Broad application prospects of bone turnover markers in pediatrics

Bone turnover markers (BTMs) have been studied for application in clinical medicine. However, BTMs in children are challenging, and few studies explore these BTMs in children. The application of BTMs is complicated mainly due to pre‐analytical factors, variable reference intervals of age‐ and sex‐related BTMs for adolescents and children in different regions and laboratories. Therefore, laboratory testing of BTMs is critical for understanding pediatric bone development and metabolism, which provides additional information about bone development and diseases.

accelerated bone loss in adulthood. Thus, even if osteoporosis does not occur in childhood, the achievement of bone mass during adolescent development can influence skeletal development in adulthood.
Dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (CT) using densitometry techniques are recognized as gold standards for assessing bone mineral contents and density for children. Normally, DXA and QCT had low radiation exposure, with the advantages of high precision and fast detection. However, there are some problems when using DXA and QCT to diagnosis for children, such as fussiness, irritability, and crying during their examination, resulting in poor reproducibility, and inability to reflect the real-time biology of the bone at that time point. 3,4 Therefore, BMD measurements have limited the use of skeletal health assessment in childhood growth disorders for a long time.
As they are minimally invasive and can be dynamically monitored, bone metabolic marker assays have been widely used in the field of childhood growth and development-related diseases, and their levels not only reflect the skeletal metabolic health of children but indirectly reflect the growth and development of children. Thus, BTMs provide a basis for early identification, diagnosis, and treatment monitoring of childhood growth and development diseases.
In this article, the characteristics of BTMs are discussed, including their classification, application in monitoring bone growth in children and adolescents. This review also discusses the relevant important studies and application of bone metabolism biomarkers in energy metabolism, the endocrine environment, osteoporosis, and childhood diseases.

| Specific BTMs provided by clinical laboratories
Bone turnover markers (BTMs) are biochemical or cellular compounds produced during the continuum of bone resorption or formation. 5 BTMs can be divided into two categories: bone formation and bone resorption markers ( Figure 1 and Table 1). The former represents the activity of osteoblasts and the state of bone formation, while the latter mainly reflects the activity of osteoclasts and the level of bone resorption. Procollagen type I N-terminal propeptide (PINP), procollagen type I C-terminal propeptide (PICP), and osteocalcin (OC) are three frequently used markers of bone formation.
Pyridinoline (PYD), deoxypyridinoline (DPD), N-terminal cross-linked telopeptide (NTX), and C-terminal cross-linked telopeptide (CTX) are bone resorption markers. In addition, some important cytokines implicated in the regulation of bone turnover by controlling the activity of osteoblasts or osteoclasts, such as osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL), and these can be considered regulators of bone turnover rather than classical biochemical markers of bone turnover.
As BTMs are mostly excreted by the kidneys, their levels are frequently detected in urine. 6 Due to the difficulty of collecting 24-hour urine samples and the need to adjust for creatinine, blood testing is a preferable sample collection technique over urine sampling. 5 Laboratory testing of BTMs is minimally invasive and relatively affordable and, if used and interpreted properly, it can be a useful tool for assessing metabolic bone disease, treatment outcomes, and patient compliance. 7,8 CTX and PINP are the commonly used bone turnover markers in clinical research and are recommended by the International Osteoporosis Foundation (IOF) and the International Federation of clinical laboratory medicine (IFCC). 9 With the development of fully automated platforms, the analytical variability of bone markers has been greatly improved. The European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has shown the biological variability (CV%) of some BTMs, such as ALP, PINP, CTX, and osteocalcin, and with small interlaboratory variation. 10 In this review, we describe several of the most widely used BTMs in clinical pediatrics.

| Procollagen type I N-terminal propeptide (PINP)
Type I collagen is a procollagen secreted by osteoblasts, which creates a triple helix consisting of PINP and PICP. Since these propeptides are cleaved in their extracellular area and released into the circulation as metabolites, PINP and PICP concentrations can represent the level of bone formation. 11 Although the clearance of PINP may be less susceptible to hormonal changes than PICP, 12 PINP is a more sensitive marker compared with PICP. 13 Several studies have noted that PINP remains stable even after repeated freezing and thawing, 6 and its levels are not affected by circadian rhythms, so it is not necessary to consider the timing of sampling. 14 F I G U R E 1 Biochemical markers of bone remodeling. BAP, bone alkaline phosphatase; CTX, C-terminal cross-linked telopeptide; DPD, deoxypyridinoline; NTX, N-terminal crosslinked telopeptide; OC, osteocalcin; PICP, procollagen type I C-terminal propeptide; PINP, procollagen type I N-terminal propeptide; PYD, pyridinoline.

| Osteocalcin
Osteocalcin is the most common noncollagenous protein in bone, and it is secreted primarily by mature osteoblasts, although it is also secreted during bone resorption. Thus, serum osteocalcin is an important indicator of bone turnover, indicating bone formation and bone resorption. 6,15 Because serum osteocalcin concentrations have a distinct circadian rhythm and are highest in the morning, 16 blood samples must be taken in the morning to ensure the accuracy and comparability of the test results. In addition, several studies have shown that seasonal changes and diet did not affect PINP or osteocalcin levels. [17][18][19][20]

| Bone alkaline phosphatase (BALP)
Human ALP is classified as tissue non-specific ALP (TNSALP), intestinal type, placental type, and placental-like type. 21 TNSALP activity is represented by the products of the ALPL gene, which indicates bone anabolic activity. ALPL gene mutation leads to abnormal skeletal mineralization. BALP is a common biochemical marker of bone formation and a specific marker for osteogenesis, as well as one type of TNSALP, along with liver-and kidney-type ALP. The expression of bone ALP occurs early in the development of osteoblasts from mesenchymal progenitors and is vital in the degradation of pyrophosphate, a natural inhibitor of mineralization. 15 It is hard to identify bone-type from livertype ALP using current immunoassays, as they share the same amino acid sequences. Clinically, the application and interpretation of bone ALP as a BTM should be unaffected by liver diseases because bone ALP is normally cleared from the serum by liver. 22 Children have higher bone ALP activity than adults due to higher bone formation rates.
Determination of bone ALP activity also seems to be helpful for the diagnosis of hypophosphatasia and hyperphosphatasia. 23

| C-and N-Terminal Telopeptides of Type I Collagen (CTX and NTX)
During bone degradation, osteocalcin breaks down the bone matrix and releases CTX and NTX. 8 Although both CTX and NTX can be measured from urine samples, CTX has gained prominence because it can also be determined from blood tests on some automated platforms. Additionally, it is the preferred biomarker for detecting bone resorption activity. 6 The designed method determined the particular amino acid sequence of telopeptide type I collagen is known as cross lap, and β-aspartic acid was called β-CTX. 18 The International Osteoporosis Foundation recommends CTX as an appropriate bone marker for investigating bone resorption in clinical and research settings. 24 Since circadian rhythms and food consumption have effects on circulation β-CTX, 25 it should be collected during the period of fasting in the morning. 17

| APPLI C ATI ON OF BTMs
Many clinical and osseous manifestations of metabolic bone disease are more prevalent in children. However, too few studies on the use of these BTMs in children have potential use implications. Here, we summarize the role of BTMs in clinical pediatric care.

| Application of BTMs in growth and development
Children and adolescents are important periods of skeletal growth and exhibit high rates of bone growth and rapid bone turnover. BTMs reach the first peak within 1 year after birth, with little differences between boys and girls. Then, BTMs begin to show a downward trend and reach the second peak in early adolescence at the age of eight, with gender differences. 26 The peak of BTMs occurring in girls is earlier than that in boys, while the magnitude of the peak was lower than in boys. This could be related to secondary sexual characteristics and hormone levels. The decline in BTMs levels occurred earlier in girls than in boys and was more significant than in boys during late adolescence, which can explain the differences in bone peak and bone mineral content (PBC) between boys and girls during puberty. 27 A comparison of the reference interval ranges of BTMs in adults and children showed that the levels of BTMs were higher in childhood than those in adults and did not approach adult levels until late adolescence. 26

| Application of BTMs in rickets
Rickets is a childhood disorder associated with mineralization and ossification defects, the most common of which is vitamin D deficiency. 38 Despite significant improvements in early screening and quality of life, epidemiological surveys have revealed that the prevalence of nutritional rickets in children in rural areas remains as high as 10%. 39,40 Due to the dramatic increase in serum ALP in children with this disease, studies have confirmed the use of total serum ALP or BALP as an early screening indicator for differential nutritional rickets due to the good correlation between total serum ALP and BALP in childhood, with normal levels suggesting a low likelihood of rickets. 39,41 In addition, Chatterjee et al. 42,43 discovered that ALP is strongly expressed in nutritional rickets and is a more reliable marker than osteocalcin, PICP, and NTX and that its expression level may be utilized clinically to predict disease severity and prognosis.
However, P1NP, β-CTX, PTH, and 25(OH)D3 have hardly been studied in the field of nutritional rickets in children, and their application value needs to be further discovered and evaluated.

| Primary osteoporosis
Children's primary osteoporosis is a genetic disease caused by mutations, and osteogenesis imperfect is the most common disease caused by 17 identified genetic defects. 44 The main manifestation is increased bone fragility. 45

| Secondary osteoporosis
Secondary osteoporosis can be caused by multiple factors, such as primary disease and associated therapy. Harada and Rodan firstly found that osteoblasts and osteoclasts release active compounds are important to the physiological activity of other organs. 48 Previous studies have indicated that bone is not only the structural scaffold of the human body, but also can be important endocrine and hormone target organ. 49,50 In addition, many young patients develop secondary osteoporosis due to chronic diseases and the medications used. 51

| Application of BTMs in diabetes
According to the International Diabetes Federation, Type 1 diabetes In the long run, Delvin's view is that 251 individuals who had been cured of leukemia did not show abnormalities in bone turnover markers. 67 For thalassemia major patients, the decrease in osteocalcin may be due to osteoblastosis caused by iron overload. Nevertheless, no significant difference was found in serum alkaline phosphatase levels. 68 In another research group, normal 25(OH)D concentrations may maintain normal calcium homeostasis in patients with thalassemia, suggesting that a normal vitamin D level is important in the pathogenesis of thalassemia bone disease. 69 Furthermore, in patients with chronic ITP, OC, and type I collagen C-terminal propeptide (PICP) concentrations were lower, urine DPD output was higher, and bone mineral density (BMD) was significantly lower in both the spine and hip Z-scores. 70   Therefore, we need to further study to make sure a reliable combination of them in clinical application. We look forward to conducting more analysis on the detection and application of BTMs in children and adolescents, this will enable us to have a more comprehensive application in the growth and prevention of bone metabolic disorders. Therefore, pediatrician should be pay more attention on the use of BTMs in pediatrics.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.