Introduction

Bone has a very dynamic metabolism with continuous bone formation and resorption processes occurring, at different rates, throughout the entire life. In children and adolescents, bone formation is particularly predominant to respond to skeletal growth [1]. Teenage years are characterized by a progressive increase of sex hormones and the appearance of secondary sexual characteristics leading to a peak of bone mineral density (BMD) at Tanner stage III [2]. Once this achieved, BMD normally remains relatively stable in young adults by maintaining a balance between time- and space-coordinated bone formation and resorption. This equilibrium progressively vanishes with age, and particularly at menopause, to be replaced by a dominant bone resorption process [3].

Serum markers of bone turnover are designed to reflect the systemic activity of the osteoblast or of the osteoclast system. As these markers originate from the entire skeleton, they reflect the cumulative activity of bone cells from all skeletal locations (e.g., axial skeleton, appendicular skeleton) and from all skeletal compartments (e.g., cortical bone, trabecular bone). Biochemical markers of bone metabolism are, therefore, mostly used to assess systemically active disorders. Nevertheless, local processes, such as fracture healing, can influence systemic bone marker levels if they are sufficiently metabolically active or involve a sufficient amount of bone tissue [4]. While imaging and DXA measurements remain the gold standards to diagnose bone diseases in adults, bone turnover markers (BTMs) have found a place in the follow-up of treatment of diseases such as osteoporosis [5]. For the time being, in pediatrics, BTMs remain mainly research tools [6, 7]. However, there are genetic, chronic, or therapy-induced bone disorders that may require BMD and BTMs measurements. Indeed, disorders that may concern both adults and children such as Paget disease, renal osteodystrophy, or glucocorticoid-induced osteoporosis are requiring a close follow-up [8].

From a laboratory point of view, a typical bone metabolism investigation is composed of creatinine, calcium, phosphate, parathyroid hormone (PTH), and 25-hydroxy-vitamin D measurements [6]. BTMs such as osteocalcin (OC) or bone alkaline phosphatase (BALP) are considered as different entities kept for specific investigations. BTMs are usually considered as either bone formation or bone resorption markers. They can further be categorized in collagenous or non-collagenous markers (Fig. 1). Noticeably, age, gender, and ethnicity are major peak BMD determinants, and by extension of BTMs. Historically, most of BTMs measurements were developed in urine samples [9]. However, blood samples being easier to handle and facing less pre-analytical variations, serum, or plasma BTMs are now preferred in laboratory medicine [5].

Fig. 1
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BTMs: Pre-analytical impacts and general expression profile

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Initially developed to study adult bone diseases such as osteoporosis, BTM measurement in children faces additional challenges, the establishment of pediatric reference values being an important one. Indeed, the establishment of proper reference ranges requires to handle very large cohorts [10, 11]. Besides the difficulty of recruiting large cohorts of children, the ethical issue of blood collection in very young infants and toddlers for research purposes should not be underestimated [12]. Finally, because reference values are assay dependent, these studies need to be regularly up-dated to fit with the new laboratory devices [13].

Since definition of relevant blood biomarkers includes adequate reference ranges, a PRISMA approach was performed to search for articles providing pediatric reference ranges for BTMs. Pubmed and Embase databases were searched using “children or adolescent”; “bone turnover markers”; and “reference ranges” as terms. We found 181 records from which 82 were directly removed being either duplicates, published in languages other than English or non-research articles. Out of the 99 remaining records, 84 were excluded based on the following criteria “cohort of unhealthy individuals,” “cohorts too small (< 120 individuals),” “inappropriate age of the cohort,” “urine samples,” “insufficiently described method,” “inappropriate calculation of reference ranges.” Table 1 summarizes the pediatric reference ranges reported in the 15 accepted articles. This review will discuss on blood biomarkers identified using this approach. The first part will focus on the expression profile of each BTM and their physiological variations and the second part their variations in pathological conditions.

Table 1 Studies providing pediatric reference ranges
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Bone Turnover Markers: Physiological Insights and Measurement

Procollagen Type I N-Propeptide (PINP)

Procollagen type I-N-propeptide (PINP) is a 35 kDa bone formation marker. PINP is cleaved from extracellular matrix procollagen type I molecules secreted by osteoblasts before their assembly into organized fibers. Procollagen type I-C-propeptide (PICP), is its C-terminal counterpart. Although both show similar physio-pathological characteristics, PINP has taken the lead in the field. Indeed, as no automated method have been developed for PICP, PINP has been defined as the preferred bone formation marker for the follow-up of postmenopausal women by the National Bone Alliance in collaboration with the American Association for Clinical Chemistry and by the IFCC-IOF joint Committee for Bone Markers (C-BM) [14, 15]. These PINP automated methods have recently been shown to provide harmonized results [16, 17].

Several studies have established pediatric reference intervals for PINP [2, 18,19,20,21] but data on PICP are much more scarce [22]. In spite of this, studies demonstrate a nonlinear relationship between age and PINP or PICP concentrations (Fig. 1). From birth until 3–4 years, PINP concentration decreases. It then stabilizes or slightly increases until the mid-puberty where it starts a new decrease. There are several links between puberty and PINP levels. First, PINP pubertal peak is earlier in girls than in boys, in line with the earlier onset of puberty for girls [21]. Second, PINP pubertal peak matches with the II/III Tanner stages of breast development in girls and the III Tanner stage of genital development in boys [2]. Finally, correlation has been made between PINP levels, testosterone, and Ghrelin concentration in boys [23].

Whether there is an association between PINP and growth velocity or bone mineral content (BMC) is still under debate. PINP was correlated to leg growth velocity in very low-birth-weight infants [24] but studies in older healthy children are lacking. Regarding BMC, Van Coeverden and colleagues showed a PINP direct correlation with BMC at four different sites in boys but not in girls [25]. Still, a correlation between low bone mineral density (BMD), PINP, and amenorrhea was observed in adolescent athlete girls [26]. More recently, Zürcher and colleagues showed that, although PINP was correlated with BMC, it explained a small percentage (Pearson coefficient varying from 0.13 to 0.24 depending of the anatomical site) of bone mineral changes [27].

PINP is present in serum either as a trimer (“intact”) or as a monomer. Assays that detect only the trimeric form are called intact assays (e.g., IDS intact PINP) whereas assays that detect both monomeric and trimeric forms are defined as total assays (e.g., Roche P1NP assay). The monomeric form is cleared by the kidneys and the trimeric forms by the hepatic endothelium, it follows that liver or kidney diseases will impact the interpretation of the results depending on the type of assay [28]. Hence, in patients suffering from chronic-kidney diseases, PINP concentrations will be particularly overestimated when measured with the total assay [29]. Procedures to limit pre-analytical impacts on PINP measurement state a limited impact of fasting and circadian rhythm-related blood procurement [30].

Total Alkaline Phosphatase (ALP) and Bone-Specific Alkaline Phosphatase (BALP)

Total alkaline phosphatase (ALP) combines four isoforms: the placental, the germ cell, the intestinal, and the tissue-nonspecific isoforms (expressed in liver, bone, and kidney) [31]. Bone-specific alkaline phosphatase (BALP) differs from the kidney and the liver isoforms by post-translational modifications. BALP, itself, can be further categorized in more isoforms (sometimes called B/I, B1, B1x, and B2) [32, 33]. As ALP combines all these isoforms its specificity to bone diseases is relatively limited. Therefore, total alkaline phosphatase is not recommended for the management of postmenopausal osteoporosis [5]. However, in children, given the low prevalence of hepatic diseases, its use has been recommended by the European Society for Paediatric Nephrology CKD-MBD and Dialysis working groups and CKD-MBD working group of the ERA-EDTA [6].

BALP, as its name suggests, is an enzyme active at alkaline pH that participates to the growth of hydroxyapatite crystals through the hydrolysis of pyridoxal phosphate (an inhibitor of the hydroxyapatite formation) [31]. BALP is also responsible for the inactivation of the calcification inhibitor osteopontin [34]. BALP is released in the blood stream after disruption of its membrane anchorage to the osteoblasts [31]. Thus, BALP activity is increased in physiological or pathological processes that involve high osteoblastic activity [35]. Therefore, it is commonly accepted as a non-collagenous biomarker of bone formation.

ALP and BALP concentrations during childhood and puberty show the same trends as PINP concentration with a valley during childhood and a peak at beginning/mid-puberty (Fig. 1) [20, 22, 36,37,38,39]. As data are scarce in infants, the contribution of the placental isoform to the level of ALP in the 0–1 year olds is unclear. Although ALP and BALP values are the same for both genders during infancy, pubertal peak is higher and occurs later in boys [38, 39]. Thus, ALP and BALP concentrations are clearly age-related although BALP, on its own, is not a good age predictor in boys [40]. Noticeably, cases of transient hyperphosphatasemia have been reported in infants under 5 years old [41, 42]. In these cases, ALP including BALP and liver isoforms is transiently increased but return to normal after a few weeks without any symptoms.

As PINP, BALP is also tightly associated with height gain velocity [36]. Still, the proportion of BALP isoforms identified by HPLC varies during puberty [43] suggesting a different impact of each isoforms in matrix mineralization.

Bone metabolism can be modulated during childhood by environmental and anthropometric factors [44,45,46,47]. Notably, weight-loss interventions have shown modifications in BALP activity associated with modified BMD [48]. Because Body Mass Index (BMI) and Vitamin D are tightly linked, the question of an association between BALP concentration and Vitamin D has also emerged. Yet, the existence of an association between BALP concentration and Vitamin D remains unclear. Indeed, many interventional studies have BMD as outcome rather than bone biomarkers [46], and observational studies based on specific diet have shown contradicting results. For example, two studies conducted by the same group 7 years apart, reported decreased BALP in vegetarian children and increased BALP activity in children with cow’s milk allergy, both groups with deficits in nutritional calcium and vitamin D intakes [49, 50]. A Danish study conducted in adults showed higher BALP and PINP concentrations in vegans compared to omnivore but no differences in β-CTX and osteocalcin (OC) [51].

From an analytical point of view, ALP is most frequently measured through enzymatic assay based on the hydrolysis of para-nitrophenyl phosphate. This method based on chemical properties of ALP is not isoform specific. Regarding BALP, there are three kinds of BALP assays. (1) Pure enzymatic assays that measure the activity of the enzyme (e.g., Quidel Microvue), (2) immunoassays that measure the mass of the enzyme (e.g., Diasorin Liaison Ostase) and (3) assays that measure activity of the enzyme but provide results in mass units after calibration (e.g., IDS iSYS and Beckman-Coulter Access Ostase) [52]. In addition, high-performance liquid chromatography (HPLC) is sometimes used to investigate specifically BALP isoforms [53]. Because of the high homology between tissue nonspecific isoforms, current immunoassays exhibit different degrees of cross-reactivity with liver alkaline phosphatase [54]. Thus, caution is needed when interpreting results from patients with severe hepatic diseases. Yet, as BALP is almost not influenced by the renal function, BALP was defined as the biomarker of choice in hemodialyzed patient [55]. From a pre-analytical point of view, fasting or circadian rhythm does not impact blood BALP values [56].

Osteocalcin (OC)

Osteocalcin, also called bone Gla protein, is a 49 kDa protein secreted by osteoblasts into the bone matrix where it is bound to the hydroxyapatite crystals through its three γ-carboxyglutamate residues. The carboxylation of these three glutamate residues is Vitamin K-dependent and is modulated by anti-Vitamin K drugs (e.g., warfarin) [57]. The fraction of OC that enters the blood circulation is dependent on the OC carboxylation levels. In the circulation, the uncarboxylated form of OC has a role in glucose metabolism [58]. Still, because secreted OC correlates to histomorphometric bone formation measurements [59], OC is generally considered as a non-collagenous bone formation marker. Yet, caution must be exercised since a fraction of bone matrix-bound OC can be released by osteoclasts during bone resorption, impact of which is still controversial [54].

Data regarding OC expression profiles vary in the literature. While some found it quite stable [36], other have shown that OC is elevated during infancy, decreases during childhood before reaching a peak at early puberty [2, 20, 21] (Fig. 1). Contrary to PINP and BALP that have the same values for boys and girls during childhood, OC levels may be slightly lower in boys during that period. Moreover, the ratio between the uncarboxylated and the carboxylated OC forms is also increased in children compared to adults [60]. Given the role of the uncarboxylated form of OC in glucose metabolism, the clinical significance of an elevated ratio in children remains controversial and might only be due to a distinct bone/glucose homeostasis during childhood. In adults, this ratio is usually admitted to reflect Vitamin K bone status and suggest that individuals with higher levels of uncarboxylated OC might be Vitamin K deficient [61]. While a meta-analysis has shown an inverse relationship between Vitamin K1 intakes and risk of fractures [62], interventional studies with Vitamin K supplementation show conflicting results [63].

Still, because OC is implicated in glucose metabolism, several studies focused on its expression in overweight individuals. OC tends to be diminished in overweight postmenopausal women and elderly men [64,65,66,67]. In children, BMI and homeostasis model assessment of insulin resistance (HOMA-IR) were found to be independent predictors of serum OC concentration together with age [67]. Also, in children, an OC concentration < 44.5 ng/ml was reported to be a good predictor of the progression of non-alcoholic steatosis, a disease typically linked to obesity [68]. Of note, children with glucocorticoid-induced osteoporosis show lower OC concentration [69]. However, whether this decrease depends on the action of glucocorticoids on glucose metabolism or on bone homeostasis remains unclear.

From an analytical point of view, OC is an unstable protein. To circumvent this problem, standardized pre-analytical settings are recommended [54]. In addition, the C-terminal sequence of OC being the least stable, immunoassays targeting the NH2-terminal mid-fragment (N-mid OC) are definitively preferred [70] and are now commonly used in routine. Furthermore, this more stable fragment accumulates less in uremic samples compared to the entire protein [71, 72].

Cross-Linked Telopeptides

Pyridinoline- and deoxypyrydinoline-Cross-linked telopeptides are bone resorption markers derived from the type I collagen that represents more than 90% of the bone matrix collagen. These telopeptides are classified as NTXs or CTXs depending whether they derive from the N- or C-terminal end. While CTXs are released from collagen through cathepsin K-mediated cleavage, the C-terminal cross-linked telopeptides of type I collagen (ICTP) are derived from metalloproteinase digestion [73]. Since the original radioimmunoassay developed for ICTP [74] has never been automated, its usefulness in routine laboratories is limited and recent pediatric studies are scarce. Therefore, nowadays, assays that measure CTXs, regardless of the isomers are grouped under the generic name of CrossLaps and are considered as the reference resorption biomarker for osteoporosis in postmenopausal women [14, 15]. The most currently used automated assays have antibodies against β-CTX epitopes. As reported by Chubb et al. [75], there is significant disagreement and limited commutability between the different methods, impeding their value as a clinical tool. The IFCC-IOF Committee for Bone Metabolism recently reinforced these observations and, in addition, showed substantial within- and between-assay variation, concluding that harmonization is a must [76].

In terms of development, CTX expression profile during childhood differs from PINP and ICTP, not showing a peak in early infancy. During childhood, CTX expression is relatively stable, slightly increasing to reach a peak at early puberty followed by a decrease [18, 20, 21, 36, 77] (Fig. 1).

Several possible determinants of CTX levels have been assessed. First, β-CTX is independently associated with age and height in children [14, 28, 67]. This is also the case for girls with central precocious puberty [78]. Yet, contrarily to PINP, no association with BMC or DXA measurements was found [27, 79]. Vitamin D is another possible determinant as a season-related inverse relationship between serum 25-hydroxyvitamin D (25(OH)D) and CTX concentrations has been observed after adjustment for several confounding factors [80]. Interventional studies have also confirmed this association by showing that Vitamin D supplementation could decrease both PINP and CTX levels in Indian Vitamin D-deficient children [81]. Finally, the link between CTX and BMI has also been evaluated with contradicting data. While some show positive association [82, 83], others show no or negative association between CTX and BMI [18, 21, 84].

From a pre-analytical point of view, CTX expression is more influenced by fasting, circadian rhythm, and renal failure than PINP [85]. Thus, a fasting morning blood collection is strongly recommend [54].

Tartrate Resistant Acid Phosphatase (TRAP)

TRAP, measured 40 years ago in osteoclasts [86], is an enzyme secreted not only by differentiating osteoclasts but also by other cells from the monocyte-macrophage lineage. Of the two TRAP isoforms (5a and dimer 5b), the second, located at the ruffled border, is the osteoclast specific one [87]. In vitro, TRAP-5b activity correlating with the number of osteoclasts [88], with CTX-I in vivo and In vitro [89] and with BMD [87, 90], is considered a good bone resorption marker. Of clinical interest, being insensitive to kidney function [91, 92], TRAP-5b serum concentrations correlate with the number of osteoclasts and bone formation rate not only in patients with normal kidney function but also in uremic patients [93].

At the present time, despite considerable efforts, pediatric data in population-representative samples are sorely lacking. There are, however, few articles reporting TRAP-5b pediatric reference ranges [36, 38, 94] that set the scene. Positive correlation with BALP activity and its gender-dependent concentrations in two studies [28, 30] provides additional support for its role as a valid biomarker. Compared to other BTMs, TRAP-5b has a very specific expression profile in children with the highest values at birth followed by a constant decline that accelerates at puberty [36]. Although Chen et al. [94] observed a possible peak at early puberty, this was not confirmed by other studies.

Lau et al. [95] and later Scarnecchia et al. [96] evaluated more than 30 years ago the potential of serum TRAP measured spectrophotometrically as a marker of bone resorption. Since then major methodological improvements have been brought. Today, assays based on immune-immobilization capturing both isoforms, or singly TRAP-5b, followed by enzyme activity assessment are available [89, 97, 98]. TRAP-5b can be measured with an automated method (IDS iSYS) and a manual EIA (Nittobo Medical), both methods showing a good agreement [98].

Interpretation of Bone Marker Results

The interpretation of serum bone marker levels in children is more complex than in adults. Indeed, in adults, osteoblasts and osteoclasts are mostly dedicated to bone remodeling [99] while in children, bone cells are also participating in bone growth, which involves processes other than remodeling, such as endochondral ossification and cortical bone modeling [100]. Bone markers in children, therefore, correlate with height velocity, as has already been noted more than 70 years ago for total ALP [101] and for other bone markers that have been developed thereafter.

As bone markers in children reflect the cumulative effect of growth and remodeling processes, they may not be as accurate as expected in assessing the bone remodeling activity. One such instance occurs upon accelerated remodeling coupled to slow growth as sometimes observed in severe forms of osteogenesis imperfecta (OI). Children with severe OI have markedly increased bone remodeling activity, as demonstrated by static and dynamic iliac bone histomorphometry, a technique that evaluates bone remodeling independent of bone growth in length [102, 103]. In this situation, despite increased remodeling, serum bone marker concentrations are typically normal or low [104], a discrepancy that may partially be explained by the slower growth of these children [105].

Apart from skeletal growth, systemic bone cell activity in children is influenced by a myriad of other factors, many yet unknown, which may influence bone marker results. However, a few key factors are particularly relevant for the clinician interpreting bone marker results. In many situations, parathyroid hormone (PTH) plays an important role, as PTH directly stimulates bone remodeling activity and PTH serum concentrations vary with vitamin D status [106]. Another consideration is the effect of immobilization. Bed-rest studies have shown that in healthy people, bone resorption markers increase within 24 to 48 h after immobilization [107, 108]. Acute or chronic lack of physical activity is frequent in children who are undergoing bone health assessments and, therefore, may influence bone marker results. Finally, bone marker levels can be influenced by drug therapies, as discussed in the following section.

Drug Effects on Bone Markers

Glucocorticoids

High-dose glucocorticoids are used to treat a wide variety of disorders and have adverse effects on the skeleton, such as suppression of longitudinal growth, osteoblast activity, and bone turnover [109, 110]. These effects are consistently observed across a wide range of clinical situations. Bone formation markers decreased within 24 to 48 h after intravenous prednisolone injections in children with asthma [111]. Dexamethasone treatment in preterm infants, to manage the development of chronic lung disease, and led to a marked suppression of bone formation and bone resorption markers within 24 h after the first dose [112]. In another study on preterm infants with bronchopulmonary dysplasia, dexamethasone at a daily dose of 500 µg/kg led to a marked decrease in bone formation and bone resorption markers within three days of starting treatment [113]. In children and adolescents with inflammatory bowel disease, glucocorticoid treatment led to reduction in bone turnover markers [114]. In a study on 24 boys with Duchenne muscular dystrophy treated with glucocorticoids, serum levels of bone formation (PINP, BALP, osteocalcin) and bone resorption (CTX, TRAP-5b) were all decreased [115]. Thus, high-dose systemic glucocorticoid treatment consistently decreases bone marker levels.

The effect of inhaled glucocorticoids on bone development is more subtle than that of systemic glucocorticoids but still detectable by biochemical bone markers. A cross-sectional study of bone markers found that children with asthma had lower bone formation markers when they received inhaled glucocorticoids [116]. One month of treatment with 200 µg of inhaled budesonide (a corticosteroid analog) per day resulted in significantly decreased levels of bone formation and resorption markers [117].

Antiresorptive Therapies

In contrast to glucocorticoids, bone is usually the intended target of bisphosphonate therapy. These drugs inactivate osteoclasts and thereby decrease bone resorption and increase bone mass [118]. Bisphosphonates are widely used in children who suffer from bone fragility disorders such as OI. Consistent with the mechanism of action, intravenous infusions with the bisphosphonate Pamidronate lead, almost immediately, to a marked decrease in bone resorption activity [119]. A more gradual decline in bone resorption markers was observed when children with OI were treated with oral Alendronate [120]. The bone resorption marker CTX remains low in children and adolescents with OI even after many years of bisphosphonates therapy [121], reflecting persistent bone resorption long after discontinuation of bisphosphonate treatment [122].

Apart from bisphosphonates, bone resorption can also be targeted with denosumab, a drug that uses RANKL antibodies to inhibit osteoclasts. While bisphosphonates lead to long-term suppression of bone resorption, RANKL antibody treatment has a more transient effect [123]. A recent example of the use of bone markers in pediatric settings is the detection of the ‘rebound’ phenomenon in children treated with RANKL antibody. It was observed that the effect of an injection with RANKL antibody on bone resorption markers persistent for a much shorter duration in children than what had previously been seen in adults receiving the same treatment [124]. Eventually it was found that this rapid rebound in bone resorption was associated with hypercalciuria and, in some patients, hypercalcemia [125, 126]. Thus, the bone markers can be helpful to monitor treatment effects of drugs that have an effect on bone metabolism.

Growth Hormone

Many studies have examined markers of bone turnover in growth hormone deficient children. In untreated patients, bone formation and bone resorption marker levels are lower than in healthy controls [127, 128]. Once growth hormone treatment is started, levels of all markers of bone metabolism rise within a few weeks [127,128,129,130,131] and bone marker changes often correlate with the longer-term growth response [127, 129, 130, 132]. Similar observations have been made in short children without growth hormone deficiency who received growth hormone [129, 130, 133,134,135]. Given the correlation between height velocity and bone marker levels, many authors during the past three decades have suggested that bone markers can be used to predict the response to growth hormone treatment [56, 127,128,129,130,131]. However, the use of bone markers for growth prediction models has not been widely adopted in clinical practice, presumably because of a cost/effectiveness perspective, of the somewhat cumbersome approach and of the insufficient accuracy for clinical decision making [136].

Vitamin D Deficiency and Vitamin D Deficiency Rickets

A study on 468 vitamin D-deficient (serum 25-OH vitamin D < 50 nmol/l) school children in India revealed that a 6-month supplementation with vitamin D was associated with decreasing serum PINP and CTX levels [81]. These children had initially elevated serum PTH concentrations but they did not have rickets. As PTH stimulates bone turnover, it is expected that the correction of secondary hyperparathyroidism by vitamin D supplementation leads to a decrease in bone makers. Accordingly, the study found that the decrease in PINP and CTX serum concentrations depended on the baseline serum level of PTH.

The time course of bone markers after vitamin D supplementation is different when vitamin D deficiency is sufficiently severe to cause rickets. In that situation, vitamin D supplementation leads to an initial increase in bone turnover markers for a few weeks, followed by an eventual decline [137]. However, it is not clear that determining markers of bone metabolism beyond total ALP is of clinical use in the context of vitamin D deficiency rickets. As the contribution of the liver isoform to total ALP activity is small in children with increased bone turnover, the determination of bone-specific ALP does not offer a clear advantage in rickets [138].

Secondary Bone Disease in Chronic Disorders

Many serious diseases in children associated with slow skeletal growth have an inflammatory component, or are associated with immobilization. These factors tend to decrease bone formation, but they may have different effects on bone resorption. Slow growth is expected to decrease bone resorption, whereas inflammation and acute immobilization often lead to transient increases in bone resorption [1]. The following provides some examples of studies that have used bone markers in the context of secondary bone disease in children.

Low bone formation markers have been observed in disease states as diverse as malnutrition, phenylketonuria, cholestatic liver disease, active rheumatic diseases, and severe burns [139,140,141,142,143,144,145]. Bone formation markers also reflected the acceleration of bone metabolism during the successful therapy of these disorders [140, 142, 146]. A 2-year longitudinal study on bone development in 220 children living with HIV and receiving antiretroviral therapy found persistently lower bone turnover markers PINP, CTX, OC than in age- and sex-matched controls, even though height velocity was similar in the two groups [147], suggesting that low bone turnover was caused either by the ongoing infection or the drug therapy.

One of the chronic childhood conditions where bone involvement has been studied in some detail is inflammatory bowel disease [148]. Untreated inflammatory bowel disease is characterized by low bone turnover, as can be observed both in biochemical bone markers and in iliac bone histomorphometry analyses [114, 149, 150]. Effective treatment can quickly reverse these disease effects, as shown in a study on 103 children Crohn’s disease, where 10 weeks of treatment with infliximab led to a marked increase in serum levels of BALP, PINP and CTX [151].

The time course of bone metabolism in acute lymphoblastic leukemia is somewhat similar to Crohn’s disease. Several studies found that in newly diagnosed children, bone turnover markers were very low and decreased further with treatment regimens that included high doses of glucocorticoids [145, 152, 153]. After glucocorticoids were discontinued, bone markers levels increased. Similar phases of decrease and recovery of markers occurred during intensification cycles that included corticosteroids [154]. In the long run, survivors of childhood leukemia seem to have normal bone metabolism. A study on 251 individuals who had been cured of leukemia an average of 13 years before did not show abnormalities in bone turnover markers [155].

Chronic-kidney disease in children is often associated with slow growth and impaired bone mineralization as well as abnormalities of calcium, phosphorus, PTH, fibroblast growth factor 23, and vitamin D metabolism, a constellation of features that is termed chronic-kidney disease—mineral and bone disorder [156]. CTX and OC are not particularly useful markers of bone metabolism in this context, as these markers are cleared by the kidneys and, therefore, are inevitably increased when renal clearance is reduced [157]. A cross-sectional study in 63 children with chronic-kidney disease found that PINP and BALP are correlated with serum levels of tumor necrosis factor alpha [158]. One 5-year prospective study in 15 children with chronic-kidney disease found mildly elevated total ALP and PINP levels in a subgroup of patients and normal TRAP-5b results throughout the study period in all patients [159]. Kidney transplantation had no consistent effect on PINP and TRAP-5b levels [160]. Given the paucity of information on markers of bone turnover in the context of chronic-kidney disease in children, total ALP activity remains the marker of bone turnover that is currently recommended for clinical use [6].

Conclusion

During the past three decades, biochemical markers of bone turnover have helped in the characterization of bone disorders in children and in the assessment of the effects of drug therapies that target bone or have adverse effects on bone. However, many questions remain opened, and it is still unclear whether BTMs could help identifying children at risk of fracture or could be used in the follow-up of bone therapies in children. As such, Despite being important tools in the context of research in the area of pediatric bone disorders, their utility in clinical pediatric practice is not yet well established. Consequently, the clinical use of bone markers is currently mostly limited to highly specialized centers. Indeed in pediatric metabolic bone disorders, such as rickets and mineral and bone disorder of chronic-kidney disease, serum total ALP still is the parameter of choice for an informed clinical management. Conversely, in young adults, a flow chart for determining causes of bone fragility that includes BTMs has been proposed [161].

Several gaps have yet to be filled to improve the interpretation of BTMs concentration. One is the establishment of population-driven pediatric reference ranges. This requires multi-centric international endeavors. As Tahamasebi et al. [10] have stated “the use of inappropriate reference intervals impacts clinical decision making and is potentially detrimental on the quality of patient healthcare.” This statement holds for BTMs for which in addition, measurements, harmonization is imperative, a task that the IFCC C-BM is currently tackling [162]. Another approach in using BTMs is the recently proposed “Bone Turnover Index” based on the calculation of z-scores [134]. This statistical tool could provide additional information on the trajectory of bone metabolism. It, however, requires the establishment of proper age-, gender-, and ethnicity-matched reference values as mentioned above.