Evaluation of serum 25-OH vitamin D and parathyroid hormone levels in patients with beta thalassemia major in relation to clinical status

Authors

Abstract

Aim Thalassemia is a severe hereditary condition characterized by anemia, hepatosplenomegaly, growth delay, jaundice, and skeletal changes, typically diagnosed within the first year of life due to mutations affecting beta-globin synthesis. Standard treatments include transfusion, chelation, and hematopoietic stem cell transplantation. While advancements in these modalities have improved life expectancy, long-term management has increased complications, particularly affecting vitamin D metabolism secondary to iron deposition in the liver and skin. This study aims to evaluate transfusion and chelation characteristics in thalassemia patients and to compare body mass index (BMI), calcium, phosphorus, alkaline phosphatase, ferritin, albumin, parathyroid hormone (PTH), and 25-OH vitamin D levels between patients and controls.
Materials and Methods Thirty-three patients with thalassemia major and 33 healthy controls were included. Age, sex, weight, height, BMI, transfusion frequency, chelation type and duration, and biochemical parameters were recorded. Serum 25-OH vitamin D and PTH levels were analyzed.
Results Among patients, 69.7% were male, with a mean age of 23.02 ± 8.18 years. Vitamin D insufficiency was identified in 33.3% of the patients, and vitamin D deficiency in 30.3% of the patients. Mean 25-OH vitamin D levels were significantly higher in the patient group than the control group (p < 0.05), while PTH levels were significantly lower in the patient group (p < 0.001).
Discussion Thalassemia patients exhibit altered vitamin D and PTH regulation, highlighting the need for routine monitoring. Larger studies are required to better understand the underlying mechanisms and additional contributing factors.

Keywords

Introduction

Beta thalassemia major was first defined in 1925 by Cooley and Lee in Italian children. It is a serious disease primarily characterized by anemia, hepatosplenomegaly, growth retardation, jaundice, and bone changes, usually diagnosed in the first year of life [1]. The cause is a genetic mutation that reduces or eliminates beta-globin synthesis [1, 2]. According to the World Health Organization, there are at least 70 million carriers worldwide, with at least 42,000 homozygous children born each year [3]. Turkey falls within the demographic with a high prevalence of the disease, with carrier frequencies ranging from 2-2.5% nationwide, reaching up to 10% in some regions [1, 4]. If untreated, beta thalassemia major severely impairs the quality of life and can result in rapid mortality. Successful outcomes have been achieved with treatment modalities such as transfusions, chelation therapy, and stem cell transplantation [5]. During the course of the disease, complications due to chronic iron overload are encountered (6-8). Endocrine disorders, commonly seen complications, are more prevalent in patients with inadequate chelation therapy and transfusion- related iron overload [7]. Several studies indicate that low vitamin D levels and parathyroid hormone levels in thalassemia major patients are implicated in the development of bone disease and iron overload; however, there is insufficient data on this topic. Vitamin D metabolism is particularly affected in thalassemia patients due to iron accumulation in the skin and liver. Despite abundant sunlight and vitamin D supplementation in many countries, vitamin D deficiency and insufficiency are reported at high rates in thalassemia patients [8].
In this study, we aimed to evaluate the transfusion and chelation therapy characteristics of thalassemia patients, assess their body mass index (BMI), calcium, phosphorus, alkaline phosphatase, ferritin, and albumin levels, and compare parathyroid hormone and 25-OH vitamin D levels between the patient and control groups.

Materials and Methods

A total of 66 cases were included in the study between October 2013 and December 2013. This comprised 33 patients (23 males, 10 females) diagnosed through hemoglobin electrophoresis and genetic analysis at the Pediatric Hematology Clinic of Okmeydanı Training and Research Hospital, and 33 individuals from the same age group as the control group (23 males, 10 females). After explaining the purpose of the research to the families and patients, consent was obtained for measuring 25- OH vitamin D and parathyroid hormone (PTH) levels. A physical examination of the patients was conducted, and data were recorded on a standardized form. The standard information forms included patient number, age, gender, date of birth, age at first diagnosis, weight, height, type of chelation therapy, monthly transfusion frequency, and serum levels of calcium (Ca), phosphorus (P), alkaline phosphatase (ALP), ferritin, and albumin obtained over the last six months. Biochemical blood values were retrospectively obtained from the patients’ files. Measurements of 25-OH vitamin D and PTH levels were conducted at the Biochemistry Laboratory of Okmeydanı Training and Research Hospital using kits purchased by the researcher. Blood samples of 4-5 mL taken from patients before transfusion were centrifuged at 4000 rpm for 10 minutes. For the 25-OH vitamin D assay, 400 µL of plasma was separated, and for the PTH assay, approximately 500 µL of serum was stored in Eppendorf tubes at -20 °C until the analysis date. Plasma 25-OH vitamin D2/D3 levels were measured using high-performance liquid chromatography (HPLC) on the Zivak ONH-100A device, while serum parathyroid hormone levels were measured using the chemiluminescence method on the Beckman Coulter UniCel DXI 800 device. The study utilized the HPLC method for measuring the 25-OH vitamin D level. Vitamin D deficiency was defined as <20 ng/mL (40-50 nmol/L), and vitamin D insufficiency as 20-30 ng/mL (50-75 nmol/L). PTH levels were considered normal if between 10-65 pg/mL and low if <10 pg/mL. Normal serum calcium levels were defined as 8.2–10.4 mg/dL, phosphorus levels as 2.5–4.6 mg/dL, and ferritin levels as 10–340 ng/mL. For statistical analysis of the data obtained in the study, SPSS (Statistical Package for Social Sciences) version 15.0 and GraphPad InStat demo version were utilized. Descriptive statistical methods (mean, standard deviation, minimum, maximum, etc.) were applied. For group comparisons, the Chi-square test and Fisher’s exact test were used for categorical variables, the Mann-Whitney U test for comparing means between two groups, and the Kruskal-Wallis test for comparing means among three groups, followed by Dunn’s test for pairwise comparisons. Correlations were assessed using the Pearson correlation test. Results were evaluated at a significance level of p < 0.05 with a 95% confidence interval ('p < 0.05, ''p < 0.01, '''p < 0.001).
Ethical Approval This study was approved by the Ethics Committee of Okmeydanı Training and Research Hospital (Date: 2013-10-08, No: 129). Written informed consent was obtained from all participants or their guardians.

Results

A total of 66 cases, comprising 33 patients and 33 controls aged between 2 and 37 years, were included in the study. The mean age of the patient group was 23.02 ± 8.18 years, while the mean age of the control group was 23.12 ± 8.16 years. There were no statistically significant differences between the patient and control groups in terms of age and gender. The mean Body Mass Index (BMI) of the patient group was 20.59 ± 3.03 (range: 11.40-25.70) (Table 1). The age at first diagnosis varied between 2 months and 4.5 years, with a mean of 1.33 ± 1.06 years (median: 1.00).
Chelation therapy with Deferoxamine was administered to 87.9% (n = 29) of the patients, Deferiprone to 60.6% (n = 20), and Deferasirox to 84.8% (n = 28). The transfusion intervals were 2 weeks for 3 patients (9.1%), 3 weeks for 29 patients (87.9%), and 6 weeks for only 1 patient (3.0%). All three chelation therapies were applied to 45.5% (n = 15) of the patients, whereas 30.3% (n = 10) received both Deferoxamine and Deferasirox, 9.1% (n = 3) received both Deferoxamine and Deferiprone, another 9.1% (n = 3) received both Deferiprone and Deferasirox, and 3.0% (n = 1) received only Deferoxamine. One patient, diagnosed at 11 months of age, did not receive any chelation therapy. The duration of chelation therapy varied: Deferoxamine was administered for 3 to 30 years (mean: 14.79 ± 6.49 years), Deferiprone for 5 months to 17 years (mean: 6.32 ± 4.66 years), and Deferasirox for 1 to 7 years (mean: 3.11 ± 1.72 years). The average transfusion interval was found to be 21.00 ± 4.29 days (range: 14-42 days). The average calcium level in the patient group was 9.64 ± 0.61 (range: 7.60- 10.50), the average phosphorus level was 4.71 ± 1.02 (range: 3.00-7.60), the average alkaline phosphatase (ALP) level was 138.70 ± 81.62 (range: 38.00-415.00), the average ferritin level was 1094.15 ± 940.81 (range: 72.30-4186.00), and the average albumin level was 4.28 ± 0.38 (range: 3.50-4.90). In the patient group, calcium levels were normal in 87.9% (n = 29), low in 3% (n = 1), and high in 9.1% (n = 3); phosphorus levels were normal in 51.5% (n = 17) and high in 48.5% (n = 16); and ferritin levels were normal in 21.2% (n = 7) and high in 78.8% (n = 26). The average ALP level was 136.80 ± 81.62, and the average albumin level was 4.14 ± 0.22. A positive moderate statistical correlation was found between age and the duration of Deferoxamine treatment (p < 0.001), as well as between age and the duration of Deferiprone treatment (p < 0.01). A weak positive correlation was observed between BMI and the duration of Deferoxamine treatment (p < 0.05). No statistically significant correlation was found between 25-OH vitamin D levels and other parameters, or between PTH levels and other parameters (p > 0.05). The average 25-OH vitamin D level was 26.82 ± 12.77 ng/ml (median: 25.30) in the patient group and 20.60 ± 7.00 (median: 19.20) in the control group, indicating a statistically significant difference favoring the patient group (p < 0.05). The average PTH level was 16.68 ± 6.69 pg/ml (median: 15.80) in the patient group and 44.92 ± 32.10 (median: 37.40) in the control group, with the patient group showing a statistically significantly lower level (p < 0.001) (Table 1). In the patient group, 36.4% (n = 12) had normal 25-OH vitamin D levels, 33.3% (n = 11) had vitamin D insufficiency, and 30.3% (n = 10) had vitamin D deficiency. In the control group, 12.1% (n = 4) had normal 25-OH vitamin D levels, 33.3% (n = 11) had vitamin D insufficiency, and 54.5% (n = 18) had vitamin D deficiency. The proportion of individuals with vitamin D deficiency in the control group was statistically significantly higher than in the patient group (Chi-square: 6.286, p < 0.05) (Figure 1).
In the patient group, 81.8% (n = 27) had normal PTH levels, while 18.2% (n = 6) had low PTH levels; in the control group, 84.8% (n = 28) had normal PTH levels, 3% (n = 1) had low PTH levels, and 12.1% (n = 4) had high PTH levels. The proportion of individuals with elevated PTH levels in the control group was statistically significantly higher than in the patient group (p < 0.05). No statistically significant correlation was found between age and 25-OH vitamin D levels, or between 25-OH vitamin D levels and PTH levels in the control group (p > 0.05), while a weak positive correlation was observed between age and PTH levels (p < 0.05). In the patient group, the average PTH level for those with normal 25-OH vitamin D levels was 17.53 ± 8.15 pg/ ml, for those with vitamin D insufficiency it was 16.49 ± 6.28 pg/ml, and for those with vitamin D deficiency it was 15.87 ± 5.71 pg/ml, with no statistically significant differences among these groups (p > 0.05). There were no statistically significant differences in age, age at first diagnosis, BMI, calcium, phosphorus, ALP, ferritin, and albumin levels, duration of Deferoxamine, duration of Deferiprone, duration of Deferasirox, or transfusion intervals between the groups categorized by 25-OH vitamin D levels in the patient group (p >. 0.05). No statistically significant differences were found in the rates of Deferoxamine, Deferiprone, and Deferasirox treatments among those with normal 25-OH vitamin D levels, those with insufficiency, and those with deficiency (p > 0.05). In the patient group, those with low PTH levels had higher ages and ages at first diagnosis, lower BMI, lower calcium, phosphorus, and ALP levels, higher albumin and 25-OH vitamin D levels, longer durations of Deferoxamine, Deferiprone, and Deferasirox treatments, and shorter transfusion intervals compared to those with normal PTH levels, although no statistically significant differences were found (p > 0.05). Ferritin levels were statistically significantly lower in the patient group with low PTH levels compared to those with normal PTH levels (p < 0.05). Additionally, the rate of Deferoxamine treatment was higher in the patient group with low PTH levels, while the rates of Deferiprone and Deferasirox treatments were lower, but no statistically significant differences were found (p > 0.05).

Discussion

In patients with thalassemia syndromes, frequent blood transfusions, chelation therapy, and bone marrow transplantation have led to an increase in life expectancy [1, 6, 9]. Along with the increased life expectancy, complications have also begun to be commonly observed in these patients (6-8). Endocrine problems are one of the frequent complications. Despite abundant sunlight and vitamin D supplementation in many countries, high rates of vitamin D deficiency and insufficiency are reported among thalassemia patients [6]. Although the exact reasons for vitamin D deficiency in thalassemia patients are not fully understood, possible mechanisms include inadequate dietary intake, poor gastrointestinal absorption, genetic and ethnocultural factors, aging, skin thickness, darker skin, or clothing that covers the skin leading to decreased synthesis of 25-OH vitamin D, reduced outdoor activities, anemia, iron overload, hemosiderosis, and hepatic dysfunction, which are associated with impaired 25-hydroxylation (6, 10- 17). 25-OH vitamin D levels are particularly reported to be lower in thalassemia patients during winter compared to summer, influenced by factors such as geographic location, daylight hours, and sunscreen usage [10, 14]. Even during the summer, lower 25-OH vitamin D levels have been detected in thalassemia patients compared to the control group [9, 14]. Therefore, vitamin D supplementation in thalassemia patients, especially during winter months, is considered clinically significant [9]. Although numerous studies have been conducted regarding vitamin D deficiency in thalassemia patients, the results of these studies show considerable variability. For example, Voskaridou et al. found vitamin D deficiency in very few adult thalassemia patients [15], whereas many other studies reported a high prevalence of vitamin D deficiency in thalassemia patients (10-12, 16, 18, 21-24). Among patients with β-thalassemia and chronic transfusions, despite calcium and vitamin D supplementation, over 60% were found to be vitamin D deficient [17]. In thalassemia patients with iron overload, 25-OH vitamin D levels were significantly lower compared to the control group, with some patients having levels below normal or undetectable; however, 1,25(OH)2D levels were found to be normal and similar to the control group in all patients [14]. Studies have reported vitamin D deficiency rates of 12% [8], 37% [7], 37.2% [18], 43% (<20 ng/ml) [19], 45.5% [2], 54.2% [16], and 80% [20] while vitamin D insufficiency rates were reported as 24.7% [2], 30% (20–29 ng/ml) [19], 54% [7], and 69.8% [8]. In thalassemia patients aged 4-15 years, the average serum 25-OH vitamin D level (10.4 ± 4.6 mcg/dl) was significantly lower compared to the control group (40.2 ± 12.3 mcg/dl) [11]. Another study also found significantly lower 25-OH vitamin D levels in thalassemia patients compared to the control group [21]. In the study by Vogiatzi et al., 12% of participants had vitamin D levels below 27 nmol/L, and 82% had levels below 75 nmol/L [12]. Lower vitamin D levels were found in adolescents compared to children and adults [22, 23]. Since adolescence is a critical period for optimal bone development, low vitamin D levels are considered a risk [12, 22]. In a study with an average age of 14.7 ± 7.6 years, 25-OH vitamin D levels were below 50 nmol/L in 13 of 24 patients and below 75 nmol/L in 23 patients (mean: 42.7 ± 21.2 nmol/L), whereas 1,25OH vitamin D levels were found to be normal or elevated. This was thought to be due to inappropriate elevation of PTH levels (secondary hyperparathyroidism compounded by primary hyperparathyroidism) or upregulation of 1,25OH hydroxylase in extra-renal tissues [24]. Despite abundant sunlight in Thailand, only 10.1% of children with Hb E/β-thalassemia, including both transfusion-dependent and non-dependent patients, had normal vitamin D levels, indicating that non-transfusion-dependent children were significantly more prone to vitamin D deficiency than their transfusion-dependent counterparts [19]. Fung et al. also found a higher prevalence of vitamin D deficiency in non- transfusion-dependent children in their study involving various types of thalassemia [23]. In a study comparing 90 patients with thalassemia major and 35 with thalassemia intermedia, serum 25-OH vitamin D was found to be below 10.4 ng/ml in 8 (10.1%) patients with thalassemia major and in 4 (11.4%) patients with thalassemia intermedia, indicating absolute vitamin D deficiency [24]. In the same study, the average 25-OH vitamin D levels were significantly lower in both patient groups compared to the healthy control group; 1,25-OH vitamin D levels were found to be normal to low, similar to previous studies [9, 14, 24]. Similarly, another study reported low 25-OH vitamin D levels in both thalassemia major and thalassemia intermedia patients. In a study involving 62 patients, low serum 25(OH)D3 levels were reported in 37 patients (60%) and borderline levels (50-75 nmol/L) in 7 patients [10, 13].

Limitations

Moreover, the relatively small sample size and absence of comprehensive data on potential confounders such as vitamin D supplementation, diet, and sunlight exposure precluded the application of multivariate regression analysis in this study. Incorporating multivariate models in future studies would provide a more robust understanding of independent predictors of vitamin D and PTH levels in patients with thalassemia major. The limitation of this study is the lack of assessment of factors that can affect vitamin D levels, such as the amount of daily sun exposure, level of outdoor physical activity, skin thickness, dietary intake of vitamin D, use of vitamin D supplementation, liver function, and comorbid conditions. Furthermore, the small sample size and the fact that 100% of the patients were transfusion-dependent and 97% were receiving chelation therapy limit the generalizability of the findings to the broader thalassemia population.

Conclusion

The findings of this study suggest that monitoring and control of vitamin D levels are also important in healthy individuals in our country. We believe that larger-scale studies evaluating additional factors influencing vitamin D levels are needed, and that in thalassemia patients, early intervention and appropriate treatment planning require not only monitoring of vitamin D levels but also parathyroid hormone (PTH) levels. Therefore, regular endocrine evaluation is considered essential.

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