European Journal of Cardiovascular Medicine

Diabetes Mellitus, a chronic metabolic disorder  characterised by elevated blood glucose values, due to decreased insulin secretion and increased resistance to action of Insulin in peripheral tissue. Diabetes affects an estimated 537 million adults worldwide between the age of 20 to 79 (10.5% of all adults in this age range). By 2030, 643 million people will have diabetes globally, increasing to 783 million by 2045. The estimated prevalence of diabetes in adults (20 to 79 years) has more than tripled since the first edition in 2000, rising from an estimated 151 million (4.6% of the world’s population at the time) to 537.5 million (10.5%) of the world’s population today. The prevalence rate will be higher than 12.8% by 2045. In addition, this study indicates that the incidence of diabetes in the world, Southeast Asia, and India was 10.5%, 8.8%, and 9.6%, respectively, throughout 2021 and will rise to 12.5%, 11.5%, and 10.9%, respectively by 2045.[1]

Type 2 diabetes, which constitutes 90% of all cases of diabetes, earlier considered to be a disease of the affluent “Western” countries, has now spread globally, and has become a major cause of disability and death. [2] Vitamin D, cholecalciferol and ergocalciferol , is a fat soluble vitamin produced in Malpighian layers of skin on exposure to UV-B light . Vitamin D has a physiological role of maintaining Calcium and Phosphorous levels in blood and it is important for inflammation, immune function, cell proliferation and differentiation. Vitamin D deficiency has been associated with increased risk of  type 2 diabetes mellitus, insulin resistance, and decreased insulin  production, and hence, it has been associated with syndrome X. T2DM is associated with decreased 25-OH Vit D compared to controls Vit D deficiency in diabetes is also associated with poor glycaemic control.   Evidence reveals that Vitamin D reduces the risk of progression and development of type 2  diabetes mellitus.[3,4,5,].

Vitamin B12 is an essential micronutrient required for optimal haemopoietic, neuro-cognitive and cardiovascular function. Biochemical and clinical vitamin B12 deficiency has been demonstrated to be highly prevalent among patients with type 1 and type 2 diabetes mellitus. [6]  Vitamin B12 deficiency is a potential comorbidity that is often overlooked, despite the fact many diabetic patients are at risk for this specific disorder. For example, many diabetic patients are treated with metformin, a medication that lowers serum vitamin B12 levels and is associated with vitamin B12 deficiency  affecting even younger age group.[7-8] 

Ferritin, one of the key proteins regulating iron homeostasis, is a widely available clinical biomarker to evaluate iron status and especially important for detecting iron deficiency.  Serum ferritin is also a marker of systematic inflammation . However, growing evidence has shown that even moderately increased iron stores, represented by high-normal ferritin concentrations, are associated with diabetes. There is positive association between elevated iron stores due to increased ferritin levels and the risks of T2DM. There is positive correlation between increased HbA1C levels and Increased Ferritin levels  [9].

Several studies have found increased ferritin levels in association with such diabetic complications as retinopathy, nephropathy, and vascular dysfunction in patients with DM and with elevated FBG.[10,11] Hence the present study is undertaken as comparative cross sectional study in tertiary  care hospital    with aim to  assess Serum Vit D  ,  Serum Vit B 12  and Ferritin levels in type 2 Diabetes mellitus patients  and to compare them with healthy controls  to understand the prevalence of Vit D deficiency, Vit B12 deficiency and Hyperferritinaemia  and correlate to Glycated Hemoglobin, Glycaemic status marker in diabetic patients attending our hospital.

Objectives: The present study aims to assess vitamin D , Vitamin B12 deficiency and increased ferritin levels in diabetic population and correlate to glycaemic status marker

The objectives for this study are .

A comparative cross sectional study was conducted in the Department of Biochemistry,  24 hrs Laboratory- RMC , KKD over a period of 6  months, March  2024-  August 2024.

Grouping newly diagnosed Diabetic patients in group I and normoglycemic controls  in group II as controls. 50 newly diagnosed type 2 DM  patients  constitute group I, 50 age and sex matched Healthy individuals are enrolled in group II.

Selection of cases :

Inclusion criteria:

1. 50 newly diagnosed diabetes patients during the study period , attending the OPD are enrolled into study after taking their informed consent.
2. Age group: 25-40 years.

               Exclusion criteria:

1. Diabetes Mellitus patients on Medication.
2. H/O CRF, CAD, DM, Hypertension, Liver disorder, Chronic diseases.
3. Pregnancy
4. On iron supplementation , Vit D and Vit B 12 supplementation

Controls selection :

Inclusion Criteria:

1. 50 healthy individuals with no history of thyroid disorders .( euthyroid status is confirmed by doing the thyroid profile test and  report values are within the reference range)
2. Age group 30-45 years ,

               Exclusion Criteria:

1. H/O recent hospitalisation, blood donation.
2. On iron supplementation
3. H/O chronic disorders,

Data collection:

Demographical information (age and  gender) of cases and controls are noted in Data collection Tables. Study participants are advised to be on overnight fast and blood sample is collected in morning  in labelled red topped  vacutainer for  Fasting Plasma Glucose (FPG),  Serum Vit B12, Serum Vit D ,  Serum  Ferritin) and violet topped vacutainer for glycated Hemoglobin  and also in Post Prandial conditions ( 1 hr 30 min  after breakfast for Post Prandial Plasma Glucose  under strict aseptic conditions.

Sample Processing: After properly mixing the sample in vacutainer, it is left at room temperature for 15-20 minutes for clot formation. The vacutainer is then centrifuged at 3000-5000 rpm for 15 minutes for separation of serum. The serum is aliquoted into labelled Eppendorf tubes. They are analysed immediately and stored at refrigeration at -20C till completion of study process.

Biochemical analysis: Beckman coulter Access 2  immunoassay analyser and AU 480 clinical chemistry analyser are used for analysis. The instruments are calibrated and calibration is checked by using appropriate controls.

1. Vit B12 Analysis: Vit B12 is analysed in serum sample by Paramagnetic particle Electro chemiluminescent Immnuoassay on Access 2 Instrument
2. Vit D analysis: 25- OH Vit D is analysed in serum sample by paramagnetic particle enhanced electrochemiluminescent immunoassay on Access 2 instrument.
3. Ferritin analysis: Ferritin is analysed in serum sample by paramagnetic particle, chemiluminescent , two site immune enzymatic method on Access 2 instrument.
4. Glucose estimation : Glucose is estimated in serum sample by Hexokinase method , on AU480 clinical chemistry analyser.
5. Glycated Hemoglobin is estimated in whole blood by turbidometric immunoinhibition method. on AU 480 clinical chemistry analyser.

Statistical Analysis:  All the participants data after collection and analysis is saved on the spreadsheets in MS  excel. SPSS software is used for data analysis. After testing for normal distribution of data of measured variables, Mean + SD is used to summarise this data. ANOVA and Student t test are used for comparison of means among the different  variables for detection of significant difference. p value <0.05 is used for identification of statistically significant difference of variables among groups. Pearson’s  correlation test is used to calculation of r value to determine the strength of association between variables

In graph 1, Age (Years): Controls are significantly younger (mean 34.43 ± 11.05) than Cases (mean 44.5 ± 12.7). The p-value of 0.018 (< 0.05) indicates this age difference is statistically significant.

Graph 1: Comparison of mean age in cases and controls

Graph 2: Distribution of Males and Females in Cases and Controls

In graph 2, Gender distribution between the two groups was comparable, with no significant difference in the number of males (17 in cases vs. 16 in controls) and females (13 in cases vs. 14 in controls), as shown by a p-value of 0.79. This indicates that gender was well matched and did not influence the biochemical comparisons.

Graph 3: Comparison  of mean FPG and PPPG in cases and controls

In graph 3, Fasting Plasma Glucose (FPG) levels were markedly elevated in the diabetic group (134.1 ± 50.1 mg/dL) compared to controls (75.3 ± 19.8 mg/dL), with a highly significant p-value of 0.0001. Similarly, Postprandial Plasma Glucose (PPPG) was significantly higher among diabetic patients (217.8 ± 60.1 mg/dL) than in controls (120.1 ± 21.5 mg/dL), also with a p-value of 0.0001. These findings confirm the presence of both fasting and postprandial hyperglycemia in T2DM patients.

Graph 4: Comparison of  mean HbA1C (%) values in cases and controls

In Graph 4, HbA1c levels—a marker of long-term glycemic control—were substantially elevated in the diabetic group (11.2 ± 3.1%) versus the control group (5.1 ± 0.7%), with a p-value of 0.0001, further validating the diagnosis of chronic hyperglycemia.

Graph 5: Comparison of mean Ferritin (ng/mL) in cases and controls

In graph 5, Ferritin levels, often associated with both iron storage and inflammation, were significantly lower in the diabetic group (196.0 ± 91.9 ng/mL) than in controls (279.2 ± 88.4 ng/mL), with a p-value of 0.0007.

Graph 6: Comparison of mean Vitamin B12 Values (pg/mL) in cases and controls

Graph 7: Comparison of mean Vitamin D Values ( ng/mL) in Cases and Controls

In graph 5, Serum Vitamin D levels were significantly lower in diabetics (23.9 ± 15.4 ng/mL) compared to controls (33.9 ± 16.6 ng/mL), with a p-value of 0.0187. This finding supports existing evidence linking Vitamin D deficiency with insulin resistance and impaired glucose metabolism. Furthermore, Vitamin B12 levels were markedly reduced in the diabetic group (462.4 ± 189.6 pg/mL) relative to controls (706.1 ± 123.7 pg/mL), with a p-value of 0.0001.

This study aimed to evaluate and compare glycemic markers and micronutrient profiles—specifically Vitamin D, Vitamin B12, and Ferritin—between newly diagnosed type 2 diabetes mellitus (T2DM) patients and normoglycemic controls. The findings clearly demonstrate significant differences between the two groups, supporting the growing evidence that micronutrient imbalances are associated with the early stages of diabetes pathogenesis.

As expected, fasting plasma glucose (FPG), postprandial plasma glucose (PPPG), and glycated hemoglobin (HbA1c) levels were significantly elevated in the diabetic group. These parameters are the cornerstone of diabetes diagnosis, and their elevation reflects poor glucose regulation due to insulin resistance or inadequate insulin secretion. Our results are consistent with diagnostic thresholds established by the American Diabetes Association (ADA), and similar patterns have been observed in earlier Indian cohort studies, including those by Mohan et al. (2018), reinforcing the reliability of these markers in identifying glycemic status. [13]

Importantly, the study also revealed significantly lower levels of Vitamin D in the diabetic group compared to controls. Vitamin D has been increasingly recognized for its role beyond bone health, including in glucose metabolism and immune regulation. Several studies, including those by Forouhi et al. (2008) and Muscogiuri et al. (2017), have reported an inverse relationship between Vitamin D levels and insulin resistance. [14,15] Vitamin D receptors are expressed in pancreatic β-cells, and its deficiency is thought to impair insulin secretion and increase systemic inflammation, both of which contribute to the development of T2DM. [16] The deficiency observed in our newly diagnosed patients suggests that hypovitaminosis D may be a contributing factor rather than merely a consequence of diabetes. [17]

Similarly, Vitamin B12 levels were significantly lower in diabetic patients. While B12 deficiency is classically associated with prolonged metformin use, all participants in our diabetic cohort were newly diagnosed and not on medication, suggesting a pre-existing or metabolic basis for the deficiency. Reinstatler et al. (2012) reported widespread subclinical B12 deficiency in U.S. adults with diabetes, while studies in India have shown a strong association between vegetarian diets, low socioeconomic status, and B12 deficiency. [18] Vitamin B12 plays a key role in neurological function and erythropoiesis, and its deficiency may further exacerbate diabetic neuropathy and anemia. [20]

An interesting finding in our study was the significantly lower serum ferritin levels among diabetics. This contrasts with some previous studies, such as Aregbesola et al. (2016), which observed hyperferritinemia in diabetics and proposed that elevated ferritin could be a marker of insulin resistance or inflammation. [21] However, our study excluded participants with chronic diseases and comorbidities, which are known to elevate ferritin as an acute-phase reactant. Thus, our findings may reflect true iron storage levels rather than inflammation-related elevations. The relatively lower ferritin levels could also be related to poor dietary iron intake or gastrointestinal losses, which are often unrecognized in early-stage diabetics. [22]

Overall, our results suggest that newly diagnosed T2DM patients are not only hyperglycemic but also prone to micronutrient deficiencies. These alterations may contribute to or exacerbate the progression of diabetes and its complications. Early screening and correction of such deficiencies could serve as a cost-effective strategy to improve metabolic control and reduce long-term morbidity. Further large-scale studies are warranted to validate these findings and explore the benefits of early micronutrient supplementation.

Elevated fasting and postprandial glucose levels, along with increased HbA1c, reaffirm the hyperglycemic state characteristic of diabetes. Importantly, the study also revealed significant deficiencies in Vitamin D and Vitamin B12, as well as reduced ferritin levels in diabetic individuals—even before the initiation of any pharmacological treatment. These findings underscore the role of micronutrient imbalances in the pathophysiology of diabetes and suggest that nutritional evaluation should be an integral part of early diabetes management. Routine screening for Vitamin D, B12, and iron status may help in timely intervention, potentially improving glycemic control and reducing the risk of complications.

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