Yang et al. BMC Pediatrics(2024) 24:577
https://doi.org/10.1186/s12887-024-05072-8

BMC Pediatrics

RESEARCH

Open Access

Differential performance regarding therelationship of C3-epi-25(OH)D3 levels
and %C3-epi-25(OH)D3 with common
pediatric diseases: a case control study
Tao Yang1†, Xiaohong Chen2†, Miyan Wang3†, Shaohua Xu1, Dong Hu2, Jie Tang2* and Yuwei Yang2*
Abstract
Background Recently, the C3-epimer of 25-hydroxyvitamin D [C3-epi-25(OH)D] has become a topic of interest
among 25-hydroxyvitamin D [25(OH)D] metabolites. Although it can lead to an overestimation of vitamin D storage,
its relationship with disease occurrence remains controversial, possibly related to the great extent of tracking of25(OH)D by C3-epi-25(OH)D over time. This study aimed to investigate the differential performance of C3-epi-25(OH)D3 and its percentage [%C3-epi-25(OH)D3] with respect to 20 common paediatric diseases.
Methods This study involved 805 healthy children and adolescents and 2962 patients with common paediatricdiseases. We investigated sex, age, and seasonal differences in C3-epi-25(OH)D3 and %C3-epi-25(OH)D3 levels; theirvariations on 20 common paediatric diseases; and their degree of correlation with 25(OH)D3 levels and various
diseases.
Results Among the healthy underage participants, C3-epi-25(OH)D3 and %C3-epi-25(OH)D3 changed similarly, withno sex differences. Moreover, their levels were higher in the infant period than in the other periods (t = 5.329–5.833,
t = 4.640–5.711, all Padj < 0.001), and in spring and summer than in autumn and winter (t = 3.495–6.061, t = 3.495–
5.658, all Padj < 0.01). Under healthy and disease conditions, C3-epi-25(OH)D3 was positively correlated with 25(OH)D3(ρ = 0.318 ~ 0.678, all P < 0.017), whereas %C3-epi-25(OH)D3 was not, except in patients with nephrotic syndrome (ρ=0.393, P = 0.001). Before and after adjusting for 25(OH)D3, the relationship of C3-epi-25(OH)D3 with the diseases wasnotably different. However, it was almost consistent for %C3-epi-25(OH)D3. Our results indicated that %C3-epi-25(OH)D3 was associated with short stature, nephrotic syndrome, lymphocytic leukaemia, rickets, paediatric malnutrition,and hypovitaminosis D (OR = 0.80 ~ 1.21, all P < 0.05).

†

Tao Yang, Xiaohong Chen and Miyan Wang contributed equally tothis work.
*Correspondence:
Jie Tang
tjandbetty@sc-mch.cn
Yuwei Yang
yyw318@sc-mch.cn
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Yang et al. BMC Pediatrics(2024) 24:577

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Conclusions The %C3-epi-25(OH)D3 can correct the properties of C3-epi-25(OH)D3 to better track 25(OH)D3 andmay be more suitable for exploring its pathological relevance. Further detailed studies of each disease should be
conducted.
Keywords C3-epimer, 25(OH)D3, Influence factor, Correlation, Paediatric diseases

Background
The level of 25-hydroxyvitamin D [25(OH)D] in vivo isa recognized criterion for determining vitamin D storage and is also recommended by the Institute of Medicine (IOM) [1, 2] and the Endocrine Society (EnS)/Japan
Endocrine Society (JES) [3, 4]. However, these criteria
are controversial. The IOM and EnS/JES defined vitamin D deficiency as 25(OH)D < 12 ng/mL (30 nmol/L)and < 20 ng/mL (50 nmol/L), respectively. Moreover, theIOM indicates that 25(OH)D of 50 nmol/L is sufficient toensure bone health in the general population [5].
The physiological roles of vitamin D include the regulation of calcium and phosphate metabolism [6], theregulation of neuromuscular function [7], immunemodulatory function [8], antioxidation [9], and antiproliferative effects [10], among others. Thus, vitamin D
deficiency is associated with impaired absorption of calcium and phosphorus, inflammation, immune dysfunction, oxidative stress, and tumorigenesis, resulting in an
increased risk of various diseases, such as rickets, osteomalacia, cardiovascular disease, and autoimmune diseases [11, 12].
The level of the C3-epimer of 25(OH)D [C3-epi25(OH)D] in vivo has also raised concerns based on the
nutritional evaluation of vitamin D [11]; that is, there
are concerns regarding the correct evaluation of vitamin
D storage. C3-epi-25(OH)D, another form of 25(OH)D,is generated mainly from 3β→3α epimerization at the
C3 spatial conformation of 25(OH)D by the 3-epimerase enzyme. Owing to its low or no bioactivity, C3-epi25(OH)D can cause an overestimation of vitamin D
storage [13]. However, there are two urgent problems inthe measurement of C3-epi-25(OH)D: (1) owing to thesimilar spectroscopic patterns and identical mass andfragmentation patterns of 25(OH)D and C3-epi-25(OH)D, almost no spectroscopy detection technique can distinguish them; (2) owing to the very low levels in the general population, C3-epi-25(OH)D is very easily identified
as signal noise using ordinary mass-spectrography techniques, except when its content is high. In 2006, it wasreported that the in vivo concentration of C3-epi-25(OH)D could be determined via high-performance liquid
chromatography-tandem mass spectrometry (HPLCMS/MS) [14]. However, assessments of C3-epi-25(OH)Din vivo have been in progress for less than 20 years andhave been conducted in few developed countries [15, 16].
Nevertheless, C3-epi-25(OH)D remains undetectable inmany individuals.

C3-epi-25(OH)D has become a topic of interest in thefield of vitamin D nutrition, particularly regarding its
controversial pathological relevance. Studies have shownthat C3-epi-25(OH)D levels increase in various conditions and diseases, such as infants, children, pregnant
women, autoimmune diseases, arthritis, diabetes mellitus, Alzheimer’s disease, and thyroid disorders [17–19].
One study revealed that the C3-epi-25(OH)D level tracks
the 25(OH)D level to a great extent over time [20]. Thesefindings indicate that the C3-epi-25(OH)D3 level isstrongly affected by the 25(OH)D3 level in the peripheral blood. Therefore, most studies have postulated that
C3-epi-25(OH)D plays a role in the overestimation ofvitamin D storage [21]. However, little is known regarding the relationship between C3-epi-25(OH)D and diseases. Recent genome-wide studies have confirmed thatgenetic factors can influence various pathways of vitamin D metabolism and that vitamin D metabolism gene
polymorphisms can influence the epimerization processitself [22, 23]. Therefore, the level of C3-epi-25(OH)D in
peripheral blood not only causes an overestimation ofvitamin D storage but also may have pathological significance. Our previous study revealed for the first time that
C3-epi-25(OH)D may be a superior marker to 25(OH)
D for predicting the severity of chronic kidney diseasein patients with rheumatoid arthritis [24]. Therefore, wehypothesized that C3-epi-25(OH)D tracks 25(OH)D to agreat extent and may explain the difficulty in determining
the pathological value of C3-epi-25(OH)D.
In this regard, we used an HPLC‒MS/MS method toassess C3-epi-25(OH)D3 levels in vivo and adopted theC3-epi-25(OH)D3% [%C3-epi-25(OH)D3] to correct
for the influence of 25(OH)D3 levels, then compared
the differential performance of C3-epi-25(OH)D3 level
and %C3-epi-25(OH)D3 in common paediatric diseases.
C3-epi-25(OH)D2 could not be analysed via our detection technique because of its relatively low content.

Methods
Participants

From November 2020 to June 2022, 4662 healthy paediatric participants and patients were involved in thedetermination of 25(OH)D metabolites. According to
the inclusion and exclusion criteria, of the 4662 subjects
screened for this study, 805 healthy underage participantsand 2962 patients with common paediatric diseases wereenrolled, including 2076 males and 1691 females aged
1 − 18 years (average age, 7.5 ± 3.7 years). The flowchart

Yang et al. BMC Pediatrics(2024) 24:577

of the participants included in the analysis is shown in
Fig. 1.
Inclusion and exclusion criteria for healthy paediatriccontrols

The inclusion criteria were as follows: (1) aged above 1
year and less than 18 years; (2) healthy participants who
attended the Child Health Center; (3) no visible physicalimpairment or disability; (4) normal cardiac, pulmonary,
hepatic, and renal function and no abnormalities on routine blood and urine examination.
The exclusion criteria were as follows: (1) age less than
1 year and above 18 years; (2) diagnosis of any definite
disease or illness after general medical examination; (3)obvious abnormalities in hepatic and renal function tests,
complete blood count, and routine urine test; (4) obvious
abnormalities on ultrasound or imaging examination (ifchecked); (5) taking any medication or vitamin D supplementation within 1 month.
Inclusion and exclusion criteria for paediatric patients

The inclusion criteria were as follows: (1) age above 1 year
and less than 18 years; (2) patients who were followed

Fig. 1 The participant flowchart

Page 3 of 13

up at the Child Health Center or the Paediatric Specialist Clinic or prepared for hospital admission; (3) diseasediagnosis that strictly adhered to the cluster coding of the
WHO International Classification of Diseases [25]; and(4) single disease diagnosis whenever possible, without
comorbid diseases or severe complications.
The exclusion criteria were as follows: (1) age less than
1 year and above 18 years, (2) presence of more than two
comorbid diseases, (3) presence of severe complicationsin other tissues or organs, (4) presence of uncommon
paediatric diseases, and (5) long-term treatment withgastrointestinal drugs (containing sucralfate or aluminium hydroxide), taking glucocorticoids within 1 week, or
taking vitamin D supplementation within 1 month.
Sample collection and processing

Approximately 3.0 mL of fasting venous blood was collected from all participants in SST-II vacuum tubes(BD, USA) early in the morning. The blood samples
were centrifuged at approximately 1500 ×g for 10 min to
separate the serum, which was then used for 25(OH)D
metabolite determination. When not used for immediate

Yang et al. BMC Pediatrics(2024) 24:577

assessment, the serum samples were stored at 2–8℃ for
less than 7 days.
On the day of assessment, the serum samples werepretreated as follows. Specifically, 200 µL of the serum
sample was mixed with 10 µL of internal standard (amixture of 25[OH]D3-d6 and C3-epi-25[OH]D3-[2H3]).
The mixture was then vortexed with 1.0 ml of tert-butyl
methyl ether release agent (CNW, Germany) for 5 min
and centrifuged at 12,000 ×g for 5 min. Then, 800 µLof the supernatant was collected and dried using an
MD200-1 A Nitrogen Evaporator (Allsheng, China). The
blow-dried substance was redissolved in 100 µL of methanol solution containing 0.1% formic acid, vortexed for
2 min, and centrifuged at 12,000 ×g for 5 min. Then, 50µL of the supernatant was collected to measure the levelsof 25(OH)D metabolites.
Isolation, identification, and measurement of 25(OH)D3
metabolites

The 25(OH)D3 metabolite levels were measured using alipid-soluble vitamin assay kit (FINDBiotech, Chengdu)
on a Jasper™ HPLC-MS/MS (Shimadzu, Japan)/AB
SCIEX™ 4500MD triple quadrupole mass spectrometer (ABI, USA). This method has been reported in ourprevious study [24]. After rigorous performance verification tests were performed, the lower limits of quantitation for 25(OH)D3 and C3-epi-25(OH)D3 were
determined to be 0.95 ng/ml and 0.40 ng/ml, respectively; the recoveries of standard addition for three levelsof 25(OH)D3 (5.0ng/mL, 30ng/mL, and 70ng/mL) during the five repeated measurements were observed to bewithin the ranges of 92.0%~114.8%, 88.2%~113.8%, and90.6%~114.1%, respectively. Furthermore, the monthly
coefficient of variation for 25(OH)D3 dual-level quality
control (15.19ng/mL and 55.34ng/mL) was calculated
during the study period, yielding results of 2.46%~5.95%
and 2.39%~5.36%, respectively. The separate monitoringof C3-epi-25(OH)D3 was not conducted due to its highly
similar mass spectrometry characteristics to 25(OH)D3.
On the basis of the measured results, %C3-epi-25(OH)D3 was defined as the C3-epi-25(OH)D3 concentration
divided by the sum of the C3-epi-25(OH)D3 and 25(OH)D3 concentrations.
Statistical analysis

Data analysis was performed using the SPSS software
v22.0 (SPSS, USA) and MedCalc software v20.1 (MedCalc, Belgium). After confirming a nonnormal distribution via a one-sample Kolmogorov‒Smirnov test, themeasurement data were described as medians (P25, P75)
[min, max]. The overall differences in sex, age, and season were analysed via the covariance analysis. To correct
for the influence of confounding factors, pairwise comparisons were performed using multiple comparisonsPage 4 of 13

of covariance analysis with Bonferroni correction, andthe significance was determined by the adjusted P value(Padj). Spearman correlation analysis was used to investigate the correlation of C3-epi-25(OH)D3 levels and%C3-epi-25(OH)D3 with 25(OH)D3 levels. Multinomial
logistic regression was used to investigate the correlations of C3-epi-25(OH)D3 levels and %C3-epi-25(OH)D3
with diseases, and odds ratios (ORs) and 95% confidence
intervals (CIs) were used to assess the degrees of theirrelationships. A P or Padj < 0.05 was considered statistically significant except for Spearman’s correlation analysis (P < 0.017).

Results
Degrees of influence of sex, age, and season on C3-epi25(OH)D3 generation

First, we focused on the degree of influence of age, sex,
and season on C3-epi-25(OH)D3 generation in 805
healthy underage individuals. The group settings forsex, age, and season were as follows: male (n = 486) andfemale (n = 377); infant period (n = 203, 1 to less than
3 years old); preschool period (n = 224, 3 to less than 6
years old); child period (n = 295, 6 to less than 12 yearsold); pubertal period (n = 83, 12 to less than 18 yearsold); spring (n = 189, Mar. to May); summer (n = 360,
Jun. to Aug.); autumn (n = 137, Sep. to Nov.); and winter(n = 119, Dec. to Feb.). To correct for the influence of age,sex, or seasonal factors, we performed covariance analysis for each factor while controlling for the remaining
two factors along with 25(OH)D3 levels as confounding
factors (shown in Fig. 2). The C3-epi-25(OH)D3 levels
and %C3-epi-25(OH)D3 did not differ according to sex(F = 1.107 and 2.164, P = 0.293 and 0.142) but did differ
according to age (χ2 = 14.981 and 13.052, both P < 0.001)
and season (F = 13.784 and 12.460, both P < 0.001).
Multiple comparisons using Bonferroni correction
revealed that the C3-epi-25(OH)D3 levels were higher
in the infant period than in the other periods (t = 5.329–
5.833, all Padj < 0.001). They were also higher in springand summer than in autumn and winter (t = 3.495–
6.061, all Padj < 0.01). Moreover, the %C3-epi-25(OH)D3 was higher in the infant period (t = 4.640–5.711,
all Padj < 0.001) than in the other periods. It was also
higher in spring and summer than in autumn and winter(t = 3.495–5.658, all Padj < 0.01). Our results confirmed
the physiological increase in C3-epi-25(OH)D3 levels and%C3-epi-25(OH)D3 during infancy, as well as in springand summer. Therefore, both can reflect the physiological
generation of the 25(OH)D3 C3-epimer.
C3-epi-25(OH)D3 levels and %C3-epi-25(OH)D3 in common
paediatric diseases

A total of 2962 cases of common paediatric diseases wereassessed in this study. Moreover, the disease categories

Yang et al. BMC Pediatrics(2024) 24:577

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Fig. 2 Gender, age, and season differences in C3-epi-25(OH)D3 levels and percentages among 805 healthy underage individuals. Note * After the Bonferroni correction, Padj < 0.05. The 25(OH)D3 distribution is only shown without analysis. The violin plot presents a density trace of detailed distributional
characteristics. The longer horizontal line represents the median, and the top and bottom horizontal lines of I shape represent P75 and P25, respectively.
Both the C3-epi-25(OH)D3 level and percentage are greater in the infant period than in other periods and are greater in spring and summer than inautumn and winter(specific diseases) included skeletal system diseases(hypovitaminosis D, short stature, rickets, and growing arthralgia), respiratory system diseases (pneumonia, atopic cough, trachitis/bronchitis, upper respiratory
infection, and cough variant asthma), endocrine systemdiseases (thyroid disorder, growth hormone deficiency,
and sexual precocity), neurological system diseases (ticdisorder, epilepsy, attention-deficit/hyperactivity disorder, mental retardation/developmental disabilities, andsleep disorders), and other system diseases (nephrotic
syndrome, paediatric malnutrition, and lymphocytic
leukaemia). Short stature was defined as a height thatfell 2 standard deviations below the mean height of thepeer population, without any identifiable aetiology [26].
Atopic cough was defined as a nonproductive coughwith an atopic tendency and cough hypersensitivity but

without nonspecific bronchial hyperresponsiveness. Thyroid disorders were mainly referred to as hyperthyroidism and hypothyroidism in this study.
The distributions of C3-epi-25(OH)D3 levels and%C3-epi-25(OH)D3 for these 20 common paediatricdiseases are described in terms of the median (P25, P75)
[Min, Max] (Table 1). With age, sex, season, and 25(OH)D3 levels as confounding factors, we performed pairwise
comparisons via post hoc covariance analysis. Compared with those in healthy controls, C3-epi-25(OH)D3 levels were lower only in patients with atopic cough(t=-3.925, Padj = 0.037) and short stature (t=-4.094,
Padj = 0.029). %C3-epi-25(OH)D3 decreased in patientswith atopic cough (t=-3.753, Padj = 0.046), short stature(t=-4.242, Padj = 0.024), hypovitaminosis D (t=-4.152,
Padj = 0.027), trachitis/bronchitis (t=-3.827, Padj = 0.042),

Yang et al. BMC Pediatrics(2024) 24:577

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Table 1 The C3-epi-25(OH)D3 levels and percentages in 20 pediatric common diseases {Median (P25, P75) [min, max]}, and theirdifference significances after adjusting 25(OH)D3 level especially
DiseaseHealthy control
Hypovitaminosis D
Short stature
Rickets
Growing arthralgia
Pneumonia
Atopic cough

n
805
196
623
83
46
44
170

25(OH)D3 (ng/mL)
27.08 (22.42, 32.64) [9.23, 54.66]
14.98 (11.89, 17.60) [5.65, 19.35]
30.05 (25.48, 34.44) [16.00, 54.48]
15.33 (12.61, 17.98) [6.26, 19.65]
19.22 (14.43, 28.43) [6.48, 45.98]
25.70 (21.27, 34.65) [8.32, 53.44]
25.41 (20.28, 31.23) [9.95, 50.05]

Trachitis/ bronchitis
Upper respiratory infection
Cough variant asthma
Nephrotic syndrome
Thyroid disorder
Growth hormone deficiency
Sexual precocity
Tic distic disorder
Epilepsy
Attention-deficit/ hyperactivity disorder
Mental retardation/ developmental
disabilities
Sleep disorders
Paediatric malnutrition

126
190
401
69
26
38
329
218
121
107
54

24.54 (19.11, 31.60) [4.03, 53.19]
25.46 (19.13, 32.58) [8.32, 53.93]
27.65 (21.64, 35.01) [6.64, 54.71]
17.49 (7.97, 28.77) [0.95, 40.77]
23.22 (16.50, 30.45) [5.84, 46.18]
25.67 (22.23, 29.97) [13.84, 44.52]
24.38 (19.94, 29.79) [6.77, 50.96]
24.04 (18.80, 28.86) [7.69, 44.64]
24.49 (19.03, 32.07) [5.70, 50.42]
23.19 (18.55, 28.45) [7.39, 45.62]
30.56 (24.78, 36.62) [15.68, 52.70]

66
29

23.20 (19.74, 28.61) [8.24, 44.85]
25.41 (18.43, 34.95) [6.94, 44.85]

Lymphocytic leukemia

26

28.30 (21.87, 30.95) [7.48, 41.84]

C3-epi-25(OH)D3 (ng/mL)
1.59 (1.08, 2.31) [0.10, 7.05]
0.79 (0.54, 1.06) [0.12, 2.52]
1.68 (1.22, 2.34) [0.28, 7.79]
0.94 (0.58, 1.27) [0.19, 3.30]
1.01 (0.64, 1.54) [0.17, 3.20]
1.43 (1.06, 2.04) [0.35, 4.84]
1.42 (0.89, 2.14) [0.19,
4.85] *
1.32 (0.91, 1.92) [0.11, 6.21]
1.43 (0.95, 2.25) [0.26, 7.71]
1.67 (1.08, 2.59) [0.22, 7.45]
1.10 (0.62, 2.15) [0.12, 7.21]
1.32 (0.74, 1.92) [0.28, 4.12]
1.30 (1.03, 1.72) [0.38, 4.03]
1.23 (0.78, 1.74) [0.15, 5.68]
1.31 (0.85, 1.92) [0.21, 4.88]
1.30 (0.87, 1.95) [0.30, 9.42]
1.28 (0.88, 1.73) [0.22, 3.68]
2.31 (1.43, 3.16) [0.18, 4.81]%C3-epi-25(OH)D3 (%)
5.71 (4.00, 7.50) [0.39, 15.61]
5.25 (3.82, 6.99) [1.05, 11.97] *5.39 (4.05, 7.04) [0.87, 15.99]
5.93 (4.44, 7.57) [1.40, 15.62] *4.91 (3.30, 7.58) [1.59, 10.42]
5.09 (4.18, 6.72) [2.48, 11.95]
5.41 (3.66, 7.34) [0.72, 14.07] *1.23 (0.87, 1.69) [0.20, 4.29]
1.11 (0.62, 1.72) [0.18,
3.81] *
1.87 (1.57, 2.18) [0.67, 4.23]

4.72 (3.72, 6.97) [1.68, 12.14]
4.65 (3.33, 5.83) [0.80, 9.96] *5.13 (4.08, 6.93) [0.64, 13.55] *5.71 (3.99, 7.50) [0.99, 14.73]
5.89 (4.10, 8.06) [0.96, 15.81]
6.89 (5.01, 9.45) [1.44, 20.25] *5.21 (3.45, 7.32) [0.87, 14.82]
5.14 (4.04, 6.65) [1.66, 10.92]
4.91 (3.24, 6.87) [0.45, 15.41]
5.57 (4.07, 7.71) [0.69, 15.07]
5.18 (3.90, 7.16) [1.41, 17.22]
5.61 (3.68, 6.70) [0.97, 11.71] *6.20 (5.02, 9.33) [0.54, 15.40]

6.88 (5.29, 7.73) [4.25, 11.74] *Note * Compared to healthy controls with Bonferroni correction, Padj < 0.05. 25(OH)D3 levels were only exhibited and not compared. After adjusted for gender, age,season, and 25(OH)D3 level, C3-epi-25(OH)D3 levels changed significantly in only two diseases, compared with eight diseases for %C3-epi-25(OH)D3. In Rickets
and Nephrotic syndrome, a separation phenomenon of decreased C3-epi-25(OH)D3 level due to tracking 25(OH)D3 level and increased %C3-epi-25(OH)D3 wereobserved, suggesting that %C3-epi-25(OH)D3 might have an ability to reflect the pathological generation of C3-epi-25(OH)D3

and attention-deficit/hyperactivity disorder (t=-3.719,
Padj = 0.048), whereas it increased in patients with rickets (t = 4.199, Padj = 0.026), nephrotic syndrome (t = 6.795,
Padj < 0.001), and lymphocytic leukaemia (t = 3.919,
Padj = 0.037). This finding showed that %C3-epi-25(OH)D3 could reveal the change in C3-epi-25(OH)D3 generation under those disease conditions more clearly than its
levels.
Bivariate relationship analysis among C3-epi-25(OH)D3
levels, %C3-epi-25(OH)D3, and 25(OH)D3 levels

We observed Spearman bivariate correlations between
25(OH)D3, C3-epi-25(OH)D3, and %C3-epi-25(OH)D3. Moreover, significance was determined by employing a Bonferroni-corrected significance level of
α = 0.05/3 ≈ 0.017. The results are shown in Table 2.
Under healthy and disease conditions, C3-epi-25(OH)D3 levels were positively correlated with 25(OH)D3 levels (ρ = 0.318–0.678, all P < 0.017). Moreover, %C3-epi25(OH)D3 was positively correlated with C3-epi-25(OH)D3 levels (ρ = 0.637–0.880, all P < 0.001) but was notcorrelated with 25(OH)D3 levels (ρ=-0.219–0.162, all
P > 0.017), except for those associated with nephrotic
syndrome (shown in Fig. 3: vs. C3-epi-25(OH)D3:

ρ = 0.331, P = 0.005; vs. 25(OH)D3: ρ=-0.393, P = 0.001).
These results suggested that the high traceability of
C3-epi-25(OH)D3 levels to 25(OH)D3 levels was always
present in healthy and diseased conditions. Thus, the useof C3-epi-25(OH)D3 levels to explore the relationshipbetween C3-epi-25(OH)D3 and diseases may yield inappropriate results. However, the use of %C3-epi-25(OH)D3 may curb this problem because %C3-epi-25(OH)D3
demonstrated a high correlation with C3-epi-25(OH)D3 levels but not with 25(OH)D3 levels under mostconditions.
Degree of the relationship of C3-epi-25(OH)D3 levels and%C3-epi-25(OH)D3 with the occurrence of various diseases

Multivariate logistic regression was used to analyse
the degree of the correlation of C3-epi-25(OH)D3 or%C3-epi-25(OH)D3 with various diseases, with age, sex,
season, and 25(OH)D3 levels as confounding factors. The
same analysis with only age, sex, and season as confounding factors was also performed to compare the differenceswith and without correction for 25(OH)D3 levels. The
results showed that before and after adjusting for 25(OH)D3, the degree of correlation of C3-epi-25(OH)D3 withmultiple diseases differed significantly (Table 3); however,Yang et al. BMC Pediatrics(2024) 24:577

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Table 2 Spearman bivariate correlations between 25(OH)D3, C3-epi-25(OH)D3, and %C3-epi-25(OH)D3 {ρ(95%CI), P}
DiseasenC3-epi-25(OH)D3 vs. 25(OH)D3

Healthy control
Hypovitaminosis D
Short stature
Rickets
Growing arthralgia
Pneumonia
Atopic coughTrachitis/ bronchitis
Upper respiratory infection
Cough variant asthma
Nephrotic syndrome

805
196
623
83
46
44
170
126
190
401
69

0.441 (0.384 to 0.495), < 0.001
0.496 (0.382 to 0.595), < 0.001
0.330 (0.258 to 0.398), < 0.001
0.432 (0.238 to 0.592), < 0.001
0.635 (0.423 to 0.781), < 0.001
0.571 (0.330 to 0.742), < 0.001
0.638 (0.540 to 0.720), < 0.001
0.678 (0.571 to 0.763), < 0.001
0.666 (0.579 to 0.738), < 0.001
0.592 (0.524 to 0.652), < 0.001
0.672 (0.517 to 0.784), < 0.001

Thyroid disorder
Growth hormone deficiency
Sexual precocity
Tic distic disorder
Epilepsy
Attention-deficit/ hyperactivity disorder
Mental retardation/ developmental
disabilities
Sleep disorders
Paediatric malnutrition
Lymphocytic leukemia

26
38
329
218
121
107
54

0.468 (0.099 to 0.724), 0.016
0.464 (0.170 to 0.683), 0.003
0.344 (0.245 to 0.436), < 0.001
0.484 (0.376 to 0.580), < 0.001
0.588 (0.458 to 0.694), < 0.001
0.543 (0.393 to 0.664), < 0.001
0.367 (0.111 to 0.578), 0.006%C3-epi-25(OH)D3 vs.
25(OH)D3-0.025 (-0.094 to 0.045), 0.485
0.022 (-0.118 to 0.162), 0.759-0.113 (-0.190 to 0.035), 0.271-0.032 (-0.246 to 0.185), 0.776-0.053 (-0.338 to 0.241), 0.726-0.063 (-0.354 to 0.238), 0.682
0.162 (0.011 to 0.305), 0.035
0.044 (-0.132 to 0.217), 0.624
0.111 (-0.032 to 0.249), 0.128
0.035 (-0.0633 to 0.132), 0.486-0.393 (-0.576 to -0.172),
0.001-0.046 (-0.425 to 0.348), 0.825-0.134 (-0.435 to 0.194), 0.423-0.166 (-0.270 to -0.059), 0.025-0.066 (-0.197 to 0.068), 0.336-0.046 (-0.222 to 0.134), 0.619
0.003 (-0.187 to 0.193), 0.973-0.170 (-0.419 to 0.102), 0.218%C3-epi-25(OH)D3 vs. C3epi-25(OH)D3
0.863 (0.844 to 0.879), < 0.001
0.842 (0.795 to 0.878), < 0.001
0.880 (0.860 to 0.896), < 0.001
0.864 (0.797 to 0.910), < 0.001
0.689 (0.499 to 0.816), < 0.001
0.727 (0.548 to 0.842), < 0.001
0.848 (0.800 to 0.886), < 0.001
0.717 (0.620 to 0.793), < 0.001
0.788 (0.727 to 0.836), < 0.001
0.800 (0.762 to 0.833), < 0.001
0.331 (0.102 to 0.527), 0.005

66
29
26

0.318 (0.082 to 0.520), 0.009
0.629 (0.342 to 0.809), < 0.001
0.566 (0.228 to 0.782), 0.003-0.185 (-0.409 to 0.0598), 0.137 0.834 (0.742 to 0.895), < 0.001-0.025 (-0.388 to 0.345), 0.898 0.701 (0.450 to 0.849), < 0.001-0.219 (-0.559 to 0.184), 0.283 0.637 (0.489 to 0.865), < 0.001

0.817 (0.628 to 0.915), < 0.001
0.774 (0.604 to 0.877), < 0.001
0.834 (0.798 to 0.864), < 0.001
0.805 (0.753 to 0.847), < 0.001
0.727 (0.630 to 0.801), < 0.001
0.795 (0.712 to 0.855), < 0.001
0.802 (0.680 to 0.880), < 0.001

Note The significant level adopted Bonferroni correction as α = 0.05/3 ≈ 0.017. Within health and disease conditions, C3-epi-25(OH)D3 levels positively correlated to25(OH)D3 levels, indirectly confirming that the peculiarity of C3-epi-25(OH)D3 highly tracking 25(OH)D3 was universal; %C3-epi-25(OH)D3 strongly and positively
correlated to C3-epi-25(OH)D3 levels and not correlated to 25(OH)D3 levels except for in nephrotic syndrome, indicating that %C3-epi-25(OH)D3 could reflect C3epi-25(OH)D3 levels and was independent of 25(OH)D3 levels

that of %C3-epi-25(OH)D3 with various diseases wasvery similar (Table 4). Based on our adjusted results,
C3-epi-25(OH)D3 was only associated with the occurrence of four diseases: short stature (OR = 1.32, P < 0.001),
nephrotic syndrome (OR = 1.61, P = 0.012), lymphocytic
leukaemia (OR = 1.71, P = 0.010), and paediatric malnutrition (OR = 0.51, P = 0.031). However, %C3-epi-25(OH)D3 was associated with the occurrence of six diseases:
short stature (OR = 1.10, P < 0.001), nephrotic syndrome (OR = 1.21, P < 0.001), lymphocytic leukaemia(OR = 1.21, P = 0.007), paediatric malnutrition (OR = 0.80,
P = 0.017), rickets (OR = 1.19, P = 0.012), and hypovitaminosis D (OR = 0.92, P = 0.040). These results suggested
that %C3-epi-25(OH)D3 was more suitable than C3-epi25(OH)D3 for exploring the correlation between C3-epi25(OH)D3 generation and disease occurrence.

Discussion
C3-epi-25(OH)D has been used as a designated indicatorin nationally representative surveys in a few developed
countries, such as the National Health and Nutrition
Examination Survey [15, 16]. However, little is known
regarding the pathological importance of C3-epi-25(OH)D, except for the overestimation of vitamin D storage[27–29]. In this study, we demonstrated the importanceof correcting the properties of C3-epi-25(OH)D3, which
can sensitively track 25(OH)D3. We believe that witha simple correction (which is a calculation of %C3-epi25(OH)D3), a relatively stable degree of relationshipbetween C3-epi-25(OH)D3 generation and the occurrence of multiple paediatric diseases can be obtained.
This finding suggests that the C3-epi-25(OH)D3%, not
its level, may be a potentially better biomarker for investigating the pathological relevance of C3-epi-25(OH)D3
generation.
Prior to our disease investigations, we observed differences regarding sex, age, and season in C3-epi-25(OH)D3 levels and %C3-epi-25(OH)D3 in healthy underagecontrols. After correcting for the other two influencing
factors, we obtained the same conclusion for the C3-epi25(OH)D3 levels and %C3-epi-25(OH)D3. Both values
were greater in the infant period than in the other periods, as well as in spring and summer than in autumn andwinter. For C3-epi-25(OH)D3 levels, the age difference
was consistent with previous reports, and the seasonal
difference was similar to that reported in other studies[18, 30]. The highest level in the summer is attributed
mainly to an increase in 25(OH)D3 generation due tosunlight [31, 32], which may increase C3-epi-25(OH)D3
generation according to the properties of C3-epi-25(OH)Yang et al. BMC Pediatrics(2024) 24:577

Page 8 of 13

Fig. 3 Changes in the correlation of %C3-epi-25(OH)D3 with 25(OH)D3 and C3-epi-25(OH)D3 levels in patients with rickets or nephrotic syndrome.
Note The significance level adopted the Bonferroni correction, with α = 0.05/3 ≈ 0.017. The black solid line represents the correlation coefficient, and thered dotted line delineates the 95% confidence interval for the correlation coefficient. The correlations of %C3-epi-25(OH)D3 with 25(OH)D3 and C3-epi25(OH)D3 levels are distinctly different in patients with nephrotic syndrome than in healthy controls and those with other diseases (for example, Rickets).
The correlation between %C3-epi-25(OH)D3 and 25(OH)D3 changed from a noncorrelated correlation to a negative correlation, and the correlation
between %C3-epi-25(OH)D3 and C3-epi-25(OH)D3 weakened

D3, which is a sensitive tracker of 25(OH)D3 [20]. However, the differences in %C3-epi-25(OH)D3 based on age
and season imply that C3-epi-25(OH)D3 generation may
be physiologically increased during infancy, spring, andsummer. The specific reasons and mechanisms underlying this phenomenon require further clarification. In
addition, some studies have reported sex differences
and the reverse changes during spring and winter [33,34]. Our results suggest the opposite, possibly due tothe influence of ethnic, regional, population, and interlaboratory differences [35, 36]. We do not consider the
influence of sunscreens on the underage population [37]because sunscreens are rarely used in this population.
Currently, standardized measurement methods and reference materials for C3-epi-25(OH)D are lacking. To better assess 25(OH)D metabolites and their percentages invarious regions and countries, it is necessary to formulatea 25(OH)D standardization measurement programmeand develop a standard reference material for C3-epi25(OH)D.
The physiological changes in C3-epi-25(OH)D3 levels
and %C3-epi-25(OH)D3 suggest that the effects of age,season, and sex should be considered in relevant studies,
similar to other vitamin D metabolite studies. However,even after adjusting for sex, age, and season, the relationship between C3-epi-25(OH)D3 and disease occurrence
remains controversial [38–40]. Further consideration
revealed that the ability of C3-epi-25(OH)D3 to track
25(OH)D3 effectively could not be ignored. Our results
revealed a strong correlation between C3-epi-25(OH)D3 and 25(OH)D3 under healthy and diseased conditions, indirectly confirming the prevalence of this property. Owing to the complexities of C3-epi-25(OH)D in
terms of its properties and influencing factors [19, 41],
its contribution to disease management remains unclear.

Yang et al. BMC Pediatrics(2024) 24:577

Page 9 of 13

Table 3 The relation of C3-epi-25(OH)D3 levels with 20 pediatric common diseases
DiseaseHypovitaminosis D
Short stature
Rickets
Growing arthralgia
Pneumonia
Atopic coughTrachitis/ bronchitis
Upper respiratory infection
Cough variant asthma
Nephrotic syndrome
Thyroid disorder
Growth hormone deficiency
Sexual precocity
Tic distic disorder
Epilepsy
Attention-deficit/ hyperactivity disorder
Mental retardation/ developmental disabilities
Sleep disorders
Paediatric malnutrition
Lymphocytic leukemia

Age, gender, and season
as the confounding factors
OR (95% CI)
Wald
0.19 (0.14 to 0.27)
85.876
1.67 (1.49 to 1.88)
72.9860.38 (0.24 to 0.58)
19.6750.59 (0.36 to 0.95)
4.745
0.70 (0.49 to 0.99)
4.034
0.74 (0.61 to 0.90)
8.683
0.72 (0.57 to 0.91)
7.725
0.90 (0.76 to 1.08)
1.253
1.03 (0.91 to 1.17)
0.273
0.72 (0.51 to 1.01)
3.701
1.09 (0.66 to 1.80)
0.127
0.86 (0.57 to 1.31)
0.501
1.02 (0.85 to 1.22)
0.038
0.87 (0.72 to 1.05)
2.228
0.79 (0.63 to 1.00)
3.970
0.72 (0.54 to 0.96)
5.116
0.94 (0.73 to 1.22)
0.228
0.68 (0.48 to 0.97)
4.528
0.48 (0.27 to 0.85)
6.427
1.66 (1.15 to 2.39)
7.336

P< 0.001< 0.001< 0.001
0.029
0.045
0.003
0.005
0.263
0.601
0.054
0.722
0.479
0.845
0.136
0.046
0.024
0.633
0.033
0.011
0.007

Age, gender, season, and 25(OH)D3
as the confounding factors
OR (95% CI)
Wald
P0.74 (0.51 to 1.08)
2.461
0.117
1.32 (1.16 to 1.50)
17.088< 0.0011.42 (0.90 to 2.24)
2.214
0.137
0.81 (0.48 to 1.37)
0.596
0.440
0.80 (0.54 to 1.18)
1.260
0.262
0.88 (0.70 to 1.10)
1.303
0.254
0.84 (0.65 to 1.08)
1.809
0.179
1.07 (0.88 to 1.30)
0.478
0.490
1.11 (0.96 to 1.27)
2.104
0.147
1.61 (1.11 to 2.34)
6.311
0.012
1.16 (0.67 to 2.01)
0.266
0.606
0.84 (0.54 to 1.32)
0.558
0.455
1.01 (0.83 to 1.23)
0.008
0.928
1.04 (0.85 to 1.28)
0.144
0.705
0.88 (0.68 to 1.13)
1.023
0.312
0.86 (0.63 to 1.17)
0.935
0.333
1.00 (0.75 to 1.34)
0.000
0.992
0.77 (0.52 to 1.13)
1.795
0.180
0.51 (0.28 to 0.94)
4.649
0.031
1.71 (1.13 to 2.58)
6.554
0.010

Note The multivariate logistic regression analysis was used with healthy controls as reference. The relation assessment about C3-epi-25(OH)D3 levels with 20
pediatric common diseases after adjusting 25(OH)D3 was significantly differed from before adjusting 25(OH)D3

Table 4 The relation of %C3-epi-25(OH)D3 with 20 pediatric common diseases
DiseaseHypovitaminosis D
Short stature
Rickets
Growing arthralgia
Pneumonia
Atopic coughTrachitis/ bronchitis
Upper respiratory infection
Cough variant asthma
Nephrotic syndrome
Thyroid disorder
Growth hormone deficiency
Sexual precocity
Tic distic disorder
Epilepsy
Attention-deficit/ hyperactivity disorder
Mental retardation/ developmental disabilities
Sleep disorders
Paediatric malnutrition
Lymphocytic leukemia

Age, gender, and season
as the confounding factors
OR (95% CI)
Wald1.02 (0.95 to 1.09)
0.344
1.06 (1.01 to 1.10)
5.874
1.15 (1.05 to 1.25)
9.844
1.00 (0.88 to 1.13)
0.000
0.95 (0.83 to 1.07)
0.749
0.95 (0.89 to 1.02)
1.700
0.95 (0.88 to 1.03)
1.634
1.02 (0.95 to 1.08)
0.241
1.03 (0.98 to 1.08)
1.567
1.28 (1.17 to 1.39)
33.0871.05 (0.90 to 1.23)
0.387
0.95 (0.83 to 1.09)
0.468
1.01 (0.95 to 1.07)
0.110
1.03 (0.97 to 1.10)
1.090
0.96 (0.89 to 1.04)
0.962
0.97 (0.89 to 1.06)
0.497
1.02 (0.92 to 1.14)
0.198
0.95 (0.86 to 1.06)
0.715
0.82 (0.69 to 0.98)
4.584
1.20 (1.05 to 1.38)
6.947

P
0.558
0.015
0.002
0.984
0.387
0.192
0.201
0.624
0.211
< 0.001
0.534
0.494
0.740
0.296
0.327
0.481
0.656
0.398
0.032
0.008

Age, gender, season, and 25(OH)D3
as the confounding factors
OR (95% CI)
Wald
P
0.92 (0.86 to 1.00)
4.236
0.040
1.10 (1.05 to 1.15)
16.219< 0.001
1.19 (1.08 to 1.29)
6.926
0.012
0.96 (0.84 to 1.09)
0.362
0.547
0.94 (0.82 to 1.06)
1.061
0.303
0.94 (0.88 to 1.01)
2.749
0.097
0.93 (0.86 to 1.01)
2.789
0.095
1.00 (0.94 to 1.07)
0.006
0.938
1.03 (0.98 to 1.08)
1.318
0.251
1.21 (1.11 to 1.32)
18.095< 0.001
1.04 (0.89 to 1.22)
0.269
0.604
0.95 (0.83 to 1.09)
0.453
0.501
1.01 (0.95 to 1.07)
0.163
0.687
1.02 (0.95 to 1.08)
0.236
0.627
0.95 (0.88 to 1.03)
1.570
0.210
0.94 (0.86 to 1.03)
1.561
0.211
1.02 (0.92 to 1.14)
0.134
0.714
0.94 (0.84 to 1.05)
1.344
0.246
0.80 (0.66 to 0.96)
5.672
0.017
1.21 (1.06 to 1.40)
7.402
0.007

Note The multivariate logistic regression analysis was used with healthy controls as reference. The relation assessment about C3-epi-25(OH)D3 levels with 20
pediatric common diseases was almost perfectly consistent both before and after adjusting 25(OH)D3

Yang et al. BMC Pediatrics(2024) 24:577

However, the use of C3-epi-25(OH)D to assess disease
conditions requires more reliable methods.
Surprisingly, with a simple correction involving calculating the %C3-epi-25(OH)D, the relationship between
C3-epi-25(OH)D3 generation and disease occurrence
was better understood. Our results revealed that, after
adjusting for influencing factors, including 25(OH)D3
levels, among 20 common paediatric diseases, C3-epi25(OH)D3 levels changed significantly only in two diseases, whereas %C3-epi-25(OH)D changed in eight
diseases. This result suggested that %C3-epi-25(OH)D3
could better reflect the pathological relevance of C3-epi25(OH)D3 generation, which will be helpful for further
research on other relevant details. Subsequent analysis
revealed that the C3-epi-25(OH)D3 level was notably different, whereas the %C3-epi-25(OH)D3 level was almost
consistent in terms of its relationship with disease occurrence before and after adjusting for 25(OH)D3 levels.
Therefore, %C3-epi-25(OH)D3 may be more suitable
than the C3-epi-25(OH)D3 level for exploring the correlation between C3-epi-25(OH)D3 generation and disease
occurrence.
Our correlation results regarding %C3-epi-25(OH)D3
with disease occurrence are consistent with the pathological conditions of vitamin D metabolism during thecourse of disease occurrence and development. Amongthe 20 common paediatric diseases, we discovered a significant correlation between %C3-epi-25(OH)D3 and sixof these diseases. Specifically, it was positively correlatedwith short stature, nephrotic syndrome, lymphocytic
leukaemia, and rickets but negatively correlated withpaediatric malnutrition and hypovitaminosis D. Thesediseases involve the metabolism and function of vitamin
D, including its absorption, synthesis, physiological utilization, and immune regulation. Therefore, there was apositive correlation between increased %C3-epi-25(OH)D3 and the aforementioned four diseases. An increase
in %C3-epi-25(OH)D3 indicates a relative increasein C3-epimer levels or a relative decrease in 25(OH)D3 levels. In individuals with high %C3-epi-25(OH)D3, if the sum of C3-epimer and non-C3 epimer levels remains constant, then it may exacerbate 25(OH)D deficiency. These findings may provide a rationale forthe pathological risks associated with elevated levels of%C3-epi-25(OH)D3. Moreover, the negative correlation
between %C3-epi-25(OH)D3 and paediatric malnutrition as well as hypovitaminosis D is easily understood.
An increase in %C3-epi-25(OH)D3 indicates an increasein C3-epi-25(OH)D3 levels, which also implies anincrease in 25(OH)D3 levels due to the high tracking
ability of C3-epi-25(OH)D3 with 25(OH)D3. From thispoint, C3-epi-25(OH)D3 or %C3-epi-25(OH)D3 is more
like a follower of 25(OH)D3 and demonstrates a negative correlation with both paediatric malnutrition andPage 10 of 13

hypovitaminosis D. Thus, to correctly understand therelationship between %C3-epi-25(OH)D3 and disease
occurrence, it is necessary to consider not only the influence of the metabolic changes in C3-epi-25(OH)D3 itself
but also its ability to effectively track 25(OH)D3 levels.
Furthermore, our findings suggest that the correlations
between %C3-epi-25(OH)D and the levels of 25(OH)D3
and C3-epi-25(OH)D3 in patients with nephrotic syndrome differ significantly from those reported in healthy
controls and from those with other diseases. These distinctions may be attributed to the crucial role of the kidneys in vitamin D metabolism, as any renal impairment
can lead to alterations in the intrarenal enzymes responsible for vitamin D metabolism [13, 42].
Vitamin D metabolism genetic polymorphism is implicated in various pathways of vitamin D, and 25(OH)D3
epimerization process is also influenced by the interplayof genetic factors and various internal environmental
factors related to vitamin D metabolism [22, 23]. Due tohighly tracking to 25(OH)D levels [20], the generationof C3-epi-25(OH)D3 is also influenced by these factors.
An investigation of single nucleotide polymorphisms in
C3-epi-25(OH)D confirmed that the genetic determinants and potential factors of C3-epimers differ fromthose of non-C3-epimers [43]. These findings suggestthat the pathological generation of C3-25(OH)D3 objectively occurs in vivo. Overall, our study may help resolve
the controversy regarding the pathological relevance ofC3-epi-25(OH)D3 generation. We hypothesize that theunique tracking ability of C3-epi-25(OH)D3 for 25(OH)D3 conceals and even eliminates exposure to its pathological generation in vivo. Therefore, in relevant research
regarding C3-epi-25(OH)D3, correction should be madefor the effect of 25(OH)D3, in addition to the effects ofsex, age, and season. In contrast with the C3-epi-25(OH)D3 level, the %C3-epi-25(OH)D3 level can circumventthe influence of the 25(OH)D3 level and may be conducive to accurately exploring the pathological significanceof 25(OH)D3 epimerization. Therefore, further studies
on the causality between diseases and %C3-epi-25(OH)D3, as well as genetic studies on these diseases, which
are influenced by a higher %C3-epi-25(OH)D3, are necessary. Moreover, calculating %C3-epi-25(OH)D3 may
be a relatively simple and effective correction method;
however, its pathological relevance requires further
exploration in many in-depth studies. Currently, IOM
and EnS/JES recommend the use of mass spectrometryfor 25(OH)D3 analysis [1, 2], while the determination of
C3-epi-25(OH)D3 is still in its nascent stages. However,neither of these methods are performed routinely. Owing
to the advantages of 25(OH)D3 in terms of biologicalactivity, origin, synthesis, and nutritional assessment, as
well as its obvious effect on vitamin D storage and possible pathological value of high C3-epi-25(OH)D3 content,

Yang et al. BMC Pediatrics(2024) 24:577

further exploration of both could significantly contributeto advancements in pathology and nutrition.
This study had a few limitations. The most prominent
limitation was that it was only a preliminary and crude
relationship analysis for %C3-epi-25(OH)D3 with multiple diseases; therefore, further detailed research isneeded for each specific disease. Second, because of thedifferent types of diseases, it was impossible to match thedata regarding age, sex, and season from patients witheach disease to healthy controls; however, our results
were adjusted for age, sex, and season. Third, this wasa single-centre, cross-sectional study; therefore, causal
associations, racial differences, and regional differences
cannot be determined. In addition, other potentiallyinfluential factors, such as dietary habits, outdoor activity, and sunscreen use, were not considered.

Conclusions
To the best of our knowledge, this is the first study
to investigate the differential performance of C3-epi25(OH)D3 levels and %C3-epi-25(OH)D3 in various
common paediatric diseases. Our results emphasize that
the C3-epi-25(OH)D percentage, not the C3-epi-25(OH)D level, may be a potential biomarker for better interpretation of the relationship between C3-epi-25(OH)D3
and multiple diseases. The %C3-epi-25(OH)D3 can correct the concern regarding the C3-epi-25(OH)D3 level,
highly tracking the 25(OH)D3 level. Moreover, %C3-epi25(OH)D3 can reflect the physiological generation andpathological increase in the 25(OH)D3 C3-epimer. This
simple calculation method is expected to pave the way
for correctly exploring the details of C3-epi-25(OH)D
generation, further promoting an in-depth study of its
pathological relevance.
Abbreviations%C3	Epi-25(OH)D3
percentage of C3	Epimer of 25-hydroxyvitamin D
25(OH)D	25-hydroxyvitamin D
APCI	Atmospheric pressure chemical ionization
C3-epi-25(OH)D	C3-epimer of 25-hydroxyvitamin DCI	Confidence interval
EnS	Endocrine Society
IOM	Institute of Medicine
JES	Japan Endocrine Society
OR	Odds ratio
Acknowledgements
The authors thank the Editage (https://www.editage.cn/) for providing
professional language services, and Prof. Jiafu Feng for providing technicalguidance and testing the platform for this study.
Author contributions
Conceptualization: YW. Y., and J. T.;Data curation: T. Y., XH. C., J. T., and
YW. Y.;Formal analysis: YW. Y., and J. T.;Funding acquisition: YW. Y., and J.
T.;Investigation: T. Y., MY. W., and SH. X.;Methodology: D. H., and T. Y.;Writing original draft: T. Y., XH. C., and MY. W.; Writing - review & editing: J. T., and YW.
Y.T. Y., XH. C., and MY. W. contributed equally to this work as co-first author.

Page 11 of 13

Funding
This research was financially supported by the Basic Application Project of
the Science & Technology Department of Sichuan Province [Approval No:
2019YJ0701] and the Popular Application Project of the Health Commission of
Sichuan Province [Approval No: 20PJ256]. The funders had no role in the study
design, data collection and analysis, decision to publish, or preparation of themanuscript.
Data availability
All the data generated or analysed during this study are included in thispublished article and its supplementary information files.

Declarations
Ethical approval and consent to participate
This study adopted a cross-sectional case‒control design, and all methods
were carried out in accordance with relevant guidelines and regulations or
the Declaration of Helsinki. This study was performed at Mianyang Central
Hospital, affiliated with the School of Medicine, University of Electronic
Science and Technology of China. The Ethics Committee of Mianyang Central
Hospital approved this study (Approval No. S2018085). All participants or theirparents signed informed consent forms.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Laboratory Medicine, Santai Country People’s Hospital,
Mianyang 621100, P.R. China
2
Department of Laboratory Medicine, School of Medicine, Mianyang
Central Hospital, University of Electronic Science and Technology ofChina, Mianyang 621000, P.R. China
3
Department of Paediatric Surgery, School of Medicine, Mianyang Central
Hospital, University of Electronic Science and Technology of China,
Mianyang 621000, P.R. China
Received: 12 May 2023 / Accepted: 10 September 2024

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