Diabetes mellitus (DM) is a major and growing public health concern, with increasing prevalence attributed to population aging and changing lifestyle patterns. The overall prevalence of diabetes across 15 states in India has been reported to be 7.3% [1]. Diabetes is associated with several microvascular complications, including neuropathy, nephropathy, and retinopathy, as well as macrovascular complications such as cardiovascular disease [13]. Diabetic retinopathy (DR) is one of the most important microvascular complications of diabetes and is a major cause of visual impairment and blindness in adults [2]. Its development and progression are influenced by established risk factors such as prolonged duration of diabetes, hyperglycemia, hypertension, and dyslipidemia [13]. In addition, oxidative stress is increasingly recognized as an important contributor to diabetic vascular injury. Chronic hyperglycemia promotes excessive generation of reactive oxygen species (ROS), resulting in oxidative damage, lipid peroxidation, and endothelial dysfunction [17]. Abnormal glucose and lipid metabolism may further aggravate oxidative injury and contribute to both microvascular and macrovascular complications of diabetes [4]. The retina is particularly susceptible to oxidative stress, and increased oxidative damage has been associated with the development and severity of diabetic retinopathy. Serum 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, has been reported to be significantly elevated in patients with DR compared with diabetic individuals without retinopathy [71]. Bilirubin, a product of heme metabolism, is now recognized as an endogenous antioxidant and anti-inflammatory molecule rather than merely a metabolic waste product [5]. It can scavenge reactive oxygen species and inhibit lipid peroxidation and oxidative modification of low-density lipoprotein [7]. More than 99% of circulating bilirubin is bound to albumin, facilitating its transport in the blood [11]. Experimental studies have also demonstrated potential protective effects of bilirubin against oxidative injury. Several studies suggest that serum bilirubin concentrations within the normal physiological range may be inversely associated with diabetes and its vascular complications. Higher bilirubin levels have been associated with a lower risk of T2DM, whereas lower-normal concentrations have been linked to microalbuminuria and subclinical atherosclerosis [8, 10]. Serum bilirubin has also been inversely associated with urinary albumin excretion, suggesting a possible protective role against microvascular injury [11]. Furthermore, higher normal-range bilirubin concentrations have been associated with reduced risks of coronary and peripheral arterial disease, potentially through antioxidant and anti-atherogenic mechanisms [5, 10]. Evidence from prospective studies and meta-analyses indicates an inverse relationship between total bilirubin levels and cardiovascular disease [71]. These observations raise the possibility that bilirubin may also exert a protective effect against retinal vascular injury in patients with diabetes [12]. However, evidence regarding the association between serum bilirubin and diabetic retinopathy remains limited, particularly in the Indian population. Therefore, the present study was undertaken to compare serum bilirubin levels in patients with diabetes mellitus with and without diabetic retinopathy and to evaluate the possible association between serum bilirubin concentration and diabetic retinopathy. AIM AND OBJECTIVES 1. To correlate the serum bilirubin levels (within normal range) with severity of Diabetic Retinopathy. 2. To correlate the type of serum bilirubin with the severity of Diabetic Retinopathy.

It was a Hospital-based Prospective Cross Sectional Observational study where clinically diagnosed cases of Diabetes mellitus attending the Outpatient Department of Ophthalmology during the study period and who fulfill the inclusion and exclusion criteria were included. Convenience sampling was used. The sample size required in our study was N = 300. The following formula was used to calculate the sample size. Cr =1/2*Log (1+r)/(1-r) r = -0.34 Zα = 1.96 (5% level of significance) Zβ = 0.84 (80% power) During the study period of 3 years, a total of 315 cases were recruited. INCLUSION CRITERIA Clinically Diagnosed cases of Diabetes Mellitus. EXCLUSION CRITERIA History of pre-existing hepatobiliary abnormalities or any other acute disease affecting hepatic functions within past 6 months or chronic alcohol consumption. Intake of hepatotoxic drugs within past 6 months (anti-tubercular drugs, anti-epileptic drugs etc.). Patients who were on anti-diabetic drugs like thiazolidinedione (piaglitazone) and alpha 1 glucosidase inhibitor (acarbose). Patients with haemolytic anemia and other haemolytic conditions. Patients in whom proper evaluation of fundus cannot be done (patients with cataract, corneal opacity etc.). METHODOLOGY After taking informed consent a detailed case history was taken from all patients regarding age, gender, duration of diabetes mellitus, history of smoking, alcohol intake or hepatobiliary disorders, hypertension or cardiovascular diseases and history of medications. Blood sugar levels were investigated by Random Blood Sugar measurement. Best corrected visual acuity (BCVA) was measured at distance using Snellen’s chart and at near using Jaeger’s chart. Intra-Ocular Pressure (IOP) was measured using Non-Contact tonometry (TOPCON CT-800). Detailed Ophthalmological examination was done using Slit lamp biomicroscope. Fundus examination was done post-dilatation with Tropicacyl plus (Tropicamide + Phenylephrine) eye drops using +90D condensing lens under slit lamp biomicroscope & +20D condensing lens with Indirect Ophthalmoscope. Patients were classified as cases- without diabetic retinopathy, with Non proliferative diabetic retinopathy (mild, moderate and severe) and Proliferative Diabetic retinopathy. Three millilitres of venous blood was drawn from the patient on the day of examination and serum Total bilirubin, Direct and indirect bilirubin levels was estimated. All findings were recorded and analyzed for any change in comparison with the variables. DATA COLLECTION METHODS: All the patients underwent ophthalmic examination at out patient department who met the inclusion and exclusion criteria. Finding were reconfirmed by consultant ophthalmologist. After pupil dilation with Tropicamide + Phenylephrine (Tropicacyl plus) eye drop, screening for DR was done by +90 D slit lamp biomicroscopy and +20 D in indirect ophthalmoscopy. Blood was drawn for serum total bilirubin level, diect bilirubin, indirect bilirubin, random blood glucose level, Serum bilirubin level was measured using semi auto analyser (STAR-20, Rapid diagnostic private Ltd.) using standard approved technique. Diagnostic Reagents used were LIQUIXX (BIT &BID), Diazo method, End point. Manufactured by Transasia bio-medicals Ltd. The method was Diazo method of Pearlman & Lee. Quality controls under norms of ERBA NORM and ERBA PATH. The standard reference normal range of total bilirubin by this method was0.1 to 1.2 mg/dl. STATISTICAL ANALYSIS The data were analyzed descriptively first with mean and standard deviation estimates were derived. The Analysis of variance test (ANOVA) was used to compare the serum bilirubin level and the type of diabetic retinopathy. The values of the type of serum bilirubin i.e. direct bilirubin and indirect bilrubin were analysed in each group of diabetic retinopathy and were analysed separately for their correlation. A two-sided p value less than 0.05 was considered to be statistically significant. All statistical analysis was carried out by using the SPSS 25.0 version (SPSS, Chicago, IL, USA) software for windows.

Table 1: Age Distribution in years Age in years No. of Cases Percentage <50 years 25 7.9% 50-70 years 249 79% >70 years 41 13% Total 315 100% The majority of patients were between 50-70 years (79%). 13% of patients were more than >70 years and 7.9% of patients were less than <50 years old. Table 2: Age distribution in years Min Q1 Median Q3 Max 39 56.00 62.00 68.00 90 The mean and standard deviation of Age in years is 62.127±8.868. The median Age in years is 62 and the range is 51 (39-90). The inter-quartile range is 12 which we get from Q1 (56) and Q3 (68). Table 3: Distribution of cases according to gender Sex No. of Cases Percentage Male 182 57.8% Female 133 42.2% Total 315 100% The maximum numbers of patients were Male (57.8%). 42.2% of patients were Female. Table 4: Diabetic Retinopathy status in our study Presence of diabetic retinopathy No. of Cases Percentage No diabetic retinopathy 118 37.5% With diabetic retinopathy 197 62.5% Total 315 100% Out of 315 patients majority of them were with diabetic retinopathy 197 (62.5%). 118(37.5%) of patients were with No diabetic retinopathy. Table 5: Distribution of stages of Diabetic retinopathy Presence of diabetic retinopathy No. of Cases Percentage No diabetic retinopathy 118 37.5% Non proliferative diabetic retinopathy 137 43.5% Proliferative diabetic retinopathy 60 19% Total 315 100% The result shows that majority of patients had Non proliferative diabetic retinopathy 137(43.5%), 118(37.5%) of patients were with No diabetic retinopathy and 60(19%) of patients had Proliferative diabetic retinopathy. Table 6: Total serum Bilirubin (in mg/dl) Min Q1 Median Q3 Max 0 .60 .80 1.00 1 The mean and standard deviation of Total serum Bilirubin (in mg/dl) is 0.8±0.231. The median Total serum Bilirubin (in mg/dl) is 0.8 and the range is 0.9 (0.3-1.2). The inter-quartile range is 0.4 which we get from Q1 (0.6) and Q3 (1). Table 7: Direct Birubin (in mg/dl) Min Q1 Median Q3 Max 0 .10 .20 .20 0 The mean and standard deviation of Direct Birubin (in mg/dl) is 0.185±0.084. The median Direct Birubin (in mg/dl) is 0.2 and the range is 0.3 (0.1-0.4). The inter-quartile range is 0.1 which we get from Q1 (0.1) and Q3 (0.2). Table 8: Indrect bilirubin (in mg/dl) Min Q1 Median Q3 Max 0 .50 .60 .80 1 The mean and standard deviation of Indrect bilirubin (in mg/dl) is 0.619±0.199. The median Indrect bilirubin (in mg/dl) is 0.6 and the range is 1 (0.1-1.1). The inter-quartile range is 0.3 which we get from Q1 (0.5) and Q3 (0.8). Table 9: Relation between presence of diabetic retinopathy and total serum bilirubin Presence of diabetic retinopathy N Mean SD t Value P Value No diabetic retinopathy 118 1.031 0.1291 21.49 <0.001* With diabetic retinopathy 197 0.662 0.1572 *-Significant The independent ‘t’ test results shows that there is a significant difference in mean of total serum bilirubin with the presence of diabetic retinopathy (t value=21.49, P<0.001). The mean of total serum bilirubin is significantly higher in cases with no diabetic retinopathy (p<0.001). Figure 1: Relation between presence of diabetic retinopathy and total serum bilirubin Table 10: Relation between presence of diabetic retinopathy and total serum bilirubin Presence of diabetic retinopathy Mean SD F Value P value No diabetic retinopathy 1.031 0.1291 411.391 <0.001* Non proliferative diabetic retinopathy 0.731 0.1211 Proliferative diabetic retinopathy 0.503 0.1073 *-Significant The one way ANOVA result shows that there is a significant difference in mean of total serum bilirubin with respect to the presence of diabetic retinopathy (F value=411.391, p<0.001). The post HOC test shows that patients with proliferative diabetic retinopathy has significantly lower total serum bilirubin when comparing to other groups (p=<0.001). Figure 2: Relation between presence of diabetic retinopathy and total serum bilirubin Table 11: Correlation of stage of Diabetic Retinopathy and Total serum Bilirubin Type N Mean Variance Std. Deviation No DR 118 1.031 .017 .1291 Mild NDPR 56 .814 .010 .0999 Moderate NDPR 49 .692 .005 .0672 Severe NDPR 32 .647 .017 .1319 PDR 60 .503 .012 .1073 Total 315 .800 .054 .2314 F Value = 253.813, P Value < 0.001, Significant The one way ANOVA result shows that there is a significant difference in mean of total serum bilirubin with respect to the presence of diabetic retinopathy (F value=411.391, p<0.001). Table 12: Type of Retinopathy and Direct Bilirubin Type N Mean Variance Std. Deviation No DR 118 .238 .007 .0836 Mild NDPR 56 .193 .004 .0657 Moderate NDPR 49 .171 .004 .0645 Severe NDPR 32 .153 .005 .0671 PDR 60 .100 .000 .0000 Total 315 .185 .007 .0838 F Value = 45.084, P Value < 0.001, Significant Table 13: Type of Retinopathy and Indrect bilirubin. Type N Mean Variance Std. Deviation No DR 118 .802 .016 .1267 Mild NDPR 56 .630 .011 .1060 Moderate NDPR 49 .520 .010 .1000 Severe NDPR 32 .494 .025 .1585 PDR 60 .395 .013 .1126 Total 315 .619 .040 .1990 F Value = 136.503, P Value < 0.001, Significant Table 14: Correlation between total serum bilirubin and direct bilirubin Parameters Mean SD Pearson’s Correlation Coefficient P Value Total serum bilirubin 0.8 0.2314 0.574 <0.001* Direct bilirubin 0.185 0.0838 The mean and SD of total serum bilirubin is 0.8 and 0.2314 respectively. Also the mean and SD of direct bilirubin is 0.185 and 0.0838 respectively. The pearson’s correlation coefficient is 0.574. It shows there that there is a moderate positive correlation between total serum bilirubin and direct bilirubin. The result is a significant since the p value is greater than the significance level (α=0.05). Table 15: Regression Analysis Results R Squared Value F Value P Value 0.33 154.176 <0.001* *-Significant The R squared value is 0.33. It shows that the regression model is account for 33% of variance. The F value is 154.176. Since the p value is lesser than the significance level (α=0.05), we concluded that our regression model can significantly predict the total serum bilirubin. Regression Equation is Total serum bilirubin = 0.507 + 1.586 * (Direct bilirubin) It shows that for every unit increase of direct bilirubin, total serum bilirubin is increased an average unit of 1.586 times. Table 16: Correlation between total serum bilirubin and indirect bilirubin Parameters Mean SD Pearson’s Correlation Coefficient P Value Total serum bilirubin 0.8 0.2314 0.931 <0.001* Indirect bilirubin 0.619 0.199 *-Significant The mean and SD of total serum bilirubin is 0.8 and 0.2314 respectively. Also the mean and SD of indirect bilirubin is 0.619 and 0.199 respectively. The pearson’s correlation coefficient is 0.931. It shows there that there is a very strong positive correlation between total serum bilirubin and indirect bilirubin. The result is significant since the p value is lesser than the significance level (α=0.05). Table 17: Regression Analysis Results R Squared Value F Value P Value 0.866 2020.862 <0.001* *-Significant The R squared value is 0.866. It shows that the regression model is account for 86.6% of variance. The F value is 2020.862. Since the p value is lesser than the significance level (α=0.05), we concluded that our regression model can significantly predict the total serum bilirubin. Regression Equation is Total serum bilirubin = 0.131 + 1.082 * (Indirect bilirubin) It shows that for every unit increase of indirect bilirubin, total serum bilirubin is increased an average unit of 1.082 times. The shows that there is a very strong positive correlation between total serum bilirubin and indirect bilirubin. It means if indirect bilirubin increases, total serum bilirubin will also increase. If in direct bilirubin decreases, total serum bilirubin will also decrease.

One of the most common ocular morbidity and leading cause of adult blindness in world is diabetic retinopathy [2]. The metabolic end product of heme degradation is Bilirubin which is considered toxic [27]. But some studies have shown bilirubin having anti atherosclerotic properties, anti-oxidant and anti-inflammatory properties [7, 28, 29, 30]. Pathophysiological inhibition of cytokines, inhibition of oxidation, inhibition of monocyte migration, reduced proliferation of smooth cells and preventing thombus formation have been implied [28, 31, 32, 33, 34]. Oxidative stress has been implied in vascular complications of Diabetes. 35 Higher serum levels of bilirubin are inversely associated with the incidence of vascular diseases, such as coronary arterial disease, peripheral arterial disease etc. through properties of bilirubin [7, 36, 38]. Elevated serum bilirubin within the normal range have been linked to provide protection from thromboembolic events [39, 40]. In the present study, 315 patients with diabetes mellitus were studied. According to the presence or absence of diabetic retinopathy. Patients having retinopathy were further categorised into without diabetic retinopathy (NO DR). Non Proliferative Diabetic Retinopathy/NPDR (mild, moderate, severe NPDR) and PDR/Proliferative Diabetic Retinopathy depending on the severity of retinopathy. DEMOGRAPHIC PROFILE In the present study, age of the patients ranged from 39-90 years. Most of them (70%) belonged to the age group 50-70 years. The mean and standard deviation of Age in years is 62.127±8.868. The median Age in years is 62. Several epidemiologic studies like CURES Eye study [21] and The United Kingdom Prospective Diabetes Study (UKPDS) [31] had showed significant correlation between age and DR. Present study had a slight male predominance. Among 315 subjects, 182(57.8%) were males and 133(42.2%) were females. Similar male preponderance was seen in epidemiological studies like CURES Eye study [21], UKPDS study [31] and The Andra Pradesh Eye Disease study (APEDS) [69]. SYSTEMIC PRAMETERS In the present study, mean values of total, direct and indirect bilirubin levels were significantly (p =0.000) lower in patients with diabetic retinopathy compared to those without retinopathy. The independent ‘t’ test results shows that there is a significant difference in mean of total serum bilirubin with the presence of diabetic retinopathy (t value=21.371, P<0.001). The mean of total serum bilirubin is significantly higher in cases with no diabetic retinopathy (p<0.001). The one way ANOVA result shows that there is a significant difference in mean of total serum bilirubin with respect to the presence of diabetic retinopathy (F value = 420.678, p<0.001). The post HOC test shows that patients with proliferative diabetic retinopathy has significantly lower total serum bilirubin when comparing to other groups (p=<0.001). In Indian population, studies were assessed to correlate between bilirubin levels and DR. The study done by Apoorva Dave et al. showed that all the three parameters-namely, total, direct and indirect bilirubin levels were lower in patients with retinopathy [13]. Results of the current study were in concordance with the above study. In South India, Divya Karuppannasamy et al., investigated the association between serum bilirubin concentration and DR in patients with Type 2 Diabetes Mellitus. Total and direct bilirubin concentrations were found to be higher in patients without DR [68]. These results were in concurrence with the present study. In the present study, subjects were assigned to quartiles based on serum total bilirubin concentration (<0.3, 0.31-0.5, 0.51. 0.7 and above 0.70mg/dl). The prevalence of diabetic retinopathy was significantly lower among persons with higher bilirubin quartile compared to those with the lowest quartile (p=0.00). In the group of patients with higher total bilirubin level (above 0.7 mg/dl), percentage of patients having diabetic retinopathy was less, while in the group with lower total bilirubin level (<0.3mg/dl), 45.20% had retinopathy. Statistical significant, p = 0.000. This was in accordance with results of previous studies, which were done by Apoorva Dave et al., Divya Karuppannasamy et al., and the Hisayama study. Prevalence of Diabetic retinopathy increased from 0.00% to 92.90% as direct bilirubin level decreased from 0.5mg/dl to 0.1-0.2mg/dl (p = 0.000). Prevalence of diabetic retinopathy in persons with highest indirect bilirubin levels (>0.4mg/dl) was 0.00% compared with the prevalence of 9.1%, 31.00% and 59.50% in those with lower levels, 0.3-0.4mg/dl, 0.2-0.3mg/dl, and 0.1-0.2mg/dl respectively and this was statistically significant. (p = 0.007). Most studies on serum bilirubin and DR had focused only on total bilirubin without separating the bilirubin types. Limited studies have shown a differentially protective effect of direct bilirubin on metabolic syndrome and chronic kidney disease. Further studies on the relation between bilirubin subtypes and DR are clearly needed. In the present study, we investigated the correlation between serum bilirubin levels and severity of DR and found a significant association between these two variables. Severity of diabetic retinopathy was more in patients with lower levels of total, direct and indirect bilirubin (p= 0.000, 0.000 and 0.001respectively). Levels of total, direct and indirect bilirubin were significantly lower in patients with late stages of retinopathy. The studies conducted by Apoorva Dave et al. and Divya Karuppannasamy et al. also found that the severity of diabetic retinopathy was inversely proportional to the total bilirubin levels. SHOWING COMPARISON OF OUR STUDY WITH VARIOUS OTHER STUDIES ON SERUM BILIRUBIN AND DIABETIC RETINOPATHY Author Ethnicity Number of cases Number of controls Mean bilirubin value in without diabetic Retinopathyin mg/dl Mean bilirubin value with diabetic retinopathyin mg/dl Our study South India 315 0 1.031 o.662 Tahir et al. Pakistan 76 76 0.610 0.73 Dave A et al. South India 80 40 1.361 0.785 Najam et al. China 1761 0 >0.99 <0.99 Ho Chan Cho et al. Korea 102 0 13.7(in mol) 10.3(in mol) Divya karrupuswamy et al. South India 86 30 0.65 0.47 In the present study, the correla¬tion between levels of serum total bilirubin and diabetic reti-nopathy as a chronic microvascular complication was investi¬gated. The results showed an inverse association of bilirubin level with retinopathy, which indicates that bilirubin may be an important biomarker of diabetic reti¬nopathy as an endogenous protectant from microvascular com¬plications due to antioxidant and anti-inflammatory properties. Further studies on the relation between bilirubin subtypes and severity of DR are needed.

The study showed that the Mean values of total, direct and indirect bilirubin levels were significantly lower in patients with diabetic retinopathy compared to those without retinopathic changes. Levels of total, direct and indirect bilirubin were significantly lower in severe stages of DR. Severity of diabetic retinopathy was inversely proportional to the total, direct and indirect serum bilirubin levels. RECOMMENDATIONS The results showed an inverse association of bilirubin level with retinopathy, which indicates that bilirubin may be an important biomarker of diabetic reti¬nopathy. Further studies on the relation between bilirubin subtypes and severity of DR are needed. LIMITATIONS  We considered patients from uniform ethnic group (Malnad region only), so the effect of race and ethnicity could not be tested.  Our study was hospital-based and not population-based, hence it only depicts the tip of the ice-berg. The large submerged portion which represents the actual number of patients in the community harbouring Diabetic Retinopathy are not analyzed for the correlation.  In this study, cross-sectional design of the study prevented determination of a temporal association or the cause-effect relationship between serum bilirubin levels and the development of retinopathy.  Only long term prospective studies in larger population can reveal whether serum bilirubin predicts the development of Diabetic Retinopathy.  We performed only a single measurement of serum bilirubin which might have been within subject variation.  Test for anti-hepatitis C virus or USG on the hepatobiliary system, which may determine the bilirubin level, were not conducted.

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