Ming-Lin Liu, Kati Ylitalo, Riitta Salonen, Jukka T. Salonen and Marja-Riitta Taskinen
Intima-Media Thickness in Asymptomatic Members of Familial Combined
Circulating Oxidized Low-Density Lipoprotein and Its Association With Carotid
Print ISSN: 1079-5642. Online ISSN: 1524-4636
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2004;24:1492-1497; originally published online June 17, 2004;
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Circulating Oxidized Low-Density Lipoprotein and Its
Association With Carotid Intima-Media Thickness in
Asymptomatic Members of Familial Combined
Ming-Lin Liu, Kati Ylitalo, Riitta Salonen, Jukka T. Salonen, Marja-Riitta Taskinen
Objective—Oxidized low-density lipoprotein (Ox-LDL)is implicated in the pathogenesis of atherosclerosis. Circulating
oxidation-specific epitopes on plasma Ox-LDL has been linked with coronary artery disease, but its determinants and
its association with early development of atherosclerosis in familial combined hyperlipidemia (FCHL) has not been very
well studied. This study aimed to investigate the determinants of the circulating Ox-LDL and the association between
Ox-LDL and carotid intima-media thickness (IMT) in asymptomatic members of FCHL families.
Methods and Results—Ox-LDL, susceptibility of LDL to oxidation in vitro, plasma 8-isoprostane and antioxidants, lipids
and lipoproteins, LDL particle size, and carotid IMT were measured in 150 asymptomatic FCHL family members.
Affected FCHL family members had reduced LDL particle size and lag time for LDL oxidation, increased plasma levels
of Ox-LDL, increased plasma urate and ?-tocopherol, and a trend for the increase of 8-isoprostane as compared with
nonaffected FCHL. Ox-LDL was independently associated with serum LDL cholesterol, apoB, and 8-isoprostane in
multivariate analysis but only univariately correlated with LDL particle size and lag time for LDL oxidation. In addition,
Ox-LDL was significantly associated with carotid mean IMT independently of other clinical and biochemical variables
in a multivariate model.
Conclusion—Serum LDL cholesterol, apoB levels, and 8-isoprostane were the most important determinants of Ox-LDL.
Ox-LDL is independently associated with carotid IMT in asymptomatic FCHL family members and can be used as a
marker of early atherosclerosis in FCHL. (Arterioscler Thromb Vasc Biol. 2004;24:1492-1497.)
Key Words: carotid arteries ? hyperlipoproteinemia ? familial combined ? lipoproteins ? low-denisty lipoprotein
? oxygen radical ? ultrasonography
rotic lesions of arteries.1Under the oxidative stress, oxidative
modification of LDL may take place in the subendothelial space
of the arterial wall,1and a small amount of Ox-LDL may also be
released into the circulation.2When “fully oxidized LDL” enters
the circulation in minor quantities, it will be rapidly cleared by
the reticuloendothelial system, particularly in the liver, or it will
be removed by the preexisting circulating autoantibodies to
Ox-LDL.3In contrast, the “minimally modified LDL,” in which
oxidative modification has not been sufficient to cause changes
recognized by scavenger receptors, can be found in circula-
tion.4,5Other studies have defined the presence of oxidation-
specific epitopes on plasma LDL6–8or baseline levels of
conjugated dienes in lipids extracted from LDL (LDL-BDC) as
measures of LDL oxidation in vivo.9Recently, several groups
here is substantial evidence that oxidized low-density li-
poprotein (Ox-LDL) is present in vivo within atheroscle-
have developed several specific methods to measure circulating
Ox-LDL using different anti–Ox-LDL antibodies.6–8As a sen-
sitive biochemical marker, Ox-LDL has been related to coronary
artery disease (CAD) in several clinical studies.6,10–12Plasma
Ox-LDL has also been associated with subclinical atherosclero-
sis in clinically healthy population.13Interestingly, high plasma
and plaque levels of Ox-LDL are associated with the vulnera-
bility of the plaques.2However, the origin of plasma Ox-LDL as
well as its determinants are unknown.
Familial combined hyperlipidemia (FCHL) is character-
ized with elevated levels of plasma cholesterol and/or triglyc-
eride. FCHL associates with early atherosclerosis and is
responsible for ?10% of premature CAD.14,15Experimental
studies have observed the enhanced generation of free radi-
cals in leukocytes from hypercholesterolemic and hypertri-
glyceridemic patients.16,17Previous studies also reported the
Received April 30, 2004; accepted May 19, 2004.
From the Department of Medicine (M.-L.L., K.Y., M.-R.T.), Helsinki University Central Hospital, University of Helsinki, Finland; Research Institute
of Public Health (R.S., J.T.S.), University of Kuopio, Finland; and Inner Savo Health Centre (J.T.S.), Suonenjoki, Finland.
*M.-L.L. is currently working in Division of Endocrinology, Diabetes, and Metabolic Diseases, Department of Medicine, Thomas Jefferson University,
Correspondence to Prof Marja-Riitta Taskinen, Department of Medicine, C423b, PL 700, Biomedicum Helsinki, Haartmanninkatu 8, HUS-00029,
Helsinki, Finland. E-mail Marja-Riitta.Taskinen@helsinki.fi
© 2004 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.orgDOI: 10.1161/01.ATV.0000135982.60383.48
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increased formation of isoprostanes, markers of in vivo
oxidative stress, in hyperlipidemic patients.18,19Interestingly,
our data have shown an adaptive increase of circulating
antioxidants with increased oxidative stress in asymptomatic
FCHL patients.20In addition, LDL from FCHL patients is
characterized by a predominance of small dense LDL21and
an increased susceptibility to oxidative modification.20All
these factors may influence the metabolism of circulating
Ox-LDL in FCHL.
Carotid artery intima-media thickness (IMT), measured
noninvasively by high-resolution B-mode ultrasonography,
has been associated with the risk of CAD, stroke, and
myocardial infarction, and it predicts the progression of
CAD.22The present study was conducted to investigate the
determinants of plasma Ox-LDL and the association between
Ox-LDL and carotid IMT in asymptomatic FCHL family
The study subjects were recruited according to the study protocol as
reported previously.23All subjects gave their informed consent to the
study protocol, which was approved by the ethical committees. Briefly,
the FCHL probands were required to be 30 to 60 years of age, have
verified CAD, and have serum total cholesterol (TC) and/or triglycer-
ides (TG) age- and sex-specific levels in ?90th Finnish population
percentiles. The TC and TG percentiles used in the present study were
derived from the results of the surveys based on the Finnish popula-
lipid phenotypes were classified as FCHL. Family members who had
diabetes or history of CAD or stroke and those with lipid medication
were excluded. As described previously,26altogether 150 FCHL (75
affected and 75 nonaffected defined according to the aforementioned
lipid criteria) family members from 38 well-defined Finnish FCHL
families participated in the present study.
Venous blood samples were collected after overnight fasting for the
biochemical measurements. EDTA plasma was separated by centrifu-
gation and stored at ?80°C until analyzed. Pulse pressure was calcu-
lated as the difference between the systolic and diastolic blood pres-
sures. Pack-years were calculated by multiplying duration of smoking
by the number of cigarettes smoked per day divided by 20.
Measurement of Plasma Ox-LDL and LDL
Oxidation In Vitro
Plasma levels of Ox-LDL were measured by a competitive enzyme-
linked immunosorbent assay using a specific murine monoclonal anti-
variation for the assay was 7.4% to 8.3%. The mAb-4E6 is directed
against a conformational epitope in the apolipoprotein B-100 (apoB-
100) moiety of LDL that is generated as a consequence of substitution
of at least 60 lysine residues of apoB-100 with aldehydes. This number
of substituted lysines corresponds to the minimal number of substituted
lysines required for scavenger-mediated uptake of oxLDL. Substituting
aldehydes can be produced by peroxidation of lipids of LDL.6LDL for
the in vitro oxidation measurement was isolated by a short-run ultracen-
trifugation.27EDTA was removed from LDL using size exclusion
chromatography (PD-25 column) just before LDL oxidation in vitro.
Altogether 100 ?g LDL protein/mL was incubated with 5 ?mol/L
CuSO4in a total volume of 2 mL at 27°C in a temperature-controlled
Other Biochemical Measurements
Plasma 8-isoprostane levels were measured using an EIA kit (516351;
Cayman, Ann Arbor, Mich). Plasma vitamin C, protein-bound thiol
groups, urate, ?-tocopherol, ?-carotene, and retinol were measured as
described.27LDL particle size was determined using gradient gel
electrophoresis.21,26All lipid and lipoprotein measurements were per-
formed as described previously.23Briefly, serum TC and TG concen-
trations were determined enzymatically, serum high-density lipoprotein
(HDL) cholesterol by precipitation procedures, and serum apolipopro-
separated by sequential flotation as described.28
Carotid IMT was determined as described previously.26Briefly,
longitudinal images from 3 projections (anterolateral, lateral, and
posterolateral) were measured by Hewlett-Packard Image Point
M2410A ultrasound system for the common carotid artery, carotid
bulb, and internal carotid artery. Measurements were performed at a
total of 28 sites, both the far wall and the near wall of the arterial
segments, right and left distal 1 cm of common carotid artery, carotid
bulb, and proximal 1 cm of and internal carotid artery. All 3
projections in common carotid artery and carotid bulb, and a single
angle in and internal carotid artery with the best visibility were used.
The mean, maximum, and minimum IMT were derived from each
measurement. The average of all mean IMT measurements over 28
(or fewer) sites was chosen as the outcome variable. The coefficient
of variation for mean IMT measurements was 2.4%. Carotid IMT
examination was performed at the same visit as blood sampling or
within a difference of few weeks.
Values are given as means?SE. Variables with nonnormal distribu-
tion were log10-transformed. Differences in means between affected
and nonaffected family members or among the different Ox-LDL
tertile groups were tested by 2-way ANOVA (Figure A). The
frequency distribution of the categorical variables between 2 groups
was compared by the ?2test. Univariate association was tested by
Pearson correlation analysis. The predictors for the subsequent
multivariate analysis were selected on the basis of the correlation
analysis (P?0.20). Backward multivariate analyses were performed
to assess the predictors of Ox-LDL and carotid mean IMT. Both in
the ANOVA and the backward multivariate analyses, family number
(which indicates belonging to a certain family) was used to correct
for the dependence of the study subjects.
Ox-LDL and Other Study Variables of the FCHL
Table 1 summarizes clinical and other study variables of the
subjects.26By definition, TC, TG, LDL cholesterol, and apoB
were significantly higher in affected than in nonaffected family
members. Affected family members had significantly smaller
Ox-LDL as compared with those in nonaffected members.
Plasma 8-isoprostane levels tended to be higher in affected than
in nonaffected family members, but the difference did not reach
statistical significance. Plasma ?-tocopherol and urate were
significantly increased in affected FCHL compared with those in
nonaffected family members as reported in our previous study.26
Other plasma antioxidants (vitamin C, thiol groups, ?-carotene,
retinol) were comparable between the 2 groups (data not
shown).26As reported previously, we did not see significant
difference in carotid mean IMT between affected and nonaf-
fected family members.26
Associations Between Ox-LDL, Related Variables,
and Carotid Mean IMT
In the univariate correlation analysis, Ox-LDL was signifi-
cantly correlated with age, gender, body mass index, smoking
pack-years, log TG, LDL cholesterol, HDL cholesterol, apoB,
Liu et alOxidized LDL and Carotid IMT in FCHL
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LDL particle size, lag time for LDL oxidation, plasma urate,
?-tocopherol, and retinol, but not with 8-isoprostane, vitamin
C, thiol groups, and ?-carotene. Interestingly, plasma Ox-
LDL and 8-isoprostane were correlated in affected family
members (r?0.349, P?0.002). In the multivariate analysis,
LDL-C, apoB, and 8-isoprostane were associated with plasma
Ox-LDL independently of other variables (Table 2).
When FCHL family members were divided by tertiles of
Ox-LDL (50 subjects in each tertile group), the carotid mean
IMT increased over tertiles of Ox-LDL (Figure A). Carotid
mean IMT in highest Ox-LDL tertile group was significantly
thicker than that in lowest Ox-LDL tertile (0.78?0.02 versus
0.69?0.02 mm, P?0.003). In addition, there were more
affected subjects in the highest Ox-LDL tertile group (40/50)
than in the middle (23/50) and in the lowest (12/50) Ox-LDL
tertile groups (P?0.001, ANOVA; Figure A). Plasma Ox-
LDL correlated with carotid mean IMT in affected and
nonaffected family members as well as in the combined
group including all family members (Figure B).
Carotid mean IMT correlated significantly with age, body
mass index, smoking pack-years, pulse pressure, logTG, LDL
cholesterol, apoB, LDL size, lag time for LDL oxidation,
plasma urate, and ?-tocopherol, as well as Ox-LDL. How-
ever, we did not observe any correlation between
8-isoprostane and mean IMT in univariate analysis. In the
multivariate analysis, only age (??0.745, P?0.001), pulse
pressure (??0.158, P?0.004), LDL size (???0.169,
P?0.022), and Ox-LDL (??0.178, P?0.038) were indepen-
dent predictors for the variation of carotid mean IMT. The
association between Ox-LDL and mean IMT persisted even
after adjustment for logTG, LDL cholesterol, and apoB.
These results demonstrate that Ox-LDL is associated with
mean IMT in FCHL family members independently of
clinical and lipid variables.
The present study shows that LDL cholesterol, apoB, and
8-isoprostane were independent determinants of plasma Ox-
LDL. The Ox-LDL was associated with carotid mean IMT
independently of other variables in asymptomatic FCHL
family members. Therefore, the Ox-LDL is a useful marker
of early atherosclerosis in FCHL.
Circulating Ox-LDL, 8-Isoprostane,
There is increased oxidative stress in dyslipidemia.16–19In this
study, the plasma Ox-LDL was significantly higher in affected
FCHL family members as compared with nonaffected subjects.
Likewise, there was a trend for an increase of the plasma
8-isoprostane in affected FCHL family members. The present
data confirmed our previous results20showing an adaptive
increase of plasma antioxidants (?-tocopherol and urate) in the
presence of the increased oxidative stress in FCHL. Recent data
suggested that supplementation of ?-tocopherol cannot prevent
lipoprotein oxidation in the vessel wall with the increased levels
of ?-tocopherol in the circulation and in the arterial wall.29A
population study showed that ?-tocopherol supplementation in
healthy individuals increases plasma levels of ?-tocopherol and
reduces LDL oxidative susceptibility and circulating oxidized
LDL.30In contrast, several other clinical studies have reported
that supplementation with ?-tocopherol has no effect on auto-
antibodies against Ox-LDL in hyperlipidemic patients,31in
patients with chronic renal failure,32or in chronic smokers.33In
agreement with the latter results, the plasma levels of Ox-LDL
were increased in affected FCHL family members, despite the
elevation of plasma ?-tocopherol. In addition, the low-fat,
high-vegetable diet, which increased plasma concentrations of
vitamin C, ?-carotene, and lycopene, failed to decrease plasma
levels of Ox-LDL measured with mAb-EO6.34The positive
correlation between Ox-LDL and plasma antioxidants (Table 2)
in FCHL family members may reflect the fact that adaptive
increase in plasma antioxidants cannot protect lipoprotein lipids
against oxidation in the arterial wall.29
Box plots illustrating the distribution of values of carotid mean
IMT in tertiles of circulating Ox-LDL in FCHL family members
without clinical CAD (A). The boxes show the middle 50% of the
data for each grouping with horizontal bars at the quartiles and
the median. *Individual data points for the extremes of the data.
Relationship between carotid mean IMT and circulating Ox-LDL
in FCHL family members (B). Black circles and solid line indicate
affected family members; white circles and dash line indicate
nonaffected family members.
1494 Arterioscler Thromb Vasc Biol.
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Potential Mechanisms of Production of
The characteristics of Ox-LDL isolated from plasma of CAD
patients are comparable to those of Ox-LDL isolated from
atherosclerotic lesions.35Lysine-aldehyde adducts, which
may be a marker of Ox-LDL, are detected in the macrophage-
rich lesions of human and rabbit aorta.36In the present study,
Ox-LDL was measured by a specific enzyme-linked immu-
nosorbent assay method in which mAb-4E6 is directed
against a conformation epitope in the apoB-100 moiety of
LDL with at least 60 aldehyde-substituted lysine residues of
apoB-100.6The mAb-4E6 is specific to Ox-LDL but, to some
extent, it also detects circulating malondialdehyde (MDA)-
modified LDL.35Several clinical studies have shown that
plasma levels of circulating Ox-LDL measured by mAb-4E6
are associated with CAD6,10or subclinical carotid atheroscle-
rosis.13The potential origin of circulating Ox-LDL may be a
direct release of modified LDL from ruptured or permeable
plaques, or ischemic injury.2,3Generation of Ox-LDL in
arterial wall is probably affected by susceptibility of LDL to
oxidation, the particle size, and the number of LDL in the
circulation, the composition of LDL, and local oxidative
stress in the arterial wall. Thus, circulating Ox-LDL may
reflect the combined effect of these factors via additive and
To the best of our knowledge, this is the first study
analyzing systematically the potential determinants of circu-
lating Ox-LDL in FCHL family members without clinical
CAD (Table 2). LDL particle size, as well as plasma
triglycerides, and apoB levels correlated positively with
Ox-LDL, whereas HDL cholesterol was negatively related to
Ox-LDL. Small LDL particles penetrate into the subendothe-
lial space more easily and have high binding affinity with
proteoglycans. This process promotes LDL modification in
the arterial wall.37In keeping with our results, other studies
have shown that small dense LDL is associated with high
levels of circulating MDA-LDL38or autoantibodies against
MDA-LDL.39The negative correlation between circulating
Ox-LDL and the lag time of LDL oxidation in vitro suggests
that LDL particles susceptible to oxidation in vitro may also
be easily oxidized in vivo. The antioxidant actions of HDL40
may explain the negative correlation between circulating
Ox-LDL and HDL. In the multivariate analysis, the concen-
trations of serum LDL cholesterol, apoB, and plasma
8-isoprostane remained the independent determinants of Ox-
LDL, thus being the most important factors contributing to
the generation of circulating Ox-LDL (Table 2). In agree-
ment, a recent study demonstrated that the levels of circulat-
ing Ox-LDL in subjects with impaired glucose tolerance were
associated with components of dyslipidemia, but not with the
antioxidant parameters.41Several other studies have shown
that LDL and total cholesterol concentrations are related to
Ox-LDL measured by different antibodies.10,13,42Previous
studies have suggested that 8-isoprostane is formed during
the in vitro oxidation of LDL.43In this context, the increased
plasma levels of LDL cholesterol and apoB in FCHL indicate
FCHL Family Members
Clinical and Biochemical Characteristics and Other Study Variables in
Pulse pressure, mm Hg
LDL particle size, nm
LDL lag time, min
Circulating oxidized LDL, U/L
Carotid mean IMT, mm
Data are means?SE.
M/F indicates male/female; BMI, body mass index; U/L, unit/liter, LDL-C, low-density lipoprotein
cholesterol; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglycerides; NS,
Statistical comparisons between affected and nonaffected family members were performed by
2-way ANOVA. The frequency distribution of the categorical variables between the 2 groups was
compared by the ?2test.
Liu et al Oxidized LDL and Carotid IMT in FCHL
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the increased number of LDL particles available to penetrate
and consequently enhance invasion of LDL into the suben-
dothelium space, where the increased oxidative stress as
measured by 8-isoprostane will promote the oxidative mod-
ification of LDL.
Association Between Ox-LDL and Carotid IMT
Oxidative modification of LDL is believed to play an impor-
tant role in the development of atherosclerosis.1Susceptibility
of LDL to oxidation in vitro and autoantibodies against
Ox-LDL have been related with atherosclerotic diseases in
some, but not all, clinical studies.26,44–46Recently, circulating
Ox-LDL measured by different antibodies has been associ-
ated with cardiovascular diseases.10–12In the present study,
the carotid mean IMT increased by tertiles of Ox-LDL in the
combined group (Figure A). In addition, the relative numbers
of affected subjects were significantly increased in highest
tertile group as compared with those in the middle and in the
lowest tertile groups. These data suggest that the subjects
with an increased level of Ox-LDL have increased carotid
IMT, particularly in affected family members. Furthermore,
carotid mean IMT was independently associated with age,
pulse pressure, LDL size, and Ox-LDL in the univariate and
multivariate analyses. The association between Ox-LDL and
mean IMT persisted even after adjustment for logTG, LDL
cholesterol, and apoB. The data indicate that Ox-LDL is a
useful marker of the early stage of atherosclerosis in FCHL
family members without clinical CAD. These results are in
keeping with previous studies in which Ox-LDL was associ-
ated with the extent of CAD.6Likewise, Ox-LDL was
univariately correlated with IMT in carotid or femoral arteries
and independently associated with subclinical plaque occur-
rence in carotid and femoral arteries in healthy population.13
However, no association between carotid IMT and plasma
8-isoprostane was observed in the present study, in line with
the other studies.47,48Pulse pressure, a pulsatile component of
blood pressure determined by stiffness and elastic properties
of arterial walls, is reported to be independently associated
with carotid IMT in some studies.49In agreement, we
observed an independent association between carotid IMT
and pulse pressure.
Serum levels of LDL cholesterol, apoB, and plasma
8-isoprostane are the most important determinants of Ox-
LDL. Ox-LDL and LDL size were associated with carotid
IMT independently of other clinical and lipid variables in the
FCHL family members without clinical CAD. Therefore, our
results suggest that the quantity of LDL particles and the
oxidative stress in vivo determine the generation of circulat-
ing Ox-LDL, which may be a surrogate marker for CAD risk
in the early stage of atherosclerosis in FCHL.
The image analyses of carotid IMT were performed at Oy Jurilab Ltd
(www.jurilab.fi). Arja Malkki’s excellent work in reading the scan-
nings is gratefully acknowledged. The authors thank Hannele Hilden,
Helina ¨ Perttunen-Nio, Virve Vesterinen, Leena Lehikoinen, Ritva
Marjanen, and Tomi Silvennoinen for their skillful laboratory assis-
tance. We also appreciate the Finnish FCHL family members for
their participation in this study. This work was supported by the
Helsinki University Central Hospital Research Fund and the Finnish
Heart Foundation. The EUFAM study is supported by the European
Commission (contract number BMH4-CT96-1678).
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