F1000ResearchF1000Research2046-1402F1000 Research LimitedLondon, UK10.12688/f1000research.133652.1Research ArticleArticlesAssociation of vitamin A and zinc with the development of diabetic retinopathy
[version 1; peer review: 1 not approved]
YeasminFarjanaConceptualizationData CurationInvestigationMethodologyWriting – Original Draft Preparationhttps://orcid.org/0000-0002-9755-9956a1RahmanMd AsikurFormal AnalysisResourcesWriting – Original Draft Preparation2ChowdhuryNafisa MarzanWriting – Original Draft PreparationWriting – Review & Editing3KhadijaSheikhResourcesWriting – Original Draft Preparation4JoarderTaufiqueResourcesWriting – Original Draft PreparationWriting – Review & Editing5MishuFarzana AkonjeeSupervisionWriting – Review & Editing6
Department of Biochemistry, BIRDEM General Hospital, Dhaka, Bangladesh
Department of Forensic Medicine, Dhaka Community Medical College and Hospital, Dhaka, Bangladesh
Department of Biochemistry, Central Medical College, Cumilla, Bangladesh
Department of Biochemistry, Monowara Sikder Medical college and Hospital, Shariatpur, Bangladesh
SingHealth Duke-NUS Global Health Institute, Singapore, Singapore
Department of Biochemistry, BIRDEM General Hospital, Dhaka, Bangladeshfarjana.yeasminemc@gmail.com
Introduction: Diabetic Retinopathy is a common microvascular disorder in people with diabetes mellitus. It is characterized by a variety of lesions within the retina and is the leading cause of blindness in adults. Hyperglycemia promotes the formation of reactive oxygen species and increased concentrations locally and throughout the body, even to the point of exceeding antioxidant capacity, a condition known as oxidative stress affecting retinal integrity. Antioxidants and trace elements are responsible for reducing oxidative damage. Vitamin A and Zinc have antioxidant properties.
Objective: To evaluate vitamin A and Zinc levels in diabetic patients with and without diabetic retinopathy.
Methods: Cross-sectional study was done in BIRDEM General Hospital and the CARS, University of Dhaka, from January 2021 to December 2021. Study parameters Fasting Blood Glucose, HbA1c, Vitamin A, and Zinc were estimated by standard laboratory methods. According to the inclusion criteria, 100 subjects over 30 years were included. They were divided into two groups; 50 were patients with diabetic retinopathy, and 50 were without diabetic retinopathy. After taking informed written consent, a structured questionnaire was filled up for each subject to collect data. Data were analyzed by independent student
t-test, Chi-square test, and Pearson’s correlation test.
Results: The study showed that vitamin A was significantly lower in patients with diabetic retinopathy (8.95±8.12) than in patients without diabetic retinopathy (22.39±11.56). Zinc level was also decreased in cases of group I (1.17±0.31) than in group II (1.43±3.60). Vitamin A had a significant negative correlation between FBG and HbA
1c. But Zinc did not show a significant correlation with FBG and HbA
1c. This study also found a significant positive correlation between vitamin A with Zinc.
Conclusion: In this study, we found that vitamin A and zinc were significantly lower in patients with retinopathy than in patients without retinopathy.
Diabetic retinopathyHyperglycemiaVitamin AZincThe author(s) declared that no grants were involved in supporting this work.Introduction
Diabetes mellitus (DM) is a chronic and serious situation that affects the lives and well-being of individuals, families, and societies worldwide.
1 The most common type of DM, type 2, was named non-insulin-dependent diabetes. As one of the long-term diseases, it is related to diverse macrovascular and microvascular complications such as cerebrovascular disease, cardiovascular disease, retinopathy, nephropathy, etc.
2 DR is a common microvascular complication with numerous grades, which results in numerous issues or blindness.
3 According to a World Health Organization (WHO) study, the global number of diabetic patients reached 366 million in 2011. By 2025, there will be more than 500 million diabetic patients worldwide, and about one-third will develop DR.
4 It is the leading cause of blindness in people between the ages of 27 and 75 years.
5 There is a 76% reduction in the onset of DR through intensive control of hyperglycemia.
6 Amid previous studies, Akhter
et al.7 discovered approximately 21.6% DR among diabetic patients in Bangladesh.
A chain of elements is associated with the development and progression of DR, including the period of DM, poor glycemic control, and oxidative stress. Chronic hyperglycemia and its associated nonenzymatic glycation play an important role in the onset of microvascular diseases.
8 The retina has the highest oxygen absorption and glucose oxidation of any tissue; it is rich in polyunsaturated fatty acids. It is at risk for largely free radicals or reactive oxygen species (ROS) because it is such a high-energy-demanding organ.
9 An imbalance between the amount of ROS or oxygen radicals in a biological system and the antioxidant defenses is referred to as oxidative stress.
10 It may serve as a “unifying mechanism” in DR, linking all the harmful metabolic pathways triggered by hyperglycemia.
11
Vitamin A refers to a group of chemical compounds that are morphologically related. Retinol, found in animal tissues and is esterified with long-chain fatty acids, is the most active form.
12 By acting as an antioxidant that scavenges free radicals, vitamin A lowers damage and a proliferative alteration in the retina.
3 It is required for ocular functioning, cellular differentiation, and immunity.
13 One previous case-control study indicated that by converting all-trans-retinal to 11-cis-retinal, retinol plays a crucial part in the visual cycle. It occurs in the retinal pigment epithelium.
5
Many enzymes involved in the manufacture, storage, release, and conformational integrity of insulin contain zinc, an essential trace element.
14 After iron, it is the second most abundant trace metal in the body.
15 Zinc also has been found to protect the retina from diabetes-induced enhanced lipid peroxidation and decreased glutathione levels by maintaining membrane structure or promoting metallothionein production. Ocular neovascularization is also an important component in DR. Zinc inhibits vascular endothelial growth factor (VEGF) expression, preventing neovascularization. It stops vascular leakage in DR patients.
16 Slowing the advancement of intermediate and advanced age-related ocular degeneration with zinc and antioxidants could be marginally beneficial.
15 Serum zinc and vitamin A levels have a symbiotic connection. Zinc is a necessary component of the RBP, responsible for circulating vitamin A in the bloodstream. Vitamin A shortage in tissues can be caused by a zinc deficiency.
3
As the number of people with diabetes increases, there is an imminent increase in visual loss due to DR. Epidemiological and clinical evidence on the relationship of vitamin A and zinc with DR is still debatable. Despite the association between DR and serum vitamin A and zinc levels, our literature review found that few studies have been conducted to assess the relationship between vitamin A and zinc with DR in diabetes. This study aimed to assess the association of vitamin A and serum zinc with DR. These findings could be used to track the progression of diabetic retinopathy.
Objective
The aim of this study was to assess the association of vitamin A and serum zinc with retinopathy in diabetes.
MethodsStudy participants
Type 2 diabetic patients attending the outpatient Department of Ophthalmology at BIRDEM, Dhaka, from January 2021 to December 2021, were enrolled for this cross-sectional study. Using the American Diabetes Association’s guidelines, diabetes was described as follows: (1) Typical symptoms with random plasma glucose levels of more than 11.1 mmol/l; or (2) fasting plasma glucose levels of more than 7 mmol/l.
17 Individuals fulfilling either of the above criteria were diagnosed with diabetes. Patients with type 2 diabetes who had no ocular issues (n=50) were included as the control group. From the same center throughout this time, patients with diabetic retinopathy (n=50) were collected. Fundus photography was used to diagnose DR. Inclusion criteria were patients aged over 30 years, body mass index (BMI) of less than 35 kg/m
2 in both sexes. The exclusion criteria were patients with any visual abnormality or eye disease except DR, taking lipid-lowering agents, diuretics, antioxidant vitamins, minerals, any acute and chronic complications such as nephropathy, cerebrovascular disease, cardiovascular disease, severe infection, smoking, alcoholism, any drug abuse, pregnancy, and lactation. A semi-structured questionnaire was administered to collect data which included the age, sex, socioeconomic status, and educational level of the participants.
Ethical approval
Ethical clearance for the study was taken from the Institutional Review Board, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM). All patients signed a consent form before the study participation.
Anthropometric measurements
Using a calibrated scale (to the nearest 100 g) and light clothing, body weight was measured to the nearest 0.1 kg. Shoes were not worn. Standing without shoes and using a wall-mounted stadiometer, height was measured (to the nearest 0.5 cm). Weight/height
2 (kg/m
2) was used as the formula to compute BMI. Measurements of the waist and hips were taken, respectively, halfway between the lower rib and the iliac crest and bumps.
Biochemical assessments
With all aseptic precautions, 8 ml venous blood was collected from each study subject after overnight fasting of 8-10 hours. From this blood sample, 2 ml was delivered in a fluoride test tube for estimation of fasting blood glucose (FBG). 3 ml blood was delivered in an EDTA tube for estimation of HbA1c. FBG was assessed immediately in the Hexokinase method by Beckman Coulter Au-480 auto analyzer, and HbA1c was measured in high-performance liquid chromatography (HPLC) method by BIO-RAD Variant TM II Turbo at clinical biochemistry section, BIRDEM General Hospital, Dhaka. The remaining 3 ml was delivered in a plain test tube for vitamin A and zinc estimation. Plain tube with blood was kept in a standing position till clot formation. The serum and plasma were separated after centrifugation at 3000 rpm for 5 minutes. For vitamin A, serum was collected in microtubes, wrapped in foils, and filled with nitrogen to prevent light exposure and oxidation of vitamin A. For zinc, serum was kept in clean metal-free polypropylene tubes and stored at -20°C until analysis. Vitamin A was determined by the HPLC method. HPLC was performed on SIL 20 series prominence HPLC (Shimadzu, Japan) equipped with an autosampler, dual pumps (Model 20 AD), column oven (Model CTO-20A), and Vacuum degasser (Model DGU-20A). UV–visible detector (Model SPD-20A) and LC solution software were used. Analytical column (Discovery C18 column, 5 mm particle size, (250×4.6) mm as the stationary phase was used. 200 μl of methanol was added to each 200 μl of serum in the polypropylene tubes. After fiv minutes of vortex, 2 ml hexane was added to the tube for three times, and vortex for each time for two minutes. Each sample was centrifuged for 15 minutes (4000 rpm), and supernatants were collected and dried under nitrogen gas. Then, the residue was dissolved with diluents (methanol). Each patient’s 4 ml sample solution was injected into the HPLC system. A stock solution of vitamin A (1mg) was prepared by dissolving it into methanol. Vitamin A (retinol) was detected under the wavelength of 325 nm.
Zinc was assessed in atomic absorption spectrophotometry by Thermos Scientific iCE 3000 Series Atomic Absorption Spectrometer. Prior to sample collection, all plastic wares were made free from metallic contamination. Serum samples were diluted 5-fold with deionized water and introduced into the nebulizer burner system. Then, I diluted the standard solution by adding 5% glycerin. I did this to match the surface tension between the serum sample and the standard solution. I used the standard solution for calibration purposes. Then, I determined the zinc level at 213.9 nm wavelength. A blank was used for the setting of zero absorbance of the spectrophotometer. The calibration curve was prepared. Finally, the serum content of zinc was estimated.
Statistical analysis
Statistical analysis was performed with the help of the software SPSS version 20. Categorical data were expressed by frequencies and percentages. The chi-square test (for categorical variables) and Student’s t-test (for continuous variables) were done to compare variables. Pearson’s correlation coefficient was used to determine the study parameters’ correlation. Statistical tests were considered significant at a 5% level of significance.
Results
A total of 100 patients were enrolled in this study. There was no significant difference between group I (DR) and group II (without DR) patients with respect to age, sex, or socioeconomic factors such as their income, educational level, etc. Among the diabetic patients, the frequency of DR increased with an increasing age, while this was not the case in the group without DR (
Table 1).
Frequency distribution of duration of diabetes between diabetic patients with and without retinopathy.
Duration of Diabetes
Group I (DM with DR)
Group II (DM without DR)
Frequency (n)
Percentage (%)
Frequency (n)
Percentage (%)
0-5
1
2
18
36
6-10
13
26
20
40
>10
36
72
***
12
24
p<0.05.
p<0.01.
p<0.001.
Statistical analysis was done by Chi-Square T test.
Significant differences in glycemic status (FBS and HbA1c) between group I and group II were identified. It was observed that mean value of vitamin A and zinc were lower in group I than group II, and the difference was statistically significant (
Table 2).
Comparison of Glycemic status Fasting blood sugar (FBS), HbA1c of the study subjects.
Variables
Group I (n=50) Mean±SD
Group II (n=50) Mean±SD
FBS (mmol/L)
9.21±3.14
**
7.43±1.53
HbA
1C (%)
9.54 ±1.52
**
7.74 ±1.15
Vitamin A (retinol) μg/dL
8.95±8.12
**
22.39±11.56
Zinc (mg/L)
1.17±0.31
**
1.43±3.60
p<0.05.
p<0.01.
p<0.001.
t-test was done to elicit statistical significance. Values are expressed as the mean±SD.
Correlation between HbA1c with vitamin A and zinc
HbA1c was inversely correlated with vitamin A (
Figure 1A and
B); but there was no statistically significant correlation with zinc. There was also a positive correlation between vitamin A and zinc (
Figure 2).
Relation between HbA1c with vitamin A (retinol) and zinc in the study subjects.
Relationship between vitamin A (retinol) and zinc in the study subjects.
Discussion
Our findings suggest that vitamin A and Zinc have a statically significant effect on DR. They are important antioxidants. Both exert effects against oxidative stress. DR occurs as a chronic complication of diabetes. The present study showed that vitamin A and zinc levels were significantly lower in the DR group. A significant correlation was shown between serum vitamin A levels with FBG and HbA1c.
As far as we are aware, no research has examined the relationship of FBG and HbA1c with vitamin A and Zinc in DR patients. In our study FBG and HbA1c (%) was higher in DR patients. This finding was in concordance with Lima
et al. (2016)
18 who found them higher in DR patients than without DR. HbA1c levels in DR patients reflect a prolonged and uncontrolled glycemic state, which is a major contributor to diabetes complications.
18 The exposure to hyperglycemia and oxidative stress increased as the duration increased, potentially leading to diabetes complications.
9
Vitamin A may be involved in proper cell development and initiation of cell proliferation. Evidence that increased blood glucose concentration causes the production of damaging free radicals and reduces protective factors such as vitamin A has been reported by ZM
et al. (2004).
19 The current study showed significantly lower (p<.001) vitamin A levels in patients with DR. This finding is similar to previous reports by Rostamkhani
et al. (2019).
3 Vitamin A is a key component of the body’s antioxidant defense mechanism. This is evidence that vitamin A helps pancreatic beta cells re-establish insulin secretion. It has also boosted synthesis and glucokinase activity.
16 Its deficiency affects nerve growth factors and brain-derived neurotrophic factor levels. Which generally protects the retina from oxidative stress and stimulates healing.
17 We found a significant negative correlation (r=.332; p<.001) between HbA1c with vitamin A. This finding is similar to previous reports by Taneera
et al. (2021).
16
In this study, the values of Zinc in DR subjects were significantly higher (p<.05) when compared to those without DR patients. Our finding is supported by the studies of Kumari
et al. (2014).
14 Moreover, Luo
et al. (2015)
20 prove our idea that a shortage of antioxidants may increase the onset and progression of DR in diabetic patients. Accumulation of free radicals may play a key role in the pathogenesis of DR. This micronutrient is a potent antioxidant that aids in the elimination of oxidizing agents. There was no significant correlation (r=.025; p<.805) between Zinc and HbA1c.This finding contrasts with previous reports by Ganiger
et al. (2016)
21 which showed a significant negative correlation between vitamin A and serum Zinc. Hyperglycemia has long been recognized to hasten the production of Activated Glycation End-products (AGEs), which have been linked to DR pathophysiology. They can cause retinal pericyte apoptosis by stimulating ROS generation in them, mostly by activation of NADPH oxidase.
16 Zinc is present in the retina in high concentration and functions as an antioxidant because it inhibits Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which prevents the generation of free radicals.
22 Accordingly, HbA1c, vitamin A, and zinc levels seem to strongly impact DR patients.
Conclusions
Analyzing the findings of present study, significant alteration in vitamin A and Zinc levels were observed in DR patients. In addition, Vitamin A and zinc were also discovered to be positively associated, implying that vitamin A deficiency may result in a drop in blood zinc levels in diabetic patients. As a result, early detection of deficiency may aid the clinician in delaying the progression of diabetic retinopathy. This study was done within the context of the facilities available to us, it has got some limitations. As the study was a cross sectional study, the population size was small and the patients who participated in this study under different therapeutic agents for diabetes or its related complication which could not be excluded. Further studies with large number of study population are required to evaluate the effect of vitamin A and zinc in different stages of retinopathy.
Data availability
Open Science Framework. Written informed questionnaire. DOI:
https://osf.io/uyptc/?view_only=a7b4fba7b03e4e26adb1044742e9e446
This project contains the following data:
A semi-structured questionnaire was administered to collect data which included the age, sex, socioeconomic status, and educational level of the participants.
Data are available under the terms of the
Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).
ReferencesSaeediPPetersohnISalpeaP:
Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas.Diabetes Res. Clin. Pract.2019;157:107843.
3151865710.1016/j.diabres.2019.107843JenkinsAJJoglekarMVHardikarAA:
Biomarkers in diabetic retinopathy.Rev. Diabet. Stud.2015;12(1-2):159–195.
2667666710.1900/RDS.2015.12.159PMC5397989RostamkhaniHMellatiAATabaeiBS:
Association of serum zinc and vitamin A levels with severity of retinopathy in type 2 diabetic patients: A cross-sectional study.Biol. Trace Elem. Res.2019;192(2):123–128.
10.1007/s12011-019-01664-zHouYCaiYJiaZ:
Risk factors and prevalence of diabetic retinopathy: A protocol for meta-analysis.Medicine.2020;99(42):e22695.
3308071910.1097/MD.0000000000022695PMC7571993ZhangCLiKZhangJ:
Relationship between retinol and risk of diabetic retinopathy: a case-control study.Asia Pac. J. Clin. Nutr.2019;28(3):607–613.
3146440810.6133/apjcn.201909_28(3).0021Tabatabaei-MalazyOArdeshirlarijaniENamaziN:
Dietary antioxidative supplements and diabetic retinopathy; a systematic review.J. Diabetes Metab. Disord.2019;18(2):705–716.
3189069410.1007/s40200-019-00434-xPMC6915253AkhterAFatemaKAhmedSF:
Prevalence and associated risk indicators of retinopathy in a rural Bangladeshi population with and without diabetes.Ophthalmic Epidemiol.2013;20(4):220–227.
10.3109/09286586.2013.809770KunduDMandalTMausumiN:
Oxidative stress in diabetic patients with retinopathy.Ann. Afr. Med.2014;13(1):41–46.
10.4103/1596-3519.126951KowluruRAChanP-S:
Oxidative stress and diabetic retinopathy.Exp. Diabetes Res.2007;2007:1–12.
1764174110.1155/2007/43603PMC1880867KangQYangC:
Oxidative stress and diabetic retinopathy: Molecular mechanisms, pathogenetic role and therapeutic implications.Redox Biol.2020;37:101799.
3324893210.1016/j.redox.2020.101799PMC7767789TarrJMKaulKChopraM:
Pathophysiology of Diabetic Retinopathy.ISRN Ophthalmol.2013;2013:1–13.
2456378910.1155/2013/343560PMC3914226Valdés-RamosRAna LauraG-LBeatriz ElinaM-C:
Vitamins and type 2 diabetes mellitus. Endocrine, Metabolic & Immune Disorders-Drug Targets Formerly Current Drug Targets-Immune.Endocr. Metab. Disord.2015;15(1):54–63.
10.2174/1871530314666141111103217TaneeraJAwadallahSMohammedAK:
Vitamin A levels are decreased but not influenced by glucose-or lipid-lowering medications in subjects with type 2 diabetes.Saudi J. Biol. Sci.2021;28(1):572–577.
3342434110.1016/j.sjbs.2020.10.044PMC7783824KumariSPradhanTPandaTK:
Trace minerals and oxidative stress in diabetic retinopathy.Bangladesh J. Med. Sci.2014;13(2):175–179.
10.3329/bjms.v13i2.14963SaperRBRashR:
Zinc: an essential micronutrient.Am. Fam. Physician.2009;79(9):768–772.
20141096MiaoXSunWMiaoL:
Zinc and diabetic retinopathy.J. Diabetes Res.2013;2013:1–8.
2367187010.1155/2013/425854PMC3647550RodriguezBLAbbottRDFujimotoW:
The American Diabetes Association and World Health Organization classifications for diabetes: their impact on diabetes prevalence and total and cardiovascular disease mortality in elderly Japanese-American men.Diabetes Care.2002;25(6):951–955.
10.2337/diacare.25.6.951LimaVCCavalieriGCLimaMC:
Risk factors for diabetic retinopathy: a case–control study.Int. J. Retin. Vitr.2016;2(1):1–7.
10.1186/s40942-016-0047-6OsmanZMGomaaAMHusseinHM:
Association between retinal metabolism and diabetic retinopathy.Polish J. Food Nutr. Sci.2004;13(4):391–396.LuoY-YZhaoJHanX-Y:
Relationship between serum zinc level and microvascular complications in patients with type 2 diabetes.Chin. Med. J.2015;128(24):3276–3282.
2666814010.4103/0366-6999.171357PMC4797501GanigerASwamyKMGundalliS:
A comparative study of serum magnesium in type 2 diabetes mellitus with and without retinopathy and healthy controls and its correlation with glycemic status.Int. J. Clin. Biochem. Res.2016;3:6.
10.5958/2394-6377.2016.00002.2SharmaYSaxenaSMishraA:
Nutrition for diabetic retinopathy: plummeting the inevitable threat of diabetic vision loss.Eur. J. Nutr.2017;56(6):2013–2027.
2825830710.1007/s00394-017-1406-210.5256/f1000research.146660.r268209Reviewer response for version 1YakarBurkay1Referee
Medical School, Department of Family Medicine, Firat University,, 23119, Elazig, Turkey
Competing interests: No competing interests were disclosed.
1- Sample size was not calculated in the study. This negatively affects the validity and power of the the study.
2- It is understood that the distribution of the data was not taken into account during the statistical analysis. This led to the use of inappropriate methods in statistical analysis. (exp: Vitamin A (retinol) μg/dL Group I: 8.95
± 8.12 group II: 22.39
± 11.56) Student's t-test not applicable for comparison
3- The authors stated that "HbA1c was inversely correlated with vitamin A (Figure 1A and B)" but the results of the correlation analysis (correlation coefficient and p value) were not reported.
4-In many places in the discussion section, extreme comments by the authors stand out.
5- In the conclusion, there are extreme comments from the authors.
exp: Vitamin A and zinc were also discovered to be positively associated, implying that vitamin A deficiency may result in a drop in blood zinc levels in diabetic patients. No such conclusion can be drawn from the research design and findings
6-limitations of the study should be presented before the conclusion
Is the work clearly and accurately presented and does it cite the current literature?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are the conclusions drawn adequately supported by the results?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
Reviewer Expertise:
endocrine disorders
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.