https://admin.imcjms.com/ Ibrahim Medical College Journal of Medical Science Wasim Md Mohosin Ul Haque*Md Faruque PathanM Abu Sayeed https://admin.imcjms.com/registration/journal_full_text/600 2026-05-04 10:27:03 Original Article January 2026; Vol. 20(1):006 0.05) different (22.22 ± 3.15 kg/m² and 26.28 ± 3.72kg/m²). Detail socio-demographic and anthropometric characteristics of Group-1 and 2 study populations are shown in Table-1. All the biochemical parameters of Group-1 study populations were within normal ranges (Table-2).   Table-1: Socio-demographic and anthropometric parameters of Group-1 (n=125) and Group-2 (n=371) study population     Table-2: Biochemical parameters of Group-1 study population (n=125)     The mean serum vitamin D level of Group-1 study population was 27.36±7.27 ng/ml, (95% CI: 26.08, 28.65). No significant difference (F = 0.506, p = 0.679) and correlation (r = -0.05511, p = 0.5416, 95% CI: -0.2285, 0.1217) were found in vitamin D levels amongst different age groups. However, there was a significant difference among BMI groups (F = 3.377, p = .037). Post-hoc testing using Bonferroni correction revealed that individuals with the highest BMI (25-29.9 kg/m²) had significantly (p = 0.037) lower serum 25(OH)D levels compared to those with the lowest BMI (<18.5 kg/m²), with a mean difference of -6.82 ng/ml. However, No significant differences of vitamin D levels were observed between other BMI groups (Table-3).   Table-3: Age and BMI-specific serum Vitamin D levels of Group-1 study population (n=125)     Based on the Endocrine Society cut-off value [9], out of total 125 participants, 39 (31.2%), 63 (50.4%) and 23 (18.4%) participants had adequate (≥30 ng/ml), insufficient (21-29.9 ng/ml) and deficient ((<20 ng/ml)) levels of vitamin D respectively (Table-4). There was no significant difference in serum iPTH level and other relevant biochemical parameters amongst these three vitamin D status groups (Table-5). Also, overall no significant correlation was observed between serum 25(OH)D level and serum iPTH or other relevant biochemical parameters of the total Group-1 study population (Table-6).   Table-4: Serum Vitamin D status of the Group-1 (coastal fishermen) study population based on the Endocrine Society cut-off value [9] (9)     Table-5: Levels of serum iPTH and other relevant biochemical markers related to vitamin D status of Groups-1 study population (n=125)     Table-6: Correlation between serum 25(OH)D levels and relevant biochemical parameters of Group-1 study population     The reference interval for serum vitamin D levels of our Group-1 reference population (coastal fishermen) was calculated in accordance with the CLSI Guidelines C28-A3 [15] using a 90% confidence interval (CI). The calculated reference interval of serum vitamin D value by parametric, non-parametric and robust methods were 15.78-44.29, 15.88-45.27 and 15.69-44.68 ng/ml, respectively. Detail is shown in Table-7. Figure-1 displays the 95% reference interval for vitamin D, visually representing the ranges defined by the aforementioned methods, ensuring a comprehensive understanding of the expected vitamin D levels within this population group. This structured approach enhances the reliability of the reference intervals, providing a solid foundation for further comparative analyses within the study.   Table-7: Reference interval of vitamin D levels calculated by different methods using serum 25(OH)D level of Group-1 study population         Figure-1: Reference interval of vitamin D. (95% Reference interval, Double-sided)   In the second part of the study, Group 2 population, consisting of 371 adult urban residents, was used to determine the lower cutoff value for serum 25-hydroxyvitamin D [25(OH)D] deficiency levels in healthy Bangladeshi adult population. Urban residents, exhibited a mean serum vitamin D level of 21.53±15.98 ng/ml (95% CI: 19.90, 23.16), with a median level of 17.18 ng/ml, ranging from 3.48 to 147.32 ng/ml. Parametric tests showed no significant differences in vitamin D levels between males and females (male: 20.16±16.18 ng/ml and females: 22.76±15.74 ng/ml (p > .05, 95% CI: -5.86, 0.66). However, nonparametric tests indicated significantly higher levels in females (p = .008). An analysis of age-related trends revealed that older age groups in the urban population exhibited significantly higher mean serum vitamin D levels, demonstrating a clear positive correlation with age (r = .174, p = .001, 95% CI: 0.073, 0.271). The distribution of vitamin D levels across different age groups and BMI categories is detailed in Table-8. Despite these age-related variations, no significant differences were found in vitamin D levels across different BMI categories (F = 0.173, p > .05).   Table-8: Age and BMI-specific Serum Vitamin D Levels of Group-2 Study Population (n=371)     According to the Endocrine Society cut-off value [9], 83.6% of the urban population had below-normal (inadequate) vitamin D levels. However, using the fishermen's reference intervals established in the first phase of this study, only 44.2% of the urban population were found to have below-normal vitamin D levels (Table-9). This discrepancy highlights that nearly half of the urban residents categorized as vitamin D deficient by conventional standards might actually fall within an adequate range when using our fishermen's reference intervals. The mean serum vitamin D level was significantly (p < .001) lower in the urban group (21.53±15.98 ng/ml) compared to fishermen (27.36±7.27 ng/ml).   Table-9: Vitamin D status of the urban people based on the Endocrine society cut-off value and fishermen's reference interval     The average serum intact parathyroid hormone (iPTH) level of Group-2 urban population was 62.80 ±31.67 pg/ml, ranging widely from 10.20 to 227.54 pg/ml, with a median value of 56.4 pg/ml. A significant negative correlation was observed between serum vitamin D and iPTH levels, evidenced by a correlation coefficient (r) of -0.233, indicating that iPTH levels generally rise as vitamin D levels fall. This relationship was statistically significant, with a p-value less than .001 and a 95% confidence interval ranging from -0.3273 to -0.1347. The analysis of iPTH levels across different vitamin D status segments, as defined by the Endocrine Society, is shown in Table-10.   Table-10: Mean PTH level in different vitamin D segments based on Endocrine society cut-off value     These findings suggest a nonlinear relationship between vitamin D and iPTH, where iPTH levels decrease as vitamin D increases, with notable differences in mean iPTH levels across vitamin D segments (F=5.55, p=.001;Figure-2). The LOWESS (locally weighted scatter plot smoothing) method was employed to illustrate the relationship between iPTH and 25(OH)D. It depicts the dynamic relation between iPTH and 25(OH)D levels. The scatterplot highlights a deflection point in the relationship, initially estimated at 12.5 ng/ml, and refined to 12.16 ng/ml (95% CI: 11.04, 13.28) through piecewise linear regression. This deflection point marks a new lower cut-off for optimal serum vitamin D levels based on physiological responses observed in iPTH.     Figure-2: The scatterplot with LOESS fitting (50%) illustrates the relationship between vitamin D and PTH, with vitamin D (ng/ml) on the x-axis and serum iPTH (pg/ml) on the y-axis. The vertical line indicates the PTH deflection point using the raw data.   Discussion Presently, reference intervals used to assess the vitamin D status of Bangladeshi population, and many other similar non-Western populations, are predominantly derived from studies conducted in Western populations and therefore, may not accurately reflect the physiological needs or health outcomes of these diverse population [9,10]. Variations in genetics, skin colour, dietary habits, and sun exposure significantly impact vitamin D levels, rendering universal reference intervals potentially inappropriate for Bangladeshi as well as many non-Western populations. Therefore, in the present study was conducted to establish a reference interval for serum 25-hydroxyvitamin D of adult Bangladeshi population using a defined reference population groups. The result of this study is expected to prevent over estimation of low vitamin D status among our local population. In our study, coastal fishermen group constituted the reference population for vitamin D estimation. The coastal environment, with its pollution-free air, excellent sun exposure, and consumption of marine fish, ensured vitamin D adequacy for our reference group. Prior studies, such as the 1958 birth cohort study in Scotland, have also linked coastal climates with higher levels of 25(OH)D due to increased sun exposure [16]. In our study, only male participants were enrolled in Group-1 to ensure adequate sun exposure. Though this might be considered as a limitation, other studies indicate no significant gender-specific differences in vitamin D levels [8,17]. Overweight subjects were excluded from our Group-1, yet consistent with findings of other studies previous studies, a significant negative correlation between vitamin D and BMI was observed [18-21]. Studies consistently show an inverse relationship between BMI and vitamin D levels, highlighting the impact of body weight on vitamin D status [22-24]. The lack of correlation between age and serum vitamin D levels in Group-1 suggests the overall good health of the study population. This might obscure the typical association between lower vitamin D levels and older age, often attributed to reduced outdoor activity and impaired kidney function in older adults [25]. In the present study, health status of the Group-2 participants was not strictly controlled. Only those with acute illnesses were excluded. This approach aligns with other studies examining the correlation between iPTH and vitamin D, which often include people with a broader range of health statuses [26-32]. In the present study, irrespective of the adequacy of sun exposure, most participants in both the fishermen and urban groups had low vitamin D levels, with means of 27.36±7.27 ng/ml and 21.53±15.98 ng/ml, respectively. This high prevalence of vitamin D inadequacy aligns with recent studies reporting widespread vitamin D insufficiency or deficiency in Bangladesh [8,33-39]. For instance, Mahmood et al. (2017) found that 100% of garment workers and 97% of agricultural and construction workers had low vitamin D levels. Garment workers, in particular, are at high risk due to limited sun exposure and poor nutritional status [40]. Within urban participants, hospital staff had lower vitamin D levels and a higher prevalence of inadequacy compared to healthy volunteers. This finding is supported by multiple studies indicating a high prevalence of vitamin D inadequacy among health personnel [41-46]. The positive correlation between vitamin D and age in the urban population may be explained by higher supplement use among older individuals [43,47,48]. Vitamin D supplementation, as shown in various studies, significantly increases serum 25(OH)D levels [43,49]. Higher vitamin D levels in our reference population (coastal fishermen) compared to urban dwellers underscores the importance of sunlight exposure. Similar studies, such as those by Lee et al. [50] and Haddad and Chyu [51], also reported higher vitamin D levels in individuals with significant sun exposure. Despite this, according to the Endocrine Society's cut-off, around 70% of coastal fishermen in this study had low vitamin D levels, raising questions about the validity of the use of current threshold values. Previous studies in heavily sun-exposed regions in India, report similar findings of low vitamin D levels despite abundant sun exposure [52,53]. Thus, the reference intervals for vitamin D determined in this study using healthy coastal fishermen as reference population represent the optimal levels for the Bangladeshi population. This conclusion is based on the nature of the studied group and the limitations of previous definitions of "optimal" vitamin D status. In this study, a reference interval of 15.88 ng/mL (90% CI: 14.26–16.92) to 45.27 ng/mL (90% CI: 41.11–51.85) was determined using non-parametric methods aligning with the Clinical and Laboratory Standards Institute (CLSI) Guidelines [54]. The non-parametric approach is appropriate given the asymmetry of the data, providing a robust understanding of the distribution in the fishermen population. The upper limit of 45.27 ng/mL aligns with the commonly accepted upper limit proposed by the Endocrine Society, suggesting the upper threshold of vitamin D status is consistent across various populations. However, the lower limit of 15.88 ng/mL stands below the traditionally accepted threshold levels of 20-30 ng/mL. Other studies also proposed lower cut-off values for vitamin D sufficiency. Studies from South Korea [50], India [30], China [28], and Greece [29] have consistently shown lower thresholds of 13-20 ng/mL. The range in this study is also closer to those findings, implying that vitamin D requirements are influenced by both environmental and genetic factors, and that a universal cut-off point may not be applicable globally. Moreover, iPTH dynamics provide additional support for our observed lower thresholds of vitamin D. Vitamin D inadequacy is often marked by increased iPTH levels, which serve as a surrogate marker for deficiency. However, in this study, no significant correlation between vitamin D and iPTH levels was found in the coastal fishermen group, unlike in the urban population, where a deflection point in iPTH occurred at levels far below the Endocrine Society’s recommended level of 30 ng/mL. Studies involving other population have noted similar findings: iPTH rises as vitamin D level drops below 8 - 21.1ng/mL [26, 28 -30, 32, 55-58]. Recently, a study involving seven non-identical occupational groups, also reported significant increase in iPTH when vitamin D level dropped below <11.8ng/ml [59]. These studies suggest that a threshold for iPTH deflection, and thus vitamin D sufficiency, may indeed be lower than previously thought. The absence of iPTH deflection in our coastal fishermen is indicative of overall vitamin D adequacy in this population. Additional support in favour of our low reference interval and lower cut-off values for vitamin D sufficiency comes from recent randomized controlled trials (RCTs) and cohort studies like VITAL and ViDA [60-62]. Post hoc analyses from trials like VITAL and ViDA revealed that vitamin D supplementation benefited participants only when baseline levels were below 12-15 ng/mL [60-62]. Also, Martineau et al. (2017) noted that vitamin D supplementation was most effective in preventing respiratory infections in participants with baseline levels below 10 ng/mL [63]. These findings argue against higher supplementation thresholds and support a more moderate cut-off for deficiency. Furthermore, based on epidemiological evidence, Manson et al. and the Institute of Medicine (IOM), support a deficiency threshold closer to 12.5 ng/mL[62,64] Studies on pregnancy and diabetes prevention also point toward 15 ng/mL as a sufficient level of vitamin D for avoiding adverse outcomes [31,65]. In view of the above, it is concluded that the reference interval identified in the current study (15.88–45.27 ng/mL) truly reflects the optimal range of vitamin D level for the adult Bangladeshi population. The high sun exposure in coastal fishermen makes this population an ideal reference group, better reflecting the biological requirements of individuals in sun-rich environments like Bangladesh. Moreover, the lower limit of this range is consistent with thresholds suggested by contemporary studies that challenge higher vitamin D cut-off points. Thus, considering the iPTH dynamics and recent trial data, the present study provides a more region-specific, evidence-backed approach regarding optimal vitamin D levels. The strength of our study lies in the fat that the study used a defined population as ‘reference population’ with supposedly adequate vitamin D levels. Also, reference intervals were determined following the Clinical and Laboratory Standards Institute (CLSI) Guidelines C28-A3 for precise comparison across populations, and the lower cutoff value was identified using the iPTH deflection point. However, the study did not employ strict random sampling and gold-standard LC-MS/MS vitamin D assay.   Conclusion This study's findings on healthy coastal fishermen provide critical insights into the optimal serum vitamin D levels for adult Bangladeshi population. Additionally, the study has defined the lower normal limit of vitamin D sufficiency by determining the iPTH deflection point, suggesting a lower optimal range than currently used. Thus, the findings of the present study would help in guiding clinicians and policymakers in developing appropriate treatment and supplementation guidelines for Bangladeshi population.   Acknowledgement Authors gratefully acknowledge the support of Bangladesh Institute of Research and Rehabilitation for Diabetes Endocrine and Metabolic Disorders and The Centre for Higher Studies and Research, Bangladesh University of Professionals. We are thankful to Professor Jalaluddin Ashraful Haq for his suggestions and support in carrying out the project.   Authors’ Contributions WMMUH was the primary researcher, and mainly involved in data collection, analysis, and drafting of the manuscript. MFP, MAS and JAH, contributed equally to the conception, design, interpretation of the data, and critical revision of the manuscript.   Conflict of interest The authors declare that they have no conflict of interest.   Funding The research received funding from the Bangladesh Medical Research Council (BMRC)   Preprint statement The article was published as preprint under the title “Reference interval of optimal vitamin D level for an adult population of Bangladesh” in medRxiv. doi: https://doi.org/10.1101/2024.11.07.24316898.   Data Availability Statement The dataset of this study is available in the Zenodo repository, DOI: 10.5281/zenodo.13950605. Additional materials, such as the technical appendix and statistical code, can also be accessed at this DOI.   Referenes 1.     Layana AG, Minnella AM, Garhöfer G, Aslam T, Holz FG, Leys A, et al. Vitamin D and age-related macular degeneration. Nutrients. 2017; 9(10): 1120.doi: 10.3390/nu9101120. 2.     Nair R, Maseeh A. 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