Study of vitamin D in patients of beta thalassemia major at tertiary care hospital

Introduction

Beta (β) Thalassemia syndrome refers to a group of genetic disorders mostly inherited in an autosomal recessive manner, which results from a quantitative decrease in β-globin chain of hemoglobin (Hb). This can result due to point mutations, or deletions in the β-globin gene (HBB). Synthesis of α-globin continues as normal, resulting in accumulation of excess αglobin chains in erythroid precursors. This results in ineffective erythropoiesis and RBCs which are prone to hemolysis. The β-thalassemia trait is prevalent at rates ranging from 1.4% to 3.4% in Central India, with β-thalassemia major (β-TM) reported at 0.94%. Patients with β-thalassemia major are in a state of chronic anemia for which they require frequent blood transfusion therapy to improve the quality of life and reduce complications [1] . One of the major complications of multiple blood transfusions is iron overload which consequently leads to the deposition of iron in various tissues (Hemosiderosis) like liver, pancreas, endocrine glands (e.g. thyroid, parathyroid) and heart [2] . A major significant challenge is Thalassemia bone disease, characterized by bone deformities, growth retardation, osteoporosis. The underlying cause for thalassemia bone disease is the dysregulation of bone remodelling and calcium homeostasis which is chiefly attributed to transfusion-induced hemosiderosis of liver and parathyroid gland [3] . Vitamin D plays a crucial role in bone health, and its deficiency can exacerbate the bone problems often seen in thalassemia patients, such as osteoporosis and bone pain [4] . Calcium is a mineral that serves as an essential component of bone structure, providing strength and rigidity to bones and teeth. Beyond its structural role, calcium acts as a signalling molecule involved in various cellular processes, including muscle contraction, neurotransmitter release, enzyme activation, and blood clotting [5] . In thalassemia, calcium metabolism is hindered due to Iron overload, Vitamin D deficiency, hormonal imbalance and chelation therapy. A study by Sultana et al. concludes that transfusion dependent beta thalassemia patients have low calcium levels and high serum alkaline phosphatase level [7] . Another study by Ali et al demonstrated a high prevalence of vitamin D deficiency and low total serum calcium level in thalassemia patients which was significantly lower than the healthy group [9] . Still, paucity of information regarding effects of multiple blood transfusion on serum levels of calcium, vitamin D and alkaline phosphate amongst patients with β-thalassemia exists. This study aims to address this knowledge gap by determining the serum levels of vitamin D and attempt to establish an association between them with the number and duration of blood transfusion in patients with β-thalassemia major.

Materials and Methods

This cross-sectional study was conducted at the Department of Biochemistry and the Department of Paediatrics at M.G.M Medical College and M.Y.H Hospital in Indore, Madhya Pradesh. The study involved a minimum of 65 patients diagnosed with Beta Thalassemia major, aged between 5 and 15 years, who were attending either the outpatient or inpatient departments. The duration of the study spanned one year, following approval from the ethical committee. The primary focus was to measure and analyze serum levels of vitD in relation to the duration and number of blood transfusions received by the participants. Inclusion criteria; Diagnosed cases of Thalassemia Major, verbal assent from children aged 7 to 15 years in the presence of their parents, along with written informed consent from the parents prior to study. For children in the age group of 5 to 6 years, only written informed consent from the parents was taken. Both male and female participants were included in the study. Exclusion criteria; Individuals with Thalassemia minor were excluded, as well as those with other types of hemolytic anemia, including sickle cell disease, G6PD deficiency, and hereditary spherocytosis. Additionally, subjects with bone diseases such as rickets, osteomalacia, Paget's disease, bone fractures, or osteogenic bone cancer were excluded. Liver diseases, including intra and extrahepatic obstruction, hepatitis, and cirrhosis, were also excluded. Endocrine disorders like hyperthyroidism and hyperparathyroidism, as well as chronic conditions such as chronic kidney disease, were further grounds for exclusion from the study. Venous blood samples (5 mL) were collected under strict aseptic conditions using disposable syringes and transferred into clot activator tubes, allowed to stand for 30 minutes at room temperature. The samples were then centrifuged at 3000 rpm for 10 minutes, and the serum was extracted using a clean pipette and stored at-20 degrees Celsius for further analysis. Serum vitamin D levels were determined using an Enzyme Immunoassay (EIA) on an ELISA processor and reader in the biochemistry laboratory.

Statistical Analysis

The collected data were entered and stored in Microsoft Excel, and statistical analysis was performed using GraphPad Prism software version 10.2.3 (347). To determine the distribution of the data, the Kolmogorov-Smirnov test and Shapiro-Wilk test were utilized to classify the data as parametric or non-parametric. Parametric data were presented as means and standard deviations (SD), while non-parametric data were presented as medians and inter-quartile ranges (IQR). For parametric comparisons, the Unpaired t-test was used for two independent groups, and One-way ANOVA was employed for comparisons involving more than two independent groups. For non-parametric data, the Mann-Whitney U test was used for two independent groups, and the Kruskal-Wallis test was utilized for comparisons across more than two independent groups, with post-hoc analysis conducted using the uncorrected Dunn's test wherever required. Correlational analysis for parametric data was carried out using the Pearson correlation test, while the Spearman rank correlation test was applied for nonparametric data. A p-value of less than 0.05 was considered statistically significant for all tests.

Results

Table 1: Status of vitamin D level in serum of patients

Serum levels

Number of patients Percentage Mean concentration Vitamin D-deficiency (<10 ng/ml) 36 55% 11.65 ng/ml -insufficiency (10-30 ng/ml) 28 43% -sufficiency (30-100 ng/ml) 1 2% Among the 65 β-thalassemia major patients included in our study, 45 (69%) were males and 20 (31%) were females. No significant difference was observed in vitamin D concentrations between males (Median = 8.9) and females (Median = 7.4); p = 0.32.

Comparison of concentration of serum vitamin D in male and female subjects

Comparison of concentration of serum vitamin D in

Comparison of concentration of serum vitamin D level with duration of blood transfusion in β-thalassemia patients

All the 65 patients in our study were grouped based on their duration of BT into 3 groups: i) 1-5 years, ii) 6-10 years and iii) >10 years. difference (p = 0.001) in vitamin D concentrations between patients that had undergone <100 BT (median = 16) as compared to those with >200 BT (median = 6.9) Thalassemia as compared to the healthy controls (9). However, there is limited information regarding the association of serum vitamin D level with duration and number of blood transfusions in patients with β-Thalassemia patients.

In our study which included 65 patients with β-thalassemia major, 64 (98%) had vitamin D levels below the lower limit of reference interval, out of which 36 (55%) were found to have vitamin D deficiency (<10 ng/mL), 28 (43%) were found to have insufficient levels of vitamin D (10-30 ng/mL), with a mean concentration of serum vitamin D of 11.65 ng/ml. These findings are similar with the findings in several studies including Agrawal et al, [8] Ali et al [9] Ridha [10] et al that demonstrated decreased serum levels of vitamin D (<30 ng/mL) in 98%, 94% and 87% of patients with β-Thalassemia respectively, with a mean serum vitamin D concentration of 8.85, 11.11 and 13.54 ng/mL respectively. This indicates that serum concentration of vitamin D is decreased in majority of the patients with β-Thalassemia. Concentrations of serum VitD between male and female patients revealed no significant difference (p = 0.32). While Agrawal et al [8] , found no significant difference in serum VitD levels between males and females, Singh et al [12] , on the other hand, found serum VitD concentration to be significantly higher in males (Mean = 13.54 mg/dL) than in females (Mean = 6.32 mg/dL), (p = 0.026) [11] . We also compared the concentrations of serum VitD between two different age groups; 5-10 years and 11-15 years, which revealed a statistically significant (p=0.03) higher median values of serum VitD among patients belonging to 5-10 years age group when compared to 11-15 years age group.

On analysing serum VitD level in patients who had undergone BT for 1-5 years, 6-10 years, and >10 years, we found out that the median values of VitD levels were significantly higher in patients who had undergone BT for 1-5 years as compared to those who underwent BT for 6-10 years as well as >10 years (p = 0.02 and 0.02 respectively). We infer that serum VitD decreases significantly in patients with β-Thalassemia after 5 years of BT.

Conclusion

After analysing the results of the study, we can conclude that patients with β-Thalassemia major undergoing multiple blood transfusion (BT) have high prevalence of vitamin D deficiency. Vitamin D deficiency worsens with increased duration and number of blood transfusions. Specifically, serum VitD levels decrease significantly after 5 years of BT and continue to decline with greater numbers of transfusions (>200 BT). There is major impact of chronic transfusions on VitD status in these patients. Age-based analysis revealed higher median VitD levels in younger patients (5-10 years) compared to older ones (11-15 years). Thus, we suggest regular monitoring of VitD level in β-Thalassemia patients, particularly with increasing duration and frequency of blood transfusions, to mitigate the risk of related complications.

Acknowledgement

AcknowledgementsThe authors acknowledge the administrative and academic support given by the HOD and faculties of Department of Biochemistry and Department of Paediatrics, M.G.M Medical college, Indore.

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