Abstract
Type 2 diabetes mellitus (T2DM) has a heavy disease burden and is one of the leading causes of death worldwide. It is considered to be evolving from a complex and multifactorial metabolic disorder to an inflammatory condition. The strong link between hyperglycemia and oxidative stress has long been established. Oxidative stress leads to the generation of inflammatory mediators and reactive oxygen species, which results in an inflammatory state, which plays a key role in the pathogenesis of diabetic complications. Vitamin D is also known to affect insulin sensitivity. We aimed to correlate the levels of glycated hemoglobin (HbA1c) and vitamin D with oxidative stress. This cross-sectional study included 100 patients with T2DM. Oxidative stress was estimated by lipid peroxidation assay for activity of malondialdehyde (MDA), as well as superoxide dismutase (SOD) levels. HbA1c was estimated using ion-exchange chromatography. Vitamin D was estimated using enzyme-linked immunosorbent assay. The data were analyzed using a t test. The results showed that as the duration of T2DM increased, the levels of SOD and MDA also increased. No significant correlation was found between SOD and MDA with HbA1c (P = 0.995 and 0.195) and vitamin D (P = 0.874 and 0.051), respectively.
Keywords: Glycated hemoglobin, HbA1c, malondialdehyde, superoxide dismutase, type 2 diabetes mellitus, vitamin D
Type 2 diabetes mellitus (T2DM) has a heavy disease burden and is projected to be the seventh leading cause of mortality by 2030.1,2 Hyperglycemia activates certain upstream events that lead to mitochondrial overproduction of reactive oxygen species, which increases the oxidative stress status.3,4 Oxidative stress mediates insulin resistance and hence accelerates the progression of glucose intolerance to diabetes mellitus and its various complications.5 Malondialdehyde (MDA), a toxic stable aldehyde, is produced by the peroxidation of lipids by the reactive oxygen species.6 Several protective mechanisms are in place to prevent the histological damage it causes. One is an antioxidant enzyme, superoxide dismutase (SOD), which catalyzes the dismutation of one of the reactive oxygen species, superoxide radical, into oxygen and water.7 Both MDA and SOD can be used as biological markers to quantify oxidative stress status.8 Glycated hemoglobin (HbA1c) is the standard test to diagnose and monitor T2DM and can be used to predict the risk of microvascular complications.9,10 Vitamin D is known to perform numerous functions in our body including regulation of insulin secretion or sensitivity and attenuation of systemic inflammation.11 There is a dearth of relevant clinical data correlating vitamin D and HbA1c with parameters of oxidative stress in patients with T2DM.
METHODS
This cross-sectional study was conducted at the Department of Biochemistry, Government Medical College, Amritsar, under the aegis of the Indian Council of Medical Research, New Delhi, from August 2019 to October 2019 on 100 patients with T2DM treated at the outpatient clinic. The Institutional Ethics Committee approved the study. The study group comprised patients of various age groups, men and women, and people from different socioeconomic strata. Patients were included in the study only after obtaining informed consent in their vernacular language. Measurements such as waist circumference, hip circumference, weight, height, and body mass index were recorded following the World Health Organization STEPS surveillance guidelines. All the relevant information was obtained through a standardized questionnaire.
To be included in the study, patients had to be at least 18 years old and have T2DM. Patients with a known deficiency of vitamin D, sun exposure <3 hours a week, or on a medication that affected vitamin D levels were excluded from the study. Patients with disorders that affected vitamin D levels (osteomalacia, malabsorption syndrome, celiac disease, inflammatory bowel disease, chronic liver disease, chronic kidney disease etc.) were also excluded.
For estimation of HbA1c, 2 mL venous blood was taken in an EDTA vial, and 3 mL venous blood in a plain vial was taken for other investigations involving serum separation and estimation. Tests done for estimation of vitamin D included Erba® vitamin D, which uses a solid-phase enzyme-linked immunoassay. The test is based on the principle of competitive binding. Tests conducted for estimation of HbA1c included Erba® Glycated Hemoglobin, which is based on the principle of ion-exchange chromatography. Oxidative stress was estimated via lipid peroxidation assay, which estimated the amount of MDA, and an assay of SOD activity, which estimated the activity of SOD.
Vitamin D levels were further characterized based on the levels of 25 hydroxyvitamin D. They were classified as insufficient (<20 ng/mL), desirable (20–29 ng/mL), and sufficient (≥29 ng/mL). The normal limits for SOD and MDA were defined as 165 to 240 units/mL and 2.4 to 4.7 µmol/mL, respectively.
Oxidative stress levels were correlated with vitamin D and HbA1c. The data thus obtained was analyzed using a t test to study variation between two groups. Comparison of mean was done with ANOVA (SPSS 21 version). Pearson coefficient of correlation was calculated to study the significance of the correlation between different parameters. The level of significance was P < 0.05.
RESULTS
The study consisted of 100 diabetic patients from an outpatient department of medicine of a tertiary care hospital. Markers of oxidative stress, i.e., SOD and MDA, vitamin D, and HbA1c were measured. Of the 100 patients, 53 were men and 47 were women, with a mean age of 55 ± 10.6 years in men and 56 ± 10.7 years in women (Table 1). The patients were subdivided into three groups based on the duration of diabetes; 58 patients (32 men and 26 women) had diabetes for ≤5 years, 33 patients (19 men and 14 women) had diabetes for >5 to 15 years, and 9 patients (2 men and 7 women) had diabetes for >15 years.
Table 1.
Baseline parameters of study participants with type 2 diabetes
| Parameters | Men (n = 53) | Women (n = 47) |
|---|---|---|
| Age (years) | 55 ± 10.6 | 56 ± 10.7 |
| Waist circumference (cm) | 103.1 ± 11.5 | 100 ± 10.2 |
| Hip circumference (cm) | 101.3 ± 9.5 | 102.1 ± 9 |
| Body mass index (kg/m2) | 27.4 ± 4.3 | 29.4 ± 5.3 |
The levels of HbA1c increased from 8.5% ± 1.48% for those with diabetes <5 years to 8.8% ± 1.5% for those with diabetes for 5 to 15 years and then decreased to 8.5% ± 1.6% for those with diabetes >15 years. Although HbA1c did not follow a specific trend, it indicated unsatisfactory glycemic control in diabetic patients. As shown in Table 2, as the duration of diabetes increased, the levels of vitamin D followed a decreasing trend.
Table 2.
Levels of vitamin D in study participants with type 2 diabetes
| Duration of diabetes | Vitamin D (ng/mL) |
|---|---|
| ≤5 years | 55.4 ± 4.2 |
| >5–15 years | 49.6 ± 3.1 |
| >15 years | 36.5 ± 1.5 |
The levels of MDA ranged from 2 to 8 µmol/L, and the levels of SOD ranged from 105 to 357 units/mL. The levels of SOD (Figure 1a) and MDA (Figure 1b) followed an increasing trend with increasing duration of diabetes. The levels of SOD did not correlate significantly with HbA1c (r = 0.000, P = 0.995) and vitamin D (r = 0.133 and P = 0.195), and the levels of MDA did not correlate significantly with HbA1c (r = 0.016, P = 0.874) and vitamin D (r = −0.199, P = 0.051).
Figure 1.
Oxidative stress in diabetic patients based on duration of type 2 diabetes: (a) superoxide dismutase and (b) malondialdehyde.
DISCUSSION
Vitamin D is known to perform various functions in our body, including regulating insulin secretion and attenuation of systemic inflammation.11 HbA1c is a very useful parameter in diagnosing and monitoring T2DM and also in predicting the development of various diabetic microvascular complications.9,10 The development of various diabetic complications is also influenced greatly by oxidative stress.12 The relationship between these parameters (HbA1c, vitamin D, and oxidative stress parameters) needs to be discussed.
In our study, patients’ ages ranged from 34 to 80 years, with a mean of 55 ± 10.6 years in men and 56 ± 10.7 in women. The mean age in different studies is highly variable. The mean age of the current study is highly comparable to that of Turk et al, Al-Rawi, and Zare-Mirzaie et al.13–15 Most patients in this study (32 men and 26 women) were in group 1, with a duration of diabetes of up to 5 years. The probable cause is that recently diagnosed diabetics were in the outpatient departments and available for recruitment.
In the current study, HbA1c ranged from 6% to 12%, with means of 8.5% (duration ≤5 years), 8.8% (duration 5–15 years), and 8.5% (duration >15 years). Gradinaru et al reported a much lower value of 7.2%, which might be due to inclusion of subjects with only good and moderate glycemic control (<8.5%).16 Similarly Aoucheri et al, Zare-Mirzaie et al, and Dhas et al, reported relatively lower values.15,17,18 Sheth et al reported values of 8.36% ± 1.79%, which were comparable to those of the current study.19 In our study, levels of HbA1c indicated unsatisfactory glycemic control, which may be due to noncompliance with dietary and lifestyle modifications, along with irregular intake of prescribed medication. In rural India, due to unawareness and lack of education, the practice of alternative medicine is rampant; patients are drawn to the false belief of quick relief without the need for rigorous dietary and lifestyle modifications. Our study had a mix of urban and rural populations.
In the current study, MDA levels ranged from 2 to 8 µmol/L and SOD levels ranged from 105 to 357 units/mL. Parameters of oxidative stress (SOD) and lipid peroxidation (MDA) showed an increasing trend with a longer duration of diabetes. Exposure to high levels of insecticides and pesticides both in rural populations (while farming) and urban populations (consumption of products) is one contributing factor to increased oxidative stress. The increased levels of lipids are more susceptible to oxidation and formation of lipid peroxidation products, leading to increased levels of SOD and MDA, which are then implicated in the development of various disorders including diabetic complications.20
Study participants’ vitamin D levels ranged from 5 to >150 ng/mL, with a decreasing trend for a longer duration of diabetes. Overall, 22% had a vitamin D deficiency, 9% had insufficient levels of vitamin D, 54% had sufficient levels of vitamin D, and 15% had vitamin D toxicity. Gradinaru et al reported a considerably lower mean than the current study.16 In the study by Al-Timimi and Ali, 58.1% of the subjects had low vitamin D levels (<25 ng/mL), of which 4.4% were vitamin D deficient and the remaining insufficient.21 In the study done by Dhas et al, 70% of subjects were deficient in vitamin D and only 3.3% had sufficient vitamin D levels.17 Kumar et al and Zare-Mirazaie et al reported lower values of 10.47 ± 2.13 and 9.6 ± 5.3, respectively.15,22 The current study reported much higher values of vitamin D, with most subjects having sufficient vitamin D levels. This can be attributed to the fact that the study consisted of subjects from both urban and rural areas. Exposure to adequate or sometimes excess sunlight in rural dwellers might have contributed to the higher values observed in the study. In addition, we excluded patients with medical conditions affecting vitamin D metabolism (chronic kidney disease, chronic liver disease, malabsorption states, etc.).
Levels of vitamin D and oxidative stress markers did not follow any trend when compared among the groups. An insignificant correlation was observed between HbA1c, vitamin D, SOD, and MDA, which differed from other studies that reported a significant correlation between these parameters.18,21,22 Turk et al showed no correlation between SOD and any other parameters in their study.13 Saif-Elnasr et al showed no significant correlation between SOD and HbA1c (r = −0.138, P = 0.825) and between SOD and vitamin D levels (r = 0.205, P = 0.697).23 An insignificant positive correlation between SOD and HbA1c (P = 0.06) was reported by Bigagli et al.24 Aouacheri et al reported a significant positive correlation between levels of SOD and HbA1c in both men (P = 0.024) and women (P < 0.001).18
Some limitations of our study should be noted. First, patients were recruited from an outpatient clinic, and as such results may vary from those of population-based randomized studies. Second, with its cross-sectional design, our study has the inherent bias of observational studies. Also, due to lack of follow-up, the data presented are less representative of the general population. Despite these limitations, our study can be used as a scaffold for larger studies to be conducted in the future.
In conclusion, our study did not show any correlation between oxidative stress status and levels of vitamin D and HbA1c in patients with T2DM. Because of the small sample size, conclusions are only provisional. Hence, further studies with larger patient cohorts are required to study this correlation.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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