Parameters of Vitamin D Metabolism in Patients with Hypoparathyroidism

Only a few studies evaluating the metabolism of vitamin D in patients with hypoparathyroidism (HypoPT) have been performed thus far, and, in particular, they mainly investigated the process of vitamin D activation (specifically, 1α-hydroxylation). This study, therefore, aimed to evaluate the extended spectrum of vitamin D metabolites in patients with HypoPT compared to healthy individuals. We examined 38 adult patients with chronic HypoPT in comparison to 38 healthy adults. The assessment included biochemical parameters (total calcium, albumin, phosphorus, creatinine, and magnesium), parathyroid hormone (PTH), and vitamin D metabolites (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3, and 24,25(OH)2D3) in serum. Our data show that an adequate level of 25(OH)D3 (median 35.3 (29.6; 42.0) ng/mL) is achieved with standard doses of cholecalciferol (median 2000 (2000; 2500) IU per day) in HypoPT patients. They also presented with supraphysiological levels of 1,25(OH)2D3 (median 71 (47; 96) vs. 40 (34; 59) pg/mL, p < 0.001) and the increased production of inactive metabolite (median 24,25(OH)2D3 3.8 (3.0; 5.1) vs. 1.9 (1.3; 2.7) ng/mL, p < 0.001; median 25(OH)D3/24,25(OH)2D3 ratio 8.9 (7.6; 11.1) vs. 13.5 (11.1; 17.0), p < 0.001) as compared to the control group. This might be a consequence of the therapy received (treatment with activated vitamin D) and the pathophysiology of the disease (lack of PTH). The abnormality of vitamin D metabolism does not seem to interfere with the achievement of hypoparathyroidism compensation.


Introduction
Hypoparathyroidism (HypoPT) is a rare condition characterized by reduced parathyroid hormone (PTH) levels resulting in hypocalcemia and hyperphosphatemia. Hypocalcemia leads to increased neuromuscular irritability causing muscle spasms, tingling, and seizures. The most common cause of HypoPT is anterior neck surgery. Less commonly, HypoPT occurs as a result of autoimmune diseases and is secondary to other conditions. To date, HypoPT therapy remains a challenge, and data on optimal treatment strategies for patients with HypoPT are limited due to the lack of controlled clinical trials. Conventional therapy includes activated vitamin D and calcium supplements; however, this treatment does not adequately compensate for the lack of PTH [1]. Inadequate medical control of the disease can lead to both hypocalcemia and hypercalcemia, which might result in long-term complications such as nephrocalcinosis, kidney stones, and calcification of the brain nuclei, vasculature, and other organ systems [1,2]. Recombinant human PTH can also be used as replacement therapy in severe cases [1].
The metabolism of vitamin D occurs in three main steps-25-hydroxylation, 1αhydroxylation, and 24-hydroxylation. All steps are carried out by cytochrome P450dependent oxidases [3]. The first step of hydroxylation occurs mainly in the liver via the CYP2R1 enzyme, resulting in the formation of 25(OH)D [4]. In the second step, 1,25(OH) 2 D (calcitriol) is produced by 1α-hydroxylation in the kidneys via the CYP27B1 enzyme (1αhydroxylase) which is regulated by PTH, fibroblast growth factor 23 (FGF23), and calcitriol itself. PTH stimulates, whereas FGF23 and 1,25(OH) 2 D inhibit CYP27B1 [3,4]. The extrarenal production of calcitriol is possible and has been shown in studies on uremic and anephric patients, where circulating calcitriol levels were determined in the absence of functioning renal tissue [5][6][7]. The expression of CYP27B1 has been detected in keratinocytes, macrophages, and a number of other tissues. It is thought that locally produced 1,25(OH) 2 D acts in autocrine or paracrine manners and is regulated by cytokines [3,4]. 24-hydroxylation via CYP24A1 (24-hydroxylase) leads to the degradation of 1,25(OH) 2 D and 25(OH)D by forming 1,24,25(OH) 3 D and 24,25(OH) 2 D. As such, this step is considered necessary in order to prevent the toxic effects of vitamin D excess. Of particular interest currently are the C3-epimers of vitamin D, which are derived from the conversion of the hydroxyl group in the C3 position of the A-ring from an α to a β orientation [4]. The physiological role of epimers is not fully understood, however, it is reported that 3-epi-1,25(OH) 2 D may possess a calcimimetic effect [8,9].
There are only a few studies that have investigated vitamin D metabolism in HypoPT and were aimed predominantly at the evaluation of the 1α-hydroxylation process. In HypoPT, the 1α-hydroxylation of 25(OH)D in kidneys is impaired due to the lack of the PTH stimulatory effect [10]. However, this process might still take place due to a PTHindependent mechanism of extrarenal hydroxylation [11]. In the study by Lund et al., only a moderate decrease in 1,25(OH) 2 D level as well as its dependence on 25(OH)D concentration was observed in patients with HypoPT [12]. In addition, 25(OH)D has shown pharmacological effects that are typical for the active form of vitamin D in high concentration settings [13].
The aim of this study is thus to evaluate the extended spectrum of vitamin D metabolites in patients with HypoPT compared to healthy individuals.

Study Population and Design
This exploratory study included 38 adult patients with chronic HypoPT admitted for inpatient treatment at a single federal tertiary care center between 2016 and 2022. The diagnosis of HypoPT was established according to the guidelines [14]. The patients were further assigned to compensated (c-HypoPT, n = 13) and non-compensated (nc-HypoPT, n = 25) groups; the compensated state was defined as albumin-adjusted calcium 2.1-2.3 mmol/L and no symptoms of hypocalcemia. The control group included 38 healthy adults. The exclusion criteria were the presence of granulomatous disease, malabsorption syndrome, or liver failure; decreased glomerular filtration rate (less than 60 mL/min per 1.73 m 2 ) for both groups; or hypercalcemia, calcium, or vitamin D supplementation for 3 months prior to the study for the control group.

Sociodemographic and Anthropometric Data Collection
Anthropometric and clinical data were collected from the patient's medical records.

Laboratory Measurements
The serum samples were stored at −80 • C, avoiding repeated freeze-thaw cycles. The PTH levels were evaluated by an electrochemiluminescence immunoassay (ELEC-SYS, Roche, Basel, Switzerland). The biochemical parameters were assessed using an ARCHITECT c8000 analyzer (Abbott, Chicago, IL, USA) and the reagents from the same manufacturer via standard methods.
The levels of the vitamin D metabolites (25(OH)D 3 , 25(OH)D 2 , 1,25(OH) 2 D 3 , 3-epi-25(OH)D 3, and 24,25(OH) 2 D 3 ) were determined by ultra-high performance liquid chromatography in combination with tandem mass spectrometry (UPLC-MS/MS). The previously in-house developed method [16] has been changed by introducing SelexION Technology in order to avoid the 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization stage leading to spectrometer contamination. A detailed description of the experimental procedure is provided in Appendix A. With this technique, the laboratory participates in DEQAS (the Vitamin D External Quality Assessment Scheme) quality assurance program (lab code 2388), and the results fall within the target range for the analysis of 25(OH)D and 1,25(OH) 2 D metabolites in human serum. All the UPLC-MS/MS measurements were performed after the first successful completion (5/5 samples within the target range) of the DEQAS distributions for both analytes simultaneously. Each batch contained control samples (analytes in blank serum) with both high and low analyte concentrations. The serum samples under investigation were barcoded and randomized prior to the measurements to eliminate analyst-related errors.

Statistical Analysis
The statistical analysis was performed using Jamovi version 2.2.5.0 (The Jamovi Project, 2021). All data were analyzed with non-parametric statistics and are expressed as a median (interquartile range) unless otherwise specified. The Mann-Whitney U-test and Fisher's exact test were used for comparisons between the two groups. The Spearman rank correlation method was used to obtain coefficients of correlation among indices. A p-value of less than 0.05 was considered statistically significant.

Characteristics of the Groups
The groups were similar in age, sex, and body mass index (BMI) (p > 0.05) ( Table 1). The characteristics of the study group in terms of the underlying disease are presented in Table 2. A total of 36 patients were diagnosed with post-surgical HypoPT due to thyroidectomy, 1 patient had HypoPT due to DiGeorge syndrome, and 1 patient had autoimmune polyglandular syndrome type 1. The median duration of the disease in the study group was 61.  Abbreviations: c-HypoPT-compensated hypoparathyroidism, nc-HypoPT-non-compensated hypoparathyroidism.

Laboratory Evaluation
A comparison between HypoPT and control groups is shown in Table 3. The patients in the HypoPT group had significantly lower PTH, total calcium, albuminadjusted calcium, and magnesium levels, while their phosphorus levels were higher than in the control group (p < 0.05). The rest of the biochemical parameters showed no clinically significant difference. Patients with HypoPT also presented with higher levels of 25(OH)D 3 , 1,25(OH) 2  A comparison between the c-HypoPT group and the nc-HypoPT group is shown in Table 4.
The nc-HypoPT group had significantly lower levels of total calcium, albumin-adjusted calcium, magnesium, and creatinine (p < 0.05). There was no significant difference in the levels of PTH, vitamin D metabolites, and the rest of the biochemical parameters.  [19,20]. * Significant difference in between-group comparison. Abbreviations: c-HypoPT-compensated hypoparathyroidism, nc-HypoPT-non-compensated hypoparathyroidism, PTH-parathyroid hormone.
In the nc-HypoPT group, a negative correlation was found between the duration of HypoPT and magnesium levels (r = −0.461, p = 0.021). The significant positive correlation was found between 1,25(OH) 2

Discussion
We found that an adequate level of 25(OH)D 3 was achieved with standard doses of cholecalciferol in HypoPT patients. Almost three-quarters of the included patients had 25(OH)D 3 levels in the target range (>30 ng/mL), which is slightly higher than the data from the federal registry [21,22] and consistent with some of the previous studies in hypoparathyroidism [23], though some of the studies showed higher levels of 25(OH)D [24]. It is worth noting that the recommended doses of cholecalciferol differ in the guidelines for the general population [17,18] and HypoPT patients [14,25]. The guidelines for HypoPT patients recommend cholecalciferol supplementations in a daily dose of 400-800 IU to the patients treated with activated vitamin D which, given our results, may not be enough to achieve the target range and may need to be revised.
The observed higher concentration of 1,25(OH) 2 D 3 in the HypoPT group (more than half of the patients had calcitriol levels above the upper limit of the reference range) is presumably related to the intake of activated vitamin D, which is converted to calcitriol by 25-hydroxilase [12]. The levels observed are also higher than the data from other studies in hypoparathyroidism [23,24,26], despite similar dosages of activated vitamin D preparations, which might be influenced by the difference in the methods used for 1,25(OH) 2 D 3 assessment. This disparity emphasizes the difficulty of the direct comparison of the results obtained by different methods and the importance of using a method for the determination of 1,25(OH) 2 D 3 that has passed an external quality control system, especially in clinical practice [27]. We have not found a correlation between the 1,25(OH) 2 D 3 levels and either alfacalcidol dosages or calcium levels, which is consistent with other studies [12,23,26] The achievement of adequate levels of 25(OH)D 3 in hypoparathyroidism may be facilitated by decreased 1α-hydroxylation as a result of the lack of PTH. On the other hand, due to the large amounts of active vitamin D, an increase in 24-hydroxylase activity might be expected, leading to the increased degradation of 25(OH)D 3 in order to prevent toxicity [3,4]. This hypothesis is supported by the higher 24,25(OH) 2 D 3 levels and lower 25(OH)D 3 /24,25(OH) 2 D 3 ratios observed in the HypoPT group. Moreover, the higher levels of 25(OH)D 3 in the HypoPT group might contribute to the obtained differences.
The differences in 3-epi-25(OH)D 3 between the groups correlated with the observed differences in the 25(OH)D 3 levels. The biochemical findings were generally expected for this nosology.
We found no difference in the levels of vitamin D metabolites in HypoPT patients depending on compensation status. The observed correlations in nc-HypoPT were consistent with normal vitamin D metabolism and reflected the function of the inactivating metabolic pathways in the presence of high levels of calcitriol. Thus, we assume that the abnormality of vitamin D metabolism does not interfere with the achievement of hypoparathyroidism compensation. We also found that magnesium levels were lower in nc-HypoPT, which highlights the importance of monitoring this parameter according to clinical guidelines [14,25].
Overall, the results do not demonstrate the rationale for determining metabolites in clinical practice in HypoPT patients who fail to achieve compensation from this standpoint. At the same time, other authors conclude that the routine measurement of 1,25(OH) 2 D 3 may be useful as a biomarker to predict the absence of hypercalciuria in HypoPT patients who are receiving treatment with oral calcium and calcitriol supplements [26].
It should be noted that, to the best of the authors' knowledge, this is the first cohort study to evaluate the process of vitamin D inactivation in hypoparathyroidism, and the results should be confirmed in further studies with the assessment of a wider range of metabolites in large samples.
Our study had several strengths: the presence of the control group, the comprehensive spectrum of vitamin D metabolism parameters investigated, and participation in an external quality control program for vitamin D metabolite measurements.
However, the study also had some limitations. The small sample size of the groups cautions against the extrapolation of results obtained. In addition, the proportion of women in the groups is significantly higher than in the general population; however, this corresponds to the data from the federal HypoPT registry and can be attributed to the specificity of the disease incidence [21,22]. Some of the important parameters related to mineral metabolism (including FGF23), as well as free 25(OH)D, were not measured. Some additional insights might be obtained given the ability to assess alfacalcidol concentration, which was not possible in the current study. Finally, the proportion between 1,25(OH) 2 D 3 converted from alfacalcidol and 25(OH)D is unknown.

Conclusions
This is the first comprehensive study of vitamin D metabolites in patients with hypoparathyroidism. Our data show the supraphysiological levels of 1,25(OH) 2 D 3 and the increased production of the principal inactive metabolite (24,25(OH) 2 D 3 ) in patients with hypoparathyroidism compared to healthy adults which might be a consequence of the received therapy and the pathophysiology of the disease. The abnormality of vitamin D metabolism does not seem to interfere with the achievement of hypoparathyroidism compensation. Informed Consent Statement: Written informed consent was obtained from all individual participants included in the study.

Data Availability Statement:
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. The data are not publicly available due to due to privacy reasons. interface temperature were 3800 V, 6.8 V, −20 V, and 150 • C, and the carrier gas was nitrogen. A carrier gas modifier and the resolution enhancement mode were not used. The registration of the components was carried out in the scheduled multiple reaction monitoring. The time interval for each component was 70 s, the target scan time was 0.7 s, and the entrance potential was kept at 10 V. The other parameters were selected for each component individually and are presented in Table A2. Data were controlled and collected using the Analyst 1.6.3 software (AB Sciex). Abbreviations: Q1-quadrupole 1 mass, Q3-quadrupole 3 mass, tR-retention time, DP-declustering potential, CE-collision energy, CXP-collision energy exit potential.