Quantitative Analysis of the Steroidal Calcitriol-Mediated Regulation of the Serological Components Responsible for IDA and TSH Disorders in Reproductive and Nonproductive Women

Anemia and thyroid disorders are global health issues that affect all ages but are more apparent in women. In this case, some serological components responsible for IDA and TSH disorders in women have been found actively regulated through a complex steroidal-calcitriol mediated pathway. This research has been investigated the correlation between Calcitriol and the serological components responsible for IDA and TSH disorders in childbearing and non-child-bearing women of different health conditions. Experimental sampling from 452 women suffering from both IDA and TSH disorders were taken, aged between 0 and 70 years. Serological parameters, such as iron, total iron-binding capacity and ferritin, were assessed for IDA profiling, whereas thyroid-stimulating hormone and free thyroxin were for TSH profiling based on the individual’s serum calcitriol status. The resulted serological data were interpreted using sophisticated computer programming language and algorithms for quantitative biochemical analysis. The study resulted in a significant correlation between FT4 and Calcitriol (P<0.0001) for all age groups. TSH also showed strong interactions with the fluctuation of calcitriol level (P<0.0001), except for the children aged below 10 years (P<0.063). The iron, TIBC, TSH, and FT4 showed phenomenal regulation with the steroidal-calcitriol concentration for congenital patients. Unlike the others, ferritin has a substantial connection with Calcitriol (P<0.0064) fluctuation in the serum. To ratify, the concentrations of TSH, FT4, iron, TIBC, and ferritin were found to be significantly interconnected in terms of serum calcitriol level in women suffering from IDA and TSH disorders simultaneously. To understand the accuracy and efficacy of the Calcitriol in IDA and TSH disorders, some other inflammatory markers and parathyroid hormone analysis are need in future studies, besides a large number of samples.


Introduction
Anemia is a global public health concern nowadays that affects developing countries and developed countries with the major consequences of human health hazards. According to the previous data, it affects one-quarter of the global population, with pregnant women and young children having a higher prevalence rate than men [1]. Turning to the factors, iron deficiency is the major cause of poor nutrition, which correspondingly results in severe Anemia with the consequence of both mother and child's death [2]. Iron is vital to all biological functions, including DNA synthesis, respiration, cell proliferation, energy production, and so on [3]. Over 2 billion people are affected by iron deficiency worldwide [4,5], and the ubiquity of Anemia among pregnant women and young children due to Iron depletion has been well documented [6,7]. Age is also significantly correlated with IDA in females of childbearing and pregnancy [8].
Additionally, premenopausal women who stick to a restrictive diet and usually intake a little amount of iron are mostly at risk of iron deficiency since they also lost iron during their menstrual cycle; however, in 2002, WHO reported iron deficiency anemia (IDA) as one of the most important factors to the global burden of disease [9]. Therefore, frequent screening of IDA is very significant. Numerous iron indicators are used to screen for IDA, and a potential example is how serum ferritin can be used as a diagnostic tool in clinical practice [10]; a combination of two serum transferrin markers are used in detecting IDA in regular hemodialysis anemic patients [11].
Thyroid dysfunction is also one of the most prevalent endocrine disorders worldwide [12].
Globally about 1.6 billion people are at risk of developing thyroid disorder due to iodine deficiency [13]. There are two types of thyroid disorder means-hyperthyroidism and hypothyroidism. Iron status in humans is inextricably related to thyroid function. IDA deteriorates thyroid metabolism and retards the physical and mental development of both young and adult individuals. Based on several investigations, both ID and Anemia are interconnected with hypothyroidism which significantly increases serum TSH levels and decreases serum iron, serum ferritin, Free T4, transferrin, RBC count, and so on [14][15][16][17][18]. Ferritin is a universal protein that acts as an iron carrier, and serum ferritin level is negatively correlated with serum TSH levels [19,20]. In addition to iron and ferritin, Values of TIBC, FT3, and FT4 were significantly lower in hypothyroid patients suffering from IDA [21,22]. This lower serum ferritin level is also associated with reducing sex hormones along with TSH, which exaggerates another endocrine dysfunction [23]. To summarize the inter-connection, on the one hand, deficiency of iron can produce hypothyroidism [24]. On the other hand, alterations in thyroid status change serum iron metabolism and hematological index [16].
Calcitriol is an activated form of vitamin D, which is semi-synthetic and hormonally active.
Vitamin D is a fat-soluble steroid hormone mainly produced in the skin when exposed to sunlight. Vitamin D may also be acquired from the ingested diet to a minor extent [25]. To explain further, vitamin D biosynthesis in the skin becomes initiated by UV rays of sun lights which convert 7-dehydrocholesterol to pre-vitamin D3; then, it is thermally isomerized to vitamin D3. Calcitriol is the main vitamin D metabolite and is the best determiner of vitamin D nutritional status in the flow. However, this metabolite is not the active form of vitamin D, which requires a further activation step, and this happens in the presence of the catabolic enzyme 25OHD-1-α-hydroxylase (CYP27B1) to finally generate Calcitriol (1,25-(OH)2D3) [26,27]. In recent years, Calcitriol has taken close attention because its deficiency entails the risks of various human diseases such as gestation-associated disorders [28]. According to previous studies, Calcitriol (activated vitamin D) insufficiency affected over one billion people globally [29,30].
The lower calcitriol levels are associated with higher serum TSH levels [25] and lower hemoglobin and ferritin levels [31]. Studies highly suggested the supplementation of vitamin D in case of hypothyroidism and Anemia, along with the high recommendation for the screening of the vitamin D deficiency in all hypothyroid patients [25,32]. It is practically required for monitoring iron nutritional status as it exaggerates thyroid disorders in reproductive age and pregnant women [14]. Thyroid dysfunctions must also be considered by physicians treating Anemia to ensure early detection and proper treatment [33].

Clinical diagnosis
The current research started with diagnosing serum calcitriol levels [34]  values,' as the primary factors. In addition, least-square mean (LSM), mean difference (MD); standard error of the difference (SED); the difference between predicted means (DBPM), and 95% CI of difference (95% CID) were analyzed as the secondary parameters. In this study, Calcitriol was assigned to be considered as a serological marker only if its quantitative values belong to P<0.02 in all the primary and secondary parameters, to each of the selected serum components; so that the analysis can be more authentic as compared to the P<0.05 scale [38].

Software tools for data analysis and validation
The biostatistical analysis and computational algorithms were performed using computational 'R Programming Scripts' (version R-4.0.2, for Linux) [39,40] and 'GraphPad Prism' (version 8.1.2, for Mac OS) [41,42,43] premium software packages.

Results
In (ng/ml). This study experienced 13 congenital cases of IDA and TSH disorders among all women.
Surprisingly, according to their calcitriol profiles, all serological parameters have been found highly significant considering their two-way ANOVA and 'Tukey's multiple t-tests' of variables ( Figure 3). For the congenital patients, individual correlations of TIBC, TSH, and FT4 with their calcitriol concentration is exactly P<0.0001 ( Figure 3A). The overall results of each parameter were assessed based on the standard data generated by sampling the same serological parameters from six normal women as a control to compare their calcitriol status with the diseased women. The calcitriol level of the normal women ranged between 20ng/ml to 40ng/ml. In response to which, their iron, TIBC, ferritin, TSH and FT4 ranges found as 50-93 µg/dl, 250-423 µg/dl, 45-113 ng/ml, 0.5-4.0 µIU/ml, and 0.70-1.67 ng/dl respectively (Figure 4).

Discussion
The term "vitamin D" refers to a group of secosteroid compounds, two of which, cholecalciferol (known as vitamin D3) and ergocalciferol (also known as vitamin D2), are most commonly associated with the term [44]. The former is composed of a reduction of 7 dehydrocholesterol in the skin following the exposure to ultraviolet B (UVB) radiation from few dietary sources (mainly fish fat). At the same time, the latter is compounded by plants and fungi, which can form a dietary basis of vitamin D2 for people [45]. Both D2 and D3 are hydrolyzed with 25- (FGF23) [46]. Calcitriol attaches to the intracellular vitamin D receptor (VDR) that influences the response components of objective genes [47].
Calcitriol has long been recognized as an important hormone in regulating the musculoskeletal system, and it continues to be so today. The extra-skeletal effects of 1,25(OH)2D were also highly researched over the last decade after the presence of vitamin D receptors in almost all tissue types was established [48]. In the context of thyroid disorders, the antiproliferative and differentiating effects of Calcitriol are significant, and its function in modulating the immune system has been demonstrated in autoimmune thyroid disease (AITD) [27,44]. Several research studies have shown that vitamin D3 has vital roles in maintaining bone health, immunity, and muscles. Several studies showed a link between vitamin D deficiency and thyroid disorders [49].
IDA is a common problem and highly prevalent among Bangladeshi women, especially among pregnant women and females living in low iron water supplies [50]. Many IDA patients remain undiagnosed worldwide as the early stages show minor symptoms.
Furthermore, people with chronic diseases like CVD (cardiovascular diseases) and CKD (chronic kidney diseases) have more significant risks of suffering from IDA [51]. The function of several proteins, metabolic activity, including imbalance of thyroid hormones, may occur due to iron deficiency. Changes in ferritin levels affect thyroid functions. Low levels of TSH and high levels of FT4 occur due to an imbalance in TSI (thyroid-stimulating immunoglobulin), leading to hyperthyroidism. FT4 does not bind to proteins, which is good for diagnosing thyroid problems [52].
The potential relationship of activated vitamin D3 (Calcitriol) with the regulation of thyroidstimulating hormone (TSH) has been identified ( Figure 1A). We have collected the TSH in µIU unit and Calcitriol in ng/ml unit of serum concentration from 452 women blood who possessed the TSH and IDA disorders according to the age limit of 0 to 70. According to the current study, Iron-deficiency anemia is defined when blood levels of iron will be low, or less than 10 micromoles per liter (mmol/L) for both men and women (normally 10-30 mmol/L) (Figure 4).
Iron deficiency anemia increases susceptibility to infectious disease, increased child mortality, slowed child development, and reduced scholastic performance [55]. There is an association of calcitriol deficiency with the regulation of greater risk of Anemia, lower mean hemoglobin (LMH), and higher usage of erythrocyte-stimulating agents [56]. The research results have been depicted a strong correlation between iron and Calcitriol (P<0.0001) among the patients ( Figure   2A). The calcitriol level of the normal women ranged between 20ng/ml to 40ng/ml, and iron level is 50-93 µg/dl (Figure 4). Here Iron level is increased (150µg/dl) and decreased (9µg/dl) with the increase and decrease of calcitriol level as 54.98 ng/ml and 8 ng/ml, respectively. Total iron-binding capacity (TIBC) plays a pivotal role in indirectly measuring the percentage of transferrin situation involved in positive correlation with vitamin D [57]. Transferrin and vitamin D levels decreased, whereas TIBC levels increased during iron-deficient anemic patients [58].
According to this study, the serum TIBC range for all the patients was between 220 µg/dL to 578 µg/dL with some fluctuations.
In contrast, the activated Vitamin D (Calcitriol) value was between 8 ng/mL and 26.29 ng/mL with a little bit of oscillation. In this research, it was found that serum TIBC result was increased.
In contrast, serum calcitriol level was decreased from all the individual patients, consequently indicating iron deficiency anemia [59], as compared to the normal range (Figure 4). The ferritin level in the human body indicates the iron status and iron storage. Ferritin levels are lower in people who have iron deficiency anemia, but they may be higher in people with inflammation and chronic disease-related Anemia [60]. However, in the present analysis, the range of the ferritin level was from 21 ng/mL to 166 ng/mL with some fluctuations, and calcitriol level was between the ranges of 8 to 26.29 ng/mL respected to the normal control. It was noticed that serum calcitriol value was slightly higher, whereas serum ferritin values were significantly lower following the calcitriol scores for all the patients ( Figure 2C). The current investigation found a positive association between serum calcitriol and ferritin levels, consistent with previous findings [61,62].
It is mainly due to maternal risk factors such as inadequate dietary intake of vitamin D, insufficient exposure to sunlight, and pregnancy that occurs close to the people who suffer from congenital calcitriol deficiency [63]. Low maternal vitamin D levels may raise the risk of a newborn's deficiency [64]. Besides, spontaneous hypothyroidism affects between 1% and 2% of the population and is more prevalent in older women [65]. Therefore, a poor pregnancy outcome is associated with vitamin D deficiency or insufficiency, which leads to several disorders such as low birth weight of newborns [66]. According to the current serological assessment, the value of Calcitriol (8.19-25.3ng/ml) and iron (15-75 µg/dl) reduced significantly (Figures 3A and 3B) than the control group (20-40 ng/ml) and (50-93 µg/dl), respectively (Figure 4) Figure 3A and 3B).
The research has described the values and regulatory relationships of the same serological parameters like iron, TIBC, ferritin, TSH, and FT4, with the Calcitriol of six normal women as a control group, where the range calcitriol level from the control group was found 20ng/ml -40 ng/ml, which was within the normal limit (Figure 4). On the other hand, the range of iron, TIBC, Ferritin, TSH, and FT4 were found 50-93mcg/dl, 250-423mcg/dl, 45-113n/ml, 0.5-4.0 mcgIUml, respectively, which were also within the normal ranges ( Figure 4). The potential calcitriol-based regulation of the other serological parameters has been found in normal six women, used as the promotes erythropoiesis, which may help to prevent Anemia. As a result, all patients diagnosed with vitamin D deficiency in the outpatient hospital should be tested for iron deficiency and Anemia. Those who require replacement medication should be provided it. However, Future studies are needed to determine the accuracy and efficacy of Calcitriol in IDA and TSH disorders, in addition to larger samples, biochemicals, inflammation markers, and parathyroid hormone analysis.

Conclusion
It is reasonable to expect that significant calcitriol (activated form of vitamin D) deficiency will occur in the majority of the women suffering from various forms of thyroid autoimmunity and iron deficiency anemia (IDA), based on our previous experience the findings of our research study. The question remains on how to respond in such a circumstance. Based on the results of our serological research study, it can be concluded that the concentrations of TSH, FT4, iron, TIBC, and ferritin were correlated with the levels of serum calcitriol or activated vitamin D3 in women suffering from TSH and IDA abnormalities. Moreover, depending on a woman's reproductive status and age, Calcitriol has been validated as a biomarker for tracking the status of IDA and TSH irregularities. However, more research with larger sample size is required to understand better the serological profiles of patients with IDA and TSH disorders.