Comparison of Serum 25-Hydroxyvitamin D [25(OH)D] In Completely Edentulous Patients with and Without Diabetes Mellitus

Vitamin D, also known as the "sunshine vitamin," is an essential nutrient that plays a pivotal role in calcium and phosphorus metabolism, which are critical for maintaining bone density and structural integrity [1]. Its significance extends beyond skeletal health, as it is involved in modulating immune function, regulating inflammatory pathways and supporting overall systemic homeostasis. Recent studies have increasingly linked vitamin D deficiency to a range of chronic conditions, including cardiovascular diseases, autoimmune disorders and metabolic diseases such as Diabetes Mellitus (DM) [2]. This widespread deficiency, often termed a silent epidemic, poses a major public health challenge globally, especially in populations with limited sunlight exposure, poor dietary intake or underlying health conditions [3].

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia due to insulin resistance, impaired insulin secretion or both [4,5]. It is a condition associated with numerous systemic complications, including neuropathy, nephropathy, retinopathy and an increased susceptibility to infections [6-9]. From a dental perspective, DM is known to have a profound impact on oral health, contributing to xerostomia, delayed wound healing, periodontal disease and an elevated risk of tooth loss [7,8,10]. Advanced stages of periodontal disease in poorly controlled diabetic patients often lead to complete edentulism, which further complicates their nutritional intake and overall health status [11].

Complete edentulism, the condition of being entirely without natural teeth, is not merely a localized oral health issue but has broader implications for an individual’s systemic health, quality of life and psychological well-being [7-9,12]. Edentulous patients often face challenges in maintaining adequate nutrition due to compromised masticatory efficiency, which can lead to deficiencies in essential nutrients, including vitamin D [13]. Additionally, the absence of teeth can contribute to bone resorption in the alveolar ridge, a process that is exacerbated by both vitamin D deficiency and diabetes. Thus, the triad of edentulism, vitamin D deficiency and diabetes mellitus presents a complex interplay of factors that can significantly impact systemic and oral health [6].

The association between vitamin D status and diabetes mellitus has garnered significant attention in recent years. Studies suggest that vitamin D plays a role in insulin sensitivity, secretion and glucose metabolism and its deficiency has been linked to increased risk and severity of diabetes [3,7-9,12]. However, despite this growing body of evidence, the impact of diabetes mellitus on serum vitamin D levels in completely edentulous patients remains underexplored [7]. This is a critical gap in knowledge, as both conditions independently and synergistically influence bone metabolism, inflammatory markers and immune responses, which are crucial for the rehabilitation and management of edentulous patients [14,15].

This study seeks to address this gap by performing a comparative evaluation of serum vitamin D levels in completely edentulous patients with and without diabetes mellitus. The primary objective is to identify differences in vitamin D status between these two groups, thereby shedding light on the systemic implications of diabetes in the context of edentulism. Additionally, the findings may help clarify whether diabetic edentulous patients require specialized nutritional interventions, such as vitamin D supplementation, to optimize their oral and overall health outcomes. Ultimately, this research aims to provide valuable insights that can inform clinical decision-making and improve the quality of care for completely edentulous patients, particularly those with diabetes mellitus.

The study received ethical and scientific approval from the Institutional Ethics Committee and Review Board of Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai, Tamil Nadu, India. Written informed consent was obtained from all participants prior to enrolment.

Sample Size Estimation

The sample size was estimated based on data from previous studies evaluating serum vitamin D levels in diabetic and non-diabetic individuals, considering a confidence level of 95%, statistical power of 80% and " = 0.05. The minimum required sample size per group was calculated to be 27 participants. To enhance statistical reliability and account for potential dropouts, the final sample size was rounded to 30 participants per group, totaling 60 subjects.

This comparative cross-sectional study was conducted at the Outpatient Department of Prosthodontics, Saveetha Dental College, SIMATS, between February 2024 and December 2024. A total of 60 completely edentulous individuals aged 40-80 years were recruited and categorized into two groups based on glycemic status (Figure 1, 2):

• Group I (Diabetic edentulous individuals, n = 30): Participants with Random Blood Sugar (RBS) levels >150 mg/dL and HbA1c >7%, with complete edentulousness
• Group II (Non-diabetic edentulous individuals, n = 30): Participants with RBS levels between 70-100 mg/dL and HbA1c <6%, with complete edentulousness

Figure 1: Non Diabetic Completely Edentulous Patient

Figure 2: Diabetic Completely Edentulous Patient

Exclusion Criteria

Individuals who were smokers, had systemic conditions other than diabetes affecting oral health, were taking vitamins A, C or E or any antioxidant therapy within the past three months, those under antibiotic treatment and pregnant or lactating women were excluded from the study.

Sample Collection Procedure

Approximately 5 mL of venous blood was collected aseptically from each participant using sterile disposable syringes. The collected blood samples were allowed to clot at room temperature and were subsequently centrifuged at 3,000 rpm for 10 minutes using a Remi C-24 Plus centrifuge (Remi Instruments Ltd., India) to separate the serum. The clear serum was carefully transferred into sterile Eppendorf® microtubes and stored at -20°C until biochemical analysis. All samples were handled under aseptic conditions to preserve sample integrity and prevent degradation.

Biochemical Analysis

Serum 25-hydroxyvitamin D [25(OH)D] concentrations were estimated using a quantitative Enzyme-Linked Immunosorbent Assay (ELISA) kit. All reagents, standards, controls and serum samples were brought to room temperature before analysis. Standards and serum samples were added to ELISA microplate wells pre-coated with anti-25(OH)D antibodies, followed by incubation with a biotinylated detection antibody and enzyme-conjugated streptavidin. After appropriate washing steps to remove unbound substances, tetramethylbenzidine (TMB) substrate solution was added to produce a colorimetric reaction proportional to the serum 25(OH)D concentration. The reaction was terminated using sulfuric acid and the optical density was measured at 450 nm using a Bio-Rad iMark™ Microplate Reader (Bio-Rad Laboratories, USA) (Figure 3). Serum vitamin D concentrations were determined using a standard calibration curve generated by the ELISA software.

Based on serum 25(OH)D levels, vitamin D status was categorized as deficient (<20 ng/mL), insufficient (20-30 ng/mL) and sufficient (>30 ng/mL).

For glycemic profiling, HbA1c was measured using an automated analyzer (Bio-Rad D-10 Hemoglobin Testing System, USA) and Random Blood Sugar (RBS) levels were determined using a GlucoOne BG03 Glucometer (Dr. Morepen®, India).

Figure 3: Clinical Photograph of the Edentulous Mucosa

This Photograph Does Not Depict the Serum ELISA Procedure

Statistical Analysis

All data were analyzed using IBM SPSS Statistics software (Version 26.0, IBM Corp., USA). The descriptive statistical analysis included serum vitamin D levels, HbA1c and RBS in completely edentulous diabetic and non-diabetic patients (Table 1). The mean and Standard Deviation (SD) of serum vitamin D levels in diabetic and non-diabetic edentulous patients were calculated, along with the range (minimum-maximum) of vitamin D concentrations in both groups. Additionally, demographic data such as age, gender distribution and clinical characteristics were analyzed to compare the two groups (Table 2). The Independent t-test was used to compare serum vitamin D levels between the groups when data were normally distributed; otherwise, the Mann-Whitney U test was applied (Table 3). The Chi-square test was used to compare the frequency distribution of vitamin D deficiency categories between diabetic and non-diabetic individuals. Pearson's or Spearman's correlation coefficient, based on data normality, was used to evaluate the association between serum vitamin D levels, HbA1c and RBS (Table 4). Binary logistic regression analysis was performed to identify independent predictors of diabetic status and assess the association between vitamin D levels and diabetes after adjusting for age and gender (Table 5). A p-value <0.05 was considered statistically significant.

Table 1: Edentulous Patients with Complete Profile

Demographic Characteristics

The study comprised 60 completely edentulous individuals aged 40-80 years, divided equally into diabetic (n = 30) and non-diabetic (n = 30) groups (Table 2). The mean age of diabetic participants was 60.50±8.79 years, while that of non-diabetic participants was 61.40±8.85 years. There was no statistically significant difference in age between the two groups (p = 0.6942), indicating comparable age distribution.

In terms of gender distribution, the diabetic group included 18 males (60%) and 12 females (40%), while the non-diabetic group consisted of 15 males (50%) and 15 females (50%). The Chi-square test revealed no significant difference in gender distribution between groups (p = 0.6038), confirming demographic comparability (Table 2).

Table 2: Descriptive Statistics for Vitamin D Levels, Age and Gender Distribution

This table summarizes the Mean±Standard deviation and gender distribution in diabetic and non-diabetic edentulous patients (n = 60), along with the p-values from statistical comparisons

Table 3: Statistical Analysis and Interpretation

This table summarizes the statistical tests used to compare age, serum vitamin D levels and gender distribution between diabetic and non-diabetic edentulous patients (n = 60). The p-values and interpretation of statistical significance are provided

Comparison of Serum Vitamin D Levels Between Groups

The mean serum vitamin D level was 21.36±7.73 ng/mL in the diabetic group and 33.06±1.69 ng/mL in the non-diabetic group, with a broader range among diabetics (2.24-34.50 ng/mL) compared to non-diabetics (30.42-37.92 ng/mL). This indicates that diabetic edentulous patients exhibited considerably lower and more variable serum vitamin D levels than non-diabetic individuals, suggesting an increased prevalence of vitamin D deficiency among diabetic patients (Table 4, Figure 4).

Figure 4: Comparison of VIT-D Levels Between Groups

Table 4: Summary of Vitamin D Levels and Glycemic Correlation in Edentulous Diabetic and Non-Diabetic Patients

The Table shows among the diabetic patients, the mean Vitamin D serum level was 21.36±7.73 ng/mL, with a wide range from 2.24 to 34.50 ng/mL, indicating significant variability and a higher prevalence of deficiency. In contrast, non-diabetic patients had a significantly higher and more consistent mean Vitamin D level of 33.06±1.69 ng/mL, ranging from 30.42 to 37.92 ng/mL, with minimal deviation from the mean. An evaluation of Vitamin D status distribution showed that 30% of diabetic patients were deficient (<20 ng/mL) and 50% were insufficient (20-29 ng/mL), leaving only 20% with sufficient levels (≥30 ng/mL). Conversely, 100% of non-diabetic patients had Vitamin D levels in the sufficient range. This striking difference underscores the higher burden of Vitamin D deficiency among the diabetic edentulous population. Furthermore, correlation analysis within the diabetic group revealed a strong negative correlation (r = -0.80) between Vitamin D levels and HbA1c and a moderate negative correlation (r = -0.65) between Vitamin D levels and random blood sugar (RBS). These findings suggest that lower Vitamin D levels are significantly associated with poorer glycemic control, reinforcing the potential role of Vitamin D in glucose metabolism and diabetes management

Normality and Group Comparisons

Data established lower mean serum 25(OH)D in the diabetes group (21.36±7.73 ng/mL) than the non-diabetes group (33.06±1.69 ng/mL). The Welch mean difference was -11.70 ng/mL (95% CI -14.64 to -8.76; two-sided p = 3.16×10⁻⁹).

Correlation Between Vitamin D and Glycemic Markers

Correlation analysis demonstrated a strong negative correlation between serum vitamin D levels and HbA1c (r = -0.80), as well as a moderate negative correlation with Random Blood Sugar (RBS) (r = -0.65) in the diabetic group. These findings indicate that lower serum vitamin D concentrations were associated with poorer glycemic control, suggesting a potential relationship between vitamin D deficiency and diabetes severity (Table 4, Figure 5a, b).

Regression Analysis

Linear regression analysis was performed to evaluate the association of serum vitamin D levels with diabetes status, age and gender. None of the variables, including Group (p = 0.8117), Age (p = 0.4002) or Gender (p = 0.3129), demonstrated a statistically significant association with serum vitamin D levels. These findings suggest that the observed variation in vitamin D levels may be influenced by factors other than age, gender or diabetic status within the study population (Table 5, Figure 5a, b).

Figure 5(a-b): Boxplot of the Reported Serum 25(OH)D Distribution in Diabetic and Non-Diabetic Groups (n = 30 in Each Group) (a) Scatterplot of Serum 25(OH)D and HbA1c Among Diabetic Participants (n = 30) and (b) Scatterplot of Serum 25(OH)D and Random Blood Sugar Among Diabetic Participants (n = 30)

Table 5: Regression Analysis Summary

This regression analysis assesses the relationship between Vitamin D Serum levels (dependent variable) and three independent variables: Group (Diabetic vs. Non-Diabetic), Age and Gender. The model includes the coefficients, p-values and interpretations for each predictor. Using the data, an adjusted linear model of 25(OH)D on diabetes status, age and sex yielded a diabetes coefficient of -11.71 ng/mL (HC3-robust 95% CI -14.73 to -8.69; p<0.001). Age (β = 0.128 per year; 95% CI -0.081 to 0.337; p = 0.229) and male sex (β = 1.283; 95% CI -1.919 to 4.485; p = 0.432) were not independently associated with 25(OH)D in this model. R² was 0.559

Overall Inference

The study demonstrates that completely edentulous diabetic patients exhibited lower serum vitamin D levels than their non-diabetic counterparts and that serum vitamin D levels showed a strong negative correlation with glycemic control markers (HbA1c and RBS). Although regression analysis did not identify diabetes status, age or gender as significant independent predictors of serum vitamin D levels, the findings suggest that vitamin D deficiency is more prevalent among diabetic edentulous individuals and may be associated with poorer glycemic control. Collectively, these results support the potential role of serum vitamin D assessment as an adjunctive biomarker in the evaluation of metabolic status and oral health in completely edentulous diabetic patients.

The findings of this study underscore the significant difference in vitamin D levels between diabetic and non-diabetic completely edentulous patients, particularly in the context of oral health and glycemic control. The lower mean vitamin D levels observed in diabetic patients suggest that these individuals are at a higher risk for vitamin D deficiency and insufficiency compared to non-diabetic individuals, which is in line with previous studies indicating that diabetes may alter the body’s ability to maintain optimal vitamin D status [9].

Vitamin D is a fat-soluble vitamin that is crucial for several bodily functions, including calcium absorption, bone health and immune function [10,16]. In diabetes, the relationship between vitamin D deficiency and glucose metabolism has garnered considerable attention. Several mechanisms have been proposed through which vitamin D influences insulin sensitivity, including the modulation of pancreatic beta-cell function, regulation of inflammatory pathways and enhancement of insulin receptor sensitivity. Deficient levels of vitamin D are thought to exacerbate insulin resistance, a hallmark of type 2 diabetes and contribute to poor glycemic control, which is consistent with our findings of a negative correlation between vitamin D levels and HbA1c [17,18].

The significant negative correlation observed between vitamin D serum levels and HbA1c in diabetic patients (r = -0.80) indicates that lower vitamin D levels are associated with higher HbA1c levels, which reflect poorer long-term blood glucose control [19]. This relationship suggests that vitamin D deficiency may contribute to the pathophysiology of diabetes and that increasing vitamin D levels could potentially enhance glycemic control. The moderate negative correlation with RBS (r = -0.65) further supports this hypothesis, demonstrating that vitamin D levels may also be linked to daily fluctuations in blood glucose [20,21].

Moreover, the high prevalence of vitamin D deficiency (over 60%) and insufficiency (over 20%) in diabetic patients in this study is a cause for concern, as these conditions are associated with an increased risk of various complications, including cardiovascular disease, osteoporosis and nephropathy [3,7,8]. It is well-documented that diabetic patients with poor glycemic control are more likely to develop these complications. Thus, addressing vitamin D deficiency in this population may offer a relatively simple and low-cost intervention to improve their overall health outcomes and reduce the risk of long-term diabetic complications [22].

However, despite the compelling correlations, it is important to note that this study did not find statistically significant differences between the two groups after performing regression analysis (p = 0.8117). This finding could be attributed to the small sample size, which may not have provided sufficient power to detect a statistically significant difference, particularly when considering the many potential confounding variables that were not included in this analysis [3,7,8,10]. For example, factors such as age, dietary intake of vitamin D, sun exposure and medication use (e.g., vitamin D supplements or anti-inflammatory drugs) could all influence vitamin D levels and glycemic control but these factors were not controlled for in the study [22-24].

The lack of statistically significant differences in vitamin D levels between the two groups, despite the observed trends, might also be explained by the inherent variability in how diabetes affects vitamin D metabolism. Diabetic patients may experience impaired renal conversion of vitamin D to its active form, leading to lower serum levels [25]. Moreover, inflammation, which is common in diabetes, could reduce the bioavailability of vitamin D, further exacerbating the deficiency [26].

Despite these limitations, the findings still suggest that vitamin D deficiency is highly prevalent among diabetic patients, which has significant implications for clinical practice. Healthcare providers should routinely assess vitamin D levels in diabetic patients, especially those with poor glycemic control or who are at increased risk for diabetic complications [25,27]. Vitamin D supplementation could be considered as part of the management plan, with careful monitoring of both vitamin D levels and glycemic control to assess the efficacy of such interventions [7,28].

Recommendations for Future Research

Given the potential link between vitamin D levels and diabetes-related complications, further research is needed to explore the causality of this relationship. Longitudinal studies could help determine whether correcting vitamin D deficiency in diabetic patients leads to sustained improvements in glycemic control, reduces the risk of complications and enhances oral health outcomes, such as wound healing in edentulous patients.

Additionally, exploring the role of vitamin D in inflammatory processes related to diabetes could provide deeper insights into its role as an immunomodulatory agent. Studies that evaluate the effect of vitamin D supplementation on inflammatory markers, such as C-reactive protein (CRP), interleukins and tumor necrosis factor-alpha (TNF-"), could help elucidate its therapeutic potential beyond glycemic control.

While our study has provided valuable insights into the relationship between vitamin D and diabetes in edentulous patients, more comprehensive and larger-scale studies are required to validate these findings and fully understand the clinical implications. Specifically, future research should focus on how vitamin D supplementation impacts long-term diabetes management, the prevention of complications and the overall quality of life in diabetic patients.

In the 60 participants, participants with diabetes had lower measured serum 25(OH)D than those without diabetes, with inverse correlations between vitamin D and the recorded glycemic measures within the diabetic subgroup. Associations cannot establish that vitamin D deficiency worsened glycemic control or that supplementation would improve outcomes.

Future studies should aim to explore other factors influencing vitamin D levels in diabetic patients, including lifestyle and environmental factors such as sunlight exposure, dietary intake and physical activity. Additionally, longitudinal studies examining the long-term effects of vitamin D supplementation on glycemic control in edentulous diabetic patients would provide valuable insights into the clinical benefits of such interventions.

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