Phosphate concentrations and modifying factors in healthy children from 12 to 24 months of age

Precis: This study determined physiological phosphate concentrations and modifiers in 525 healthy children aged 12 and 24 months. Abstract Context: Phosphate homeostasis and its modifiers in early childhood are inadequately characterized. Objective: To determine physiological plasma phosphate concentration and modifying factors in healthy infants at 12 to 24 months of age. Design: This study included 525 healthy infants (53% girls), who participated in a randomized vitamin D intervention (VIDI) trial and received daily vitamin D 3 supplementation of either 10 or 30 μg from age two weeks to 24 months. Biochemical parameters were measured at 12 and 24 months. Dietary phosphate intake was determined at 12 months. Main Outcome Measures: Plasma phosphate concentrations at 12 and 24 months of age. Results: Mean (SD) phosphate concentration decreased from 12 months (1.9±0.15 mmol/L) to 24 months (1.6±0.17 mmol/L) of age (p<0.001 for repeated measurements). When adjusted by covariates, such as body size, creatinine, 25OHD, intact and C-terminal FGF23, mean plasma phosphate was higher in boys than girls during follow-up (p=0.019). Phosphate concentrations were similar in the vitamin D intervention groups (p>0.472 for all). Plasma iron was associated positively with plasma phosphate at both time points (B, 0.006 and 0.005, 95% CI 0.004 to 0.009 and 0.002 to 0.008, p<0.001 at both time points, respectively). At 24 months of age, the main modifier of phosphate concentration was plasma creatinine (B, 0.007, 95% CI 0.003 to 0.011, p<0.001). Conclusion: Plasma phosphate concentration decreased from age 12 to 24 months. In infants and toddlers, the strongest plasma phosphate modifiers were sex, iron, and creatinine, whereas vitamin D supplementation did not modify phosphate concentrations. The temporal changes and factors modifying phosphate concentrations in children 12 to 24 months of age have not been previously studied. Our study examined plasma phosphate concentrations in a large cohort of healthy young Finnish children at 12 and 24 months and studied modifying factors for phosphate concentrations at these time points. potential molecular mechanisms behind these observations. One possible explanation of higher phosphate concentrations at 12 can be age-dependent changes in expression levels of sodium-phosphate co-transporters. The expression of genes and proteins responsible for phosphate absorption decreases with age, and this could also contribute to lower phosphate levels in 24-month-old children compared with 12-month-old children with and C-terminal FGF23 showed variable association with phosphate, depending on the time and As FGF23 is a phosphate modifying factor, it seems that iron regulates phosphate metabolism through FGF23. Our study confirms the existence of the link between iron and phosphate metabolism already in early childhood. Further research on specific mechanisms of this regulation is needed. Our study presents normative phosphate concentrations in healthy children aged 12 to 24 months. The observed decrease in phosphate concentrations from 12 to 24 months is a novel finding. Iron at both time points, and creatinine at 24 months, were the key modifiers associated positively with phosphate concentrations. Vitamin D supplementation did not modify phosphate concentrations, but sex and intact and C-terminal FGF23 may impact phosphate concentrations. The reported normative data should prove useful for early detection of children with hypo- or hyperphosphatemia. Further research on phosphate metabolism in early is needed.


Introduction
Although only 0.8% of the total human body weight consists of phosphate (1), it is one of the most abundant minerals in the body, and necessary for many different vital processes (2).
Phosphate participates e.g. in energy metabolism, synthesis of DNA and RNA and regulation of proteins by phosphorylation (3). Phosphate also plays a crucial role in mineral metabolism and about 85% of phosphate is present as hydroxyapatite in bone and teeth (4). Abnormalities in phosphate metabolism may cause rickets, osteomalacia or soft tissue mineralization (5).
Generally, phosphate concentrations are higher in healthy infants and children than in adults (13)(14)(15). Published normative data, especially for young children, have thus far been based largely on small cohort studies, often including premature infants (16-18). Published reference values for phosphate concentration vary from 1.54-2.72 mmol/L for infants (aged A c c e p t e d M a n u s c r i p t 5 15 days to 1 year), 1.38-2.19 mmol/L for children (aged 1 to 5 years) and 1.33-1.92 mmol/L for older children and adolescents (aged 5 to 13 years) (13).
The temporal changes and factors modifying phosphate concentrations in children 12 to 24 months of age have not been previously studied. Our study examined plasma phosphate concentrations in a large cohort of healthy young Finnish children at 12 and 24 months and studied modifying factors for phosphate concentrations at these time points.

Study participants
This study is part of the Vitamin D Intervention trial in infants (VIDI), a prospective, doubleblinded and randomized intervention study, performed in Helsinki, Finland. In the VIDI trial, altogether 975 healthy infants were randomized to receive vitamin D supplementation either 10 µg (Group10) or 30 µg (Group30) daily from two weeks to 24 months of age (19). The infants were carefully monitored clinically and biochemically during the trial. The detailed VIDI protocol and inclusion and exclusion criteria as well as the main findings of the study have previously been reported (19,20). The study was conducted in accordance with the principles of The Declaration of Helsinki. Research permit was obtained from The Research Ethics Committee of the Hospital District of Helsinki and Uusimaa (107/13/03/03/2012) and all participating families gave informed consent before study onset. The trial protocol is registered in ClinicalTrials.gov (NCT01723852).
The present study includes data from a total of 525 VIDI participants for whom plasma phosphate concentrations were available at both 12 and 24 months of age. Subjects with incomplete data on phosphate concentrations (n=152) as well as those later diagnosed with Downloaded from https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgab495/6313245 by guest on 05 July 2021 A c c e p t e d M a n u s c r i p t 6 significant medical conditions (n=8) were excluded (21). Baseline data was collected during recruitment from the participating infants' medical records. Growth parameters and venous blood samples for analyses of biochemical parameters were obtained at follow-up visits at 12 and 24 months of age. Growth parameters were evaluated according to Finnish pediatric growth references (22). Obtained samples were stored at -80 ° C until completed analyses.

Biochemical assays
Due to the participants' young age, there was no fasting before sampling. The samples were taken between morning and early afternoon.

Nutritional data
Nutritional data on food consumption was obtained from 3-day food diaries, including two weekdays and one weekend day, as previously described (24). Dietary intake data at 12 months of age were given by the parents or daycare personnel and they were analyzed using AivoDiet software (version 2.0.2.3, Aivo Oy, Turku, Finland). Relative phosphate intake was calculated as daily total phosphate intake relative to daily total energy intake.

Statistical analysis
Results are presented as mean and standard deviation or as median and interquartile range (IQR), as appropriate. Comparisons between sexes were performed using Student's t test for parametric and Mann-Whitney U test for non-parametric variables. Chi-squared test was used for categorical variables. The dependence between variables was examined using Pearson's correlation.
Normal distribution of the variables was primarily examined by visual assessment of histograms, assessment of Skewness and Kurtosis, and secondarily by Kolmogorov Smirnov test. Logarithmic transformation was performed for the variables that were not normally distributed. Season was determined, by month of the study-visit and sampling, as winter (December, January and February), spring (March, April and May), summer (June, July and August) and fall (September, October and November). Season was used as a dichotomous variable in analyses (1=winter, 2=others), as concentrations of phosphate in winter were higher than at other times of the year, in post-hoc analyses.
The effects of various factors on phosphate concentration were studied using a linear regression model (forward method, including variables with a p-value less than 0.05). The A c c e p t e d M a n u s c r i p t 8 covariates were selected based on correlation and previous studies related on phosphate (7,8,11,12,25).
The differences in phosphate levels between the sexes and intervention groups and changes over time were studied using a mixed model with a diagonal covariance structure with heterogeneous variance. Based on Akaike's information criterion, this default covariance structure for repeated measures performed better than other options.
The mixed models included the covariates with a p-value less than 0.05 and took into account the size of the children (weight, length). Estimation method was restricted maximum likelihood.
Statistical analyses were performed with IBM SPSS Statistics 25 (IBM, Armonk, NY, USA). P values less than 0.05 were considered statistically significant.

Characteristics of participating children
Characteristics of the participating infants at 12 and 24 months are presented in Table 1.
The study included 525 infants (53% girls). Boys were longer and heavier than girls at both 12 and 24 months of age (t-test p<0.001 at both time points) ( Table 1).
Mean unadjusted calcium concentrations in girls were slightly higher than in boys at age 12 months and 24 months (t-test p<0.001 and p=0.002, respectively) ( Table 1). PTH concentrations did not differ between sexes at 12 and 24 months (Mann-Whitney U test p>0.099 for all) ( Table 1). As previously reported (34), intact FGF23 concentrations were higher in girls than in boys at 12 and 24 months of age (Mann-Whitney U test p <0.001 for A c c e p t e d M a n u s c r i p t 9 all), while C-terminal FGF23 concentration did not differ between the sexes (Mann-Whitney U test p>0.499 for all) ( Table 1). Serum 25OHD was between 50 and 125 nmol/L in 80.9% of the participants at 12 months and in 78.1% at 24 months of age. Less than 1.1% of the participants had serum 25OHD below 50 nmol/L at both time points. In the remaining children, 25OHD was above 125 nmol/L.
One third of the infants were partially breastfed at 12 months of age (24). Boys received more energy (kcal/d) and iron than girls (t-test p<0.040 for all) ( Table 1).

Circulating phosphate concentration
Mean unadjusted phosphate concentrations were at 12 months 1.9±0.15 mmol/L in boys and 1.9±0.16 mmol/L in girls, and at 24 months 1.6±0.15 mmol/L in boys and 1.6±0.17 mmol/L in girls. The phosphate concentrations for all study -participants at 12 and 24 months of age are presented in Figure 1.
Unadjusted phosphate concentrations were largely (90.1% and 99.8% at 12 and 24 months, respectively) within the previously reported age-related reference range (1.25-2.10 mmol/L) (26). However, at 12 months, phosphate concentrations were above the reference range in 9.9% of the children (n = 52). At 24 months, 0.2% of the values (n=1) were above and 2.3% (n=12) below the reference values. Unadjusted phosphate concentrations according to sex and the Vitamin D intervention group are presented in Table 2.
In unadjusted models, phosphate concentrations did not differ between the sexes (t-test p=0.416 and p=0.150 at 12 and 24 months, respectively) (

Phosphate intake and temporal change
Dietary intake of phosphate at 12 months was similar in girls and boys (t-test p=0.081) ( Table   1). When studied by intervention group, phosphate intake was higher in boys than in girls in Group10 (Post-hoc test, Bonferroni p=0.015) ( Table 3).
As unadjusted phosphate concentrations did not differ between the sexes or intervention groups at 12 or 24 months, analyses of temporal change were performed on the whole study population, and not separately by sex or intervention group. Mean phosphate concentrations decreased from 1.9±0.15 mmol/L at 12 months to 1.6±0.17 mmol/L at 24 months of age without covariates (p interaction <0.001) and by 0.33 mmol/L units with covariates

Factors modifying phosphate concentration
Iron modified phosphate concentrations positively at both studied time-points (B, 0.006 and 0.005, 95% CI 0.004 to 0.009 and 0.002 to 0.008, p<0.001 at both 12 and 24 months, respectively) ( Table 4). At 12 months of age calcium intake from food was positively associated with phosphate concentrations (B, 0.047, 95% CI 0.006 to 0.089, p=0.027) and season was found to modify phosphate, with higher concentrations observed in winter (Bonferroni p=1.000, 0.004 and 0.154 for winter vs spring, winter vs summer and winter vs autumn, respectively) than other seasons (B, -0.040, 95% CI -0.075 to -0.006, p=0.022) ( Table 4). PTH did not modify phosphate concentrations at either 12 or 24 months of age.
A c c e p t e d M a n u s c r i p t 12 At 24 months of age, plasma creatinine was a key modifying factor in both sexes (B, 0.007 and 0.013, 95% CI 0.003 to 0.011 and 0.008 to 0.017, p<0.001 for both) (

Discussion
To the best of our knowledge, this is the first study to investigate phosphate concentrations and modifying factors in healthy infants aged 12 to 24 months. Our comparatively large study population (n=525) and the longitudinal study setting allowed us to evaluate factors influencing changes in phosphate homeostasis in this age group. The current study shows that children have significantly higher phosphate concentrations at age 12 months than at 24 months. No statistically significant difference in phosphate concentrations was observed between the vitamin D intervention groups (10 µg vs 30 µg), and the primary modifying factors for plasma phosphate concentrations were plasma iron and creatinine concentrations which both associate positively with phosphate concentration.
We observed a significant decrease in phosphate concentration from age 12 months to 24 months. We did not find previous similar observations in the literature. Adeli et al. studied phosphate concentrations in children and adolescents aged 0-19 years, and 368 children aged A c c e p t e d M a n u s c r i p t 13 1-5 years participated in this cross-sectional study (13). However, their cross-sectional study did not specifically compare phosphate concentrations between different age groups in early childhood. Our results indicate that major physiological changes in phosphate homeostasis take place also after the first year of life. Our study did not evaluate potential molecular mechanisms behind these observations. One possible explanation of higher phosphate concentrations at age 12 months can be age-dependent changes in expression levels of sodium-phosphate co-transporters. The expression of genes and proteins responsible for phosphate absorption decreases with age, and this could also contribute to lower phosphate levels in 24-month-old children compared with 12-month-old children (27).
Unadjusted phosphate concentrations, or temporal change from 12 to 24 months, did not differ by sex. In the adjusted model for repeated measurements, boys had higher phosphate concentrations than girls at 12 and 24 months, and the difference would be explained by the selection of covariates. No sex differences in phosphate concentrations were observed in children aged 1-5 years in the Caliper study which also supports our results (13). In a large cohort of adults including overweight, hypertensive and diabetic patients (n=92 756) unadjusted phosphate concentrations were higher in women than in men (28). Age (young children vs adults) and cohort selection (healthy children vs adults with a chronic illness) could potentially explain the differences between these findings. Possible age-and sexdependent differences in phosphate concentrations and modifying factors during childhood warrant further investigation.
We observed no differences in phosphate concentrations between vitamin D intervention groups. Vitamin D plays a crucial role in calcium and phosphate metabolism and is essential for bone health in infants, children, and adolescents (29). Low phosphate concentration is a A c c e p t e d M a n u s c r i p t 14 common finding in children with rickets (5,30,31) or vitamin D deficiency (serum 25OHD <50 nmol/L) (30), most likely due to increased phosphate excretion in response to secondary hyperparathyroidism. In our cohort 25OHD levels were largely normal and this is a likely reason why no association between vitamin D or PTH and phosphate concentrations was detected.
In our study, 99.8% of phosphate concentrations at 24 months were within the previously described reference range [1.25-2.10 mmol/l aged 1-3 years] whereas at 12 months almost 10% of the values were above these references (26). In previous studies, reference values have not been specified separately for 12-and 24-month-olds (13,26). Our results, showing that phosphate concentrations decrease from 12 to 24 months, indicate that age-specific reference ranges should be used in young children in order to correctly early identify infants with subnormal or supranormal phosphate values. Our data can potentially be used to update reference values in this age-group.
In our study iron was the main modifying factor of phosphate concentrations at 12 months and modified phosphate concentrations also at 24 months. Previous research on the association of iron and phosphate concentrations is limited. Our findings indicate that iron is positively associated with phosphate concentrations at 12 and 24 months of age. In contrast, previous studies have shown iron administration to result in a transient decrease in phosphate (12,32). The effect depends on the type of iron: ferric carboxymaltose induces hypophosphatemia, whereas ferric dextran does not have similar effect. However, in an experiment in rats, when a high dose of iron was administered, no difference in phosphate concentrations was observed (33). We have previously reported that iron is an important modifying factor of FGF23 in healthy infants, with iron being positively associated with A c c e p t e d M a n u s c r i p t 15 intact FGF23 and inversely associated with C-terminal FGF23 (23,34). In the present study, intact and C-terminal FGF23 showed variable association with phosphate, depending on the time point and sex. As FGF23 is a phosphate modifying factor, it seems that iron regulates phosphate metabolism through FGF23. Our study confirms the existence of the link between iron and phosphate metabolism already in early childhood. Further research on specific mechanisms of this regulation is needed.
Creatinine was the second major modifying factor of phosphate at 24 months of age. In our study, creatinine concentration was positively associated with phosphate concentration. The association between phosphate and creatinine clearance is well known in patients with renal failure, creatinine being a marker of kidney function (35). Anthropometric measurements, including arm circumference, have been linked to creatinine in fully active children aged 2 to 6 years, so creatinine may associate with muscle mass growth also in young children (36).
However, our study found no statistically significant correlation between creatinine and anthropometric variables.
Other significant modifying factors were 25OHD and season. As previously published, seasonal fluctuations have been observed in phosphate concentrations (37), and this was also seen in our models. In our study, children's phosphate levels were slightly higher in winter than in other seasons, but the differences between seasons were minimal. As previously speculated, the seasonal variation of phosphate comes through vitamin D metabolism, particularly via 1,25(OH)2D (38). In our study, seasonal variation in PTH concentrations was also observed at 24 months of age. Possible explanations for the phenomenon in this age group are the use of protecting clothing against UVB radiation and staying indoors in the summertime, which may reduce the effect of seasonal variation on vitamin D status in young A c c e p t e d M a n u s c r i p t 16 children (39). There is also evidence of seasonal variation in longitudinal growth in infants (40), which may explain slightly higher phosphate levels in winter when bone growth and mineralization are reduced. PTH reduces phosphate reabsorption in the proximal renal tubules and is therefore known to be involved in phosphate regulation. Surprisingly, PTH was not a significant modifying factor of phosphate concentration in the models used in this study.
Calcium and phosphate play key roles in skeletal development (41). In our findings, relative dietary calcium intake was positively associated with phosphate concentration in children.

Data Availability
Restrictions apply to the availability of some or all data generated or analyzed during this study to preserve patient confidentiality or because they were used under license.
The corresponding author will on request detail the restrictions and any conditions under which access to some data may be provided.                 A c c e p t e d M a n u s c r i p t 33