Association of Age and Sex with Different Status of Serum Vitamin D Level among Different Grades of Diabetic Retinopathy: A Cross-sectional Study

1. Medical Officer, Department of Ophthalmology, E.S.I. Hospital, Under Labour Department, Government of West Bengal, Serampore, West Bengal, India. 2. Associate Professor, Department of Physiology, Calcutta National Medical Collage and Hospital, Kolkata, West Bengal, India. 3. Demonstrator, Department of Physiology, Calcutta National Medical Collage and Hospital, Kolkata, West Bengal, India. 4. Associate Professor, Department of Physiology, R.G.Kar Medical Collage and Hospital, Kolkata, West Bengal, India.

Abstract

Introduction: Diabetes Mellitus (DM) is rapidly escalating globally as well as in India, affecting all age and sex groups. One of the dreaded microvascular complications of DM is Diabetic Retinopathy (DR). In parallel to increase in prevalence of DM and its complications, several reports of serum 25 hydroxy (OH) Vitamin D deficiencies have been documented in India.

Aim: To establish the relation of different age and sex groups with different status of serum 25 (OH) Vitamin D level among different grading of Diabetic Retinopathy in patients of Type 2 Diabetes Mellitus.

Materials and Methods: This cross-sectional and observational study was conducted in Calcutta National Medical College and Hospital, Kolkata, West Bengal, India, from May 2019 to May 2020. Total 107 type 2 DM patients aged 40 years and above including both males and females were taken. Direct ophthalmoscopy was done for examination of retina and venous blood was taken for Fasting Blood Sugar (FBS), Post Prandial Blood Sugar (PPBS), Glycated Haemoglobin (HbA1c) and serum 25 (OH) Vitamin D level estimation aseptically. Number of patients and percentage of patients were compared across the groups using Fisher’s-exact test/Pearson’s Chi-square test for independence of attributes as appropriate. Mean, median and standard deviation were compared across the groups using Mann-Whitney’s U-Test/Kruskal-Wallis’s Test as appropriate. Spearman’s test was applied for assessing the correlation between age of diabetic patients and vitamin D levels. The p-value <0.05 was considered as statistically significant.

Results: In this study, most of the participants were under the age group of 50-59 years. No significant relationship between the age and Vitamin D levels of the subjects was observed. The association between different status of serum Vitamin D level and different age groups among different grading of Diabetic Retinopathy is statistically significant out of entire sample size, not in individual grading. The association between different sex groups and different vitamin D status among different grading of DR was not statistically significant. There was no significant difference between serum Vitamin D level in males and females with DR. Correlation between serum Vitamin D level and age was linear and positive; but strength was low and p-value was not significant (correlation coefficient=0.100 , p-value=0.306).

Conclusion: This present study showed that maximum subjects were under the age group of 6^th decade. There was a significant association between different status of serum Vitamin D level and different age groups out of whole study population, but not in individual grading of DR. No association was observed between different sex groups and different Vitamin D status among different grading or severity of DR.

Age related macular degeneration, Neovascularisation elsewhere, Neovascularisation on disc, Proliferative diabetic retinopathy, Retinal detachment

The Diabetes Mellitus (DM) is a large public health problem which affects more than 300 million individuals worldwide with significant morbidity and mortality (1). In parallel to increase in prevalence of DM, there has been resurgence of Vitamin D deficiency worldwide and it is seen across all ages, races and geographic regions (2),(3). In India, inspite of adequate sunlight exposure, several reports have documented the prevalence of Vitamin D deficiency in general population (4). Uncontrolled diabetes increases risk of microvascular complications. Diabetic Retinopathy (DR) is most common complication among them (5). Diabetic retinopathy is a microangiopathy primarily affecting the precapillary arterioles, capillaries and post capillary venules although larger vessels may also be involved. Diabetic retinopathy is characterised by features of both microvascular occlusion and leakage owing to elevated blood glucose level for long duration. In patients diagnosed with diabetes before the age of 30 years, the incidence of DR after 10 years is 50% and after 30 years is 90%. Diabetic retinopathy rarely develops within five years of the onset of diabetes or before puberty, but about 5% of Type 2 diabetic patients have DR at presentation (6). The individuals, who have high level of blood glucose chronically, are very much prone to develop moderate to severe retinopathy in comparison to individuals having lower blood glucose level. Although there is no cut-off value of HbA1c to determine the retinopathy, it has been seen that usually patients with HbA1c < 6.5% have less chance of developing DR (7). Besides its main action in mineral homeostasis and bone remodelling, Vitamin D plays a potential role in glucose homeostasis and in the pathogenesis of Type 2 Diabetes and its complications by directly stimulating insulin secretion from β cells as well as improving insulin sensitivity to peripheral tissues (8). Experimental study revealed that Vitamin D also has protective effects on DR by inhibiting Vascular Endothelial Growth Factor (VEGF) and Transforming Growth Factor- β (TGF-β) (9). There are several conflicting reports about the association between DR and hypovitaminosis of Vitamin D. Serum 25 (OH) Vitamin D level is widely accepted as a good indicator of status of Vitamin D in a subject (10). This study was done to evaluate the association between different age and sex groups and different serum Vitamin D status among different grading of DR in type 2 diabetic patients.

Material and Methods

This cross-sectional and observational study was done in the Department of Medicine (Diabetic Clinic), Ophthalmology, Physiology and Biochemistry of Calcutta National Medical College and Hospital, Kolkata, West Bengal, India, from May 2019 to April 2020 with type 2 diabetic patients attending diabetic clinic of Calcutta National Medical College and Hospital. All examinations were done after taking consent from patients and with due permission of Institutional Ethics Committee.

Sample size calculation: Sample size was determined by applying the formula 4pq/e²; where ‘p’ is the prevalence. The prevalence of DR among Type 2 diabetic patients attending Medicine Outpatient Department of a tertiary care hospital in India is presently 31.5% (11).

So, p=0.315; q=(1-p) i.e.,0.685 and e=allowable error (10% in this study)=0.1.

Thus, the final sample size (n) calculated was 86.31. To avoid bias, a total of 107 patients were included in the study.

Inclusion criteria: Males and females aged 40 years and above and clinically diagnosed Type 2 DM with unknown Vitamin D status were included in the study.

Exclusion criteria: Whereas, the subjects with the following conditions were excluded:

• History of recent Vitamin D supplementation within last six months.
• History of intake of any medication such as rifampicin, phenytoin, or phenobarbitone those alter the blood level of 25 OH Vitamin D.
• Subjects with prior diseases that suggest baseline alteration in serum 25 (OH) Vitamin D level and calcium metabolism like osteomalacia, Hyperparathyroidism etc.
• Any Cardiovascular, Hepatic disease or Renal Disease.
• Other causes of retinopathy like trauma, Central Serous Retinopathy (CSR), Age Related Macular Degeneration (ARMD), Retinal Detachment (RD), Hypertensive retinopathy etc.,
• Patients who were cognitively impaired or unable to provide informed written consent and also Type 1 DM.

Procedure

Sampling method was systematic random sampling. After collecting 107 type 2 diabetic patients, direct ophthalmoscopy (β Heine-200) was done to detect presence of DR and to perform grading of retinopathy, if present. Venous blood was taken aseptically for Fasting Blood Sugar Test (FBS), Postprandial Blood Sugar (PPBS), glycated haemoglobin (HbA1c) estimation by High Performance Liquid Chromatography (HPLC) and 25 (OH)vitamin D estimation by Enzyme Linked Immunosorbent Assay (ELISA) method. In the present study, the patients were divided into 3 groups according to serum 25(OH) Vitamin-D level (Table/Fig 1) (12)-

• Sufficient,
• Insufficient and
• Deficient

(Table/Fig 2) shows the International Clinical DR Disease Severity Scale (13).

Statistical Analysis

The statistical software- Statistical Package for the Social Sciences (SPSS; Version 20.0) was used for the analysis. Number and percentage of patients were compared across the groups using Fisher’s-Exact test/Pearson’s Chi-square test for Independence of Attributes as appropriate. Mean, median and standard deviation were compared across the groups using Mann-Whitney’s U Test/Kruskal-Walli’s Test as appropriate. Spearman’s test was applied for assessing the correlation between age of diabetic patients and Vitamin D levels. The p-value <0.05 was considered as statistically significant.

Results

(Table/Fig 3) shows that maximum patients were in the age group of 50-59 years (44.9%) and minimum (0.9%) patients were in the age group of 80-89 years. Histogram of age (Table/Fig 4) shows that maximum patients were under the age group of 50-59 years.

Most of the patients in present study were females, 59.8% patients were females and 40.2% patients were males (Table/Fig 5).

About 43% had no apparent retinopathy, 11.2% had mild NPDR; 30.8% had moderate NPDR; 12.1% had Severe NPDR and only 2.8% had PDR (Table/Fig 6).

Most patients were Vitamin D deficient (51.4%); 41.1% patients had insufficient Vitamin D level in blood and only 7.5% Patients had sufficient Vitamin D level (Table/Fig 7).

(Table/Fig 8) shows that the minimum age was 40 years, maximum age was 82 years; mean age was 56.19 years; minimum FBS was 72 mg/dL, maximum FBS was 351 mg/dL, mean FBS was 149.09 mg/dL; minimum PPBS was 88 mg/dL, maximum PPBS was 584 mg/dL, mean PPBS was 214.45 mg/dL; minimum vitamin D level 7.60 ng/mL, maximum vitamin D level was 98.83 ng/mL, mean value was 21.20 ng/mL, in respect to serum Vitamin D level; minimum level of HbA1c was 6%, maximum HbA1c was 15%, mean value was 8.30%.

Mean age was more in ‘No Retinopathy’ (56.41years) in comparison to ‘Retinopathy’ group (56.02 years), however the comparison was not statistically significant (Table/Fig 9).

(Table/Fig 10) showed that there was no statistically significant difference between mean age groups among different grading of DR. However, vitamin D and HbA1c showed significant difference among different grades of DR.

The association between different age groups and different grading of DR was not statistically significant (Table/Fig 11).

(Table/Fig 12) shows that the association between different status of Vitamin D level and different age groups was not statistically significant among different grades of DR. However, different serum Vitamin-D status showed a significant association with different age groups of diabetic patients out of whole sample size in current study. Correlation between serum Vitamin D level and age was linear and positive; but strength was low and p-value was not significant (Table/Fig 13), (Table/Fig 14). (Table/Fig 15) shows that the association between different status of serum Vitamin D levels and different sex groups among different grading of DR patients was not statistically significant. There was no significant statistical difference in Vitamin D level of female diabetic patients with retinopathy and Vitamin D level of male diabetic patients with retinopathy (Table/Fig 16).

Discussion

In current study, an attempt was made to show the association of different age and sex groups with different status of Vitamin D level among severity of DR with type 2 diabetic patients. Most of the subjects (44.9%) belonged to 50-59 years of age group. The youngest patient enrolled was 40 years and the oldest was 82 years. Mean age was 56.19±8.51 years. Histogram of frequency of age also showed that maximum percentage of patients belonged between the age group 50-59 years. The present study is in accordance with the study done by Tan CSH et al., (14).

The present study showed that age was not significantly associated with presence and severity of DR. The Correlation coefficient between age and Vitamin D level was also positive, however the strength of which was very low and p-value was not statistically significant. The distribution of different Vitamin D status in different age groups was statistically significant out of whole sample size, but not in individual grading of DR. In present study, the percentage of male patients was 40.2% in comparison to percentage of female patients (59.8%) out of total 107 patients. The association between sex groups and different Vitamin D status among different grading of DR was not statistically significant.

Kahn HA and Bradley RF found the positive association between retinopathy and age was limited to the group with diabetes of less than duration of 10 years (15). Cahill M et al., concluded that the majority of elderly type 2 diabetics (greater than 70 years at diagnosis) will not develop significant DR (16).

Wei J et al., conclusively established that mean 25 (OH) Vitamin D concentration was lower in China than in the US (45.1 vs 83.5 nmol/L) with Chinese elderly lower than American elderly for different age groups. 70.3% in China and 17.4% in the US were considered as Vitamin D deficient. Older age, females, ethnic minorities, lower income, self-rated ‘very bad’ health and never drinkers were statistically significant in predicting lower serum 25 (OH) vitamin D levels in China. In the US, males, ethnic minorities, lower income, self-rated ‘very bad’ health, physically inactive, overweight and obese were related to lower serum 25 (OH) vitamin D levels (17).

Kader S et al., showed in their study that Vitamin D levels were found to be lower in both men and women as age progresses. Deficiency of Vitamin D (<10 ng/mL) was found in 83.8% of women and 18.2% of men, while insufficiency (10-30 ng/mL) of Vitamin D in 69.6% of women and 30.4% of men among admitted patients in Karapinar Public Hospital (18).

Muscogiuri G et al., found that serum Vitamin D level is lower in females in comparison to male group due to less sun exposure, higher Fat Mass percentage (FM%), lower intake of fish, which is the main dietary source of Vitamin D, extensive use of sunscreen etc., (19). The results of the present study were not in accordance with the results of the study, done by Muscogiuri G et al., in respect to sex group. Nadri G et al., revealed that serum Vitamin D is a biomolecular biomarker for Proliferative Diabetic Retinopathy (PDR). They showed that a significant decrease in serum Vitamin D level is associated with severity of DR (20).

The most important factor leading to hyperglycaemia with increased age is deficiency of insulin secretion developing with age as well as growing insulin resistance caused by a change in body composition and sarcopenia. As age advances, decreased retinal blood flow, retinal thinning and microglial changes occur and these changes can render the retina more vulnerable to oxidative and ischaemic changes which lead to DR. As age progresses, serum Vitamin D level decreases owing to decreased concentration of 7-dehydrocholesterol in epidermis and a reduced response to ultraviolet ray and thereby decreases insulin secretion and insulin sensitivity as well as less inhibition of VEGF and other factors which lead to DR and its increased severity (8),(9),(21).

Limitation(s)

As this study is cross-sectional the study design allows only for the identification of the association between study variables at a time. Peripheral retinal lesions may be missed by direct ophthalmoscopy as field of vision is less in direct ophthalmoscopy in comparison to that in indirect ophthalmoscopy. The period of sun exposure of the participants was not determined. Though 1,25- OH Vitamin-D is active form of Vitamin D, serum 25(OH) Vitamin D is a better indicator of Vitamin D status because the hepatic 25 hydroxylase is constitutively expressed and unregulated and thereby circulating level of 25 hydroxy(OH)Vitamin D reflects the availability of precursor for 25- hydroxylation. In future, well designed prospective observational study should be conducted and the duration of sun exposure should be determined.

Conclusion

No association exists between different status of serum Vitamin D level and different age and sex groups among different grades of DR. The study showed that maximum subjects were in the age group of 50-59 years. A low positive correlation was observed between the Vitamin D status and age of the diabetics which was not significant.

Acknowledgement

Authors express their deepest gratitude to Prof.( Dr.) Manika Sadhu Ghorai; Prof. (Dr.) Surajit Kumar Mukhopadhyay; Prof. (Dr.) Asok Kumar Sau; Prof. (Dr.) Anindya Dasgupta; Prof. (Dr.) Shantunu Tapadar ; Prof. (Dr.) Sanhita Mukherjee because this study was not possible without their help and co-operation.

DOI and Others

DOI: 10.7860/JCDR/2022/54908.16210

Date of Submission: Jan 12, 2022
Date of Peer Review: Feb 04, 2022
Date of Acceptance: Mar 17, 2022
Date of Publishing: Apr 01, 2022

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

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