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. 2018 Jul 17;597(5):1311–1319. doi: 10.1113/JP275452

Using stable isotope tracers to study bone metabolism in children

Kimberly O O'Brien 1, Steven A Abrams 2,✉
PMCID: PMC6395421  PMID: 29869788

Abstract

Skeletal mineralization is initiated in utero and continues throughout childhood and adolescence. During these key periods of the life cycle, calcium retention must increase significantly to provide sufficient mineral for bone deposition and skeletal growth. Stable calcium isotopes have served as a fundamental tool to non‐invasively characterize the dynamic changes in calcium physiology that occur from infancy through adolescence. These approaches have helped define the dynamics of calcium absorption and utilization in healthy children and in children with chronic diseases. As data in this area have accumulated, new areas of emphasis are beginning to characterize the determinants of variability in mineral retention, the genetic determinants of bone turnover and calcium flux and the impact of the gut microbiome on whole body and niche specific calcium dynamics. Advances in these areas will help define calcium utilization in paediatric populations and provide information that may be useful in maximizing bone acquisition across this critical phase of the life cycle.

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Keywords: nutrient absorption, inflammation, vitamin D, microbiome, kinetics

Introduction

Childhood and adolescence are key life cycle stages that are integral to skeletal growth and mineralization. Peak skeletal calcium accretion occurs at approximately 12.5 years of age in females and at approximately 14.0 years of age in males (Bailey et al. 2000). At these ages roughly 300–400 mg of calcium per day are deposited into the growing skeleton (Bailey et al. 2000). Marked alterations in calcium physiology occur to minimize urinary losses, maximize intestinal calcium absorption and increase skeletal calcium retention in support of bone mineralization. The importance of this age period can be appreciated when one considers approximately one‐quarter of an adolescent's skeletal calcium content is accumulated within the 2‐year period of peak skeletal growth (Bailey et al. 2000). Approaches that characterize calcium dynamics in healthy paediatric populations are needed to help define optimal calcium intakes and to elucidate modifiable and non‐modifiable factors that influence calcium retention. These studies are particularly important in children with chronic diseases that often adversely impact calcium absorption and the ability to retain and deposit calcium into the growing skeleton. Studies characterizing normal and pathological changes in calcium metabolism in paediatric populations require techniques that allow calcium dynamics to be characterized without overly invasive or potentially harmful approaches. Early paediatric and adult studies utilized calcium radioisotopes as tracers to begin to characterize in vivo calcium dynamics. These approaches were pioneered by the late Dr Robert Heaney (Weaver & Lappe, 2017) and by the late Dr Felix Bronner (Bronner et al. 1954; Hall et al. 1969). Studies using calcium radiotracers are no longer widely employed to evaluate calcium absorption or metabolism in children. Approximately four decades ago, Dr Alfred Yergey and Dr James Hansen began using stable calcium isotopes and mathematical modelling to further characterize calcium absorption and rates of bone turnover in paediatric populations (Yergey et al. 1980; Moore et al. 1985). These stable calcium isotopic approaches have now been safely used in thousands of healthy children, and the technique has been employed to evaluate perturbations in calcium physiology that occur in children with disorders known to impact bone quality and calcium retention (Abrams, 1994, 1999, 2001; O'Brien & Abrams, 1994; Abrams et al. 1995b, 2000; Wastney et al. 1996; Wu et al. 2010).

Dietary calcium intake and absorption

Calcium is a threshold nutrient. Until this threshold is achieved, higher dietary calcium intakes provide additional absorbed calcium that can be utilized to support the rapid bone growth that occurs across gestation and throughout the pubertal growth period. In spite of the importance of this nutrient for bone health, low dietary intakes are common among children with chronic illnesses. While important in the management and identification of calcium flux in paediatric populations, assessing dietary calcium intake can be difficult clinically. In the United States, more than half of the calcium intake in children is obtained from milk and other dairy products (Institute of Medicine, 2011). However, intake of these foods is often challenging to quantify as milk and dairy products can be ingested as components of mixed meals and the concentration of calcium may be highly variable in different cheeses and yogurts. Research studies evaluating calcium metabolism need to accurately evaluate calcium intake and control this variable over the course of the study. Calcium absorption efficiency is generally not directly affected by bone mineral mass or serum calcium, but is affected by the chemical form of the calcium, the calcium load of the meal and the overall dietary intake and composition (Heaney, 2001). It is also greatly affected by genetic factors including race (Abrams et al. 1995a; McCormack et al. 2017). Among dietary factors, much of the interest in this topic has focused on the chemical form of calcium ingested, with considerable research using stable isotopes to look at different anions as well as calcium absorption from cow's milk, human milk and the bioavailability from vegetables (Zhao et al. 2005; Charoenkiatkul et al. 2008; Abrams, 2010). The general reference standard for these measurements is the bioavailability of calcium from cow's milk. However, even this value must be evaluated in relation to the calcium load ingested (O'Brien et al. 1996a), and to the pubertal status of the child (Abrams, 1993; Jackman et al. 1997).

Dietary calcium intakes are suboptimal in many areas of the world where dairy products are not routinely ingested. Stable calcium isotopes may be used in these settings to evaluate the degree to which calcium absorption is impacted by habitually low calcium intakes or by alterations in vitamin D status. Nigeria is one international setting where dietary calcium intakes are low and rickets is prevalent. Several paediatric stable calcium isotope studies have been undertaken to evaluate factors that may influence calcium absorption in Nigerian children. Using this approach, enhanced calcium absorption was found in children ingesting a traditional meal of maize porridge compared to calcium absorption measured without this meal (Thacher et al. 2009a). Similar approaches were used to show vitamin D supplementation had no significant effect on calcium absorption in Nigerian children (Thacher et al. 2009b), but intestinal calcium absorption was significantly increased in Nigerian children after active rickets was resolved (Graff et al. 2004).

One key issue in paediatrics is the potential impact of vitamin D status on calcium absorption. Given the concern with vitamin D insufficiency among the US population, it is of great interest to determine whether 25(OH)D status in healthy children is associated with fractional absorption of calcium, and if so, if there is an optimal concentration of this vitamin that maximizes intestinal absorption of calcium (Abrams et al. 2009). To address this question, data from 439 children who had completed stable calcium isotope studies were pooled. This analysis found no substantial effect of 25(OH)D values >28 nmol L−1 (11 ng mL−1) on calcium absorption when assuming a calcium intake of at least 600 mg day−1, an amount that is well below recommended calcium intakes (Abrams et al. 2009).

A central use of isotope‐based calcium absorption studies in healthy children is to provide data that can inform the dietary reference intakes for calcium and potentially for vitamin D. We have reviewed data on calcium bioavailability in children (Abrams, 2010), and have utilized these findings to demonstrate how these data may be used to develop dietary guidelines for healthy children (Lynch et al. 2007). Stable calcium isotopes have been utilized as tracers to evaluate the relationship between calcium intake and calcium absorption and calcium retention by the skeleton (Abrams et al. 1992; Jackman et al. 1997). Combining these data with other information, such as whole body bone mineral density measured by dual energy x‐ray absorptiometry (DXA), usual rates of calcium accretion to the skeleton can be calculated. These types of calculations are subject to substantial limitations based on variability in factors including genotype, overall composition of the diet and data accuracy. Nonetheless, use of data from stable isotope based studies has supplanted much earlier data that were compiled using mass balance techniques as detailed in the recent dietary reference intake recommendations for calcium and vitamin D (Institute of Medicine, 2011).

It is often assumed that calcium malabsorption is the cause of bone deficits observed in paediatric diseases such as cystic fibrosis and juvenile diabetes, but there is limited evidence to substantiate this hypothesis. Stable calcium isotopes provide a means of directly evaluating the potential impact of paediatric disease on intestinal calcium absorption. Using oral and intravenous stable calcium isotopes, calcium absorption was evaluated in a group of adolescent females with cystic fibrosis and was found to be similar to that reported among females of the same pubertal stage (Schulze et al. 2003). A similar finding was recently made in females with juvenile diabetes in whom calcium absorption did not appear to be lower than predicted based on reference data using the same stable isotopic approach (Weber et al. 2017). These types of studies have highlighted the need to further explore individual disease processes and their impact on other regulators of calcium metabolism and bone calcium turnover to inform targeted treatment regimens.

Non‐disease‐related conditions, such as adolescent pregnancy, may also modify calcium requirements and calcium absorption in paediatric populations. Teen pregnancy continues to be a public health concern in many areas of the world. Stable calcium isotopes have been used to evaluate the ability of adolescents to respond to the physiological challenges of pregnancy and to determine if calcium absorption and rates of bone calcium deposition are altered in relation to calcium intake, vitamin D status or gynaecological age. To date there has been one study using oral and intravenous stable calcium isotopes in pregnant US teens to evaluate calcium absorption (O'Brien et al. 2003) and calcium kinetics (O'Brien et al. 2012). There was no significant effect of maternal age on calcium absorption (across the range of 13–18 years), nor was calcium absorption higher than reported among adult women consuming similar calcium intakes. Urinary calcium excretion in the pregnant adolescents was also similar to that reported among adults suggesting that there were no unique physiological adaptations that would have allowed the pregnant adolescents to retain more calcium in support of their own continued bone consolidation and to support fetal bone mineralization (O'Brien et al. 2003). Longitudinal studies using heel ultrasound measures have reported that adolescents lose trabecular bone across gestation (Whisner et al. 2014), and this loss appears greater in adolescents when compared to adult women (Sowers et al. 2000). Low dairy or calcium intake and low maternal vitamin D status have all been associated with significant reductions in fetal long bone growth in pregnant adolescents (Chang et al. 2003; Young et al. 2012). Additional studies are needed to determine if there are permanent deficits in bone mass in teens that become pregnant prior to acquisition of peak bone mass or if the in utero competition for calcium leads to any maladaptive developmental programming effects in offspring born to skeletally immature females.

Assessment of bone acquisition and status

Longitudinal studies using DXA have evaluated changes in bone mineral content as peak bone mass is accrued (McKay et al. 1998; Bailey et al. 2000; Baxter‐Jones et al. 2011; McCormack et al. 2017). Due to the magnitude of bone acquisition that occurs across childhood, any chronic disorder or disease that limits absorption and retention of calcium can have a long‐term detrimental impact on risk of low bone mass and fracture.

Initially, bone health received relatively little attention in children with chronic diseases due to the other challenges associated with acute disease management and the limited adult lifespan expected for many children with significant illness. Long‐term survival is now common for many conditions such as childhood cancers, cystic fibrosis, HIV and others, leading to a renewed interest in bone complications that could impact these children. When evaluated, suboptimal bone acquisition is often reported in children with chronic illnesses (O'Brien et al. 2001; Abrams & O'Brien, 2004; Schulze et al. 2006).

Deficits in bone mass are frequently multifactorial and appropriate therapies must address the underlying mechanisms to be most effective. Some paediatric diseases would be expected to directly affect calcium absorption such as observed in intestinal diseases including short gut syndrome, and other intestinal failure conditions (Pichler et al. 2013). There are others where the disease therapies themselves adversely impact bone, such as the use of steroids and other medications in many conditions including severe asthma, cystic fibrosis and cancer (Schulze et al. 2004, 2006; Abrams, 2008). Moreover, with some paediatric diseases, the longer term consequences of the underlying condition on bone health may take many years to present, such as cerebral palsy and other neurological conditions, making clinical practice guidelines for bone health in these diseases challenging to develop (Ozel et al. 2016).

Other paediatric diseases influence retention of absorbed calcium via increased losses of calcium through endogenous faecal secretion (cystic fibrosis) (Schulze et al. 2006). Often these diseases and their comorbidities involve multiple abnormalities; for example, a child with severe cerebral palsy may receive medications including anti‐epileptics that affect mineral metabolism, may have less sunshine exposure leading to vitamin D deficiency and may have abnormalities in gait leading to a greater risk of bone disease from reductions in weight‐bearing activities.

Urinary calcium excretion

Fasting urinary calcium excretion (evaluated using urinary calcium to creatinine ratios) is highly variable and has been found to capture a significant amount of the variability in calcium absorption efficiency (Barger‐Lux et al. 1995). During the growth period, urinary calcium losses are not significantly related to dietary calcium intake across the range of typical calcium intakes, as the additional absorbed calcium is utilized for bone calcium accretion (O'Brien et al. 1996b). However, similar to data in adults, even among healthy children there is a large degree of variability in daily urinary calcium excretion (O'Brien et al. 1996b). Because of the large load of calcium that is filtered each day by the kidney, paediatric diseases that alter kidney function or other aspects of renal calcium handling will likely have a significant impact on net calcium retention.

The prevalence of paediatric end stage renal disease has increased over the past three decades (Kaspar et al. 2016). To date, use of stable isotopic methodology in children with chronic kidney disease (CKD) has been challenging due to issues related to the use and type of dialysis, which may necessitate methodological changes to the calcium protocols commonly employed among children. We recently evaluated the use of the dual tracer stable isotope method to assess calcium absorption in patients receiving peritoneal dialysis using both urine and serum collection methods (Ware et al. 2016). A lower than predicted enrichment of oral tracer in urine and possible differences in peak absorption times were evident in these children receiving dialysis (Ware et al. 2016). This may suggest possible alterations in the magnitude and timing of calcium absorption perhaps due to other nutrient deficiencies such as vitamin D, or to other gut abnormalities that may occur in kids receiving chronic dialysis. These preliminary findings suggest that larger doses may be needed in children receiving dialysis (Ware et al. 2016).

Most disease processes affecting bone metabolism are multifactorial and not a consequence of primary endocrine disorders. For example, CKD causes numerous abnormalities in bone metabolism and is associated with metabolic bone disease. Longitudinal studies of children with CKD have reported deficits in whole body bone mineral content z‐scores (using DXA) and cortical bone mineral content z‐scores (using peripheral quantitative computed tomography) (Griffin et al. 2012). Without additional data the causes of these deficits in bone deposition are difficult to identify and treat. Further studies are needed to define and optimize stable calcium isotopic methodology for paediatric populations with renal disease as well as to expand use of this technique to otherwise difficult to assess paediatric populations.

The microbiome and calcium metabolism

The human microbiome is increasingly linked to nutrient physiology and homeostasis. The term osteomicrobiology has now been coined to encompass the role of the microbiota in skeletal health including skeletal development and bone turnover (Jones et al. 2018). Composition of the gut microbiome influences gut permeability and gut inflammatory status and thereby impacts the availability of calcium needed for bone turnover. Many paediatric diseases have been associated with dysbiotic changes in the gut microbiome that may ultimately impact calcium absorption, retention and availability for bone deposition (Videhult & West, 2016). Nutritional therapies have been employed in healthy paediatric populations to alter the gut microbiota in support of increased calcium absorption and skeletal health (Holloway et al. 2007; Whisner, 2013; Whisner et al. 2016). Additional challenges occur when evaluating these relationships in chronically ill paediatric populations due to the many other factors, such as drug therapy, disease management and dietary alterations, that can also influence study outcomes. Stable calcium isotopes provide a means of further evaluating the impact of the microbiome on calcium utilization in children and these studies may be particularly important in children with disease‐related alterations that cause dysbiotic shifts in the gut microbiome that may alter nutrient utilization and systemic inflammatory status.

Developmental programming, calcium metabolism and bone health

The in utero environment is an important determinant of organ, brain and skeletal development and subsequent risk of chronic disease. A greater awareness of the importance of this life stage on long‐term bone outcomes has stimulated interest in techniques that can provide information on fetal utilization of calcium and vitamin D and of factors that impact fetal skeletal development. Developmental programming is also impacted by placental function and ability to traffic calcium, vitamin D and other skeletally important nutrients across the placenta to the fetus. The National Institutes of Health has recently prioritized this area of research and has set aside funding to study the role of the human placenta in health and disease (Guttmacher & Spong, 2015; Human Placenta Project, 2018). To date, little is known about how calcium or vitamin D is transferred across the human placenta or what maternal or fetal factors may regulate this process. Safe and relatively non‐invasive approaches are needed to answer these questions. At present fetal biomarkers can only realistically be evaluated at birth in the newborn using umbilical cord blood. In spite of these challenges stable calcium isotopic approaches have been applied to evaluate the determinants of calcium transfer from the maternal to the fetal circulation. For example, oral and intravenous stable calcium isotopes were administered to pregnant adolescents when in active labour, umbilical cord blood and placental tissue were obtained at birth, and enrichment of these two calcium tracers in the neonate was evaluated in relation to fetal biometry data and placental expression of vitamin D receptor (Young et al. 2014). This approach identified a significant association between enrichment of the intravenous stable calcium isotope in the neonate at birth with the expression of the vitamin D receptor in placental tissue, and with in utero measures of the fetal femur z‐score (Young et al. 2014). These techniques can be further utilized to provide key data on determinants of maternal–fetal calcium and vitamin D partitioning that may help identify the functional roles of proteins involved in calcium trafficking to the fetus and identify maternal and neonatal factors in this process.

Genetics, genetic disorders and calcium and vitamin D metabolism

Bone mass is primarily under the control of genetic factors with heritable factors explaining approximately 60–80% of the variability in bone mass (Heaney et al. 2000; Karasik et al. 2016). The majority of bone is obtained during childhood highlighting the need to characterize genetic factors that may contribute to paediatric bone phenotypes (Mitchell et al. 2016). As these genetic factors are identified, stable calcium isotope methods can evaluate the functional impact of these genotypes on parameters of calcium absorption and utilization. As examples of this approach, paediatric studies have evaluated the influence of race on calcium absorption and bone turnover in children (Abrams et al. 1995b; Bryant et al. 2003; Wu et al. 2010), and the impact of vitamin D receptor polymorphisms on calcium absorption (Ames et al. 1999).

A novel area of recent research is the use of stable calcium isotopes to evaluate calcium absorption in children with rare endocrine disorders associated with altered vitamin D metabolism. Several genetic disorders in the cytochrome P450 enzymes that activate and catabolize vitamin D have been identified (Carpenter, 2017; Thacher & Levine, 2017; Roizen et al. 2018). These disorders lead to abnormally low levels of calcitriol (such as mutations in CYP2R1 or CYP27B1) or impact the catabolism of 25(OH)D and calcitriol (CYP24A1) and can lead to persistently elevated concentrations of calcitriol (Dauber et al. 2012; Carpenter, 2017). Mutations in enzymes that activate or catabolize vitamin D may enhance or impair calcium absorption; stable isotopic approaches allow these effects to be examined and quantified. We recently studied a group of children in Israel with hereditary vitamin D‐resistant rickets and found that calcium absorption was vitamin D dependent in infancy until the end of puberty, after which there was a period of about 10 years in which other non‐vitamin D‐dependent mechanisms appeared to be primarily involved in calcium absorption (Tiosano et al. 2011). Similarly, stable calcium isotopic approaches have been used to help evaluate a child that presented with severe early life hypercalcaemia and was found to have a rare genetic defect in the vitamin D 24‐hydroxylase gene (CYP24A1) (Dauber et al. 2012). Using stable isotopic methodology, this child's mutation was found to be associated with marked increases in calcium absorption (Dauber et al. 2012). These approaches allow the impact of the defect on calcium physiology to be quantified and provide opportunities to subsequently evaluate the efficacy of interventions used to treat the calcium‐related consequences of these genetic disorders.

Summary

Skeletal growth and mineralization are regulated by myriad dietary, genetic and environmental factors. For decades stable calcium isotopes have helped characterize calcium physiology and bone turnover dynamics in healthy and chronically ill paediatric populations. Findings from these studies have also helped inform national dietary guidelines and treatment regimens for children suffering from chronic disease. As knowledge of paediatric bone acquisition evolves, and new metabolic and diagnostic approaches are developed, integration of these multidisciplinary approaches will continue to inform the literature in support of paediatric bone health.

Additional information

Competing interests

Neither of the authors have any conflicts of interest to disclose with respect to this submission.

Author contributions

Both authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

In memoriam

This review is presented in memory of Dr Alfred (Al) Yergey, who died on 27 May 2018 at the age of 76. Al spent four decades at the NIH developing the field of mass spectrometry for use in human subjects, especially children. He was the laboratory mentor for both K.O.O. and S.A.A. and was a true pioneer in the field whose visionary research led to changes in how children are cared for throughout the world.

Biography

Steven Abrams obtained his medical degree at Ohio State University and completed a neonatology fellowship at Texas Children's Hospital. He is the inaugural Chair of the Department of Pediatrics at the Dell Medical School at the University of Texas at Austin. Kimberly O'Brien obtained her doctorate in nutrition at the University of Connecticut and is currently a professor of human nutrition at Cornell University. They met while completing fellowships in Dr Alfred Yergey's laboratory at the National Institutes of Health where under his guidance they discovered the utility of stable mineral isotopes. S.A. is a world expert in the use of this technology in pediatric populations and K.O.O.’s research has employed these techniques in pediatric and higher risk obstetric populations. These stable isotopic approaches have identified new aspects of calcium utilization and have helped inform dietary guidelines and treatment regimens for pediatric populations.

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Edited by: Ole Petersen & Troy Hornberger

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