Serum Vitamin D Components and Whole Blood Trace Elements Storage in Chinese Short Stature Children

Background: Literature data on nutritional status in short stature children are unfortunately still scarce. The aim of the study was to compare the serum levels of 25(OH)D2, 25(OH)D3, total 25(OH)D and whole blood trace elements between short stature and healthy controls in Chinese children. Methods: A case-control study including 370 short stature (SS) children and 398 healthy controls (HC) was performed in Mianyang Central Hospital from January 2017 to June 2020. Serum 25(OH)D2 and 25(OH)D3 were accurately measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were summed to obtain total 25(OH)D. Whole blood concentrations of calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), iron (Fe), manganese (Mn), selenium (Se) and lead (Pb) were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) method. Results: 25(OH)D2 and total 25(OH)D levels in the SS group were signicantly lower than the HC subjects (both P<0.05). Besides, both 25(OH)D2 and 25(OH)D3 were positively correlated with total 25(OH)D in the SS and HC groups. 25(OH)D2 levels had weakly negative association with 25(OH)D3 among healthy subjects, whereas no correlation between 25(OH)D2 and 25(OH)D3 levels was found in short stature group. Otherwise, a signicant elevation was observed in Zn (P<0.001), Fe (P<0.001), and Se (P=0.027) in SS patients, while a statistically signicant decrease in Cu (P=0.002) and Mn (P<0.001) was found. Additionally, the signicant correlation of serum levels of 25(OH)D2, 25(OH)D3, total 25(OH)D and trace elements were observed between SS and HC group. Conclusion: of Cu and Mn, but elevation of Zn, Fe and Se were observed. Achieved information about vitamin D and trace element status in SS subjects could further improve the nutritional status of these patient groups.


Introduction
Short stature, which affects about 3% of the population, poses a considerable public health problem globally [1] . Short stature is de ned as a height less than or equal to 2 standard deviations (SD) below corresponding mean for a given age, sex and population or height below third percentile [1][2] . Although it is not a disease, individuals with general health but short stature are under physical and psychological stress in the modern society. Especially for children, severe short stature has been found to be vulnerable to diverse developmental, social and educational problems [3] .
Growth is not only determined by genetic factors, but also the environments [4] . Although environmental factors' in uence is relatively small compared to that of the genetic factors, it is the more important area to maximize the growth potential due to that environmental factors can be modi ed through intervention [5] . Short stature is caused by various underlying environmental factors, including nutritional problems and excess or de ciency of trace elements [6] .
Vitamin D is one of essential nutrient, which is the most crucial factor because it plays a key role in maintaining and synthesizing body tissues during the growth period [7] . Vitamin D de ciency can reduce skeletal mineralization and bone growth rate [8] . Infants and young children are the most affected individuals because of their rapid growth. The major circulating form of vitamin D is 25-hydroxyvitamin D [25(OH)D], which is the best indicator to monitor for vitamin D status with a circulating half-life of 2-3 weeks [9] . 25(OH)D is estimated as total of 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] [10][11] , but above 2 types of 25(OH)D are obtained from different sources. Generally, 25(OH)D3 is endogenously produced in the skin through the effect of UV-B on 7-dehydrocholesterol, whilst 25(OH)D2 is derived from the diet as ergosterol [12] . More and more evidences have considered 25(OH)D2 is equally as effective as 25(OH)D3 for bone health [13] . Therefore, both clinical and laboratory experts recommand 25(OH)D2 and 25(OH)D3 should be simultaneously detected, thus to ensure that vitamin D status is completely evaluated. Unfortunately, there are few studies to quantify the levels of 25(OH)D2 and 25(OH)D3 in short stature children. Thus, there is an urgent need to determine the relationship of 25(OH)D2 and 25(OH)D3 levels between subjects with and without short stature.
Essential trace elements (TEs, such as zinc or selenium) de ciency and potentially harmful TEs (such as lead or arsenic) excess are both known to have adverse consequences in general population, especially in children [14] . They are essential components of biological structures and have an important effect on and play a key role in a variety of the processes necessary for life throughout mediate vital biochemical reactions [15] . Recently, it is still unclear if the trace elements' excess or de ciency are correlated with short stature. Considering this, our study aimed to compare the levels of various TEs between short stature children and normal children visiting a growth clinic. Therefore, considering that 25(OH)D2 and 25(OH)D3 concentrations in short stature children are unclear, meanwhile nutrient status of trace elements is also yet unknown, the purpose of this study was to measure levels of 25(OH)D2, 25(OH)D3, total 25(OH)D and eight trace elements including calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), iron (Fe), manganese (Mn), selenium (Se) and lead (Pb) in short stature children in Western China using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) respectively. Achieved information about vitamin D and trace element status in short stature subjects could assist in the process of developing vitamin D supplements or microelement correction strategy, and it would further improve the nutritional status of these patient groups.

Reagents and instrumentation
Vitamin D detection reagent kit was purchased from Jinan Yingsheng Biotechnology Co. Ltd (Jinan, China). Nitric acid (HNO 3 , 65%) and TritonX-100 were both obtained from Kelong Reagent Co. Ltd (Chengdu, China). Eight elements standard solutions (Ca, Mg, Cu, Zn, Fe, Mn, Se and Pb) and internal standard (IS) solution (Germanium and Bismuth) were all provided by PerkinElmer (Shelton, USA). All solutions were prepared with deionized water provided by a water puri cation system (18.25 MΩ cm, Elix®, Merck Millipore). All chemicals used for analysis were of highest purity.
The LC-MS/MS analysis was performed on a Shimadzu LC-30AD UHPLC system (Shimadzu, Kyoto, Japan) coupled to an Applied Biosystems Sciex API 4500 Qtrap (Applied Biosystems/MDS Sciex, Toronto, CA, USA) equipped with an electrospray ionization (ESI) interface. Data acquisition and analysis were performed using the Analyst software version 1.6.2 (Applied Biosystems, Foster City, CA, USA).
Trace elements of whole blood samples were analyzed by NexION 300Q ICP-MS (PerkinElmer, Shelton, USA). Data acquisition and analysis were performed using the NexION software. Analyses were performed using a standard addition procedure. Germanium (Ge) and and Bismuth (Bi) were used as internal standards for matrix and instrument drift corrections.

Study participants
A total of 370 eligible short stature children in ages from 1 to 13 years, including 231 males and 139 females, were recruited between January 2017 and June 2020 in Mianyang Central Hospital, Mianyang, Sichuan Province, China. In order to eliminate confounding variables, patients with histories of endocrine diseases such as growth hormone de ciency or hypothyroidism, malnutrition, chromosomal abnormalities, skeletal dysplasia, psychological and emotional disorders, or family short stature were excluded from the study. 398 healthy subjects were taken as control group, including 240 males and 158 females. Chromatographic separation of the samples was performed on a Shimadzu LC-30AD UHPLC system equipped with a Kinetex 2.6 μm C8 100A column. Mobile phase A was water with 0.1% acetic acid, and mobile phase B consisted of methanol with 0.1% acetic acid. 15 μL of the sample solutions was injected into the LC system using a column temperature of 45 °C and a ow rate of 0.6 mL/min. Mass spectrometer detection and quanti cation were carried out in the positive mode using multiple reaction monitoring (MRM) mode. The optimized parameters for mass detection were as follows: curtain gas was 35 psi; temperature was 550 o C; ion spray voltage was 5500 V; gas 1 and gas 2 (nitrogen) were set both at 60 psi; the dwell time was 100 ms.

Measurement of trace elements
The concentrations of trace elements were measured using the methods validated by our laboratory previously [16] . Brie y, the whole blood samples were directly analyzed in 1/50 (v/v) dilution with the sample diluent solution which contained 0.5% (v/v) HNO3 and 0.5% (v/v) Triton X-100. All diluted samples were centrifuged at 4000 rpm for 10 min, and stored at -4 o C before analysis. TEs' analyses were performed using a standard addition procedure. Addition of IS elements' solution to sample solutions was applied via a three-way pipe.

Statistical analysis
Data were analyzed using the statistical package SPSS 22.0 (SPSS Inc, Chicago, USA). Normally distributed continuous variables were expressed as mean ± standard deviation (SD), and LSD-T test was used for comparing the mean value of data. Non-normally distributed variables were showed as median (interquartile rang, IQR). and comparisons between the two groups were analyzed by Mann-Whitney U tests. For statistical comparisons between multiple groups, one-way analysis of variance (ANOVA) was performed. The association between vitamin D status and trace elements was examined by Spearman correlation coe cients. A P-value of less than 0.05 was considered statistically signi cant.

Storage of 25(OH)D2, 25(OH)D3 and 25(OH)D in serum
There is no consensus on the optimal level of 25(OH)D in blood, which depends on the person's age, the person's sensitivity to sunlight, the latitude, the season, the time of day and how much skin is directly exposed to sunlight. Most agree that a 25(OH)D concentration <20 ng/mL is indicative of a vitamin D de ciency, whereas a 25(OH)D concentration of 21-29 ng/mL is considered to indicate a vitamin D insu ciency. Concentrations >30 ng/mL are considered to be su cient [17] . As shown in Fig.1A, the rates of the vitamin D su ciency, insu ciency and de ciency were 12.1%, 60.1% and 27.8% in SS children, and 23.1%, 48.8% and 28.1% in healthy subjects respectively. Additionally, the levels of 25(OH)D2, 25(OH)D3 and the sum of them in body serum in both SS and healthy cohorts were present in Fig.1B-1D. There were signi cant differences of 25(OH)D2 and total 25(OH)D levels between the SS and healthy control groups The relationship between the element concentrations and the genders was also performed ( Table 1). ANOVA analysis revealed that among these four subgroups, the concentrations of Ca (P=0.183) and Mg

Correlations of vitamin D components and trace elements
The correlations of serum levels of vitamin D components and trace elements were demonstrated in Table 2. The results showed that signi cant correlations varied between short status and healthy controls.

Discussion
Interest on height is greatly emphasized in the modern society due to the development of media and widespread perception that long stature is superior. Our study applied LC-MS/MS and ICP-MS methods respectively to measure serum 25(OH)D2, 25(OH)D3 and total 25(OH)D levels, as well as whole blood TE concentrations in SS children, giving more information about the nutritional status of these short patient groups.
Some data had once suggested 25(OH)D3 that accounts for approximately 95% of the total circulating 25(OH)D pool [18] , is a more potent supplement than 25(OH)D2 for increasing total vitamin D levels [19] . Several methods including chemiluminescence, radioimmunoassay (RIA), and high-performance liquid chromatography (HPLC) have been developed for 25(OH)D status measurement [9] . But there are still signi cant drawbacks of them. For example, suboptimal cross-reactivity of the antibody with 25(OH)D2 would cause under recovery of 25(OH)D2 in chemiluminescent immunoassays with unsatisfactory accuracy and precision, while RIA methods are unable to distinguish between the two metabolites of 25(OH)D2 and 25(OH)D3, which could not meet our requirement [20] . Although HPLC techniques with UV detection are capable of determining 25(OH)D2 and 25(OH)D3 levels simultaneously, most of these methods require large sample volumes (0.5-2 mL) and time-consuming procedures before quanti cation [21] . LC-MS/MS is able to overcome the defects mentioned above, and has been the "gold standard" measuring method [9] . Thus, one strength of our study was the use of sensitive and speci c LC-MS/MS vitamin D metabolite assays that separately analyze 25(OH)D2 and 25(OH)D3 concentrations.
In Pakistani children, vitamin D de ciency had been the second leading cause of short stature [22] . Vitamin D regulates circulating insulin-like growth factor 1 (IGF-1) and the gene expression of its receptor as well as various other binding proteins [23] . In addition, polymorphisms of vitamin D receptor gene which in uence biological e ciency of vitamin D, is also associated with adult or babies' height [24] .
Consistent with this result, our ndings indicated that the prevalence of vitamin D de ciency and insu ciency among children in Western China was high, and it was more severe in children with SS ( Fig. 1). Our study further evaluated the levels of different vitamin D metabolites. To our best knowledge, our ndings were the rst to show that 25(OH)D2 levels in the SS subjects were signi cantly lower than the healthy controls. The underlying reasons might be the insu cient intake of vitamin D2 were positively correlated with total 25(OH)D in both the SS group and the healthy group (Fig. 2).
Unfortunately, the ndings were not compatible with other publications, which reported higher 25(OH)D2 levels had no associations with higher levels of 25(OH)D. The possible reason is vitamin D concentrations and metabolism vary substantially by race/ethnicity [25] . Additionally, 25(OH)D2 levels had weakly negative association with 25(OH)D3 among healthy subjects in our study, which was similar to the reports that higher 25(OH)D2 was associated with lower levels of 25(OH)D3 in large healthy cohorts [21,[26][27] . No correlation between 25(OH)D2 and 25(OH)D3 levels was found in SS group. To understand possible reasons for these associations, it is helpful to remember that cholecalciferol has about a 2-fold higher a nity for vitamin D binding protein compared to ergocalciferol, and 25(OH)D3 has a higher a nity than does 25(OH)D2, likely yielding different amounts of free vitamin D metabolite with different serum half-life periods (D3 > D2) available for hydroxylation [28][29] . The short stature disease state might in uence the enzymatic preference for substrate and/or positive and negative feedback mechanisms, thus changing the rates of synthesis of 25(OH)D2 vs 25(OH)D3, as well as their associations. Moreover, 25(OH)D2 of male, 25(OH)D3 of female and total 25(OH)D of both male and female in healthy controls were all higher than the relative groups in SS group (Table 1). These ndings suggested that we should take gender into consideration when further studies were conducted.
ICP-MS exhibits a good precision, an excellent sensitivity, and multi-isotopic and multi-elemental capabilities [30] . It was the most suitable technique to obtain such reliable reference values for TEs. Compared with the control subjects, a signi cant elevation was observed in Zn (P < 0.001), Fe (P < 0.001), and Se (P = 0.027) in SS patients, while a statistically signi cant decrease was found in Cu (P = 0.002) and Mn (P < 0.001) (Fig. 3). Zn is essential for cell replication and DNA synthesis [6] , and its de ciency is considered to cause growth retardation [31][32] . Several evidences suggested SS group had signi cantly decreased Zn concentrations in whole blood and plasma [33] . In contrast, Yoshida, K. et.al claimed that low Zn level and Zn de ciency were not associated with idiopathic SS in Japanese children [6] . Multiple analytical techniques and biological uid might be one of the causes for the contradictory results [16] . Se has a wide range of pleiotropic effects including production of active thyroid hormone by incorporating into selenoproteins [34] . Fe acts as essential nutrient utilized in almost every aspect of cell function [35] , and interacts with other trace elements (Cu and Zn) [36] . Adequate dietary supply of Se and Fe is required for a healthy thyroid during development and adolescence [37] . However, our result presented higher Se and Fe levels in SS group. Excessive accumulation might be related to multiple factors, such as adequate intake and environmental exposure. Further research is still warranted to elucidate the importance of Se and Fe levels in children with SS. In the present study, Cu and Mn concentration in SS children was signi cantly lower than that in the control group, which was in accord with previous researches in hair and whole blood [6,31] . Cu de ciency induces anemia, decreases absorption of vitamin B1, thus has an effect on various biological progress including growth [33] . Lower maternal blood Mn is associated with lower birth weight [38] . And animal experiment con rmed that low Mn diet could impair fetal growth and development [39] . Physiological systems involved in metabolic homeostasis also exhibit a gender difference. Signi cant differences of trace element concentrations were found between female and male subjects in both HC and SS patients in our ndings ( Table 1). The recognition and identi cation of gender-speci c biological processes will lead to better understanding of trace element alterations in short stature, and drive novel discovery to develop corresponding element correction strategies based on gender differences.
Moreover, our results displayed that signi cant correlations between SS patients and healthy controls ( Our article was the rst to evaluate the vitamin D components and essential trace elements storage in SS children in West China. Nevertheless, our study also has some limitations. First, our study was limited by its small sample size and its retrospective nature. Second, our data were collected at a single institution. Third, we did not collect data on the use of vitamin D and trace element supplementations in our participants. Similarly, we did not collect information on the person's sensitivity to sunlight, the latitude, the season, the time of day and how much skin is directly exposed to sunlight, all of which could be associated with vitamin D status. Next step, we plan to carry out a study with large sample sizes, prospective design, multiple centers and rigorous inclusion of incident patients to re ect the nutritional status of vitamin D and trace elements in short stature patients in West of China. Although reliability of this study results was not so satisfactory, it might serve as an important reference for the design and conduction of related researches.

Conclusion
The presented study revealed essential differences between the content of vitamin D and trace elements in SS children compared with healthy children. Our results suggested that SS patients had more severe

Declarations
Ethics approval and consent to participate The study was approved by the Medical Ethics Committee of Mianyang Central Hospital (approval no. P2020040). All participants provided informed consent prior to participation.