Bone turnover markers, and growth and bone parameters in infants participating in a vitamin D intervention study

in Endocrine Connections
Authors:
Sabrina Persia Children’s Hospital Bambino Gesù, Tor Vergata University, Rome, Italy
Research Program for Clinical and Molecular Metabolism (CAMM), Faculty of Medicine, University of Helsinki, Helsinki, Finland

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Elisa Holmlund-Suila Research Program for Clinical and Molecular Metabolism (CAMM), Faculty of Medicine, University of Helsinki, Helsinki, Finland
Children’s Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

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Saara Valkama Research Program for Clinical and Molecular Metabolism (CAMM), Faculty of Medicine, University of Helsinki, Helsinki, Finland
Children’s Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

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Maria Enlund-Cerullo Research Program for Clinical and Molecular Metabolism (CAMM), Faculty of Medicine, University of Helsinki, Helsinki, Finland
Children’s Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
Folkhälsan Institute of Genetics, Helsinki, Finland

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Jenni Rosendahl Research Program for Clinical and Molecular Metabolism (CAMM), Faculty of Medicine, University of Helsinki, Helsinki, Finland
Children’s Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

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Sture Andersson Children’s Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland

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Outi Mäkitie Research Program for Clinical and Molecular Metabolism (CAMM), Faculty of Medicine, University of Helsinki, Helsinki, Finland
Children’s Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
Folkhälsan Institute of Genetics, Helsinki, Finland
Department of Moecular Medicine and Surgery, Karolinska Institutet, and Clinical Genetics, Karolinska University Laboratory, Karolinska University Hospital, Stockholm, Sweden

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Helena Hauta-alus Research Program for Clinical and Molecular Metabolism (CAMM), Faculty of Medicine, University of Helsinki, Helsinki, Finland
Children’s Hospital, Pediatric Research Center, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
Population Health Unit, National Institute for Health and Welfare (THL), Helsinki, Finland
Clinical Medicine Research Unit, MRC Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland

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https://orcid.org/0000-0002-3487-7834

Correspondence should be addressed to H Hauta-alus: helena.hauta-alus@helsinki.fi
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Amino-terminal propeptide of type 1 procollagen (P1NP) and carboxy-terminal crosslinked telopeptide of type 1 collagen (CTX-I) are markers of bone metabolism. We examined the effect of vitamin D3 supplementation on these markers and their relationship with growth and bone parameters in 12-month-old infants. In a randomized, double-blinded, vitamin D intervention in infants (VIDI) study, 987 infants received daily vitamin D3 supplementation of 10 μg (group-10) or 30 μg (group-30) from age 2 weeks to 24 months. We conducted a secondary analysis of the original VIDI trial. At 12 months of age, P1NP (n = 812) and CTX-I (n = 786) concentrations were analyzed, and anthropometrics and total bone mineral content, volumetric bone mineral density, cross-sectional area and polar moment of inertia of tibia were measured by peripheral quantitative computed tomography. The growth rate in weight and length was calculated from birth to 12 months. The vitamin D dose did not influence mean (SD) levels of CTX-I (group-10: 0.90 (0.31); group-30: 0.89 (0.31) (P > 0.53)). The mean difference of P1NP (CI 95%) comparing group-10 with group-30 was 35 (−103, 33) ng/mL (P = 0.31) in boys and −63 (−4, 130) ng/mL (P = 0.064) in girls. In group-10, girls had higher mean (SD) value of P1NP (1509 (362) ng/mL) than boys (1407 (297) ng/mL) (P = 0.003); no sex differences were observed in group-30 (girls: 1446 (359); boys: 1442 (359), P = 0.91) or CTX-I. P1NP associated positively with the growth rate in length (B (CI 95%) 0.0003 (0.0001, 0.001), P = 0.022) in the whole cohort but not in subgroups divided by the intervention group or sex, adjusted for birth size and parental heights and corrected for multiple testing. P1NP associated positively with the growth rate in weight (0.01 (0.0003, 0.01), P < 0.001). An inverse association was observed between CTX-I and length (cm) in the whole cohort (−0.90 (−1.40, −0.40), P = 0.005) and in group-30 (−1.05 (−1.72, −0.39), P = 0.011). Furthermore, CTX-I associated negatively with weight (SDS) in the whole cohort (−0.33 (−0.55, −0.12), P = 0.015) and the growth rate in weight (−0.43 (−0.66, −0.20), P = 0.005), persisting in group-30 and in boys but not in group-10 or in girls. Neither marker was associated with bone parameters. The observed sex difference in P1NP might suggest that a higher vitamin D dose resulted in a small decrease in bone collagen matrix formation in girls but not in boys. P1NP and CTX-I associate with growth and body size but not with bone mineralization in infancy.

Abstract

Amino-terminal propeptide of type 1 procollagen (P1NP) and carboxy-terminal crosslinked telopeptide of type 1 collagen (CTX-I) are markers of bone metabolism. We examined the effect of vitamin D3 supplementation on these markers and their relationship with growth and bone parameters in 12-month-old infants. In a randomized, double-blinded, vitamin D intervention in infants (VIDI) study, 987 infants received daily vitamin D3 supplementation of 10 μg (group-10) or 30 μg (group-30) from age 2 weeks to 24 months. We conducted a secondary analysis of the original VIDI trial. At 12 months of age, P1NP (n = 812) and CTX-I (n = 786) concentrations were analyzed, and anthropometrics and total bone mineral content, volumetric bone mineral density, cross-sectional area and polar moment of inertia of tibia were measured by peripheral quantitative computed tomography. The growth rate in weight and length was calculated from birth to 12 months. The vitamin D dose did not influence mean (SD) levels of CTX-I (group-10: 0.90 (0.31); group-30: 0.89 (0.31) (P > 0.53)). The mean difference of P1NP (CI 95%) comparing group-10 with group-30 was 35 (−103, 33) ng/mL (P = 0.31) in boys and −63 (−4, 130) ng/mL (P = 0.064) in girls. In group-10, girls had higher mean (SD) value of P1NP (1509 (362) ng/mL) than boys (1407 (297) ng/mL) (P = 0.003); no sex differences were observed in group-30 (girls: 1446 (359); boys: 1442 (359), P = 0.91) or CTX-I. P1NP associated positively with the growth rate in length (B (CI 95%) 0.0003 (0.0001, 0.001), P = 0.022) in the whole cohort but not in subgroups divided by the intervention group or sex, adjusted for birth size and parental heights and corrected for multiple testing. P1NP associated positively with the growth rate in weight (0.01 (0.0003, 0.01), P < 0.001). An inverse association was observed between CTX-I and length (cm) in the whole cohort (−0.90 (−1.40, −0.40), P = 0.005) and in group-30 (−1.05 (−1.72, −0.39), P = 0.011). Furthermore, CTX-I associated negatively with weight (SDS) in the whole cohort (−0.33 (−0.55, −0.12), P = 0.015) and the growth rate in weight (−0.43 (−0.66, −0.20), P = 0.005), persisting in group-30 and in boys but not in group-10 or in girls. Neither marker was associated with bone parameters. The observed sex difference in P1NP might suggest that a higher vitamin D dose resulted in a small decrease in bone collagen matrix formation in girls but not in boys. P1NP and CTX-I associate with growth and body size but not with bone mineralization in infancy.

Introduction

The likelihood of lifetime bone fractures depends not only on the rate of bone loss in adulthood but also on the peak bone mass acquired in childhood and adolescence. Bone health and normal growth in childhood are important determinants of the risk of developing osteoporosis and fractures in adulthood (1).

In early childhood, the formation of a healthy skeleton requires the synchronized coupling of osteoblast and osteoclast activity to ensure normal bone modeling and remodeling (2). However, in infants and children, there is a predominance of bone formation over bone resorption (3, 4). The balance between these two processes determines peak bone density at around 30 years of age (3). The dynamic bone turnover can be monitored using circulating biomarkers, known as bone turnover markers (BTMs). This term refers to a broad category of factors, mainly degradation products of bone formation and resorption, which are released into blood circulation or secreted in the urine.

BTMs reflect bone metabolic activity at a specific time point, and they are grouped into two categories based on the metabolic phase during which they are produced: bone formation and resorption. Two of the most studied BTMs are related to type I collagen. Both of these markers – the amino-terminal propeptide of type 1 procollagen (P1NP) and the carboxy-terminal crosslinked telopeptide of type 1 collagen (CTX-I) – are recommended as the best markers of bone formation and resorption, respectively, by the International Osteoporosis Foundation (IOF) and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) (5, 6). Currently, their clinical use focuses on estimating fracture risk and monitoring adherence and response to therapies (e.g., bisphosphonates) (7, 8). However, little is known about P1NP and CTX-I distribution and their role in bone mineral content (BMC) and skeletal growth in healthy infants (9).

Vitamin D is an important requirement for healthy development of the skeleton (10). It is well established that chronic vitamin D deficiency causes rickets in children; hence, many health authorities recommend vitamin D supplementation in infancy (11). An adequate level of blood 25-hydroxy vitamin D (25(OH)D) – an indicator of vitamin D intake – is pivotal for the growth plate because its active metabolite (1,25-dihydroxyvitamin D; 1,25(OH)2D) regulates both mineral deposition and calcium homeostasis (12, 13). Nevertheless, the role of vitamin D in BTM metabolism is not yet completely understood. The few studies that have examined this relationship have mostly focused on vitamin D deficient populations and have found conflicting results (14, 15, 16).

In the present secondary analysis, our first aim was to investigate whether vitamin D3 supplementation has an impact on P1NP and CTX-I and whether P1NP and CTX-I associate with 25(OH)D and parameters of bone strength and growth in healthy 12-month-old infants participating in the vitamin D intervention in infants (VIDI) study, a randomized controlled trial (RCT) in Finland, Northern Europe. In addition, our second aim was to describe variations and determinants of P1NP and CTX-1 concentrations, including sex differences. Participants were randomly assigned to receive either 10 or 30 μg vitamin D3 supplementation daily between 2 weeks and 24 months of age. As previously reported, higher vitamin D dosage raised infants’ 25(OH)D concentrations, but it had no effect on the primary outcomes – bone development or infections (17); however, an effect on growth was observed (18).

Materials and methods

Participants

We recruited 987 families with their newborn child at Kätilöopisto Maternity Hospital (Helsinki, Finland) to participate in the VIDI study between January 2013 and June 2014 (Fig. 1). A detailed description of the recruitment process and study protocol is available from previous reports (17, 19).

Figure 1
Figure 1

Flowchart of the study participants. CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen; P1NP, amino-terminal propeptide of type 1 procollagen.

Citation: Endocrine Connections 14, 1; 10.1530/EC-24-0482

Inclusion criteria included that infants were born between 37 and 42 weeks of gestation with birth weights appropriate for the gestational age, and mothers were of Northern European origin and had singleton pregnancy with no regular medications. Infants were randomized to receive daily vitamin D3 supplementation of either 10 μg (group-10) or 30 μg (group-30) starting at 2 weeks of age until 24 months of age. No additional vitamin D supplements were allowed. Study visits, which included anthropometric measurements, were held at the ages of 6, 12 and 24 months, and bone parameters were measured with peripheral quantitative computed tomography (pQCT) at ages 12 and 24 months.

Parents signed a written informed consent at recruitment. This study was conducted according to the guidelines put forth in the Declaration of Helsinki. Ethical approval was obtained from the Research Ethics Committee of the Hospital District of Helsinki and Uusimaa (107/13/03/03/2012), and the project protocol is registered at ClinicalTrials.gov (NCT01723852).

Family questionnaires

Parents filled self-administered questionnaires about the family background information. Parental education level was categorized into ‘lower’ and ‘higher’ education (lower, lower or upper secondary or postsecondary nontertiary education/less than a bachelor’s degree; higher, first or second stage of tertiary education/at least a bachelor’s degree). We assessed parental smoking status before pregnancy and at the 24-month visit, which was coded as smoking if either of the parents had smoked during the 24-month follow-up period. Any frequency of smoking, e.g., one cigarette once in a week, was considered as smoking. At infant age of 12 months, the breastfeeding status and mean daily dietary intake of vitamin D and calcium from food were calculated on the basis of a 3-day food record without the amount of breast milk (20).

Anthropometrics

Midwives measured the birth size according to standard procedures. The birth size was standardized according to parity-, gestational age- and sex-specific SDSs based on national references (21). Infant weight (kg), length (cm) and head circumference (cm) were measured during 6- and 12-month follow-up visits by a pediatrician or a research nurse. Length was measured using a tabletop meter in the supine position, and weight was measured on an electronic scale (Seca, Germany). Weight, length, length-adjusted weight and head circumference were expressed as SDSs using age- and sex-specific national references (22) and considered normal when falling between +2.0 SDS and −2.0 SDS.

Bone parameters

Bone parameters were measured using the pQCT technique (XCT2000L Research+, Stratec Medizintechnik GmbH, Germany) when infants were 12 months of age. The left tibia was used for all participants as the dominant leg could not be determined in this age group. The length of the tibia was measured from the medial malleolus to the medial condyle, and the scanning site at 20% distal length was marked with a colored line. A scan with slice thickness of 2.0, 0.40 mm voxel size and 25 mm/s scan speed was measured. The total BMC (mg/mm), volumetric bone mineral density (mg/cm3), cross-sectional area (CSA) (mm2) and polar moment of inertia (PMI) (mm4) were assessed for the total tibial bone during 12- and 24-month follow-up visits. Additional details about the procedure and quality assessment have been previously reported (23).

Biochemical methods

The concentrations of P1NP, CTX-I, 25(OH)D, intact parathyroid hormone (PTH), plasma phosphate and ionized calcium (iCa) were analyzed from serum samples collected from infants at the age of 12 months without fasting. Because of the intense growth rate during the first year of life, we chose to focus P1NP and CTX-I measurements on samples collected at age 12 months. IDS intact P1NP kits were employed on iSYS analyzers to analyze P1NP values. The assay is based on chemiluminescence technology. All P1NP samples were diluted manually (1:10) using the diluent supplied with the kit, as all values without dilution were above the measurement range (2–230 ng/mL). Some samples (n = 15) needed further dilution (1:50). Dilutions were accounted for in the final concentrations.

IDS-iSYS CTX-I (CrossLaps®, Immunodiagnostic Systems Ltd, UK) assays were employed for CTX-I analysis with a reportable range of 0.033–6.000 ng/mL according to the manufacturer’s protocol (24, 25). The concentrations of 25(OH)D and PTH were determined using an IDS-iSYS fully automated immunoassay system with chemiluminescence detection (Immunodiagnostic Systems Ltd, UK). The quality and accuracy of 25(OH)D analyses were ensured by adhering to the Vitamin D External Quality Assessment Scheme (DEQAS, Charing Cross Hospital, UK); 25(OH)D concentrations were on average 10% higher than the National Institute of Standards and Technology (NIST) standards. Plasma inorganic phosphate (Pi) concentrations were measured according to the standard procedure (26). The concentration of iCa (adjusted to pH 7.40) was analyzed by applying ABL 90 FLEX or ABL 835 FLEX blood gas analyzers. The details of these assessments have been reported previously (17).

Statistical analysis

Normality of the variables was visually inspected and statistical tests were applied accordingly. The differences between groups were examined using an independent samples t-test, ANOVA and ANCOVA. We employed simple unadjusted and adjusted linear regression models to assess associations between continuous variables. Covariates were chosen by selecting significant associations with dependent variables. Unadjusted results are reported in the text only if they differ significantly from the adjusted results. Linear regression analyses regarding growth parameters were adjusted for paternal height (cm), maternal height (cm) and the corresponding birth size except for tibia length, which was adjusted for birth length (cm). Regarding pQCT parameters, we identified weight (kg) as a covariate. We performed additional adjustment for parental smoking status, duration of breastfeeding, maternal education and PTH in all regression analyses; since no significant difference was found in the results, these data are not reported. We applied the Benjamini–Hochberg correction to adjust for multiple testing (27). Growth rates in length and weight at 12 months of age were calculated as the residuals from the linear regression model where body size in SDS is regressed by the corresponding body size in SDS at earlier ages (at birth and 6 months of age) (18, 28).

We categorized P1NP and CTX-I into tertiles to examine the differences in mean values of growth and bone parameters with ANCOVA after Bonferroni correction and adjusted with the same covariates as in the linear regression models. The serum P1NP/CTX-I ratio was calculated by dividing the P1NP values by the CTX-I measurements and then expressed as the mean value of all P1NP/CTX-I quotients. Multivariate linear regression analysis with a subsequent backward procedure was used to reveal the independent predictor variables of P1NP and CTX-I. In this analysis, we only included parameters that correlated significantly with P1NP and CTX-I in the simple correlation and linear regression analysis without any intercorrelation with each other. Interaction of sex and the vitamin D supplemental dose was examined in analyses; only significantly different results are reported in the text and tables.

We considered P < 0.05 statistically significant. All statistical analyses were performed using IBM’s SPSS program for Windows, version 29 (IBM, USA).

Results

Cohort characteristics

A flowchart of participants is shown in Fig. 1. Of the 987 infants recruited, we excluded 12 who did not meet the inclusion criteria. In addition, we excluded subjects without assessment of P1NP and CTX-I concentrations. This resulted in a final sample of 812 subjects, of which 786 had CTX-I values and 812 had P1NP values (Fig. 1). Current cross-sectional data include partially missing data in some of the variables, which are presented in the tables and figures.

Table 1 shows an overview of the cohort’s birth characteristics and family background. Approximately 30% of parents smoked with any frequency at some point during follow-up. On average, mothers had a normal BMI before pregnancy. The majority of infants had sufficient levels of 25(OH)D (≥50 nmol/L) (792/802, 99%) (17). As reported previously, boys had greater length, weight, BMC, CSA and PMI values compared with girls (data not shown) (23). Conversely, girls showed higher iCa concentration than boys (Table 2) (29). Infants in group-30 showed higher 25(OH)D and iCa but lower PTH concentrations than infants in group-10 (Table 2) (17). Boys had higher vitamin D intake from food than girls in group-10 but not in group-30. Boys were more commonly breastfed than girls in group-30 but not in group-10. Calcium intake from food was higher among boys compared with girls (Table 2).

Table 1

Background characteristics of the study participants.

nMean (SD)
Family characteristics
 Maternal age at delivery (years)81231.6 (4.3)
 Maternal pre-pregnancy BMI (kg/m2)80823.1 (3.6)
 Maternal education, higher1 (%)80275
 Paternal age (years)79033.2 (5.3)
 Paternal pre-pregnancy BMI (kg/m2)78525.7 (3.3)
 Paternal education, higher1 (%)79262
 Parental smoking2 (%)80730
 Duration of pregnancy (weeks)81240.1 (1.0)
Birth anthropometrics
 Weight (SDS)812−0.1 (0.8)
 Length (SDS)812−0.1 (0.9)
 Cord blood 25(OH)D (nmol/L)79482.4 (25.6)

Values are mean (SD) unless otherwise stated. Abbreviations: BMI, body mass index and SDS, standard deviation score.

Higher = first or second stage of tertiary education or at least a bachelor’s degree; lower = upper secondary or post-secondary nontertiary education or less than a bachelor’s degree.

Combined baseline and smoking status at child age of 2 years when at least one of the parents smoked.

Table 2

Descriptive data on blood biochemical compounds and diet in infants aged 12 months.

AllGroup-10Group-30BoysGirls
CTX-I (ng/mL) (range)0.90 (0.30) (0.21–2.50) n = 7850.90 (0.31) n = 3920.89 (0.31) n = 3930.88 (0.29) (0.21–1.82) n = 3840.91 (0.32) (0.24–2.50) n = 401
P1NP (ng/mL) (range)1452 (347) (466–3470) n = 8121459 (336)2 n = 4051444 (359) n = 4071425 (330) (761–3470) n = 3981478 (362) (466–3190) n = 414
Ratio of P1NP/CTX-I11794 (711) n = 7851794 (716) n = 3921794 (707) n = 3931786 (726) n = 3841802 (697) n = 401
25(OH)D (nmol/L)98.99 (29) n = 80282.8 (19.8) n = 399115.1 (27.7) n = 40398.24 (28.4) n = 39199.71 (29.5) n = 411
PTH (pg/mL)26.08 (14.43) n = 77327.5 (13.8) n = 38524.7 (14.9) n = 38825.21 (13.80) n = 37326.90 (14.96) n = 400
iCa (mmol/L)1.33 (0.03) n = 8021.33 (0.03) n = 3991.33 (0.03)*3 n = 4031.33 (0.03) n = 3921.33 (0.03)4 n = 410
Pi (mmol/L)1.91 (0.15) n = 6741.91 (0.16) n = 3371.91 (0.15) n = 3371.91 (0.15) n = 3251.90 (0.15) n = 349
Dietary vitamin D intake from food (μg/day)56.1 (3.6) n = 6836.3 (3.7)6 n = 3426.0 (3.4) n = 3416.2 (3.7) n = 3476.1 (3.4) n= 336
Dietary calcium intake from food (mg/day)5613 (306) n = 683632 (308) n = 342595 (304) n = 341634 (316) n =347592 (295)* n = 336
Breastfed (% (n/n))736 (245/683)33 (111/342)839 (134/341)36 (125/347)36 (120/336)

Values are means (SD) unless otherwise indicated. Independent samples t-test and Pearson chi-square used to determine statistical significance between vitamin D intervention groups and sexes. *P < 0.05, P < 0.01 and P < 0.001. Abbreviations: 25(OH)D, 25-hydroxy vitamin D; CTX-I, carboxy-terminal crosslinked telopeptide of type 1 collagen; group-10, vitamin D intervention group of 10 μg/day; group-30, vitamin D intervention group of 30 μg/day; iCa, ionized calcium; P1NP, amino-terminal propeptide of type 1 procollagen; Pi, inorganic phosphate; PTH, parathyroid hormone.

Ratio of P1NP and CTX-I was calculated as the mean of all the P1NP/CTX-I quotients.

An interaction was detected for sex and intervention group (P = 0.044): boys had lower P1NP (mean (SE) 1407 (25) ng/mL) than girls (1509 (24) ng/mL, P = 0.003) in group-10 but not in group-30 (girls: 1446 (24) ng/mL; boys: 1442 (25), P = 0.91).

Group-30 had a higher concentration of iCa than group-10.

Girls had a higher concentration of iCa than boys.

Mean dietary intake from food calculated from 3-day food record, excluding breast milk.

An interaction was detected for sex and intervention group (P = 0.010): boys had higher vitamin D intake from food (mean (SE) 6.7 (0.27) μg) than girls (5.9 (0.27) μg) in group-10 but not in group-30 (P = 0.09).

Breastfed at 12 months of age during food record period.

An interaction was detected for sex and intervention group: Higher proportion of boys was breastfed in group-30 than group-10 (44 vs 28%, P = 0.001) with no difference in girls (34 vs 37%, P = 0.51).

Vitamin D intervention, P1NP and CTX-I

The vitamin D supplementation did not affect P1NP (P = 0.26) and CTX-I mean concentrations (P = 0.84) at the whole cohort level (Table 2). However, in group-10, girls had higher P1NP concentrations than boys, but no sex differences were observed in group-30 (Table 2, Fig. 2). The mean difference of P1NP and CTX-I (CI 95%) comparing group-10 with group-30 was 35 (−103, 33) ng/mL (P = 0.31) and 0.03 (−0.03, 0.10) ng/mL (P = 0.28) in boys, respectively, and −63 (−4, 130) ng/mL (P = 0.064) and −0.02 (−0.08, 0.04) ng/mL (P = 0.59) in girls, respectively (Fig. 2). P1NP associated positively with 25(OH)D only in the unadjusted linear model (P = 0.04) but not when adjusted for PTH (P = 0.34). No linear association was found between CTX-I and 25(OH)D (P = 0.33). We observed no difference in mean values of P1NP (P = 0.22) or CTX-I (P = 0.23) between categories of 25(OH)D <75, 75–125 and >125 nmol/L (P = 0.22) with no interaction effect with sex adjusted for PTH.

Figure 2
Figure 2

P1NP and CTX-1 mean (95% CI) values according to the intervention group and sex (group-10 (A, C): girls, n = 207; boys, n = 198, and group-30 (B, D): girls, n = 207; boys, n = 200) in 12-month-old infants (26 with missing CTX-I values). ANCOVA **P < 0.01. CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen; P1NP, amino-terminal propeptide of type 1 procollagen; group-10, vitamin D intervention group of 10 μg/day; group-30, vitamin D intervention group of 30 μg/day.

Citation: Endocrine Connections 14, 1; 10.1530/EC-24-0482

P1NP, growth and bone parameters

In the adjusted models after P value correction, we found that P1NP associated positively with the growth rate in length and weight, and body weight across the entire cohort, whereas association with body length was seen only in girls (Table 3). In addition, the first (lowest) tertile of P1NP exhibited the lowest body length and weight, and growth rate in length and weight compared with the higher P1NP tertiles (Fig. 3). No association was observed between P1NP and bone parameters (Table 3 and Fig. 3). Interaction with vitamin D intervention was not detected.

Table 3

Linear association between P1NP, linear growth and bone strength parameters in 12-month-old infants.

P1NP, 10 ng/mLAllGroup-10Group-30BoysGirls
Length (cm)10.003 (−0.002, 0.01)0.004 (−0.003, 0.01)0.001 (−0.004, 0.01)−0.001 (−0.01, 0.01)0.01 (0.003, 0.01)
 Adjusted P value; corrected P value3; n0.26; 0.52; 7910.26; 0.50; 3930.66; 0.85; 3980.69; 0.86; 3870.002; 0.02; 404
Length (SDS)10.002 (0.0004, 0.004)0.003 (0.001, 0.01)0.001 (−0.001, 0.003)0.0001 (−0.003, 0.003)0.004 (0.002, 0.01)
 Adjusted P value; corrected P value3; n0.018; 0.07; 7910.021; 0.08; 3930.30; 0.56; 3980.96; 0.89; 387<0.001; 0.009; 404
Growth rate in length (SD unit)10.003 (0.001, 0.01)0.002 (−0.001, 0.01)0.003 (0.001, 0.01)0.003 (0.001, 0.01)0.003 (0.0003, 0.01)
 Adjusted P value; corrected P value3; n0.004; 0.022; 7910.10; 0.31; 3930.017; 0.07; 3980.021; 0.07; 3870.028; 0.08; 404
Weight (kg)10.002 (0.0001, 0.004)0.002 (−0.001, 0.01)0.002 (−0.001, 0.01)0.002 (−0.001, 0.01)0.004 (0.001, 0.01)
 Adjusted P value; corrected P value3; n0.042; 0.12; 7920.16; 0.36; 3940.13; 0.33; 3980.23; 0.48; 3880.004; 0.025; 404
Weight (SDS)10.003 (0.001, 0.01)0.003 (0.001, 0.01)0.002 (0.000001, 0.01)0.002 (−0.001, 0.01)0.004 (0.001, 0.01)
 Adjusted P value; corrected P value3; n0.003; 0.021; 7920.022; 0.07; 3940.050; 0.13; 3980.18; 0.39; 3880.002; 0.017; 404
Growth rate in weight (SDS)10.01 (0.003, 0.01)0.004 (0.001, 0.01)0.01 (0.003, 0.01)0.01 (0.004, 0.01)0.004 (0.001, 0.01)
 Adjusted P value; corrected P value3; n<0.001; <0.001; 7920.009; 0.041; 394<0.001; <0.001; 398<0.001; <0.001; 3880.004; 0.020; 404
Tibial length (mm)10.004 (−0.02, 0.03)0.01 (−0.03, 0.05)0.003 (−0.02, 0.02)0.01 (−0.03, 0.05)0.01 (−0.02, 0.03)
 Adjusted P value; corrected P value3; n0.72; 0.82; 6200.70; 0.85; 3020.77; 0.82; 3180.61; 0.85; 2960.72; 0.84; 324
BMC (mg/mm)20.001 (−0.02, 0.02)0.01 (−0.02,0.03)−0.004 (−0.03, 0.02)−0.004 (−0.03, 0.03)0.01 (−0.01, 0.03)
 Adjusted P value; corrected P value3; n0.88; 0.83; 6380.57; 0.84; 3140.76; 0.83; 3240.80; 0.80; 3050.45; 0.78; 333
vBMD (mm/cm3)20.04 (−0.13, 0.21)0.00 (−0.24, 0.24)0.08 (−0.15, 0.32)−0.04 (−0.31, 0.23)0.11 (−0.11, 0.34)
 Adjusted P value; corrected P value3; n0.63; 0.85; 6380.98; 0.89; 3140.49; 0.77; 3240.78; 0.81; 3050.32; 0.57; 333
CSA (mm2)2−0.01 (−0.08, 0.06)0.02 (−0.09, 0.13)−0.04 (−0.14, 0.06)0.01 (−0.11, 0.13)−0.03 (−0.12, 0.07)
 Adjusted P value; corrected P value3; n0.78; 0.80; 6380.71; 0.85; 3140.46; 0.74; 3240.85; 0.82; 3050.59; 0.84; 333
PMI (mm4)2−0.36 (−3.90, 3.18)1.27 (−3.86, 6.40)−1.90 (−6.84, 3.05)1.08 (−5.09, 7.25)1.33 (−5.62, 2.96)
 Adjusted P value; corrected P value3; n0.84; 0.82; 6380.63; 0.83; 3140.45; 0.75; 3240.73; 0.81; 3050.54; 0.82; 333

Values are adjusted B coefficients with 95% confidence intervals (CI 95%) per 10 ng/mL higher in P1NP concentration. Multivariate simple linear regression applied. Significant P values are shown in bold. Abbreviations: BMC, bone mineral content; CSA, total cross-sectional area; group-10, vitamin D intervention group of 10 μg/day; FDR, false discovery rate; group-30, vitamin D intervention group of 30 μg/day; P1NP, amino-terminal propeptide of type 1 procollagen; PMI, total polar moment of inertia; SDS, standard deviation score (based on Finnish sex- and age-specific norms for child size); vBMD, total volumetric bone mineral density.

Adjusted for parental heights and the corresponding birth size (except for tibial length, which was adjusted for birth length).

Adjusted for body weight (kg) at age 12 months.

To account for multiple testing, FDR Benjamini–Hochberg correction was applied to adjusted P values.

Figure 3
Figure 3

Adjusted mean (95% CI) of (A) length (n = 791), (B) growth rate in length (n = 791), (C) weight (n = 792), (D) growth rate in weight (n = 792), (E) BMC (n = 638) and (F) BMD (n = 638) according to P1NP tertiles in 12-month-old infants. P1NP concentrations in the first (lowest) tertile: 466–1,295 ng/mL, second (intermediate) tertile: 1,296–1,553 ng/mL and third (highest) tertile: 1,554–3,470 ng/mL. Covariates in growth models were parental heights and corresponding birth size (except in analyses of growth rates) and covariate in bone models was body weight (kg) at the age of 12 months. ANCOVA *P < 0.05, **P < 0.01 and ***P < 0.001. BMC, bone mineral content; BMD, bone mineral density; P1NP, amino-terminal propeptide of type 1 procollagen.

Citation: Endocrine Connections 14, 1; 10.1530/EC-24-0482

Infants with weight below −1 SDS had lower mean (SD) P1NP (n = 186, 1,386 (293) ng/mL) than those with weight ≥−1 SDS (n = 626, 1,471 (360) ng/mL) (P = 0.001). Likewise, when dividing length SDS as below −1 SDS and ≥−1 SDS, mean values in P1NP differed (n = 265, 1,404 (375) ng/mL; n = 546, 1,474 (331) ng/mL, respectively, P = 0.007).

CTX-I, growth and bone parameters

CTX-I associated inversely with body length (cm) and weight (kg and SDS), and growth rate in weight across the entire cohort after adjustments with P value correction (Table 4). After categorizing CTX-I values into tertiles, infants in the third (highest) tertile of CTX-I had slower growth rate in weight and lower body weight compared with lower tertiles (Fig. 4). We observed no interaction with vitamin D intervention.

Table 4

Linear association between CTX-I, linear growth and bone strength parameters in 12-month-old infants.

CTX-I, ng/mLAllGroup-10Group-30BoysGirls
Length (cm)1−0.90 (−1.40, −0.40)−0.69 (−1.43, 0.06)−1.05 (−1.72, −0.39)−0.84 (−1.53, −0.15)−0.61 (−1.25, 0.03)
 Adjusted P value; corrected P value3; n<0.001; 0.005; 7640.07; 0.20; 3800.002; 0.011; 3840.017; 0.07; 3730.06; 0.18; 391
Length (SDS)1−0.21 (−0.41, −0.02)−0.19 (−0.49, 0.10)−0.22 (−0.49, 0.05)−0.23 (−0.52, 0.06)−0.19 (−0.47, 0.08)
 Adjusted P value; corrected P value3; n0.034; 0.13; 7640.20; 0.38; 3800.11; 0.29; 3840.12; 0.30; 3730.17; 0.37; 391
Growth rate in length (SD unit)1−0.09 (−0.32, 0.14)−0.19 (−0.52, 0.13)0.01 (−0.31, 0.34)−0.04 (−0.36, 0.29)−0.09 (−0.41, 0.23)
 Adjusted P value; corrected P value3; n0.46; 0.58; 7640.24; 0.41; 3800.94; 0.94; 3840.83; 0.88; 3730.60; 0.69; 391
Weight (kg)1−0.47 (−0.71, −0.23)−0.19 (−0.55, 0.18)−0.74 (−1.05, −0.43)−0.57 (−0.91, −0.23)−0.21 (−0.51, 0.09)
 Adjusted P value; corrected P value3; n<0.001; 0.004; 7650.32; 0.49; 381<0.001; <0.001; 3840.001; 0.010; 3740.16; 0.37; 391
Weight (SDS)1−0.33 (−0.55, −0.12)−0.13 (−0.45, 0.20)−0.53 (−0.82, −0.24)−0.52 (−0.83, −0.20)−0.15 (−0.45, 0.15)
 Adjusted P value; corrected P value3; n0.003; 0.015; 7650.45; 0.58; 381<0.001; 0.005; 3840.001; 0.009; 3740.33; 0.48; 391
Growth rate in weight (SD unit)1−0.43 (−0.66, −0.20)−0.34 (−0.67, −0.02)−0.54 (−0.87, −0.21)−0.58 (−0.94, −0.23)−0.29 (−0.59, 0.01)
 Adjusted P value; corrected P value3; n<0.001; 0.005; 7650.038; 0.13; 3810.001; 0.010; 3840.001; 0.008; 3740.06; 0.19; 391
Tibia length (mm)1−3.02 (−5.65, −0.39)−2.39 (−7.25, 2.48)−2.79 (−5.02, −0.47)−3.45 (−7.34, 0.44)−2.26 (−5.86, 1.34)
 Adjusted P value; corrected P value3; n0.025; 0.10; 6000.33; 0.49; 2920.014; 0.06; 3080.08; 0.22; 2860.22; 0.39; 314
BMC (mg/mm)21.12 (−0.80, 3.03)1.18 (−1.52, 3.88)1.01 (−1.74, 3.76)2.08 (−1.08, 5.25)0.40 (−1.93, 2.73)
 Adjusted P value; corrected P value3; n0.25; 0.42; 6180.39; 0.52; 3040.47; 0.57; 3140.20; 0.39; 2950.74; 0.80; 323
vBMD (mm/cm3)2−7.96 (−27.1, 11.2)−12.8 (−40.4, 14.8)−4.55 (−31.4, 22.3)8.12 (−20.5, 36.8)−19.7 (−45.5, 6.17)
 Unadjusted P value; corrected P value3; n0.42; 0.55; 6180.36; 0.51; 3040.74; 0.81; 3140.58; 0.68; 2950.14; 0.34; 323
CSA (mm2)25.60 (−2.84, 14.0)8.10 (−4.24, 20.4)−3.39 (−8.29, 15.1)2.68 (−10.3, 15.6)7.61 (−3.55, 18.76)
 Unadjusted P value; corrected P value3; n0.19; 0.39; 6180.20; 0.37; 3040.57; 0.68; 3140.69; 0.77; 2950.18; 0.38; 323
PMI (mm4)2179 (−224, 582)327 (−268, 922)37.2 (−515, 590)39.2 (−609, 688)278 (−231, 786)
 Unadjusted P value; corrected P value3; n0.38; 0.52; 6180.28; 0.45; 3040.90; 0.93; 3140.91; 0.93; 2950.28; 0.44; 323

Values are adjusted B coefficients with 95% CI per 1 ng/mL higher in CTX-I concentration. Multivariate simple linear regression applied. Significant P values are shown in bold. Abbreviations: BMC, bone mineral content; CSA, total cross-sectional area; CTX, carboxy-terminal crosslinked telopeptide of type 1 collagen; FDR, false discovery rate; group-10, vitamin D intervention group of 10 μg/day; group-30, vitamin D intervention group of 30 μg/day; PMI, total polar moment of inertia; SDS, standard deviation score (based on Finnish sex- and age-specific norms for child size); vBMD, total volumetric bone mineral density.

Adjusted for parental heights and the corresponding birth size (except for tibial length, which was adjusted for birth length).

Adjusted for body weight (kg) at age 12 months.

To account for multiple testing, FDR Benjamini–Hochberg correction was applied to adjusted P values.

Figure 4
Figure 4

Adjusted mean (95% CI) of (A) length (n = 764), (B) growth rate in length (n = 764), (C) weight (n = 765), (D) growth rate in weight (n = 765), (E) BMC (n = 618) and (F) BMD (n = 618) according to CTX-I tertiles in 12-month-old infants. CTX-I concentration in the first (lowest) tertile: 0.21–0.74 ng/mL; second (intermediate) tertile: >0.74–0.99 ng/mL and third (highest) tertile: >0.99–2.50 ng/mL. Covariates in growth models were parental heights and the corresponding birth size (except in analyses of growth rates) and covariate in bone models was body weight (kg) at the age of 12 months. ANCOVA *P < 0.05, **P < 0.01 and ***P < 0.001. BMC, bone mineral content; BMD, bone mineral density; CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen.

Citation: Endocrine Connections 14, 1; 10.1530/EC-24-0482

Infants with weight SDS below −1 SDS or ≥−1 SDS had similar mean CTX-I (n = 181, 0.94 (0.35) ng/mL; n = 604, 0.89 (0.29) ng/mL, respectively, P = 0.08). When dividing length SDS as below −1 SDS and ≥−1 SDS, no differences in mean values of CTX-I were observed (n = 254, 0.91 (0.33) ng/mL; n = 530, 0.89 (0.29) ng/mL, respectively, P = 0.56).

P1NP and CTX-I

Table 2 presents the mean values of P1NP and CTX-1 stratified by sex and intervention group. There was a positive linear association between P1NP and CTX-I (Fig. 5). We calculated P1NP/CTX-I ratio, showing a predominance of bone formation processes at this specific age cohort without differences between sexes or vitamin D intervention groups (Table 2). PTH associated positively with both P1NP (B (CI 95%) 3.8 (2.21, 5.54), P < 0.001) and CTX-I (0.01 (0.01, 0.01), P < 0.001); there was also a positive linear association between P1NP and Pi (414 (256, 572), P = 0.01).

Figure 5
Figure 5

Scatter plot of linear relation between P1NP and CTX-1 in 12-month-old infants. Line represents the simple linear regression with 95% confidence bands of the best-fit line (β 0.26, P < 0.001 and n = 785). CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen; P1NP, amino-terminal propeptide of type 1 procollagen.

Citation: Endocrine Connections 14, 1; 10.1530/EC-24-0482

P1NP and CTX-I determinants

In our cohort, the determinants of P1NP were sex in group-10, PTH and CTX-I (Table 5). Furthermore, we found that determinants of CTX-I were PTH, parental smoking and P1NP (Table 6). The different models of multivariate regression explained between 3 and 7% of the variance of P1NP and between 6 and 9% of the variance of CTX-I. Parental smoking status was associated with lower offspring CTX-I levels at 12 months of age.

Table 5

Key determinants of P1NP concentration at 12 months of age in vitamin D intervention groups.

P1NP predictorsUnstandardized BConfidence intervalP valueAdjusted R square
Model 11
Group-10
 PTH (pg/mL)3.30.9, 5.70.0070.04
 Girls vs boys106.740.2, 173.20.002
Group-30
 PTH (pg/mL)4.42.0, 6.7<0.0010.03
Model 22
Group-10
 CTX (ng/mL)296.4171.1, 421.6<0.0010.07
Group-30
 CTX (ng/mL)262.6142.2, 382.9<0.0010.06

Multivariate linear regression with backward procedure applied for possible determinants. Only significant determinants are shown. Interaction between the vitamin D intervention group and sex was observed; thus, results are reported separately in intervention groups. Abbreviations: BMC, bone mineral content; CTX, carboxy-terminal crosslinked telopeptide of type 1 collagen; group-10, vitamin D intervention group of 10 μg/day; group-30, vitamin D intervention group of 30 μg/day; P1NP, amino-terminal propeptide of type 1 procollagen; PTH, parathormone.

Model 1 included PTH, parental smoking status and parental education.

Model 2 included BMC, CTX and maternal age at delivery.

Table 6

Key determinants of CTX-I concentration at 12 months of age.

CTX-I predictorsUnstandardized BConfidence intervalP valueAdjusted R square
Model 110.09
 Parental smoking−0.05−0.09, −0.0030.035
 PTH (pg/mL)0.0060.005, 0.008<0.001
Model 220.06
 P1NP (ng/mL)0.00020.0002, 0.0003<0.001

Multivariate linear regression with backward procedure applied for possible determinants. Only significant determinants are shown. Interaction with the vitamin D intervention group was not detected. Abbreviations:. BMC, total bone mineral content; CTX, carboxy-terminal crosslinked telopeptide of type 1 collagen; group-10, vitamin D intervention group of 10 μg/day; group-30, vitamin D intervention group of 30 μg/day; P1NP, amino-terminal propeptide of type 1 procollagen; PTH, parathormone.

Model 1 included parental smoking status, PTH, sex, vitamin D intervention group and parental education.

Model 2 included BMC, P1NP and maternal age at delivery.

Discussion

We report detailed data on the variation in the concentration of BTMs, P1NP and CTX-I, in a large cohort of healthy 12-month-old infants who participated in a vitamin D trial. Our data provide insight into the complex relation between bone modeling and remodeling processes at this specific age. Vitamin D supplementation did not influence CTX-I values. We observed that in infants receiving standard dose of 10 μg vitamin D, girls had higher P1NP mean values than boys. This sex difference was further demonstrated as a trend toward a decrease in P1NP levels in girls in the higher vitamin D dose group (30 μg/day) compared with lower dose group (10 μg/day). P1NP associated positively with growth rate in length and weight, whereas CTX-I showed an inverse association with weight and length, as well as the growth rate in weight. No strong linear associations were found between either P1NP or CTX-I and bone parameters (BMD, BMC, CSA and PMI). Interestingly, we observed that parental smoking associated with lower CTX-I levels in the offspring.

Rapidly growing infants experience a predominance of bone formation processes over resorption (3) compared with adults (30, 31). This premise is confirmed in the current study by a uniformly high P1NP/CTX-I ratio in participant infants. There are no official reference data for P1NP and CTX-I in this specific age group, as multicentric international studies are still lacking. In a recent review, Ladang and coworkers (32) reported all of the studies that have provided pediatric reference ranges known in the literature thus far. P1NP and CTX-I are strongly age-dependent (33). Both European-origin (24, 34, 35) and Asian-origin studies (36, 37) have also reported the highest levels of P1NP and CTX-I during the first year of life, with similar values as in our cohort.

To date, the effect of vitamin D supplementation on bone markers in children has not been well studied. In our cohort, in which most infants were vitamin D-sufficient (17), a higher vitamin D dosage had a possible decreasing effect in girls’ P1NP values but no effect on CTX-I concentrations. Affected P1NP in our study might suggest one possible pathway underlining our previous findings on higher vitamin D and slower infant growth (18, 38). However, many studies have reported no change in BTMs after vitamin D supplementation in children, adolescents (39, 40, 41, 42) and adults (16, 41, 43, 44). Notably, in most of these cohorts, subjects were vitamin D-deficient. In contrast, in an Indian RCT, 468 children and adolescents with vitamin D insufficiency (25(OH)D < 50 nmol/L) were given 600, 1,000 or 2,000 IU of daily vitamin D supplementation for 6 months. After that period, a decrease was observed in both bone formation (P1NP) and resorption (CTX-I) in all of the intervention groups (45). Interestingly, Kuchuk and coworkers suggested that BTMs might only be affected by vitamin D supplementation at baseline 25(OH)D levels below 40 nmol/L (46); however, only few studies exist with infants of high 25(OH)D concentrations such as in our study (38), thus possibly explaining inconsistent findings.

The reason why girls had higher P1NP and were affected differently by vitamin D dosage than boys might be related to a different stage of ‘minipuberty’ at this age (47). It is well known that P1NP is strongly dependent on estrogen stimulation (48). Since girls usually have a longer minipuberty period than boys, the prolonged exposure to the estrogen activity could be a possible explanation for the sex difference.

We found that P1NP is positively associated with the growth rate in length and body size, in line with the previous report from the current cohort in which collagen X biomarker also correlated with growth (49). In a previous report on 30 prepubertal children with idiopathic short stature, P1NP correlated positively with the growth rate in length (50). In another report, P1NP correlated with leg growth velocity in a very low birth weight cohort (51). Concerning CTX-I and growth-related parameters, we found, interestingly, a negative correlation not only with weight and growth rates in weight but also with length. Other reports have investigated the negative correlation of CTX-I with weight (52) but to the best of our knowledge, not with length.

Against our expectations, we did not find linear associations between P1NP, CTX-I and pQCT-derived tibial bone parameters. In the literature, it is still debated whether there is a correlation between P1NP and BMC. In a semi-longitudinal study that included 303 healthy youths, P1NP correlated with BMC measured with dual-energy X-ray absorptiometry (DXA) at the lumbar spine, femur and total body in pubertal boys but not in girls (53). However, a weak correlation between low BMD, P1NP and amenorrhea was observed in adolescent athlete girls (54). Regarding CTX-I, no correlation was reported with any of the DXA-derived bone measures in 395 7-year-old children (55).

Finally, regarding P1NP and CTX-I determinants, we highlight the novel finding of parental smoking as a predictor of offspring CTX-I at 12 months of age. A number of studies have examined the relation between smoking and bone turnover in adults (56, 57). In these reports, the authors observed a decrease in bone formation and resorption markers after exposure to smoking, indicating lower bone turnover as well as associating with lower bone mineral density. Limited studies analyzing the association of maternal smoking and child bone health exhibited conflicting results (58, 59). Maternal smoking during pregnancy, however, has known effects on fetal development (60) such as reduction of birth weight (61).

Our study has several strengths and some limitations. No previous study has explored P1NP and CTX-I measurements in such an extended cohort of infants. The VIDI trial is a large, randomized trial in healthy term infants with prospective comprehensive longitudinal data on mineral and bone metabolism, growth and imaging data in a homogeneous cohort. However, we do not have repeated BTM measurements, which could have offered more precision. Smoking status was applied as a crude dichotomized factor with any frequency of usage considered as a smoker by either of the parent; thus, smoking might reflect lower socioeconomic status in the current cohort; however, including parental education in analyses did not attenuate our findings on the association between parental smoking and infant CTX-I.

Conclusion

In conclusion, in vitamin D-sufficient infants, higher vitamin D supplementation did not affect bone resorption marker CTX-I concentrations, but possibly decreased bone formation marker P1NP concentrations in girls but not in boys compared with a lower dose of vitamin D. Vitamin D dosage of 10 μg daily is adequate for bone mineralization in these infants. We found that in healthy 12-month-old infants, the two BTMs – P1NP and CTX-I – associated with growth but not bone parameters. We also found that parental smoking associated with reduced offspring’s CTX-I levels. Our results suggest that during the rapid growth phase in infancy, these specific BTMs reflect processes in collagen matrix development rather than direct volume of bone mineralization. More studies including longitudinal measurements of P1NP and CTX-I at different ages, as well as the comparison between healthy infants and infants with impaired growth, would offer possibilities for clinical application of bone markers.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work.

Funding

This study was funded by the Academy of Finland (OM), Sigrid Jusélius Foundation (OM), Folkhälsan Research Foundation (OM), Foundation for Pediatric Research (OM and HH), Novo Nordisk Foundation (OM), Finska Läkaresällskapet (SA), a special governmental subsidy for clinical research (OM), Erasmus traineeship and pediatrics specialization scholarship from the University of Tor Vergata (SP), Juho Vainio Foundation (HH) and the Finnish Cultural Foundation (HH).

Acknowledgments

We express our gratitude to all the children and families who participated in this study. Furthermore, we acknowledge nurses S Nolvi, R Paajanen, P Turunen and N Boman and technician S Lindén, as well as the personnel of the Kätilöopisto Maternity Hospital for their important contribution to this study. We are also grateful to H Granroth-Wilding and F d’Amore for consultancy in statistics.

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  • Figure 1

    Flowchart of the study participants. CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen; P1NP, amino-terminal propeptide of type 1 procollagen.

  • Figure 2

    P1NP and CTX-1 mean (95% CI) values according to the intervention group and sex (group-10 (A, C): girls, n = 207; boys, n = 198, and group-30 (B, D): girls, n = 207; boys, n = 200) in 12-month-old infants (26 with missing CTX-I values). ANCOVA **P < 0.01. CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen; P1NP, amino-terminal propeptide of type 1 procollagen; group-10, vitamin D intervention group of 10 μg/day; group-30, vitamin D intervention group of 30 μg/day.

  • Figure 3

    Adjusted mean (95% CI) of (A) length (n = 791), (B) growth rate in length (n = 791), (C) weight (n = 792), (D) growth rate in weight (n = 792), (E) BMC (n = 638) and (F) BMD (n = 638) according to P1NP tertiles in 12-month-old infants. P1NP concentrations in the first (lowest) tertile: 466–1,295 ng/mL, second (intermediate) tertile: 1,296–1,553 ng/mL and third (highest) tertile: 1,554–3,470 ng/mL. Covariates in growth models were parental heights and corresponding birth size (except in analyses of growth rates) and covariate in bone models was body weight (kg) at the age of 12 months. ANCOVA *P < 0.05, **P < 0.01 and ***P < 0.001. BMC, bone mineral content; BMD, bone mineral density; P1NP, amino-terminal propeptide of type 1 procollagen.

  • Figure 4

    Adjusted mean (95% CI) of (A) length (n = 764), (B) growth rate in length (n = 764), (C) weight (n = 765), (D) growth rate in weight (n = 765), (E) BMC (n = 618) and (F) BMD (n = 618) according to CTX-I tertiles in 12-month-old infants. CTX-I concentration in the first (lowest) tertile: 0.21–0.74 ng/mL; second (intermediate) tertile: >0.74–0.99 ng/mL and third (highest) tertile: >0.99–2.50 ng/mL. Covariates in growth models were parental heights and the corresponding birth size (except in analyses of growth rates) and covariate in bone models was body weight (kg) at the age of 12 months. ANCOVA *P < 0.05, **P < 0.01 and ***P < 0.001. BMC, bone mineral content; BMD, bone mineral density; CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen.

  • Figure 5

    Scatter plot of linear relation between P1NP and CTX-1 in 12-month-old infants. Line represents the simple linear regression with 95% confidence bands of the best-fit line (β 0.26, P < 0.001 and n = 785). CTX-1, carboxy-terminal crosslinked telopeptide of type 1 collagen; P1NP, amino-terminal propeptide of type 1 procollagen.

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