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Lucie Levaillant, Agnès Linglart, Vincent Gajdos, Alexandra Benachi, Jean-Claude Souberbielle, Reference Values for Serum Calcium in Neonates Should Be Established in a Population of Vitamin D–Replete Subjects, The Journal of Clinical Endocrinology & Metabolism, Volume 110, Issue 1, January 2025, Pages e68–e71, https://doi.org/10.1210/clinem/dgae167
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Abstract
Serum calcium is frequently measured during the neonatal period, and it is known to be influenced by the vitamin D status.
We hypothesized that the 25-hydroxyvitamin D (25OHD) concentration may influence the lower limit of the serum calcium normal range in neonates.
We included in our prospective cohort study 1002 mother-newborn pair recruited from April 2012 to July 2014, in 2 centers located in the neighborhoods of Paris, France, whose serum calcium was measured at 3 days of life. We established, after exclusion of outliers, a 95% CI for serum calcium (i) in our whole population of 1002 neonates; (ii) in neonates with a cord blood 25OHD concentration ≥ 30 nmol/L; and (iii) in those with a 25OHD ≥ 50 nmol/L.
The mean serum total calcium was 2.46 ± 0.13 nmol/L (95% CI: 2.19-2.72 mmol/L), 2.47 ± 0.25 mmol/L (95% CI: 2.22-2.72 mmol/L), and 2.50 ± 0.25 mmol/L (95% CI: 2.25-2.75 mmol/L) in the whole group, in the 514 neonates with 25OHD ≥ 30 nmol/L, and in the 202 neonates with 25OHD ≥ 50 nmol/L respectively. The lower limit of the 95% range was significantly higher in neonates with 25 OHD ≥ 30 nmol/L (P < 0.05) and ≥ 50 nmol/L (P < .001) than in the entire cohort.
We show that the lower limit of the normal serum calcium range is higher in groups with a higher 25OHD than in unselected subjects. We propose that the reference range for serum calcium in neonates is 2.25 to 2.75 mmol/L.
Serum calcium is frequently measured during the neonatal period, even in newborns who have no specific symptoms of hypo- or hypercalcemia. This is done, for example, in case of prematurity, low birth weight, or gestational diabetes mellitus (1). Establishing a specific normal range for serum calcium in neonates is thus of paramount importance to avoid misdiagnosis of hypo- or hypercalcemia, which could lead to unnecessary further examinations and follow-up. We recently published the results of serum total calcium range in more than 1000 healthy neonates at 3 days of life (2). As the distribution of serum calcium was not Gaussian in this population, we used the nonparametric method to calculate the normal range which varied from 2.06 mmol/L to 2.73 mmol/L (2.5 and 97.5 percentiles), with a mean of 2.45 mmol/L and a median of 2.45 mmol/L. However, in this study, we included all the cohort and did not take the vitamin D status into account, whereas it is well-known to influence both calcium and phosphorus homeostasis. Hypovitaminosis D, defined by a serum 25-hydroxyvitamin D (25OHD) concentration < 50 nmol/L, is common in French pregnant women (3, 4) and children (5). The binding of calcitriol, the active metabolite of 25OHD, to the vitamin D receptor elicits the expression of calcium transporters in the gut, thus allowing the absorption of calcium. Insufficient levels of 25OHD lead to reduced calcium absorption and hypocalcemia (6, 7). Several authors have reported lower serum calcium concentrations in vitamin D–deficient neonates in comparison to those with normal vitamin D concentrations (6). As vitamin D deficiency is an unwanted condition with possible harmful consequences such as the increased risk of hypocalcemia, it is relevant to exclude vitamin D–deficient subjects from a population of neonates recruited to establish reference values for serum calcium. This has previously been proposed by Roizen et al, who reported data from a large cohort of children of various ages showing that the lower limit of normal of serum calcium is higher in their vitamin D–repleted children than in those with vitamin D insufficiency and deficiency (8).
According to these results, we therefore hypothesized that the 25OHD status may influence the serum total calcium normal range in our cohort of 1002 healthy neonates. To test this hypothesis, we used the same protocol to establish a 95% CI for serum calcium: (i) in the whole population of 1002 neonates, that is, without any selection according to their 25OHD serum level; (ii) in neonates with a serum 25OHD concentration ≥ 30 nmol/L, a threshold that defines severe vitamin D deficiency; and (iii) in neonates with a serum 25OHD concentration ≥ 50 nmol/L, which is the definition of vitamin D sufficiency.
Methods
Study Design
This study is an ancillary study of the prospective observational FEPED cohort of pregnant women, which was designed to evaluate the relationship between vitamin D status in pregnancy and the risk for preeclampsia (9). Written informed consent was obtained from each woman before inclusion in the study. The study protocol was conducted in accordance with the Declaration of Helsinki and was reviewed and approved by Assistance Publique-Hôpitaux de Paris (AP-HP) ethics committee (approval number 2011/13NICB). The study was sponsored by the AP-HP and was funded by a grant from the Programme Hospitalier de Recherche Publique national 2010 (Ministry of Health-AOM10113). It is registered with the ClinicalTrials.gov identifier NCT01648842.
Briefly, women were included during the first consultation following the diagnosis of pregnancy if they were at between 10 and 14 weeks post last menstrual period (LMP) of a singleton pregnancy. Women were not included in the case of hypercalcemia (serum calcium over 2.65 mmol/L) or any other calcium-phosphorus imbalance. Further documentation can be found in Courbebaisse et al (3), Benachi et al (9), and our recent report (2).
Participants and Data Collection
We used the samples obtained during the FEPED study in 2 recruiting centers located in Paris neighborhoods (AP-HP, Antoine Béclère and Bicêtre, Saclay University Hospitals) that included mother-newborn pairs from April 2012 to July 2014. Mothers received a prescription for an oral bolus vitamin D dose (100 000 IU vitamin D3) at the seventh month of pregnancy according to current French recommendations (10). They were asked at 34 weeks post LMP whether they received or not the vial. Pairs were included if the delivery occurred at 36 weeks post LMP and thereafter.
Cord blood for the measurement of 25OHD was available for all cases included in the present study. In addition to the data collected during the pregnancies, a blood sample of 250 μL was collected by venipuncture from the newborns in the maternity hospital at 3 days of life for serum calcium measurement, at the time of neonatal screening.
All blood samples were centrifuged within 1 hour of sampling. Serum total calcium was measured locally by a colorimetric assay with the Ortho VITROS 5.1 FS automated chemistry system (Ortho Clinical Diagnostics). Serum aliquots were stored locally at −20 °C and transferred monthly to the department of Physiology of Necker University hospital (Paris, France) where 25OHD levels were measured in duplicate by radioimmunoassay after pretreatment with acetonitrile to allow the denaturation of any protein that may interfere in the assay (Diasorin, Stillwater, MN, USA) (11). This method was based on an antibody that was cospecific for 25OHD2 and 25OHD3 and did not cross-react with the 25OHD C3-epimer which is largely present infants. In our hands, intra- and interassay CVs were < 10% over a large range of concentrations (2.5-100 ng/mL). When the measurements were performed, this assay had been recently calibrated against the NIST reference material. Quality control was ensured through the participation of this laboratory in the DEQAS quality assurance program and in the French national control of quality.
Statistical Analysis
Statistics were performed using R software (Vienna, Austria) (12). Results for quantitative variables were expressed as mean ± SD or median (interquartile range [IQR]) according to the Gaussian or non-Gaussian distribution of the variable, and as number of patients and percentages for qualitative variables. Correlation between cord blood 25OHD concentration and serum calcium at 3 days of life was assessed by simple regression. To determine the 95% serum total calcium range in 3 groups of neonates that we considered according to their 25OHD level: (i) all subjects; (ii) 25OHD ≥ 30 nmol/L; and (iii) 25OHD concentration ≥ 50 nmol/L (using the Horn method) (13). We first detected outliers, defined as serum calcium concentrations below (quartile 1 [Q1] − 1.5 × IQR) and above (quartile 3 [Q3] + 1.5 × IQR), after log transformation of the raw values. We then tested the normality of the remaining values (Shapiro-Wilk normality test), and we calculated the mean ± 2 SD to define the 95% range in case of normal distribution or we used the nonparametric method in case of non-Gaussian distribution. A P value < .05 was considered significant. The Welch Two Sample t test was used to compare 95% intervals.
Results
Among the whole group of 1002 cases, 911 (90.9%) of the mothers ingested the 100 000 IU vitamin D3 dose around the seventh month of pregnancy. Serum 25OHD concentrations were higher in cord blood from neonates of supplemented mothers than in nonsupplemented ones, 37.4 nmol/L (IQR, 25.0) and 34.9 nmol/L (IQR, 25.0), respectively (P = .022). Serum calcium at 3 days of life was significantly associated with neonatal cord blood 25OHD concentrations (r = 0.19; P < .001). After excluding 39 outliers (6 high values and 33 low values) mean and median serum calcium were 2.46 ± 0.13 nmol/L and 2.46 nmol/L (IQR 0.19) respectively. As the distribution of the remaining 963 serum calcium was non-Gaussian, we used the nonparametric method to determine the 95% CI of serum calcium, which was 2.19 to 2.72 mmol/L (2.5-97.5th percentile).
We found that 532 neonates (53.1% of the whole group) had a cord blood 25OHD level ≥ 30 nmol/L. After excluding 18 outliers (14 high values and 4 low value) and verifying that the distribution of the remaining 514 calcium measures was normal, the mean ± 2 SD serum total calcium measured at 3 days of life was 2.47 ± 0.25 mmol/L, corresponding to a 95% interval of 2.22 to 2.72 mmol/L.
We found that 208 neonates (20.8% of the whole group) had a cord blood 25OHD level ≥ 50 nmol/L. After excluding 6 outliers (5 high values and 1 low value) and verifying that the distribution of the remaining 202 serum calcium was normal, the mean ± 2 SD serum total calcium measured at 3 days of life was 2.50 ± 0.25 mmol/L, corresponding to a 95% interval of 2.25 to 2.75 mmol/L.
The above results are summarized in Table 1. According to the Welch Two Sample t test, the lower limit of the 95% range was significantly higher in neonates with cord blood ≥ 30 nmol/L (P < .05), and in those with 25OHD ≥ 50 nmol/L (P < .001) than in the entire cohort.
95% CI for serum calcium (2.5-97.5 percentile) in 3 groups of neonates selected according to their vitamin D status; the lower limits of these ranges increase with increasing vitamin D status of the group
| . | n . | Serum calcium after exclusion of outliers: Mean ± SD; (IQR) . | 95% CI for serum calcium (2.5-97.5 percentile) . |
|---|---|---|---|
| Whole cohort of neonates unselected according to their vitamin D status | 963 | a2.46 ± 0.13 nmol/L; (0.19) | 2.19-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 30 nmol/L | 532 | a2.47 ± 0.13 nmol/L; (0.18) | 2.22-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 50 nmol/L | 208 | a2.50 ± 0.13 nmol/L; (0.18) | 2.25-2.75 mmol/L |
| . | n . | Serum calcium after exclusion of outliers: Mean ± SD; (IQR) . | 95% CI for serum calcium (2.5-97.5 percentile) . |
|---|---|---|---|
| Whole cohort of neonates unselected according to their vitamin D status | 963 | a2.46 ± 0.13 nmol/L; (0.19) | 2.19-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 30 nmol/L | 532 | a2.47 ± 0.13 nmol/L; (0.18) | 2.22-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 50 nmol/L | 208 | a2.50 ± 0.13 nmol/L; (0.18) | 2.25-2.75 mmol/L |
aIn these cases, median and mean were the same.
95% CI for serum calcium (2.5-97.5 percentile) in 3 groups of neonates selected according to their vitamin D status; the lower limits of these ranges increase with increasing vitamin D status of the group
| . | n . | Serum calcium after exclusion of outliers: Mean ± SD; (IQR) . | 95% CI for serum calcium (2.5-97.5 percentile) . |
|---|---|---|---|
| Whole cohort of neonates unselected according to their vitamin D status | 963 | a2.46 ± 0.13 nmol/L; (0.19) | 2.19-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 30 nmol/L | 532 | a2.47 ± 0.13 nmol/L; (0.18) | 2.22-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 50 nmol/L | 208 | a2.50 ± 0.13 nmol/L; (0.18) | 2.25-2.75 mmol/L |
| . | n . | Serum calcium after exclusion of outliers: Mean ± SD; (IQR) . | 95% CI for serum calcium (2.5-97.5 percentile) . |
|---|---|---|---|
| Whole cohort of neonates unselected according to their vitamin D status | 963 | a2.46 ± 0.13 nmol/L; (0.19) | 2.19-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 30 nmol/L | 532 | a2.47 ± 0.13 nmol/L; (0.18) | 2.22-2.72 mmol/L |
| Neonates with a serum 25OHD ≥ 50 nmol/L | 208 | a2.50 ± 0.13 nmol/L; (0.18) | 2.25-2.75 mmol/L |
aIn these cases, median and mean were the same.
Discussion
In the present study, we assessed ranges of serum calcium concentration in groups of healthy neonates at 3 days of life selected according to their vitamin D status. We show that the lower limit of these ranges is dependent on the cord blood 25OHD level of the neonates and is higher in groups with a higher 25OHD concentration than in unselected subjects. This supports the idea to include only vitamin D–repleted neonates in a population recruited to establish reference values for serum calcium.
Like in the present study, several authors have reported a significant correlation between serum 25OHD and calcium levels in neonates and/or a lower serum calcium in neonates with low 25OHD concentration (14-18). Roizen et al tested whether reference intervals for serum total calcium in a pediatric population (0 to 19 years) were different in vitamin D–replete children and in children unselected for their vitamin D status (8). Like in the neonates in the present study, they found that at all ages, the lower limit, but not the higher limit, of the reference intervals for normal subjects with vitamin D sufficiency, that is, 25OHD levels ≥ 50 nmol/L, were significantly higher than those for unselected subjects. They found a reference interval for serum calcium of 2.0 to 2.8 mmol/L in their group of children aged 0 to 90 days with a 25OHD serum concentration between 50 and 200 nmol/L. It is worth noting that the lower limit of this range is clearly lower than the one we characterized, that is, 2.25 mmol/L, in our cohort of neonates with a 25OHD ≥ 50 nmol/L. We suggest several possible explanations for this finding. First, the cohort of Roizen et al was made of hospitalized children, many of them being acutely ill. Although none of their patients were known to have an anomaly of calcium and phosphorus homeostasis, it remains possible that some patients had asymptomatic moderate hypocalcemia. Second, contrary to Roizen et al, we identified and excluded a certain number of outliers from our groups of subjects. This had a significant influence on the lower limit of the ranges of serum calcium, which was 2.06 mmol/L in the whole cohort of 1002 unselected neonates (2) and 2.19 mmol/L in the selected cohort of 963 neonates after exclusion of 39 outliers. Excluding outliers from a population recruited to establish the reference values of a given biological parameter may be critical and has direct consequences on decision taking, and thus patient management. Third, Roizen et al did not focus on the first few days after birth, a period at high risk of disturbances in calcium homeostasis due to the physiological changes occurring at birth, which includes the brutal interruption of the calcium flux from the cord blood and the need of a reliable digestive absorption of calcium. Finally, Roizen et al found that children with a 25OHD concentration above 75 nmol/L had a higher serum calcium than those with a 25OHD level between 50 and 75 nmol/L, suggesting that 75 nmol/L was a more relevant threshold than 50 nmol/L to define vitamin D–replete children. Our analysis was limited by the number of neonates with a cord blood 25OHD level ≥ 75 nmol/L (n = 47; 0.05% of the entire cohort) which did not allow us to test this hypothesis.
Some limitations of this study are that we only measured serum calcium, without taking into account factors that may interfere with this calcium measurement (for example, pH, protein concentration, or venipuncture technique) (19, 20). We were also unable to measure free 25OHD or other parameters of calcium metabolism due to the small volume of the blood samples.
In conclusion, we propose to include only vitamin D–replete neonates in a population intended to establish reference values for the serum calcium concentration. This has a strong influence on the reference range, leading to a narrowing of the reference interval, especially by increasing the lower value. Given our findings, we suggest that the reference range for serum calcium in neonates is 2.50 ± 0.25 mmol/L (95% interval of 2.25 to 2.75 mmol/L).
Acknowledgments
We would like to thank A. Bellino and M. Delattre for coordinating the study. We are grateful to the doctors, midwives, and nurses for management of patients. We would also like to thank the “Centre de Ressources Biologiques, Centre Hospitalier Intercommunal de Créteil”, for the management and storage of biospecimens.
Disclosures
The authors have no conflicts of interest relevant to this article to disclose.
Data Availability
The data that support the findings of this study are available from the corresponding author, upon reasonable request.
Clinical Trial Information
ClinicalTrials.gov registration no. NCT01648842.
