Introduction

Childhood obesity has become a global health concern not only due to its increasing prevalence but also because of its association with numerous metabolic disorders. Among the various micronutrient deficiencies observed in obese individuals, vitamin B12 is particularly important due to its potential impact on metabolic dysfunction, neurocognitive development, and long-term cardiovascular risk1,2.

Vitamin B12 plays a fundamental role in DNA synthesis, optimal hematopoiesis, and neurological function; thus, its deficiency can result in clinically significant consequences. Vitamin B12 deficiency has been associated with inadequate dietary intake, intestinal malabsorption, and conditions such as pernicious anemia, and it is well-documented, particularly in adults. Furthermore, B12 malabsorption has been linked to both metformin treatment, an insulin-sensitizing agent commonly used in the management of type 2 diabetes, and increasingly to obesity itself3,4,5,6,7,8.

Several studies have investigated the vitamin B12 status concerning obesity and metabolic parameters, showing that while the prevalence of B12 deficiency in healthy individuals is approximately 12.5%, it ranges between 20 and 35% among obese individuals4. In obese children, low B12 levels have been reported to be up to four times more common compared to their normal-weight counterparts. Each unit increase in body mass index standard deviation score (BMI SDS) has been associated with a 1.6- to 4-fold increased risk of B12 deficiency4,9. This association may be explained by factors such as chronic low-grade inflammation accompanying obesity, insulin resistance, altered gastrointestinal absorption, and decreased intake of B12-rich foods, as well as increased demand due to greater body surface area and growth requirements2. Moreover, children exhibiting features of metabolic syndrome (MetS) have been found to have significantly lower B12 levels compared to their normal-weight peers, with insulin resistance identified as a contributing factor in this deficiency4,5,6. Although several studies have reported lower vitamin B12 levels in obese children and adolescents, the evidence remains inconsistent. A recent meta-analysis found no significant difference in serum vitamin B12 or folate levels between obese and non-obese children/adolescents. However, the presence of publication bias and the trim-and-fill correction suggested that a true difference may still exist10.

More than 40 different definitions have been proposed for identifying MetS in childhood, most of which are adapted from adult criteria. In general, the presence of at least three parameters affecting metabolic health is required for the diagnosis of MetS. However, observations that some obese individuals exhibit a significantly lower risk for cardiometabolic abnormalities have led to the emergence of the concept of “metabolically healthy obesity” (MHO)11,12,13,14. MHO is defined as the absence of any MetS criteria in obese individuals. Studies have shown that MHO individuals tend to have lower visceral and hepatic fat accumulation levels, higher leg fat deposition, preserved subcutaneous adipose tissue expansion capacity, maintained beta-cell function, and increased insulin sensitivity15,16,17,18. This subgroup appears to be protected from the adverse cardiometabolic outcomes typically associated with obesity, which raises the question of whether the MHO phenotype is also protective against micronutrient deficiencies such as vitamin B12.

However, no studies have directly compared vitamin B12 deficiency between metabolically healthy and unhealthy obese individuals in the pediatric population. This study aims to compare vitamin B12 levels according to metabolic health status in children and adolescents with obesity and to assess the relationship between vitamin B12 deficiency and metabolic health. Additionally, we aim to examine the prevalence of vitamin B12 deficiency in overweight/obese individuals and healthy controls, and to evaluate the impact of vitamin B12 levels on indicators of metabolic health. In this context, the relationship between vitamin B12 levels and metabolic health parameters, as well as body adiposity distribution, will be investigated through multivariate analyses.

Methods

This retrospective cross-sectional study was conducted at the Pediatric Endocrinology Department of Zonguldak Bulent Ecevit University by reviewing the medical records of children aged 2–18 years who were evaluated for obesity, as well as healthy controls, between January 1, 2022, and April 1, 2025. This study was approved by the Zonguldak Bulent Ecevit University Clinic Research Ethics Committee and adhered to the ethical standards of the Declaration of Helsinki (approval date: April 30, 2025; approval number: 2025/09). Due to the retrospective nature of the study, Zonguldak Bulent Ecevit University Clinic Research Ethics Committee waived the need of obtaining informed consent. The study population consisted of obese cases with BMI SDS ≥ 2 (corresponding approximately to the 97th percentile) according to national age- and sex-specific BMI reference curves. Patients with syndromic obesity, hypothalamic obesity, coexisting autoimmune or chronic systemic diseases, or those receiving medical treatment were excluded. Additionally, individuals with underlying conditions or medication use that could cause or influence vitamin B12 deficiency (e.g., inherited intrinsic factor deficiency, Imerslund-Grasbeck syndrome, transcobalamin receptor deficiency, inflammatory bowel disease, celiac disease, pancreatic insufficiency, use of proton pump inhibitors, H2 blockers, or vitamin B12 supplementation) were also excluded.

At the time of admission, anthropometric measurements and laboratory parameters including triglycerides, high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), total cholesterol, fasting blood glucose, fasting insulin, alanine transaminase (ALT), aspartate transaminase (AST), thyroid-stimulating hormone (TSH), free thyroxine (FT4), vitamin B12, and 25-hydroxyvitamin D3 levels were recorded. Absolute values for BMI, weight, and height were presented descriptively, whereas all evaluations and analyses were performed using Z-scores (SDS) according to age- and sex-specific reference curves. Biochemical parameters were measured using a spectrophotometric method with a chemical autoanalyzer, and hormone levels (TSH, FT4) and vitamin B12 levels were measured using an electrochemiluminescence immunoassay [Cobas 6000 (Roche Diagnostics, Mannheim, Germany)].

Patients were categorized into two groups based on their metabolic health status. Those meeting at least one of the following criteria were classified as metabolically unhealthy obese (MUO): fasting glucose ≥ 100 mg/dL, triglycerides ≥ 150 mg/dL, HDL cholesterol ≤ 40 mg/dL, or systolic/diastolic blood pressure above the 95th percentile. Individuals not meeting any of these criteria were classified as MHO14. Serum vitamin B12 levels, measured by chemiluminescent immunoassay, were classified according to age-appropriate reference ranges: <200 pg/mL (148 pmol/L) as deficient, 200–300 pg/mL (148–221 pmol/L) as borderline, and > 300 pg/mL (> 221 pmol/L) as sufficient19,20. The frequency of vitamin B12 deficiency was compared between MHO, MUO, and healthy control groups. The relationship between vitamin B12 levels and metabolic health parameters and body adiposity indices was evaluated using multivariate regression analyses.

The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated using the formula: fasting glucose × fasting insulin / 405. To eliminate the potential effect of insulin resistance, HOMA-IR cutoff values derived from studies conducted in our population were applied according to pubertal stage and sex: 2.22 and 2.67 for prepubertal girls and boys, respectively, and 3.82 and 5.22 for pubertal girls and boys, respectively21. Using these thresholds, the presence of insulin resistance was incorporated into the classification of metabolic health markers, and a secondary evaluation was performed.

Statistical analysis

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 29.0 for Windows (SPSS Inc., Chicago, IL, USA). The distribution characteristics of the data were assessed using the Kolmogorov–Smirnov test. Continuous variables with normal distribution were expressed as mean ± standard deviation, while those not normally distributed were presented as median (minimum–maximum).

For group comparisons, the Independent Samples t-test was used to compare two groups for normally distributed variables, and one-way ANOVA was used for comparisons involving more than two groups. For non-normally distributed variables, the Mann–Whitney U test was used for two-group comparisons, and the Kruskal–Wallis test was used for three or more groups. When a statistically significant difference was identified with the Kruskal–Wallis test, post hoc multiple comparisons were conducted using Dunn’s (1964) procedure with Bonferroni adjustment. Correlations between continuous variables were evaluated using Spearman correlation analysis for non-normally distributed data. Factors associated with vitamin B12 deficiency were analyzed using binary logistic regression analysis. A p-value of < 0.05 was considered statistically significant.

Results

A total of 291 obese children and 138 healthy controls were included in the study. Age and sex distribution were similar between groups. The mean age of the obese group (MHO: n = 133; MUO: n = 158) was 13.3 years (range: 2.5–19), while the control group had a mean age of 11.7 ± 2.6 years (p = 0.061). Among all obese participants, 54.3% were classified as MUO. No statistically significant differences were observed between the groups regarding height and height Z-scores (p = 0.052 and p = 0.825, respectively); however, the MUO group had significantly shorter height than the control group (p = 0.001). Although there was no significant difference in BMI SDS between MHO and MUO groups (p = 0.587), both obese groups had significantly higher BMI SDS than the control group (p = 0.001) (Table 1).

Table 1 Anthropometric characteristics and micronutrient levels of all groups.
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Between the MHO and MUO groups, statistically significant differences were found in triglycerides, HDL cholesterol, total cholesterol, TG/HDL ratio, glucose, insulin, HOMA-IR, TSH, and ALT levels (p < 0.05). In contrast, no statistically significant differences were observed in LDL cholesterol, HbA1c, free T4, AST, uric acid, vitamin B12, and 25(OH)D levels (Table 2).

Table 2 Comparison of biochemical parameters between metabolically healthy obese and metabolically unhealthy obese groups.
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In the evaluation of micronutrient status, 34.3% of the obese children had vitamin B12 deficiency, and 26.4% had subclinical deficiency; 39.1% had sufficient levels. In contrast, 16.1% of the control group had B12 deficiency (p = 0.015). The mean B12 level in the MHO group was 281.6 pg/mL, compared to 388.5 pg/mL in the control group, showing a statistically significant difference (p = 0.001). However, B12 and 25(OH)D levels were similar between the MHO and MUO groups (Table 1). When vitamin B12 levels were classified as deficient, intermediate, and sufficient, no significant difference was observed between the two obese groups (p = 0.517). The prevalence of vitamin B12 deficiency was 37.5% in the MHO group and 31.6% in the MUO group (Table 3).

Table 3 Distribution of vitamin B12 status in metabolically healthy obese (MHO) and metabolically unhealthy obese (MUO) groups.
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In the regression analysis conducted to examine the relationship between vitamin B12 deficiency and anthropometric variables, a statistically significant negative association was found between weight SDS and B12 deficiency (B = -0.835, p = 0.038), indicating that individuals with lower weight SDS had a reduced risk of B12 deficiency (OR = 0.434). Conversely, a positive and statistically significant association was observed between BMI SDS and B12 deficiency (B = 1.460, p = 0.024), suggesting that higher BMI SDS increased the risk of B12 deficiency approximately 4.3-fold (OR = 4.307). No significant associations were found between B12 deficiency and sex, age, body weight, height, height SDS, or BMI values (p > 0.05) (Table 4).

Table 4 Logistic regression analysis of anthropometric factors associated with vitamin B12 deficiency.
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In the multivariate logistic regression analysis conducted to identify independent biochemical predictors of vitamin B12 deficiency, free T4 (p < 0.001, OR: 0.001), AST (p = 0.011, OR: 0.897), triglycerides (p = 0.032, OR: 1.054), HDL cholesterol (p = 0.029, OR: 0.847), TG/HDL ratio (p = 0.017, OR: 0.092), and uric acid (p = 0.047, OR: 1.491) were found to be statistically significant. Increases in free T4 and HDL levels were statistically significantly associated with a reduced risk of B12 deficiency, whereas elevated triglycerides, TG/HDL ratio, and uric acid levels showed a positive association with B12 deficiency (Table 5) (Fig. 1).

Table 5 Associations between metabolic parameters and vitamin B12 deficiency: logistic regression results.
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Fig. 1
figure 1

Forest Plot of Significant Variables Related to Vitamin B12 Deficiency. Forest plot illustrating the odds ratios and 95% confidence intervals of significant variables associated with vitamin B12 deficiency based on logistic regression analysis. Variables with odds ratios > 1 indicate increased risk, while those < 1 indicate reduced risk. ST4: free thyroxine, TG/HDL: triglyceride-to-HDL cholesterol ratio, HDL: high-density lipoprotein cholesterol.

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Spearman correlation analysis revealed a significant, positive correlation between vitamin B12 and free T4 levels (r = 0.480, p < 0.001), and a weak but statistically significant negative correlation between B12 and uric acid levels (r = -0.206, p = 0.034). No statistically significant correlations were found between vitamin B12 and other metabolic parameters, including triglycerides, fasting glucose, fasting insulin, HOMA-IR, HbA1c, AST, ALT, TSH, total cholesterol, HDL, and LDL (p > 0.05). When insulin resistance was included in the definition of MHO, 28.1% of participants were classified as MHO and 71.8% as MUO. However, no significant difference was observed between the two groups regarding vitamin B12 deficiency (p = 0.421).

Discussion

Our study evaluated the prevalence of vitamin B12 deficiency among obese children and adolescents, its distribution across MHO and MUO groups, and its relationship with metabolic parameters. Our findings revealed that the prevalence of vitamin B12 deficiency was significantly higher in obese children than in healthy controls. While 16.1% of healthy children had B12 deficiency, this rate was 34.3% among obese children, with an additional 26.4% showing subclinical deficiency. These results support the view that obese children and adolescents are at a higher risk for vitamin B12 deficiency compared to the general population. While our study found a higher prevalence of vitamin B12 deficiency among obese children compared to healthy controls, a previous meta-analysis of Ulloque-Badaracco et al. reported no significant differences in serum vitamin B12 levels between children/adolescents with and without obesity10. Notably, that meta-analysis detected publication bias, and correction with the trim-and-fill method suggested that a significant difference may in fact exist. This implies that the true relationship between obesity and vitamin B12 status remains uncertain and may be shaped by study design, population characteristics, and reporting bias.

Our results indicating lower vitamin B12 status in obese children suggest a possible genuine association, though cross-study inconsistencies warrant cautious interpretation. Moreover, as our assessment relied solely on serum concentrations, without functional biomarkers, this limitation must be considered when interpreting the findings.

Prevalence of vitamin B12 deficiency and etiological factors

Previous studies have reported a similar association between obesity and vitamin B12 deficiency. Our findings are consistent with the previously reported prevalence range of 10–30% in obese children2,3,4,5,9,10,18. In a review by Demirtaş et al., which included seven studies involving children aged 8–18, one study reported B12 deficiency in 34.1% of obese individuals compared to 12.5% of controls5. The same review indicated that obesity could lead to a 1.6-fold reduction in vitamin B12 levels. Similarly, Pinhas-Hamiel et al. reported that low B12 status was up to 4.3 times more common in obese Israeli children and adolescents compared to their normal-weight peers, and that each 1-unit increase in BMI SDS raised the risk of B12 deficiency by 1.4 times9. Comparable results have also been reported in large-scale studies22.

Vitamin B12 deficiency in obese individuals has been associated with various physiological, environmental, and behavioral factors, although the exact mechanisms remain unclear. Diets high in energy but poor in micronutrients are considered a primary contributing factor1,23,24. The World Health Organization (WHO) defines obesity as a “double burden of malnutrition”. It emphasizes that in populations with lower socioeconomic status, undernutrition may coexist with obesity, increasing the risk of chronic diseases25.

The widespread consumption of fast food and processed foods may lead to inadequate intake of animal-based products such as red meat, dairy, and eggs, which are rich sources of vitamin B1226. In addition, gastrointestinal disorders leading to malabsorption, the use of antacid medications, treatment with metformin—a commonly used insulin sensitizer in type 2 diabetes27—and bariatric surgery28 have all been well-documented as contributing factors to B12 deficiency.

Furthermore, obesity-related dysbiosis of the gut microbiota and increased intestinal permeability have been shown to impair intestinal barrier integrity through chronic inflammation, indirectly affecting vitamin B12 absorption29,30.

Additional mechanisms and the role of defining metabolic health status

In addition to dietary deficiencies and gastrointestinal issues, other mechanisms that may contribute to vitamin B12 deficiency in individuals with obesity include chronic low-grade inflammation, non-alcoholic fatty liver disease, and oxidative stress. Obesity is characterized by chronic low-grade inflammation driven by proinflammatory cytokines secreted from adipose tissue, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). This inflammatory state has been suggested to impair intestinal barrier function, negatively affecting vitamin B12 absorption31,32.

Vitamin B12 deficiency is a multifactorial condition, and existing literature indicates that it may also be associated with obesity during childhood. However, the evidence regarding its relationship with parameters such as MetS and insulin resistance remains conflicting. Notably, no study has directly compared MHO and MUO individuals regarding vitamin B12 status. Therefore, our study makes a significant contribution to the current body of knowledge in this area. In our study, vitamin B12 deficiency was found in 36% of the MHO group and 31% of the MUO group. Similarly, the proportions of moderate and sufficient B12 levels were comparable between the two groups.

Given the unpredictable global rise in pediatric obesity and the associated cardiovascular and metabolic risks, the American Academy of Pediatrics recommends that each component of the MetS be assessed and managed as an independent cardiovascular risk factor33. To date, more than 40 different definitions—mostly derived from adult populations—have been proposed for diagnosing MetS in obese individuals. At present, as reflected in the diversity of recent studies, there is no consensus on the criteria used to define metabolic syndrome in children. In a study comparing the use of different diagnostic criteria for metabolic syndrome in children aged 6–13 years, the IDF criteria were found to be the most commonly applied34,35.

This variability in diagnostic criteria, combined with findings that some obese individuals exhibit low cardiometabolic risk, has led to the emergence of the concept of “metabolically healthy obesity”. MHO refers to a phenotype in which individuals, despite being obese, exhibit metabolic parameters within normal limits. In contrast, “metabolically unhealthy obesity” is characterized by at least one metabolic abnormality, such as hyperglycemia, dyslipidemia, or hypertension36,37.

The literature suggests that MHO individuals have less visceral and hepatic fat accumulation, greater lower extremity adipose tissue development, preserved subcutaneous fat expandability, better beta-cell function, and higher insulin sensitivity11,12,13,14,15,16. However, our study indicates that metabolic health status does not appear to be a determining factor in the development of B12 deficiency among obese individuals. Although obesity is associated with lower vitamin B12 levels, the metabolically healthy phenotype does not seem to protect against B12 deficiency in obese children.

Another important aspect to consider is the criteria used to define the term MHO. In the literature, consensus has largely been reached on the four main components outlined in the IDF definition of metabolic syndrome. In children, following a meta-analysis published in 201814, Aureli et al. re-evaluated these criteria using the Delphi process and concluded that there is no agreement on the inclusion of insulin resistance markers such as HOMA-IR14,37. The challenge of defining metabolic health is further complicated during puberty, where physiologic insulin resistance occurs. Similarly, in adult populations, international collaborations such as the BioShare-EU project, involving European and Canadian cohorts, have suggested considering four additional parameters beyond the WHO and NCEP ATP III definitions38. Although HOMA-IR is widely used as a practical screening tool for insulin resistance in adults, its sensitivity in pediatric cohorts is relatively low, and reported cut-off values vary substantially with age, sex, and ethnicity21,34,35. Pubertal insulin resistance is a transient phenomenon driven by increased growth hormone and sex steroid secretion, resulting in reduced insulin sensitivity. While this typically resolves in normal-weight children, in obese children, it may exacerbate metabolic disturbances and mimic features of MetS. Notably, current definitions of MUO do not incorporate insulin levels or direct measures of insulin resistance, so classification should be interpreted cautiously, taking pubertal stage into account11,14,16.

Importantly, the concept of MHO aims to address cardiometabolic risk beyond insulin resistance alone. Current research supports a conceptual shift from BMI or total fat mass to adipose tissue function and liporegulatory capacity as the central determinants of metabolic health11. In our study, when insulin resistance, defined by HOMA-IR, was added to the MHO definition, 28.1% of participants were classified as MHO, while the remainder were considered MUO. However, no significant differences were observed in the prevalence of vitamin B12 deficiency between these groups (p = 0.421). Thus, even when insulin resistance markers were incorporated, being metabolically healthy did not appear to confer protection against vitamin B12 deficiency.

Relationship between vitamin B12 deficiency and MetS

Most studies investigating the relationship between vitamin B12 deficiency and MetS have been conducted in adult populations. In these studies, when individuals with MetS were compared with control groups, the increased risk of B12 deficiency associated with obesity was emphasized. Narang et al. evaluated serum homocysteine, vitamin B12, and folic acid levels in individuals with MetS and reported significantly lower B12 levels in this group39. In a meta-analysis where 66 studies were evaluated, it was determined that high B12 levels were associated with MetS10.

In our study, we examined the relationship between insulin resistance and vitamin B12 deficiency, and no statistically significant associations were found between B12 levels and parameters such as fasting glucose, insulin, HOMA-IR, and HbA1c. The findings in the literature are inconsistent: while some studies have not reported a relationship between insulin resistance and B12 deficiency18, others have suggested a possible link between the two conditions2,6.

In a study conducted by Baltaci et al. involving 976 participants, the effect of MetS and insulin resistance on vitamin B12 levels was investigated in obese, overweight, and normal-weight individuals. Although vitamin B12 levels were reported to be lower in both MetS (+/-) and IR (+/-) groups, the differences did not reach statistical significance (p = 0.075 and p = 0.058, respectively). These discrepancies across studies may be attributed to various factors, such as using different methods to assess insulin resistance in children, inadequate consideration of the physiological insulin resistance observed during puberty, and the prevalence of diets low in vitamin B12 within the population studied18.

On the other hand, in our study, statistically significant associations were found between vitamin B12 deficiency and some other components of MetS. In particular, elevated triglyceride levels (OR = 1.054; p = 0.032), low HDL cholesterol levels (OR = 0.847; p = 0.029), and elevated uric acid levels (OR = 1.491; p = 0.047) were significantly associated with B12 deficiency. These findings suggest that the impact of metabolic dysfunction on B12 deficiency may be mediated more by mechanisms such as lipid metabolism disorders and chronic inflammation rather than insulin resistance1,10,18,40.

Similarly, a study by Li et al. reported that vitamin B12 deficiency was associated with the number of MetS components, particularly among individuals with morbid obesity41.

Vitamin B12 status in relation to adiposity, lipid profile, liver enzymes and thyroid function

However, in our study, the presence of metabolic disorders did not have a significant effect on vitamin B12 levels. In contrast, B12 deficiency was found to be more prominent in children with older age and higher BMI. This finding suggests that increasing obesity heightens the risk of B12 deficiency, but this effect may occur independently of metabolic health status.

When the relationships between vitamin B12 levels and anthropometric variables were examined, it was determined that higher BMI SDS values increased the risk of B12 deficiency by approximately 4.3 times. On the other hand, a negative association was observed between weight SDS and B12 deficiency. These findings suggest that increased adiposity may have an adverse effect on B12 levels. It is considered that sequestration of vitamin B12 in adipose tissue or inflammatory processes associated with obesity may contribute to these mechanisms.

In our logistic regression analyses, an increase in serum free T4 levels was found to significantly reduce the risk of B12 deficiency (OR = 0.001; p < 0.001), whereas elevated triglyceride levels (OR = 1.054; p = 0.032) and serum uric acid levels (OR = 1.491; p = 0.047) were shown to increase the risk of B12 deficiency. Additionally, high HDL levels (OR = 0.847; p = 0.029) and a low TG/HDL ratio (OR = 0.092; p = 0.017) were found to protect against B12 deficiency.

These findings indicate that vitamin B12 deficiency is not only related to insufficient intake or dietary factors but is also closely associated with metabolic health indicators. In particular, inflammatory and oxidative stress mechanisms related to increased adiposity may affect B12 metabolism. Indeed, studies in the literature have reported associations between vitamin B12 deficiency and oxidative stress, endothelial dysfunction, and inflammation42.

Moreover, the strong associations observed with free T4 and AST warrant particular attention. Hypothyroidism has been linked to impaired vitamin B12 absorption, possibly due to slowed gastrointestinal motility, atrophic gastritis, or autoimmune mechanisms affecting intrinsic factor production, all of which may compromise B12 bioavailability43. Recent evidence indicates that children and adults with thyroid dysfunction, especially hypothyroidism, frequently exhibit lower vitamin B12 levels compared to euthyroid individuals. On the other hand, elevated AST levels may reflect hepatic stress, particularly non-alcoholic fatty liver disease, which is common in obese populations. As the liver is the primary storage site for vitamin B12, hepatocellular injury and impaired hepatic metabolism could reduce B12 mobilization and availability44. Taken together, these mechanisms suggest that both thyroid dysfunction and hepatic alterations may play important roles in modulating vitamin B12 status in obese children, beyond dietary intake and adiposity-related effects.

Association between vitamin B12 and vitamin D deficiencies in obese children

Our study also observed low vitamin D levels in cases with vitamin B12 deficiency. However, this vitamin D reduction was statistically significant only in the B12-deficient group and did not show a meaningful difference in the other groups. This may suggest that B12 and vitamin D deficiencies often coexist in obese children and may be influenced by similar mechanisms. While decreased vitamin D levels in obese individuals are frequently attributed to sequestration in adipose tissue, B12 deficiency has also been associated with the “volumetric dilution” hypothesis, which is linked not only to inadequate intake or malabsorption but also to changes in body compartment volumes2,46.

Volumetric Dilution hypothesis and serum micronutrient levels

In obese individuals, increased total body water, particularly a disproportionately larger expansion of extracellular fluid (ECF), has been suggested to dilute serum vitamin levels1,2,45,46. Aasheim et al. reported that, despite low serum vitamin levels in morbidly obese individuals, intracellular (erythrocyte) vitamin concentrations were normal or elevated. Similarly, other studies report that increased ECF volume in obese individuals could dilute serum micronutrient concentrations. In a systematic review by Pannu et al., it was stated that an increase in 25(OH)D was observed with weight loss in some studies, and in metaregression analysis, there may be an average increase of 6.0 nmol/L in 25(OH)D vitamin level for every 10 kg of average weight loss45.

This hypothesis highlights that low serum vitamin levels in obese individuals may not always reflect actual deficiencies. In our study, while serum B12 levels were lower in metabolically unhealthy obese children, it remains uncertain whether this represents a functional deficiency or merely a dilutional effect. If dilution contributes to lower serum B12, the clinical significance of these findings should be interpreted with caution. Therefore, clinical decisions should not rely solely on serum B12 levels; functional biomarkers (e.g., methylmalonic acid, homocysteine) and the presence of clinical symptoms should guide the need for intervention, particularly in indeterminate cases. An important limitation of our work is that its retrospective design precluded the assessment of functional vitamin status.

Biological role and active forms of vitamin B12

The term vitamin B12 is used to describe all cobalamin derivatives that are active in the human body, including cyanocobalamin, hydroxocobalamin, methylcobalamin, and adenosylcobalamin. Maintaining sufficient folate and cobalamin levels during childhood and adolescence is critical for cellular processes, particularly nucleic acid synthesis, cell division, amino acid and lipid metabolism, red blood cell formation, and nervous system myelination47. Vitamin B12 exerts its biological functions by converting into active forms—methylcobalamin and adenosylcobalamin.

It plays a vital role in the conversion of methylmalonyl-CoA to succinyl-CoA. Disruption of this process results in the accumulation of methylmalonic acid (MMA), which can impair fatty acid synthesis in neuronal membranes. It also serves as a cofactor for methionine synthase, which transfers a methyl group from methyl-tetrahydrofolate to homocysteine, thus producing tetrahydrofolate and methionine48.

Diagnostic challenges in assessing vitamin B12 deficiency

Studies on optimal serum concentrations of vitamin B12 in healthy European children remain limited, and universally accepted cut-off values have yet to be established49. In studies conducted on patients with megaloblastic anemia, Bolann et al. suggested a B12 deficiency threshold of 148 pmol/L (200 pg/mL), which showed 90% sensitivity, although 3–5% of deficient cases were still missed50. Another study found that 200 pmol/L as the cut-off yielded a 72–75% specificity for diagnosing vitamin B12 deficiency51.

However, serum vitamin B12 levels may not always reflect actual vitamin status. Metabolic B12 deficiency can occur in individuals with serum concentrations of 148–221 pmol/L (200–300 pg/mL), even without classic hematologic or neurologic symptoms. Therefore, in this “gray zone,” measuring functional biomarkers such as MMA and homocysteine may provide a more accurate assessment52. In our study, the active forms of vitamin B12 were not measured, which may have limited the accurate identification of true deficiencies. Nevertheless, previous research suggests that serum B12 levels are generally reliable indicators in epidemiological studies.

Study limitations and strengths

This study has several important limitations. First, due to its retrospective design, we were unable to evaluate more sensitive indicators of vitamin B12 status, such as holotranscobalamin (active B12), or functional metabolic markers like MMA and homocysteine. Furthermore, the cross-sectional nature of the study prevents any inference of causality. Key potential confounders, including socioeconomic status, detailed dietary intake, physical activity, and markers of inflammation, could not be accounted for in the analysis due to the retrospective nature of the data. The long-term clinical effects of observed low or borderline vitamin B12 levels remain unclear, warranting cautious interpretation of the findings.

Nevertheless, the study also has notable strengths. Most importantly, it included well-defined subgroups that allowed for comparisons between metabolically healthy and unhealthy obese children and their healthy peers. This approach made it possible to assess the relationship of vitamin B12 deficiency not only with obesity but also with metabolic phenotype in greater detail. In addition, the relatively large sample size and reliable laboratory data obtained from electronic medical records enhance the study’s internal validity.

Conclusion

In conclusion, our study demonstrated that vitamin B12 levels were similar between MHO and MUO children. However, when both obese groups were compared to healthy controls, vitamin B12 deficiency was found to be more common among obese individuals. Moreover, the observed associations between vitamin B12 levels and metabolic parameters such as triglycerides, uric acid, HDL cholesterol, and TG/HDL ratio suggest that B12 deficiency may be influenced not only by inadequate intake or malabsorption but also by metabolic alterations associated with increased adiposity. These findings emphasize the importance of monitoring vitamin B12 levels in childhood obesity and indicate that the metabolically healthy obese phenotype may not be protective regarding micronutrient status. In conclusion, our study highlights the importance of routine monitoring of micronutrient deficiencies, including vitamin B12, in children with obesity. While our study assessed serum vitamin B12 levels, functional testing was not performed. In clinical practice, functional assessment (e.g., methylmalonic acid, homocysteine) may be considered in cases with borderline or ambiguous serum B12 results, particularly in the context of the volumetric dilution hypothesis. These considerations can help guide targeted interventions beyond routine monitoring.