Journal of Diabetes and Its Complications 29 (2015) 218–221
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
Serum total bilirubin concentration is negatively associated with increasing severity of retinopathy in patients with type 2 diabetes mellitus Risa Sekioka ⁎, 1, Masami Tanaka, Takeshi Nishimura, Hiroshi Itoh Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 25 November 2014 Accepted 1 December 2014 Available online 5 December 2014 Keywords: Total bilirubin Diabetic retinopathy Type 2 diabetes mellitus Hypertension Human
a b s t r a c t Aims: Serum bilirubin concentration is associated with diabetic retinopathy in patients with type 2 diabetes. This study investigated the relationships between serum bilirubin concentration and the severity of diabetic retinopathy. In addition, the importance of bilirubin was compared with factors that were previously shown to be associated with the incidence of diabetic retinopathy. Methods: A total of 674 patients with type 2 diabetes were investigated in this cross-sectional study. Serum total bilirubin concentration was compared between patients with and without diabetic retinopathy, and according to the severity of retinopathy. Univariate and multivariate analyses were performed to evaluate the association of retinopathy with total bilirubin concentration, duration of diabetes, body mass index, systolic blood pressure, and haemoglobin A1c. Results: Serum total bilirubin concentration was significantly lower in patients with retinopathy than in those without. Patients with severer retinopathy showed lower total bilirubin concentration, longer diabetes duration, and higher systolic blood pressure. These three parameters were independent explanatory factors for diabetic retinopathy. Conclusions: Total bilirubin concentration is lower in patients with type 2 diabetes complicated with severer retinopathy. Thus, bilirubin might protect against retinopathy in patients with type 2 diabetes. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Hyperglycaemia is the hallmark of diabetes; it activates certain biochemical pathways leading to macro- and microvascular complications (Takayanagi, Inoguchi, & Ohnaka, 2010). Diabetic retinopathy is a common microvascular complication of diabetes and a leading cause of legal blindness in working-age adults. Bilirubin has long been considered to be a useless or potentially toxic metabolite in jaundice (especially in high doses), particularly in neonates. However, recent studies revealed that serum bilirubin level was inversely associated with atherosclerosis in a cross-sectional analysis (Kim, Mo, Moon, & Han, 2014) and meta-analysis (Novotný & Vítek, 2003). In addition, serum total bilirubin level was negatively associated with incidence of metabolic syndrome (Lee et al., 2014) Conflict of interest: Hiroshi ITOH has received compensation from NIPRO, lecture fees from Takeda and MSD, and scholarship grants from Sanofi-Aventis, Astellas, Takeda, MSD, and Daiichi Sankyo. The other authors have no conflict of interest. ⁎ Corresponding author. Tel.: + 81 3 5363 3797; fax: + 81 3 3359 2745. E-mail addresses: [email protected] (R. Sekioka), [email protected] (M. Tanaka), [email protected] (T. Nishimura), [email protected] (H. Itoh) 1 Present Address: Department of Internal Medicine, Jujo Takeda Rehabilitation Hospital, 32 Kisshoinhatsutandacho, Kyoto Minami-ku, Kyoto, 601-8325, Japan. http://dx.doi.org/10.1016/j.jdiacomp.2014.12.002 1056-8727/$© 2015 Elsevier Inc. All rights reserved.
and lower-limb amputation events in type 2 diabetes (Chan et al., 2013). Furthermore, there have been several reports demonstrating that type 2 diabetic patients with high serum total bilirubin concentration tend to have less complication of diabetic retinopathy (Cho, 2011; Inoguchi, Sasaki, Kobayashi, Takayanagi, & Yamada, 2007; Najam et al., 2014; Yasuda et al., 2011). Although this observation might suggest a protective role of bilirubin against diabetic retinopathy, the detailed association between bilirubin and retinopathy, such as the severity of retinopathy, remains to be elucidated. Therefore, this study was undertaken to clarify the relationship between serum bilirubin concentration and the severity of diabetic retinopathy. In addition, the importance of bilirubin was evaluated by comparing it with factors previously shown to be significantly associated with the incidence of diabetic retinopathy in The Japan Diabetes Complications Study (JDCS) (Kawasaki et al., 2011). 2. Materials and methods 2.1. Patients The subjects included 674 patients with type 2 diabetes admitted to Keio University Hospital from January 2008 to December 2013. The
R. Sekioka et al. / Journal of Diabetes and Its Complications 29 (2015) 218–221
exclusion criteria were as follows: malignant disease under treatment, liver cirrhosis, haematological disease, abnormal liver function test results (i.e., serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) level N 3 times the upper limit of the normal range), and anti-glutamate decarboxylase antibody level ≥ 1.5 U/mL. This study was approved by the ethical committee of Keio University School of Medicine and performed in accordance with the Declaration of Helsinki. Retinopathy was evaluated by an ophthalmologist using fundoscopy after pupillary dilation. Classification was based on the modified Davis classification as follows (Davis, Kern, & Rand, 1997): no diabetic retinopathy (NDR), simple diabetic retinopathy (SDR), preproliferative diabetic retinopathy (PPDR), and proliferative diabetic retinopathy (PDR); PPDR and PDR were combined into a PPDR + PDR category for analysis. Nephropathy was defined as urinary albumin excretion ≥ 30 mg/g creatinine or estimated glomerular filtration rate (eGFR) b 30 mL∙min − 1∙ 1.73 m − 2. Cerebrovascular and cardiovascular disease was defined as a history of these diseases. Smoking was defined as previous or current tobacco usage. Hypertension was defined as blood pressure ≥ 140/90 mmHg or the use of antihypertensive drugs. Dyslipidaemia was defined as serum LDL cholesterol ≥ 140 mg/dL, HDL cholesterol b 40 mg/dL, triglycerides ≥ 150 mg/dL, or taking a hypolipidemic agent.
2.2. Data collection For this cross-sectional study, basic demographic data from all patients were collected from medical records, including sex, age, height, weight, duration of diabetes, total bilirubin, AST, ALT, haemoglobin A1c (HbA1c), eGFR, and lipid levels. Blood and urine samples taken on admission during the study period were used for analysis. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. HbA1c was expressed in accordance with the National Glycohemoglobin Standardization Program guidelines as recommended by the Japanese Diabetes Society (Committee of the Japan Diabetes Society on the Diagnostic Criteria of Diabetes Mellitus et al., 2010). eGFR was calculated using the following formula established by the working group of the Japanese Chronic Kidney Disease Initiative (Horio, Imai, Yasuda, Watanabe, & Matsuo, 2010; Matsuo et al., 2009): eGFR (mL min−1 1.73 m −2) = 194 × (serum creatinine) −1.094 × (age)−0.287 (×0.739 for women).
2.3. Statistical analyses Serum total bilirubin concentration was compared between patients with and without diabetic retinopathy with paired t-test. It was also compared according to the severity of retinopathy using the Bonferroni test. The associations of diabetes duration and systolic blood pressure with the severity of retinopathy were evaluated using the Jonckheere–Terpstra test. Univariate analyses were performed to evaluate the association of retinopathy with total bilirubin concentration and factors previously shown to be important in the JDCS (Kawasaki et al., 2011) — namely, sex, age, BMI, duration of diabetes, systolic blood pressure, and smoking. Multivariate logistic regression analysis was performed to determine the effects of factors examined in univariate analyses on diabetic retinopathy. Variables are expressed as mean ± standard deviation. All statistical analyses were performed using SPSS (version 21.0; SPSS, Chicago, IL, USA). The level of significance was set at p b 0.05.
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3. Results 3.1. Patient characteristics Patient demographic and laboratory data are shown in Table 1. The mean concentration of serum total bilirubin in all patients was 0.74 ± 0.36 mg/dL. No patient had a serum bilirubin level greater than twice the upper limit of the normal range. Of 674 patients with type 2 diabetes mellitus, 58.6% (395) were classified as NDR; of these, 279 were complicated with diabetic retinopathy, with 19.6% (n = 132), 6.4% (n = 43), and 15.4% (n = 104) classified as SDR, PPDR, and PDR, respectively. 3.2. Association between total bilirubin concentration and diabetic retinopathy Patients with diabetic retinopathy had a significantly lower serum total bilirubin concentration than those without retinopathy (NDR, 0.65 ± 0.30 mg/dL vs. 0.80 ± 0.39 mg/dL, p b 0.001; Fig. 1). As for the severity of retinopathy, patients with SDR and PPDR + PDR had significantly lower bilirubin concentrations than those without retinopathy (SDR, 0.71 ± 0.34 mg/dL, p = 0.048; PPDR + PDR, 0.59 ± 0.24 mg/dL, b 0.001 vs. NDR), and patients with PPDR + PDR
Table 1 Patient demographic and laboratory data. N
674
Age (years) Sex (male/female) Duration of diabetes (years) BMI (kg/m2) HbA1c (%) Total bilirubin (mg/dL) AST (IU/L) ALT (IU/L) eGFR (mL min−1 1.73 m−2) Smoking, n (%) Hypertension, n (%) Dyslipidaemia, n (%) Diabetic retinopathy, n (%) NDR/SDR/PPDR/PDR, n (%) Diabetic nephropathy, n (%) Cerebrovascular disease, n (%) Cardiovascular disease, n (%) Concomitant medication, n (%) SU Metformin Thiazolidinedione α-GI Glinide DPP-4 inhibitor Insulin GLP1 analogue Antihypertensive agent, n (%) ARB ACEI CCB α-blocker β-blocker Diuretic Hypolipidemic agent, n (%) Statin Fibrate Ezetimibe Eicosapentaenoic acid
64.7 ± 13.9 446/228 13.9 ± 10.9 25.5 ± 6.3 9.13 ± 2.16 0.74 ± 0.36 26.8 ± 17.0 26.8 ± 20.1 61.7 ± 31.6 324 (48.1) 498 (73.9) 520 (77.2) 279 (41.4) 395 (58.6)/132 (19.6)/43 (6.4)/104 (21.8) 323 (47.9) 101 (15.0) 128 (19.0) 198 (29.4) 104 (15.4) 50 (7.4) 159 (23.6) 31 (4.6) 107 (15.9) 257 (38.1) 6 (0.89) 287 (42.6) 34 (5.0) 269 (39.9) 16 (2.4) 115 (17.1) 124 (18.4) 264 (39.2) 18 (2.7) 18 (2.7) 16 (2.4)
Values are expressed as number (%) or mean ± SD. Abbreviations: BMI, body mass index; HbA1c, haemoglobin A1c; AST, aspartate aminotransferase; ALT, alanine aminotransferase; eGFR, estimated glomerular filtration rate; NDR, no diabetic retinopathy; SDR, simple diabetic retinopathy; PPDR, preproliferative diabetic retinopathy; PDR, proliferative diabetic retinopathy; SU, sulfonylurea; α-GI, α-glucosidase inhibitor; ARB, angiotensin receptor blocker; ACEI, angiotensin-converting enzyme inhibitor; CCB, calcium channel blocker.
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R. Sekioka et al. / Journal of Diabetes and Its Complications 29 (2015) 218–221 Table 2 Univariate logistic regression analysis of diabetic retinopathy.
Sex (Female = 1, Male = 0) Age BMI Duration of diabetes Systolic blood pressure HbA1c Total bilirubin Smoking
Wald
Odds ratio
95% CI
p
0.188 14.318 0.125 82.556 14.853 8.811 27.113 0.613
1.074 1.023 0.996 1.082 1.014 0.895 0.233 1.133
0.777–1.484 1.011–1.034 0.971–1.020 1.064–1.100 1.007–1.021 0.831–0.963 0.134–0.403 0.829–1.549
0.665 0.000 0.724 0.000 0.000 0.003 0.000 0.434
Abbreviations: CI, confidence interval. BMI, body mass index; HbA1c, haemoglobin A1c.
4. Discussion Fig. 1. Total serum bilirubin concentration of patients with and without retinopathy. ** p b 0.001.
had a significantly lower bilirubin concentration than those with SDR (p = 0.011; Fig. 2).
3.3. Association between diabetes duration, blood pressure, and severity of diabetic retinopathy The Jonckheere–Terpstra test showed that a longer duration of diabetes (PPDR + PDR, 19.7 ± 11.6; SDR, 17.8 ± 9.3; NDR, 10.5 ± 9.7 years; p b 0.001) and a higher systolic blood pressure (PPDR + PDR, 139 ± 25; SDR, 136 ± 24; NDR, 131 ± 20 mmHg; p b 0.001) were significantly associated with severer retinopathy.
3.4. Determinants of diabetic retinopathy Univariate logistic regression analyses demonstrated significant difference in total bilirubin concentration (odds ratio [OR] = 0.233; 95% confidence interval [CI], 0.134–0.403; p b 0.001), duration of diabetes (OR = 1.082; 95% CI, 1.064–1.100; p b 0.001), systolic blood pressure (OR = 1.014; 95% CI, 1.007–1.021; p b 0.001), HbA1c (OR = 0.895; 95% CI, 0.831–0.963; p = 0.003), and age (OR = 1.023; 95% CI, 1.011–1.034; p b 0.001) between patients with and without diabetic retinopathy (Table 2). Multivariate logistic regression analysis demonstrated that total bilirubin concentration (OR = 0.262; 95% CI, 0.142–0.486; p b 0.001), duration of diabetes (OR = 1.079; 95% CI, 1.058–1.100; p b 0.001), and systolic blood pressure (OR = 1.012; 95% CI, 1.004–1.021; p = 0.004) were significant independent explanatory factors for retinopathy (Table 3).
In this observational study, it was shown that type 2 diabetic patients with retinopathy had lower serum total bilirubin concentration than those without retinopathy, which is in line with other studies (Cho, 2011; Inoguchi et al., 2007; Najam et al., 2014; Yasuda et al., 2011). Furthermore, it was also shown that total bilirubin concentration is negatively related with diabetic retinopathy from the results of univariate (OR = 0.233; p b 0.001) and multivariate (OR = 0.262; p b 0.001) logistic analyses. Judging from OR, each mg/dL decrease of serum bilirubin was associated with 4 times higher risk of diabetic retinopathy. In addition, we demonstrated that total bilirubin concentration was negatively associated with increasing severity of retinopathy. To our knowledge, this has not been demonstrated in the past. Our observation that the patients with severer diabetic retinopathy showed lower total bilirubin concentration suggests the possibility that bilirubin is deeply involved in the prevention of diabetic retinopathy. However, the precise mechanism underlying this phenomenon is unknown. Several reports support the idea that chronic hyperglycaemia-associated reactive oxygen species stress and low-grade inflammation play critical roles in the development of diabetic retinopathy. Animal studies have revealed elevated levels of superoxide and hydrogen peroxide in the retina of diabetic rats (Cui et al., 2006; Ellis, Guberski, Somogyi-Mann, & Grant, 2000). Human studies using materials obtained at vitrectomy demonstrated that the vitreal levels of pro-inflammatory cytokines (interleukin-8 and monocyte chemoattractant protein-1) were higher in diabetic patients with PDR than in non-diabetic controls (Hernández et al., 2005). Moreover, vitreous levels of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin were significantly higher in eyes with PDR than in eyes of non-diabetic controls (Limb, Hickman-Casey, Hollifield, & Chignell, 1999). On the other hand, bilirubin is known to exhibit not only potent antioxidant properties by scavenging reactive oxygen species (Sedlak et al., 2009; Stocker, Yamamoto, McDonagh, Glazer, & Ames, 1987), but also anti-inflammatory properties by inhibiting the TNFα-related induction of three endothelial adhesion molecules: E-selectin, VCAM1, and ICAM-1 (Mazzone et al., 2009). In animal study about diabetic nephropathy, bilirubin significantly inhibited nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide production, Table 3 Multivariate logistic regression analysis of diabetic retinopathy.
Fig. 2. Total serum bilirubin concentration of patients with NDR, SDR, and PPDR + PDR. ** p b 0.001, * p b 0.05.
Sex (Female = 1, Male = 0) Age BMI Duration of diabetes Systolic blood pressure HbA1c Total bilirubin Smoking
Wald
Odds ratio
95% CI
p
2.620 0.327 0.295 57.419 8.100 0.002 18.112 1.005
1.419 0.995 0.992 1.079 1.012 1.002 0.262 1.225
0.929–2.168 0.980–1.011 0.962–1.022 1.058–1.100 1.004–1.021 0.917–1.095 0.142–0.486 0.824–1.822
0.106 0.568 0.587 0.000 0.004 0.961 0.000 0.316
Abbreviations: CI, confidence interval. BMI, body mass index; HbA1c, haemoglobin A1c.
R. Sekioka et al. / Journal of Diabetes and Its Complications 29 (2015) 218–221
and both high glucose- and angiotensin II-induced production of reactive oxygen species (Fujii et al., 2010), which suggests a protective role of bilirubin against diabetic microangiopathy. Furthermore, experimental studies have demonstrated that bilirubin interferes with expression of cell adhesion molecules, activity of complements, and differentiation of T cell (Vítek, 2012). Therefore, bilirubin might prevent diabetic retinopathy through its anti-oxidant and anti-inflammatory properties. The results of univariate and multivariate analyses in this study showed that not only total bilirubin concentration, but also diabetes duration and systolic blood pressure are significant explanatory factors for the complication of diabetic retinopathy. The results of the Jonckheere–Terpstra test are in line with this observation. There were also tendencies for patients with severer retinopathy to have a longer diabetes duration and higher systolic blood pressure. UKPDS 38 (UK Prospective Diabetes Study Group., 1998) and UKPDS 69 (Matthews et al., 2004) demonstrated the importance of strict blood pressure control for the prevention of loss of vision and retinopathy progression. The animal experiments of Hammes et al. (Hammes et al., 1994) showed that the frequency of acellular capillaries, the gold standard morphological marker of diabetic retinopathy, is nearly twice as high in diabetic spontaneous hypertensive rats as in diabetic normotensive rats. This study has some limitations that should be mentioned. First, the cross-sectional design precludes the establishment of a causal relationship between bilirubin concentration and diabetic microangiopathy. In addition, the patients were admitted to university hospitals. Therefore, the results may not be applicable to the general population or patients with type 2 diabetes in primary care settings. Accordingly, further cross-sectional or prospective studies with larger sample sizes are required to confirm the present findings. In conclusion, serum total bilirubin concentration is significantly associated with the severity of diabetic retinopathy. Bilirubin might play protective roles against the incidence and/or progression of retinopathy in patients with type 2 diabetes. Acknowledgements Risa SEKIOKA researched data and wrote the manuscript. Masami TANAKA, Takeshi NISHIMURA, and Hiroshi ITOH researched data and reviewed and edited the manuscript. Masami TANAKA conceived and designed the study and reviewed and edited the manuscript. Risa SEKIOKA is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. References Chan, K. H., O'Connell, R. L., Sullivan, D. R., Hoffmann, L. S., Rajamani, K., Whiting, M., et al. (2013). Plasma total bilirubin levels predict amputation events in type 2 diabetes mellitus: The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study. Diabetologia, 56, 724–736. Cho, H. C. (2011). The relationship among homocysteine, bilirubin, and diabetic retinopathy. Diabetes & metabolism journal, 35, 595–601. Committee of the Japan Diabetes Society on the Diagnostic Criteria of Diabetes Mellitus, Seino, Y., Nanjo, K., Tajima, N., Kadowaki, T., Kashiwagi, A., et al. (2010). Report of the committee on the classification and diagnostic criteria of diabetes mellitus. Journal of Diabetes Investigation, 19, 212–228.
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Cui, Y., Xu, X., Bi, H., Zhu, Q., Wu, J., Xia, X., et al. (2006). Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: The role of reactive oxygen species in diabetic retinopathy. Experimental Eye Research, 83, 807–816. Davis, M. D., Kern, T. S., & Rand, L. I. (1997). Diabetic retinopathy. In K. G. Alberti, P. Zimmet, & R. DeFronzo (Eds.), International textbook of diabetes mellitus (pp. 1413–1446) (2nd ed.). Chichester, UK: John Wiley & Sons. Ellis, E. A., Guberski, D. L., Somogyi-Mann, M., & Grant, M. B. (2000). Increased H2O2, vascular endothelial growth factor and receptors in the retina of the BBZ/Wor diabetic rat. Free Radical Biology and Medicine, 28, 91–101. Fujii, M., Inoguchi, T., Sasaki, S., Maeda, Y., Zheng, J., Kobayashi, K., et al. (2010). Bilirubin and biliverdin protect rodents against diabetic nephropathy by downregulating NAD(P)H oxidase. Kidney International, 78, 905–919. Hammes, H. P., Brownlee, M., Edelstein, D., Saleck, M., Martin, S., & Federlin, K. (1994). Aminoguanidine inhibits the development of accelerated diabetic retinopathy in the spontaneous hypertensive rat. Diabetologia, 37, 32–35. Hernández, C., Segura, R. M., Fonollosa, A., Carrasco, E., Francisco, G., & Simó, R. (2005). Interleukin-8, monocyte chemoattractant protein-1 and IL-10 in the vitreous fluid of patients with proliferative diabetic retinopathy. Diabetic Medicine, 22, 719–722. Horio, M., Imai, E., Yasuda, Y., Watanabe, T., & Matsuo, S. (2010). Modification of the CKD epidemiology collaboration (CKD-EPI) equation for Japanese: Accuracy and use for population estimates. American Journal of Kidney Diseases, 56, 32–38. Inoguchi, T., Sasaki, S., Kobayashi, K., Takayanagi, R., & Yamada, T. (2007). Relationship between Gilbert syndrome and prevalence of vascular complications in patients with diabetes. JAMA, 298, 1396–1400. Kawasaki, R., Tanaka, S., Tanaka, S., Yamamoto, T., Sone, H., Ohashi, Y., et al. (2011). Incidence and progression of diabetic retinopathy in Japanese adults with type 2 diabetes: 8 Year follow-up study of the Japan Diabetes Complications Study (JDCS). Diabetologia, 54, 2288–2294. Kim, E. S., Mo, E. Y., Moon, S. D., & Han, J. H. (2014). Inverse association between serum bilirubin levels and arterial stiffness in Korean women with type 2 diabetes. PLoS one, 9(10), e109251. Lee, M. J., Jung, C. H., Kang, Y. M., Hwang, J. Y., Jang, J. E., Leem, J., et al. (2014). Serum bilirubin as a predictor of incident metabolic syndrome: A 4-year retrospective longitudinal study of 6205 initially healthy Korean men. Diabetes & Metabolism, 40, 305–309. Limb, G. A., Hickman-Casey, J., Hollifield, R. D., & Chignell, A. H. (1999). Vascular adhesion molecules in vitreous from eyes with proliferative diabetic retinopathy. Investigative Ophthalmology & Visual Science, 40, 2453–2457. Matsuo, S., Imai, E., Horio, M., Yasuda, Y., Tomita, K., Nitta, K., et al. (2009). Revised equations for estimated GFR from serum creatinine in Japan. American Journal of Kidney Diseases, 53, 982–992. Matthews, D. R., Stratton, I. M., Aldington, S. J., Holman, R. R., Kohner, E. M., & UK Prospective Diabetes Study Group (2004). Risks of progression of retinopathy and vision loss related to tight blood pressure control in type 2 diabetes mellitus: UKPDS 69. Archives of Ophthalmology, 122, 1631–1640. Mazzone, G. L., Rigato, I., Ostrow, J. D., Bossi, F., Bortoluzzi, A., Sukowati, C. H., et al. (2009). Bilirubin inhibits the TNFalpha-related induction of three endothelial adhesion molecules. Biochemical and Biophysical Research Communications, 386, 338–344. Najam, S. S., Sun, J., Zhang, J., Xu, M., Lu, J., Sun, K., et al. (2014). Serum total bilirubin levels and prevalence of diabetic retinopathy in a Chinese population. Journal of Diabetes, 6, 221–227. Novotný, L., & Vítek, L. (2003). Inverse relationship between serum bilirubin and atherosclerosis in men: A meta-analysis of published studies. Experimental Biology and Medicine, 228, 568–571. Sedlak, T. W., Saleh, M., Higginson, D. S., Paul, B. D., Juluri, K. R., & Snyder, S. H. (2009). Bilirubin and glutathione have complementary antioxidant and cytoprotective roles. Proceedings of the National Academy of Sciences of the United States of America, 106, 5171–5176. Stocker, R., Yamamoto, Y., McDonagh, A. F., Glazer, A. N., & Ames, B. N. (1987). Bilirubin is an antioxidant of possible physiological importance. Science, 235, 1043–1046. Takayanagi, R., Inoguchi, T., & Ohnaka, K. (2010). Clinical and experimental evidence for oxidative stress as an exacerbating factor of diabetes mellitus. Journal of Clinical Biochemistry and Nutrition, 48, 72–77. UK Prospective Diabetes Study Group. (1998). Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. BMJ, 317, 703–713. Vítek, L. (2012). The role of bilirubin in diabetes, metabolic syndrome, and cardiovascular diseases. Frontiers in Pharmacology, 3, 55. Yasuda, M., Kiyohara, Y., Wang, J. J., Arakawa, S., Yonemoto, K., Doi, Y., et al. (2011). High serum bilirubin levels and diabetic retinopathy: The Hisayama Study. Ophthalmology, 118, 1423–1428.
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications journal homepage: WWW.JDCJOURNAL.COM
Serum total bilirubin concentration is negatively associated with increasing severity of retinopathy in patients with type 2 diabetes mellitus Risa Sekioka ⁎, 1, Masami Tanaka, Takeshi Nishimura, Hiroshi Itoh Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 25 November 2014 Accepted 1 December 2014 Available online 5 December 2014 Keywords: Total bilirubin Diabetic retinopathy Type 2 diabetes mellitus Hypertension Human
a b s t r a c t Aims: Serum bilirubin concentration is associated with diabetic retinopathy in patients with type 2 diabetes. This study investigated the relationships between serum bilirubin concentration and the severity of diabetic retinopathy. In addition, the importance of bilirubin was compared with factors that were previously shown to be associated with the incidence of diabetic retinopathy. Methods: A total of 674 patients with type 2 diabetes were investigated in this cross-sectional study. Serum total bilirubin concentration was compared between patients with and without diabetic retinopathy, and according to the severity of retinopathy. Univariate and multivariate analyses were performed to evaluate the association of retinopathy with total bilirubin concentration, duration of diabetes, body mass index, systolic blood pressure, and haemoglobin A1c. Results: Serum total bilirubin concentration was significantly lower in patients with retinopathy than in those without. Patients with severer retinopathy showed lower total bilirubin concentration, longer diabetes duration, and higher systolic blood pressure. These three parameters were independent explanatory factors for diabetic retinopathy. Conclusions: Total bilirubin concentration is lower in patients with type 2 diabetes complicated with severer retinopathy. Thus, bilirubin might protect against retinopathy in patients with type 2 diabetes. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Hyperglycaemia is the hallmark of diabetes; it activates certain biochemical pathways leading to macro- and microvascular complications (Takayanagi, Inoguchi, & Ohnaka, 2010). Diabetic retinopathy is a common microvascular complication of diabetes and a leading cause of legal blindness in working-age adults. Bilirubin has long been considered to be a useless or potentially toxic metabolite in jaundice (especially in high doses), particularly in neonates. However, recent studies revealed that serum bilirubin level was inversely associated with atherosclerosis in a cross-sectional analysis (Kim, Mo, Moon, & Han, 2014) and meta-analysis (Novotný & Vítek, 2003). In addition, serum total bilirubin level was negatively associated with incidence of metabolic syndrome (Lee et al., 2014) Conflict of interest: Hiroshi ITOH has received compensation from NIPRO, lecture fees from Takeda and MSD, and scholarship grants from Sanofi-Aventis, Astellas, Takeda, MSD, and Daiichi Sankyo. The other authors have no conflict of interest. ⁎ Corresponding author. Tel.: + 81 3 5363 3797; fax: + 81 3 3359 2745. E-mail addresses: [email protected] (R. Sekioka), [email protected] (M. Tanaka), [email protected] (T. Nishimura), [email protected] (H. Itoh) 1 Present Address: Department of Internal Medicine, Jujo Takeda Rehabilitation Hospital, 32 Kisshoinhatsutandacho, Kyoto Minami-ku, Kyoto, 601-8325, Japan. http://dx.doi.org/10.1016/j.jdiacomp.2014.12.002 1056-8727/$© 2015 Elsevier Inc. All rights reserved.
and lower-limb amputation events in type 2 diabetes (Chan et al., 2013). Furthermore, there have been several reports demonstrating that type 2 diabetic patients with high serum total bilirubin concentration tend to have less complication of diabetic retinopathy (Cho, 2011; Inoguchi, Sasaki, Kobayashi, Takayanagi, & Yamada, 2007; Najam et al., 2014; Yasuda et al., 2011). Although this observation might suggest a protective role of bilirubin against diabetic retinopathy, the detailed association between bilirubin and retinopathy, such as the severity of retinopathy, remains to be elucidated. Therefore, this study was undertaken to clarify the relationship between serum bilirubin concentration and the severity of diabetic retinopathy. In addition, the importance of bilirubin was evaluated by comparing it with factors previously shown to be significantly associated with the incidence of diabetic retinopathy in The Japan Diabetes Complications Study (JDCS) (Kawasaki et al., 2011). 2. Materials and methods 2.1. Patients The subjects included 674 patients with type 2 diabetes admitted to Keio University Hospital from January 2008 to December 2013. The
R. Sekioka et al. / Journal of Diabetes and Its Complications 29 (2015) 218–221
exclusion criteria were as follows: malignant disease under treatment, liver cirrhosis, haematological disease, abnormal liver function test results (i.e., serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) level N 3 times the upper limit of the normal range), and anti-glutamate decarboxylase antibody level ≥ 1.5 U/mL. This study was approved by the ethical committee of Keio University School of Medicine and performed in accordance with the Declaration of Helsinki. Retinopathy was evaluated by an ophthalmologist using fundoscopy after pupillary dilation. Classification was based on the modified Davis classification as follows (Davis, Kern, & Rand, 1997): no diabetic retinopathy (NDR), simple diabetic retinopathy (SDR), preproliferative diabetic retinopathy (PPDR), and proliferative diabetic retinopathy (PDR); PPDR and PDR were combined into a PPDR + PDR category for analysis. Nephropathy was defined as urinary albumin excretion ≥ 30 mg/g creatinine or estimated glomerular filtration rate (eGFR) b 30 mL∙min − 1∙ 1.73 m − 2. Cerebrovascular and cardiovascular disease was defined as a history of these diseases. Smoking was defined as previous or current tobacco usage. Hypertension was defined as blood pressure ≥ 140/90 mmHg or the use of antihypertensive drugs. Dyslipidaemia was defined as serum LDL cholesterol ≥ 140 mg/dL, HDL cholesterol b 40 mg/dL, triglycerides ≥ 150 mg/dL, or taking a hypolipidemic agent.
2.2. Data collection For this cross-sectional study, basic demographic data from all patients were collected from medical records, including sex, age, height, weight, duration of diabetes, total bilirubin, AST, ALT, haemoglobin A1c (HbA1c), eGFR, and lipid levels. Blood and urine samples taken on admission during the study period were used for analysis. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. HbA1c was expressed in accordance with the National Glycohemoglobin Standardization Program guidelines as recommended by the Japanese Diabetes Society (Committee of the Japan Diabetes Society on the Diagnostic Criteria of Diabetes Mellitus et al., 2010). eGFR was calculated using the following formula established by the working group of the Japanese Chronic Kidney Disease Initiative (Horio, Imai, Yasuda, Watanabe, & Matsuo, 2010; Matsuo et al., 2009): eGFR (mL min−1 1.73 m −2) = 194 × (serum creatinine) −1.094 × (age)−0.287 (×0.739 for women).
2.3. Statistical analyses Serum total bilirubin concentration was compared between patients with and without diabetic retinopathy with paired t-test. It was also compared according to the severity of retinopathy using the Bonferroni test. The associations of diabetes duration and systolic blood pressure with the severity of retinopathy were evaluated using the Jonckheere–Terpstra test. Univariate analyses were performed to evaluate the association of retinopathy with total bilirubin concentration and factors previously shown to be important in the JDCS (Kawasaki et al., 2011) — namely, sex, age, BMI, duration of diabetes, systolic blood pressure, and smoking. Multivariate logistic regression analysis was performed to determine the effects of factors examined in univariate analyses on diabetic retinopathy. Variables are expressed as mean ± standard deviation. All statistical analyses were performed using SPSS (version 21.0; SPSS, Chicago, IL, USA). The level of significance was set at p b 0.05.
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3. Results 3.1. Patient characteristics Patient demographic and laboratory data are shown in Table 1. The mean concentration of serum total bilirubin in all patients was 0.74 ± 0.36 mg/dL. No patient had a serum bilirubin level greater than twice the upper limit of the normal range. Of 674 patients with type 2 diabetes mellitus, 58.6% (395) were classified as NDR; of these, 279 were complicated with diabetic retinopathy, with 19.6% (n = 132), 6.4% (n = 43), and 15.4% (n = 104) classified as SDR, PPDR, and PDR, respectively. 3.2. Association between total bilirubin concentration and diabetic retinopathy Patients with diabetic retinopathy had a significantly lower serum total bilirubin concentration than those without retinopathy (NDR, 0.65 ± 0.30 mg/dL vs. 0.80 ± 0.39 mg/dL, p b 0.001; Fig. 1). As for the severity of retinopathy, patients with SDR and PPDR + PDR had significantly lower bilirubin concentrations than those without retinopathy (SDR, 0.71 ± 0.34 mg/dL, p = 0.048; PPDR + PDR, 0.59 ± 0.24 mg/dL, b 0.001 vs. NDR), and patients with PPDR + PDR
Table 1 Patient demographic and laboratory data. N
674
Age (years) Sex (male/female) Duration of diabetes (years) BMI (kg/m2) HbA1c (%) Total bilirubin (mg/dL) AST (IU/L) ALT (IU/L) eGFR (mL min−1 1.73 m−2) Smoking, n (%) Hypertension, n (%) Dyslipidaemia, n (%) Diabetic retinopathy, n (%) NDR/SDR/PPDR/PDR, n (%) Diabetic nephropathy, n (%) Cerebrovascular disease, n (%) Cardiovascular disease, n (%) Concomitant medication, n (%) SU Metformin Thiazolidinedione α-GI Glinide DPP-4 inhibitor Insulin GLP1 analogue Antihypertensive agent, n (%) ARB ACEI CCB α-blocker β-blocker Diuretic Hypolipidemic agent, n (%) Statin Fibrate Ezetimibe Eicosapentaenoic acid
64.7 ± 13.9 446/228 13.9 ± 10.9 25.5 ± 6.3 9.13 ± 2.16 0.74 ± 0.36 26.8 ± 17.0 26.8 ± 20.1 61.7 ± 31.6 324 (48.1) 498 (73.9) 520 (77.2) 279 (41.4) 395 (58.6)/132 (19.6)/43 (6.4)/104 (21.8) 323 (47.9) 101 (15.0) 128 (19.0) 198 (29.4) 104 (15.4) 50 (7.4) 159 (23.6) 31 (4.6) 107 (15.9) 257 (38.1) 6 (0.89) 287 (42.6) 34 (5.0) 269 (39.9) 16 (2.4) 115 (17.1) 124 (18.4) 264 (39.2) 18 (2.7) 18 (2.7) 16 (2.4)
Values are expressed as number (%) or mean ± SD. Abbreviations: BMI, body mass index; HbA1c, haemoglobin A1c; AST, aspartate aminotransferase; ALT, alanine aminotransferase; eGFR, estimated glomerular filtration rate; NDR, no diabetic retinopathy; SDR, simple diabetic retinopathy; PPDR, preproliferative diabetic retinopathy; PDR, proliferative diabetic retinopathy; SU, sulfonylurea; α-GI, α-glucosidase inhibitor; ARB, angiotensin receptor blocker; ACEI, angiotensin-converting enzyme inhibitor; CCB, calcium channel blocker.
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R. Sekioka et al. / Journal of Diabetes and Its Complications 29 (2015) 218–221 Table 2 Univariate logistic regression analysis of diabetic retinopathy.
Sex (Female = 1, Male = 0) Age BMI Duration of diabetes Systolic blood pressure HbA1c Total bilirubin Smoking
Wald
Odds ratio
95% CI
p
0.188 14.318 0.125 82.556 14.853 8.811 27.113 0.613
1.074 1.023 0.996 1.082 1.014 0.895 0.233 1.133
0.777–1.484 1.011–1.034 0.971–1.020 1.064–1.100 1.007–1.021 0.831–0.963 0.134–0.403 0.829–1.549
0.665 0.000 0.724 0.000 0.000 0.003 0.000 0.434
Abbreviations: CI, confidence interval. BMI, body mass index; HbA1c, haemoglobin A1c.
4. Discussion Fig. 1. Total serum bilirubin concentration of patients with and without retinopathy. ** p b 0.001.
had a significantly lower bilirubin concentration than those with SDR (p = 0.011; Fig. 2).
3.3. Association between diabetes duration, blood pressure, and severity of diabetic retinopathy The Jonckheere–Terpstra test showed that a longer duration of diabetes (PPDR + PDR, 19.7 ± 11.6; SDR, 17.8 ± 9.3; NDR, 10.5 ± 9.7 years; p b 0.001) and a higher systolic blood pressure (PPDR + PDR, 139 ± 25; SDR, 136 ± 24; NDR, 131 ± 20 mmHg; p b 0.001) were significantly associated with severer retinopathy.
3.4. Determinants of diabetic retinopathy Univariate logistic regression analyses demonstrated significant difference in total bilirubin concentration (odds ratio [OR] = 0.233; 95% confidence interval [CI], 0.134–0.403; p b 0.001), duration of diabetes (OR = 1.082; 95% CI, 1.064–1.100; p b 0.001), systolic blood pressure (OR = 1.014; 95% CI, 1.007–1.021; p b 0.001), HbA1c (OR = 0.895; 95% CI, 0.831–0.963; p = 0.003), and age (OR = 1.023; 95% CI, 1.011–1.034; p b 0.001) between patients with and without diabetic retinopathy (Table 2). Multivariate logistic regression analysis demonstrated that total bilirubin concentration (OR = 0.262; 95% CI, 0.142–0.486; p b 0.001), duration of diabetes (OR = 1.079; 95% CI, 1.058–1.100; p b 0.001), and systolic blood pressure (OR = 1.012; 95% CI, 1.004–1.021; p = 0.004) were significant independent explanatory factors for retinopathy (Table 3).
In this observational study, it was shown that type 2 diabetic patients with retinopathy had lower serum total bilirubin concentration than those without retinopathy, which is in line with other studies (Cho, 2011; Inoguchi et al., 2007; Najam et al., 2014; Yasuda et al., 2011). Furthermore, it was also shown that total bilirubin concentration is negatively related with diabetic retinopathy from the results of univariate (OR = 0.233; p b 0.001) and multivariate (OR = 0.262; p b 0.001) logistic analyses. Judging from OR, each mg/dL decrease of serum bilirubin was associated with 4 times higher risk of diabetic retinopathy. In addition, we demonstrated that total bilirubin concentration was negatively associated with increasing severity of retinopathy. To our knowledge, this has not been demonstrated in the past. Our observation that the patients with severer diabetic retinopathy showed lower total bilirubin concentration suggests the possibility that bilirubin is deeply involved in the prevention of diabetic retinopathy. However, the precise mechanism underlying this phenomenon is unknown. Several reports support the idea that chronic hyperglycaemia-associated reactive oxygen species stress and low-grade inflammation play critical roles in the development of diabetic retinopathy. Animal studies have revealed elevated levels of superoxide and hydrogen peroxide in the retina of diabetic rats (Cui et al., 2006; Ellis, Guberski, Somogyi-Mann, & Grant, 2000). Human studies using materials obtained at vitrectomy demonstrated that the vitreal levels of pro-inflammatory cytokines (interleukin-8 and monocyte chemoattractant protein-1) were higher in diabetic patients with PDR than in non-diabetic controls (Hernández et al., 2005). Moreover, vitreous levels of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin were significantly higher in eyes with PDR than in eyes of non-diabetic controls (Limb, Hickman-Casey, Hollifield, & Chignell, 1999). On the other hand, bilirubin is known to exhibit not only potent antioxidant properties by scavenging reactive oxygen species (Sedlak et al., 2009; Stocker, Yamamoto, McDonagh, Glazer, & Ames, 1987), but also anti-inflammatory properties by inhibiting the TNFα-related induction of three endothelial adhesion molecules: E-selectin, VCAM1, and ICAM-1 (Mazzone et al., 2009). In animal study about diabetic nephropathy, bilirubin significantly inhibited nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide production, Table 3 Multivariate logistic regression analysis of diabetic retinopathy.
Fig. 2. Total serum bilirubin concentration of patients with NDR, SDR, and PPDR + PDR. ** p b 0.001, * p b 0.05.
Sex (Female = 1, Male = 0) Age BMI Duration of diabetes Systolic blood pressure HbA1c Total bilirubin Smoking
Wald
Odds ratio
95% CI
p
2.620 0.327 0.295 57.419 8.100 0.002 18.112 1.005
1.419 0.995 0.992 1.079 1.012 1.002 0.262 1.225
0.929–2.168 0.980–1.011 0.962–1.022 1.058–1.100 1.004–1.021 0.917–1.095 0.142–0.486 0.824–1.822
0.106 0.568 0.587 0.000 0.004 0.961 0.000 0.316
Abbreviations: CI, confidence interval. BMI, body mass index; HbA1c, haemoglobin A1c.
R. Sekioka et al. / Journal of Diabetes and Its Complications 29 (2015) 218–221
and both high glucose- and angiotensin II-induced production of reactive oxygen species (Fujii et al., 2010), which suggests a protective role of bilirubin against diabetic microangiopathy. Furthermore, experimental studies have demonstrated that bilirubin interferes with expression of cell adhesion molecules, activity of complements, and differentiation of T cell (Vítek, 2012). Therefore, bilirubin might prevent diabetic retinopathy through its anti-oxidant and anti-inflammatory properties. The results of univariate and multivariate analyses in this study showed that not only total bilirubin concentration, but also diabetes duration and systolic blood pressure are significant explanatory factors for the complication of diabetic retinopathy. The results of the Jonckheere–Terpstra test are in line with this observation. There were also tendencies for patients with severer retinopathy to have a longer diabetes duration and higher systolic blood pressure. UKPDS 38 (UK Prospective Diabetes Study Group., 1998) and UKPDS 69 (Matthews et al., 2004) demonstrated the importance of strict blood pressure control for the prevention of loss of vision and retinopathy progression. The animal experiments of Hammes et al. (Hammes et al., 1994) showed that the frequency of acellular capillaries, the gold standard morphological marker of diabetic retinopathy, is nearly twice as high in diabetic spontaneous hypertensive rats as in diabetic normotensive rats. This study has some limitations that should be mentioned. First, the cross-sectional design precludes the establishment of a causal relationship between bilirubin concentration and diabetic microangiopathy. In addition, the patients were admitted to university hospitals. Therefore, the results may not be applicable to the general population or patients with type 2 diabetes in primary care settings. Accordingly, further cross-sectional or prospective studies with larger sample sizes are required to confirm the present findings. In conclusion, serum total bilirubin concentration is significantly associated with the severity of diabetic retinopathy. Bilirubin might play protective roles against the incidence and/or progression of retinopathy in patients with type 2 diabetes. Acknowledgements Risa SEKIOKA researched data and wrote the manuscript. Masami TANAKA, Takeshi NISHIMURA, and Hiroshi ITOH researched data and reviewed and edited the manuscript. Masami TANAKA conceived and designed the study and reviewed and edited the manuscript. Risa SEKIOKA is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. References Chan, K. H., O'Connell, R. L., Sullivan, D. R., Hoffmann, L. S., Rajamani, K., Whiting, M., et al. (2013). Plasma total bilirubin levels predict amputation events in type 2 diabetes mellitus: The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study. Diabetologia, 56, 724–736. Cho, H. C. (2011). The relationship among homocysteine, bilirubin, and diabetic retinopathy. Diabetes & metabolism journal, 35, 595–601. Committee of the Japan Diabetes Society on the Diagnostic Criteria of Diabetes Mellitus, Seino, Y., Nanjo, K., Tajima, N., Kadowaki, T., Kashiwagi, A., et al. (2010). Report of the committee on the classification and diagnostic criteria of diabetes mellitus. Journal of Diabetes Investigation, 19, 212–228.
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