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Serum Total Bilirubin Concentration Is Inversely Correlated with Framingham Risk Score in Koreans
Archives of Medical Research 43 (2012) 288e293
ORIGINAL ARTICLE
Serum Total Bilirubin Concentration Is Inversely Correlated with Framingham Risk Score in Koreans Kwang-Min Kim, Bom-Taeck Kim, Sat-Byul Park, Doo-Yeoun Cho, Sang Hyeon Je, and Kyu-Nam Kim Department of Family Practice and Community Health, Ajou University School of Medicine, Suwon, Republic of Korea Received for publication December 4, 2011; accepted May 2, 2012 (ARCMED-D-11-00606).
Background and Aims. Compelling evidence suggests that bilirubin, via its antioxidant potential, has anti-atherogenic properties, and that serum bilirubin concentrations within the reference range for the general population may provide some protection against coronary artery disease (CAD). This study examined the association between serum total bilirubin concentration and Framingham risk score (FRS) in the Korean population. Methods. This cross-sectional study was performed on 19,792 Koreans. In addition to FRS, data on body mass index, fasting blood glucose, liver enzymes, lipid profile, uric acid, gamma glutamyltransferase, high-sensitive C-reactive protein and total bilirubin were used. Results. Negative correlations were established between log-transformed total bilirubin concentration and FRS (females; r 5 0.067, p !0.001, males; r 5 0.128, p !0.001). Analyses relating total bilirubin to FRS $10% utilized multiple confounder adjusted logistic regression. Unadjusted odd ratios for FRS $10% were 0.325 (95% CI: 0.160e0.659, p 5 0.002) and 0.342 (95% CI: 0.281e0.417, p !0.001) for log-transformed total bilirubin in females and males, respectively. These inverse relationships remained significant after adjustments for multiple confounders in both genders. Conclusions. Increased total bilirubin concentrations are associated with the decrease in FRS. Serum total bilirubin may be helpful to decrease the future risk of CAD. Ó 2012 IMSS. Published by Elsevier Inc. Key Words: Total bilirubin, Framingham risk score, Oxidative stress.
Introduction Oxidative stress is one of the most significant aspects in the pathogenesis of coronary artery disease (CAD), which is among the leading causes of death worldwide (1) and which is thought to play an important role in the progression of atherosclerosis (2,3). Several studies have implicated bilirubin as a biomarker of an antioxidant under physiological conditions (4e6). Parallel evidence from epidemiological studies suggest that low serum concentrations of total bilirubin are associated with development of CAD risk
Address reprint requests to: Kyu-Nam Kim, Department of Family Practice and Community Health, Ajou University School of Medicine, 164 Worldcup-ro, Youngtong-gu, Suwon, Gyeonggi-do, Republic of Korea; Phone: 82-031-219-5324; FAX: 82-31-219-5218; E-mail: ktwonm@ hanmail.net
factors including diabetes (7,8), hypertension (9) and metabolic syndrome (10,11). In addition, several studies have reported that low serum total bilirubin concentrations are related to an increased risk of CAD (12,13) as well as increased carotid intima-media thickness and peripheral arterial disease (14,15). Furthermore, a study reported a significant negative correlation even between bilirubin and CAD risk calculated using the Framingham Equation (16). However, this study targeted smoking status or was reported without adjusting multiple confounding factors such as the traditional risk factors or other novel CAD risk factors. In spite of the clinical impact these decreased total bilirubin levels have in many studies, little is known about the effect of total bilirubin levels in the prediction of CAD risk after controlling for multiple potential confounding factors in the general population. The Framingham Risk Score (FRS) is a mathematical model for predicting CAD
0188-4409/$ - see front matter. Copyright Ó 2012 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2012.05.003
Serum Total Bilirubin and Framingham Risk Score
during a 10-year period. The FRS has become a widely used clinical tool to guide the delivery of preventive medicine (17,18). The present study was grounded in the hypothesis that increasing serum total bilirubin is associated with a decreased risk of CAD, calculated using the FRS modified by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) guideline and may contribute to reduce CAD risk prediction. The aim of this study was to examine whether serum total bilirubin levels are associated with FRS in Koreans.
Subjects and Methods Subjects From January 2007 to May 2009, data of 33,906 Koreans aged 20e86 years who visited the Health Promotion Center, Ajou University Hospital, Suwon, Gyeonggi-do, Republic of Korea were reviewed for inclusion in the study. Medical history, demographics, and anthropometric and laboratory data were collected. Data on cigarette smoking and alcohol consumption were collected by a self-reported questionnaire. Subjects who, at the time of the survey, had smoked cigarettes regularly within the prior year were considered to be current smokers and weekly alcohol intake was calculated and then converted to weekly alcohol consumption (grams of ethanol per week) by the graduated frequency method (19). Of the initial 33,906 subjects, we excluded 14,114 subjects who met one of the following criteria: 8,221 subjects had missing data values for any component of FRS or serum total bilirubin or alcohol history; 831 subjects were positive for antibody to hepatitis B surface antigen, antibody to hepatitis B virus core antigen or anti-hepatitis C virus; 2,314 subjects had a medical history of chronic liver disease or liver cirrhosis or were taking drugs influencing liver function (hepatotonics); 293 subjects were on medication (statins or other lipid-lowering drugs) for hyperlipidemia; 1,842 subjects had been diagnosed with diabetes mellitus or a CAD; 1,310 subjects had abnormal liver function tests defined as O41 IU/L for males and O31 IU/L for females for alanine aminotransferase, O40 IU/L for aspartate aminotransferase, and O66 IU/L for males and O39 IU/L for females for gamma glutamyltransferase (GGT) or O1.2 mg/dL for serum total bilirubin level, which were established as the cutoff values based on 95% confidence limits for 705 healthy adults; 299 subjects had highsensitive C-reactive protein (hs-CRP) $10.0 mg/L to preclude any possible occult inflammatory or infectious disease. Thus, a total of 19,792 healthy Korean subjects (11,206 females, 8,586 males) were included in the final analyses. Written informed consent was obtained from all participants and the institutional review board of Ajou University Hospital approved this study.
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Materials and Methods Before collection of blood, each subject fasted for 10 h or more. After overnight fasting, a venous blood sample was obtained between 08:00 and 10:00 am to measure total bilirubin concentrations, fasting blood glucose, liver enzymes, total cholesterol, triglycerides, HDL (high-density lipoprotein) cholesterol, LDL (low-density lipoprotein) cholesterol, hsCRP and uric acid. Serum total bilirubin, fasting blood glucose, liver enzymes, lipid levels and uric acid were assayed by the standard method recommended by the International Federation for Clinical Chemistry using a Toshiba200FR automatic analyzer (Toshiba Medical Systems, Tokyo, Japan). Fractions of bilirubin were not assessed. hs-CRP was measured by a high-sensitivity nephelometric method (Dade Behring Marburg GMBH, Marburg, Germany). Blood pressure (BP) was measured using a standard mercury manometer with the participant in a sitting position for 5 min prior to measurement; the average of two measurements was recorded. Hypertension was defined as a systolic BP $140 mm Hg or a diastolic BP $90 mm Hg or by the use of antihypertensive medication. Diabetes was defined by fasting blood glucose $126 mg/dL or the use of oral hypoglycemic agents or insulin. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). Statistical Analyses The distribution of total bilirubin, hs-CRP, GGT values and alcohol consumption were right-skewed; therefore, a natural log-transformation was applied. To assess the relationship between biomarkers and the individual components of the FRS, Pearson correlation coefficients relating individual risk factor scores and the total of FRS to log-transformed total bilirubin levels were analyzed. The FRS was calculated from the NCEP ATP III algorithm based on six coronary risk factors: gender, age, total cholesterol, HDLcholesterol, systolic BP and smoking habit (20). Among these factors, age, BP, and cholesterol levels were categorized according to their values. Smoking status was classified as either current smoker or non-smoker. Framingham risk equations were used to predict the risk of developing coronary disease events (myocardial infarction or coronary heart disease death) over the next 10 years for adults aged 20 and older without heart disease or diabetes. Participants were divided into three groups (20): low risk (#9% risk of developing a coronary heart disease event over the next 10 years), intermediate risk (10e20% risk), and high risk (O20% risk). For analyses relating total bilirubin to the intermediate-risk group and beyond (FRS $10%) or high risk (FRS O20%), we constructed an adjusted logistic regression analyses that considered alcohol consumption þ BMI þ LDL cholesterol, and (alcohol consumption þ BMI þ LDL cholesterol) þ novel risk factors including hs-CRP, GGT and uric acid. Results of group data are expressed as mean standard deviation (SD). All statistical
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analyses were performed using SPSS v.13.0 software (SPSS, Chicago, IL); p !0.05 was considered statistically significant.
Results Table 1 shows the characteristics of the excluded subjects including missing data. Numbers of excluded males were more than numbers of females in this study because various metabolic parameters including diabetes and elevated liver enzymes for exclusion criteria in men were higher than those in women. Table 2 shows the characteristics of the selected subjects. Compared with females, males presented with more CAD risk factors such as hypertension, dyslipidemia, alcohol consumption and current smoking. Therefore, the total Framingham point score and 10-year CAD risk were higher in men than in women. The correlation coefficients between log-transformed total bilirubin levels and 10-year CAD risk were r 5 0.067 ( p !0.001) and r 5 0.128 ( p !0.001) in females and males, respectively (Table 3). Of the Framingham individual components, serum total bilirubin also was negatively correlated with age score, smoking score, HDL cholesterol score and systolic BP score in both genders. Unadjusted odds ratio (OR) of progressing from the low-risk group to the intermediate-risk group and beyond was 0.325 (95% CI: Table 1. General characteristics of the excluded subjects
Characteristic Age, years BMI (kg/m2) Current smoker, n (%) Alcohol (g/week) SBP (mmHg) DBP (mmHg) Hypertension, n (%) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) AST (IU/L) ALT (IU/L) GGT (IU/L) Fasting blood glucose (mg/dL) Diabetes mellitus, n (%) hs-CRP (mg/dL) Uric acid (mg/dL) Total bilirubin (mg/dL)
All patients (n 5 14,114)
Females (n 5 6,775)
Males (n 5 7,339)
45.1 9.1 23.1 3.0 3,246 (23.1) 58.4 54.1 116.0 14.1 75.7 11.1 1,961 (13.9) 187.2 31.9 53.7 12.0 110.2 29.1 105.5 63.8 21.0 12.5 21.8 10.6 23.6 23.3 93.3 15.7
44.5 9.5 22.1 2.8 210 (3.1) 40.55 48.1 113.5 12.9 73.3 9.9 684 (10.1) 183.2 30.5 57.2 12.4 107.5 28.9 90.3 57.5 19.3 10.4 17.4 11.7 16.6 16.4 90.2 12.9
45.9 8.8 23.9 2.8 3,036 (41.3) 64.2 10.61 119.8 12.7 79.8 10.1 1,277 (17.4) 190.3 32.1 49.5 10.7 112.7 25.8 125.5 65.7. 23.7 12.9 24.5 10.5 30.0 24.1 96.2 17.5
Table 2. General characteristics of the study subjects
Characteristic Age, years BMI (kg/m2) Current smoker, n (%) Alcohol (g/week) SBP (mmHg) DBP (mmHg) Hypertension, n (%) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) AST (IU/L) ALT (IU/L) GGT (IU/L) Fasting blood glucose (mg/dL) hs-CRP (mg/dL) Uric acid (mg/dL) Total bilirubin (mg/dL) Total Framingham point score 10 year CAD risk (%)
496 (2.9) 0.2 0.4 3.9 0.8 0.8 0.3
1,056 (14.3) 0.3 0.5 5.8 1.2 1.1 0.4
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; hs-CRP, high-sensitivity C-reactive protein; CAD, coronary artery disease. Data are expressed as mean standard deviations (SD) or as number (percentage).
Females (n 5 11,206)
Males (n 5 8,586)
44.7 10.2 23.1 2.9 4,067 (20.5) 55.6 54.1 116.7 14.4 75.7 11.0 2,710 (13.7) 185.1 32.5 53.2 12.9 108.4 28.8 100.5 64.4 19.9 5.1 18.5 7.5 21.3 12.3 91.5 8.7
44.3 10.4 22.6 2.9 373 (3.3) 39.9 43.9 114.6 14.9 73.2 10.7 1,122 (10.0) 182.3 33.7 57.8 13.0 106.6 29.6 86.2 56.0 19.1 5.0 16.0 6.5 15.9 8.4 90.0 8.5
45.3 10.0 23.8 2.6 3,694 (43.0) 63.1 56.8 119.5 13.3 79.4 10.3 1,588 (18.5) 187.0 31.5 49.3 11.5 110.7 27.6 119.2 69.7 21.0 5.1 21.9 7.4 28.4 12.9 93.3 8.6
0.1 4.7 0.8 5.8
0.5 1.3 0.2 6.1
0.1 4.0 0.7 4.7
3.0 4.3
0.3 0.9 0.2 6.5
0.2 5.7 0.8 7.3
1.0 1.6
0.6 1.2 0.1 5.2
5.7 5.3
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; hs-CRP, high-sensitivity C-reactive protein; CAD, coronary artery disease. Data are expressed as mean standard deviations (SD) or as number (percentage).
0.160e0.659, p 5 0.002) and 0.345 (95% CI: 0.281e0.417, p !0.001) for log-transformed total bilirubin in females and males, respectively (Table 4). After additional adjustments for BMI, log-transformed weekly alcohol consumption and other conventional and novel cardiovascular risk factors such as LDL cholesterol, GGT, hs-CRP and uric acid, we discovered that increases in the Table 3. Pearson correlation coefficients relating individual components and the total score of Framingham point to log-transformed total bilirubin concentration in each gender Females
Variables 1,552 (11.0) 0.2 0.4 4.9 1.4 0.9 0.4
All patients (n 5 19,792)
Age score Total cholesterol score Smoking score HDL cholesterol score Systolic BP score Total Framingham point score 10 year CAD risk
Males
Correlation coefficient
p
Correlation coefficient
p
0.070 0.028 0.038 0.121 0.058 0.086
!0.001 0.003 !0.001 !0.001 !0.001 !0.001
0.055 0.051 0.070 0.116 0.023 0.104
!0.001 !0.001 !0.001 !0.001 0.035 !0.001
0.067
!0.001
0.128
!0.001
HDL, high-density lipoprotein; BP, blood pressure; CAD, coronary artery disease.
Serum Total Bilirubin and Framingham Risk Score Table 4. Logistic regression analysis of log-transformed total bilirubin as an independent variable and intermediate and high-risk for CAD (10-year risk $10%) as a dependent variable in females and males
Females Model Model Model Males Model Model Model
B
SE
p
OR (95% CI)
1 2 3
1.124 2.133 3.466
0.360 1.146 1.644
0.002 0.010 0.035
0.325 (0.160e0.659) 0.119 (0.013e0.589) 0.031 (0.001e0.784)
1 2 3
1.072 1.068 0.955
0.101 0.135 0.206
!0.001 !0.001 !0.001
0.342 (0.281e0.417) 0.344 (0.264e0.448) 0.385 (0.257e0.577)
CAD, coronary artery disease; LDL, low-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; GGT, gamma glutamyltransferase; CI, confidence interval. Model 1: unadjusted; Model 2: after adjustment for body mass index, logtransformed weekly alcohol consumption and LDL cholesterol; Model 3: model 2 plus adjustment for log-transformed hs-CRP, log-transformed GGT and uric acid.
concentrations of total bilirubin decreased the risk of progressing from the low-risk group to the intermediate-risk group and beyond at a statistically significant level in both genders. In addition, the relationship also remained statistically significant in the risk model of progressing from the low- and intermediate-risk groups to the high-risk group in men (Table 5). We could not apply logistic regression analysis to total bilirubin concentration in females because the number of subjects with high CAD risk (O20%) in this gender group was too small.
Discussion The present data demonstrate that serum total bilirubin levels inversely correlate with the risk of CAD calculated using the FRS. The association was not confounded by the traditional risk factors (LDL cholesterol and BMI) or the other novel CAD risk factors (hs-CRP, uric acid and GGT). Although bilirubin has been widely utilized as a marker for various hepatobiliary diseases, increasing evidence Table 5. Logistic regression analysis of log-transformed total bilirubin as an independent variable and high-risk for CAD (10 year risk O20%) as a dependent variable in males
Model 1 Model 2 Model 3
B
SE
p
OR (95% CI)
1.382 1.397 1.037
0.205 0.289 0.418
!0.001 !0.001 0.013
0.251 (0.168e0.375) 0.247 (0.140e0.436) 0.355 (0.156e0.804)
CAD, coronary artery disease; LDL, low-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; GGT, gamma glutamyltransferase; CI, confidence interval. Model 1: unadjusted. Model 2: after adjustment for body mass index, logtransformed weekly alcohol consumption and LDL cholesterol. Model 3: model 2 plus adjustment for log-transformed hs-CRP, log-transformed GGT and uric acid.
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suggests that bilirubin is not only a cellular marker for antioxidants (5,21,22) but also is a decreased favorable factor of cardiovascular disease and metabolic syndrome (10e15). Bilirubin is created by the activity of biliverdin reductase on biliverdin, a green tetrapyrrolic bile pigment that is also a product of heme catabolism. Bilirubin, when oxidized, reverts to again become biliverdin. This cycle, in addition to the demonstration of the potent antioxidant activity of bilirubin, has led to the hypothesis that the main physiological role of bilirubin is as a cytoprotectant (5,21). Thus, it was hypothesized that increasing total bilirubin could be a decreased marker for CAD risk prediction. Our data show that the OR of FRS $10% decreased significantly with increased serum total bilirubin levels. After adjustment for BMI, alcohol consumption and other known or novel CAD risk factors, the inverse relationship between total bilirubin and FRS $10% remained significant. In addition, the increase in the concentrations of total bilirubin also significantly decreased the risk of progressing from the low- and intermediate-risk groups to the high-risk group, even after adjusting for age and other known or novel cardiovascular risk factors. These findings suggest that increased bilirubin levels are a favorable condition from a metabolic standpoint. What is the possible underlying mechanism to explain the observed link between serum total bilirubin levels and CAD risk prediction? First, although the mechanisms that explain the contribution of bilirubin to reducing CAD have not been fully elucidated, bilirubin is a potent antioxidant within the physiological range that suppresses oxidation of lipids and lipoproteins (5,21). Bilirubin makes up |10% of the total antioxidant capacity in normobilirubinemic adults and is inversely correlated with serum markers of oxidative stress (23,24). These data provide a biological basis for the negative relationship between serum total bilirubin within the reference range and FRS. In addition to being an effective antioxidant, bilirubin has been reported to be involved in the insulin receptor /insulin growth factor-1 signaling cascade through the biliverdin reductase cycle (25). Low total bilirubin levels may be related with dysregulation of insulin signaling (26). This possibility may also explain the finding of an inverse association between serum total bilirubin levels and CAD risk prediction. Third, bilirubin also has anticomplement properties that defend against inflammation. It is possible that bilirubin is an endogenous tissue protector by virtue of its involvements in immune reactions and inflammatory processes (6). Several studies suggests that the beneficial effect of bilirubin is related with not only the antioxidant effect but also with the anti-inflammatory effect of bilirubin (10,27). On the other hands, two prospective studies demonstrated that a U-shaped relationship between total bilirubin and CAD incidence was observed in middle-aged males (13,28). However, Gilbert syndrome, a genetic variant characterized by moderate hyperbilirubinemia, is associated
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with a reduced risk for CAD (29). Thus, the U-shape relationship may be associated with underlying liver disease in middle-aged males (30). Recent studies also reported that individuals with the UDP-glucuronosyltransferase (UGT1A1)*28 allele that have decreased hepatic bilirubin UDP-glucuronosyltransferase activity, decreased bilirubin clearance, and increased serum bilirubin concentrations may be associated with a lower CAD risk (30e32). However, an association of the UGT1A1*28 allele and CAD is still obscure because of differing results among studies (32e35). Therefore, further studies will be needed to evaluate the relationship between subjects with the UGT1A1*28 allele and CAD risk. This study has several strengths and limitations. One of the strengths is the large scale of the study with subjects of both genders and all age groups. Therefore, results can be generalized into the entire Korean population. Second, we used the categories of the estimated 10-year CAD risk calculated using the NCEP ATP III, which is considered the current standard for prediction of CAD risk. Although the FRS is a generalized tool, this study endpoint is itself a mathematical surrogate for the presence of CAD. In addition, its direct application in some populations may overestimate CAD risk, especially in Asian-Pacific populations such as the Japanese and the Chinese (36,37). However, at present, there is no other validated and reliable CAD risk assessment tool for Koreans, and many Korean physicians utilize the FRS. Therefore, further longitudinal cohort studies are needed to evaluate the predictive value of biomarkers for the decreased risk of CAD. The present study was cross-sectional and we did not investigate oxidative stress markers, and thus could not examine any association of oxidative stress with serum total bilirubin levels. Neither serum direct nor indirect bilirubin was included in the analysis. However, this limitation may not have affected our results because all forms of bilirubin (free or albumin-bound) inhibit hyperoxidation of LDLcholesterol (38). In conclusion, the present study demonstrates that serum total bilirubin concentrations are inversely associated with the estimated 10-year CAD risk calculated with NCEP ATP III in Koreans. These significant inverse associations in serum total bilirubin concentrations under physiological conditions appear to have an additional benefit in the prediction of future development of CAD. Thus, serum total bilirubin may be helpful to decrease the future risk of CAD.
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ORIGINAL ARTICLE
Serum Total Bilirubin Concentration Is Inversely Correlated with Framingham Risk Score in Koreans Kwang-Min Kim, Bom-Taeck Kim, Sat-Byul Park, Doo-Yeoun Cho, Sang Hyeon Je, and Kyu-Nam Kim Department of Family Practice and Community Health, Ajou University School of Medicine, Suwon, Republic of Korea Received for publication December 4, 2011; accepted May 2, 2012 (ARCMED-D-11-00606).
Background and Aims. Compelling evidence suggests that bilirubin, via its antioxidant potential, has anti-atherogenic properties, and that serum bilirubin concentrations within the reference range for the general population may provide some protection against coronary artery disease (CAD). This study examined the association between serum total bilirubin concentration and Framingham risk score (FRS) in the Korean population. Methods. This cross-sectional study was performed on 19,792 Koreans. In addition to FRS, data on body mass index, fasting blood glucose, liver enzymes, lipid profile, uric acid, gamma glutamyltransferase, high-sensitive C-reactive protein and total bilirubin were used. Results. Negative correlations were established between log-transformed total bilirubin concentration and FRS (females; r 5 0.067, p !0.001, males; r 5 0.128, p !0.001). Analyses relating total bilirubin to FRS $10% utilized multiple confounder adjusted logistic regression. Unadjusted odd ratios for FRS $10% were 0.325 (95% CI: 0.160e0.659, p 5 0.002) and 0.342 (95% CI: 0.281e0.417, p !0.001) for log-transformed total bilirubin in females and males, respectively. These inverse relationships remained significant after adjustments for multiple confounders in both genders. Conclusions. Increased total bilirubin concentrations are associated with the decrease in FRS. Serum total bilirubin may be helpful to decrease the future risk of CAD. Ó 2012 IMSS. Published by Elsevier Inc. Key Words: Total bilirubin, Framingham risk score, Oxidative stress.
Introduction Oxidative stress is one of the most significant aspects in the pathogenesis of coronary artery disease (CAD), which is among the leading causes of death worldwide (1) and which is thought to play an important role in the progression of atherosclerosis (2,3). Several studies have implicated bilirubin as a biomarker of an antioxidant under physiological conditions (4e6). Parallel evidence from epidemiological studies suggest that low serum concentrations of total bilirubin are associated with development of CAD risk
Address reprint requests to: Kyu-Nam Kim, Department of Family Practice and Community Health, Ajou University School of Medicine, 164 Worldcup-ro, Youngtong-gu, Suwon, Gyeonggi-do, Republic of Korea; Phone: 82-031-219-5324; FAX: 82-31-219-5218; E-mail: ktwonm@ hanmail.net
factors including diabetes (7,8), hypertension (9) and metabolic syndrome (10,11). In addition, several studies have reported that low serum total bilirubin concentrations are related to an increased risk of CAD (12,13) as well as increased carotid intima-media thickness and peripheral arterial disease (14,15). Furthermore, a study reported a significant negative correlation even between bilirubin and CAD risk calculated using the Framingham Equation (16). However, this study targeted smoking status or was reported without adjusting multiple confounding factors such as the traditional risk factors or other novel CAD risk factors. In spite of the clinical impact these decreased total bilirubin levels have in many studies, little is known about the effect of total bilirubin levels in the prediction of CAD risk after controlling for multiple potential confounding factors in the general population. The Framingham Risk Score (FRS) is a mathematical model for predicting CAD
0188-4409/$ - see front matter. Copyright Ó 2012 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2012.05.003
Serum Total Bilirubin and Framingham Risk Score
during a 10-year period. The FRS has become a widely used clinical tool to guide the delivery of preventive medicine (17,18). The present study was grounded in the hypothesis that increasing serum total bilirubin is associated with a decreased risk of CAD, calculated using the FRS modified by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) guideline and may contribute to reduce CAD risk prediction. The aim of this study was to examine whether serum total bilirubin levels are associated with FRS in Koreans.
Subjects and Methods Subjects From January 2007 to May 2009, data of 33,906 Koreans aged 20e86 years who visited the Health Promotion Center, Ajou University Hospital, Suwon, Gyeonggi-do, Republic of Korea were reviewed for inclusion in the study. Medical history, demographics, and anthropometric and laboratory data were collected. Data on cigarette smoking and alcohol consumption were collected by a self-reported questionnaire. Subjects who, at the time of the survey, had smoked cigarettes regularly within the prior year were considered to be current smokers and weekly alcohol intake was calculated and then converted to weekly alcohol consumption (grams of ethanol per week) by the graduated frequency method (19). Of the initial 33,906 subjects, we excluded 14,114 subjects who met one of the following criteria: 8,221 subjects had missing data values for any component of FRS or serum total bilirubin or alcohol history; 831 subjects were positive for antibody to hepatitis B surface antigen, antibody to hepatitis B virus core antigen or anti-hepatitis C virus; 2,314 subjects had a medical history of chronic liver disease or liver cirrhosis or were taking drugs influencing liver function (hepatotonics); 293 subjects were on medication (statins or other lipid-lowering drugs) for hyperlipidemia; 1,842 subjects had been diagnosed with diabetes mellitus or a CAD; 1,310 subjects had abnormal liver function tests defined as O41 IU/L for males and O31 IU/L for females for alanine aminotransferase, O40 IU/L for aspartate aminotransferase, and O66 IU/L for males and O39 IU/L for females for gamma glutamyltransferase (GGT) or O1.2 mg/dL for serum total bilirubin level, which were established as the cutoff values based on 95% confidence limits for 705 healthy adults; 299 subjects had highsensitive C-reactive protein (hs-CRP) $10.0 mg/L to preclude any possible occult inflammatory or infectious disease. Thus, a total of 19,792 healthy Korean subjects (11,206 females, 8,586 males) were included in the final analyses. Written informed consent was obtained from all participants and the institutional review board of Ajou University Hospital approved this study.
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Materials and Methods Before collection of blood, each subject fasted for 10 h or more. After overnight fasting, a venous blood sample was obtained between 08:00 and 10:00 am to measure total bilirubin concentrations, fasting blood glucose, liver enzymes, total cholesterol, triglycerides, HDL (high-density lipoprotein) cholesterol, LDL (low-density lipoprotein) cholesterol, hsCRP and uric acid. Serum total bilirubin, fasting blood glucose, liver enzymes, lipid levels and uric acid were assayed by the standard method recommended by the International Federation for Clinical Chemistry using a Toshiba200FR automatic analyzer (Toshiba Medical Systems, Tokyo, Japan). Fractions of bilirubin were not assessed. hs-CRP was measured by a high-sensitivity nephelometric method (Dade Behring Marburg GMBH, Marburg, Germany). Blood pressure (BP) was measured using a standard mercury manometer with the participant in a sitting position for 5 min prior to measurement; the average of two measurements was recorded. Hypertension was defined as a systolic BP $140 mm Hg or a diastolic BP $90 mm Hg or by the use of antihypertensive medication. Diabetes was defined by fasting blood glucose $126 mg/dL or the use of oral hypoglycemic agents or insulin. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). Statistical Analyses The distribution of total bilirubin, hs-CRP, GGT values and alcohol consumption were right-skewed; therefore, a natural log-transformation was applied. To assess the relationship between biomarkers and the individual components of the FRS, Pearson correlation coefficients relating individual risk factor scores and the total of FRS to log-transformed total bilirubin levels were analyzed. The FRS was calculated from the NCEP ATP III algorithm based on six coronary risk factors: gender, age, total cholesterol, HDLcholesterol, systolic BP and smoking habit (20). Among these factors, age, BP, and cholesterol levels were categorized according to their values. Smoking status was classified as either current smoker or non-smoker. Framingham risk equations were used to predict the risk of developing coronary disease events (myocardial infarction or coronary heart disease death) over the next 10 years for adults aged 20 and older without heart disease or diabetes. Participants were divided into three groups (20): low risk (#9% risk of developing a coronary heart disease event over the next 10 years), intermediate risk (10e20% risk), and high risk (O20% risk). For analyses relating total bilirubin to the intermediate-risk group and beyond (FRS $10%) or high risk (FRS O20%), we constructed an adjusted logistic regression analyses that considered alcohol consumption þ BMI þ LDL cholesterol, and (alcohol consumption þ BMI þ LDL cholesterol) þ novel risk factors including hs-CRP, GGT and uric acid. Results of group data are expressed as mean standard deviation (SD). All statistical
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analyses were performed using SPSS v.13.0 software (SPSS, Chicago, IL); p !0.05 was considered statistically significant.
Results Table 1 shows the characteristics of the excluded subjects including missing data. Numbers of excluded males were more than numbers of females in this study because various metabolic parameters including diabetes and elevated liver enzymes for exclusion criteria in men were higher than those in women. Table 2 shows the characteristics of the selected subjects. Compared with females, males presented with more CAD risk factors such as hypertension, dyslipidemia, alcohol consumption and current smoking. Therefore, the total Framingham point score and 10-year CAD risk were higher in men than in women. The correlation coefficients between log-transformed total bilirubin levels and 10-year CAD risk were r 5 0.067 ( p !0.001) and r 5 0.128 ( p !0.001) in females and males, respectively (Table 3). Of the Framingham individual components, serum total bilirubin also was negatively correlated with age score, smoking score, HDL cholesterol score and systolic BP score in both genders. Unadjusted odds ratio (OR) of progressing from the low-risk group to the intermediate-risk group and beyond was 0.325 (95% CI: Table 1. General characteristics of the excluded subjects
Characteristic Age, years BMI (kg/m2) Current smoker, n (%) Alcohol (g/week) SBP (mmHg) DBP (mmHg) Hypertension, n (%) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) AST (IU/L) ALT (IU/L) GGT (IU/L) Fasting blood glucose (mg/dL) Diabetes mellitus, n (%) hs-CRP (mg/dL) Uric acid (mg/dL) Total bilirubin (mg/dL)
All patients (n 5 14,114)
Females (n 5 6,775)
Males (n 5 7,339)
45.1 9.1 23.1 3.0 3,246 (23.1) 58.4 54.1 116.0 14.1 75.7 11.1 1,961 (13.9) 187.2 31.9 53.7 12.0 110.2 29.1 105.5 63.8 21.0 12.5 21.8 10.6 23.6 23.3 93.3 15.7
44.5 9.5 22.1 2.8 210 (3.1) 40.55 48.1 113.5 12.9 73.3 9.9 684 (10.1) 183.2 30.5 57.2 12.4 107.5 28.9 90.3 57.5 19.3 10.4 17.4 11.7 16.6 16.4 90.2 12.9
45.9 8.8 23.9 2.8 3,036 (41.3) 64.2 10.61 119.8 12.7 79.8 10.1 1,277 (17.4) 190.3 32.1 49.5 10.7 112.7 25.8 125.5 65.7. 23.7 12.9 24.5 10.5 30.0 24.1 96.2 17.5
Table 2. General characteristics of the study subjects
Characteristic Age, years BMI (kg/m2) Current smoker, n (%) Alcohol (g/week) SBP (mmHg) DBP (mmHg) Hypertension, n (%) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) AST (IU/L) ALT (IU/L) GGT (IU/L) Fasting blood glucose (mg/dL) hs-CRP (mg/dL) Uric acid (mg/dL) Total bilirubin (mg/dL) Total Framingham point score 10 year CAD risk (%)
496 (2.9) 0.2 0.4 3.9 0.8 0.8 0.3
1,056 (14.3) 0.3 0.5 5.8 1.2 1.1 0.4
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; hs-CRP, high-sensitivity C-reactive protein; CAD, coronary artery disease. Data are expressed as mean standard deviations (SD) or as number (percentage).
Females (n 5 11,206)
Males (n 5 8,586)
44.7 10.2 23.1 2.9 4,067 (20.5) 55.6 54.1 116.7 14.4 75.7 11.0 2,710 (13.7) 185.1 32.5 53.2 12.9 108.4 28.8 100.5 64.4 19.9 5.1 18.5 7.5 21.3 12.3 91.5 8.7
44.3 10.4 22.6 2.9 373 (3.3) 39.9 43.9 114.6 14.9 73.2 10.7 1,122 (10.0) 182.3 33.7 57.8 13.0 106.6 29.6 86.2 56.0 19.1 5.0 16.0 6.5 15.9 8.4 90.0 8.5
45.3 10.0 23.8 2.6 3,694 (43.0) 63.1 56.8 119.5 13.3 79.4 10.3 1,588 (18.5) 187.0 31.5 49.3 11.5 110.7 27.6 119.2 69.7 21.0 5.1 21.9 7.4 28.4 12.9 93.3 8.6
0.1 4.7 0.8 5.8
0.5 1.3 0.2 6.1
0.1 4.0 0.7 4.7
3.0 4.3
0.3 0.9 0.2 6.5
0.2 5.7 0.8 7.3
1.0 1.6
0.6 1.2 0.1 5.2
5.7 5.3
BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transferase; hs-CRP, high-sensitivity C-reactive protein; CAD, coronary artery disease. Data are expressed as mean standard deviations (SD) or as number (percentage).
0.160e0.659, p 5 0.002) and 0.345 (95% CI: 0.281e0.417, p !0.001) for log-transformed total bilirubin in females and males, respectively (Table 4). After additional adjustments for BMI, log-transformed weekly alcohol consumption and other conventional and novel cardiovascular risk factors such as LDL cholesterol, GGT, hs-CRP and uric acid, we discovered that increases in the Table 3. Pearson correlation coefficients relating individual components and the total score of Framingham point to log-transformed total bilirubin concentration in each gender Females
Variables 1,552 (11.0) 0.2 0.4 4.9 1.4 0.9 0.4
All patients (n 5 19,792)
Age score Total cholesterol score Smoking score HDL cholesterol score Systolic BP score Total Framingham point score 10 year CAD risk
Males
Correlation coefficient
p
Correlation coefficient
p
0.070 0.028 0.038 0.121 0.058 0.086
!0.001 0.003 !0.001 !0.001 !0.001 !0.001
0.055 0.051 0.070 0.116 0.023 0.104
!0.001 !0.001 !0.001 !0.001 0.035 !0.001
0.067
!0.001
0.128
!0.001
HDL, high-density lipoprotein; BP, blood pressure; CAD, coronary artery disease.
Serum Total Bilirubin and Framingham Risk Score Table 4. Logistic regression analysis of log-transformed total bilirubin as an independent variable and intermediate and high-risk for CAD (10-year risk $10%) as a dependent variable in females and males
Females Model Model Model Males Model Model Model
B
SE
p
OR (95% CI)
1 2 3
1.124 2.133 3.466
0.360 1.146 1.644
0.002 0.010 0.035
0.325 (0.160e0.659) 0.119 (0.013e0.589) 0.031 (0.001e0.784)
1 2 3
1.072 1.068 0.955
0.101 0.135 0.206
!0.001 !0.001 !0.001
0.342 (0.281e0.417) 0.344 (0.264e0.448) 0.385 (0.257e0.577)
CAD, coronary artery disease; LDL, low-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; GGT, gamma glutamyltransferase; CI, confidence interval. Model 1: unadjusted; Model 2: after adjustment for body mass index, logtransformed weekly alcohol consumption and LDL cholesterol; Model 3: model 2 plus adjustment for log-transformed hs-CRP, log-transformed GGT and uric acid.
concentrations of total bilirubin decreased the risk of progressing from the low-risk group to the intermediate-risk group and beyond at a statistically significant level in both genders. In addition, the relationship also remained statistically significant in the risk model of progressing from the low- and intermediate-risk groups to the high-risk group in men (Table 5). We could not apply logistic regression analysis to total bilirubin concentration in females because the number of subjects with high CAD risk (O20%) in this gender group was too small.
Discussion The present data demonstrate that serum total bilirubin levels inversely correlate with the risk of CAD calculated using the FRS. The association was not confounded by the traditional risk factors (LDL cholesterol and BMI) or the other novel CAD risk factors (hs-CRP, uric acid and GGT). Although bilirubin has been widely utilized as a marker for various hepatobiliary diseases, increasing evidence Table 5. Logistic regression analysis of log-transformed total bilirubin as an independent variable and high-risk for CAD (10 year risk O20%) as a dependent variable in males
Model 1 Model 2 Model 3
B
SE
p
OR (95% CI)
1.382 1.397 1.037
0.205 0.289 0.418
!0.001 !0.001 0.013
0.251 (0.168e0.375) 0.247 (0.140e0.436) 0.355 (0.156e0.804)
CAD, coronary artery disease; LDL, low-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; GGT, gamma glutamyltransferase; CI, confidence interval. Model 1: unadjusted. Model 2: after adjustment for body mass index, logtransformed weekly alcohol consumption and LDL cholesterol. Model 3: model 2 plus adjustment for log-transformed hs-CRP, log-transformed GGT and uric acid.
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suggests that bilirubin is not only a cellular marker for antioxidants (5,21,22) but also is a decreased favorable factor of cardiovascular disease and metabolic syndrome (10e15). Bilirubin is created by the activity of biliverdin reductase on biliverdin, a green tetrapyrrolic bile pigment that is also a product of heme catabolism. Bilirubin, when oxidized, reverts to again become biliverdin. This cycle, in addition to the demonstration of the potent antioxidant activity of bilirubin, has led to the hypothesis that the main physiological role of bilirubin is as a cytoprotectant (5,21). Thus, it was hypothesized that increasing total bilirubin could be a decreased marker for CAD risk prediction. Our data show that the OR of FRS $10% decreased significantly with increased serum total bilirubin levels. After adjustment for BMI, alcohol consumption and other known or novel CAD risk factors, the inverse relationship between total bilirubin and FRS $10% remained significant. In addition, the increase in the concentrations of total bilirubin also significantly decreased the risk of progressing from the low- and intermediate-risk groups to the high-risk group, even after adjusting for age and other known or novel cardiovascular risk factors. These findings suggest that increased bilirubin levels are a favorable condition from a metabolic standpoint. What is the possible underlying mechanism to explain the observed link between serum total bilirubin levels and CAD risk prediction? First, although the mechanisms that explain the contribution of bilirubin to reducing CAD have not been fully elucidated, bilirubin is a potent antioxidant within the physiological range that suppresses oxidation of lipids and lipoproteins (5,21). Bilirubin makes up |10% of the total antioxidant capacity in normobilirubinemic adults and is inversely correlated with serum markers of oxidative stress (23,24). These data provide a biological basis for the negative relationship between serum total bilirubin within the reference range and FRS. In addition to being an effective antioxidant, bilirubin has been reported to be involved in the insulin receptor /insulin growth factor-1 signaling cascade through the biliverdin reductase cycle (25). Low total bilirubin levels may be related with dysregulation of insulin signaling (26). This possibility may also explain the finding of an inverse association between serum total bilirubin levels and CAD risk prediction. Third, bilirubin also has anticomplement properties that defend against inflammation. It is possible that bilirubin is an endogenous tissue protector by virtue of its involvements in immune reactions and inflammatory processes (6). Several studies suggests that the beneficial effect of bilirubin is related with not only the antioxidant effect but also with the anti-inflammatory effect of bilirubin (10,27). On the other hands, two prospective studies demonstrated that a U-shaped relationship between total bilirubin and CAD incidence was observed in middle-aged males (13,28). However, Gilbert syndrome, a genetic variant characterized by moderate hyperbilirubinemia, is associated
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with a reduced risk for CAD (29). Thus, the U-shape relationship may be associated with underlying liver disease in middle-aged males (30). Recent studies also reported that individuals with the UDP-glucuronosyltransferase (UGT1A1)*28 allele that have decreased hepatic bilirubin UDP-glucuronosyltransferase activity, decreased bilirubin clearance, and increased serum bilirubin concentrations may be associated with a lower CAD risk (30e32). However, an association of the UGT1A1*28 allele and CAD is still obscure because of differing results among studies (32e35). Therefore, further studies will be needed to evaluate the relationship between subjects with the UGT1A1*28 allele and CAD risk. This study has several strengths and limitations. One of the strengths is the large scale of the study with subjects of both genders and all age groups. Therefore, results can be generalized into the entire Korean population. Second, we used the categories of the estimated 10-year CAD risk calculated using the NCEP ATP III, which is considered the current standard for prediction of CAD risk. Although the FRS is a generalized tool, this study endpoint is itself a mathematical surrogate for the presence of CAD. In addition, its direct application in some populations may overestimate CAD risk, especially in Asian-Pacific populations such as the Japanese and the Chinese (36,37). However, at present, there is no other validated and reliable CAD risk assessment tool for Koreans, and many Korean physicians utilize the FRS. Therefore, further longitudinal cohort studies are needed to evaluate the predictive value of biomarkers for the decreased risk of CAD. The present study was cross-sectional and we did not investigate oxidative stress markers, and thus could not examine any association of oxidative stress with serum total bilirubin levels. Neither serum direct nor indirect bilirubin was included in the analysis. However, this limitation may not have affected our results because all forms of bilirubin (free or albumin-bound) inhibit hyperoxidation of LDLcholesterol (38). In conclusion, the present study demonstrates that serum total bilirubin concentrations are inversely associated with the estimated 10-year CAD risk calculated with NCEP ATP III in Koreans. These significant inverse associations in serum total bilirubin concentrations under physiological conditions appear to have an additional benefit in the prediction of future development of CAD. Thus, serum total bilirubin may be helpful to decrease the future risk of CAD.
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