Edited by: Rodolfo A. Rey, Hospital de Niños Ricardo Gutiérrez, Argentina
Reviewed by: Paulo Collett-Solberg, Rio de Janeiro State University, Brazil; Oscar Brunetto, Hospital Pedro de Elizalde, Argentina
*Correspondence: Jiafu Feng,
†These authors have contributed equally to this work
This article was submitted to Pediatric Endocrinology, a section of the journal Frontiers in Endocrinology
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Vitamin D is critical for calcium and bone metabolism. Vitamin D insufficiency impairs skeletal mineralization and bone growth rate during childhood, thus affecting height and health. Vitamin D status in children with short stature is sparsely reported. The purpose of the current study was to investigate various vitamin D components by high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) to better explore vitamin D storage of short-stature children
Serum circulating levels of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], and 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3, C3-epi] were accurately computed using the LC-MS/MS method. Total 25(OH)D [t-25(OH)D] and ratios of 25(OH)D2/25(OH)D3 and C3-epi/25(OH)D3 were then respectively calculated. Free 25(OH)D [f-25(OH)D] was also measured.
25(OH)D3 and f-25(OH)D levels in short-stature subgroups 2 (school age: 7~12 years old) and 3 (adolescence: 13~18 years old) were significantly lower compared with those of healthy controls. By contrast, C3-epi levels and C3-epi/25(OH)D3 ratios in all the three short-stature subgroups were markedly higher than the corresponding healthy cases. Based on cutoff values developed by Endocrine Society Recommendation (but not suitable for methods 2 and 3), sufficient storage capacities of vitamin D in short-stature subgroups 1, 2, and 3 were 42.8%, 23.8%, and 9.0% as determined by Method 3 [25(OH)D2/3+25(OH)D3], which were lower than those of 57.1%, 28.6%, and 18.2% as determined by Method 1 [25(OH)D2+25(OH)D3+C3-epi] and 45.7%, 28.5%, and 13.6% as determined by Method 2 [25(OH)D2/3+25(OH)D3+C3-epi]. Levels of 25(OH)D2 were found to be weakly negatively correlated with those of 25(OH)D3, and higher 25(OH)D3 levels were positively correlated with higher levels of C3-epi in both short-stature and healthy control cohorts. Furthermore, f-25(OH)D levels were positively associated with 25(OH)D3 and C3-epi levels in children.
The current LC-MS/MS technique can not only separate 25(OH)D2 from 25(OH)D3 but also distinguish C3-epi from 25(OH)D3. Measurement of t-25(OH)D [25(OH)D2+25(OH)D3] alone may overestimate vitamin D storage in children, and short-stature children had lower vitamin D levels compared with healthy subjects. Ratios of C3-epi/25(OH)D3 and 25(OH)D2/25(OH)D3 might be alternative markers for vitamin D catabolism/storage in short-stature children. Further studies are needed to explore the relationships and physiological roles of various vitamin D metabolites.
Short stature is a global public health problem (
Stature is hereditary trait regulated by both genetic and environmental factors. Manipulation of environmental factors may be an effective strategy to maximize the growth potential of children (
Circulating 25-hydroxyvitamin D [25(OH)D] is currently widely used as a functional indicator for vitamin D status (
Recent studies reported that vitamin D3 metabolites are further metabolized through the C3-epimerization pathway (
The current study recruited patients who visited the child healthcare department for short-stature problems between January 2017 and January 2021 in Mianyang Central Hospital, Sichuan Province, China, as study participants. Clinically, individual diagnostic categories are often indistinguishable, and the demarcation of diagnoses often leads to joint diagnoses. Therefore, the current study summarized all subtypes under general term and boundary definition of short stature but excluded those caused by genetic, syndromic, organic, and psychosocial conditions.
The diagnosis of short stature was based on a previous diagnostic guideline (
Exclusion criteria included children with other conditions such as growth hormone deficiency, multiple pituitary hormone deficiency, hypothyroidism, skeletal development disorder, intracranial tumor, chromosomal disease, chronic systemic disease, familial short stature, physical puberty delay, severe malnutrition, and other known causes of short stature. Participants who had received growth hormone, gonadotropin releasing hormone, or antihypertensive treatment were also excluded by a qualified pediatrician.
Healthy participants were assigned into the control group. The current study was approved by the Medical Ethics Committee of Mianyang Central Hospital.
Venous blood was collected between 6:00 and 10:00 a.m. after overnight fasting to eliminate the influence of diet on serum measurements. The blood was centrifuged at 3,000 rpm for 15 min to obtain serum.
This was undertaken based on our previously described study (
Chromatographic analysis was performed on a Shimadzu LC-30AD UHPLC system equipped with a Kinetex 2.6 μm C8 100A column. Mobile phase A consisted of water with 0.1% acetic acid, and mobile phase B consisted of methanol with 0.1% acetic acid. Fifteen microliters of the sample solutions was injected into the LC system using a column temperature of 45°C and a flow rate of 0.6 ml/min. Mass spectrometer detection and quantification were undertaken in positive mode using multiple reaction monitoring (MRM) mode. Optimized parameters for mass detection were as follows: curtain gas was 35 psi; temperature was 550°C; ion spray voltage was 5,500 V; gas 1 and gas 2 (nitrogen) were both set at 60 psi; and the dwell time was 100 ms. Analyst® MD software (version number: 1.6.3) was used for chromatogram output, and MultiQuant™ MD software (version number: 3.0.2) was performed for data processing.
The free 25(OH)D ELISA kit was obtained from DIAsource ImmunoAssays SA (Belgium) to detect f-25(OH)D levels. The assay was calibrated against Rate Dialysis, which is the gold standard method for the determination of free hormones. Final concentrations were analyzed using the RT-6100 enzyme label analyzer (Redu Life Science Co., Ltd., Shenzhen, China) based on kit instructions.
The capacity of vitamin D3 to store vitamin D is two to three times higher compared with that of vitamin D2. To provide alternative methods for accurate or sufficient vitamin D storage converted into active vitamin D [1,25(OH)2D], three different computation methods were applied, including 25(OH)D2+25(OH)D3+C3-epi (Method 1), 25(OH)D2/3+25(OH)D3+C3-epi (Method 2), and 25(OH)D2/3+25(OH)D3 (Method 3) (
Statistical analyses were performed using SPSS 25.0 software (International Business Machines Corp., USA). Data were expressed as mean ± standard deviation (SD) for normally distributed continuous data and analyzed using Student’s t-test between two study groups. The M=median and interquartile range (IQR) were selected for non-normally distributed variables and analyzed by Mann–Whitney U tests. One-way ANOVA was used to analyze differences between means of more than two groups for equal variances. Welch’s approximate analysis was used followed by Dunnett’s T3 test if the variances are uneven. The strength of the relationship between selected metabolite parameters and commonly used fasting lipid profiles was determined using Pearson or Spearman bivariate correlation analysis for normal or skew distribution. p < 0.05 was considered statistically significant.
A total of 99 eligible short-stature children aged between 1 and 18 years, including 45 males and 54 females, were recruited in the current study. In addition, 186 healthy participants were assigned to the control group, among whom were 86 males and 100 females. Influence of age on outcomes was minimized by grouping children into three subgroups: Subgroup 1 (preschool age) aged between 1 and 6 years; Subgroup 2 (school age) aged between 7 and 12 years; and Subgroup 3 (adolescence) aged between 13 and 18 years.
General characteristics of the study cohort.
Items | Healthy control (n = 186) | Short stature (n = 99) | p values |
---|---|---|---|
Sex (male/female) | 86/100 | 45/54 |
|
Age (years) | 8.5 ± 2.5 | 8.3 ± 1.9 |
|
Height (cm) | 139.32 ± 17.03 | 125.84 ± 20.04 |
|
Height SDS | 0.5 (-0.2, 1.0) | -2.87 (-2.7, -3.1) |
|
Weight (kg) | 34.54 ± 11.17 | 29.03 ± 11.19 |
|
Weight SDS | 0.65 ± 0.21 | -0.81 ± 0.24 |
|
BMI (kg/m2) | 17.20 ± 1.87 | 17.48 ± 1.69 |
|
BMI SDS | 0.20 (-0.19, 0.73) | 0.31 (-0.12, 0.68) |
|
Ca (mmol/L) | 2.51 ± 0.13 | 2.52 ± 0.10 |
|
PHOS (mmol/L) | 1.66 ± 0.21 | 1.68 ± 0.16 |
|
FT3 (pg/mL) | 3.69 ± 0.48 | 3.95 ± 0.46 |
|
FT4 (ng/dL) | 1.10 ± 0.11 | 0.99 ± 0.22 |
|
HTSH (μIU/mL) | 2.30 ± 1.19 | 2.57 ± 1.51 |
|
PTH (pg/mL) | 32.56 ± 17.92 | 36.87 ± 18.72 |
|
ALP (U/L) | 254.52 ± 78.15 | 249.81 ± 76.29 |
|
Ca, calcium; PHOS, phosphate; FT3, free triiodothyronine; FT4, free thyroxine; HTSH, thyroid-stimulating hormone; PTH, parathyroid hormone; ALP, alkaline phosphatase.
Data were expressed by mean ± SD, or median (P25, P75).
The p value determines statistical significance between the two compared groups. P<0.001 was considered statistically significant.
Findings of the current study showed that both serum levels of 25(OH)D3 (t = 3.825, p < 0.001; t = 3.121, p = 0.003) and f-25(OH)D (t = 3.848, p = 0.002; t = 2.282, p = 0.017) in subgroups 2 and 3 of short-stature patients were significantly lower compared with those of healthy controls, whereas C3-epi levels (z = 2.548, p = 0.023; z = 3.282, z = 0.007; z = 4.848, p < 0.001) and C3-epi/25(OH)D3 ratios (z = 2.845, p = 0.022; z = 2.285, z = 0.027; z = 3.788, p = 0.002) were all markedly higher in subgroups 1, 2, and 3 than those of healthy participants for the corresponding control subgroups (
Comparison of various vitamin D components between short stature and healthy control subgroups. Levels of 25(OH)D2
Percentages of vitamin D status among subgroups in short-stature and healthy participants are presented in
Evaluation of the subject’s vitamin D nutritional status [%(case/total)].
Subjects | Healthy Control | Short Stature | |||||
---|---|---|---|---|---|---|---|
Subgroup 1 | Subgroup 2 | Subgroup 3 | Subgroup 1 | Subgroup 2 | Subgroup 3 | ||
Deficiency | 0 (0/32) | 10.9 (14/128) | 3.8 (1/26) | 14.3 (5/35) | 35.7 (15/42) | 22.7 (5/22) | |
|
Insufficiency | 25.0 (8/32) | 34.4 (44/128) | 46.2 (12/26) | 28.6 (10/35) | 35.7 (15/42) | 59.1 (13/22) |
Sufficiency | 75.0 (24/32) | 54.7 (70/128) | 50.0 (13/26) | 57.1 (20/35) | 28.6 (12/42) | 18.2 (4/22) | |
Deficiency | 3.1 (1/32) | 13.3 (17/128) | 15.4 (4/26) | 22.9 (8/35) | 40.5 (17/42) | 45.5 (10/22) | |
|
Insufficiency | 25.0 (8/32) | 38.3 (49/128) | 46.2 (12/26) | 31.4 (11/35) | 31.0 (13/42) | 40.9 (9/22) |
Sufficiency | 71.9 (23/32) | 48.4 (62/128) | 38.4 (10/26) | 45.7 (16/35) | 28.5 (12/42) | 13.6 (3/22) | |
Deficiency | 3.1 (1/32) | 14.9 (19/128) | 15.4 (4/26) | 28.6 (10/35) | 42.9 (18/42) | 45.5 (10/22) | |
|
Insufficiency | 34.4 (11/32) | 45.3 (58/128) | 50.0 (13/26) | 28.6 (10/35) | 33.3 (14/42) | 45.5 (10/22) |
Sufficiency | 62.5 (20/32) | 39.8 (51/128) | 34.6 (9/26) | 42.8 (15/35) | 23.8 (10/42) | 9.0 (2/22) |
Method 1 = 25(OH)D2+25(OH)D3+C3-epi. Method 2 = 25(OH)D2/3 + 25(OH)D3+C3-epi. Method 3 = 25(OH)D2/3 + 25(OH)D3.
Vitamin D storage in all subgroups was determined, and results are presented in
Evaluation of vitamin D storage in subjects.
Subjects | Healthy control | Short stature | F, p value | ||||
---|---|---|---|---|---|---|---|
Subgroup 1 | Subgroup 2 | Subgroup 3 | Subgroup 1 | Subgroup 2 | Subgroup 3 | ||
VitD storage by Method 1 (ng/mL) | 35.45 ± 6.91 | 31.14 ± 9.151 | 30.39 ± 7.452 | 33.08 ± 12.27 | 24.76 ± 8.893,
|
25.06 ± 9.244,
|
7.316, |
VitD storage by Method 2 (ng/mL) | 33.70 ± 7.58 | 30.05 ± 8.871 | 28.69 ± 7.112 | 30.85 ± 12.34 | 23.84 ± 8.833,
|
21.55 ± 9.104 | 7.964, |
VitD storage by Method 3 (ng/mL) | 32.05 ± 7.15 | 28.58 ± 8.341 | 27.63 ± 6.892 | 29.30 ± 11.60 | 22.81 ± 8.393,
|
20.98 ± 8.744,
|
7.517, |
Compared with healthy children in subgroup 2, p < 0.05.
Compared with healthy children in subgroup 3, p < 0.05.
1,2Compared with children in subgroup 1 in the healthy cohort, p < 0.05.
3,4Compared with children in subgroup 1 in the short stature group, p<0.05.
Analysis of the results shown in
Correlation analysis of various vitamin D components in short stature and healthy control groups.
Several methods including radioimmunoassay, ELISA, and chemiluminescence have been utilized for determination of t-25(OH)D. However, they are incapable of separating 25(OH)D2, 25(OH)D3, and C3-epi from t-25(OH)D effectively (
Several previous studies have reported inverse associations between 25(OH)D concentrations and PTH levels in humans, but these findings were commonly observed in adults and old people (
Our results indicated that serum 25(OH)D3 levels in short-stature patients aged 7–12 and 13–18 years were lower compared with healthy participants during the same periods. Conversely, C3-epi levels and ratios of C3-epi/25(OH)D3 in all age ranges of short-stature children were higher than those in healthy subjects. C3-epi is the isomeric form of vitamin D3. Its active form, 3-epi-1α,25(OH)2D3, appears to have reduced calcemic effects than non-epimeric forms and can activate bone gamma-carboxy glutamic acid-containing protein (BGLAP, also called osteocalcin) at a much lower rate compared with 1α,25(OH)2D3 (
The current study showed a weak positive correlation between C3-epi and 25(OH)D3 values in both short-stature and healthy children, which was consistent with previous studies on adults. However, this relationship could not hold in infant populations because the relative C3-epimer concentration is high in neonates and declines rapidly across infancy (
Approximately 0.03% of total 25(OH)D and 0.4% of total 1,25(OH)2D are free in circulation in healthy non-pregnant subjects. Its capacity depends on its physiological effects and body demands for vitamin D, rather than complex individual influencing factors (
Total 25(OH)D comprising 25(OH)D2 and 25(OH)D3 is recommended by guidelines as the best indicator of vitamin D storage (
C3-epi currently accounts for a significant proportion in neonates (
Nevertheless, the current study had some limitations. First, it was limited by its retrospective nature with single academic center and relatively small sample size. Second, data on use of vitamin D supplements by participants were not collected. Similarly, information on sensitivity to sunlight, latitude, season, time of day, and how much direct sunlight that skin is exposed to was not included, all of which could be related to vitamin D status. In addition, methods for determining vitamin D metabolites were not standardized. High sensitivity of LC-MS/MS and poor reproducibility of ELISA may have led to certain variations in the obtained findings. Although reliability of the current study was not entirely satisfactory, it provides important reference for design and implementation of related studies.
The current study revealed essential differences between various vitamin D contents in short-stature children compared with healthy ones. The findings indicated that short-stature patients had lower levels of vitamin D storage compared with healthy subjects. To accurately assess vitamin D nutritional status, kinds of vitamin D components in circulation including 25(OH)D2, 25(OH)D3, f-25(OH)D, t-25(OH)D, and C3-epi and ratios of C3-epi/25(OH)D3 and 25(OH)D2/25(OH)D3 should be determined extensively, in order to provide a scientific evidence-based basis for the diagnosis and treatment evaluation of short-stature individuals.
Datasets analyzed during the current study are available from corresponding author on reasonable request.
The current study was approved by Medical Ethics Committee of Mianyang Central Hospital (approval no. P2020040). Written informed consent to participate in this study was provided by legal guardian/next of kin of participants.
All authors contributed to the current study conception and design and take responsibility for the integrity of data and accuracy of data analyses. Data collection was undertaken by BX, LG, and YZ, and analysis was undertaken by YF. Material preparation was done by and the first draft of the manuscript written by WJ, JF, and LY, and all authors commented on the previous versions of manuscript. All authors contributed to the article and approved the submitted version.
The current work was financially supported by the Sichuan Health and Health Committee Support Program (20PJ255) and the Incubation Project of Mianyang Central Hospital (2019FH01 and 2019YJ22).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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