Abstract
Objectives
The primary objective was to determine levels of C3-epi-25(OH)D in very low birth weight infants. The secondary objective was to evaluate the possible influence of preterm birth, intrauterine growth restriction (IUGR), and season of birth on the production of C3-epimers.
Methods
A total of 127 infants with birth weight less than 1,500 g met the inclusion criteria of the study. We examined 25-hydroxyvitamin-D [25(OH)D] levels and C3-epi-25(OH)D in maternal serum before labor, and in cord blood and infants’ serum on days 14 and 28, and at discharge.
Results
The mean levels (±SD) of C3-epi-25(OH)D of the cord, on day 14, on day 28, and at discharge were 2.2 (2.9), 7.7 (5.5), 11.7 (7.6) and 14.9 (11.7) nmol/L respectively. The proportion of total 25(OH)D as the C3-epimer was 6.9% (cord), 16.3% (day 14), 22.4% (day 28) and 23.3% (discharge). A statistically significant correlation between 25(OH)D and C3-epi-25(OH)D can be demonstrated from birth. The severity of immaturity and IUGR did not affect the production of C3-epimers. In summer/autumn vs. winter/spring, the mean (SD) percentage of total 25(OH)D as the C3-epimer significantly differs only in maternal serum samples and umbilical cord samples (p value <0.001).
Conclusions
The production of C3-epi-25(OH)D is functional even in the most immature newborns, has fetal origins, and is largely dependent on circulating 25(OH)D. At the end of the first month of life, C3-epimers make up more than 20% of 25(OH)D.
Introduction
Hypovitaminosis D (<50 nmol/L) during pregnancy is generally frequent with a high prevalence [1], [2], [3], and has been associated with a number of health-related issues such as miscarriage [4], inadequate placental development [5], and pre-eclampsia [6]. In offspring, hypovitaminosis D during pregnancy has been associated with increased risk of autoimmune diseases [7, 8] and impaired neurocognitive development [9] in some. As fetal vitamin D stores depend exclusively on maternal vitamin D status, newborns from mothers with vitamin D deficiency are also at increased risk of hypovitaminosis D [3, 10]. After birth, vitamin D deficiency (<25 nmol/L) may lead to rickets and osteomalacia [11]. However, the interpretation of serum 25(OH)D levels in pregnant women and in infants is complicated by the presence of C3 epimers.
It has been discovered that vitamin D can alternatively be metabolized through a C3-epimerization pathway that parallels the standard metabolic pathway. This process was first described in neonatal human keratinocytes [12, 13]. In the C-3 epimerization pathway, the hydroxyl group at position C-3 of the A-ring is converted from the alpha to beta orientation. Both in vitro and in vivo studies support that all major vitamin D intermediate metabolites can be epimerized and that these epimers can be further metabolized by the same enzymes responsible for hydroxylation and oxidation events as in the standard pathway, thereby producing 3-epi-25(OH)D, 3-epi-1α,25(OH)2D, and 3-epi-24(R),25(OH)2D [12, 14]. The epimerase enzyme is present in the endoplasmic reticulum of a range of cell types/tissues including liver, bone, and skin, but not kidney. However, it is poorly characterized and surprisingly the gene responsible for encoding it has not yet been discovered [15]. The activity of epimerase enzyme appears to be unidirectional, and is independent of the presence of hydroxyl groups at positions C-1 or C-25. It is also independent of cytochrome P450 enzymes (CYP24, CYP27A1, and CYP27B1). It appears to occur in extrarenal tissues and to employ enzymes distinct from the classical hepatic enzyme systems involved in vitamin D metabolism [12].
While 25(OH)D concentrations appear to increase within the first year of life and stabilize after 1 year of age in term newborns, C3-epi-25(OH)D concentrations remain constant across the same period and drop abruptly around 12 months, continuing to fall gradually throughout childhood [16]. Clinical studies have shown the presence of C3-epi-25(OH)D in infant, pediatric, and adult populations. An analysis of 9 studies revealed that the weighted mean of C3-epi-25(OH)D level was 18.2 nmol/L in infants up to one year of age (accounting for 21.4% of total calcidiol). The weighted mean of C3-epi-25(OH)D in the pediatric population (1–18 years) and in adults was 2.5 and 4.3 nmol/L, respectively (5.9 and 6.1% of total calcidiol, respectively) [12]. The clinical significance of C3-epimers is still unknown and C3-epimers should not be included in the calculation of total 25(OH)D [17]. The separation of C3-epimers from metabolites 25(OH)D2 and 25(OH)D3 is very important for samples from infants under the age of 1 year, to avoid overestimation of circulating 25(OH)D [18, 19]. Commercially available immunoassay techniques are not able to differentiate C3-epimers [12, 20, 21]. It is known that LC–MS/MS is the most recommended method for analysis of vitamin D, especially in young infants, due to its ability to detect and quantify epimeric and non-epimeric metabolites separately in plasma [14, 22, 23].
There is a paucity of information about production of C3-epimers in preterm infants. The aim of this study is to determine levels of C3-epi-25(OH)D from birth to discharge from hospital stay in very low and extremely low birth weight infants. The secondary objective is to evaluate the possible influence of prematurity, intrauterine growth restriction (IUGR), and season of birth on the production of C-epimers in these preterm infants.
Materials and methods
Design
This was a single-center, prospective, observational cohort study.
Study population
The study was approved by the local Ethics Committee of The University Hospital Hradec Kralove (reference number 201510 S14P). The study in the Perinatology Center of the University Hospital Hradec Kralove included all newborns with birth weight under 1,500 g admitted to the neonatal intensive care unit in the period from January 2015 till December 2016. Informed written consent was obtained from every participating pregnant woman before preterm delivery, and afterwards both parents signed informed consent before inclusion of the newborn into the study. Exclusion criteria were: missing umbilical cord vitamin D; informed consent withheld; outborn neonates; major congenital anomalies. The state of prenatal supplementation of vitamin D in the mothers of included preterm newborns is not available.
Study protocol
Blood samples were taken from the mothers shortly before labor, and for the infants from the umbilical cord, at age 14 and 28 days, and at discharge. Blood withdrawals were made between 7 and 8 am. Blood was collected to a standard tube and was centrifuged for at least 30 min after blood draw. Serum was separated and was immediately taken to −80 °C and stored until 25(OH)D analysis. The maternal serum sample, the umbilical cord blood, and serum samples of preterm infants were analysed for 25(OH)D2 and 25(OH)D3 levels and their C3-epimers. 25-hydroxyvitamin D and C3-epimers were determined using a modified method of isotope-dilution liquid chromatography-tandem mass spectrometry ID-LC MS/MS [24]. In order to resolve the isomers by LC-MS/MS, we used a pentafluorophenyl stationary phase [12, 25]. The measurement was performed on an Agilent 6,490 Triple Quadrupole connected to an Agilent Infinity 1,290 liquid chromatography system. The assay quantitatively measured both major vitamin D metabolites 25(OH)D2 and 25(OH)D3 independently, as well as C3 epimers. The sample preparation included the precipitation of 100 µL serum samples using a mixture of acetonitrile, isopropanol and methanol enriched with the internal standards 25(OH)D2(6,19,19-d3) and 25(OH)D3(6,19,19-d3). After centrifugation the analytes in the supernatant were separated on an Ascentis Express F5 column (150 × 3 mm I.D., 2.7 µm particle size, Supelco) at flow rate 0.35 mL/min and run time 12 min. The mobile phase was composed of 23% ammonium formate buffer (5 mmol/L) with addition of 0.1% formic acid and 77% methanol. Fragments of vitamin D metabolites were detected by multiple reaction monitoring using the following mass-to-charge transitions: 401>383 for 25(OH)D3 and its C3 epimer; 404>386 for 25(OH)D3 (6,19,19-d3); 413>395 for 25(OH)D2 and its C3 epimer; 416>398 for 25(OH)D2 (6,19,19-d3). The ID-LC MS/MS method was validated and for the internal quality control were used samples of DEQAS and MassCheck 3-epi-25-OH-Vitamin D3/D2 and 25-OH-Vitamin D3/D2 Serum Control (Chromsystems). The validation parameters were confirmed using standard reference material SRM 972a from the National Institute of Standards and Technology (NIST). The range of applicability of the analytical method was enclosed within the lower limits of quantification (LLOQ) 3.12 nmol/L for 25(OH)D3 and its C3 epimer, and 3.03 nmol/L for 25(OH)D2 and its C3 epimer, and the upper limits of quantification (ULOQ). ULOQ was 625 nmol/L for 25(OH)D3 and its C3 epimer and 605 nmol/L for 25(OH)D2 and its C3 epimer. Parameters of precision expressed as intra- and inter-assay coefficients of variation were 1.2–10% for 25(OH)D3 and 2.2–6.6% for its C3 epimer; and 2.1–6.6% for 25(OH)D2 and 2.3–9.1% for its C3 epimer. The intra- and inter-assay accuracy expressed as recovery reached acceptable values of 87.6–106.4% for 25(OH)D3, and 89.3–104.5% for its C3 epimer; and 97.3–104.9% for 25(OH)D2, and 98.8–107% for its C3 epimer. For the purpose of this study, we define total 25(OH)D as the sum of 25(OH)D2 and 25(OH)D3 levels. Likewise, total C3-epi-25(OH)D is the sum of C3-epi-25(OH)D2 and C3-epi-25(OH)D3 levels.
Parenteral and enteral nutrition of the included newborns was administered according to the nutritional protocol in respect of the current nutrition recommendations for preterm newborns [26], [27], [28]. Enteral nutrition was started with 30 mL of milk/kg/day and increased by 30 mL of milk/kg/day. The children were fed with expressed breast milk fortified with Nestlé FM85 or preterm milk formula (Nutricia Nutrilon Nenatal – Danone, Paris, France; Nestlé PreBeba – Biessenhofen, Germany). The target amount of milk was 175–185 mL/kg/day with appropriate daily intake of calcium of 190–210 mg/kg/day and phosphorus 105–140 mg/kg/day. Enteral and parenteral vitamin D supply to all newborns included in the study was consistent with the ESPGHAN recommendation [27]. After birth the delivery of vitamin D was ensured via parenteral nutrition containing vitamin D2 (Vitalipid N Infant, Fresenius Kabi AB, Uppsala, Sweden). When the enteral dose of milk had reached more than 150 mL/kg/day, supplementation with 500 IU of vitamin D3 was begun (Vigantol gtt./drops/, Merck KGaA, Darmstadt, Germany). At the target dose of 175–185 mL/kg/day of fortified breast milk or preterm formula, the total daily vitamin D intake including extra supply was between 800 and 1000 IU.
Although there is no global consensus on optimal levels of serum 25(OH)D, by many authors a serum concentration <50 nmol/L indicates deficiency, and a serum concentration <25 nmol/L indicates severe deficiency for the entire paediatric population [1, 29], [30], [31], [32].
Outcome measures
The primary outcome was to determine levels of C3-epimers of vitamin D2 and vitamin D3 from birth to hospital discharge in very low birth weight infants (including newborns with intrauterine growth restriction).
The secondary objective was to evaluate the possible influence of preterm birth, intrauterine growth restriction (IUGR) and the season of birth on the production of C-epimers in these preterm infants. Season was assessed as comparison between summer/autumn vs. winter/spring.
Diagnosis of IUGR was made prenatally by obstetricians using a stage-based approach [33]. Newborns with IUGR were not considered when assessing the effect of preterm birth on C3-epimer levels.
Statistical analysis
Data were analyzed using NCSS 2019 Statistical Software (2019) (NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss). Qualitative data were presented as numbers and percentages while quantitative data were presented as mean with standard deviation (SD) or as median with interquartile ranges (IQR). The term “proportion of total 25(OH)D as the C3-epimer” is used to express the amount of C3-epi-25(OH)D as a percentage of the sum of 25-hydroxy vitamin D2 and D3 and C3-epi-25(OH)D2 and D3. Two-sample T-test or nonparametric Mann–Whitney, Kolmogorov-Smirnov tests were used to compare quantitative parameters between groups. Spearman’s Rank Correlation was used as a measure of linear correlation. The level of significance was p<0.05.
Results
Between January 2015 and December 2016 there were 183 newborns admitted to NICU with birth weight below 1,500 g. Of these, 127 met the entry criteria for this study. 17 newborns failed to complete the study (missing blood sample at discharge) (Figure 1 – flow diagram of the study group). The infants’ median (IQR) of gestational age was 29 weeks (27–31), and the median (IQR) of birth weight was 1,170 g (870–1,325). 36.2% (46) of preterm newborns had IUGR. All infants were of Caucasian origin. 56.7% (72) of the study group were boys, and 43.3% (55) were girls. The infants’ median (IQR) of age at discharge from hospital stay was 8 weeks (6–11). More demographic and clinical characteristics are summarized in Table 1.
Flow chart of the study group.
Demographic and clinical characteristics of study group (n=127).
| Characteristics | Values |
|---|---|
| Birth weight, g, median (IQR) | 1,170 (870–1,325) |
| Gestational week, median (IQR) | 29 (27–31) |
| Complete course of corticosteroids for a lung maturity, n (%) | 78 (61.4) |
| Male gender, n (%) | 72 (56.7) |
| Intrauterine growth restriction, n (%) | 46 (36.2) |
| Respiratory distress syndrome, n (%) | 98 (77.2) |
| nCPAP, days, median (IQR) | 16 (4–45) |
| Mechanical ventilation, days, median (IQR) | 0 (0–3) |
| Bronchopulmonary dysplasia (mild form), n (%) | 45 (35.4) |
| Bronchopulmonary dysplasia (moderate, severe form), n (%) | 10 (7.9) |
| Pneumothorax, n (%) | 4 (3.2) |
| Early onset sepsis, n (%) | 7 (5.5) |
| Late onset sepsis, n (%) | 19 (15.0) |
| Necrotizing enterocolitis | 12 (9.5) |
| Intraventricular haemorrhage grade II-IV., n (%) | 16 (12.6) |
| Periventricular leukomalacia, n (%) | 1 (0.8) |
| Retinopathy of prematurity grade I., n (%) | 1 (5) |
| Retinopathy of prematurity requiring invasive procedure, n (%) | 4 (3.2) |
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nCPAP, nasal continuous positive airway pressure.
The mean (±SD) total vitamin D [total 25(OH)D] level of all available maternal serum samples before delivery (n=94; 33 samples were missing) was 41.2 (±25.2) nmol/L. The mean (±SD) C3-epi-25(OH)D level was 1.4 (2.0) nmol/L. C3-epi-25(OH)D accounted for 3.2% in maternal serum samples.
The mean (±SD) total 25(OH)D levels in serum of cord, day 14, day 28, and discharge were 29.9 (17.8), 39.5 (17.2), 40.3 (12.7) and 49.2 (17.9) nmol/L respectively. The mean (±SD) total C3-epi-25(OH)D levels were 2.2 (2.9), 7.7 (5.5), 11.7 (7.6) and 14.9 (11.7) nmol/L respectively. The proportion of total 25(OH)D as the C3-epimer was 6.9% (cord), 16.3% (day 14), 22.4% (day 28), and 23.3% (discharge). Detailed postnatal development of vitamin D2, D3, total vitamin D and their C3-epimers are shown in Figures 2–4.
Postnatal development of the level of 25(OH)vitamin D2 and its C3-epimers. Postnatal rise in mean levels of vitamin D2 and its C3-epimers corresponds to the use of lipid emulsion in parenteral nutrition during first weeks after birth. 25(OH)D2 and C3-epi-25(OH)D2 almost disappeared until discharge.
Postnatal development of the mean level of 25(OH)vitamin D3 and its C3-epimers. Postnatal slight decrease in mean levels of vitamin D3 corresponds with parenteral nutrition (vitamin D2) and delayed initiation of enteral supplementation of vitamin D3. C3-epi-25(OH)D3 gradually increased from birth to discharge.
Postnatal development of the mean level of total 25(OH)D and its C3-epimers. 25(OH)D2 + 25(OH)D3=Total 25(OH)D. The mean levels of total vitamin D and its C3-epimers gradually increased from birth to discharge.
A statistically significant correlation between 25(OH)D and C3-epi-25(OH)D can be demonstrated from birth with correlation coefficient for 25(OH)D rho=0.60 (p= <0.001, n=440). In preterm neonates the increasing vitamin D level is accompanied by an increase in production of C3-epimers (Figure 5).
The correlation between total 25(OH)D and total C3-epi-25(OH)D in preterm newborns. The point graph shows the measurements of total 25(0H)D (axis x) in relation to values of C3-epi-25(OH)D (axis y) from the birth to discharge from hospital stay. A statistically significant correlation between 25-hydroxyvitamin D and its C3-epimers can be demonstrated from birth with correlation coefficient for 25(OH)D rho=0.60 (p= <0.001, n=440). In preterm neonates the increasing vitamin D level is accompanied by the increase production of C3-epimers.
Production of C3-epimers did not differ between preterm newborns grouped according to PMA (PMA≤28 vs. PMA>28) (Table 2).
Vitamin D status according to the season of birth, PMA, and intrauterine growth.
| Vitamin D levels nmol/L; MED (IQR) | Summer/autumn | Winter/spring | p-Value | ≤28th PMA | >28th PMA | p-Value | IUGR | Non-IUGR | p-Value |
|---|---|---|---|---|---|---|---|---|---|
| Total 25(OH)D | |||||||||
| – Mother | 58 (32–76) (n=39) |
26 (21–38) (n=55) |
<0.001 | 47 (32–76) (n=28) |
33 (22–51) (n=30) |
0.242 | 30 (22–37) (n=36) |
39 (24–66) (n=58) |
0.027 |
| – Cord blood | 43 (27–52) (n=59) |
17 (14–27) (n=68) |
<0.001 | 32 (21–50) (n=35) |
24 (15–44) (n=46) |
0.069 | 21 (14–34) (n=46) |
29 (17–48) (n=81) |
0.093 |
| – Day 14 | 46 (36–54) (n=49) |
31 (25–39) (n=54) |
<0.001 | 44 (29–53) (n=24) |
40 (32–48) (n=39) |
0.610 | 31 (24–39) (n=40) |
41 (31–51) (n=63) |
0.001 |
| – Day 28 | 43 (36–52) (n=46) |
37 (30–45) (n=55) |
0.037 | 43 (35–54) (n=24) |
41 (32–48) (n=36) |
0.337 | 38 (31–44) (n=41) |
42 (33–49) (n=60) |
0.086 |
| – Discharge | 48 (38–60) (n=52) |
45 (37–60) (n=58) |
0.749 | 55 (35–69) (n=25) |
48 (38–57) (n=44) |
0.225 | 44 (37–56) (n=41) |
48 (37–63) (n=69) |
0.357 |
|
|
|||||||||
| Total C3-epi-25(OH)D | |||||||||
|
|
|||||||||
| – Mother | 2 (0–3) (n=40) |
0 (0–1) (n=56) |
<0.001 | 1 (0–3) (n=28) |
1 (0–2) (n=30) |
0.549 | 0 (0–1) (n=36) |
1 (0–3) (n=58) |
0.059 |
| – Cord blood | 3 (1–5) (n=59) |
1 (0–1) (n=68) |
<0.001 | 2 (1–5) (n=35) |
1 (0–3) (n=46) |
0.070 | 1 (0–2) (n=46) |
1 (0–4) (n=81) |
0.197 |
| – Day 14 | 9 (5–16) (n=49) |
6 (3–8) (n=54) |
<0.001 | 11 (5–15) (n=24) |
7 (6–9) (n=39) |
0.053 | 5 (3–8) (n=40) |
8 (5–12) (n=63) |
0.016 |
| – Day 28 | 13 (5–20) (n=46) |
10 (7–13) (n=55) |
0.044 | 14 (10–19) (n=24) |
10 (7–13) (n=36) |
0.112 | 9 (6–13) (n=41) |
12 (7–16) (n=60) |
0.098 |
| – Discharge | 13 (8–18) (n=52) |
12 (6–19) (n=58) |
0.860 | 14 (8–24) (n=25) |
11 (8–20) (n=44) |
0.565 | 13 (6–16) (n=41) |
12 (8–22) (n=69) |
0.406 |
|
|
|||||||||
| % of total 25(OH)D as C3 epimers mean (SD) | |||||||||
|
|
|||||||||
| – Mother | 3.7 (2.6) (n=39) |
1.7 (3.4) (n=55) |
<0.001 | 3.2 (3.6) (n=28) |
2.5 (2.8) (n=30) |
0.679 | 1.7 (2.2) (n=36) |
2.9 (3.2) (n=58) |
0.315 |
| – Cord blood | 8 (6.8) (n=59) |
4.5 (6.9) (n=68) |
<0.001 | 6.1 (4.6) (n=35) |
5.3 (6.0) (n=46) |
0.122 | 5.1 (5.6) (n=46) |
5.6 (5.4) (n=81) |
0.508 |
| – Day 14 | 22.0 (12.7) (n=49) |
17.7 (11.7) (n=54) |
0.127 | 17.2 (8.5) (n=24) |
16.0 (6.6) (n=39) |
0.208 | 14.3 (9.6) (n=40) |
16.5 (7.3) (n=63) |
0.077 |
| – Day 28 | 29.4 (16.3) (n=46) |
26.3 (10.6) (n=55) |
0.270 | 22.4 (9.9) (n=24) |
20.8 (6.5) (n=36) |
0.069 | 20.0 (8.9) (n=41) |
21.5 (8.0) (n=60) |
0.319 |
| – Discharge | 31.8 (19.5) (n=52) |
28.0 (19.5) (n=58) |
0.276 | 22.4 (10.8) (n=25) |
22.1 (10.5) (n=44) |
0.920 | 20.1 (10.7) (n=41) |
22.2 (10.6) (n=69) |
0.440 |
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PMA, postmenstrual age; IUGR, intrauterine growth restriction; p-values in bold indicate statistical significance (p<0.05).
The median (IQR) 25(OH)D level of maternal serum samples in pregnancies complicated by IUGR was 30 nmol/L (22–37). For eutrophic fetuses, the median (IQR) 25(OH)D level of maternal serum samples was 39 nmol/L (24–66). In the preterm newborns with IUGR, the median (IQR) total 25(OH)D level in cord samples and serum samples at day 14 were 21 (14–34) and 31 (24–39) nmol/L respectively. The corresponding values in the non-IUGR group were 29 (17–48) and 41 (31–51) nmol/L respectively. Statistical significance is shown in Table 2. The percentage contribution of C3-epimers did not differ statistically between IUGR and non-IUGR newborns at all monitored points (Table 2).
In summer/autumn the median (IQR) of total 25(OH)D and of total C3-epi-25(OH)D in maternal serum samples and in serum samples of preterm newborns up to one month was significantly higher than in winter/spring (p= <0.001; for details see Table 2). However, in summer/autumn vs. winter/spring the mean (SD) proportion of total 25(OH)D as the C3-epimer significantly differs only in maternal serum samples and umbilical cord samples (p value <0.001; see Table 2).
Discussion
We found significantly increasing C3-epi-25(OH)D levels and proportion of total 25(OH)D as the C3-epimer in preterm infants from birth (6.9%) to discharge (22.3%). The results are in agreement with several previous studies involving C3-epimer levels in term and preterm neonates. In maternal serum samples and cord blood samples of term newborns, the proportion of total 25(OH)D as the C3-epimer was 2.2–6.5% and 1.5–13.2% respectively [17, 19, 34, 35]. To the best of our knowledge this study has examined the largest number of preterm newborns (n=127) concerning levels of C3-epi-25(OH)D. Previously, Hanson et al. [36] reported that in preterm infants C3-epi-25(OH)D increased significantly over time, and the percentage of total 25(OH)D concentration that was 3-epi-25(OH)D also increased significantly (7.2 vs. 29.7%, p<0.001 for cord blood vs. 8 weeks of life). In a small cohort of 10 preterm newborns, Fatani et al. [37] found an even higher C3-epimer level as percentage of 25(OH)D (32.2%) in the first week of life. Finally, Ooms et al. [38] reported that in 15 preterm infants (aged<34 week), MED (IQR) of the relative contribution (%) of C3-epimers to total 25(OH)D was 33 (18–45) from the second week to 3 months of age. In addition, in 163 infants (full-term and preterm together), from 3 months of life, a sharp decline in plasma C3-epimer level is seen, thereafter remaining at a relatively constant level up to age 2 years [mean (range) 8 (1–62) nmol/L, <10% of 25(OH)D].
Recently we also discovered that preterm infants produce C3-epimers of 25(OH)D2, especially during the first two weeks after birth. The source of vitamin D2 is from Vitalipid, a solution for parenteral nutrition used in these preterm newborns. As infants reached total enteral nutrition with enteral vitamin D3 supply, vitamin D2 disappeared from serum samples in infants.
The origin of C3-epimers has been the subject of speculation. In addition, it has been published that vitamin D supplementation increases the C3-epimer level in maternal and cord blood serum samples [34, 39]. Yazdanpanah et al. examined liquid vitamin D3 supplements that are widely prescribed to breast-fed infants. As these supplements were found to possess almost exclusively cholecalciferol, they are unlikely to be an exogenous source of C3-epi-25(OH)D [16]. Thus, endogenous factors must contribute predominantly to the increased production of C3-epi-25(OH)D in newborns. According to our findings of higher C3-epimer concentration in cord blood than in maternal serum samples, and because the majority of production of C3-epimers occurs during the first 4 weeks of life, we conclude, in agreement with previous authors, that it is made endogenously in fetuses/neonates.
Previously we published that umbilical cord 25(OH)D levels in preterm infants were strongly correlated with maternal serum levels of 25(OH)D [3]. In this report we showed that the increasing vitamin D level is accompanied by an increased production of C3-epimers. This result is comparable to reports by other authors, and also as previously reported, total concentrations of 25(OH)D and C3-epimer of 25(OH)D correlated positively in adults and infants [17, 19, 35, 38], [39], [40], [41]. Epimer levels increased linearly with increasing 25(OH)D levels after supplementation in adults [40, 41]. It has not yet been revealed why production of C3-epimers is beneficial for infants less than one year old. According to an animal study, vitamin D epimers have positive effects on growth and bone mineral density. The authors concluded that C3-epimers clearly have biological activity [42].
It has been speculated by Ooms et al. [38] that prematurity could be a factor influencing the formation of C3-epimers. They based their claim on a higher relative % contribution of C3-epimers [MED (IQR) 33 (18–45)] in preterm infants (aged<34 week) compared with that of full-term infants [MED (IQR) 8 (6–12)] (p= <0.001). According to this hepatic immaturity hypothesis, it would be reasonable to expect that more premature infants have higher production of C3-epimers due to more immature metabolic pathways. Our results do not support this conjecture. Production of C3-epimers did not differ between groups of preterm newborns according to PMA (PMA≤28 vs. PMA>28).
In preterm newborns with IUGR, the median (IQR) total 25(OH)D level from cord blood samples up to serum samples of day 28 was lower than in preterm newborns without IUGR. It is related to the significantly lower maternal levels of vitamin D for preterm newborns with IUGR than maternal levels of vitamin D for eutrophic (non-IUGR) preterm newborns. Absolute C3-epimer levels were also lower in the IUGR infants. However, the percentage contribution of C3-epimers did not differ statistically between IUGR and non-IUGR newborns. The postnatal production of vitamin D epimers is not affected by development of IUGR during pregnancy. Production is influenced only by vitamin D level, which is consistent with our previous findings.
According to expectation [43], in summer/spring season we found significantly higher levels in preterm infants of total 25(OH)D and total C3-epi-25(OH)D from birth to the age of one month. This is undoubtedly a result of higher vitamin D levels in mothers during pregnancy in the summer/spring season. A strong correlation has previously been reported between maternal and umbilical cord 25(OH)D levels [3], the same as the correlation between 25(OH)D and C3-epimers levels presented in this study. The proportion of total 25(OH)D as the C3-epimer, when comparing summer/autumn vs. winter/spring, differed significantly only in maternal serum samples and in umbilical cord samples. From the second week of life, there was no difference. It is reasonable to assume that after interruption of exposure to sunlight, the production of C3-epimers in preterm infants is largely dependent on circulating 25(OH)D. Mydtskov et al. reported that the C3-epimers were independently correlated to season and considered it as a direct effect of sunlight on the skin [39]. It is well known that human keratinocytes are the only cells in the body capable of vitamin D synthesis, with the whole pathway from 7-dehydrocholesterol to 1,25(OH2)D, and the C3 epimerization pathway first described in human keratinocytes [39]. In recent animal/human study, vitamin D C3-epimer levels are proportionally higher with oral vitamin D supplementation compared to ultraviolet irradiation of skin in mice, but not in humans [44].
Our study has some limitations. We did not assess maternal socio-economic status, dietary vitamin D intake, and serum 25(OH)D3 levels during pregnancy. In addition, we did not evaluate cord blood 25(OH)D3 levels by chemiluminescence immunoassay to better quantify possible vitamin D status misclassification due to inability to separate C3-epimers.
However, our study has several strengths. In particular the enrollment of 127 preterm infants only, with birth weight below 1500 g, has improved understanding of the C3-epimer metabolic pathway. Furthermore, vitamin D status in all samples was assayed by LC-MS-MS, which represents the gold standard for quantification and separation of C3-epimers.
Conclusions
The production of C3-epi-25(OH)D is functional even in the most immature newborns, has fetal origins, and is largely determined by levels of circulating 25(OH)D. At the end of the first month of life, they make up more than 20% of 25(OH)D. The severity of immaturity and IUGR did not affect the production of C3-epimers. In summer/spring season we found significantly higher levels in preterm infants of total 25(OH)D and total C3-epi-25(OH)D from birth to the age of one month. However, from the second week of life in preterm infants the proportion of total 25(OH)D as the C3-epimer did not differ between summer/autumn and winter/spring. The aim of future studies must be to elucidate the clinical significance of higher levels of C3-epimers in newborns compared to older children and adults.
Funding source: Ministerstvo ZdravotnictvÃ- Ceské Republiky
Award Identifier / Grant number: MH CZ – DRO (UHHK, 00179906)
Funding source: Univerzita Karlova v Praze
Award Identifier / Grant number: Specific University Research Programme (SVV 260396
Acknowledgments
The authors are grateful to Ian McColl MD, PhD for assistance with the manuscript and to RNDr. Eva Cermakova for statistical analysis.
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Research funding: This work was supported by University Hospital Hradec Kralove, Czech Republic project MH CZ – DRO (UHHK, 00179906). The study was supported by the Specific University Research Programme (SVV 260396) from Charles University, Faculty of Medicine in Hradec Kralove.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: The authors have no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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Informed consent: Informed written consent was obtained from every participating pregnant woman before preterm delivery, and afterwards both parents signed informed consent before inclusion of the newborn into the study.
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Ethical approval: The study was approved by the local Ethics Committee of The University Hospital Hradec Kralove (reference number 201510 S14P).
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