectively) in TGRL.
Correlations
The fasting apoB-48 level was significantly correlated
with the concentrations of squalene (r l 0.71), triacylglycerols (r l 0.63) and cholesterol (r l 0.58) in the
TGRL fraction. Basal serum triacylglycerols, TGRL
squalene and VLDL cholesterol were positively correlated with the postprandial AUICs of triacylglycerols,
squalene, retinyl palmitate and apoB-48 in the TGRL
fraction (Table 4). The postprandial AUIC for
TGRL apoB-48 was also correlated positively with the
fasting concentrations of TGRL apoB-48 and LDL
cholesterol. The AUICs for squalene and apoB-48 were
significantly correlated with the AUICs for triacylglycerols (r l 0.72 and r l 0.61 respectively) and retinyl
palmitate (r l 0.82 and r l 0.53 respectively) in the
TGRL fraction. The higher the fasting serum and LDL
cholesterol and squalene concentrations, the higher were
the squalene AUICs in TGRL or in chylomicrons.
DISCUSSION
The present study shows for the first time that postmenopausal women with angiographically demonstrated
CAD without hormone replacement therapy have altered
postprandial metabolism of squalene and apoB-48 in the
TGRL fraction as compared with healthy control women
matched for age and body mass index. The findings
extend a previous study, which demonstrated that the
apoB-48 level only in postprandial IDL particles was
abnormally high in women with CAD compared with
controls with valvular heart disease [15]. Other major
findings of the present study were that postprandial
VLDL accumulated in women with CAD, and that
hormone replacement therapy did not affect the post-
ApoB-48
0.36**
0.26
0.34*
k0.18
0.49***
0.50***
0.33*
prandial metabolism of squalene, apoB-48 and retinyl
palmitate in healthy post-menopausal women. Squalene
could label postprandial lipoproteins with higher specificity than vitamin A, since the squalene AUICs were
higher for chylomicrons and VLDL in women with
CAD. In addition, postprandial squalene in d 1.006
lipoprotein peaked earlier than retinyl palmitate, and had
returned to normal at 24 h, in both groups.
A greater postprandial rise in intestinally derived
lipoproteins could be attributed either to effective and
prolonged intestinal absorption of lipids or to their
retarded clearance. Lipoprotein lipase hydrolyses triacylglycerols of large chylomicrons or VLDL particles,
followed by hepatic catabolism of the resulting remnants.
The similar triacylglycerol responses in the plasma and
chylomicrons in the women with CAD and the controls
after the fat meal suggest that the subjects with CAD are
unlikely to have delayed fat absorption or inefficient
lipolytic activity. Identical postprandial 4 h curves of
apoB-48 in the CAD and control groups (Figure 1,
bottom panel) suggest that the formation of intestinal
lipoproteins was similar. The higher postprandial AUICs
in the CAD group were mostly accounted for by the
increased areas under the 4–24 h curves for squalene and
the 4–9 h curves for apoB-48 (Figure 1). Thus it can be
inferred that the clearance of intestinally derived lipoproteins is diminished in women with CAD. Lipoprotein
lipase activity appears to be similar in women with and
without CAD [15], but chylomicrons and VLDL particles compete for the same lipolytic activity [27].
Oestrogens may up-regulate hepatic LDL receptors [28]
and result in the rapid clearance of intestinal lipoproteins
[29,30], whereas progestin, in contrast with oestrogen,
enhances hepatic lipase activity [31]. The present study
showed no effect of hormone replacement therapy
(oestrogen in the presence or absence of progestin), in
agreement with a previous study [32].
The diminished postprandial clearance of lipoproteins
in the women with CAD was strongly substantiated by
the responses of squalene and apoB-48, weakly by that of
# 1999 The Biochemical Society and the Medical Research Society
189
190
R. A. Rajaratnam, H. Gylling and T. A. Miettinen
retinyl palmitate, and insignificantly by that of triacylglycerol. Patients with CAD have been shown to exhibit
similar postprandial chylomicron triacylglycerol AUICs,
but higher retinyl palmitate AUIC responses of chylomicron remnants, in a case control follow-up study [13].
On the other hand, similar AUICs for chylomicron
retinyl palmitate and TGRL apoB-48, but higher respective chylomicron triacylglycerol values, have been
demonstrated previously in women with CAD compared
with controls [15]. As intestinal particles are hydrolysed
before hepatic uptake and are mixed with endogenous
VLDL or its remnants, the ability of postprandial
triacylglycerols to label intestinal lipoproteins should be
considered carefully. Postprandial responses of squalene
and retinyl palmitate were correlated significantly with
each other in the TGRL fraction, similar to earlier studies
[19]. However, in d 1.006 lipoproteins, which also
contain remnants of IDL density, postprandial squalene
peaked almost 3 h earlier than retinyl palmitate (Figure 1 ;
P 0.001). In contrast with the retinyl palmitate levels,
the 24 h postprandial concentration of squalene had
returned to baseline in both groups, and in postprandial
lipoproteins the concentration of squalene was more
frequently increased than that of retinyl palmitate in the
women with CAD, indicating that squalene may be a
better marker than vitamin A for intestinally derived
lipoproteins.
More rapid attainment of peak postprandial triacylglycerol and apoB-48 concentrations compared with
those of squalene and retinyl palmitate (Figure 1) suggests
that the absorption of squalene and vitamin A is slower
than that of fat. In addition, squalene and retinyl esters, in
contrast with triacylglycerols, remain in the chylomicrons and chylomicron remnants until hepatic uptake
occurs. Consequently, lipoprotein particles of intestinal
origin should become triacylglycerol-depleted and
squalene- and retinyl ester-enriched in the later phases
of the postprandial period. In fact, the squalene\
triacylglycerol and, in particular, retinyl palmitate\
triacylglycerol ratios were clearly higher in chylomicrons
than in the VLDL fractions, probably due in part to
hepatic VLDL, indicating that, in proportion to triacylglycerols, the two markers accumulated mainly in chylomicrons throughout the postprandial period (Table 3).
On the other hand, the earlier peaking of squalene and
retinyl palmitate concentrations in chylomicrons compared with VLDL in the control subjects only suggested
that VLDL particles appeared more rapidly in the
circulation of the CAD women than of the controls.
Thus chylomicrons were converted rapidly into VLDL
remnants, or the latter were secreted directly from the
intestine, as has been shown previously [33,34].
The magnitude of postprandial lipaemia is usually
associated with basal lipid and lipoprotein levels [35–37].
Although the postprandial AUICs for squalene and
apoB-48 were positively associated with their baseline
# 1999 The Biochemical Society and the Medical Research Society
concentrations, adjusted AUICs for the fasting values
were still significantly higher in the women with CAD
than in the controls. In addition, the possible confounding effects of diabetes [38], obesity [39], smoking
[40] and apoE phenotype distribution [41,42] on postprandial lipaemia were avoided in the present study,
because these parameters were similar in the two groups.
The subjects with CAD taking beta-blocker medication
tended to have lower postprandial AUICs for squalene
and apoB-48 ; thus, among the non-users, the CAD
patients showed higher postprandial responses than the
controls.
ApoB-48 is essential for the synthesis of intestinally
derived lipoproteins, and the circulating pool of this
protein is regulated by the intestine, depending on fat
intake. Biliary lipids, intestinal fatty acid uptake and the
microsomal triacylglycerol content of mucosal cells
actually regulate intestinal apoB-48 synthesis in rats [43].
Elevated apoB-48 concentrations in humans could also
be explained by increased apoB mRNA editing activity
[44]. Interestingly, in the present study the fasting apoB48 concentration was strongly correlated with the
squalene concentration in the TGRL fraction. The
increased fasting squalene concentration in the women
with CAD could not be explained by the higher LDL
cholesterol level, because the squalene\cholesterol ratio
was also increased. Whether this depends on enhanced
cholesterol synthesis or altered metabolism of squalene
should be shown by further non-cholesterol sterol and
sterol balance studies.
In summary, the retarded postprandial clearance of
lipoproteins and squalene, particularly of VLDL density,
in women with CAD may have significant implications
for the development of CAD in women.
ACKNOWLEDGMENTS
This study was supported by grants from the Clinical
Research Institute of Helsinki University Central Hospital, the Research Foundation of Orion Corp., Ida
Montin’s Foundation, the Finnish Academy of Medical
Sciences and Helsinki University Central Hospital. The
valuable technical assistance of Leena Kaipiainen, Anja
Salolainen, Jaana Tuomikangas, Leena Saikko, Pia
Hoffstro$ m, Orvokki Ahlroos and Elli Kampas is gratefully acknowledged.
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Received 12 January 1999/18 March 1999; accepted 23 April 1999
# 1999 The Biochemical Society and the Medical Research Society