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The Association between Glycemic Control with Oxidant Status Parameters in Type 2 Diabetic Patients

Purpose: Glycemic control is important in order to avoid LDLs increased susceptibility to oxidation in diabetic patients. This study assess the relationship between diabetes control with serum prooxidant-antioxidant balance (PAB), oxidized LDL cholesterol (oxLDLc), homocysteine and vitamin D levels in patients with type 2 diabetes. Material and methods: This was a cross-sectional study on three groups including 80 subjects as well (WGC) and poor (PGC) glycemic control and 40 healthy subjects. Presence of nephropathy and retinopathy were determined using IDF criteria. HbA1c level was determined with columnar chromatography using BioSystems kit. Serum PAB, homocysteine, oxLDLc and vitamin D levels were measured by the standard tests. Results: There was a significant association between PAB with PGC (P< 0.001), diabetic retinopathy (P< 0.01) and nephropathy (P< 0.01) in type 2 diabetic patients. Moreover, the results showed that vitamin D serum levels was significantly lower in PGC patients (P< 0.01), and diabetic patients with retinopathy (P< 0.01). Multiple linear regression analysis revealed that the vitamin D deficiency can predict the HbA1c variations by 77.7% (β=- 0.775) in subjects with type 2 diabetes mellitus (P<0.001). Conclusions: There was a significant association between prooxidant-antioxidant balance and vitamin D serum levels with diabetic complications. (www.actabiomedica.it)


Introduction
Diabetes mellitus can be considered as one of the serious and costly multifactorial diseases with micro and macrovascular complications (1). According to the International Diabetes Federation (IDF), 285 million people worldwide are currently living with diabetes and 90-95% have type 2 diabetes (2). This number is expected to reach over 300 million by the year 2025 (3).
It was shown increased risk of cardiovascular disease (CVD) can explain the mortality in type 2 diabetes. Lipid peroxidation is high in type 2 diabetic patients and could be due to hyperglycemia, hyperhomocysteinemia and probably decreased antioxidant enzymes activity (4). It has been shown that hyperglycaemia has an important role in LDL susceptibility to oxidation (5). Elevated levels of oxidized LDL cholesterol (oxLDLc), which consider as a biochemical risk marker for CVD, have found in diabetic patients (6). Disruption of the prooxidant-antioxidant balance (PAB) can cause oxidative stress (7) which is well-documented in groups of patients with type 2 diabetes (8). Many studies reported conflicting results regarding the LDL cholesterol oxidation (9,10) and homocysteine level (11,12) in patients with type 2 diabetes. Epidemiological evidence supports the link between hypovitaminosis D and increased risk of mortality due to diabetes (13). It has been suggested that vitamin D exerts its anti-oxidant features by activating the MEKs/ERKs/SirT-1 axis (14).
Diabetes microvascular complications including retinopathy and nephropathy were associated with higher levels of HbA1c (15). The major cause of type 2 diabetes mellitus complications has not yet been clarified. Understanding the role of various nutritional or other modifiable risk factors that may contribute to the pathogenesis of diabetes, is important in the effort to combat the rising tide of diabetes and CVD worldwide (16). This study was conducted to assess the relationship between diabetes control, serum PAB, oxidized LDL cholesterol, homocysteine and vitamin D levels and microvascular diabetic complications in patients with type 2 diabetes mellitus in Iran.

Study population
This cross-sectional study was approved by Ethics Committee of Mashhad University of Medical Sciences, Mashhad, Iran. One hundred and twenty volunteers were included (80 with and 40 without type 2 diabetes). Questionnaires containing past medical history, social and family history were obtained and written informed consent was obtained from all individuals.
Venous blood samples (8-10 ml of whole blood) were obtained after a 10-h overnight fasting. EDTA was used as anticoagulant. HbA 1 c was determined in whole blood samples with columnar chromatography using BioSystems kit (Barcelona, Spain). Volunteers were divided into three age-and sex-matched groups including 40 subjects as well glycemic control: (HbA1c < 8%), 40 subjects as poor glycemic control, (HbA1c >10%) and 40 healthy subjects.

Biochemical measurements
The whole blood samples were centrifuged at 3000 rpm for 10 min, and the serum aliquots were separated and stored at -20˚C for the subsequent measurement of PAB, homocysteine, oxidized LDL cholesterol and vitamin D levels.
The PAB assay is based on 3,3',5,5'-tetramethylbenzidine (TMB) and its cation (17), and measure the balance of oxidants and antioxidants simultaneously in one experiment. It uses two different kinds of reactions: one is an enzymatic reaction where the chromogen TMB is oxidized to a color cation by peroxides and the second is a chemical reaction where the TMB cation is reduced to a colorless compound by antioxidants. The photometric absorbance is then compared with the absorbances given by a series of standard solutions (17). The standard solutions were prepared by mixing varying proportions (0-100%) of 250 mM hydrogen peroxide with 3mM uric acid (in 10mM NaOH). A low PAB value means that antioxidants are present at greater concentration than oxidants, while a high PAB value means more oxidants are present than antioxidants (18).
Serum homocysteine level were measured using immunoturbidometry based on assays to co-substrate conversion product (Demeditec Diagnostic GmbH, Lise-Meitner-strabe 2, D-24145 Kiel Germany). Serum homocysteine level was determined with an intraassay precision CV of 5.08%.
Oxidized LDL cholesterol levels were measured using a specific immunometric assay for human ox-LDLc using an enzyme-linked immunosorbent assay kit (Mercodia Oxidized LDL ELIZA; Mercodia AB, Uppsala, Sweden) and results were multiplied by dilution factor (6561) for this factor. Intra-assay precision CV was 6.2% for measuring serum oxidized LDL cholesterol levels.
Serum 25-hydroxy vitamin D (25OHD) levels were measured using a competitive electroluminescence protein binding assay (Roche Diagnostics vitamin D total assay kit; Roche Diagnostics, Mannheim, Germany) on a Cobas e411 analyzer. Serum 25OHD was measured with an intra-assay precision CV of 3.07%. Vitamin D insufficiency and deficiency were defined as a serum 25OHD of 50-75 nmol/L and <50 nmol/L, respectively (19). Presence of nephropathy and retinopathy were determined using the IDF criteria (20).

Statistical analysis
All data were analyzed using the Statistical Package for Social Sciences (SPSS version 11.5). The normality of distribution was assessed by the Kolmogorov Smirnov test. Categorical variables were compared using Chi-square test. Quantitative data were expressed as the mean ± SD for normally distributed variables or the median and IQR for not normally distributed variables. Analysis of normally distributed variables is included independent-samples T tests, One-Way ANOWA tests. Mann-Whitney U test and Kruskal-Wallis one-way analysis of variance used for not normal distribute variables. In order to assess the independent effects of vitamin D, homocysteine, PAB, oxidized LDL cholesterol, smoking, sex and age on measures of HbA1c, multiple regression analyses was performed separately in each group. A P-value <0.05 is considered as level of statistical significance for all tests.

Basic characteristics
There were three groups in this study including 40 subjects as well glycemic control, (HbA1c <8%, comprised 11 male and 29 female, aged 50.27±8.00 years), 40 subjects as poor glycemic control, (HbA1c >10%, included 15 male and 25 female, aged 48.5±11.00 years) and 40 healthy subjects comprised 11 male and 29 female (aged 46.02±7.71years). There was no significant difference in gender distribution between the groups (P= 0.535). Demographic and some clinical characteristics in each group of study are shown in Table 1. Data indicate significant differences in clinical factors such as cardiovascular disease, retinopathy and nephropathy between three groups. Poor control diabetic patients had a significantly higher CVD, diabetic retinopathy and nephropathy (P <0.001, for all), (Table 1).

Biochemical factors
Level of biochemical factors in the three groups of the study are shown in Table 1. Poor control diabetic patients had a significantly lower vitamin D concentration and higher PAB, homocysteine and oxidized LDL cholesterol levels (P <0.001, for all), (Table 1). There was a significant difference in frequency of vitamin D deficiency between the study groups. The frequency of vitamin D deficiency was 100% and 95% in subjects with poor glycemic control and healthy subjects, respectively. The frequency of vitamin D insufficiency was 100% in well control glyceamic subjects (Table 1).

Prooxidants antioxidants levels
There was a significant higher PAB in poor glycemic control than well glycemic control (p <0.001). Also, positive retinopathy (p <0.01), and nephropathy (p <0.01) diabetic patients had higher serum PAB levels compared to without retinopathy and nephropathy subjects, (Table 2).

Homocysteine levels
Average homocysteine level in poor glycemic control patients was higher than other groups, (p <0.001), ( Table 1). As shown in Table 2, poor glycemic control patients had a significantly higher serum homocysteine concentration than well glycemic control subjects, (p <0.01). Positive nephropathy patients had a higher serum homocysteine compared to without nephropathy diabetic subjects (p <0.05).

Oxidized LDL cholesterol levels
Average oxidized LDL cholesterol was higher in poor control subjects compared to well control group (p <0.05), ( Table 2). As shown in Table 2, there was no difference in serum oxidized LDL cholesterol level between subjects with and without retinopathy, or nephropathy (p = 0.134 and p = 0.407, respectively).

Vitamin D
There was a significant difference in serum vitamin D levels between well glycemic control diabetic patients, poor glycemic control diabetic patients and control group (p <0.001), (Table 1). Serum vitamin D levels in well glycemic control patients were higher than poor glycemic control ones; this rate was 58.00 (56.79-59.73) and 19.57 (14.25-26.84) nmol/L, respectively (p <0.001). There was a significantly lower in serum vitamin D levels in positive retinopathy diabetic patients than no-retinopathy diabetic patients (p <0.001), (Table 2).

Discussion
In current study, some oxidant status parameters including serum pro-oxidant-antioxidant balance, oxidized LDL cholesterol, homocysteine and vitamin D levels determined in well and poor control diabetic patients and healthy subjects. Our results are largely confirmatory in a small number of patients. The most important finding of this study was the fact that the poor control diabetic patients had significantly higher serum PAB, homocysteine and oxidized LDL cholesterol levels compared to the well control diabetic patients. Additionally, the serum level of vitamin D was lower in poor control diabetic patients than the well control diabetic patients.
Another interesting finding was the high frequency of vitamin D deficiency in our subjects that has not been widely reported. The major reasons for low vitamin D levels have not been known in the Iranian population. Clothing due to cultural issues can decrease sun exposure in Iran (21). Daneshvar et al. have reported that the intake of vitamin D is lower than recommended dietary allowances in adults in Isfahan (22). Bonakdaran et al. have suggested that sedentary life style and sunscreen use are other reasons for high frequency of vitamin D deficiency in Iranian population. They have shown that the frequency of vitamin D deficiency was 80.7% in patients with metabolic syndrome in Iranian population (23).
In current study, there was a significant lower in serum vitamin D levels in positive retinopathy diabetic patients compared to no-retinopathy ones. Additionally, multiple linear regression analysis showed that the vitamin D deficiency can predict the HbA1c variations by 77.7%. A number of studies have reported that vitamin D supplementation has no significant effects on HbA1c level in type 2 diabetic patients (24,25). Calvo-Romero et al. have found a small non-significant reduction in HbA1c in patients with type 2 diabetes mellitus after supplementation with vitamin D at least for 8 week (26). While, Ahmadieh et al. have reported hypovitaminosis D is an independent predictor of HbA1c, diabetic neuropathy and retinopathy in patients with type 2 diabetes (27). It seems that low vitamin D receptor signaling is the potential mechanism by which vitamin D deficiency can mediate risk of cardiovascular disease in type 2 diabetic patients (28). One study showed serum vitamin D level were lower in subjects with diabetic retinopathy than subjects without diabetic retinopathy (29). This result is in concordance with our finding. In current study, average PAB and oxidized LDL cholesterol levels were higher in poor control subjects compared to those well glycemic control diabetic patients. A high PAB value means more oxidants are present than antioxidants (18). Also, positive retinopathy and nephropathy diabetic patients had higher serum PAB levels compared to without retinopathy and nephropathy subjects. It seems that change in PAB levels is thought to be the cause of chronic diabetic complications, consistent with the literature. Kumawat et al. have reported that alteration in anti-oxidant status can help to predict the risk of diabetic retinopathy (30). Our results is consistent with a previous study shown that glycemic control is very important in order to avoid an increased susceptibility of LDLs to oxidation in diabetic patients (31). It is well documented that oxidized LDL cholesterol can activate the pathways associated with innate immunity and trigger proinflammatory events (32). Alkalin et al. showed that oxidized LDL cholesterol has been increased in diabetic patients and this may contribute to the increased atherogenesis in diabetes (33). Therefore, the outcome of our study shows the supportive role of well glycemic control decreasing the level of oxidant status parameters in type 2 diabetic patients.
Timar et al., have reported that high dose vitamin D supplementation (50000IU) once weekly during 9 weeks can increase the serum PAB levels in both groups of adolescents girls (aged 12-18) years with BMI <25 and ≥25 (kg/m 2 ). This result may be because of the high dose, or long-term of vitamin D in this study (34). Therefore, whether mega dose is better than low dose is controversial among scholars yet.

Study limitations
Present study was a cross-sectional analysis which had some limitations. Firstly, it lacks baseline data on seasonal changes, sun exposure and thus vitamin D level. Secondly, we don't have enough information about drug use, vitamin D injection and nutritional status of volunteers.

Conclusions
This study indicates that there was a significant association between glycemic control with prooxidantantioxidant balance and vitamin D serum levels.