Type 1 diabetes mellitus (T1DM) is the most common type of DM in children. The pathogenesis of T1DM has not been elucidated, but it is considered to be mainly related to the destruction of pancreatic β cells by autoimmune responses and oxidative stress caused by genetic and environmental factors (1,2). The global incidence of T1DM has been increasing steadily at an average annual rate of 3-4% over the past 30 years, and the role of environmental factors in the occurrence of T1DM is increasingly being emphasized (1).

Vitamin D is a fat-soluble steroid hormone derivative. Under normal circumstances, 70-80% of vitamin D in the body is synthesized from 7-dehydrocholesterol in the human skin after ultraviolet irradiation and becomes biologically active after two hydroxylations. Serum 25-(OH)D₃ levels are typically used as an evaluation index of vitamin D levels in vivo. Vitamin D not only regulates calcium and phosphorus metabolism but also cell proliferation, differentiation, metabolism and immune functions (3,4). Vitamin D levels have been reported to be positively associated with the function of pancreatic β cells due to their immunoregulatory, anti-inflammatory and anti-oxidative effects, and vitamin D insufficiency or deficiency has been associated with the development of T1DM and its complications (3-5). In a meta-analysis, among children of different ethnicities and ages, those with T1DM had a higher incidence of vitamin D deficiency and lower 25-(OH)D₃ levels than healthy children (6). By contrast, vitamin D or active vitamin D supplementation has been suggested to be effective in preventing the incidence of T1DM (7). Therefore, the role of vitamin D in the onset, prevention and treatment of diabetes in children is receiving increasing attention.

In the present study, the 25-(OH)D₃ levels between children with T1DM and healthy children were collected and compared, and various factors that may affect 25-(OH)D₃ levels were analyzed to provide new ideas and a basis for the clinical prevention and treatment of T1DM in children.

In the present study, inpatients with T1DM, especially those who were newly diagnosed or whose cases were complicated with DKA/DK or infection, admitted in the third quarter had lower 25-(OH)D₃ levels, with a higher proportion of patients with vitamin D insufficiency or deficiency. Age, month of admission, duration of disease, sex, place of residence and HbA₁C were the factors significantly associated with 25-(OH)D₃ levels.

Vitamin D insufficiency or deficiency in childhood may be an important risk factor for the onset and progression of T1DM and development of its complications (2,5,7-9). The mechanism by which vitamin D affects the development of T1DM may be as follows: i) Polymorphisms of vitamin D metabolism genes: Genetic susceptibility to T1DM is affected by different polymorphisms of genes encoding the vitamin D receptor (VDR), vitamin D-dependent calcium-binding protein (VDBP) and vitamin D hydroxylase (10,11). Vitamin D regulates the amount of VDR and the level of VDBP in pancreatic islet β cells, acting as a transcription factor to regulate the expression of cell cycle, cell differentiation, apoptosis- and insulin-related genes, and promotes insulin synthesis and secretion (11,12). ii) Immunomodulatory effects: Vitamin D inhibits the expression of antigen-presenting cell surface costimulatory and MHC II molecules, inhibits the differentiation, maturation and antigen presentation ability of dendritic cells, and increases their apoptosis (3,11). Vitamin D inhibits the proliferation, differentiation and activation of T lymphocytes and promotes immune tolerance, as well as inhibiting the secretion of pro-inflammatory cytokines, and enhancing the production of anti-inflammatory cytokines (3,13). The vitamin reduces the proliferation of B lymphocytes and the production of autoantibodies, induces B cell apoptosis, prevents the overactivation of the immune system and reduces the destruction of normal islet β cells (3,11,13). iii) Antioxidant effect: Vitamin D can fight intracellular oxidative stress by reducing the myeloperoxidase content, protein carbonyl groups and glycation end products, and by upregulating the mRNA expression of superoxide dismutase and other anti-oxidative pathways (14,15). Vitamin D reduces the oxidative damage of chromosomal telomere sequences and mitochondria in pancreatic islet β cells, and reduces insulin resistance (11,14,15).

Although a high-quality ongoing prospective cohort study in Finland (16) showed that no significant differences were observed in the serum 25(OH)D₃ levels between 126 children who progressed to T1DM and 126 matched controls, and that the occurrence of T1DM was not associated with vitamin D status, the study had limited samples with vitamin D deficiency and left possible variables affecting vitamin D metabolism, such as dietary vitamin D intake, physical activity, and genetic polymorphisms of VDR and VDBP, unassessed. The present findings, derived from a larger case-control sample, contribute additional descriptive data on the association between T1DM and vitamin D status in a different geographic and clinical context, but do not resolve the causal question addressed by prospective designs (16). Serum 25(OH)D₃ levels in T1DM children were lower than those in controls (8,10,17), even when exposed to abundant sunlight. However, the reported differences in 25(OH)D₃ levels appeared after the diagnosis of T1DM and the associated complications may affect vitamin D metabolism. Therefore, changes in vitamin D levels during the development of T1DM need to be detected and analyzed in more ongoing prospective case-cohort studies.

Some prospective studies (18,19) have shown that vitamin D intake or 25(OH)D₃ levels during pregnancy and childhood are not significantly associated with the development of T1DM, as concluded in a review (1). Some observational studies have found that timely vitamin D supplementation in early childhood, especially infancy, reduces the risk of developing T1DM later in life. The effect of vitamin D on reducing the incidence of T1DM was dose-dependent, and the incidence of T1DM was the lowest in participants with a daily intake of vitamin D >2,000 IU (20,21). Therefore, the efficacy of vitamin D intake in preventing T1DM remains controversial and requires additional randomized controlled trials (RCTs) to provide evidence. However, for children at a high risk of developing T1DM, such as those with a T1DM family history, islet autoantibodies and high-risk HLA genotypes, vitamin D insufficiency and deficiency should be diagnosed and treated promptly in the first few years of life, given the greater benefits of vitamin D and the fact that prevention is better than a cure.

Many direct and indirect factors affect vitamin D in children, such as related gene polymorphisms, exposure time to sunlight, regular and appropriate vitamin D supplementation, diet, medications, underlying medical conditions, gestational age and maternal vitamin D status at birth (22). The present analysis was limited by the absence of data on several important confounders, including BMI, dietary vitamin D and calcium intake, daily outdoor activity time, sun protection habits, history of vitamin D supplementation, family history of diabetes and birth history (22-24). Failure to account for these factors means that the observed association between T1DM and low 25-(OH)D₃ levels may be subject to residual confounding. The present study found that age, month of admission, sex, place of residence, duration of disease and HbA₁C levels were the significant factors affecting 25-(OH)D₃ levels. With increasing age, 25-(OH)D₃ levels and the proportion of children with normal 25-(OH)D₃ levels decreased, and 25-(OH)D₃ levels were negatively correlated with age. Although the traditional peak incidence of T1DM is in children aged 10-14 years, recent studies indicate a shift toward younger age at onset, with the peak now occurring at 4-5.9 years and significant changes in age distribution observed more recently (12,14,25-27). The present study also found that the number of children aged 5-10 years was relatively high among newly-diagnosed children with T1DM. Studies have shown an overall downward trend in the proportion of children taking outdoor activities and regular/moderate and irregular/insufficient vitamin D supplementation with increasing age, which may lead to more severe vitamin D insufficiency and deficiency and a higher risk of T1DM (23,24,28). In addition, according to the admission quarter, in the present study, the 25-(OH)D₃ level and the proportion of children with normal 25-(OH)D₃ levels were highest in the third quarter, which may be related to the greater accumulation of vitamin D in the summer. Shanxi Province is located at a latitude between 35 and 40° north, and July and August are the hottest months of the third quarter, as well as being summer vacation time. During this period, the sunlight hours are long, ultraviolet rays are strong, children wear less and have more skin exposure, and outdoor activities are long, which can greatly increase the synthesis and storage of vitamin D in the body. In addition, the present study found that girls had lower 25-(OH)D₃ levels, and a lower proportion of girls had normal 25-(OH)D₃ levels, compared with boys, which may be related to the fact that girls usually spend less time outdoors (24). Although place of residence was also a significant factor affecting 25(OH)D₃ levels, no significant differences were observed in 25(OH)D₃ levels and the proportion of patients with 25(OH)D₃ levels <50 nmol/l between different place of residence subgroups within the control group. A significant difference was observed only in the proportion of patients with 25(OH)D₃ level <50 nmol/l between different place of residence subgroups in the T1DM group, which may require more sample data for analysis.

In addition to the aforementioned factors, T1DM can affect 25-(OH)D₃ levels, which may be related to oxidative stress caused by blood glucose fluctuations (2). Oxidative stress can lead to decreased 25-hydroxylase levels and 25-(OH)D₃ synthesis (11). In addition, some pancreatic islets can produce vitamin D, which binds to VDR to promote insulin synthesis and secretion (28,29). Oxidative stress can also damage the pancreatic islet cells (2) and reduce vitamin D production. The present study found that the RBG and HbA1C levels of newly-diagnosed T1DM children were significantly higher than the normal ranges and the levels of those children with established T1DM, whereas the 25-(OH)D₃ levels and the proportion of children with normal 25-(OH)D₃ levels were lower. 25-(OH)D₃ levels were negatively correlated with RBG and HbA1C, indicating that lower 25-(OH)D₃ levels in newly-diagnosed children with T1DM may be due to larger blood glucose fluctuations and more serious oxidative stress damage, which is consistent with the results of some studies (15,17,30). In addition, Vitamin D levels are inversely correlated with oxidative stress in patients with T1DM (31). Therefore, 25-(OH)D₃ may serve as an indirect indicator of oxidative stress in patients with T1DM.

In addition, DKA may affect vitamin D metabolism, and low vitamin D levels may contribute to a higher risk of DKA presentation in patients with T1DM (13,32-34). DKA can inactivate 1-α-hydroxylase, increase renal excretion of VDBP and decrease vitamin D levels (32). The present study found that the RBG and HbA1C levels in children with DKA/DK were significantly higher than those in children without DKA/DK, and the 25-(OH)D₃ levels and the proportion of children with normal 25-(OH)D₃ levels were lower, which is also consistent with the aforementioned study (32). Although, whether the patient case was complicated with DKA/DK was not a variable affecting 25-(OH)D₃ levels in the multiple linear regression analysis, univariate analysis revealed significantly lower 25-(OH)D₃ levels in children with DKA/DK, a clinically meaningful difference. Thus, clinical attention to vitamin D status in T1DM children with DKA/DK remains warranted.

Children with vitamin D insufficiency or deficiency are more susceptible to infections (3,34). When cases in children are complicated by infection, glycemic control is affected, and acute complications such as DKA and DK are more likely to occur, thus affecting 25-(OH)D₃ levels. In the present study, 106 of 132 co-infected children also experienced complications with DKA/DK. The mean RBG level was as high as 23.12 mmol/l, while the median 25-(OH)D₃ level was only 39.66 nmol/l, and the proportion of patients with 25-(OH)D₃ <50 nmol/l was up to 78/106 (73.6%). Therefore, prompt vitamin D supplementation in children with T1DM may reduce the occurrence of infections and the risks of DKA and DK.

Vitamin D deficiency can affect blood glucose control and the development of metabolic diseases in patients with T1DM, while vitamin D or active vitamin D supplementation is effective in preventing the incident of T1DM and improving the prognosis, including improving the function of residual pancreatic β cells and insulin resistance, reducing blood glucose and HbA1c levels in diabetic patients, and preventing and improving acute and chronic complications (5,35-39). Owing to the shorter observation duration in most studies, most children with diabetes have not yet developed chronic complications at the time of the study; therefore, relevant studies, long-term observation and follow-up are lacking (25). However, as a relatively safe, reliable, effective and inexpensive drug, vitamin D as an additional therapy may open new perspectives for the control of T1DM and the improvement of patient health (7,28).

The present study has certain limitations. First, the main limitation was the lack of dynamic and long-term monitoring of 25-(OH)D₃ levels in the patients. Second, the retrospective, observational design means that the findings only support associations, not causality. Reverse causation (i.e., disease state affecting vitamin D levels rather than the converse) and confounding by disease severity, acute metabolic stress (such as DKA) and hospitalization status are important limitations that must be considered when interpreting the observed associations (17,29,30). Specifically, it is plausible that the metabolic disturbances associated with T1DM, particularly during acute presentation with DKA, directly contribute to lower measured 25-(OH)D₃ levels, rather than vitamin D deficiency predisposing to T1DM onset. Third, the control group was not matched to the case group for age, and significant age differences existed between groups. Although adjustments were made for age in the logistic regression models, residual confounding may persist. Additionally, the control group (n=136) was substantially smaller than the case group (n=757). Consequently, subgroup comparisons between T1DM subgroups and their corresponding control subgroups (for example, stratifying by age or sex) may be too underpowered to detect small-to-moderate effects. Therefore, findings from underpowered subgroup analyses should be interpreted cautiously and considered hypothesis-generating rather than conclusive. Fourth, Due to the retrospective design, several key covariates, including dietary vitamin D/calcium intake, daily outdoor activity time, sun protection habits and history of vitamin D supplementation, were not routinely documented in the electronic medical records of Shanxi Children's Hospital (Taiyuan, China). Covariates such as BMI, family history of diabetes and birth history (for example, prematurity) should have been recorded in standard clinical practice; however, due to incomplete documentation, high missing rates or unstructured text formats, they could not be reliably extracted for analysis. An additional complexity was that BMI in children must be interpreted via age- and sex-specific percentiles or z-scores, requiring precise age, sex, height and weight data fields that were often incomplete or inconsistently recorded in this retrospective cohort. While such standardization is feasible in well-curated prospective datasets, applying it here would risk imputation errors, misclassification (especially at age extremes where BMI changes rapidly) and further sample size reduction. Forcing their inclusion would have led to a substantial reduction in sample size and introduced selection bias. Notably, any residual confounding from these unmeasured factors would likely bias the effect estimate toward the null, as lower vitamin D levels tend to cluster with unfavorable profiles of these variables (for example, higher BMI, less outdoor activity and no supplementation), all of which are independently associated with increased T1DM risk. Thus, the observed association is likely conservative. Moreover, all participants were recruited from the same geographic region during a similar calendar period, and lifestyle factors are relatively homogeneous across this pediatric population. Consequently, differential confounding between groups is unlikely. Prospective studies with standardized covariate collection are required to counter this limitation.. Fifth, this was a single-center, hospital-based study in a specific region of Shanxi Province. Vitamin D status is strongly influenced by geographic, climatic and local practice factors, which limits the generalizability of the findings to other populations or outpatient settings. Finally, the effects of vitamin D supplementation on patients with T1DM were not investigated.

In conclusion, in the present single-center retrospective case-control study, children with T1DM had significantly lower serum 25-(OH)D₃ levels and a higher prevalence of vitamin D insufficiency or deficiency compared with healthy controls, even after adjusting for age, sex and admission quarter. These associations were particularly pronounced in children with newly-diagnosed T1DM, those with DKA/DK and those with concurrent infections. Age, admission quarter, disease duration, sex, place of residence and HbA1c levels were significantly associated with 25-(OH)D3 levels. However, due to the observational design, the absence of matching for key confounders, and the lack of data on important covariates such as BMI, dietary intake and sun exposure, the findings support only associations and cannot establish causality. The possibility of reverse causation, whereby T1DM or its acute complications lead to reduced vitamin D levels, remains a major alternative explanation. Given the various benefits of vitamin D, the timely diagnosis and treatment of vitamin D insufficiency and deficiency are necessary, especially in infants and adolescents at the peak of growth and at high risk of diabetes. Future high-quality, large-scale prospective interventional RCTs and well-designed prospective cohort studies with comprehensive covariate assessment are needed to fully elucidate the role of vitamin D in T1DM, to clarify the direction of the association and to guide clinical practice.