Association of lipoprotein subclasses and carotid intima-media thickness in the Chinese population

Abstract

Aims: To determine whether lipoprotein subclasses are associated with the carotid intima-media thickness (IMT) in Chinese population. Patients & methods: Total cholesterol (TC), triglycerides, high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were measured by standard enzymatic methods. The subclasses of LDL were determined by 1D gradient gel electrophoresis and subclasses of HDL were determined by 2D gradient gel electrophoresis in 342 participants. Carotid IMT was measured by means of high-resolution vascular ultrasound. Results: In multivariate analysis, TC and LDL-C levels were positively correlated with the carotid IMT (p < 0.001). In stepwise multiple regression analysis, small dense LDL was found to be significantly associated with IMT after adjustment for other metabolic risks and traditional lipids such as triglycerides, TC and HDL-C. Additionally, HDL subclasses were not correlated with IMT. Conclusion: Small dense LDL was a stronger predictor for carotid atherosclerosis in the general Chinese population when compared with HDL-C or HDL subclasses, which is independent of lipids and other related metabolic risks.

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ISSN 1758-4299
Clin. Lipidol. (2014) 9(4), 407– 415
part of
Research Article Clinical Lipidology
Association of lipoprotein subclasses and
carotid intima-media thickness in the
Chinese population
Aims: To determine whether lipoprotein subclasses are associated with the carotid
intima-media thickness (IMT) in Chinese population. Patients & methods: Tot al
cholesterol (TC), triglycerides, high-density lipoprotein-cholesterol (HDL-C) and
low-density lipoprotein-cholesterol (LDL-C) were measured by standard enzymatic
methods. The subclasses of LDL were determined by 1D gradient gel electrophoresis
and subclasses of HDL were determined by 2D gradient gel electrophoresis in
342 participants. Carotid IMT was measured by means of high-resolution vascular
ultrasound. Results: In multivariate ana lysis, TC and LDL-C levels were positively
correlated with the carotid IMT (p < 0.001). In stepwise multiple regression ana lysis,
small dense LDL was found to be significantly associated with IMT after adjustment
for other metabolic risks and traditional lipids such as triglycerides, TC and HDL-C.
Additionally, HDL subclasses were not correlated with IMT. Conclusion: Small dense
LDL was a stronger predictor for carotid atherosclerosis in the general Chinese
population when compared with HDL-C or HDL subclasses, which is independent of
lipids and other related metabolic risks.
KEYWORDS: carotid intima-media thickness • Chinese • high-density lipoprotein subclasses
• lipid • low-density lipoprotein subclasses
Low-density lipoprotein-cholesterol (LDL-
C), a hallmark for risk assessment, has been
thought to be the primary target of lipid-
lowering therapy in the National Choles-
terol Education Program – Adult Treatment
Panel III. However, using the indicator of
LDL-C alone is insufficient to identify indi-
viduals with severe stable coronary heart dis-
ease (CHD), since these subjects have lower
LDL-C concentrations [1] . Thus, small dense
LDL (sdLDL) particles have been considered
to be another important factor in monitor-
ing the adequacy of a patient’s lipid-lowering
therapy. Many studies even revealed that car-
diovascular disease (CVD) is more strongly
related to sdLDL than LDL-C [2] . Interest-
ingly, several studies have shown that sdLDL
measured by nondenaturing polyacrylamide
gradient gel electrophoresis (GGE) was also
significantly associated with asymptomatic
carotid atherosclerosis in Type 2 diabetes [3] .
In addition, the inverse relation-
ship between high-density lipoprotein–C
(HDL-C) and CVD risk is well accepted in
epidemiological surveys [4]. However, contro-
versy still remains as to whether HDL sub-
classes play anti-inflammatory roles in anti-
atherogenesis. HDL is a heterogeneous class
of lipoproteins, which differ by their density,
size, electrophoretic mobility, apolipoprotein
content and associated enzymes. The multi-
ple enzymes associated with HDL particles,
including paraoxonase, glutathione peroxidase
and platelet-activating factor acetylhydrolase,
normally have antioxidant properties [5] . They
can modify atherogenic reactive oxygen spe-
cies in the vessel wall. While in the presence
of systemic inflammation, these enzymes were
then modified by reactive oxygen to be inac-
tive. Thus, the enzymatic changes in HDL
particles can impede cholesterol efflux and
induce the proinflammation in atherosclerosis.
Fangfang Yan1,2, Li Tian2,
Huangdao Yu2, Attit Baskota1,
Yun Gao1, Sheyu Li1, Mingde
Fu2 & Haoming Tian*,1
1Department of Endocrinology
& Metabolism, West China Hospital of
Sichuan University, Chengdu,
610041 Sichuan, P. R. China
2Laboratory of Endocrinology
& Metabolism, West China Hospital of
Sichuan University, Chengdu,
610041 Sichuan, P. R. China
*Author for correspondence:
Tel.: +86 02885422982
Fax: +8 6 02885423459
haomingtian666@126.com
Research Article
10.2217/CLP.14.22 © 2014 Future Medicine Ltd
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408 Clin. Lipidol. (2014) 9(4)
Furthermore, their physical characteristics were relevant
to their differential effects on the risk of CHD [6] . Stud-
ies revealed that pre-beta1-HDL was positively corre-
lated with carotid atherosclerosis [7] , while HDL2 and
HDL3 were inversely correlated with vascular disease
[8]. Recently, this concept has been challenged by sev-
eral studies demonstrating that the large HDL particles
showed a negative association with retinopathy, but no
relationship was seen to macrovascular risk [9].
Indeed, the standardization for the various analytic
methodologies achieving HDL separation is still lack-
ing. The early work with the analytical ultracentrifu-
gation led to the characterization of lipoprotein sub-
classes based on their ultracentrifugal flotation rates in
high-salt solutions. Recently, GGE delineated multiple
physically distinguished LDL and HDL subclasses,
ranging from very large to very small particles. There-
fore, it is reasonable to expect less than perfect inte-
rassay agreement. This may explain in part the con-
flicting results on the correlation of HDL subclasses
with vascular disease in epidemiological studies and
clinical trials [10 ] .
To date, only a few cross-sectional studies have
directly assessed the association of lipoprotein sub-
classes and carotid atherosclerosis. Alagona et al.
measured HDL subfractions by ultracentrifugation
and found that neither HDL2-C nor HDL3-C was
significantly related to carotid intima-media thick-
ness (IMT) [11] . In addition, a 481 Japanese-Ameri-
can study also revealed that higher HDL2-C level
measured by precipitation was better protected from
atherosclerosis. However, the significant relation-
ship between HDL2-C and carotid atherosclerosis
disappeared after further adjustment for age and sex
[12 ] . Therefore, our study aims to observe the distri-
bution of lipoprotein particles and the relationship
of lipoprotein particles subclasses with the carotid
IMT in the Chinese population, and to confirm that
this relationship is independent of lipids and other
metabolic risks.
Participants & methods
Participants
This study is a part of a long-term prospective epide-
miological survey initiated in 2007. Hereby, we pre-
sent the data that investigate social determinants of
health, specifically the prevalence of diabetes and vas-
cular diseases in Chinese patients during follow-up
within the year of 2009. The epidemiological survey
was previously described in detail elsewhere [13 ] . After
a 12-h fasting, venous blood was drawn and samples
were stored at -70°C, plasma lipid concentrations
(total cholesterol [TC], triglycerides [TG] and HDL-
C) were measured as described previously [1 4] . When
TG ≥4.52 mmol/l, LDL-C was measured by standard
enzymatic methods (Roche Diagnostics). The Fried-
wald formula was used when TG <4.52 mmol/l [15 ] .
ApoA1 and apoB100 were assessed by immunoneph-
elometric methods (Roche Diagnostics). To study the
correlation between the LDL-C/HDL-C ratio and
lipoprotein subclass distribution, subjects were strati-
fied into three subgroups according to the tertiles
of LDL-C/HDL-C. The cut-off points were: lower
than 1.5, 1.5 to 2.0 and higher than 2.0.
Our study included 342 subjects, which consisted
of 168 men and 174 women, aged 20–75 years. Eli-
gible participants were free of any drugs affecting
the lipid metabolism and were recruited from the
same community at Chengdu, Sichuan province
in 2009. Diagnosis of diabetes mellitus was based
on the revised 1999 WHO criteria as follows: fast-
ing plasma glucose level ≥126 mg/dl; plasma glucose
≥200 mg/dl 2 h after a 75 g oral glucose load as in a
glucose tolerance test. A repeat of any of the above
methods should be taken on a different day. Hyper-
tension is defined as a systolic blood pressure consist-
ently higher than 140 mmHg and/or a diastolic blood
pressure consistently higher than 90 mmHg. Smok-
ers were defined as having smoked at least 100 ciga-
rettes during their lifetimes. Alcohol drinking was
defined as the consumption of at least 30 g of alco-
hol per week for 1 year or more. All subjects signed
informed consent forms. The Drugs/Medical Appa-
ratus & Instruments Ethics Committee at China
Japan Friendship Hospital (07020470055) approved
the study protocol and the registration number is
#TR-CCH-Chi CTR-CCH-00000361.
Carotid ultrasound measurements
Carotid IMT was measured by means of high-resolu-
tion vascular ultrasound (Philips ATL 3500 device).
We used the mean of eight measurements of maximal
IMT (right and left common carotid arteries, three
longitudinal views of each internal carotid arteries)
[16 ] . All imaging studies were transmitted to a central
reading center for interpretation.
Lipoprotein subclasses measurement
HDL subclasses were measured by 2D GGE coupled
with immunoblotting, as previously described [1 4] .
Then, HDL subclasses can be classified into small
discoidal HDL (Preb-1HDL and Preb-2HDL),
intermediate spherical HDL3 (HDL3c, HDL3b
and HDL3a) and large mature HDL2 (HDL2a
and HDL2b).
LDL subclasses size was measured by 2–16%
gradient polyacrylamide gel electrophoresis (GGE),
as described [17] . LDL subclasses were classified by
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Research Article Yan, Tian, Yu et al.

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their diameters into small (<25.5 nm), intermedi-
ate (25.5–26.0 nm) and large (>26.0 nm), as LDL1,
LDL2 and LDL3, respectively.
Statistical methods
Data are presented as mean + SD. TG and IMT
measurements were log-transformed to normalized
distribution. A multiple comparison test (least sig-
nificant difference) was used to compare lipopro-
tein subclass distribution across the tertiles of TG
or LDL-C/HDL-C. Univariate ana lysis was used to
assess the independent associations of baseline clini-
cal and laboratory characteristics with carotid IMT.
Additionally, three multivariate regression models
were then performed using the mean IMT as the
dependent variable. The first model included lipopro-
tein subclasses, age, sex, BMI, smoking and alcohol
drinking. A second multivariate regression model also
included blood pressure and hemoglobin A1c, and a
third model also included lipid parameters (TG, TC
and HDL-C). Statistical significance was considered
at p < 0.05.
Results
Characteristic of subgroups classified based on gender
are presented in Ta ble 1. The prevalence of diabetes,
hypertension and dyslipidemia in the male group was
similar to that in the female group. The smoking rate
among males was 46.7%, while it was 3.7% in females.
The percentage of alcohol drinking in males was four-
to five-times more than that in females. The levels of
TG in the male group was significantly higher than
that in the female group, and the females also had the
lower HDL-C levels. 18 patients with diabetes received
insulin or oral antidiabetic drugs, 42 patients with
hypertension received antihypertensive drugs, and
seven dyslipidemic patients received statins or fibrates
therapy.
Table 1. Characteristics of participants.
Characteristic Male (n = 152) Female (n = 190)
Age (years) 49.9 ± 13. 8 47. 7 ± 12.8
BMI ( kg /cm2)24.2 ± 3.6 23.3 ± 3.4*
Smokers, n (%) 71 (4 6.7) 7 (3.7)***
Alcohol drinking, n (%) 36 (23.6）8 (4.2)***
Diabetes, n (%) 15 (9.9 ) 16 (8.4)
Hypertension, n (%) 29 (19.0 ) 32 (16.8)
Dyslipidemia, n (%) 18 (11 . 8 ) 24 (12 .6 )
Hb A1c (%) 5.7 ± 1.0 5.5 ± 0.4
SBP (mmHg) 122.1 ± 18.3 115 . 2 ± 21.5
DBP (mmHg) 79.3 ± 9.8 73.6 ± 10.9
TG (mg /dl）132.8 ± 97. 4 106.2 ± 70.8*
TC (mg /dl）181.7 ± 30.9 185.6 ± 38.7
HD L- C ( m g / dl）5 7.7 ± 15.4 61.5 ± 11.5**
LD L- C (mg/dl）111.9 ± 30.5 108.0 ± 34 .7
Small dense LDL (%) 38.2 ± 17.7 34.4 ± 17.7
Intermediate dense LDL (%) 27. 8 ± 7.1 27. 9 ± 7.3
Large dense LDL (%) 34.1 ± 17. 7 3 7.7 ± 17.7
Preb1-HDL (mg /dl) 93.2 ± 16.5 86.5 ± 14.0***
Preb2-HDL (mg /dl) 42.4 ± 9.1 41.9 ± 8.1
HDL3c (mg/dl) 7 7. 0 ± 9.5 76.0 ± 9.3
HDL3b (mg/dl) 120.0 ± 21.2 118.2 ± 21.2
HDL3a (mg/ dl) 173 .5 ± 25.3 172 .1 ± 22.2
HDL2a (mg/ dl) 19 7.9 ± 17.4 201.6 ± 16 .4*
HDL2b (mg/dl) 281.2 ± 30.6 289.5 ± 29.8*
*p < 0.05; **p < 0.01; ***p < 0.001.
DBP: Diastolic blood pressure; HbA1c: Hemoglo bin A1c; HDL-C: High-density lipoprotein-cholesterol; L DL-C: Low-density
lipoprotein -cholesterol; SBP: Systolic blo od pressure ; TC: Total cholesterol ; TG: Triglycerides.
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Association of lipoprotein subclasses & carotid intima-media thickness in the Chinese population Research Article

410 Clin. Lipidol.(2014) 9(4)
Relationship of lipoprotein particles subclasses
with levels of TG
The size of LDL particles was inversely correlated with
the levels of TG in Ta bl e 2. TG levels were significantly
positively correlated with sdLDL, but inversely
correlated with large dense LDL. In addition, the dis-
tribution of HDL subclasses was not related to the
levels of TG.
Relationship between the concentrations of
lipids, lipoprotein subclasses & the LDL-C/HDL-C
ratio
The relationships of the distribution of lipoprotein
particles subclasses with serum LDL-C/HDL-C ter-
tiles are shown in Tab l e 3 . Compared with those in the
lower tertile of LDL-C/HDL-C ratios, participants in
the upper tertile had higher levels of TC, TG and LDL-
Table 2. Association of lipoprotein particles subclasses and triglycerides.
TG ≤77. 9 (mg /dl ) 77.9 < TG <118.6 (mg /dl) TG ≥118.6 ( mg/dl)
Small dense LDL (%) 29.2 ± 15.0 33.6 ± 16.3 44.0 ± 19.0†***‡***
Intermediate dense LDL (%) 28.0 ± 7. 6 28.1 ± 6.0 27. 3 ± 7. 8
Large dense LDL (%) 42 .1 ± 17.1 38.1 ± 16.9 28.8 ± 16.8†***‡***
Preβ1-HDL (mg /dl) 89.2 ± 12.7 86.7 ± 15. 2 91.0 ± 17.0‡*
Preβ2-HDL (mg /dl) 40.8 ± 9.3 43 .1 ± 8.3†*42.5 ± 7.6
HDL3c (mg/dl) 77. 3 ± 9.1 75.9 ± 9.7 76.0 ± 9.3
HDL3b (mg/dl) 119.7 ± 21.6 119.1 ± 21.6 118.8 ± 20.0
HDL3a (mg/ dl) 174 .5 ± 22.3 172.2 ± 22.8 171.8 ± 25.0
HDL2a (mg/ dl) 199.1 ± 16.9 200.9 ± 16.8 200.6 ± 16.9
HDL2b (mg/dl) 285.8 ± 31.6 288.9 ± 29.5 283.4 ± 30.0
*p < 0.05; ***p < 0.001.
†Signicant difference between TG ≥118.6 group and TG ≤77.9 group.
‡Signicant difference between TG ≥118.6 and 77.9 < TG <118.6 group.
HDL: High -densit y lipoprotein; LDL: Low -densit y lipoprotein; TG: Triglyceride s.
Table 3. Association of lipids and lipoprotein particles subclasses and low-density
lipoprotein-cholesterol /high-density lipoprotein-cholesterol.
LDL-C/HDL-C ≤1.5
(n = 118)
1.5 <LDL-C/HDL-C <2.0
(n = 104)
LDL-C/HDL-C ≥2.0
(n = 120)
TG (mg /dl)†88.5 ± 44.3 106. 2 ± 53.1 159.4 ± 115.1‡***§***
TC (mg /dl) 162.4 ± 30.9 17 7. 9 ± 27.1‡*** 205.0 ± 38.7‡***§***
LD L- C ( m g / dl)†81.1 ± 19.3 104.3 ± 19.3‡*** 13 9.1 ± 30.9‡***§***
HD L- C ( m g / dl) 73.1 ± 11. 5 61.5 ± 11. 5‡*** 53.8± 7.7 ‡***§***
Small dense LDL (%) 26.0 ± 14.2 32 .1 ± 12. 2‡** 47. 9 ± 18.2‡***§***
Intermediate dense LDL (%) 27.1 ± 6.5 30.2 ± 6.5‡** 26.2 ± 7.8 §***
Large dense LDL (%) 46.9 ± 16.4 37.7 ± 14.6‡*** 25.9 ± 15.7‡***§***
Pre-beta1-HDL (mg/dl) 83.9 ± 14.8 89.2 ± 13 .3‡** 93.4 ± 16 .0‡***§*
Pre-beta2- HDL (mg/dl) 41.5 ± 8.2 42.1 ± 8 .1 42.6 ± 9.0
HDL3c (mg/dl) 76.8 ± 9.0 76.0 ± 9.1 76.4 ± 10.0
HDL3b (mg/dl) 116 . 6 ± 22.4 120.5 ± 20.6 119.5 ± 20.5
HDL3a (mg/ dl) 171.4 ± 23.3 174.0 ± 22.2 172 .5 ± 24.5
HDL2a (mg/ dl) 203.5 ± 17.1 199.8 ± 15.9 197.7 ± 17.1‡*
HDL2b (mg/dl) 2 97.1 ± 3 0 .1 283.8 ± 29.9‡** 279.0 ± 27. 8 ‡***
Values are expre ssed as mean ± s tandard deviation.
*p < 0.05; **p < 0.01; ***p < 0.001.
†These variables were analyzed with log10 transformation.
‡Signicantly different from LDL-c/ HDL-c ≤1.5 group.
§Signicantly different from 1.5 <LDL-C/ HDL-c <2.0 group.
HDL-C: High- density lipoprotein- cholesterol; LDL-C, Low-densit y lipoprotein -cholesterol; TC: Total cholesterol; TG: Triglycerides.
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C, and lower HDL-C levels. Participants in the upper
tertile also had an increased level of pre-beta1-HDL
and decreased levels of HDL2 (HDL2a and HDL2b).
They also had an elevated level of LDL1.
Relationship between concentrations of lipids
& carotid IMT
To investigate the factors of vascular risk, we performed
correlation ana lysis with carotid IMT as a dependent
variable and the levels of lipid parameters including
lipoprotein subclasses as independent variables. We
found that IMT was significantly positively correlated
with TC and LDL, but no correlation with TG and
HDL-C was seen (Figure 1A –D) .
Relationship between lipoprotein subclasses
& carotid IMT
The lipoprotein subclasses distribution is shown in
Tab le 4. The major subclasses of HDL were HDL2
(HDL2a and HDL2b) and HDL3a, whereas there
was no significant difference in LDL subclasses
distribution. In univariate correlations, the spearman’s
1.40
1.20
1.00
0.80
0.60
0.40
IMT (mm)
LDL-C (mmol/l)
2.00 4.00 6.00 8.00
r = 0.28
p < 0.001
1.40
1.20
1.00
0.80
0.60
0.40
IMT (mm)
HDL-C (mmol/l)
0.50 1.00 1.50 2.00 2.50 3.00
r = -0.02
p = 0.07
1.40
1.20
1.00
0.80
0.60
0.40
IMT (mm)
TC (mmol/l)
2.000.00 4.00 6.00 8.00 10.00
r = 0.28
p < 0.001
1.40
1.20
1.00
0.80
0.60
0.40
IMT (mm)
TG (mmol/l)
2.000.00 4.00 6.00 8.00
r = 0.09
p = 0.11
AB
C D
Figure 1. Univariate correlation of intima -media thickness with lipids in all subjects. (A) Positive correlation
between LDL-C and carotid IMT. (B) Positive correlation between TC and carotid IMT. (C) No correlation between
HDL-C and carotid IMT. (D) No significant correlation between TG and carotid IMT.
HDL-C: High-density lipoprotein- cholesterol; IMT: Intima-media thickness; LDL-C: Low-density
lipoprotein-cholesterol ; TC: Total cholesterol; TG: Triglycerides.
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Association of lipoprotein subclasses & carotid intima-media thickness in the Chinese population Research Article

412 Clin. Lipidol.(2014) 9(4)
correlation coeffi cients of IMT with lipoprotein
subclasses were 0.3 for LDL1 and -0.24 for LDL3, as
shown in Tabl e 2 . No association was found between
HDL subclasses and IMT.
Additionally, the independent association of LDL
subclasses with carotid IMT was tested by several
multivariate regression models. Higher serum LDL1
levels were associated with increased IMT, independ-
ent of age and sex. When this association was also
adjusted for conventional risk factors (model 3), higher
serum LDL1 remained a significant positive predic-
tor of carotid atherosclerosis. The adjusted standard
regression coefficient (b values) was 0.107 for LDL1
(Tab le 5 ). Interestingly, an independent association
between LDL3 and IMT disappeared.
Discussion
Several epidemiologic studies have demonstrated that
LDL-C is an important marker for CHD. However,
almost half of all patients with CHD have normal
LDL-C levels. The Japanese study has shown that
LDL-C/HDL-C ratios have predictive value for
residual risk of acute myocardial infarction/sudden
death when LDL-C levels <120 mg/dl [18].
In our results, we also observed that TC, TG and
LDL-C levels increased, but HDL-C level decreased
significantly when the ratio of LDL-C/HDL-C
increased. The present results also showed the high-
est LDL-C/HDL-C tertile was significantly associated
with smaller LDL and HDL subclasses.
The alteration of lipoprotein subclasses distribu-
tion may be explained by the impaired cholesterol ester
transfer protein (CETP) activity and enhanced hepatic
lipase activity. It is reported that a significant increase
of TG is observed in high LDL-C/HDL-C ratio. At
the same time, several epidemiological studies revealed
that high levels of TG were associated with impaired
CETP activity [19 ] . CETP mainly transfers CE from
HDL2 to LDL in exchange for TG. Thus, CETP
plays an important role in the conversion of HDL2 to
Table 4. Association of lipoprotein subclasses by univariate correlation analysis with intima-media
thickness.
Lipoprotein subclasses Concentrations Rp-value
LD L- C / H DL-C 1.9 ± 0.7 0.254 < 0.001
LDL subclasses
Small dense LDL (%) 35.8 ± 17.7 0.30 <0.001
Intermediate dense LDL (%) 27.8 ± 7. 2 - 0.13 0.02
Large dense LDL (%) 36.4 ± 17.8 -0.24 <0.001
HDL subclasses
Pre-beta1-HDL (mg/l) 89.0 ± 15.3 -0.01 >0.05
Pre-beta2-HDL (mg /l) 42.1 ± 8.5 -0.06 >0.05
HDL3c (mg/l) 76.4 ± 9.4 - 0 .11 0.06
HDL3b (mg/l) 119.0 ± 21.1 0.01 >0.05
HDL3a (mg/l) 172.7 ± 23.3 0 >0.05
HDL2a (mg/l) 200.2 ± 16.8 0 >0.05
HDL2b (mg/ l) 286.4 ± 30.4 0 >0.05
Values are presented as mean ± standard deviation.
HDL-C: High- density lipoprotein- cholesterol; LDL-C: Low-density lipoprotein-choles terol.
Table 5. Association of l ow-density lipoprotein 1 levels by stepwise multiple regression analysis with
intima-media thickness after adjusting for clinical characteristics and other lipids.
Adjustment Beta p-value
Small dense LDL 0.3 <0.001
Adjusted for age, sex, BMI, smoking and alcohol drinking 0.117 0 .013
Adjusted for age, sex, BMI, smoking, alcohol drinking, blood
pressure and HbA1c
0.1 07 0.030
Adjusted for all above plus lipids (TG, TC, HDL-C) 0 .107 0.031
HbA1c: Hemoglobin A1c; HDL-C: High-d ensity lipoprotein-cholestero l; LDL: Low-density lipoprotein; TC: Total cholesterol; TG: Triglycerides.
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pre-beta1-HDL [20] . On the other hand, hydrolysis of
LDL-TG by hepatic lipase increases sdLDL (LDL1)
concentration. In this regard, the LDL-C/HDL-C
ratio may be a good indicator for the distribution of
lipoprotein subclasses.
Furthermore, our results also showed that TC and
LDL levels had positive correlations with the risk of
carotid atherosclerosis. Additionally, we investigated
the relationship between lipoprotein subclasses and
carotid atherosclerosis after adjustment for smok-
ing, alcohol drinking, lipid, age, pressure, BMI and
hemoglobin A1c. As a result, sdLDL was found to be
significantly and independently associated with IMT.
These findings support previous research, which
showed that LDL-C is the primary goal of treatment in
lipid management. A number of well-designed epidemi-
ological and clinical studies reported that LDL-C is an
independent risk in macrovascular disease. A Heart Pro-
tection Study Collaborative Group trial also reported
that reducing LDL-C level by allocating simvastatin
can reduce the incidence of vascular disease, irrespective
of their initial cholesterol concentrations. [ 21] . However,
the effect of LDL-C on the process of atherosclerosis
may depend on lipoprotein subclasses. It is more likely
for sdLDL to enter the endothelium, become oxidized
and trigger the atherosclerosis progression. In the cur-
rent study, sdLDL is also an established independent
indicator for carotid IMT in the general Chinese popu-
lation, which is identical to that which was observed
in a Genetics of Coronary Artery Disease in Alaska
Natives study [2 2]. The Framingham Offspring cohort
also found that LDL particle number (LDL-P) is a more
sensitive indicator of CVD risk than either LDL-C or
non-HDL-C, which measured LDL particles based on
nuclear magnetic resonance in 3066 middle-aged white
participants (53% women) without CVD [2] . In another
study, nuclear magnetic resonance-measured LDL-P
was decreased 5% by gemfibrozil. This reduction was
caused by a significant 20% decrease in the number of
small LDL particles, though no changes in LDL-C were
observed [23]. Moreover, a growing body of evidence
suggests that fibrates increase LDL particle size and
modify LDL subclasses distribution from small, dense
particles to large LDL particles, and reduce the number
of small LDL particles in patients with hypertriglyceri-
demia and the metabolic syndrome [24] . Unfortunately,
the effect of statins on the LDL-P is limited. All these
observations provide support for the concept that there
still remains a high residual risk for vascular disease even
when LDL-C is well-controlled. In this sense, sdLDL
should be considered as a stronger predictor for mac-
rovascular disease than LDL-C. Therefore, although
achieving the LDL target level is the first priority for
therapy in most current guidelines for lipid treatment,
more attention should be given to sdLDL in the Chinese
population [25] .
The third major finding in our study was that
carotid IMT was not correlated with HDL-C or HDL
particle subclasses. Our findings agreed with the
results that higher levels of HDL2 prevent the pro-
gression of carotid IMT in Japanese-Americans [1 2] .
Recent studies have also shown that the inverse asso-
ciation of HDL-C with CHD disappeared after adjust-
ment of apoAI and apoB, while HDL particle number
(HDL-P) remained inversely related with CHD. Thus,
the anti-atherogenic effects of HDL particles may be
related to HDL’s protein or antioxidant enzyme rather
than to its cholesterol concentration [26 ]. HDL asso-
ciated enzyme had the properties of antioxidant anti-
atherosclerosis. Indeed, in the European Prospective
Investigation into Cancer (EPIC)-Norfolk, the HDL-
associated antioxidant paroxonase-1 was positively cor-
related with HDL-P more strongly than with HDL-C
or apoAI [2 7]. Furthermore, in the clinical trials, the
roles of HDL-C and HDL subclasses on vascular dis-
ease were still inconclusive. In a Action to Control
Cardiovascular Risk in Diabetes (ACCORD)-lipid
trial, the combination of fenofibrate plus simvastatin
was compared with simvastatin monotherapy, sug-
gesting that fenofibrate has been associated with a
significant reduction of microvascular complications
in patients with Type 2 diabetes, including the pro-
gression of nephropathy and retinopathy [9] . This is
also in accordance with the Fenofibrate Intervention
and Event Lowering in Diabetes study and Diabetes
Atherosclerosis Intervention Study, which showed that
the progression for microalbuminuria and the need for
laser treatment for diabetic retinopathy could be fur-
ther reduced [2 8, 29] . Taken together, these studies sug-
gest that fenofibrate may prevent microvascular events
but has little effect on macrovascular disease as a whole
[9]. It seems possible that these results are due to the
redistribution of HDL subclasses by fenofibrate. A fur-
ther study with more focus on the relationship between
HDL subclasses and microvascular events is therefore
suggested. However, a 31% reduction in cardiovascu-
lar event rate with combination therapy was observed
in a predefined subgroup of participants with the com-
bination of significant hypertriglyceridemia and low
HDL-C experienced [30] . Unfortunately, it has not
been adjusted by the redistribution of LDL particles by
fenofibrate. Thus, the relationship between HDL sub-
classes and macrovascular disease still remains highly
uncertain [22].
Conclusion
sdLDL was a strong predictor for carotid atherosclerosis
in the general Chinese population. Moreover, HDL-C
future science group
Association of lipoprotein subclasses & carotid intima-media thickness in the Chinese population Research Article

414 Clin. Lipidol.(2014) 9(4)
or HDL subclasses remained free from carotid
atherosclerosis.
Acknowledgements
We would like to thank China Japan Friendship Hospital and
the First People’s Hospital of Longquan for par ticipating in this
st udy, an d fo r col lec t in g dem o gra ph ic da ta an d blo od sa m pl e s.
Financial & competing interests disclosure
This study was suppor ted by grants from the Chinese Medical
Association Foundation and Chinese Diabetes Society
(No. 07020 470055 ). The authors have no other relevant
afliations or nancial involvement with any organization or
entity with a nancial interest in or nancial conict with the
subject matter or materials discussed in the manuscript apart
from those disclosed.
No writing assistance was utilized in the production of this
manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate
insti tutional review board approval or have followed the
princi ples outlined in the Declaration of Helsinki for all
human or animal experimental investigations. In addition, for
investi gations involving human subjects, informed consent
has been obtained from the participants involved.
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Research Article Yan, Tian, Yu et al.
Executive summary
• Low-density lipoprotein-cholesterol (LDL-C) /high-density lipoprotein-C (HDL-C) ratio is a good indicator for
the distribution of lipoprotein subclasses.
• Small dense LDL was positively correlated with triglycerides levels.
• Total cholesterol and LDL-C were significantly correlated with intima-media thickness.
• Triglycerides and HDL-C were not related with intima-media thickness.
• HDL subclasses did not correlate with carotid atherosclerosis.
• Small dense LDL was a stronger predictor for carotid atherosclerosis than LDL-C.

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