Advance Publication by J-STAGE Circulation Journal Official Journal of the Japanese Circulation Society http://www.j-circ.or.jp Bilirubin Is Negatively Associated With Hemoglobin A1c Independently of Other Cardiovascular Risk Factors in Apparently Healthy Japanese Men and Women Eiji Oda, MD; Ryu Kawai, MD Background: Serum total bilirubin (TB) is a potent antioxidant and may be a negative risk factor of cardiovas- cular disease. In non-diabetic adults, hemoglobin A1c (HbA1c), but not fasting plasma glucose, is an independent risk factor of cardiovascular disease. Methods and Results: Linear regression using TB as a dependent variable and cardiovascular risk factors, in- cluding HbA1c, as independent variables, linear regression using HbA1c as a dependent variable and other cardio- vascular risk factors, including TB, as independent variables, and logistic regression using the highest decile (≥5.4%) of HbA1c as a dependent variable and TB and other cardiovascular risk factors as independent variables were performed for 893 apparently healthy male smokers, 1,607 male nonsmokers, and 1,680 women. The standardized regression coefficient of HbA1c for TB and that of TB for HbA1c was –0.12 (P=0.007) and –0.06 (P=0.02), respec- tively, in the smokers, –0.20 (P<0.0001) and –0.07 (P<0.0001), respectively, in the nonsmokers, and –0.21 (P< 0.0001) and –0.14 (P<0.0001), respectively, in the women. The odds ratio of 1 SD increment in TB for HbA1c ≥5.4% was not significant in the smokers, 0.67 (P=0.002) in the nonsmokers, and 0.55 (P<0.0001) in the women. Conclusions: Bilirubin was negatively associated with HbA1c independently of other cardiovascular risk factors in apparently healthy Japanese men and women. The association was weak in male smokers. Key Words: Antioxidant; Bilirubin; Cardiovascular disease; Diabetes; Hemoglobin A1c ilirubin is a potent antioxidant in the human body.1 for CVD. In nondiabetic adults, however, fasting plasma Increased serum levels of total bilirubin (TB) have glucose (FPG) was not significantly associated with risk of B been reported to be associated with reduced risk for CVD and of death from any cause, adjusting for hemoglobin cardiovascular disease (CVD) in some epidemiological studies A1c (HbA1c). In contrast, HbA1c was significantly associated and TB is suggested to be an independent risk factor for with risk of CVD and of death from any cause, adjusting for CVD.2 Healthy subjects with lower TB levels have been found FPG.10 HbA1c levels appear to increase with age11 and will to have significant endothelial dysfunction and increased be influenced by any condition that changes red cell turnover, intima – media thickness (IMT) of the carotid artery, which such as iron deficiency, hemolytic anemia, chronic malaria, are predictors for CVD.3 TB has been suggested to be a novel major blood loss, or blood transfusions.12 Other than age and risk marker of CVD in middle-aged men.4 Increased TB is hemoglobin metabolism, some cardiovascular risk factors associated with reduced peripheral artery disease prevalence.5 may have an independent association with HbA1c. Thus, we TB may have some protective function against stroke risk.6 hypothesized that the stronger association of HbA1c, than of Low TB is associated with coronary artery calcification, which FPG, with CVD, in nondiabetic adults may at least partly be is a predictor of coronary artery events in Japanese people.7 mediated by the association between HbA1c and bilirubin in Gilbert syndrome, a genetic variant characterized by moder- nondiabetic subjects. The aim of the present study was there- ate hyperbilirubinemia, was reported to be associated with fore to examine the cross-sectional association between TB a reduced risk for CVD.8 In subjects with Gilbert syndrome, and HbA1c in apparently healthy Japanese men and women elevated TB levels are associated with lower levels of who had no history of CVD and who were not on antidiabetic, advanced glycation endproducts that promote oxidant stress antihypertensive, or antihyperlipidemic medication. and inflammation.9 Thus, TB may be a negative risk factor Received July 4, 2010; revised manuscript received August 13, 2010; accepted September 3, 2010; released online December 2, 2010 Time for primary review: 28 days Medical Check-up Center, Tachikawa Medical Center, Nagaoka, Japan Mailing address: Eiji Oda, MD, Medical Check-up Center, Tachikawa Medical Center, 2-2-16 Nagachou, Nagaoka 940-0053, Japan. E-mail: [email protected] ISSN-1346-9843 doi: 10.1253/circj.CJ-10-0645 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Advance Publication by J-STAGE ODA E et al. Table 1. Baseline Data Smoking men Non-smoking men Women (n=893) (n=1,607) (n=1,680) Age (years) 48.2±8.7 49.5±9.9* 48.5±9.5 BMI (kg/m2) 23.1±2.9 23.0±2.7 21.4±3.0 WC (cm) 83.9±8.0 83.4±7.8 77.9±8.3 Systolic BP (mmHg) 118.2±15.4 120.7±17.2* 109.8±15.4 Diastolic BP (mmHg) 75.6±10.3 77.1±11.0* 68.5±9.9 TB (mg/dl) 0.76±0.30 0.93±0.38* 0.73±0.28 HbA1c (%) 5.02±0.51 4.99±0.41 4.96±0.31 FPG (mg/dl) 94.6±15.9 94.1±11.4 88.1±8.5 Triglyceride (mg/dl) 112 [80, 162] 95 [69, 137]# 68 [52, 92] HDL-C (mg/dl) 55.0±13.9 59.2±14.5* 68.0±14.6 LDL-C (mg/dl) 122.4±30.8 122.8±29.0 119.4±30.9 hs-CRP (mg/L) 0.35 [0.20, 0.78] 0.28 [0.15, 0.56]# 0.21 [0.11, 0.46] FPG >– 100 mg/dl 21.1 20.2 7.2 FPG >– 126 mg/dl 2.7 1.8 0.5 HbA1c >– 5.2% 24.1 21.8 22.9 HbA1c >– 5.4% 11.4 9.4 9.1 Current smoker 100 0 6.6 Data are mean ± SD, median [25, 75 percentiles], or %. *P<0.01 compared with smoking men, #P<0.01 (log-transformed) compared with smoking men. BMI, body mass index; WC, waist circumference; BP, blood pressure; TB, total bilirubin; HbA1c, hemoglobin A1c; FPG, fasting plasma glucose; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein. factant method using Choletest-LDL (Sekisui Medical, Tokyo, Methods Japan). The measurement limit of hs-CRP was 0.02 mg/L, Subjects and any results under this measurement limit were taken as Between April 2008 and March 2010, 3,401 men and 2,095 being 0.01 mg/L. Average systolic blood pressure (SBP) and women visited Medical Check-up Center, Tachikawa Medical diastolic blood pressure (DBP) were calculated from 2 mea- Center, for general health screening. Among them 3,375 men surements done while the subjects were seated after a 5-min and 2,069 women signed informed consent. Excluding sub- rest. Body weight was measured with the subjects wearing jects with a history of CVD or who were taking antidiabetic, light clothes provided by the Center and the weight of the antihypertensive, or antihyperlipidemic medication from those clothing was subtracted from the measured body weight. who gave consent, the resulting 2,500 men and 1,680 women Waist circumference (WC) was measured at the level of the comprised the subjects in the present study, which is part of umbilicus with the subject standing. Body mass index (BMI) the study on obesity-related cardiovascular risk factors at was calculated as weight in kilograms divided by the square Tachikawa Medical Center. Data collected at the first visit of height in meters. were used for subjects who visited 2 or more times. The pro- tocol for the study was approved by the ethics committee at Statistical Analysis Tachikawa Medical Center and written informed consent Pearson correlation coefficients between age, BMI, SBP, was given by each subject. DBP, FPG, triglycerides, HDL-C, LDL-C, hs-CRP, TB, and HbA1c, and both TB and HbA1c were calculated separately in Measurements male smokers and nonsmokers, and women. Linear regres- After an overnight fast, blood samples were obtained to mea- sions with stepwise exclusion of independent variables with sure levels of HbA1c, FPG, triglycerides, high-density lipo- P>0.05, using TB as a dependent variable and age, BMI, SBP, protein cholesterol (HDL-C), low-density lipoprotein choles- FPG, triglycerides, HDL-C, LDL-C, hs-CRP, and HbA1c as terol (LDL-C), high-sensitivity C-reactive protein (hs-CRP), initial independent variables; linear regressions with stepwise and TB. Chemistry was assessed at BML Nagaoka (Nagaoka, exclusion of independent variables with P>0.05, using HbA1c Japan) using routine laboratory methods except for hs-CRP, as a dependent variable and age, BMI, SBP, FPG, triglycer- which was measured at BML General Laboratory (Tokyo, ides, HDL-C, LDL-C, hs-CRP, and TB as initial independent Japan) with nephelometry using N-latex CRP-2 (Siemens variables; and logistic regressions with stepwise exclusion of Healthcare Japan, Tokyo, Japan). HbA1c was measured with independent variables with P>0.05, using the highest decile a latex aggregation immunoassay using Determiner HbA1c (≥5.4%) of HbA1c as a dependent variable and age, BMI, SBP, (Kyowa Medex, Tokyo, Japan). This assay is not affected by FPG, triglycerides, HDL-C, LDL-C, hs-CRP, TB, and smok- the presence of bilirubin when conjugated bilirubin concen- ing status as initial independent variables, were performed trations are <40 mg/dl. In Japan, HbA1c levels are expressed separately in the men, and women.