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Relations of plasma high-sensitivity C-reactive protein to traditional cardiovascular risk factors
Atherosclerosis 167 (2003) 73 /79 www.elsevier.com/locate/atherosclerosis
Relations of plasma high-sensitivity C-reactive protein to traditional cardiovascular risk factors Mayumi Saito a, Toshihiko Ishimitsu a,*, Junichi Minami a, Hidehiko Ono a, Masami Ohrui b, Hiroaki Matsuoka a a
Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan b Department of Health Care, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan Received 25 March 2002; received in revised form 21 August 2002; accepted 30 September 2002
Abstract Variations of circulating C-reactive protein (CRP) levels are supposed to reflect chronic inflammatory process of the cardiovascular system. In particular, it has been reported that high-sensitivity CRP (hsCRP) is a promising marker of coronary heart disease. In the present study, we assessed the relationship between hsCRP and classic cardiovascular risk factors, such as age, blood pressure, smoking habit and serum lipids. Plasma hsCRP was measured by ELISA in 908 subjects, aged 30 /79 years, who entered our health-check program. Plasma hsCRP level was 0.549/0.02 mg/l in 566 subjects without any disease currently treated. The level was significantly higher in patients treated for hypertension (0.749/0.06 mg/l, P/0.002), diabetes mellitus (0.779/0.09 mg/ l, P/0.016) or coronary artery disease (0.999/0.16 mg/l, P/0.008) than in subjects without diseases. In a simple regression analyses of the 566 subjects without diseases, plasma hsCRP positively correlated with male gender, smoking, body mass index, systolic blood pressure, white blood cell count, blood hemoglobin, fasting blood glucose, serum g-GTP, uric acid and triglycerides, and inversely correlated with serum albumin and HDL-cholesterol. In multiple regression analysis, white blood cell count (r/0.276, P B/0.001), body mass index (r /0.246, P B/0.001), age (r/0.122, P/0.001) and smoking (r /0.112, P /0.009) showed independent correlations with plasma hsCRP. It is suggested that variation of circulating hsCRP, even within normal range, is involved in the interrelation of cardiovascular risk factors, such as age, smoking, obesity, high blood pressure and dyslipidemia, which are supposed to promote atherosclerosis and ultimately provoke cardiovascular diseases, such as coronary artery disease. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C-reactive protein; Obesity; Smoking; Dyslipidemia; Hypertension
1. Introduction Traditional risk factors of cardiovascular diseases include aging, hypertension, dyslipidemia, smoking and diabetes mellitus. Among these risk factors, dyslipidemia plays an important role in the formation of atheromatous lesions, which ultimately cause stenosis or occlusion of arterial lumens. In addition to lipid accumulation, laboratory and experimental evidence indicate that chronic inflammatory process takes part in the development of atherosclerosis [1]. A number of studies have examined various circulating markers of
* Corresponding author. Tel.: /81-282-86-1111x2735; fax: /81282-86-1596 E-mail address: [email protected] (T. Ishimitsu).
inflammations, such as cytokines and adhesion molecules, as potential predictors of the present and the future risk of cardiovascular diseases. Of the numerous circulating markers thus far studied, high-sensitivity Creactive protein (hsCRP) seems to have the most consistent relation to the risk of cardiovascular diseases in a variety of clinical settings, including healthy subjects [2,3], selected high-risk subjects with traditional risk factors [4] and patients with cardiovascular diseases [5]. In order to prevent the incidence of cardiovascular events effectively, it is important to weigh the influence of each risk factor on the cardiovascular system. In this context, it must be taken into consideration that various cardiovascular factors are not independent of one another, but have direct or indirect relations. For instance, the prevalence of hypertension increases with
0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 2 ) 0 0 3 8 0 - 5
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age and the treatment of hypertension possibly causes glucose intolerance or hyperlipidemia as the adverse effects of antihypertensive drugs. Effective prevention of cardiovascular diseases should be started with unraveling the network of multiple risk factors. In this respect, the relation of inflammation markers to other risk factors are not well understood. This study was undertaken to clarify the relationship between circulating hsCRP and traditional risk factors in the health-check program participants.
2. Subjects and methods All 908 subjects who entered the 2-day hospitalized health check program from December 1999 to November 2000 were involved in this study. They are residents of a suburban area located 100 km north of Tokyo. They are supposedly interested in their health state because they voluntarily applied to the charged medical checkup program of our university hospital. The program included the taking of a full medical history and physical examinations, urinalysis, a stool examination, blood cell counts, blood chemistry, a glucose tolerance test, serological tests for hepatitis viruses, a chest roentgenogram, an electrocardiogram, a respiratory function, an alimentary examination of the upper gastrointestinal tract and an abdominal ultrasonography. The physical examination was performed by an internist, a surgeon, an ophthalmologist, an otorhinolaryngologist, a dentist and a gynecologist for women. Information about current habits of cigarette smoking and alcohol consumption was obtained by a selfadministered questionnaire. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). Blood pressure was measured twice on separate days by a sphygmomanometer with the subject in a sitting position and the averaged value was used for analyses. Antecubital venous blood was collected into a plain tube and a tube containing EDTA (1 mg/ml) in the morning after the subjects had fasted overnight. Plasma was separated by centrifugation at 4 8C and stored at / 80 8C until assayed. Routine laboratory tests, including blood cell counts and blood chemistry, were performed using automatic analyzers. Serum high density lipoprotein (HDL) cholesterol was assayed in a supernatant of polyethylene glycol precipitation [6]. In addition, plasma levels of CRP were measured by a highly sensitive sandwich ELISA technique using the anti-human-CRP goat antibody (first antibody) and rabbit antibody (second antibody) and the anti-rabbit-IgG goat IgG conjugated with horse radish peroxidase (third antibody). The 96-well microplate was coated with the first antibody and the sample plasma was diluted with 50 mM sodium phosphate buffer (pH 7.4) 100 times. A 50
ml aliquot of the diluted sample or the standard CRP solution was added to the well of 96-well microplate already containing 100 ml of the buffer with 0.05% Tween-20. The plate was shaken for 2 h and then incubated at 25 8C overnight. On the following day, the wells were washed three times with physiological saline containing 0.05% Tween-20 and a 100 ml of the second antibody solution was added to each well. Again the plate was shaken for 2 h at 25 8C and the wells were washed three times. Next, a 100 ml of the third antibody solution was added to each well and the plate was shaken for 2 h at 25 8C. Then, the wells were washed three times and a 100 ml solution containing 1 mg/ml ophenylenediamine (OPD) and 0.015% H2O2 in 0.1 M sodium citrate buffer (pH 5.0) was added to each well. The plate was incubated for 10 min at 25 8C and the reaction was stopped by adding 100 ml of 2 N sulfuric acid solution to each well. Optical density of the well was measured at 492 nm and the value was subtracted by the optical density at 690 nm. Sensitivity of the assay was 0.2 mg/l and the inter- and the intra-assay variations were 2.5 and 5.0%, respectively. Albumin concentration in the first urine in the morning was determined by the immunoturbidimetric method [7] and the value was expressed as a ratio to the urinary creatinine concentration measured by colorimetry. In evaluating the physical and laboratory findings of the subjects, hypertension was diagnosed when the subject was given antihypertensive drugs or two blood pressure measurements on separate days both exceeded 140 mmHg in systole and/or 90 mmHg in diastole. Diabetes mellitus was diagnosed when the subject was medicated for diabetes or the fasting blood glucose exceeded 126 mg/dl. Hyperlipidemia was diagnosed when the subject was taking lipid-lowering drugs or the serum total cholesterol exceeded 260 mg/dl, upper limit of the normal range of our institutional laboratory. The study protocol was in accordance with the recommendations of the World Medical Association for biomedical research involving human subjects (Somerset West version, 1996) and the informed consent was obtained from each subject. Data are presented as means9/S.D., except in Fig. 1. The horizontal bars on the columns of Fig. 1 indicate S.E. Clinical characteristics between the two groups were compared by unpaired Student’s t -test for parametric data and by x2-test for categoric data. Parametric data of three or more groups were compared using analysis of variance (ANOVA) followed by Scheffe’s post hoc test. Linear regression analysis was used to evaluate correlations between the two variables. Stepwise multiple regression analysis was performed to evaluate dependency between variables. The dichotomous variable, such as sex, was put into categories, 0 (women) and 1 (men). Smoking was evaluated as the number of cigarettes consumed per day and alcohol
M. Saito et al. / Atherosclerosis 167 (2003) 73 /79
Fig. 1. Plasma levels of hsCRP in subjects without diseases and subjects with diseases being treated. CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease. P values indicate significance compared with subjects without diseases.
intake was expressed by the number of drinks per day. A P value of B/0.05 was considered to indicate statistical significance.
3. Results Of 908 subjects examined in this study, 296 had diseases currently treated. Table 1 lists these diseases and the numbers of patients. The most frequent disease Table 1 Diseases in study subjects which were being treated Disease
No. of patients
Hypertension Arrhythmia Coronary artery disease Other cardiovascular diseases Cerebrovascular disease Other neurological diseases Hepatitis Gastroduodenal ulcer Other digestive diseases Chronic obstructive lung disease Diabetes mellitus Thyroid diseases Hyperlipidemia Hyperuricemia Chronic glomerulonephritis Prostatic diseases Urolithiasis Hematological diseases Bone and joint diseases Gynecological diseases Glaucoma
124 24 20 4 4 6 20 8 3 11 57 13 32 15 4 11 3 4 5 2 5
Total
296
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was hypertension followed by diabetes mellitus and hyperlipidemia. Fifty-five subjects had two or more diseases. Some 296 subjects were excluded in the following correlation analyses. In routine blood chemistry, the serum C-reactive protein (CRP) levels of 46 subjects exceeded the upper limit of normal range, 3 mg/ l. They were also excluded in the following analyses as apparently having infectious or inflammatory disorders. Factors relating to the plasma hsCRP were analyzed in the remaining 566 subjects. These subjects did not have any inflammatory disorders through the comprehensive checkup. Basic characteristics and the laboratory findings of the 566 subjects, 447 men and 119 women, are listed in Tables 2 and 3. There were well-expected differences in physical findings, habits, blood cell counts and blood chemistry between men and women. In particular, men had higher blood pressure, more frequent smoking habits and less favorable profiles of glucose and lipid metabolism. In addition, men showed a higher plasma hsCRP level by 26.8% than women. Table 4 shows the results of univariate correlation analyses of factors relating to plasma hsCRP level. White blood cell count (WBC) showed the highest correlation coefficient value and this was followed by BMI, smoking and serum HDL-cholesterol. Plasma hsCRP also had significant correlation with other classical cardiovascular risk factors, such as age and blood pressure. Alcohol intake did not significantly correlate with hsCRP. Plasma hsCRP positively correlated with blood hemoglobin, serum albumin and liver enzymes, such as g-GTP. Although the correlation between plasma hsCRP and serum total cholesterol was not significant, serum triglycerides, uric acid and fasting plasma glucose had positive correlation with plasma hsCRP. The correlation of hsCRP with serum creatinine or urinary albumin was insignificant. The results of stepwise multiple regression analysis is shown in Table 5. In the multiple regression analysis, Table 2 Background characteristics of the 566 subjects without diseases
Age (year) Body length (cm) Body weight (kg) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Habitual smoking (yes/no) Habitual alcohol intake (yes/no)
Men (n 447)
Women (n 119)
P value
5198 16895 67.298.6 23.892.6 122915
5397 15595 55.498.6 23.193.3 117915
0.022 B 0.001 B 0.001 NS B 0.001
79910
7399
B 0.001
180/267 236/211
9/110 13/106
B 0.001 B 0.001
Data are mean9S.D. NS, not significant.
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Table 3 Laboratory findings of the 566 subjects without diseases
White blood cell count ( 103/ml) Red blood cell count ( 106/ml) Blood hemoglobin (g/dl) Hematocrit (%) Platelet count ( 103/ml) Serum AST (U/l) Serum ALT (U/l) Serum g-GTP (U/l) Serum total protein (g/dl) Serum albumin (g/dl) Serum uric acid (mg/dl) Serum creatinine (mg/dl) Serum total cholesterol (mg/dl) Serum HDL-cholesterol (mg/dl) Serum triglycerides (mg/dl) Fasting plasma glucose (mg/dl) Plasma C-reactive protein (mg/l) Urinary albumin (mg/g creatinine)
Men (n 447)
Women (n 119)
P value
5.8791.57
4.9991.27
B 0.001
4.7890.38
4.3290.31
B 0.001
15.391.03 46.093.1 226949 2599 29917 30927 7.290.4 4.790.2 5.891.1 0.890.1 197931
13.291.07 40.192.9 232950 2196 1998 14912 7.290.4 4.590.2 4.391.0 0.690.1 209931
B 0.001 B 0.001 NS B 0.001 B 0.001 B 0.001 NS B 0.001 B 0.001 B 0.001 B 0.001
54912
65915
B 0.001
1469107 98912
103962 9498
B 0.001 B 0.001
0.5690.52
0.4490.41
8.8915.5
10.4915.1
0.003 NS
Table 4 Univariate correlations of plasma high-sensitivity C-reactive protein with physical and laboratory findings in the 566 subjects without diseases Parameter
Coefficient of correlation
P value
Age Sex Body mass index Systolic blood pressure Diastolic blood pressure Smoking Alcohol intake White blood cell count Red blood cell count Blood hemoglobin Hematocrit Platelet count Serum AST Serum ALT Serum g-GTP Serum total protein Serum albumin Serum uric acid Serum creatinine Serum total cholesterol Serum HDL-cholesterol Serum triglycerides Fasting plasma glucose Urinary albumin
0.088 0.102 0.276 0.132 0.094 0.226 0.002 0.338 0.092 0.132 0.126 0.057 0.072 0.085 0.118 0.000 0.108 0.111 0.038 0.015 0.217 0.153 0.092 0.018
0.037 0.014 B 0.001 0.001 0.025 B 0.001 NS B 0.001 0.028 0.001 0.002 NS NS 0.044 0.005 NS 0.009 0.008 NS NS B 0.001 B 0.001 0.029 NS
Data are mean9S.D. AST, aspartate aminotransferase; ALT, alanine aminotransferase; g-GTP, g-glutamyl transpeptidase; HDL, high density lipoprotein; NS, not significant.
AST, aspartate aminotransferase; ALT, alanine aminotransferase; g-GTP, g-glutamyl transpeptidase; HDL, high density lipoprotein; NS, not significant.
age, body mass index, smoking and white blood cell count remained as factors showing independent correlation with plasma hsCRP. Fig. 1 depicts plasma hsCRP levels in 566 subjects without diseases and patients grouped by currently treated diseases. Patients treated for hypertension, diabetes mellitus or coronary artery disease had significantly higher plasma hsCRP than subjects without diseases. Of the 124 patients treated for hypertension, 100 were taking antihypertensive drugs and 24 were not. Plasma hsCRP levels were not significantly different between the former and the latter (0.759/0.73 vs. 0.789/ 0.47 mg/l). Three diabetic patients were on insulin therapy, 25 were taking antidiabetic drugs and 29 were on diet therapy. Plasma hsCRP did not differ between the patients on pharmacological therapy and those on diet therapy (0.819/0.76 vs. 0.739/0.57 mg/l). Plasma hsCRP in patients treated for hyperlipidemia or hyperuricemia was not increased. Of 32 hyperlipidemic patients, 21 were taking lipid-lowering drugs and 11 were not. Plasma hsCRP levels were not significantly different between the former and the latter (0.509/0.50 vs. 0.639/0.44 mg/l). The increase of hsCRP in patients with chronic obstructive pulmonary disease was not significant compared with subjects without diseases.
Table 5 Stepwise multiple regression analysis of relations of various parameters to plasma high-sensitivity C-reactive protein in the 566 subjects without diseases Parameter
Standardized coefficient of correlation
P value
Age Body mass index Smoking White blood cell count
0.122 0.246 0.112 0.276
0.001 B 0.001 0.009 B 0.001
4. Discussion 4.1. Inflamation and cardiovascular risk Additional markers or better markers of cardiovascular risk are still needed because evaluation of traditional risk factors can insufficiently predict the incidence of cardiovascular diseases [8]. For instance, half of all myocardial infarctions occur in individuals with moderate to low risk based on assessment of lipid profile [9,10]. With regard to the pathogenesis of cardiovascular diseases, the development of atherosclerosis is now considered to be due, in part, to an inflammatory response [1]. Furthermore, inflammatory components
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are believed to contribute greatly to instability and rupture of atheromatous plaque that leads to atherothrombotic events [11 /14]. Thus, it has been hypothesized that inflammatory markers, such as hsCRP, may improve cardiovascular risk prediction adjunctive to the assessment of traditional risk factors. In support of this hypothesis, several large-scale epidemiological studies have shown that plasma hsCRP levels are an independent predictor of risk of future cardiovascular events, such as myocardial infarction, stroke and peripheral arterial disease [2,15 /17]. Risk factors of cardiovascular diseases are often related to one another. Namely, hypertension is often associated with glucose intolerance and dyslipidemia. The pathogenesis of these disorders seems to have relation to the state of insulin resistance [18 /20]. Simple reduction of blood pressure or lowering of serum lipids alone may reduce the risk of cardiovascular diseases to some extent. However, in order to prevent the occurrence of cardiovascular diseases more effectively, comprehensive reductions of cardiovascular risks and improvement of insulin resistance should be considered. Therefore, it is of critical importance to understand the interrelationship of each risk factor. In this respect, relations between inflammatory markers and other cardiovascular risk factors have not been fully elucidated. 4.2. Factors relating to plasma hsCRP In the present study, we investigated the factors relating to the plasma hsCRP level. As a result of our analysis in health-check program participants, WBC, BMI, age and smoking are the independent factors relating to plasma hsCRP levels. In addition, blood pressure, hematocrit, blood glucose and serum lipids and uric acid also had significant correlations with plasma hsCRP. Among these factors, it is quite plausible that WBC showed the highest value of correlation coefficient. Smoking is well supposed to give chemical and oxidative stimuli to the cardiovascular system and cause inflammation. It has recently been reported that moderate alcohol consumption reduces circulating CRP [21]. In the present study, when we divided the subjects according to the number of drinks per day, the plasma hsCRP level was not significantly affected by the quantity of alcohol consumption; 0 drink (n /316) 0.529/0.49 mg/l, one drink (n /93) 0.499/0.46 mg/l, two drinks (n/104) 0.609/0.54 mg/l, three drinks or more (n /53) 0.559/0.58 mg/l. On the other hand, it is known that alcoholic liver injury induces inflammatory cytokines [22]. Even if moderate alcohol consumption reduces plasma CRP, the effect may be canceled by the development of liver injury. Indeed, the subjects of this study consuming two drinks or more showed higher serum g-glutamyl transpeptitase than non-drinkers; 0
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drink 209/17 U/l, one drink 279/28 U/l, two drinks 359/ 26 U/l (P B/0.001 versus 0 drink), three drinks or more 619/66 U/l (P B/0.001 versus 0 drink). Plasma hsCRP was negatively correlated with serum albumin, but not with total protein in this study. Among serum proteins, g-globulin is expected to correlate positively with CRP. An increase in g-globulin with increasing CRP may be canceled by a decrease in albumin resulting in the lack of significant correlation between plasma hsCRP and serum total protein. 4.3. Plasma hsCRP in various disorders It is also conceivable that components of insulin resistance syndrome, such as hypertension, glucose intolerance and dyslipidemia promote atherosclerosis which involves inflammatory process. In our study, subjects who were treated for hypertension, diabetes mellitus and coronary artery disease had increased levels of plasma hsCRP. These patients have an increased risk of developing atherosclerosis. However, the plasma hsCRP in subjects being treated for hyperlipidemia was on a par with the subjects without diseases. This is supposedly because these hyperlipidemia patients were mostly given hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor which has been shown to reduce circulating hsCRP levels [23,24]. However, in the present study, plasma hsCRP of the hyperlipidemic patients on pharmacological treatment was insignificantly lower than those on diet therapy. A greater number of patients may be needed to statistically reveal the anti-inflammatory effect of HMG-CoA reductase inhibitors. It has been reported that C-reactive protein levels are elevated in overweight adults [25]. Because obesity founds a predisposition to hypertension, hyperlipidemia and diabetes mellitus, the relationship between obesity and atherosclerosis seems rather indirect. However, judging from the values of correlation coefficients, hsCRP seems to have a closer correlation with BMI than with blood pressure, plasma glucose or serum lipids. It should be borne in mind that BMI does not fluctuate, whereas blood pressure, glucose and lipids vary all the time, so that the correlation with these variables may tend to be lower. However, adipose tissue is known to secrete interleukin-6 (IL-6), the primary stimulant of CRP synthesis [26]. This suggests the existence of direct mechanism by which obesity increases CRP independently of the effects of insulin resistance. Hepatic production of CRP is increased with acute infection and trauma. Although the subjects having inflammatory disorders are excluded from the correlation analysis through the comprehensive checkup in the present study, the influence of non-cardiovascular minor inflammatory disorders may not be completely negated because this study concerns the variation of CRP within
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normal range. However, except for the effect of such disorders, circulating levels of hsCRP are stable within a person over long periods and are supposed to reflect chronic inflammatory process of the cardiovascular system [23,27]. Indeed, histological studies have shown that CRP localizes to the foam cells in atheromatous plaques [28,29]. Among other circulating markers of inflammation, IL-6 is the major stimulus for production of most acute phase proteins and appears to play a pivotal role as a mediator of inflammation [30,31]. IL-6 contributes to atherogenesis by inducing adhesion molecules, stimulating smooth muscle cell proliferation and increasing endothelial permeability [32]. It has also been demonstrated that plasma IL-6 levels are elevated and have a predictive value in patients with coronary artery disease [33 /35]. However, the plasma concentrations of IL-6 are far lower than those of CRP and are known to show a great circadian variation, while such circadian variation does not exist for hsCRP [36,37]. Therefore, currently, measurement of circulating hsCRP seems to provide a reproducible, reliable and feasible index of inflammation and cardiovascular risk compared with other inflammatory markers.
5. Conclusion Assuming that plasma hsCRP reflects future risk of cardiovascular diseases, intervention which reduces CRP may be effective in preventing the occurrence of cardiovascular events. In this context, a number of studies have shown that long-term therapy with aspirin, vitamin E or HMG-CoA reductase inhibitor, which has anti-inflammatory action, reduces hsCRP levels and the incidence of cardiovascular events [2,4,23,24,38 /43]. The results of the present study suggest that correction of overweight may be effective in reducing plasma hsCRP. Indeed, a recent study has reported that weight loss markedly reduces plasma CRP in obese postmenopausal women [44]. It has been also reported that moderate exercise reduces inflammation markers [45,46]. Taken together, these physical and dietary approaches to correct obesity may be promising in inhibiting cardiovascular inflammation and future risk of developing cardiovascular diseases.
Acknowledgements The authors thank Yasuko Mamada, Masako Minato, Mika Nomura and Machiko Sakata for technological assistance in executing the study. This study was supported in part by grant-in-aid for scientific research (10218209) from the Ministry of Education, Science and Culture of Japan.
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[33] Ridker PM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation 2000;101:1767 /72. [34] Biasucci LM, Vitelli A, Liuzzo G, Altamura S, Caligiuri G, Monaco C, Rebuzzi AG, Ciliberto G, Maseri A. Elevated levels of interleukin-6 in unstable angina. Circulation 1996;94:874 /7. [35] Biasucci LM, Liuzzo G, Fantuzzi G, Caligiuri G, Rebuzzi AG, Ginnetti F, Dinarello CA, Maseri A. Increasing levels of interleukin (IL)-1Ra and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased risk of in-hospital coronary events. Circulation 1999;99:2079 /84. [36] Sothern RB, Roitman-Johnson B, Kanabrocki EL, Yager JG, Roodell MM, Weatherbee JA, Young MR, Nenchausky BM, Scheving LE. Circadian characteristics of circulating interleukin-6 in men. J Allergy Clin Immunol 1995;95:1029 /35. [37] Meier-Ewert HK, Ridker PM, Rifai N, Price N, Dinges DF, Mullington JM. Absence of diurnal variation of C-reactive protein concentrations in healthy human subjects. Clin Chem 2001;47:426 /30. [38] Hansson L, Zanchetti A, Carruthers SG, Dahlof B, Elmfeldt D, Julius S, Menard J, Rahn KH, Wedel H, Westerling S. Effects of intensive blood-pressure lowering and low-dose aspirin in patients with hypertension: principal results of the Hypertension Optimal Treatment (HOT) randomised trial. HOT Study Group. Lancet 1998;351:1755 /62. [39] Devaraj S, Jialal I. Alpha tocopherol supplementation decreases serum C-reactive protein and monocyte interleukin-6 levels in normal volunteers and type 2 diabetic patients. Free Radic Biol Med 2000;29:790 /2. [40] Pryor WA. Vitamin E and heart disease: basic science to clinical intervention trials. Free Radic Biol Med 2000;28:141 /64. [41] Jialal I, Devaraj S. Vitamin E supplementation and cardiovascular events in high-risk patients. New Engl J Med 2000;342:1917 /8. [42] Maron DJ, Fazio S, Linton MF. Current perspectives on statins. Circulation 2000;101:207 /13. [43] Ross SD, Allen IE, Connelly JE, Korenblat BM, Smith ME, Bishop D, Luo D. Clinical outcomes in statin treatment trials: a meta-analysis. Arch Intern Med 1999;159:1793 /802. [44] Tchernof A, Nolan A, Sites CK, Ades PA, Poehlman ET. Weight loss reduces C-reactive protein levels in obese postmenopausal women. Circulation 2002;105:564 /9. [45] Smith JKDR, Douglas JE, Krishnaswamy G, Berk S. Long-term exercise and atherogenic activity of blood mononuclear cells in persons at risk of developing ischemic heart disease. J Am Med Assoc 1999;281:1722 /7. [46] Manson JE, Hu FB, Rich-Edwards JW, Colditz GA, Stampfer MJ, Willett WC, Speizer FE, Hennekens CH. A prospective study of walking as compared with vigorous exercise in the prevention of coronary heart disease in women. New Engl J Med 1999;341:650 /8.
Relations of plasma high-sensitivity C-reactive protein to traditional cardiovascular risk factors Mayumi Saito a, Toshihiko Ishimitsu a,*, Junichi Minami a, Hidehiko Ono a, Masami Ohrui b, Hiroaki Matsuoka a a
Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan b Department of Health Care, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan Received 25 March 2002; received in revised form 21 August 2002; accepted 30 September 2002
Abstract Variations of circulating C-reactive protein (CRP) levels are supposed to reflect chronic inflammatory process of the cardiovascular system. In particular, it has been reported that high-sensitivity CRP (hsCRP) is a promising marker of coronary heart disease. In the present study, we assessed the relationship between hsCRP and classic cardiovascular risk factors, such as age, blood pressure, smoking habit and serum lipids. Plasma hsCRP was measured by ELISA in 908 subjects, aged 30 /79 years, who entered our health-check program. Plasma hsCRP level was 0.549/0.02 mg/l in 566 subjects without any disease currently treated. The level was significantly higher in patients treated for hypertension (0.749/0.06 mg/l, P/0.002), diabetes mellitus (0.779/0.09 mg/ l, P/0.016) or coronary artery disease (0.999/0.16 mg/l, P/0.008) than in subjects without diseases. In a simple regression analyses of the 566 subjects without diseases, plasma hsCRP positively correlated with male gender, smoking, body mass index, systolic blood pressure, white blood cell count, blood hemoglobin, fasting blood glucose, serum g-GTP, uric acid and triglycerides, and inversely correlated with serum albumin and HDL-cholesterol. In multiple regression analysis, white blood cell count (r/0.276, P B/0.001), body mass index (r /0.246, P B/0.001), age (r/0.122, P/0.001) and smoking (r /0.112, P /0.009) showed independent correlations with plasma hsCRP. It is suggested that variation of circulating hsCRP, even within normal range, is involved in the interrelation of cardiovascular risk factors, such as age, smoking, obesity, high blood pressure and dyslipidemia, which are supposed to promote atherosclerosis and ultimately provoke cardiovascular diseases, such as coronary artery disease. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: C-reactive protein; Obesity; Smoking; Dyslipidemia; Hypertension
1. Introduction Traditional risk factors of cardiovascular diseases include aging, hypertension, dyslipidemia, smoking and diabetes mellitus. Among these risk factors, dyslipidemia plays an important role in the formation of atheromatous lesions, which ultimately cause stenosis or occlusion of arterial lumens. In addition to lipid accumulation, laboratory and experimental evidence indicate that chronic inflammatory process takes part in the development of atherosclerosis [1]. A number of studies have examined various circulating markers of
* Corresponding author. Tel.: /81-282-86-1111x2735; fax: /81282-86-1596 E-mail address: [email protected] (T. Ishimitsu).
inflammations, such as cytokines and adhesion molecules, as potential predictors of the present and the future risk of cardiovascular diseases. Of the numerous circulating markers thus far studied, high-sensitivity Creactive protein (hsCRP) seems to have the most consistent relation to the risk of cardiovascular diseases in a variety of clinical settings, including healthy subjects [2,3], selected high-risk subjects with traditional risk factors [4] and patients with cardiovascular diseases [5]. In order to prevent the incidence of cardiovascular events effectively, it is important to weigh the influence of each risk factor on the cardiovascular system. In this context, it must be taken into consideration that various cardiovascular factors are not independent of one another, but have direct or indirect relations. For instance, the prevalence of hypertension increases with
0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 2 ) 0 0 3 8 0 - 5
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age and the treatment of hypertension possibly causes glucose intolerance or hyperlipidemia as the adverse effects of antihypertensive drugs. Effective prevention of cardiovascular diseases should be started with unraveling the network of multiple risk factors. In this respect, the relation of inflammation markers to other risk factors are not well understood. This study was undertaken to clarify the relationship between circulating hsCRP and traditional risk factors in the health-check program participants.
2. Subjects and methods All 908 subjects who entered the 2-day hospitalized health check program from December 1999 to November 2000 were involved in this study. They are residents of a suburban area located 100 km north of Tokyo. They are supposedly interested in their health state because they voluntarily applied to the charged medical checkup program of our university hospital. The program included the taking of a full medical history and physical examinations, urinalysis, a stool examination, blood cell counts, blood chemistry, a glucose tolerance test, serological tests for hepatitis viruses, a chest roentgenogram, an electrocardiogram, a respiratory function, an alimentary examination of the upper gastrointestinal tract and an abdominal ultrasonography. The physical examination was performed by an internist, a surgeon, an ophthalmologist, an otorhinolaryngologist, a dentist and a gynecologist for women. Information about current habits of cigarette smoking and alcohol consumption was obtained by a selfadministered questionnaire. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). Blood pressure was measured twice on separate days by a sphygmomanometer with the subject in a sitting position and the averaged value was used for analyses. Antecubital venous blood was collected into a plain tube and a tube containing EDTA (1 mg/ml) in the morning after the subjects had fasted overnight. Plasma was separated by centrifugation at 4 8C and stored at / 80 8C until assayed. Routine laboratory tests, including blood cell counts and blood chemistry, were performed using automatic analyzers. Serum high density lipoprotein (HDL) cholesterol was assayed in a supernatant of polyethylene glycol precipitation [6]. In addition, plasma levels of CRP were measured by a highly sensitive sandwich ELISA technique using the anti-human-CRP goat antibody (first antibody) and rabbit antibody (second antibody) and the anti-rabbit-IgG goat IgG conjugated with horse radish peroxidase (third antibody). The 96-well microplate was coated with the first antibody and the sample plasma was diluted with 50 mM sodium phosphate buffer (pH 7.4) 100 times. A 50
ml aliquot of the diluted sample or the standard CRP solution was added to the well of 96-well microplate already containing 100 ml of the buffer with 0.05% Tween-20. The plate was shaken for 2 h and then incubated at 25 8C overnight. On the following day, the wells were washed three times with physiological saline containing 0.05% Tween-20 and a 100 ml of the second antibody solution was added to each well. Again the plate was shaken for 2 h at 25 8C and the wells were washed three times. Next, a 100 ml of the third antibody solution was added to each well and the plate was shaken for 2 h at 25 8C. Then, the wells were washed three times and a 100 ml solution containing 1 mg/ml ophenylenediamine (OPD) and 0.015% H2O2 in 0.1 M sodium citrate buffer (pH 5.0) was added to each well. The plate was incubated for 10 min at 25 8C and the reaction was stopped by adding 100 ml of 2 N sulfuric acid solution to each well. Optical density of the well was measured at 492 nm and the value was subtracted by the optical density at 690 nm. Sensitivity of the assay was 0.2 mg/l and the inter- and the intra-assay variations were 2.5 and 5.0%, respectively. Albumin concentration in the first urine in the morning was determined by the immunoturbidimetric method [7] and the value was expressed as a ratio to the urinary creatinine concentration measured by colorimetry. In evaluating the physical and laboratory findings of the subjects, hypertension was diagnosed when the subject was given antihypertensive drugs or two blood pressure measurements on separate days both exceeded 140 mmHg in systole and/or 90 mmHg in diastole. Diabetes mellitus was diagnosed when the subject was medicated for diabetes or the fasting blood glucose exceeded 126 mg/dl. Hyperlipidemia was diagnosed when the subject was taking lipid-lowering drugs or the serum total cholesterol exceeded 260 mg/dl, upper limit of the normal range of our institutional laboratory. The study protocol was in accordance with the recommendations of the World Medical Association for biomedical research involving human subjects (Somerset West version, 1996) and the informed consent was obtained from each subject. Data are presented as means9/S.D., except in Fig. 1. The horizontal bars on the columns of Fig. 1 indicate S.E. Clinical characteristics between the two groups were compared by unpaired Student’s t -test for parametric data and by x2-test for categoric data. Parametric data of three or more groups were compared using analysis of variance (ANOVA) followed by Scheffe’s post hoc test. Linear regression analysis was used to evaluate correlations between the two variables. Stepwise multiple regression analysis was performed to evaluate dependency between variables. The dichotomous variable, such as sex, was put into categories, 0 (women) and 1 (men). Smoking was evaluated as the number of cigarettes consumed per day and alcohol
M. Saito et al. / Atherosclerosis 167 (2003) 73 /79
Fig. 1. Plasma levels of hsCRP in subjects without diseases and subjects with diseases being treated. CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease. P values indicate significance compared with subjects without diseases.
intake was expressed by the number of drinks per day. A P value of B/0.05 was considered to indicate statistical significance.
3. Results Of 908 subjects examined in this study, 296 had diseases currently treated. Table 1 lists these diseases and the numbers of patients. The most frequent disease Table 1 Diseases in study subjects which were being treated Disease
No. of patients
Hypertension Arrhythmia Coronary artery disease Other cardiovascular diseases Cerebrovascular disease Other neurological diseases Hepatitis Gastroduodenal ulcer Other digestive diseases Chronic obstructive lung disease Diabetes mellitus Thyroid diseases Hyperlipidemia Hyperuricemia Chronic glomerulonephritis Prostatic diseases Urolithiasis Hematological diseases Bone and joint diseases Gynecological diseases Glaucoma
124 24 20 4 4 6 20 8 3 11 57 13 32 15 4 11 3 4 5 2 5
Total
296
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was hypertension followed by diabetes mellitus and hyperlipidemia. Fifty-five subjects had two or more diseases. Some 296 subjects were excluded in the following correlation analyses. In routine blood chemistry, the serum C-reactive protein (CRP) levels of 46 subjects exceeded the upper limit of normal range, 3 mg/ l. They were also excluded in the following analyses as apparently having infectious or inflammatory disorders. Factors relating to the plasma hsCRP were analyzed in the remaining 566 subjects. These subjects did not have any inflammatory disorders through the comprehensive checkup. Basic characteristics and the laboratory findings of the 566 subjects, 447 men and 119 women, are listed in Tables 2 and 3. There were well-expected differences in physical findings, habits, blood cell counts and blood chemistry between men and women. In particular, men had higher blood pressure, more frequent smoking habits and less favorable profiles of glucose and lipid metabolism. In addition, men showed a higher plasma hsCRP level by 26.8% than women. Table 4 shows the results of univariate correlation analyses of factors relating to plasma hsCRP level. White blood cell count (WBC) showed the highest correlation coefficient value and this was followed by BMI, smoking and serum HDL-cholesterol. Plasma hsCRP also had significant correlation with other classical cardiovascular risk factors, such as age and blood pressure. Alcohol intake did not significantly correlate with hsCRP. Plasma hsCRP positively correlated with blood hemoglobin, serum albumin and liver enzymes, such as g-GTP. Although the correlation between plasma hsCRP and serum total cholesterol was not significant, serum triglycerides, uric acid and fasting plasma glucose had positive correlation with plasma hsCRP. The correlation of hsCRP with serum creatinine or urinary albumin was insignificant. The results of stepwise multiple regression analysis is shown in Table 5. In the multiple regression analysis, Table 2 Background characteristics of the 566 subjects without diseases
Age (year) Body length (cm) Body weight (kg) Body mass index (kg/m2) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Habitual smoking (yes/no) Habitual alcohol intake (yes/no)
Men (n 447)
Women (n 119)
P value
5198 16895 67.298.6 23.892.6 122915
5397 15595 55.498.6 23.193.3 117915
0.022 B 0.001 B 0.001 NS B 0.001
79910
7399
B 0.001
180/267 236/211
9/110 13/106
B 0.001 B 0.001
Data are mean9S.D. NS, not significant.
M. Saito et al. / Atherosclerosis 167 (2003) 73 /79
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Table 3 Laboratory findings of the 566 subjects without diseases
White blood cell count ( 103/ml) Red blood cell count ( 106/ml) Blood hemoglobin (g/dl) Hematocrit (%) Platelet count ( 103/ml) Serum AST (U/l) Serum ALT (U/l) Serum g-GTP (U/l) Serum total protein (g/dl) Serum albumin (g/dl) Serum uric acid (mg/dl) Serum creatinine (mg/dl) Serum total cholesterol (mg/dl) Serum HDL-cholesterol (mg/dl) Serum triglycerides (mg/dl) Fasting plasma glucose (mg/dl) Plasma C-reactive protein (mg/l) Urinary albumin (mg/g creatinine)
Men (n 447)
Women (n 119)
P value
5.8791.57
4.9991.27
B 0.001
4.7890.38
4.3290.31
B 0.001
15.391.03 46.093.1 226949 2599 29917 30927 7.290.4 4.790.2 5.891.1 0.890.1 197931
13.291.07 40.192.9 232950 2196 1998 14912 7.290.4 4.590.2 4.391.0 0.690.1 209931
B 0.001 B 0.001 NS B 0.001 B 0.001 B 0.001 NS B 0.001 B 0.001 B 0.001 B 0.001
54912
65915
B 0.001
1469107 98912
103962 9498
B 0.001 B 0.001
0.5690.52
0.4490.41
8.8915.5
10.4915.1
0.003 NS
Table 4 Univariate correlations of plasma high-sensitivity C-reactive protein with physical and laboratory findings in the 566 subjects without diseases Parameter
Coefficient of correlation
P value
Age Sex Body mass index Systolic blood pressure Diastolic blood pressure Smoking Alcohol intake White blood cell count Red blood cell count Blood hemoglobin Hematocrit Platelet count Serum AST Serum ALT Serum g-GTP Serum total protein Serum albumin Serum uric acid Serum creatinine Serum total cholesterol Serum HDL-cholesterol Serum triglycerides Fasting plasma glucose Urinary albumin
0.088 0.102 0.276 0.132 0.094 0.226 0.002 0.338 0.092 0.132 0.126 0.057 0.072 0.085 0.118 0.000 0.108 0.111 0.038 0.015 0.217 0.153 0.092 0.018
0.037 0.014 B 0.001 0.001 0.025 B 0.001 NS B 0.001 0.028 0.001 0.002 NS NS 0.044 0.005 NS 0.009 0.008 NS NS B 0.001 B 0.001 0.029 NS
Data are mean9S.D. AST, aspartate aminotransferase; ALT, alanine aminotransferase; g-GTP, g-glutamyl transpeptidase; HDL, high density lipoprotein; NS, not significant.
AST, aspartate aminotransferase; ALT, alanine aminotransferase; g-GTP, g-glutamyl transpeptidase; HDL, high density lipoprotein; NS, not significant.
age, body mass index, smoking and white blood cell count remained as factors showing independent correlation with plasma hsCRP. Fig. 1 depicts plasma hsCRP levels in 566 subjects without diseases and patients grouped by currently treated diseases. Patients treated for hypertension, diabetes mellitus or coronary artery disease had significantly higher plasma hsCRP than subjects without diseases. Of the 124 patients treated for hypertension, 100 were taking antihypertensive drugs and 24 were not. Plasma hsCRP levels were not significantly different between the former and the latter (0.759/0.73 vs. 0.789/ 0.47 mg/l). Three diabetic patients were on insulin therapy, 25 were taking antidiabetic drugs and 29 were on diet therapy. Plasma hsCRP did not differ between the patients on pharmacological therapy and those on diet therapy (0.819/0.76 vs. 0.739/0.57 mg/l). Plasma hsCRP in patients treated for hyperlipidemia or hyperuricemia was not increased. Of 32 hyperlipidemic patients, 21 were taking lipid-lowering drugs and 11 were not. Plasma hsCRP levels were not significantly different between the former and the latter (0.509/0.50 vs. 0.639/0.44 mg/l). The increase of hsCRP in patients with chronic obstructive pulmonary disease was not significant compared with subjects without diseases.
Table 5 Stepwise multiple regression analysis of relations of various parameters to plasma high-sensitivity C-reactive protein in the 566 subjects without diseases Parameter
Standardized coefficient of correlation
P value
Age Body mass index Smoking White blood cell count
0.122 0.246 0.112 0.276
0.001 B 0.001 0.009 B 0.001
4. Discussion 4.1. Inflamation and cardiovascular risk Additional markers or better markers of cardiovascular risk are still needed because evaluation of traditional risk factors can insufficiently predict the incidence of cardiovascular diseases [8]. For instance, half of all myocardial infarctions occur in individuals with moderate to low risk based on assessment of lipid profile [9,10]. With regard to the pathogenesis of cardiovascular diseases, the development of atherosclerosis is now considered to be due, in part, to an inflammatory response [1]. Furthermore, inflammatory components
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are believed to contribute greatly to instability and rupture of atheromatous plaque that leads to atherothrombotic events [11 /14]. Thus, it has been hypothesized that inflammatory markers, such as hsCRP, may improve cardiovascular risk prediction adjunctive to the assessment of traditional risk factors. In support of this hypothesis, several large-scale epidemiological studies have shown that plasma hsCRP levels are an independent predictor of risk of future cardiovascular events, such as myocardial infarction, stroke and peripheral arterial disease [2,15 /17]. Risk factors of cardiovascular diseases are often related to one another. Namely, hypertension is often associated with glucose intolerance and dyslipidemia. The pathogenesis of these disorders seems to have relation to the state of insulin resistance [18 /20]. Simple reduction of blood pressure or lowering of serum lipids alone may reduce the risk of cardiovascular diseases to some extent. However, in order to prevent the occurrence of cardiovascular diseases more effectively, comprehensive reductions of cardiovascular risks and improvement of insulin resistance should be considered. Therefore, it is of critical importance to understand the interrelationship of each risk factor. In this respect, relations between inflammatory markers and other cardiovascular risk factors have not been fully elucidated. 4.2. Factors relating to plasma hsCRP In the present study, we investigated the factors relating to the plasma hsCRP level. As a result of our analysis in health-check program participants, WBC, BMI, age and smoking are the independent factors relating to plasma hsCRP levels. In addition, blood pressure, hematocrit, blood glucose and serum lipids and uric acid also had significant correlations with plasma hsCRP. Among these factors, it is quite plausible that WBC showed the highest value of correlation coefficient. Smoking is well supposed to give chemical and oxidative stimuli to the cardiovascular system and cause inflammation. It has recently been reported that moderate alcohol consumption reduces circulating CRP [21]. In the present study, when we divided the subjects according to the number of drinks per day, the plasma hsCRP level was not significantly affected by the quantity of alcohol consumption; 0 drink (n /316) 0.529/0.49 mg/l, one drink (n /93) 0.499/0.46 mg/l, two drinks (n/104) 0.609/0.54 mg/l, three drinks or more (n /53) 0.559/0.58 mg/l. On the other hand, it is known that alcoholic liver injury induces inflammatory cytokines [22]. Even if moderate alcohol consumption reduces plasma CRP, the effect may be canceled by the development of liver injury. Indeed, the subjects of this study consuming two drinks or more showed higher serum g-glutamyl transpeptitase than non-drinkers; 0
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drink 209/17 U/l, one drink 279/28 U/l, two drinks 359/ 26 U/l (P B/0.001 versus 0 drink), three drinks or more 619/66 U/l (P B/0.001 versus 0 drink). Plasma hsCRP was negatively correlated with serum albumin, but not with total protein in this study. Among serum proteins, g-globulin is expected to correlate positively with CRP. An increase in g-globulin with increasing CRP may be canceled by a decrease in albumin resulting in the lack of significant correlation between plasma hsCRP and serum total protein. 4.3. Plasma hsCRP in various disorders It is also conceivable that components of insulin resistance syndrome, such as hypertension, glucose intolerance and dyslipidemia promote atherosclerosis which involves inflammatory process. In our study, subjects who were treated for hypertension, diabetes mellitus and coronary artery disease had increased levels of plasma hsCRP. These patients have an increased risk of developing atherosclerosis. However, the plasma hsCRP in subjects being treated for hyperlipidemia was on a par with the subjects without diseases. This is supposedly because these hyperlipidemia patients were mostly given hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor which has been shown to reduce circulating hsCRP levels [23,24]. However, in the present study, plasma hsCRP of the hyperlipidemic patients on pharmacological treatment was insignificantly lower than those on diet therapy. A greater number of patients may be needed to statistically reveal the anti-inflammatory effect of HMG-CoA reductase inhibitors. It has been reported that C-reactive protein levels are elevated in overweight adults [25]. Because obesity founds a predisposition to hypertension, hyperlipidemia and diabetes mellitus, the relationship between obesity and atherosclerosis seems rather indirect. However, judging from the values of correlation coefficients, hsCRP seems to have a closer correlation with BMI than with blood pressure, plasma glucose or serum lipids. It should be borne in mind that BMI does not fluctuate, whereas blood pressure, glucose and lipids vary all the time, so that the correlation with these variables may tend to be lower. However, adipose tissue is known to secrete interleukin-6 (IL-6), the primary stimulant of CRP synthesis [26]. This suggests the existence of direct mechanism by which obesity increases CRP independently of the effects of insulin resistance. Hepatic production of CRP is increased with acute infection and trauma. Although the subjects having inflammatory disorders are excluded from the correlation analysis through the comprehensive checkup in the present study, the influence of non-cardiovascular minor inflammatory disorders may not be completely negated because this study concerns the variation of CRP within
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normal range. However, except for the effect of such disorders, circulating levels of hsCRP are stable within a person over long periods and are supposed to reflect chronic inflammatory process of the cardiovascular system [23,27]. Indeed, histological studies have shown that CRP localizes to the foam cells in atheromatous plaques [28,29]. Among other circulating markers of inflammation, IL-6 is the major stimulus for production of most acute phase proteins and appears to play a pivotal role as a mediator of inflammation [30,31]. IL-6 contributes to atherogenesis by inducing adhesion molecules, stimulating smooth muscle cell proliferation and increasing endothelial permeability [32]. It has also been demonstrated that plasma IL-6 levels are elevated and have a predictive value in patients with coronary artery disease [33 /35]. However, the plasma concentrations of IL-6 are far lower than those of CRP and are known to show a great circadian variation, while such circadian variation does not exist for hsCRP [36,37]. Therefore, currently, measurement of circulating hsCRP seems to provide a reproducible, reliable and feasible index of inflammation and cardiovascular risk compared with other inflammatory markers.
5. Conclusion Assuming that plasma hsCRP reflects future risk of cardiovascular diseases, intervention which reduces CRP may be effective in preventing the occurrence of cardiovascular events. In this context, a number of studies have shown that long-term therapy with aspirin, vitamin E or HMG-CoA reductase inhibitor, which has anti-inflammatory action, reduces hsCRP levels and the incidence of cardiovascular events [2,4,23,24,38 /43]. The results of the present study suggest that correction of overweight may be effective in reducing plasma hsCRP. Indeed, a recent study has reported that weight loss markedly reduces plasma CRP in obese postmenopausal women [44]. It has been also reported that moderate exercise reduces inflammation markers [45,46]. Taken together, these physical and dietary approaches to correct obesity may be promising in inhibiting cardiovascular inflammation and future risk of developing cardiovascular diseases.
Acknowledgements The authors thank Yasuko Mamada, Masako Minato, Mika Nomura and Machiko Sakata for technological assistance in executing the study. This study was supported in part by grant-in-aid for scientific research (10218209) from the Ministry of Education, Science and Culture of Japan.
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