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Atherosclerosis and Lipoproteins |
From the Department of Epidemiology and Biostatistics (A.E.H., M.L.B., I.C.D.W., A.H., J.C.M.W.), Erasmus University Medical School, Rotterdam; the Department of Internal Medicine (C.D.A.S., K.H.P.), University Hospital Vrije Universiteit and Institute for Cardiovascular Research Vrije Universiteit, Amsterdam; the Julius Center for Patient Oriented Research (M.L.B.), University Medical Center Utrecht, Utrecht; and the Department of Clinical Chemistry (C.G.S.), University Hospital Vrije Universiteit and Institute for Cardiovascular Research Vrije Universiteit, Amsterdam, the Netherlands.
Correspondence to J.C.M. Witteman, PhD, Department of Epidemiology and Biostatistics, Erasmus University Medical School, PO Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail witteman{at}epib.fgg.eur.nl
| Abstract |
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Key Words: C-reactive protein obesity insulin resistance carotid artery intima-media thickness women
| Introduction |
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Recent data also indicate that the insulin resistance syndrome is accompanied by an increased acute-phase response.16 17 A link between the insulin resistance syndrome and the inflammatory state is further suggested by increased levels of the acute-phase proteins plasminogen activator inhibitor-1 (PAI-1) and fibrinogen in the insulin resistance syndrome18 19 20 and by the finding that dyslipidemia in the insulin resistance syndrome and during the acute-phase response shows strong similarities.21 22 23
Obesity, the insulin resistance syndrome, and atherosclerotic disease are closely linked and may all be determinants of an increased acute-phase response. However, it is not clear whether these factors are independently associated with the inflammatory state. Previous studies on associations between CRP level as a measure of inflammation and cardiovascular risk factors were conducted in middle-aged men and elderly men and women, all of whom are at relatively high risk for atherosclerosis.14 15 Atherosclerosis and smoking are potential sources of inflammation and possibly obscure the relation of CRP with other risk factors.
In the present study, we investigated the relationship between CRP and measures of obesity, the insulin resistance syndrome, and subclinical atherosclerosis in a population of healthy, middle-aged women with a low exposure to tobacco smoke.
| Methods |
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Measurements
During a visit at the research center, a medical history was
taken by a physician. Height, weight, and waist and hip circumferences
were measured while the subjects wore indoor clothes without shoes.
Body mass index (BMI, weight divided by height squared) and
waist-to-hip ratio (WHR) were computed. Cigarette smoking history was
obtained by a standardized questionnaire. Blood pressure was assessed
with a DINAMAP automatic blood pressure recorder (Critikon, Inc).
After a 5-minutes rest in the supine position, blood pressure was
measured 4 times at the right upper arm with an appropriately sized
cuff, and the mean was used in the analyses. Venous blood
samples were drawn from each subject after a 12-hour fast. The samples
were stored at -80°C. Total cholesterol was measured
with an automated enzymatic method using the CHOD-PAP
high-performance reagent kit from Boehringer Mannheim.
HDL cholesterol was measured by the phosphotungstate
method. LDL cholesterol was computed by the Friedewald
formula.24 Triglycerides were determined by
using a reagent kit from Boehringer Mannheim after enzymatic
hydrolysis of the triglycerides and subsequent
determination of liberated glycerol by colorimetry. No
correction was made for serum free glycerol. Apolipoproteins A1 and B
were measured by an automated turbidimetric immunoassay with reagent
kits from Orion Diagnostics. Glucose was enzymatically
determined by the hexokinase method (Instruchemie). Serum insulin was
determined by metric assay (Biosource Diagnostics). This
assay has no cross-reactivity with either proinsulin or C-peptide.
PAI-1 antigen and tissue-type plasminogen
activator (tPA) antigen levels were determined by ELISA
(Innotest PAI-1, Innogenetics NV, and Imulyse, Biopool, respectively).
CRP was measured by an in-house ELISA with rabbit anti-CRP (Dako) as
the catching and tagging antibody.25 Intra-assay and
interassay CVs for CRP were 3.8% and 4.7%, respectively. Fasting
insulin levels were used as a measure of insulin
resistance.26 In addition, insulin sensitivity was
calculated according to the formula of the homeostasis model assessment
method (HOMA): insulin resistance=fasting insulinxfasting
glucose/22.5.27
Carotid Artery Intima-Media Thickness (IMT)
Common carotid artery IMT was used as an indicator of
generalized
atherosclerosis.28
Ultrasonography of the right common carotid artery was performed with a
7.5-MHz linear-array transducer (ATL UltraMark IV) as described in
detail previously.29 For each individual, the common
carotid artery IMT was determined as the average of near- and far-wall
measurements. Carotid artery IMT measurements have been shown to be
reproducible.30 In short, mean differences (and SDs) in
far-wall IMT of the common carotid arteries between paired measurements
of sonographers, readers, and visits were 0.040 mm (0.07),
0.069 mm (0.04), and 0.071 mm (0.09), respectively. The
intraclass correlation coefficients were 0.63, 0.88, and 0.74,
respectively. These results are in agreement with the reproducibility
of IMT measurements found in other studies.31 In the
present study, all measurements were conducted by 1 sonographer and
1 reader.
Statistical Analysis
The clinical and biochemical features of the population are
presented as mean±SD, median (and interquartile range) for
variables with a skewed distribution, or percentages. Because the
distribution of CRP was highly skewed, it was natural-logtransformed
for all analyses. The strength of the associations between CRP
and clinical and biochemical variables was assessed by linear
regression of ln CRP on each variable separately, adjusted for age.
Because strong associations were found between CRP and measures of
obesity, we adjusted for them in additional models. Regression
analysis was further used to estimate the explained proportion
of variance in CRP (R2). The
difference in CRP between premenopausal and postmenopausal women
adjusted for age and measures of obesity was studied with regression
analysis. A probability value <0.05 (2-tailed test) was
considered significant. SPSS 7.5 for Windows was
used for all analyses.
| Results |
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CRP was significantly associated with measures of obesity: BMI, waist
and hip circumferences, and WHR (Table 2
). Associations with CRP were stronger
for BMI and waist and hip circumferences than for WHR
(r=0.54 for BMI, r=0.55 for waist circumference,
r=0.53 for hip circumference, and r=0.33 for WHR,
all adjusted for age). After adjustment for BMI, hip circumference and
WHR were no longer associated with CRP, whereas waist circumference
still was. We next visualized this relationship between BMI, WHR, and
CRP by subdividing the study population by the median BMI (23.9
kg/m2) and WHR (0.77) (the
Figure
, geometric means). BMI explained
29.7% of the variance of CRP; waist circumference, 31.3%; hip
circumference, 28.7%; and WHR, 11.4%, after adjustment for age.
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The other variables included in or associated with the insulin
resistance syndrome were also significantly associated with CRP: blood
pressure, insulin, HDL cholesterol,
triglycerides, apolipoprotein A1 (inversely), PAI-1
antigen, and tPA antigen (Table 3
). No
associations were found with glucose or with total and LDL
cholesterol, whereas an association with apolipoprotein B
was present. Separate analyses after exclusion of subjects
with levels of CRP >10 mg/L did not affect the results (data not
shown).
|
After controlling for BMI, the associations between CRP and
variables of the insulin resistance syndrome disappeared except for
the association with PAI-1 antigen, although there was a substantial
decline in the magnitude of this association (Table 3
).
Controlling for waist circumference gave the same results, whereas
controlling for hip circumference decreased the described associations
to a somewhat smaller extent. Controlling for WHR, on the other hand,
had only a small influence on the described associations (data not
shown). To evaluate whether the clustering of variables belonging
to the insulin resistance syndrome might be a reflection of a general
acute-phase response, associations between measures of insulin
resistance (insulin and HOMA) and the other variables of the
insulin resistance syndrome were adjusted for CRP. This adjustment did
not modify the relation between insulin, HOMA, and the other
variables (data not shown).
Measures of obesity and CRP in premenopausal and postmenopausal women
separately are shown in Table 4
. CRP did
not differ significantly between premenopausal and postmenopausal
women. Age-adjusted geometric means were 0.61 and 0.71 mg/L
respectively (15% increase with menopause, 95% CI, 15% to 45%).
Because menopause may be associated with changes in measures of
obesity, we adjusted for these variables, which slightly influenced
the results. Postmenopausal women had an age-adjusted level of
cholesterol of 6.48 mmol/L versus 5.89 mmol/L in
premenopausal women (10% difference; 95% CI, 5% to 14%). PAI-1
antigen increased with menopause, but the difference lacked statistical
significance. In premenopausal women, the age-adjusted geometric mean
of PAI-1 antigen was 52.9 ng/mL versus 61.1 ng/mL in postmenopausal
women (13% increase with menopause; 95% CI, 8% to 33%). Because
cholesterol and PAI-1 antigen are known to increase with
menopause, these results indicate a correct selection of menopausal
groups. The associations between CRP on the one hand and both measures
of obesity and other variables of the insulin resistance syndrome
on the other were found to be identical when examined in premenopausal
and postmenopausal women separately (data not shown).
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CRP was significantly associated with common carotid artery IMT. After
stratification by smoking status, associations between CRP and common
carotid artery IMT appeared to be present in ever-smokers only
(Table 5
). Common carotid artery IMT
explained 3.7% of the variance of CRP after adjustment for age.
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| Discussion |
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One hypothesis explaining these results is that adipose tissue might be
the common antecedent of both CRP and insulin resistance. The
associations between CRP and variables of the insulin resistance
syndrome may thus be due to the association of BMI with both insulin
resistance and the acute-phase response. This idea is
consistent with experimental evidence indicating that
adipocytes produce tumor necrosis factor (TNF)-
.33
TNF-
induces interleukin-6 (IL-6) synthesis,34 a prime
regulator of CRP synthesis.35 36 Additional support for
this hypothesis comes from the observation that weight reduction leads
to a decrease of TNF-
mRNA expression37 and of serum
levels of TNF-
in diabetic subjects.38 We found that
CRP was strongly related to BMI and to waist and hip circumferences
separately, but less to WHR. These results are compatible with previous
studies, in which BMI but not WHR was related to TNF-
expression or
TNF-
levels.33 39 However, after adjustment for BMI,
waist circumference was still related to CRP, whereas hip circumference
was not. This suggests that abdominal fat deposition is most important
in inducing inflammation.
Associations between CRP concentrations and fasting serum insulin concentrations, which persisted after adjustment for BMI, have been observed in a population of male patients with angina pectoris.18 In addition, in healthy, middle-aged men, relationships between CRP and cardiovascular risk factors like HDL cholesterol and triglycerides persisted after adjustment for BMI.14 One possible explanation for these discrepant results might be that the relationships between obesity, the insulin resistance syndrome, and the acute-phase response are different between men and women. Support for this hypothesis comes from the observation that sex steroids influence the metabolic activity of adipose tissue.40 Additionally, the described studies differ from ours in that those subjects were likely to suffer from more pronounced atherosclerosis because they were male or suffering from angina pectoris. Atherosclerosis might have spuriously induced the relation between CRP and other cardiovascular risk factors. Because in our population women had a low burden of atherosclerosis, as estimated from carotid artery IMT, the potential for confounding by atherosclerosis in our study is less likely.
Associations between measures of insulin resistance and other
variables included in the insulin resistance syndrome were not
attenuated by adjusting for CRP levels. Therefore, our data do not
suggest that the clustering of variables belonging to the insulin
resistance syndrome might be a reflection of a general acute-phase
response.16 Also, because the association between insulin
resistance and measures of obesity was not affected by adjustment for
CRP, our data do not support the hypothesis that
adipose-tissuederived cytokines may mediate the relation
between obesity and the insulin resistance
syndrome.17 33 37 39 However, this hypothesis encompasses
a causal role for TNF-
; therefore, this inference might have been
more valid had we adjusted for TNF-
instead of CRP.
The selection of premenopausal and postmenopausal women is likely to be
accurate, as reflected by an age-adjusted increase of
cholesterol of 10%, which is in agreement with other
studies.41 42 We did not find a clear influence of
menopause on CRP levels. Both age- and age-and-BMIadjusted levels of
CRP were slightly higher in postmenopausal (0.71 mg/L) than in
premenopausal (0.61 mg/L) women, but this 15% difference was not
statistically significant. This result can probably be attributed to
the large variation of this measure. To the best of our knowledge, no
published data on the association between menopause and CRP levels are
available from other studies. Estrogen replacement therapy in
postmenopausal women has been shown to lower TNF-
43 and
acute-phase reactants other than CRP.44 Experimental data
suggest an inhibitory effect of estrogens on IL-6 gene
expression.45 Recent data from the
Cardiovascular Health Study, however, suggest an
increase of CRP with hormone replacement therapy.46
Further studies are needed to address the association between
inflammation, estrogens, and menopause.
We are the first to describe an association between CRP and common
carotid artery IMT in healthy, middle-aged women, which association was
limited to ever-smokers (Table 4
). In a study by Tracy et
al15 in a population of elderly men and women, CRP was not
related to internal carotid wall thickness but was related to ankle-arm
index in ever-smokers only Data from the MRFIT (Multiple Risk Factor
Intervention Trial) study also show a stronger association of CRP with
coronary heart disease deaths in middle-aged male smokers than
in nonsmokers, as defined at baseline.8 Taken together,
these and the present data suggest that CRP may mark permanent,
underlying vascular damage due in part to smoking. This may explain why
the associations between inflammation and
atherosclerosis are more pronounced not only in current
but also in former smokers. In the Physicians' Health Study, however,
smoking did not modify the relation between CRP and the risk of
cardiovascular events.7
Some issues of our study need to be addressed. First, we did not measure exposure to infectious agents such as Helicobacter pylori and Chlamydia pneumoniae, which may be weak determinants of CRP levels.12 17 However, it appears unlikely that exposure to these agents would confound the association between BMI and CRP level. Second, in this study we measured atherosclerosis at only 1 location in the vascular system. Although we assume that common carotid artery IMT is a measure of generalized atherosclerosis,28 assessment of the degree of atherosclerosis might have been more accurate had we used measurements at multiple locations. Finally, this study was conducted in healthy, middle-aged women without clinical cardiovascular disease, no medication use, and a low, current exposure to tobacco. Smoking and atherosclerosis are potential determinants of CRP, and therefore, the choice of our population facilitates the investigation of other factors associated with CRP. However, in this population, ever-smoking was also found to modify the association between CRP and atherosclerosis.
In summary, our results indicate that adipose tissue is strongly associated with CRP in healthy, middle-aged women. In this population with a low burden of atherosclerosis and current smoking, BMI accounts for the association between CRP and variables of the insulin resistance syndrome. Because inflammatory mediators may be directly involved in atherogenesis, these results suggest an important mechanism through which obesity might affect the risk of coronary heart disease. Further studies should determine whether losing weight ameliorates the inflammatory state.
| Acknowledgments |
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Received September 23, 1998; accepted December 8, 1998.
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