Vitamin D status in type 2 diabetic patients and its association with glycemic control, lipids & microalbuminuria: A pilot study

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1 Vitamin D status in type 2 diabetic patients and its association with glycemic control, lipids & microalbuminuria: A pilot study Shaafie IA 1 *, Hesham RA 2, Basha AA 3 1 Department of Biochemistry, 2 College of Graduate Studies, Gulf Medical University, Ajman, UAE 3 Department of Internal Medicine, Gulf Medical College Hospital, Ajman, UAE * Presenting author ABSTRACT Objective: The objective of the present study was to determine the vitamin D status of type 2 diabetic patients and correlate the levels with glycemic control, lipids and microalbuminuria. Materials & Methods: A pilot study was conducted on 192 randomly selected type 2 diabetic patients of both sexes from different ethnic backgrounds, in the age group of years, with or without hypertension and dyslipidemia, who attended the medical OPD at GMC Hospital Ajman from January to April A questionnaire was used to obtain sociodemographic and clinical details about the patients. American Diabetic Association Criteria (2013) were used for diagnosis. Patients were classified into three groups according to their total vitamin D status: deficient (10-<20 ng/ml), insufficient (20-30 ng/ml), Sufficient (optimum >30-50 ng/ml). Fasting blood specimens were analysed for glucose, total vitamin D, calcium, phosphorus, lipoproteins and HbA1C levels in addition to routine kidney functions using Cobas 601 analyzer (Roche). Morning urine specimens were analyzed for Urinary Albumin Excretion Rate (UAER) using DCA 2000 analyzer (Bayer) employing Turbidimetric Inhibition Immunoassay. UAER between µg/mg creatinine was described as microalbuminuria. Results & Conclusions: Vitamin D deficiency & insufficiency was found among 96.8% of type 2 diabetic patients. Serum calcium and phosphorus levels and routine kidney function tests were normal. Plasma glucose, lipoprotein and HbA1C levels were significantly elevated (p <0.05) in patients with raised UAER compared to patients with normal UAER. HbA1C and LDLc showed a negative correlation with vitamin D levels. The presence of elevated UAER, HbA1C and LDLc levels in type 2 diabetic patients with low vitamin D levels suggest the role of vitamin D deficiency in the pathogenesis of type 2 diabetes mellitus and its complications. The results of the pilot study will be used in carrying out our study on large cohort of type 2 diabetics and identify the role of vitamin D deficiency in its pathogenesis and complications. In the meantime, it is suggested that proper exposure to sun in addition to regular intake of vitamin D and calcium may delay the development of type 2 diabetes and its complications. Keywords: T2DM, ADA criteria, microalbuminuria, UAER, HbA 1 C, dyslipidemia, egfr, lipid profile, renal profile, ECLIA, turbidimetric inhibition immunoassay INTRODUCTION Type 2 Diabetes mellitus (T2DM) is a major chronic non-communicable disorder having a high morbidity and mortality due to its complications 1. Diabetes continues to be a major public health problem. Currently 371 million people worldwide have diabetes 1.It has been estimated that 552 million individuals would be affected with diabetes worldwide by the year More than 80% of diabetes deaths occur among the population of low and middle income groups. It has been projected that diabetes deaths will increase by two thirds between 2008 and In spite of the widespread knowledge about the risk factors associated with T2DM, the etiopathogenesis of the disease and its complications are still being debated. Oxidative stress, inflammation and autoimmune reactions have emerged as 6

2 major pathogenic effectors for diabetes 3. The increasing burden of T2DM has led to the search for various predisposing factors for the disease. Identification and control of these factors will reduce the prevalence of diabetes mellitus and its complications. Obesity is the most common and well known predisposing factor for T2DM. Recently, vitamin D has generated widespread interest for its role in the pathogenesis and prevention of diabetes mellitus 4. The first indication about this role was the observation of poor glycemic control during winter months when hypovitaminosis D is common 5. Many researchers suggest that vitamin D may be a modifiable risk factor for diabetes mellitus 6,7,8. The major evidence about the role of vitamin D in prevention of T2DM came from a Finnish study in which researchers collected health data from men and women in the age range of years. None of the individuals had diabetes at the start of the study. The subjects were followed for 22 years to see the pattern of development of T2DM.It was found that the subjects with high level of vitamin D were less likely to develop T2DM, thereby indicating that the vitamin had a protective effect against the development of T2DM 2. A number of studies have shown that vitamin D deficiency affects insulin levels and thus the glucose metabolism 9,10,11. The use of vitamin D with calcium supplementation led to a significant fall in the insulin resistance and glucose levels in patients with impaired fasting glucose levels 12. The studies on human 13,14 and animal models 15 of type I diabetes suggest that vitamin D supplementation may improve and even prevent the development of diabetes. Vitamin D is involved in multiple physiological functions in additional to its classic effect on calcium homeostasis. These diverse actions may be due to the presence of vitamin D receptors in almost all the tissues in the body. Many studies have shown that vitamin D plays a complex physiological role in human body including regulation of insulin synthesis and secretion, modulation of the inflammatory response, cell maturation and cell differentiation 16,17. A number of cross-sectional studies have shown an inverse relationship between vitamin D status and prevalent hyperglycemia in T2DM patients 18.Low vitamin D status has been associated with increase in the markers of impaired glucose metabolism like HbA1C 19,20. A number of studies have shown an association between microalbuminuria and vitamin D deficiency in T2DM 21,22. Vitamin D acts as a negative regulator of reninangiotensin system and thereby protects kidney from the endothelial damage which leads to microalbuminuria 23. A few epidemiological studies conducted in UAE show that T2DM is highly prevalent in the country (19.2%) and a large number of diabetics develop chronic complications 1. A study conducted by Fatima Al Anouti et al. (2011) showed that 68% of the students from Sheikh Zaid University had low serum vitamin D levels 24. Another study conducted by Shamma JM, et al (2012) involving adolescent students of Al-Ain schools showed that 19.7 % of them were vitamin D deficient and 45.4% had vitamin D insufficiency 25.However,there is no documented study till date showing a relationship between vitamin D deficiency and type 2 diabetes mellitus in UAE population. OBJECTIVES 1. To study the vitamin D status in Type II Diabetic patients (T2DM) with and without co-morbidities. 2. To determine any association of vitamin D status with glycemic control, lipid profile and albumin excretion in the type 2 DM patients studied. HYPOTHESIS The study is based on hypothesis that Vitamin D deficiency does not have any association with Type 2 Diabetes Mellitus and does not play any role in the pathogenesis and complications of the disease. 7

3 MATERIALS AND METHODS Study Population: A pilot study was conducted on 192 randomly selected Type 2 Diabetic (T2DM) patients of both genders(m141,f51;m/f ratio 2.76:1) with different ethnic background(south Asia 45%,Middle East 53%,Others 2%), in the age group of years who attended the medical OPD at GMC Hospital Ajman from January to April, Majority of the patients (79.7%) were in the age group of years. In addition to diabetes, 60 patients(31%) had hypertension,103 patients(54%) dyslipidemia,8 patients (4%) neuropathy and 3 patients (2%) retinopathy. 18 patients were excluded from the initial group of 210 patients because of incomplete clinical and/ or laboratory data. The patients excluded from the study were Type I Diabetics, patients with known renal and cardiovascular disease, pregnant and lactating women, patients on vitamin and calcium supplements, hypertensive patients on calcium channel blockers and patients with recurrent infections. All known diabetic patient were on oral hypoglycemic drugs and patients with hypertension and dyslipidemia were taking statins regularly. Study instrument: A questionnaire was used to obtain socio-demographic (age, gender, weight, ethnicity, weight, height, BMI) and clinical details (duration of diabetes, medications, presence and duration of diabetic complications) of patients. The questions related to dietary history were adopted according to the recommendations of MRC, UK. The questionnaire was validated by the subject experts and pilot tested before use. Patients were diagnosed as type 2 diabetics using the American Diabetic Association (ADA) Criteria, Ethical issues: The study was conducted after approval from GMU Ethics Committee. Informed written consent was obtained from each patient regarding participation in the study. Patient confidentiality was respected and any specification reflecting the identity of the person was omitted from the data. METHODOLOGY After clinical diagnosis of T2DM by the attending physician the patients were recruited in the study. Consent was taken and questionnaire filled up. Fasting blood and urine specimens were collected. Blood analysis was done for glucose, lipids, routine kidney functions, calcium and phosphorus using the standard enzymatic-colorimetric procedures on Cobas 601 Analyzer (Roche). The inter- and intra-assay variation for the biochemical parameters was less than 3%.Vitamin D was estimated by Electro- Chemiluminiscence-Immunoassay (ECLIA) technique on Cobas 601 analyser ( Roche). The inter-assay CV for vitamin D was 6% for low control and 8% for high control. HbA 1 C and Albumin Excretion Rates were estimated by Turbidimetric Inhibition Immunoassay using DCA 2000+Analyzer System (Siemens, USA). Reference Ranges: The laboratory reference ranges were used to interpret the results of routine biochemical investigations. The reference ranges for vitamin D as recommended by the Institute of Medicine (USA) and American Association of Clinical Endocrinologists and applicable to our patients were used27,28. Vitamin D level 10-<20 ng/ ml was described as deficient, ng/ ml as insufficient and >30-50 ng/ml as sufficient (Optimum). These reference ranges are based on the observation of the levels required for prevention of fractures and other diseases, minimal stimulation of parathyroid hormone and maximum intestinal absorption of calcium. Urinary Albumin Excretion Rate (UAER) was expressed as ug albumin excreted/mg of creatinine in urine and classified as: Normal(<30), Mild(30-100), 8

4 Moderate(> ), Severe(> ), Macroalbuminuria(>300) ug/mg. The term microalbuminuria was used to describe urinary albumin excretion between ug/mg creatinine. HbA1C levels (%) were described as: Excellent control ( ),Good control( ),fair control( ) and Poor control(>8.0)26. Estimated GFR (egfr) was calculated according to the new equation proposed by levey et al29. Statistical analysis: The data was analysed using t test, Pearson correlation co-efficient and ANOVA.All statistical calculations were performed using SPSS for Windows, Version 20.0 (IBM Chicago,USA). RESULTS & DISCUSSION 1. Baseline Characteristics of Patients: Table 1 shows the mean age of the T2DM patients studied (both males and females) was ± 8.56 years. The mean BMI was ± 5.34 Kg/m 2 with 25% patients being overweight and 62% obese. The duration of diabetes varied from zero (newly diagnosed) to 23 years with a median value of 7.74 ± 5.68 years. Vitamin D level varied from 3.0 to 32 ng/ ml with a median value of ± 6.88 ng/ml. Vitamin D deficiency was seen in 32.3% patients and insufficiency in 54.7% patients in spite of normal dietary intake of calcium ( mg/ day) and vitamin D ( IU/day). Routine kidney function tests and egfr were within the reference ranges. The mean HbA1C level was % and Fasting Plasma Glucose mg/dl indicating that diabetes was not well controlled. 2. Correlation of Vitamin D Levels with other Biochemical Parameters: Table 2 shows that vitamin D is negatively and weakly correlated with HbA1C, Total Cholesterol (TC), LDLc, TAG and AER. However, a significant positive correlation (r=0.43) was observed between HbA1C and AER. Table 4 shows that T2DM patients with microalbuminuria have significantly raised levels of TC, LDLc and TAG when compared to patients without Table 1: Baseline characteristics of Type 2 Diabetic Patients (n=192) Parameters Age* Units years Mean/Median SD BMI Kg/m Duration of Diabetes** Vitamin D*** Calcium Phosphorus Fasting Plasma Glucose Urea Creatinine Uric acid years ng/ml egfr ml/min/1.73 m HbA 1 C % Calcium Intake mg/day Vitamin D intake IU/day *Age: years;**duration of Diabetes:0-23 years;***vitamin D 3-32 ng/ml. 9

5 microalbuminuria(p<0.05). LDLc levels are markedly elevated when compared to TC and TAG levels. HDLc levels do not show any difference in patients with or without microalbuminuria. Table 2: Correlation of Vitamin D levels with other Biochemical Parameters in T2DM Patients Parameters Correlation coefficient Hb A1c TC HDL LDL TAG * AER Note: There was a significant correlation between HbA1C and AER (r=0.43) 3. Relationship between Vitamin D Status and Urinary Albumin Excretion Rate in T2DM Patients: Table 3 shows that the majority of patients (103/192; 54%) had vitamin D deficiency with microalbuminuria(mild).none of the patients with optimum vitamin D level had microalbuminuria.29% of patients with vitamin D deficiency or insufficiency but with normal AER may be the freshly diagnosed and/or well controlled diabetics. 4. Relationship between HbA1C and Vitamin D Status in T2DM Patients: A great variation is observed in the glycemic control of T2DM patients when the results are compared to their vitamin D status.table 5 shows that the majority of patients (140/192; 73%) with vitamin Table 3: Distribution of T2DM Patients according to their Vitamin D Status and Urinary Albumin Excretion Rate (UAER) UAER (ug Albumin/mg Cr) Vitamin D Status(ng/mL) Deficient Insufficient Sufficient (10-<20) (20-30) (Optimum) Total (>30-50) Normal < Mild Moderate Severe Macro > Total Table 4: Comparison of Serum Lipid Profiles in T2DM Patients with and without Microalbuminuria (n=192) Tests Mean (All patients) Microalbuminuria P- value Present Absent TC () <0.05 TAG () <0.05 LDLc () <0.05 HDLc () NS 10

6 Table 5: Relationship between HbA1C and Vitamin D Levels in T2DM HbA1C(%) Vitamin D Status(ng/mL) Total Deficient (<20) Insufficient (20-30) Optimum (>30) <= > Total D deficiency have poor glycemic control. Although all the patients were on oral hypoglycemic, only 16% of them showed good control of diabetes. 5. Relation between HbA1C and Urinary Albumin Excretion Rate(UAER) in T2DM Patients: The patients were categorized according to their HbA1C levels and Albumin Excretion Rate. Table 6 shows that Table 6: Relationship between HbA1C(%) Levels and Urinary Albumin Excretion Rate(ug/mg) in T2DM(r=0.43) HbA1C (%) Normal <30 Mild Moderate Severe Macroalb. >301 <= > Total Total UAER (µg alb/mg Cr.) Figure 1: Correlation of Urinary Albumin Excretion Rate (UAER) with Hb A 1 C in T2DM Patients 11

7 only 26% patients had normal AER but varying HbA1C levels. 70% of patients had microalbuminuria (AER µg/mg) with poor control of diabetes. A positive correlation was observed between HbA1C and UAER in the patients (Figure 1). CONCLUSIONS AND SUGGESTIONS 1. Vitamin D deficiency is prevalent among T2DM patients in spite of apparent normal dietary intake of calcium and vitamin D. 2. The presence of normal serum calcium and phosphorus levels in spite of vitamin D deficiency may be due to associated secondary hyperparathyroidism. 3. A positive correlation between HbA 1 C and urinary albumin excretion levels indicates that effective control of diabetes mellitus is essential in reducing microalbuminuria thereby delaying the development of nephropathy. Hypertension seems to be a contributing factor for microalbuminuria in our patients. 4. The presence of normal kidney functions and egfr in spite of increased urinary albumin excretion suggests that the two conditions are unrelated and may be acting independently in the pathogenesis of nephropathy. Vitamin D has been shown to have a renoprotective role and its deficiency is characterized by activation of a number of proinflammatory factors damaging the glomerular basement membrane leading to microalbuminuria. 5. The presence of high LDLc levels in T2DM patients with microalbuminuria suggests that the former may have a direct effect on the glomerular epithelial and mesengial cells leading to their damage. Some studies show that small LDL particles (sldl) may be involved in the pathogenesis of microalbuminuria. 6. Improvement in glycemic control and albumin excretion in diabetic patients by vitamin D supplementation has not been proved yet and the work is still in its experimental stage. 7. General screening of UAE population for their Vitamin D status may be carried out and the relationship of Vitamin D status with the development and complications of diabetes mellitus may be studied. 8. Proper exposure to sunlight remains the best option at present to synthesize optimal levels of endogenous vitamin D for carrying out the physiological functions. This is the natural way to prevent vitamin D deficiency in UAE, a sun rich country, where 18.9 % of population lives with diabetes 1. REFERENCES 1. Diabetes atlas. Available from: URL: www. idf.org/diabetesatlas/5e/the global burden, Assessed on Knekt P, Laeksonan M, Mattila C, et al. Serum vitamin D and subsequent occurance of type 2 diabetes. Epidemiology Sep;19(5): Pickup JC. Inflammation and activate innate immunity in the pathogenesis of type 2 diabetes. Diabetes Care. 2004;27(3): DeLuca HF. Overview of general physiologic features and functions of vitamin D. Am J ClinNutr. 2004;80(6 Suppl): CampbellIT, Jarrett RJ, Keen H. Diurnal and seasonal variations in oral glucose tolerance. Diabetologia. 1975;11: Mathieu C, Waer M, Casteels K, et al. Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1, 25-dihydroxyvitamin D3, Kh1060.Endocrinology. 1995;136: Zella JB, DeLuca HF. Vitamin D and autoimmune diabetes. Cell J Biochem. 2003;88: Gregori S, GiarratanaN, Smiroldo S, et al. A 1, 25-Dihydroxyvitamin D 3 Analog Enhances Regulatory T-Cells and Arrests Autoimmune Diabetes in NOD Mice Diabetes. 2002;51: Zipitis CS, Akobeng AK. Vitamin D supplementation in early childhood and risk of type 1 diabetes: a systematic review and meta-analysis. Arch Dis Child. 2008;93:

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