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Articles |
1
Department of Laboratory Medicine, Children's Hospital, and Department of Pathology, Harvard Medical School, Boston, MA 02115.
2
Department of Biochemistry, School of Pharmacy,
University of Damascus, Damascus, Syria.
3
Department of Cardiology, University of Damascus
Hospitals, Damascus, Syria.
a Address correspondence to this author at: Children's Hospital, Department of Laboratory Medicine, 300 Longwood Ave., Boston, MA 02115. Fax 617-355-6081; e-mail rifai{at}a1.tch.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: The concentrations of C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6), and soluble intracellular adhesion molecule (sICAM-1) were examined in 100 men with angiographically documented CHD and 100 age-, gender-, and smoking-matched controls with no history of CHD. We assessed the association of these markers with severity of disease as indicated by >50% obstruction in one vessel (n = 30), two vessels (n = 39), or three vessels (n = 31).
Results: Significant increases were noted in serum CRP (median for cases vs controls, 3.4 vs 1.5 mg/L; P <0.0001), SAA (5.9 vs 3.7 mg/L; P <0.005), and IL-6 (2.3 vs 1.7 ng/L; P <0.013) in patients with CHD compared with controls. These differences remained significant after correction for age, smoking, hypertension, diabetes, and lipid and homocysteine concentrations. Plasma sICAM-1 was not significantly different between the two groups (335 vs 339 µg/L). No significant correlation was seen between these markers and the severity of coronary disease.
Conclusions: Concentrations of CRP, SAA, and IL-6 were increased in patients with CHD but failed to correlate with severity of coronary disease. These markers might reflect the diffuse atherosclerotic process in the vascular system rather than the degree of localized obstruction from coronary lesions.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interleukin-6 (IL-6) is produced mainly by activated monocytes and smooth muscle cells. This cytokine causes the de novo hepatic synthesis of acute phase reactants (12), and its concentration is increased in patients with unstable angina compared with those with stable angina (13). Soluble cellular adhesion molecules such as soluble intercellular adhesion molecule-1 (sICAM-1) are expressed on the surface of activated endothelial cells, in response to several inflammatory cytokines, to capture circulating leukocytes and increase the migration of white cells into the vascular intima. Recently, it has been shown that sICAM-1 concentrations are associated with ultrasound-based carotid intimal-medial thickness, an early index of atherosclerosis, thus providing additional support for the role of systemic inflammation in the development of the atherosclerotic lesion (14). Furthermore, increased sICAM-1 concentrations have been associated with increased risk of future MI in apparently healthy men (15).
To further explore the role of the systemic markers of inflammation and coronary disease, we examined the plasma concentrations of CRP, SAA, IL-6, and sICAM-1 in men with angiographically documented CHD and compared them with appropriate controls. In addition, we studied the possible association of these markers with the severity of coronary stenosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prior medical histories and personal characteristics and habits were obtained from all participants via a questionnaire. Smoking was characterized by the current smoking status and the number of cigarettes smoked per day. Hypertension was defined as resting systolic blood pressure >160 mmHg and/or diastolic blood pressure >95 mmHg; diabetes mellitus was established by preexisting diagnosis, a fasting blood glucose concentration >1.20 g/L, or glycosylated hemoglobin concentrations >6%.
Blood samples were collected after 12 h fast for the determination of cholesterol, high density lipoprotein cholesterol (HDL-C), triglycerides, homocysteine, CRP, SAA, IL-6, and sICAM-1 concentrations. In cases, blood samples were obtain on the day before the angiographic procedure. Blood samples were also collected in the morning from all controls after 12 h fast, as has been done in cases. Serum and plasma samples were separated immediately after collection by centrifugation at 2000 g for 15 min and stored at -70 °C until analysis. Serum samples were used for the measurement of lipids and lipoproteins at the University of Damascus Hospitals and plasma EDTA samples were used for the determination of homocysteine and the inflammatory markers at Children's Hospital in Boston.
laboratory measurements
Cholesterol and triglycerides were determined on a Hitachi 911
automated analyzer (Roche Diagnostics) according to the
manufacturer's recommendations. Triglyceride measurements were
corrected for endogenous glycerol. HDL-cholesterol (HDL-C) was
determined after precipitation of the apolipoprotein B-containing
particles by phosphotungstic acid-MgCl2 (Roche
Diagnostics). LDL-cholesterol (LDL-C) was estimated using the
Friedewald equation (16). The total homocysteine
concentration was measured by HPLC with fluorometric detection
as reported previously (17). The concentrations of CRP and
SAA were determined using the BN II nephelometer (Dade Behring)
(18), and the concentrations of IL-6 and sICAM-1 were
measured by ELISA (R&D Systems), according to the manufacturers'
specifications. The run-to-run imprecision of the CRP assay at
concentrations of 0.47, 54.9, and 138 mg/L was 6.4%, 2.9%, and 3.6%,
respectively; for SAA at concentrations of 11.5, 81.6, and 301 mg/L it
was 7.3%, 7.0%, and 8.5%, respectively; for IL-6 at concentrations
of 2.6 and 4.1 ng/L it was 5.8% and 6.9%, respectively; and for
sICAM-1 at concentrations of 257.1 and 320.2 µg/L it was 8.7% and
4.7%, respectively.
statistical analysis
All data are presented as the mean ± SD, with the exception
of triglycerides, the inflammatory markers, and homocysteine, which are
presented as median (20th, 80th percentiles). The CRP, IL-6, sICAM-1,
and SAA concentrations were treated as continuous variables and as
quintiles defined by the distributions in the control group. Because
triglycerides, CRP, IL-6, sICAM-1, SAA, and homocysteine were skewed
rightward, a natural log transformation was applied to normalize the
data for analysis. The unpaired Student t-test, Pearson
correlation, and analysis of variance were performed on normally
distributed and transformed data. Case-control differences in nominal
data (diabetes, hypertension, and smoking) were evaluated with the
2 test. A general linear model was used to
consider case-control differences after adjustment for other CHD risk
factors and covariates. All P values were two-tailed, and
P <0.05 was deemed statistically significant. All analyses
were performed with SPSS 8.0 (SPSS Inc.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Because of matching criteria, no statistically significant
difference was noted in age and smoking between the two groups. The
incidence of hypertension and diabetes, however, was higher in the case
group. Furthermore, the total cholesterol, triglycerides, and
homocysteine concentrations and the total cholesterol/HDL-C ratio were
higher in the case group compared with the control group.
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The frequency distributions of CRP, SAA, IL-6, and sICAM-1 are
presented in Fig. 1
. The median concentrations of CRP, SAA, and IL-6 were
significantly higher in the case group than in the control group (Table 2
). These differences remained statistically significant after
correction for age, smoking, diabetes, hypertension, cholesterol,
LDL-C, HDL-C, triglycerides, and homocysteine. Two-thirds of the cases
had CRP, SAA, and IL-6 concentrations in the fourth or fifth quintile,
as defined by the distributions in controls (Fig. 2
). No significant difference was seen in sICAM-1 concentrations
between the two groups. Fewer than 50% of the cases had sICAM-1
concentrations in the fourth or fifth quintile (Fig. 2
). For both cases
and controls, the concentrations of all four inflammatory markers were
higher in smokers and hypertensives. However, these differences reached
statistical significance only for CRP in cases with hypertension (6.8
vs 3.3 mg/L; P <0.04) and for sICAM-1 in control smokers
(366 vs 318 µg/L; P <0.001).
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No significant correlation was seen between any of the inflammatory
markers and measured lipids or homocysteine. In contrast, modest but
statistically significant correlations were noted between IL-6 and SAA
(r = 0.480; P <0.0001), sICAM-1 and CRP
(r = 0.213; P <0.002), and sICAM-1 and IL-6
(r = 0.303; P <0.0001). The strongest
correlations were seen between CRP and IL-6 (r = 0.643;
P <0.0001) and CRP and SAA (r = 0.697;
P <0.0001; Fig. 3
).
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The relationship between the inflammatory markers and the severity of
coronary disease was examined. Case subjects, who have undergone
coronary angiography, were divided into three categories: those with
>50% obstruction in one vessel (n = 30), two vessels (n =
39), or three vessels (n = 31). Plasma CRP, SAA, IL-6, and sICAM-1
concentrations were examined in these three subgroups and compared with
the control group (Table 3
). None of these markers showed any significant correlation with
the severity of disease. Similar findings were also noted for the
measured lipids. In contrast, homocysteine showed a significant
association (P <0.002).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence from laboratory, clinical, and epidemiological studies regarding the role of inflammation in atherogenesis is accumulating rapidly. These findings suggest that atherosclerosis is a chronic inflammatory condition that evolves as a result of a combination of biochemical, physical, and possibly infectious processes. Biochemical measures, such as acute phase reactants, have been used for detecting and assessing severity of systemic inflammation. Of the four inflammatory markers examined in this report, CRP has been studied the most in a variety of atherosclerotic diseases because of the availability of sensitive and automated analytical methods for its measurement. The de novo hepatic synthesis of CRP is triggered by the pleiotropic cytokine IL-6 (12). Therefore, CRP acts as a reliable surrogate marker for IL-6 and other mediators such as tumor necrosis factor. Data from prospective studies have shown that plasma CRP concentrations are increased many years in advance of the first coronary and cerebrovascular events in healthy (7)(8)(9)(11) and high-risk (10)(19)(20) individuals. In addition, sICAM-1 has been reported to predict MI and stroke many years before these events in asymptomatic healthy men (15). Studies have also shown that concentrations of CRP, SAA, and IL-6 are increased in patients with MI or unstable angina (1)(13)(21). In fact, in patients with unstable angina, CRP and SAA are good predictors of mortality (4)(5)(6) and IL-6 is associated with poor in-hospital prognosis (13), which seems to be independent of plaque rupture or myocardial necrosis (22).
In this study of men with angiographically documented CHD, plasma concentrations of CRP, SAA, and IL-6 were significantly increased when compared with age-, gender-, and smoking-matched apparently healthy controls. The distributions of CRP, SAA (7)(18), and IL-6 (Dr. Paul Ridker, personal communication) of our controlswere comparable to those reported earlier. The CRP and SAA medians of our cases were lower than those seen in subjects with unstable angina (CRP, 3.4 vs 5.8 mg/L; SAA, 5.9 vs 7.6 mg/L) when the same noncompetitive immunoassays were used (4)(5), but CRP and IL-6 medians were higher than those reported in asymptomatic men who later developed MI [CRP, 3.4 vs 1.5 mg/L; IL-6, 2.3 vs 1.8 ng/L; Ref. (7), and personal communication from Dr. Paul Ridker for IL-6]. The median concentrations of sICAM-1 did not differ significantly among cases and controls (335 vs 339 µg/L). This adhesion molecule is thought to mediate the binding of circulating leukocytes to the endothelial cell and the subsequent transendothelial migration, a pivotal process in atherogenesis. An increased sICAM-1 concentration has been shown to be significantly associated with risk of future MI in men (15). In the latter study, sICAM-1 values in controls were ~30% lower, over a wide range of encountered concentrations, than those seen in our controls. Whether this difference could be attributed to methodological or physiological causes is unclear at present.
The high correlation seen between CRP, SAA, and IL-6 in this study raises the question of whether additional information would be obtained by measuring more than one marker. We previously have shown that CRP and SAA are both equal predictors of mortality in patients with unstable angina and non-Q-wave MI (4)(5) and of recurrent coronary events in patients who suffered acute MI (3). The addition of SAA to CRP in those studies did not improve its predictive power. In this report, when IL-6 and SAA values were adjusted for CRP, their expected differences among cases and controls fell short of statistical significance. In contrast, the difference in CRP values between the two groups remained significant (P <0.01) after correcting for SAA and IL-6, thus suggesting that CRP may be a stronger discriminator between patients with CHD and controls than SAA or IL-6.
The severity of coronary disease may be assessed angiographically by examining the number of vessels with >50% stenosis. In this study, none of the inflammatory markers, however, showed a significant correlation with severity of disease. The increases seen in CRP, SAA, and IL-6 of cases may, therefore, reflect the diffuse atherosclerotic process in the vascular system rather than the degree of localized obstruction from lesions. The small sample size in this study could not also be ruled out as the cause of this negative finding.
The potential limitations of this study merit consideration. The control group did not include diabetics, which potentially could influence the differences in measured inflammatory markers. In the case group, diabetics had higher CRP (median, 7.8 vs 2.9 mg/L; P <0.004) and IL-6 (2.7 vs 2.2 ng/L; P <0.024) than nondiabetics. However, all measured changes in biochemical markers were similar and remained statistically significant when nondiabetics were considered separately or when the data were adjusted for diabetes. Further work is required to investigate inflammatory markers in diabetics with and without CHD. Because all participants in this study were men, the effect of gender could not be ascertained. Lipids and CRP were shown to be predictors of future coronary events in both men and women. However, the literature supports gender-specific differences in these biochemical markers (11). Therefore, it is unclear at present whether it is appropriate to extrapolate these findings to women. The presence of occult ischemic coronary disease in our controls could not be excluded and may have attenuated the differences seen among the two examined groups. Therefore, more stringent evaluation of controls likely would have enhanced the encountered differences.
In conclusion, in this case-control study, concentrations of CRP, SAA, and IL-6 are increased in patients with CHD but failed to correlate with severity of coronary disease. These findings suggest that biochemical markers of inflammation might reflect the diffuse atherosclerotic process in the vascular system rather than the degree of localized obstruction from coronary plaques.
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Footnotes |
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