DOI: 10.5281/zenodo.13950605
title: Reference interval of optimal vitamin D level for an adult population of Bangladesh

description: Description of the data and file structure

The study collected data from healthy fishermen in Bangladesh to determine the reference interval for vitamin D levels in this population. Additionally, data from an urban population were gathered to identify the parathyroid hormone (PTH) deflection point in relation to vitamin D levels

Files and variables

File: Urban_final.sav

Description: This file contains data from an urban population aimed at identifying the parathyroid hormone (PTH) deflection point in relation to vitamin D levels.

File: Fishermen_final.sav

Description: This file contains data from healthy fishermen in Bangladesh, collected to determine the reference interval for vitamin D levels in this population.

Code/software

IBM SPSS v25 with Python plug-in software was used to analyse the data.

Medcalc version-14 statistical software was used to calculate reference intervals.
description: Abstract

Introduction: Vitamin D deficiency is a significant public health concern globally, including in Bangladesh, despite its favorable geographical location. Current reference intervals for vitamin D, predominantly derived from Western populations, may not accurately reflect the physiological needs of diverse global populations due to variations in genetics, skin pigmentation, dietary habits, and sun exposure. This study aimed to find out the population-specific reference intervals for serum 25-hydroxyvitamin D [25(OH)D] levels in healthy adult population of Bangladesh. Also, the study determined the   cut-off value of vitamin D deficiency level in Bangladeshi adults by detecting the  deflection point of parathyroid hormone (PTH) level compared to 25-hydroxy vitamin D [25(OH)D] level.  

Methods: A cross-sectional study was conducted involving two population groups. Group-1 comprised of adequately sun exposed 125 healthy coastal fishermen and Group-2 included 371 healthy urban dwellers. Group-1 represented an ideal population for assessing the reference serum vitamin D level and Group-2 population were used to  detect the cut-off value of vitamin D deficiency level in adult Bangladeshi population. The reference interval of vitamin D was determined based on Clinical and Laboratory Standards Institute (CLSI) Guidelines C28-A3 The lower cut-off value of vitamin D deficiency level was determined by detecting deflection point of parathyroid hormone (PTH) level compared to serum 25(OH)D level.

Results: The reference interval for vitamin D levels was found as 15.88-45.27 ng/ml.  The first increased response of iPTH level was observed when serum 25(OH)D level fell below 12.16 ng/ml (95% CI: 11.04, 13.28).

Conclusion: The findings suggest that current Western-based vitamin D reference intervals may not be appropriate for the Bangladeshi population. Establishing specific reference intervals for Bangladesh is crucial for accurate diagnosis and effective management of vitamin D deficiency. Depending on iPTH response, the cut-off value for vitamin D deficiency in Bangladeshi adults should be 12.16 ng/ml. This study provides a foundation for developing tailored public health strategies to address vitamin D deficiency in Bangladesh.
description: Methodology

The study was conducted from January 1, 2017, to December 31, 2021. The study protocol was approved by the institutional ethics review board. Informed consent was obtained from all participants, and data confidentiality was strictly maintained. 

Study site and population: The study was conducted in two geographical locations: Cox’s Bazar, a coastal district, about 298 km south of Dhaka city along the coast of Bay of Bengal with abundant sunlight; and Dhaka, the capital city, characterized by limited sun exposure of its inhabitants due to urbanization. The study was conducted from January 1, 2017, to December 31, 2021. 

Cross-sectional study design was employed to assess vitamin D levels in two population groups: adequately sun-exposed healthy coastal fishermen (Group-1) and urban residents with limited sun exposure (Group-2). Group-1 consisted of healthy adult coastal fishermen aged 18 years and above from Cox’s Bazar, who are regularly and adequately exposed to sunlight and consume marine fish, a rich source of vitamin D. Adequate sun exposure was defined as exposure to direct sunlight at least for 30 minutes, between 11 am and 2 pm, three times a week (13)  . Group-1 individuals (coastal fishermen) were  considered as reference population for determining the vitamin D reference interval of local population.  Group-2 comprised of ostensibly healthy urban residents from Dhaka, aged 18 years and above, with limited sun exposure due to lifestyle and environmental factors. 

Exclusion Criteria:  Any individual who refused to participate in the study or who were physically and psychologically handicapped, had known acute or chronic illnesses like diabetes mellitus, hypertension, liver disease or gastrointestinal disorders, kidney disease, malignancy, metabolic bone disease, primary hyperparathyroidism or lactose intolerance were excluded.   Anyone with relevant biochemical abnormalities and on anti-epileptic drugs was also excluded. 

For Group-1, any one who was consuming either vitamin D or calcium-containing products or who had ever consumed such drugs in the past was excluded.

Sampling technique and sample size: Convenience sampling was used to select participants from both groups. The sample size was determined based on the desired confidence limits and the variability in vitamin D levels observed in preliminary studies. Adequate sample sizes were ensured to provide statistically significant results.

Data collection tools and procedures: Sociodemographic  and clinical data were collected using structured questionnaires.. Height, weight, and blood pressure were measured, and blood samples were collected for estimation of serum 25(OH)D, iPTH levels and relevant biochemical parameters . The biochemical analyses were conducted using standardized procedures to ensure accuracy and reliability.

Collection of blood and biochemical investigations: About 10 ml ofvenous blood was collected aseptically from each participant after counselling and thorough clinical evaluation. Serum was separated immediately and was preserved at -600C for biochemical analysis. Chemiluminescent micro-particle immunoassay (CMIA) was used to estimate serum 25(OH)D level (14) by Architect analyser. Based on the  Endocrine Society guideline (9), vitamin D level was categorised as:  adequate/normal range: 75-100 nmol/L (30-40ng/ml), insufficient: 50 – 74.9 nmol/L (20ng/ml-29.99ng/ml) and deficient: <50 nmol/L  (<20ng/ml). Samples were  tested for  random blood glucose (RBG), serum creatinine, calcium, PO4, albumin, alkaline phosphatase and iPTH by Architect autoanalyser at the biochemistry laboratory of BIRDEM.

 Statistical Analyses: Statistical analyses were conducted using descriptive and inferential methods to summarize the data and assess factors influencing vitamin D levels. Chi-square tests, correlation analyses, and regression models were employed to explore these relationships. MedCalc version 14 statistical software was utilized to calculate reference intervals (95%, double-sided) following parametric, non-parametric, and robust methods as outlined in the CLSI C28-A3 guideline(15). A 90% confidence interval (CI) was used for these reference intervals, applying the appropriate statistical techniques. 

To determine the lower cutoff value of vitamin D deficiency, the deflection point of intact parathyroid hormone (iPTH) levels was identified using a non-linear model. Piecewise linear regression, applied through the Python plugin, was used to fit two separate lines before and after the deflection point (or “break” point), where the relationship between vitamin D and PTH shifts.

The equation used in this case represented as follows:






y= {



k1⋅x+y0−k1⋅x0



for x<x0





k2⋅x+y0−k2⋅x0



for x≥x0





 

In this model:



x represents Vitamin D level (independent variable).

y represents PTH level (dependent variable).

x0 is the deflection point where the two lines meet.

y0 is the PTH level at the deflection point.

k1 is the slope of the line before the deflection point (for x<x0).

K2is the slope of the line after the deflection point (for x≥x0).


The steps for calculating the deflection point include:



Initial estimation: A starting guess for x0 is made based on visual inspection or prior knowledge.

Curve fitting: The SciPy curve fit function optimizes x0, y0, k1and k2 by minimizing the difference between the model and observed data.

Confidence intervals: Standard errors are used to calculate 95% confidence intervals for the estimates.


This approach effectively identifies the point where the relationship between vitamin D and PTH levels changes.