{"id":58248,"date":"2024-06-25T10:08:09","date_gmt":"2024-06-25T10:08:09","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=58248"},"modified":"2024-07-03T18:18:27","modified_gmt":"2024-07-03T18:18:27","slug":"serum-total-bilirubin-and-oxidative-stress-status-in-diabetic-retinopathy-a-hospital-based-observational-study","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/serum-total-bilirubin-and-oxidative-stress-status-in-diabetic-retinopathy-a-hospital-based-observational-study\/","title":{"rendered":"Serum Total Bilirubin and Oxidative Stress Status in Diabetic Retinopathy \u2013A Hospital-Based Observational Study"},"content":{"rendered":"\n

Introduction <\/strong><\/p>\n\n\n\n

Type\n2 Diabetes mellitus (T2DM) is characterized by hyperglycemia due to defects in\ninsulin secretion, action, or both. Global data of diabetes prevalence in 2021\nin the age group of 20\u201379 years is estimated at around 10.5% (536.6 million\npeople), with an expected rise to 12.2% (783.2 million) by 2045. The Prevalence\nof Diabetes in either of the genders was highest in the age group of 75\u201379\nyears1<\/sup>. Data from 2021 has predicted that there may be a\npreponderance in middle-income countries of 21.1% compared to high and\nlow-income countries of 11.9% and 12.2%, respectively, by 20451<\/sup>.\nAccording to 2019 estimates, the number of diabetics in India could double\napproximately from 77 million to 134 million by 20452<\/sup>.<\/p>\n\n\n\n

Among\nthe major microvascular complications of diabetes, Diabetic retinopathy (DR) is\nthe cause of vision loss among adults who are in the earning age group. A\ncombined survey of the R. P. Center for Ophthalmic Sciences, National Diabetic\nRetinopathy Rapid Assessment of Avoidable Blindness (RAAB) Survey, and the\nMinistry of Health and Family Welfare, Government of India between 2015- 2019\nhas predicted the prevalence of DR to be 16.9%3<\/sup>.<\/p>\n\n\n\n

One\nof the crucial factors in the development of DR is Oxidative Stress (OS).\nChronicity of hyperglycemia plays a pivotal role in the formation of reactive\noxygen species (ROS), activation of the polyol, protein kinase C (PKC), and\noveractivity of the hexosamine pathways. Oxidative stress results in\ninflammation, mitochondrial dysfunction, pyroptosis, apoptosis, or autophagy.\nThe consequential effect of oxidative stress in conjunction with\nneurodegeneration leads to neural, vascular, and retinal tissue damage. DR is a\nconsequence of the synthesis of Advanced Glycation End products (AGEs) and\nexpression of Receptors for Advanced Glycation End products (RAGEs), which\ngenerate free radicals with sequential oxidative tissue damage and glutathione\n(GSH) depletion4<\/sup>. Malondialdehyde (MDA), a marker of lipid\nperoxidation, affects the cell membrane phospholipids and correlates well with\nhigher oxidative stress5<\/sup>.<\/p>\n\n\n\n

The\nUnited States National Health and Nutrition Examination Survey (NHANES) data of\n1999-2006 on sixteen thousand subjects documented the upper bilirubin range in\nthe Biological reference interval. This elevation observed is beneficial to the\nsubjects, with a 26% reduction in the risk of developing T2DM6<\/sup>. Once\nconsidered a biological waste product of heme catabolism, bilirubin has been\nrecognized as a potential endogenous antioxidant under physiological\nconditions. Bilirubin is been documented to have anti-inflammatory activity on\nthe vasculature7<\/sup>.<\/p>\n\n\n\n

A\nstudy has reported that T2DM patients with higher serum bilirubin, however,\nwithin the biological reference interval, will have a lower risk of developing\nretinopathy8<\/sup>. These factors made us study the role of oxidative\nstress in the pathogenesis of DR and its association with the antioxidant\neffect of bilirubin.<\/p>\n\n\n\n

Materials and Methods <\/strong><\/p>\n\n\n\n

Study Design<\/strong><\/p>\n\n\n\n

A\nHospital-based case-control study was conducted from 2015 to 2018 at R L\nJalappa Hospital and Research Centre, a tertiary care rural referral hospital\nattached to Sri Devaraj Urs Medical College, affiliated to Sri Devaraj Urs\nAcademy of Higher Education and Research, Kolar.<\/p>\n\n\n\n

The\ncentral ethical committee of SDUAHER, Kolar, approved the study, Ref. No.:\nSDUAHER \/ Res. Project \/ 89 \/2013-14. Written informed consent obtained from\nall study subjects. All the parameters analyzed at the Central Diagnostic\nLaboratory Services’ biochemistry section at RLJH and RC.<\/p>\n\n\n\n

A\ntotal of two hundred eighty-eight subjects of either gender, in the age group\nof 30-70 years were enrolled. Subjects, divided into three groups. Group\nI: 96 clinically proven healthy individuals, Group II: 96 clinically proven\nT2DM subjects without retinopathy (T2DM), and Group III: 96 clinically proven\ncases of T2DM with retinopathy (DR) of all stages. Group III categorization\nbased on fundoscopy changes. Factors that affect or alter the cases or controls\nwere excluded from the study.<\/p>\n\n\n\n

Sample collection<\/strong><\/p>\n\n\n\n

After\nthe individuals had fasted for eight hours the previous night, 5 mL of blood\nwas extracted from the median cubital vein with complete aseptic precautions in\nthe supine position. Precautions were taken to prevent sample hemolysis. The\nstandard sample collection protocol prevented the factors affecting the\nparameters, such as bilirubin. The samples centrifuged for 10 minutes at 3000\nrpm. The supernatant was separated, and carried out the analysis. <\/p>\n\n\n\n

Methods<\/strong><\/p>\n\n\n\n

Plasma\nglucose estimated by glucose oxidase peroxidase method9<\/sup>, serum urea\nby urease method,10<\/sup> serum creatinine by enzymatic\ncreatinine-amidohydrolase method10<\/sup>, total cholesterol(TC) by\ncholesterol oxidase peroxidase method11<\/sup>, triglycerides(TG) by\nenzymatic colorimetric test GPO-PAP12<\/sup>,high-density lipoproteins\ncholesterol(HDL-C) by phosphotungstic acid enzymatic method13<\/sup>,total\nBilirubin and direct bilirubin by azobilirubin and duly wavelength\nspectrophotometric method14<\/sup> and other liver function test by\nstandard methods15<\/sup> using Vitros 5.1 FS, Ortho Clinical Diagnostic\ndry chemistry analyzer instrumentation, based on reflectance photometry.\nLow-density lipoprotein cholesterol (LDL-C) was calculated using Friedewald’s\nequation, considering its limitations. HbA1c was analyzed by the HPLC method\nusing a Bio-Rad D10 analyzer (Biorad, Hemel Hempstead, UK) as a Laboratory\nreference method. Erythrocyte-reduced glutathione was assayed by\nspectrophotometer using chromogen 5,5′- di thiobis 2-nitrobenzoic acid (DTNB)16\n<\/sup>and MDA assayed by thiobarbituric acid reactive substances (TBARS) method17<\/sup>.<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

Data was analyzed using the licensed version of Statistical Product and Service Solutions (SPSS) software version 22 for statistical significance. Results were expressed as mean \u00b1 standard deviation. ANOVA test was used to determine significance, and posthoc Bonferroni was used to validate results. The p-value of <0.05 was considered statistically significant. Pearson’s correlation was performed for the association of serum total bilirubin and oxidative stress markers in DR subjects.<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

In\nthis study, 288 subjects enrolled, and there was male preponderance in all\nthree groups (62.5%, 64.6%, 57.3%) compared to female subjects (37.5%, 35.4%,\n42.7%).<\/p>\n\n\n\n

The mean age of the subjects and duration of diabetes are shown in Table 1. Duration of diabetes was significantly higher in DR patients compared to T2DM patients (p<0.001).<\/p>\n\n\n\n

Table 1: Mean age and Duration of diabetes of study groups<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Group I<\/strong><\/p>\n

No.=96<\/strong><\/p>\n<\/td>\n

\n

Group II<\/strong><\/p>\n

No.=96<\/strong><\/p>\n<\/td>\n

\n

Group III<\/strong><\/p>\n

No.=96<\/strong><\/p>\n<\/td>\n

\n

p- value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Mean age (years)<\/p>\n<\/td>\n

\n

52.31 \u00b1 12.09<\/p>\n<\/td>\n

\n

56.36 \u00b1 8.65<\/p>\n<\/td>\n

\n

57.12 \u00b1 7.33<\/p>\n<\/td>\n

\n

>0.05<\/p>\n<\/td>\n<\/tr>\n

\n

Duration of DM (years)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

5.31 \u00b1 0.97<\/p>\n<\/td>\n

\n

12.79 \u00b1 3.92<\/p>\n<\/td>\n

\n

< 0.001*<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are expressed as Mean + SD. *p value < 0.001 is highly significant.<\/p>\n

Group I (Controls), Group II (T2DM) and Group III (DR).<\/p>\n\n\n

Out\nof 96 DR subjects considered for the study, 36 had mild Non-Proliferative\nDiabetic Retinopathy (Mild NPDR), 29 had moderate Non-Proliferative Diabetic\nRetinopathy (Moderate NPDR), 17 had severe Non-Proliferative Diabetic\nRetinopathy (Severe NPDR), and 14 had Proliferative Diabetic Retinopathy (PDR).\nBased on the serum total bilirubin levels, the study groups were divided into\nfour quartiles18<\/sup>. I quartile (< 0.45), II quartile (0.46 -0.55),\nIII quartile (0.56- 0.65), and IV quartile (> 0.66) as represented in Table\n2, with the first quartile representing the lowest and the fourth quartile\ndenoting the highest.<\/p>\n\n\n\n

We\nobserved the highest percentage of PDR cases in the I quartile, moderate and\nsevere NPDR cases in the II quartile, mild NPDR cases in the III quartile, and\nT2DM and control subjects in the IV quartile, indicating an inverse relation of\nserum total bilirubin levels with severity of DR (p<0.001).<\/p>\n\n\n\n

Table\n3 depicts the biochemical and oxidant-antioxidant parameters of the study\ngroups. Serum total bilirubin levels significantly decreased in DR cases\ncompared to control and T2DM subjects (p<0.001). Results on the levels of\nGSH showed a significant decrease in T2DM and DR patients compared to the\ncontrol group (p<0.001). However, significantly increased serum MDA levels\nand GGT in the T2DM and DR groups compared to the control (p<0.001). We did\nnot observe significant differences between the three groups, comparing urea,\ncreatinine, SGPT, SGOT, total proteins, albumin, and alkaline phosphatase.<\/p>\n\n\n\n

Post\nHoc analysis using Bonferroni correction for significance indicates that Group\nI Vs. Group II, Group II Vs. Group III and Group I Vs. Group III showed\nincreased FBS, HbA1c, GGT, and MDA levels and decreased GSH levels with highly\nsignificant p<0.001. Total Bilirubin in Group I Vs. Group II did not show\nany significance; however, the increase in Group II Vs. Group III and Group I\nVs. Group III was highly significant with p<0.001. Increased total\ncholesterol, TG, and LDL showed a highly significant p-value <0.001 in Group\nI Vs. Group II and Group I Vs. Group III, however, increased levels did not\nshow any significance (p=0.355, p=0.336, p=0.300) in Group II Vs. Group III.<\/p>\n\n\n\n

Tables\n4 and 5 depict the correlation analysis of serum total bilirubin in T2DM (Group\nII) and DR (Group III), which showed a significant positive correlation with\nGSH and a significant negative correlation with FBS, HbA1C, and MDA. On the\nother hand, there was no significant correlation of serum total bilirubin with\nlipid profile parameters. However, no significant correlation was observed\nbetween the duration of diabetes mellitus with fasting blood sugars, glycated\nhemoglobin, liver function test, lipid profile parameters, GSH, and MDA levels\nin T2DM and DR cases.<\/p>\n\n\n\n

DR is caused by microangiopathy, leading to microvascular leakage and occlusion of the retinal veins, arteries, and capillaries. Prolonged hyperglycemia, dyslipidemia, aging, and oxidative stress are major risk factors associated with the progression of retinopathy in diabetic patients19<\/sup>. The present study demonstrated a male preponderance of 57.3% versus 42.7% females for early development of DR. Our findings are consistent with a study by Cherchi and his coworkers20<\/sup>. However, a previous study reported female predominance21<\/sup> and Yau and his team documented equal distribution across both genders22<\/sup>.<\/p>\n\n\n\n

Our\nstudy showed an increase in the prevalence of DR correlating positively with\nthe disease duration. These findings are consistent with the previous study23<\/sup>.\nWe observed the mean duration of diabetes at 5.31 years and retinopathy following\ndiabetes at 12.79 years (Table 1). This finding implies the importance of\nregular fundus examinations and tight diabetic control in T2DM.<\/p>\n\n\n\n

The frequency quartile distribution of DR subjects documented in Table 2 predicted that 64.3% of PDR cases were in the I quartile, with severe NPDR of 70.6 % and 100% of moderate NPDR in the II quartile. A mild NPDR of 83.3% was observed in the III quartile. We observed serum bilirubin values of 93.8% in the IV quartile in T2DM cases. The IV quartile values correlate well with the control group, indicating strict diabetes control shall enable delay in developing either NPDR or PDR or both. Observed findings are on par with previous studies which demonstrated that serum total bilirubin levels are inversely proportional to the severity of DR24,25<\/sup>.<\/p>\n\n\n\n

Table 2: Prevalence of DR by quartiles of serum concentration of total bilirubin<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Cases and Controls<\/strong><\/p>\n<\/td>\n

\n

quartiles based on serum total bilirubin(mg\/dL)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n<\/td>\n

\n

I quartile<\/strong><\/p>\n<\/td>\n

\n

II quartile<\/strong><\/p>\n<\/td>\n

\n

III quartile<\/strong><\/p>\n<\/td>\n

\n

IV quartile<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

<<\/u> 0.45(mg\/dL)<\/p>\n<\/td>\n

\n

0.46 \u2013 0.55(mg\/dL)<\/p>\n<\/td>\n

\n

0.56-0.65(mg\/dL)<\/p>\n<\/td>\n

\n

> <\/u>0.66(mg\/dL)<\/p>\n<\/td>\n<\/tr>\n

\n

No.<\/strong><\/p>\n<\/td>\n

\n

%<\/strong><\/p>\n<\/td>\n

\n

No.<\/strong><\/p>\n<\/td>\n

\n

%<\/strong><\/p>\n<\/td>\n

\n

No.<\/strong><\/p>\n<\/td>\n

\n

%<\/strong><\/p>\n<\/td>\n

\n

No.<\/strong><\/p>\n<\/td>\n

\n

%<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Controls (96)<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

95<\/p>\n<\/td>\n

\n

99<\/p>\n<\/td>\n<\/tr>\n

\n

T2DM (96)<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

6.2<\/p>\n<\/td>\n

\n

90<\/p>\n<\/td>\n

\n

93.8<\/p>\n<\/td>\n<\/tr>\n

\n

Mild NPDR(36)<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

13.9<\/p>\n<\/td>\n

\n

30<\/p>\n<\/td>\n

\n

83.3<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

2.8<\/p>\n<\/td>\n<\/tr>\n

\n

Moderate NPDR(29)<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

29<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Severe NPDR(17)<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

29.4<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

70.6<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

PDR(14)<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

64.3<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

35.7<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

T2DM: Type 2 Diabetes Mellitus; NPDR: Non Proliferative Diabetic Retinopathy;                     <\/p>\n

PDR: Proliferative Diabetic Retinopathy<\/p>\n\n\n

The present study showed a significant increase in FBS and HbA1c levels in Group III and Group II compared to Group I (Table 3) and is similar to the conducted study by Hadeel in 202026<\/sup>. In T2DM, the early development and progression of micro and macrovascular complications is mainly due to chronic hyperglycemia. HbA1c has a unique affinity for oxygen, leading to tissue anoxia, and plays a vital role in causing micro and macroangiopathy27<\/sup>.<\/p>\n\n\n\n

Table 3: ANOVA comparing HbA1c, FBS, Renal function test, Liver function test, Lipid profile, GSH and MDA in Group I (Controls), Group II (T2DM) and Group III (DR)<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Parameters<\/strong><\/p>\n<\/td>\n

\n

Group  I<\/strong><\/p>\n<\/td>\n

\n

Group II<\/strong><\/p>\n<\/td>\n

\n

Group III<\/strong><\/p>\n<\/td>\n

\n

 P value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

FBS (mg\/dL)<\/p>\n<\/td>\n

\n

89.01 +<\/u> 9.81<\/p>\n<\/td>\n

\n

152.32 +<\/u>29.51<\/p>\n<\/td>\n

\n

206.34 +<\/u>42.46<\/p>\n<\/td>\n

\n

p<0.001*a, b, c<\/p>\n<\/td>\n<\/tr>\n

\n

HbA1c %<\/p>\n<\/td>\n

\n

5.49+<\/u>0.58<\/p>\n<\/td>\n

\n

8.37+<\/u>1.16<\/p>\n<\/td>\n

\n

10.68+<\/u>1.80<\/p>\n<\/td>\n

\n

p<0.001* a, b, c<\/p>\n<\/td>\n<\/tr>\n

\n

Total Bilirubin (mg\/dL)<\/p>\n<\/td>\n

\n

1.02+<\/u>0.20<\/p>\n<\/td>\n

\n

1.00+<\/u>0.01<\/p>\n<\/td>\n

\n

0.63+<\/u>0.48<\/p>\n<\/td>\n

\n

p<0.001* b, c<\/p>\n<\/td>\n<\/tr>\n

\n

Direct Bilirubin (mg\/dL)<\/p>\n<\/td>\n

\n

0.2+<\/u>0.02<\/p>\n<\/td>\n

\n

0.12+<\/u>0.01<\/p>\n<\/td>\n

\n

0.02+<\/u>0.01<\/p>\n<\/td>\n

\n

p=0.062<\/p>\n<\/td>\n<\/tr>\n

\n

GSH(mg\/Gm of Hb)<\/p>\n<\/td>\n

\n

16.14+<\/u>0.90<\/p>\n<\/td>\n

\n

9.07+<\/u>1.13<\/p>\n<\/td>\n

\n

5.97+<\/u>1.14<\/p>\n<\/td>\n

\n

p<0.001* a, b, c<\/p>\n<\/td>\n<\/tr>\n

\n

MDA(nmol\/mL)<\/p>\n<\/td>\n

\n

1.90+0.57<\/p>\n<\/td>\n

\n

6.43 +<\/u> 1.72<\/p>\n<\/td>\n

\n

10.88 + <\/u>1.36<\/p>\n<\/td>\n

\n

p<0.001* a, b, c<\/p>\n<\/td>\n<\/tr>\n

\n

Total Cholesterol(TC) (mg\/dL)<\/p>\n<\/td>\n

\n

160.89+<\/u>20.27<\/p>\n<\/td>\n

\n

192.59 +<\/u> 25.99<\/p>\n<\/td>\n

\n

199.80 +<\/u> 29.47<\/p>\n<\/td>\n

\n

p<0.001* a, c<\/p>\n<\/td>\n<\/tr>\n

\n

Triglycerides(TG)  (mg\/dl)<\/p>\n<\/td>\n

\n

136.72+<\/u>27.59<\/p>\n<\/td>\n

\n

222.81+<\/u>45.40<\/p>\n<\/td>\n

\n

239.22+<\/u>57.17<\/p>\n<\/td>\n

\n

p<0.001* a, c<\/p>\n<\/td>\n<\/tr>\n

\n

HDL – C(mg\/dL)<\/p>\n<\/td>\n

\n

40.49+<\/u>7.44<\/p>\n<\/td>\n

\n

40.15+<\/u>7.04<\/p>\n<\/td>\n

\n

39.63+<\/u>7.41<\/p>\n<\/td>\n

\n

p=0.717<\/p>\n<\/td>\n<\/tr>\n

\n

LDL -C(mg\/dL)<\/p>\n<\/td>\n

\n

95.00+<\/u>21.39<\/p>\n<\/td>\n

\n

114.32+<\/u>22.70<\/p>\n<\/td>\n

\n

108.27+<\/u>31.06<\/p>\n<\/td>\n

\n

p<0.001* a, c<\/p>\n<\/td>\n<\/tr>\n

\n

AST\/ SGOT (IU\/L)<\/p>\n<\/td>\n

\n

28.75+<\/u>7.83<\/p>\n<\/td>\n

\n

27.05+<\/u>7.0<\/p>\n<\/td>\n

\n

25.04+<\/u>5.94<\/p>\n<\/td>\n

\n

p=0.071<\/p>\n<\/td>\n<\/tr>\n

\n

ALT \/SGPT(IU\/L)<\/p>\n<\/td>\n

\n

31.69+<\/u>7.45<\/p>\n<\/td>\n

\n

30.05+<\/u>6.72<\/p>\n<\/td>\n

\n

30.96+<\/u>6.82<\/p>\n<\/td>\n

\n

p=0.271<\/p>\n<\/td>\n<\/tr>\n

\n

ALP(IU\/L)<\/p>\n<\/td>\n

\n

160.01+<\/u>30.82<\/p>\n<\/td>\n

\n

159.96+<\/u>34.59<\/p>\n<\/td>\n

\n

165.20 + <\/u>54.93<\/p>\n<\/td>\n

\n

p=0.603<\/p>\n<\/td>\n<\/tr>\n

\n

Total protein (g\/dL)<\/p>\n<\/td>\n

\n

7.27+<\/u>0.73<\/p>\n<\/td>\n

\n

7.28+<\/u>0.73<\/p>\n<\/td>\n

\n

7.36+<\/u>0.79<\/p>\n<\/td>\n

\n

p=0.642<\/p>\n<\/td>\n<\/tr>\n

\n

Albumin (g\/dL)<\/p>\n<\/td>\n

\n

4.33+<\/u>0.51<\/p>\n<\/td>\n

\n

4.44+<\/u>0.61<\/p>\n<\/td>\n

\n

4.81+<\/u>0.60<\/p>\n<\/td>\n

\n

p=0.081<\/p>\n<\/td>\n<\/tr>\n

\n

Globulin (g\/dL)<\/p>\n<\/td>\n

\n

2.86+<\/u>0.57<\/p>\n<\/td>\n

\n

2.86+<\/u>0.4<\/p>\n<\/td>\n

\n

2.61+<\/u>0.48<\/p>\n<\/td>\n

\n

p=0.073<\/p>\n<\/td>\n<\/tr>\n

\n

A\/G ratio<\/p>\n<\/td>\n

\n

1.56+<\/u>0.32<\/p>\n<\/td>\n

\n

1.58+<\/u>0.26<\/p>\n<\/td>\n

\n

1.82+<\/u>0.33<\/p>\n<\/td>\n

\n

p=0.062<\/p>\n<\/td>\n<\/tr>\n

\n

GGT (IU\/L)<\/p>\n<\/td>\n

\n

24.01+<\/u>7.79<\/p>\n<\/td>\n

\n

40.60+<\/u>6.20<\/p>\n<\/td>\n

\n

51.33+<\/u>6.66<\/p>\n<\/td>\n

\n

p<0.001* a, b, c<\/p>\n<\/td>\n<\/tr>\n

\n

Blood Urea (mg\/dL)<\/p>\n<\/td>\n

\n

22.43+<\/u>7.66<\/p>\n<\/td>\n

\n

23.41+<\/u>8.00<\/p>\n<\/td>\n

\n

21.44+<\/u>6.85<\/p>\n<\/td>\n

\n

p=0.195<\/p>\n<\/td>\n<\/tr>\n

\n

Serum Creatinine (mg\/dL)<\/p>\n<\/td>\n

\n

0.83+<\/u>0.25<\/p>\n<\/td>\n

\n

0.90+<\/u>0.19<\/p>\n<\/td>\n

\n

0.90+<\/u>0.22<\/p>\n<\/td>\n

\n

p=0.711<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are expressed as Mean + SD. *p-value <0.001 is highly significant: <\/p>\n

a for Group I Vs. Group II, b for Group II Vs. Group III and c for Group III Vs. Group I.<\/p>\n

FBS: Fasting blood sugar; HbA1c: Glycated haemoglobin; MDA: Malondialdehyde; GSH: Glutathione; HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein cholesterol; AST: Aspartate transaminase; ALT: Alanine transaminase; ALP: Alkaline phosphatase; GGT: Gamma glutamyl transferase<\/p>\n

<\/p>\n\n\n

Bilirubin, intended as a toxic substance, is an end product of heme breakdown. Studies have demonstrated that a higher total bilirubin level within the biological reference interval protects against cardiovascular diseases, stroke, and peripheral vascular disease28,29<\/sup>. Our results of the serum total bilirubin revealed significantly decreased levels in DR subjects compared to T2DM subjects, which concords with the study by Yasuda and coworkers and our previous in-house study30,8<\/sup>. A study conducted in Netherlands population demonstrated an increase in serum total bilirubin level interrupts the pathways leading to the progression of DR by inhibiting inflammation processes and oxidative stress31<\/sup>. Possible mechanisms of the protective role of bilirubin may be through its cytoprotective, anti-inflammatory, and antioxidant action on retinal vasculature31<\/sup>.<\/p>\n\n\n\n

Table 4: Correlation of serum total bilirubin with FBS, HbA1c, GGT, GSH, MDA and Lipid Profile in T2DM (Group II)<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

 <\/em><\/strong><\/p>\n<\/td>\n

\n

FBS<\/strong><\/p>\n<\/td>\n

\n

HbA1c<\/strong><\/p>\n<\/td>\n

\n

GGT<\/strong><\/p>\n<\/td>\n

\n

GSH<\/strong><\/p>\n<\/td>\n

\n

MDA<\/strong><\/p>\n<\/td>\n

\n

TC<\/strong><\/p>\n<\/td>\n

\n

TG<\/strong><\/p>\n<\/td>\n

\n

HDL-C<\/strong><\/p>\n<\/td>\n

\n

LDL-C<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

serum total bilirubin<\/p>\n<\/td>\n

\n

Pearson Correlation<\/p>\n

(r value)<\/p>\n<\/td>\n

\n

-0.338<\/p>\n<\/td>\n

\n

-0.533<\/p>\n<\/td>\n

\n

0.221<\/p>\n<\/td>\n

\n

0.130<\/p>\n<\/td>\n

\n

-0.308<\/p>\n<\/td>\n

\n

0.130<\/p>\n<\/td>\n

\n

-0.154<\/p>\n<\/td>\n

\n

-0.154<\/p>\n<\/td>\n

\n

0.130<\/p>\n<\/td>\n<\/tr>\n

\n

p- value<\/p>\n

Sig.(2-tailed)<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n

\n

0.031<\/p>\n<\/td>\n

\n

0.206<\/p>\n<\/td>\n

\n

0.002<\/p>\n<\/td>\n

\n

0.206<\/p>\n<\/td>\n

\n

0.134<\/p>\n<\/td>\n

\n

0.134<\/p>\n<\/td>\n

\n

0.206<\/p>\n<\/td>\n<\/tr>\n

\n

No.<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*Correlation is highly significant at p value 0.001 level<\/p>\n

FBS: Fasting blood sugar; HbA1c: Glycated haemoglobin; GGT: Gamma glutamyl transferase; GSH: Glutathione; MDA: Malondialdehyde; TC: Total Cholesterol; TG: Triglycerides; HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein cholesterol<\/p>\n\n\n

Vital factors considered in the pathogenesis of DR are oxidative stress and inflammation. Studies have suggested the critical role of oxidative stress in the pathogenesis of diabetic retinopathy. Chronic hyperglycemia plays a vital role in the formation of Reactive Oxygen Species (ROS) due to the activation of the secondary pathways viz, polyol, protein kinase C (PKC) pathways, and overactivity of hexosamine pathways, leading to structural and functional changes in the retinal microvasculature32,33,34<\/sup>.<\/p>\n\n\n\n

Table 5: Correlation of serum total bilirubin with FBS, HbA1c, GGT, GSH, MDA and Lipid Profile, in DR (Group III)<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n<\/td>\n

\n

FBS<\/strong><\/p>\n<\/td>\n

\n

HbA1c<\/strong><\/p>\n<\/td>\n

\n

GGT<\/strong><\/p>\n<\/td>\n

\n

GSH<\/strong><\/p>\n<\/td>\n

\n

MDA<\/strong><\/p>\n<\/td>\n

\n

TC<\/strong><\/p>\n<\/td>\n

\n

TG<\/strong><\/p>\n<\/td>\n

\n

HDL-C<\/strong><\/p>\n<\/td>\n

\n

LDL-C<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

serum
total bilirubin<\/p>\n<\/td>\n

\n

Pearson Correlation<\/p>\n

 (r value)<\/p>\n<\/td>\n

\n

-0.375<\/p>\n<\/td>\n

\n

-0.351<\/p>\n<\/td>\n

\n

0.335<\/p>\n<\/td>\n

\n

-0.323<\/p>\n<\/td>\n

\n

-0.323<\/p>\n<\/td>\n

\n

0.159<\/p>\n<\/td>\n

\n

0.097<\/p>\n<\/td>\n

\n

-0.056<\/p>\n<\/td>\n

\n

0.104<\/p>\n<\/td>\n<\/tr>\n

\n

p- value
Sig.(2-tailed)<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n

\n

0.122<\/p>\n<\/td>\n

\n

0.347<\/p>\n<\/td>\n

\n

0.587<\/p>\n<\/td>\n

\n

0.313<\/p>\n<\/td>\n<\/tr>\n

\n

No.<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n

\n

96<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*Correlation is significant at p- value 0.001 level<\/p>\n

FBS: Fasting blood sugar; HbA1c: Glycated haemoglobin; GGT: Gamma glutamyl  transferase; GSH: Glutathione; MDA: Malondialdehyde; TC: Total Cholesterol; TG: Triglycerides; HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein cholesterol<\/p>\n\n\n

ROS\ndamages crucial biomolecules such as DNA, proteins, and lipid membranes. Lipids\nare one of the primary targets of ROS, and oxidized lipids generate MDA35,36<\/sup>.\nIncreased MDA in plasma, serum, and other tissues observed in diabetic patients37<\/sup>.\nIn the present study, there was increased lipid peroxidation, expressed as\nsignificantly increased levels of MDA in T2DM and DR compared with clinically\nproven controls. Our results are on par with few studies, who have demonstrated\nhigher MDA levels in the DR compared with DM and controls38,39<\/sup>. The\nbiochemical mechanisms for increased levels of MDA in DR are mainly based on\nthe degree of lipolysis, with peroxidative damage of the membrane lipids\nresulting in increased levels of free fatty acids in the blood, leading to\nincreased production of MDA levels and suggesting it as lipid peroxidation\nmarker for retinal complications of diabetes18<\/sup>.<\/p>\n\n\n\n

The\nbody has natural antioxidant systems to protect against the harmful effects of\nROS. These systems include enzymes such as catalase, glutathione peroxidase,\nsuperoxide dismutase, and non-enzymatic antioxidants such as glutathione and\nvitamin E40<\/sup>. In the present study, there was a statistically\nsignificant decrease in levels of GSH in DR and T2DM groups compared with that\nof clinically proven healthy controls. Similar findings were found in the study\nby Kundu and his coworkers41<\/sup>.<\/p>\n\n\n\n

A\nstudy conducted in 2018 observed the elevation in circulating levels of\npro-inflammatory cytokines, reactive oxidative species, and a decrease in GSH\nlevels. The possible mechanism is that an increase in polyol pathway activity\nin DR causes increased usage of nicotinamide adenine dinucleotide phosphate\n(NADPH) by the enzyme aldose reductase (AR), which further reduces the\navailability of NADPH for regenerating the intracellular antioxidant GSH and\nthereby decreasing the antioxidant capacity of the cells40<\/sup>. Irreversible\nloss and diminished GSH synthesis may reduce the concentration of GSH42,43<\/sup>.\nFrom these findings, it is proposed that upper levels of serum total bilirubin\nlevels in the physiological range may inhibit inflammation processes, decrease\noxidative stress, and thereby interrupt or delay the development of DR.<\/p>\n\n\n\n

We\nobserved elevated total cholesterol, LDL-cholesterol, and triglyceride values\nin Group II and Group III and HDL-C values in biological reference intervals.\nOur findings are on par with few studies 44,45<\/sup>. An International\nstudy showed no significant association between hyperlipidemia and DR46<\/sup>.\nHyperlipidemia is found in poorly controlled diabetes and causes increased\nviscosity of blood with alterations in the fibrinolytic system, leading to the\nformation of hard exudates. There may also be an assimilation of serum\ntriglycerides into the cell membrane, which causes changes in membrane\nfluidity, leading to plasma leakage into the retina and resulting in hemorrhage\nand edema in the retina46<\/sup>. Few studies demonstrated that decreased\nserum lipids due to oral statins may help prevent retinal hard exudate\nformation and loss of vision47,48<\/sup>. \nGamma-glutamyl transferase (GGT) is a recognized marker of alcohol\nintake and liver-related diseases. The present study showed a significant\nincrease in serum GGT levels in DR subjects compared to clinically proven\nhealthy controls and T2DM. Similar observations found in the study conducted in\nPakistan & Iran population49,50<\/sup>.<\/p>\n\n\n\n

A\nstudy in 2019 demonstrated that serum GGT levels were inversely proportional to\nglutathione and glutathione reductase in people with T2DM and DR, showing\ndecreased antioxidant defenses51<\/sup>. <\/p>\n\n\n\n

A\ncross-sectional study conducted in the third U.S. National Health and Nutrition\nExamination Survey demonstrated that serum GGT values elevated along with serum\nMDA levels and further indicates that GGT is potentially a pro-oxidant, and its\neffect is expressed in the presence of transition metals or iron. The cysteinyl\nglycine, a product of GGT, reduces ferric ions to ferrous, which promotes free\nradical production52<\/sup>.<\/p>\n\n\n\n

We\nobserved no significant differences between the three Groups when comparing\nnon-nitrogenous substances and hepatic markers. Previous study reported that increased\nlevels of urea and creatinine, which were associated with an increased risk for\nprogression to DR53<\/sup>. Decreased serum albumin levels in DR cases were\ndemonstrated in a previous study54<\/sup>. A study by Gupta and his team\nshowed deranged levels of SGOT, SGPT, and ALP in DR cases55<\/sup>.<\/p>\n\n\n\n

The\ncorrelation of duration of diabetes with fasting blood sugars, glycated\nhemoglobin, lipid profile parameters, renal and liver function test parameters,\nGSH, and MDA in Groups II and III did not show any significant positive or\nnegative association.<\/p>\n\n\n\n

In\nGroups II and III, we observed a significant negative correlation of serum\ntotal bilirubin with FBS and glycated hemoglobin. These findings are concurrent\nwith two international studies conducted in Japan population and our previous\nstudy56,57,8<\/sup>. In Groups II and III, we observed a significant positive\ncorrelation of serum total bilirubin levels with GSH and a negative correlation\nwith MDA. Our observations are inconsistent with our previous study and a study\nconducted in 2017 by Shumaila and coworkers 8,58<\/sup>. A study conducted in\n2014 showed no correlation between GSH and other parameters in all three groups41<\/sup>.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

This study demonstrated that increased levels of MDA and decreased\nlevels of GSH and serum total bilirubin were associated with increased risk of\nT2DM to DR development. Serum total bilirubin in upper levels in the\nphysiological range may protect against the development of retinopathy in\nsubjects with T2DM, and these findings suggest that serum total bilirubin\nlevels may be used as a biomarker to expect the risk of development of\nretinopathy. Estimating serum total bilirubin in T2DM on regular check-ups\nhelps the physician to predict DR and initiate early treatment.<\/p>\n\n\n\n

Limitations<\/strong><\/p>\n\n\n\n

The fundus examination and one-time measurement of serum total\nbilirubin served as the foundation for our investigation. Concordant readings\nof total bilirubin within the physiological range are relevant considering the\nrheological variations. Insulin estimation to assess the resistance in the\nsubjects would have been better. Dietary habits or medications that may alter\nliver function or bilirubin levels are pertinent information. As this study was\ncarried out in a semi-urban tertiary care hospital in a Kolar population of\nKarnataka state and our findings apply to other ethnic groups, it has to be\nconsidered and proved with prospective multi-centric studies.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

The authors would like to express their gratitude to Sri Devaraj Urs Academy of Higher Education and Research, Kolar, for funding this project<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors have no conflicts of interest to declare.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Funded this project. Reference No. SDUAHER\/KLR\/R&D\/261\/2013-14 Dated 18-12-2013<\/p>\n\n\n\n

Ethical clearance<\/strong><\/p>\n\n\n\n

No. SDUAHER\/KLR\/R&D\/242\/2013-14 dated 03-12-2013<\/p>\n\n\n\n

Approval of university research project for funding <\/strong><\/p>\n\n\n\n

Project no. SDUAHER\/Res.Proj\/89\/2013-14<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

  1. Sun H, Saeedi P, Karuranga S, et al. IDF Diabetes Atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045 [published correction appears in Diabetes Res Clin Pract. 2023 Oct;204:110945]. Diabetes Res Clin Pract<\/em>. 2022;183:109119. doi:10.1016\/j.diabres.2021.109119.
    CrossRef<\/a><\/li>
  2. Pradeepa R, Mohan V. Epidemiology of type 2 diabetes in India. Indian J Ophthalmol<\/em>. 2021;69(11):2932-2938. doi:10.4103\/ijo.IJO_1627_21
     CrossRef <\/a><\/li>
  3. Kumar A, Vashist P. Indian community eye care in 2020: Achievements and challenges. Indian J Ophthalmol<\/em>. 2020;68(2):291-293. doi:10.4103\/ijo.IJO_2381_19
    CrossRef <\/a><\/li>
  4. Cecilia O M, Jos\u00e9 Alberto C G, Jos\u00e9 N P, Ernesto Germ\u00e1n, C. M., Ana Karen, L. C., Luis Miguel, R. P., Ricardo Ra\u00fal, R. R., & Adolfo Daniel, R. C. Oxidative Stress as the Main Target in Diabetic Retinopathy Pathophysiology. J Diabetes Res<\/em>. 2019:8562408. Published 2019 Aug 14. doi:10.1155\/2019\/8562408
     CrossRef <\/a><\/li>
  5. Shawki HA, Elzehery R, Shahin M, Abo-Hashem EM, Youssef MM. Evaluation of some oxidative markers in diabetes and diabetic retinopathy. Diabetol Int<\/em>. 2020;12(1):108-117. Published 2020 Jun 27. doi:10.1007\/s13340-020-00450-w
    CrossRef <\/a><\/li>
  6. Cheriyath P, Gorrepati VS, Peters I, Nookala, V., Murphy, M. E., Srouji, N., & Fischman, D. High Total Bilirubin as a Protective Factor for Diabetes Mellitus: An Analysis of NHANES Data From 1999 – 2006. J Clin Med Res<\/em>. 2010;2(5):201-206. doi:10.4021\/jocmr425w
     CrossRef <\/a><\/li>
  7. Cho HC. The Relationship among Homocysteine, Bilirubin, and Diabetic Retinopathy. Diabetes Metab J<\/em>. 2011;35(6):595-601. doi:10.4093\/dmj.2011.35.6.595
     CrossRef <\/a><\/li>
  8. Prabhavathi K, Kunder M, Shashidhar KN, et al.Serum total bilirubin levels in diabetic retinopathy\u2014a case control study. IOSR J Pharm. 2013;4(8):1\u20136. https :\/\/doi.org\/10.9790\/3013-04080 106.<\/li>
  9. Trinder P. Determination of Glucose in Blood Using Glucose Oxidase with an Alternative Oxygen Acceptor. Annals of Clinical Biochemistry<\/em>. 1969;6(1):24-27. doi:10.1177\/000456326900600108
     CrossRef <\/a><\/li>
  10. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem<\/em>. 1974;20(4):470-475.
     CrossRef <\/a><\/li>
  11. Trinder, P. Enzymatic Calorimetric Edmund Lamb, David J, Cristopher P. Estimation of Creatinine and Urea. In. Carl A Burtis, Edward R Ashwood and David E Bruns, Tietz text book of Clinical Chemistry 4th ed, Saunders. New Delhi: Elsevier Co.:798-803(2006). <\/li>
  12. Trinder P.  Enzymatic Calorimetric Determination of Triglycerides by GOP-PAP Method. Annals of Clinical Biochemistry<\/em>,1969; 6:24-27.
    https:\/\/doi.org\/10.1177\/000456326900600108
     CrossRef <\/a><\/li>
  13. Naito, H.K.  HDL Cholesterol. Kaplan A et al. Clin Chem<\/em>. The C.V. Mosby Co., St Louis, Toronto, Princeton.1984; 437 :1207-1213.<\/li>
  14. Weiss JS, Sundberg MW, Dappen GM, et al. Diazo-based assay for total bilirubin in a coated thin film evaluated. Clin Chem<\/em>. 1984;30(8):1310-1313.
     CrossRef <\/a><\/li>
  15. Panteghini M, Bais R and Van soling WW. Enzymes In. Carl A Burtis , Edward R Ashwood and  David E Bruns, Tietz text book of Clinical Chemistry<\/em> 4th ed, Saunders.NewDelhi: ElsevierCo.: 604-613(2006).<\/li>
  16. Beutler E, Duron O. Kelly B.M. Improved method for the determination of blood glutathione. J Lab Clin Med<\/em>. 1963; 61: 882-888.<\/li>
  17. Gutteridge JM, Quinlan GJ. Malondialdehyde formation from lipid peroxides in the thiobarbituric acid test: the role of lipid radicals, iron salts, and metal chelators. J Appl Biochem<\/em>. 1983;5(4-5):293-299. PMID: 6679543 <\/li>
  18. Dave A, Kalra P, Gowda BH, Krishnaswamy M. Association of bilirubin and malondialdehyde levels with retinopathy in type 2 diabetes mellitus. Indian J Endocrinol Metab<\/em>. 2015;19(3):373-377. doi:10.4103\/2230-8210.152777
     CrossRef <\/a><\/li>
  19. Aldosari DI, Malik A, Alhomida AS, Ola MS. Implications of Diabetes-Induced Altered Metabolites on Retinal Neurodegeneration. Front Neurosci<\/em>. 2022;16:938029. Published 2022 Jul 13. doi:10.3389\/fnins.2022.938029
     CrossRef <\/a><\/li>
  20. Cherchi S, Gigante A, Spanu MA, Contini P, Meloni G, Fois MA, Pistis D, Pilosu RM, Lai A, Ruiu S, et al. Sex-Gender Differences in Diabetic Retinopathy. Diabetology<\/em>. 2020; 1(1):1-10. https:\/\/doi.org\/10.3390\/diabetology1010001
     CrossRef <\/a><\/li>
  21. Li M, Wang Y, Liu Z, et al. Females with Type 2 Diabetes Mellitus Are Prone to Diabetic Retinopathy: A Twelve-Province Cross-Sectional Study in China. J Diabetes Res<\/em>. 2020;2020:5814296. Published 2020 Apr 21. doi:10.1155\/2020\/5814296
     CrossRef <\/a><\/li>
  22. Yau JW, Rogers SL, Kawasaki R, et al. Global prevalence and major risk factors of diabetic retinopathy. Diabetes Care<\/em>. 2012;35(3):556-564. doi:10.2337\/dc11-1909
     CrossRef <\/a><\/li>
  23. Mehta R , Punjabi S , Bedi N , Prevalence of diabetic retinopathy: A tertiary care centre based study. Indian J Clin Exp Ophthalmol.<\/em> 2020;6(3):383-386
     CrossRef <\/a><\/li>
  24. Ghaffar T, Marwat ZI, Ullah F, Khan S, Hassan Aamir AU. Association Of Serum Total Bilirubin Level With Diabetic Retinopathy In Type 2 Diabetes Mellitus. J Ayub Med Coll Abbottabad<\/em>. 2016;28(3):537-541.
     CrossRef <\/a> <\/li>
  25. Wan H, Zhu H, Wang Y, et al. Associations between different bilirubin subtypes and diabetic microvascular complications in middle-aged and elderly individuals. Ther Adv Endocrinol Metab<\/em>. 2020;11:2042018820937897. Published 2020 Jul 11. doi:10.1177\/2042018820937897<\/li>
  26. Shawki HA, Elzehery R, Shahin M, Abo-Hashem EM, Youssef MM. Evaluation of some oxidative markers in diabetes and diabetic retinopathy. Diabetol Int<\/em>. 2020;12(1):108-117. Published 2020 Jun 27. doi:10.1007\/s13340-020-00450-w
     CrossRef <\/a><\/li>
  27. Sinclair H S, Luttrull K J. Diabetes Mellitus Associated Progressive Neurovascular Retinal Injury: Recommendations for Imaging and Functional Testing and Potential Role for Early Intervention with Modern Retinal Laser Therapy. Journal of Ophthalmology Research Reviews and Reports<\/em>. 2022;3:2-17. Doi 10. 47363\/ JORRR\/ 2022(3) 130.
     CrossRef <\/a><\/li>
  28. Gazzin S, Vitek L, Watchko J, Shapiro SM, Tiribelli C. A Novel Perspective on the Biology of Bilirubin in Health and Disease. Trends Mol Med.<\/em> 2016;22(9):758-768. doi:10.1016\/j.molmed.2016.07.004
    CrossRef <\/a><\/li>
  29. Ding Y, Zhao J, Liu G, et al. Total Bilirubin Predicts Severe Progression of Diabetic Retinopathy and the Possible Causal Mechanism. J Diabetes Res<\/em>. 2020;2020:7219852. Published 2020 Jul 31. doi:10.1155\/2020\/7219852
     CrossRef <\/a><\/li>
  30. Yasuda M, Kiyohara Y, Wang JJ, et al. High serum bilirubin levels and diabetic retinopathy: the Hisayama Study. Ophthalmology<\/em>. 2011;118(7):1423-1428. doi:10.1016\/j.ophtha.2010.12.009
     CrossRef <\/a><\/li>
  31. Abbasi A, Deetman PE, Corpeleijn E, et al. Bilirubin as a potential causal factor in type 2 diabetes risk: a Mendelian randomization study. Diabetes<\/em>. 2015;64(4):1459-1469. doi:10.2337\/db14-0228
     CrossRef <\/a><\/li>
  32. Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circ Res<\/em>. 2010;107(9):1058-1070. doi:10.1161\/CIRCRESAHA.110.223545
    CrossRef <\/a><\/li>
  33. Safi SZ, Qvist R, Kumar S, Batumalaie K, Ismail IS. Molecular mechanisms of diabetic retinopathy, general preventive strategies, and novel therapeutic targets. Biomed Res Int<\/em>. 2014;2014:801269. doi:10.1155\/2014\/801269
     CrossRef <\/a><\/li>
  34. Rodr\u00edguez ML, P\u00e9rez S, Mena-Moll\u00e1 S, Desco MC, Ortega \u00c1L. Oxidative Stress and Microvascular Alterations in Diabetic Retinopathy: Future Therapies. Oxid Med Cell Longev<\/em>. 2019;2019:4940825. Published 2019 Nov 11. doi:10.1155\/2019\/4940825
     CrossRef <\/a><\/li>
  35. Juan CA, P\u00e9rez de la Lastra JM, Plou FJ, P\u00e9rez-Lebe\u00f1a E. The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies. Int J Mol Sci<\/em>. 2021;22(9):4642. Published 2021 Apr 28. doi:10.3390\/ijms22094642
    CrossRef <\/a><\/li>
  36. Nita M, Grzybowski A. The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults. Oxid Med Cell Longev<\/em>. 2016;1-23:2016:3164734. doi:10.1155\/2016\/3164734
     CrossRef <\/a><\/li>
  37. Shalash M, Badra M, Imbaby S, Elbanna E. Malondialdehyde in type 2 diabetics and association with cardiovascular risk factors. Journal of the Medical Research Institute<\/em>, 2020; 41(2):21-30. Doi:10.21608\/jmalexu.2020.147116
     CrossRef <\/a><\/li>
  38. de Souza Bastos A, Graves DT, de Melo Loureiro AP, et al. Diabetes and increased lipid peroxidation are associated with systemic inflammation even in well-controlled patients. J Diabetes Complications<\/em>. 2016;30(8):1593-1599. doi:10.1016\/j.jdiacomp.2016.07.011
     CrossRef <\/a><\/li>
  39. Hou Y, Lin M, Qiu X , He M. Zhang Y, Guo F. Effect of Type-2 diabetes mellitus in retinopathy patients on MDA, SOD activity and its correlation with HbA1c.Brazilian Archives of Biology and Technology. 2021; 64. Doi:10.1590 \/1678-4324-2021200075.
     CrossRef <\/a><\/li>
  40. Prabhakar PK. Pathophysiology of Diabetic Secondary Complication and their Management. Curr Diabetes Rev<\/em>. 2021;17(4):395-396. doi:10.2174\/157339981704210326092455
     CrossRef <\/a><\/li>
  41. Kundu D, Mandal T, Nandi M, Osta M, Bandyopadhyay U, Ray D. Oxidative stress in diabetic patients with retinopathy. Ann Afr Med<\/em>. 2014;13(1):41-46. doi:10.4103\/1596-3519.126951
     CrossRef <\/a> <\/li>
  42. Lutchmansingh FK, Hsu JW, Bennett FI, et al. Glutathione metabolism in type 2 diabetes and its relationship with microvascular complications and glycemia. PLoS One<\/em>. 2018;13(6):e0198626. Published 2018 Jun 7. doi:10.1371\/journal.pone.0198626
     CrossRef <\/a> <\/li>
  43. Zhu B, Wu X, Ning K, Jiang F, Zhang L. The Negative Relationship between Bilirubin Level and Diabetic Retinopathy: A Meta-Analysis. PLoS One<\/em>. 2016;11(8):e0161649. Published 2016 Aug 29. doi:10.1371\/journal.pone.0161649
     CrossRef <\/a> <\/li>
  44. Badikillaya V U, Potluri R, Venkataramana G,  Pernenkil SR. (2013). Serum Gamma Glutamyl Transferase as a marker of oxidative stress in Type 2 Diabetic Retinopathy. Ijpbs<\/em>.2013; 3(2) :496-503. <\/li>
  45. Khubchandani A S.  Sanghani H. Study of serum magnesium and HbA1C in diabetic patients along with changes in their lipid profiles. IJCP<\/em>.2013;23(11):717-719. <\/li>
  46. Chou Y, Ma J, Su X, Zhong Y. Emerging insights into the relationship between hyperlipidemia and the risk of diabetic retinopathy. Lipids Health Dis<\/em>. 2020;19(1):241. Published 2020 Nov 19. doi:10.1186\/s12944-020-01415-3
    CrossRef <\/a><\/li>
  47. Kang EY, Chen TH, Garg SJ, et al. Association of Statin Therapy With Prevention of Vision-Threatening Diabetic Retinopathy. JAMA Ophthalmol<\/em>. 2019;137(4):363-371. doi:10.1001\/jamaophthalmol. 2018.6399
    CrossRef <\/a> <\/li>
  48. Kawasaki R, Konta T, Nishida K. Lipid-lowering medication is associated with decreased risk of diabetic retinopathy and the need for treatment in patients with type 2 diabetes: A real-world observational analysis of a health claims database. Diabetes Obes Metab<\/em>. 2018;20(10):2351-2360. doi:10.1111\/dom.13372
     CrossRef <\/a> <\/li>
  49. Javaid H, Haroon MA, Javaid M, Abidin FU, Masud H, Khalid UB. Association of serum gamma glutamyl transferase as biomarker of oxidative stress in type-II diabetics with and without diabetic retinopathy. Pak J Pathol<\/em>. 2021; 32(4): 165-169.<\/li>
  50. Valizadeh N, Mohammadi R, Mehdizadeh A, Motarjemizadeh Q, Khalkhali H R. Evaluation of serum gamma glutamyl transferase levels in diabetic mellitus patients with and without retinopathy. Shiraz E- med J.<\/em> 2018;19(7):e 64073.https:doi.org\/10.5812\/semj.64073.
     CrossRef <\/a> <\/li>
  51. R Divya, V Ashok. Evaluation of serum gamma glutamyl transferase levels as a marker of oxidative stress in type 2 diabetes patients with and without retinopathy. MedPulse International Journal of Physiology<\/em>. 2019; 9:30\u201334.  DOI:10.26611\/103935.
     CrossRef <\/a> <\/li>
  52. Lim JS, Yang JH, Chun BY, Kam S, Jacobs DR Jr, Lee DH. Is serum gamma-glutamyltransferase inversely associated with serum antioxidants as a marker of oxidative stress?. Free Radic Biol Med<\/em>. 2004;37(7):1018-1023. doi:10.1016\/j.freeradbiomed.2004.06.032
     CrossRef <\/a><\/li>
  53. Hsieh YT, Tsai MJ, Tu ST, Hsieh MC. Association of Abnormal Renal Profiles and Proliferative Diabetic Retinopathy and Diabetic Macular Edema in an Asian Population With Type 2 Diabetes [published correction appears in JAMA Ophthalmol. 2019 Feb 1;137(2):233]. JAMA Ophthalmol<\/em>. 2018;136(1):68-74. doi:10.1001\/jamaophthalmol.2017.5202
     CrossRef <\/a><\/li>
  54. Wang GX, Fang ZB, Li JT, et al. The correlation between serum albumin and diabetic retinopathy among people with type 2 diabetes mellitus: NHANES 2011-2020. PLoS One<\/em>. 2022;17(6):e0270019. Published 2022 Jun 16. doi:10.1371\/journal.pone.0270019
     CrossRef <\/a> <\/li>
  55. Gupta R, Bhat SPS, Gangadhar PR, et al. Study of liver function tests in patients with long standing type 2 diabetes mellitus in comparison to healthy individuals. J Evolution Med Dent Sci<\/em> 2021;10(05):289- 293, DOI: 10.14260\/jemds\/2021\/64
     CrossRef <\/a><\/li>
  56. Ohnaka K, Kono S, Inoguchi T, et al. Inverse associations of serum bilirubin with high sensitivity C-reactive protein, glycated hemoglobin, and prevalence of type 2 diabetes in middle-aged and elderly Japanese men and women. Diabetes Res Clin Pract<\/em>. 2010;88(1):103-110. doi:10.1016\/j.diabres.2009.12.022
     CrossRef <\/a><\/li>
  57. Shichiri M, Kishikawa H, Ohkubo Y, Wake N. Long-term results of the Kumamoto Study on optimal diabetes control in type 2 diabetic patients. Diabetes Care<\/em>. 2000;23 Suppl 2:B21-B29.<\/li>
  58. Shaikh S, Memon A, Ata M A, Hina, Khoharo HK. Association of Serum Bilirubin, Serum Malondialdehyde and Glycemic Control with Retinopathy in Type 2 Diabetic Subjects. Int J Diabetes Endocrinol<\/em>. (2017);2(1):10-14. doi: 10.11648\/j.ijde.20170201.13.<\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"

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