Redefining normal bone and mineral clinical biochemistry reference intervals for healthy infants in Canada

Clinical Biochemistry 47 (2014) 27–32

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Redefining normal bone and mineral clinical biochemistry reference intervals for healthy infants in Canada☆,☆☆ Sina Gallo a,c, Kathyrn Comeau a, Atul Sharma b,d, Catherine A. Vanstone a, Sherry Agellon a, John Mitchell b, Hope A. Weiler a,1, Celia Rodd b,d,⁎,1 a

School of Dietetics and Human Nutrition, McGill University, Montréal, Québec H9X 3V9, Canada McGill University Health Center, Montréal, Québec H3G 1A4, Canada Nutrition and Food Studies, George Mason University, Fairfax, VA 22030, USA d Winnipeg Children's Hospital, University of Manitoba, Winnipeg, Manitoba R3E 0Z2, Canada b c

a r t i c l e

i n f o

Article history: Received 10 April 2014 Received in revised form 12 July 2014 Accepted 15 July 2014 Available online 23 July 2014 Keywords: Normative Breastfed Infants Minerals Calcium Phosphate Creatinine Growth

a b s t r a c t Background: Few normative data exist for routine clinical chemistry in healthy term infants, that is, during a time of rapid development. Biochemical markers are significantly affected by these physiological changes and the lack of appropriate reference intervals may impede diagnostics in infants. Objective: To define reference intervals for calcium, phosphate, creatinine, and alkaline phosphatase in infants from 1 to 12 months of age. Design and methods: This was an unblinded secondary analysis of 132 breastfeeding infants participating in a vitamin D3 supplementation trial (400–1600 IU/d) followed prospectively until 1 year of age (NCT00381914). Serial non-fasting capillary and spot urine samples were collected for the measurement of plasma calcium, phosphate, creatinine, and alkaline phosphatase; urinary calcium, phosphate and creatinine (DxC600 Beckman Coulter); and whole-blood ionized calcium (ABL 725 Radiometer). All visits were conducted at McGill University in Montréal, Canada. Results: All analytes changed significantly over time (p b 0.05), but there was no effect of sex. From 1 to 12 months, values decreased for whole-blood ionized calcium; plasma calcium, phosphate, and alkaline phosphatase; and urinary calcium:creatinine. Plasma creatinine increased. For some analytes, particularly calcium and alkaline phosphatase, values were often above the ‘typical’ adult or older child reference limits. Smoothed centile curves (LMS method) were developed to fill existing gaps in normative data for these analytes. Conclusions: Most analytes showed a significant change from 1 to 12 months, confirming the need for age-specific reference values. These data can assist in the generation of new reference intervals for healthy term infants and ultimately improve the care of children. © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Abbreviations: d, day; mo, month; n, number; y, year; 25(OH)D, 25-hydroxyvitamin D; CV, coefficient of variation; CLSI, Clinical and Laboratory Standards Institute; CALIPER, Canadian Laboratory Initiative on PEdiatric Reference Intervals; GAMLSS, Generalized Additive Models for Location, Scale and Shape; SAS, Statistical Analysis System; LMS, lambda–mu–sigma = L curve (Box–Cox power to remove skewness), M curve (median) and S curve (coefficient of variation); LOQ, limit of quantification; WHO, World Health Organization. ☆ Disclosure: HAW is a Canada Research Chair with infrastructure funding from the Canadian Foundation for Innovation. ☆☆ Previous presentations: As a poster at the 2008 Experimental Biology and 2012 Clinic Chemistry meetings. ⁎ Corresponding author at: Section of Pediatric Endocrinology & Metabolism, Winnipeg Children's Hospital, 685 William Avenue, Room FW302, Winnipeg, Manitoba R3E 0Z2, Canada. Fax: +1 204 787 1655. E-mail address: [email protected] (C. Rodd). 1 Senior authors.

Providing standard-of-care health services requires access to laboratory facilities and age- and sex-specific reference intervals [1,2]. Pediatric health care providers have long been disadvantaged relative to adult health care providers because of the relative paucity of norms; data for some age groups – e.g. infants – have been particularly limited even for routine clinical chemistries [2]. Moreover, the biologic samples used to develop reference intervals have often been derived from hospitalized children or leftover material from specific outpatient clinics [3–5]. Ideally, more than 120 specimens are required to construct each confidence reference interval as per the Clinical and Laboratory Standards Institute (CLSI) [6]. Additional information such as ethnicity, age and sex is desirable for association analyses [7]. Because of rapid maturation in the pediatric age range [8], the biochemical analyses are frequently partitioned by age (usually graphically).

http://dx.doi.org/10.1016/j.clinbiochem.2014.07.012 0009-9120/© 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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S. Gallo et al. / Clinical Biochemistry 47 (2014) 27–32

Often, all infants are grouped as a single category despite infancy being a notable period of growth. The CALIPER group (Canadian Laboratory Initiative on PEdiatric Reference Intervals) [7,9] has helped this field by developing reference intervals for some common pediatric clinical chemistry analytes on at least 2 platforms (Abbott ARCHITECT, Roche Cobas) [7,9]. Although data are available for N2000 pediatric participants (newborn to 18 y), the data are pooled for infants (0–365 d) and the ‘n’ in this age ranges from ~ 100 to 250 per analyte. Understandably, partitions in infancy have been difficult to implement given the limited access to samples from healthy individuals. Notwithstanding these major advances, significant lacunae still exist. For example there are few normative data for ionized calcium concentrations between the ages of 7 d and 12 mo of life [10–13]. The objective of this report was to assist in generating normative reference data for healthy infants for several routine analytes, with emphasis on determining whether age-specific partitions were required from 1 to 12 mo of age. Measured values for whole blood ionized calcium; plasma calcium, phosphate, creatinine, and total alkaline phosphatase; and urinary calcium, phosphate and creatinine were tabulated from a cohort of healthy, breastfed, appropriate-for-gestational age infants followed longitudinally and prospectively as part of a vitamin D dose–response trial from 1 to 12 mo of age [14].

Children's Hospital laboratory participates 3 times per year in a proficiency testing program organized by the Laboratoire de Santé Publique du Québec. For the Radiometer, the intra-individual % CV was b 2.2% and the inter-individual % CV was b4.2% based on internal quality controls for ionized calcium. For the Beckman Coulter analyzer, the intraindividual % CV was b3.8% and the inter-individual % CV was b 5.5% based on external controls for calcium, phosphate, alkaline phosphatase and creatinine (Bio-Rad Laboratories, Inc.). Remaining plasma was stored frozen at − 80 °C for batch analysis of 25-hydroxyvitamin D (25(OH)D) measured using liquid chromatography tandem mass spectrometry (Warnex Bioanalytical Services) [14]. As the primary driver of calcium homeostasis is parathyroid hormone (PTH), samples were batched for analysis using an ELISA (Immutopics International). However, these results are presented elsewhere [14].

Materials and methods

All analytes are expressed as median and range (minimum, maximum) at each time point. Given the design of the dose–response study, associations with 25(OH)D, time, and sex were explored for each analyte using a mixed effects model with a random subject effect to account for repeated measures over time (Statistical Analysis System, SAS, Proc Mixed). Feeding status (breast vs. mixed/formula feeding) was also explored as a possible covariate although the sample size in the mixed/formula fed group was limited by study design. Regression assumptions for the mixed effects models were checked by standard diagnostic methods. Overall statistical significance was set at p ≤ 0.05 (two-tailed). Data were analyzed using SAS version 9.2 (SAS Institute Inc., Cary, NC). Reference curves were generated for all analytes using the LMS method [20] in the Generalized Additive Models for Location, Scale and Shape (GAMLSS) [21] statistical package. The LMS method fits a 3 parameter skew normal distribution at each time, with cubic splines used to smooth the resulting curves [20,22]. The 3 parameters represent the median (M), the coefficient of variation (S), and the power in the Box–Cox transformation (L) that vary as a function of age, with centiles calculated using the following formula: M(1 + L · S · Z)1 / L [20,22], where L, M, and S are age-specific and Z is the Zscore that corresponds to a given percentile. Model fit was verified through standard diagnostic procedures and comparison with empiric centiles [23].

As described in Gallo et al. [14], in a vitamin D3 dose–response trial (400, 800, 1200 or 1600 IU/d), 132 healthy predominately breastfeeding infants from Montréal, Québec, Canada were recruited between 2007 and 2010 (ClinicalTrials.gov # NCT00381914). All infants were closely monitored by a safety monitoring officer and vitamin D supplementation was stopped if plasma 25(OH)D concentrations exceeded 250 nmol/L and then vitamin D supplements were re-initiated when 25(OH)D was b100 nmol/L (see [14]). Vitamin D supplementation for the 1600 IU/d group was discontinued because of high plasma 25(OH)D on screening, resulting in the lower sample size in this group (see [14]); the data from this group were included in this study. Healthy, term, singleton infants, born appropriate-forgestational age (between the 5th and the 95th percentile, 2000 Centers for Disease Control growth charts) and breastfeeding (consuming N 80% of total milk volume) were included. Infants of mothers with gestational diabetes, hypertension in pregnancy, chronic alcohol use, or malabsorption syndromes were excluded. Serial visits took place at approximately 1, 2, 3, 6, 9 and 12 mo of age. At each visit, weight, length, and head circumference were taken and mothers were asked if they continued to breastfeed. Any breast milk was defined as ‘breastfeeding’ and a combination of breast-and-formula feeding and/or formula-only feeding were considered as ‘mixed/formula feeding’. Capillary blood samples, in the fed state, were collected at each visit by heel lance or finger prick. Whole blood ionized calcium was analyzed (ABL 725 series blood gas analyzer; Radiometer America) within 4 h of collection at the Montréal Children's Hospital. Blood was centrifuged at 2235 ×g for 20 min at 4 °C; plasma total calcium (indirect ion selective electrode method), phosphate (phosphomolybdate method), total alkaline phosphatase (AACC reference method) and creatinine measurements were performed, on the same day within 4 h of collection, using a Beckman Coulter UniCel DxC600 auto-analyzer. Creatinine was initially measured using the Jaffe method and switched to an enzymatic method in June 2009; calibration was traceable to an isotope dilution mass spectrometry (IDMS) reference procedure [15–17]. An equation (pl. creatinine × 1.04 − 20.4) was developed to reconcile differences between the two methods, with r = .99 [15–17]. A spot urine sample was collected for the assessment of calcium, creatinine and phosphate (Beckman Coulter UniCel DxC600). Values below the assay limit of quantification (LOQ) for all analytes were not included [18,19] and all values above the LOQ were reported separately. The Montréal

Ethics The original research study was approved by the Institutional Review Board of McGill University and permission for secondary analyses (present study) was approved by McGill, George Mason University, and the University of Manitoba. Statistical analyses

Results Samples from 56 female and 76 male infants were collected prospectively over the first year of life [range 24–390 d]; attrition was 26% (see [14]). Fewer samples were available for some analytes secondary to insufficient volume. Sample sizes ranged from 89 to 132 (Tables 1–2). Demographic characteristics were previously described [14]. In brief, the large majority of parents were white (82% of mothers and 81% of fathers). Infants were all breastfed from birth (receiving 80% of their total needs from breast milk); 88% were still receiving some breast milk to 6 mo and 35% to 12 mo. All infants were healthy and growing: mean weight-for-age and weight-for-length Z-scores at each time point were within ±0.5 Z-score from the WHO growth standard [14]. All plasma analytes had values within the quantification limits of the assay except for creatinine (LOQ = limit of quantification = 7 μmol/L; % b LOQ: 1 mo: 16%, 2 mo: 5%, 3 mo: 8%, 6 mo: 6%, 9 mo: 2% and 12 mo: 0%, which were not included in analyses). Similarly, urinary samples with values for calcium b LOQ (b0.5 nmol/L) or creatinine

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Table 1 Plasma analytes for infants over time. Presented as median and range (no. of samples tested). Analyte

Age (mo) 1

2

3

6

9

12

Ionized calcium (mmol/L)a

1.40 1.29–1.52 (127) 2.53 2.32–2.69 (132) 2.06 1.75–2.39 (132) 13 8–28 (92) 302 141–608 (128) 0

1.39 1.22–1.48 (111) 2.54 2.37–2.72 (116) 1.95 1.61–2.25 (111) 13 7–24 (96) 272 140–540 (114) 0

1.38 1.30–1.49 (109) 2.55 2.32–2.76 (114) 1.87 1.56–2.31 (110) 14 7–26 (98) 248 2–541 (112) 0

1.35 1.16–1.42 (109) 2.51 2.32–2.71 (108) 1.77 1.29–2.18 (108) 15 7–31 (97) 190 77–362 (109) 0

1.33 1.26–1.43 (96) 2.48 2.18–2.65 (97) 1.73 1.44–2.14 (96) 15 8–31 (92) 203 94–488 (93) 2

1.33 1.24–1.39 (92) 2.47 2.25–2.65 (98) 1.77 1.32–2.20 (98) 14 7–37 (89) 210 86–745 (95) 3

Total calcium (mmol/L)

Phosphate (mmol/L)

Creatinine (μmol/L)

Alkaline phosphatase (U/L)

Alkaline phosphatase, % N 1000 U/L b a b

Whole blood ionized calcium. Transient hyperphosphatasemia as defined in the text, participants with values N1000 U/L were not included in the preceding line.

b LOQ (b 0.88 nmol/L) were not included in analyses; % b LOQ at 1 mo: 17%, 2 mo: 19%, 3 mo: 15%, 6 mo: 6%, 9 mo: 14%, and 12 mo: 23%. Urinary phosphate values are not presented, as the large majority of values for younger infants (b6 mo) were b LOQ of the assay (% b LOQ = 3.24 nmol/L, 1 mo: 73%, 2 mo: 90%, 3 mo: 88%, 6 mo: 45%, 9 mo: 4%, 12 mo: 0%). As a result, tubular reabsorption of phosphate could not be calculated with precision. Infants with transient hyperphosphatasemia, defined as plasma alkaline phosphatase N1000 U/L, were not included in the analysis due to the possibility of residual illness (2% of sample at 9 mo and 3% at 12 mo only, see Table 1). All analytes varied significantly from 1 to 12 mo of age (p b 0.05). Ionized calcium, total plasma calcium, phosphate and alkaline phosphatase all declined significantly over the course of the study, while creatinine increased significantly (Table 1). With the exception of urine calcium:creatinine, there was no effect of sex or 25(OH)D, allowing the data to be pooled. There was a small positive effect of increasing plasma 25(OH)D concentrations on urinary calcium:creatinine (β = 0.002, 95% CI: 0.000, 0.005). The median and range measurements of urinary calcium:creatinine were thus presented in Table 2 with and without stratification by plasma 25(OH)D above or below the median. However, it was clear that urine calcium:creatinine decreased significantly with increasing age. Except for a small difference in plasma phosphate at 6 mo between breastfed and mixed/formula fed infants (p b 0.01), there was no effect of feeding status on analytes. The smoothed percentile curves for all analytes are shown in Fig. 1a–e, which depicts centiles 3, 25, 50, 75 and 97.

Discussion Currently, there are no reports on normative clinical biochemistry for infants followed longitudinally in the first year of life. This study provides much needed mineral status data from healthy, primarily breastfed infants as assayed on the Beckman platform. These norms will aid in the assessment of infantile disorders. Most analytes decreased significantly over this 11 mo period. With the exception of urine calcium:creatinine, there were no sex or plasma 25(OH)D effects, which allowed us to unify the data across treatment groups. Ionized calcium was notably elevated at 1 mo with a range (1.29–1.52 mmol/L) that generally exceeds the upper limits for adults (1.15–1.29 mmol/L, Radiometer America). This decreased over time, but 25% of infants still exceeded centile 97.5 of reference ranges at 12 mo. Our concentrations were higher than those reported in the literature for neonates (Supplemental Table 1), prompting repeated testing and electrocardiography for signs of hypercalcemia [14]. All safety evaluations were normal and thus provided confidence that these results are likely normal physiological values. Similarly, total calcium concentrations were elevated compared to reference data for older children, with about 25% of children over the upper adult/child limits (2.23– 2.58 mmol/L, Beckman Coulter). The decline in total calcium occurred later than that in ionized calcium (Table 1). Plasma phosphate also decreased over time, particularly during the first 6 mo. Others have also described a decline from infancy to childhood [24]. This steady decline over the first year is consistent with changes in diet (switch from breastfed to formula fed) [25]. Phosphate

Table 2 Urinary calcium:creatinine (Ca:Cr) ratio for all infants over time. The data are presented as median and range (no. of samples tested) for low or high 25(OH)D concentration for each time point. Age (mo)

Urinary Ca:Cr (mmol:mmol), unstratified

Median 25(OH)D at each time point, nmol/L Urinary Ca/Cr below median 25(OH)D

Urinary Ca/Cr above median 25(OH)D

1

2

3

6

9

12

1.76 0.24–2.50 (119) 58.5 1.79 0.60–4.60 (52) 1.88 0.24–3.8 (52)

1.60 0.31–5.08 (104) 87.5 1.46 0.31–3.07 (51) 1.88 0.46–5.07 (46)

1.63 0.31–4.66 (105) 102.8 1.57 0.36–3.66 (51) 2.04 0.54–4.66 (50)

1.40 0.11–3.63 (106) 104.1 1.23 0.24–3.48 (50) 1.76 0.34–3.70 (50)

0.71 0.09–2.47 (86) 88.1 0.79 0.09–2.33 (49) 0.67 0.22–2.47 (49)

0.46 0.01–2.15 (94) 79.9 0.58 0.08–2.11 (48) 0.54 0.10–2.15 (48)

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Fig. 1. Reference percentile values of (a) ionized calcium (mmol/L), plasma (b) total calcium (mmol/L), (c) phosphate (mmol/L), (d) creatinine (μmol/L) and (e) alkaline phosphatase (U/L) of all age groups from 1 to 12 mo of age. Sample size ranged from 89 to 132 (see Tables 1–2). Curves were generated using the LMS method [18] in the GAMLSS [19] statistical package.

absorption is higher with human milk vs. formula; however, higher amounts of phosphate offset the lower mineral absorption of formula (cow or soy based) relative to human milk [26]. Although a significant increase (p b 0.01) in plasma phosphate in mixed/formula-feeding (1.94, 95% CI: 1.84–2.03 nmol/L, n = 12) vs. breastfeeding infants (1.77, 95% CI: 1.74–1.81 nmol/L, n = 95) was found at 6 mo, the proportion receiving mixed/formula feeds was small and these data are not applicable to exclusively formula fed infants. The decline in phosphate over time coincided with almost undetectable levels of urine phosphate; this may simply be a dilution effect (high urine volume), a relatively high LOQ, or a consequence of renal maturation. There are few reports of urine phosphate norms for this age group [27,28]. Alkaline phosphatase followed a similar trend in being relatively elevated in the first few months of life. The range we report is similar to that of other infants [29,30] and is several-fold higher than that of adult concentrations (32–91 U/L; Beckman Coulter). We noted several episodes of markedly elevated alkaline phosphatase (N 1000 U/L; hyperphosphatasemia), particularly in children 9 mo or older. We only measured total alkaline phosphatase and thus the relative contribution of bone and gastrointestinal isoforms is unclear. Elevated

concentrations resolved quickly and were often preceded by a transient viral illness, typical of this benign disorder [31,32]. Our rate of hyperphosphatasemia was 2% at 9 mo and 3% at 12 mo, similar to that of other reports (2.8%) [33]. There are likely numerous reasons for the observed changes over infancy and ranges that do not fully overlap with older children or adults. Concentrations for most analytes decreased over time, most apparent for ionized calcium. This may reflect non-fasting samples or it may be physiologic, as infancy represents a period of rapid bone growth and bone mass accrual [34]. The observed decline may also reflect the maturation of renal function [8]. On the other hand, plasma creatinine concentrations increased slightly over time with absolute values in keeping with other publications. Boer et al. [35] reported on a large number of children under 1 y of age and found a striking decline from birth to 100 d of life (~ 3 mo of age). Higher concentrations were seen with Roche enzymatic analyses across infancy [35,36; see Supplemental Table 1]. However, similar plasma creatinine concentrations were reported by Colantonio et al. [7] using an enzymatic technique on a different platform (Supplemental Table 1). The use of serum creatinine as a

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semi-quantitative method of assessing estimated glomerular filtration rate in this age group may need to be re-visited. The only analyte that appeared to vary with plasma 25(OH)D concentration was the urinary calcium:creatinine ratio. The effect of increasing 25(OH)D was small, but statistically significant. Our concentrations were significantly higher than those noted in other countries (Supplemental Table 1), prompting us to search for nephrocalcinosis [31]. All ultrasound results (n = 6) were normal. Higher concentrations might be related to vitamin D status, feeding method, or genetic variation (given that our sample was largely white). This study addresses a dearth of data from a large group of healthy, community-based infants followed longitudinally, with good retention, from 1 to 12 mo. The longitudinal nature of this study permitted robust statistical methods to determine the effect of age (i.e., maturation) compared to most data collections, which were cross-sectional. Feeding and vitamin D status were well described in this group and nearly all had adequate vitamin D status. This is particularly helpful when describing normal mineral status, unlike the study by Carpenter et al. [29] which looked at inner city children (15% were considered vitamin D insufficient). Moreover, we were able to confirm that for most analytes there was no association with plasma 25(OH)D and no effect of sex. Thus, this dataset – which examined changes in blood and urine minerals simultaneously – fills important gaps in knowledge, particularly for ionized calcium and for the Beckman (DxC600 model) and Radiometer platforms. The study was not without limitations, as the initial neonatal period (0–1 mo) was not captured; ethnic differences were not tested due to the homogeneity of the cohort. Ethnicity may not be an important covariate for some analytes (i.e., plasma calcium) [7], but it may be for others, including urinary calcium:creatinine [37,38]. A larger, more multiethnic population would permit reference intervals consistent with CLSI guidelines [6]. Our group was primarily breastfed from birth and hence breastfeeding rates in the present sample were higher than the Canadian average [39]. Thus, these results are not applicable to formula fed infants or those with inadequate vitamin D status (25(OH)D concentrations b 50 nmol/L). The study was longitudinal in nature. However, not all participants provided samples at all collection times due to sample attrition; nonetheless, the effect of time (i.e., maturation) was clear, as most analytes decreased with time. The number of values below the limit of quantification in this infant sample was high, particularly for the urine analytes creatinine, calcium, and phosphate. Given the potential for bias with standard methods for dealing with nondetectable values [40], better assays are required to develop normative ranges for these analytes in infants. Conclusion These data represent reference values for healthy, term, breastfed, Canadian infants collected prospectively from 1 to 12 mo of age. Most analytes decreased with time and were not associated with sex or vitamin D status. Concentrations for some were higher than those reported in the literature, possibly reflecting differences in platform, feeding, genetics and vitamin D status compared to earlier studies [1,2,7,9,40]. Additional samples will be required to generate complete reference intervals per CLSI guidelines for the Beckman and Radiometer platforms (ionized calcium). Nonetheless, our study provides helpful insights. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.clinbiochem.2014.07.012. Acknowledgments and funding This work was supported by the Canadian Institutes of Health Research (MOP-82763), Nutricia Research Foundation and the Canadian Foundation for Innovation (Hope Weiler). The authors wish to thank

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Drs. Fabienne Parente and David Blank, Ms. Kristen Mckenzie and staff from the Montreal Children's central laboratory for help with this study.

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