Bone turnover markers (BTM) may give information on bone formation and resorption, risk of fracture and response to treatments [1]. BTMs have been extensively studied as markers in the diagnosis and monitoring of osteoporosis (OP), and resulted potentially useful as tools to evaluate the estimation of fracture future risk, although their significance was essentially demonstrated helpful to monitor efficacy of anti-OP treatments [2]. Other possible application includes the prediction rate of bone loss, the identification of secondary OP, the improvement of targeted treatments and patient compliance, although other data are needed in such areas [3]. However, they are influenced by a number of pathophysiological factors, and by analytical aspects, still need to be overcome to extend their application and significance in the clinical practice [1]. Thus, BTMs practical use requires careful awareness of their advantages as well as their limitations to interpret results produced by the laboratory.

The present review aim to describe the most commonly used serum bone formation and resorption biochemical markers, discuss their advantages and disadvantages and give practical information on their use and result interpretation in the laboratory and clinical settings according to current recommendations from the International Osteoporosis Foundation (IOP) and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), as well as the US National Bone Health Alliance (NBHA) [4,5].

Bone is a dynamic tissue, characterized by a continuously renewed through processes of bone removal parallel to bone formation and replacement, which occur in the so-called basic multicellular units (BMU). Main cells in the BMU are osteoblasts, deputed to bone formation, and osteoclasts, to bone resorption.

Osteoblasts not only release a variety of factors, which regulate bone formation, but also drive osteoclast maturation, which requires stimulation by RANKL expressed on osteoblasts [3,6]. The different proteins or released degradation products can be measured in blood or urine samples, representing reliable tools to assess the dynamic nature of bone tissue (Table 1).

Table 1: Traditional, most actually used and new proposed bone turnover biomarkers.

Bone resorption markers include collagen breakdown products, but also the less used pyridinoline rings, or osteoclast-specific enzymes (tartrate-resistant acid phosphatase) [3,6]. Conversely, bone formation markers include non-collagenous matrix proteins, such as osteocalcin (OC), precursor molecules of collagen type I synthesized by osteoblasts, and osteoblast-specific enzyme [3,6].

However, as of today the most utilized BTMs in the clinical settings included OC, bone specific alkaline phosphatase (BALP), procollagen type I N propeptide (P1NP), and carboxy-terminal cross-linking telopeptide of type I collagen (CTX) [3,6]. In particular, serum PINP (s-PINP) and serum CTX (s-CTX) have been suggested by main International Scientific Societies (IOF, IFCC, and NBHA) as reference biomarkers of bone formation and resorption respectively in OP, although further data are needed to expand their laboratory and clinical use [4-6].

Biological and pre-analytical variability

Recent literature confirms that serum BTMs are still subjected to a variety of pre-analytical sources of variability that included uncontrollable as well as controllable variables (Table 2). The first group includes aging, gender, menopausal status, pregnancy and lactation, diseases that impact bone health (as diabetes, liver disease, and renal impairment), and recent fracture and immobility (Table 2). Conversely, controllable factors include sampling time (circadian variability) and sample processing (collection, handing and storage), fasting status and exercise (Table 2).

Table 2: Main sources of variability of the most utilized BTM.

In particular, during the menopause transition, a decline in ovarian function beginning about 2 years before the final menstrual period is follow by an increase in bone resorption and subsequently by bone loss [7]. For it concerns variation related to menstrual cycle, CTX resulted highest at the start of the cycle (when oestrogens are lowest), decreased significantly from T 0 to T 26 (when oestrogens are highest, in the pre-ovulatory period, and when progesterone activity is highest, in the advanced luteal phase) and then increased again from T 26 to T 01 [8]. Accordingly, other recent data suggested that during 17-β estradiol administration CTX levels decreased, whereas P1NP concentration increased [9]. Serum OC and BALP seem not to significantly vary during the menstrual cycle [10,11].

Physiological bone turnover has a circadian rhythm, and OC as well as P1NP and CTX are higher in early morning [6]. Postprandial suppression of bone resorption is considered one of the main contributors in the circadian rhythm of bone turnover markers. Accordingly, after oral glucose load, a reduction was observed for BTM, especially CTX (46.9% for β-CTX, 7.9% for P1NP, and 8% for OC) [12].

Seasonal variation may account for a significant percentage of BTMs variability, almost in part related to vitamin D decreased in winter, as evidenced by recent data in children and adults [13-15]. In particular, 25(OH) vitamin D was found higher and CTX lower during summer when compared to winter, especially in black children (15). In fact, although black children reported less sunscreen use and less travel to more sunny locations during holidays compared to whites, being exposure to sunlight (UV irradiation) the major source of vitamin D is derived from, skin pigmentation may significantly reduce UVB effects [16].

Elite athletes (who performs at a high level in a competitive sport, or professional athletes) showed an higher bone turnover than sedentary subjects, although a single session of exercise is insufficient to modify BTM concentration, and effects largely depends on the type, intensity and duration of physical activity, with bone formation markers more sensitive than bone resorption markers [17-20]. Conversely, immobility exacerbates bone loss and increased bone resorption and CTX levels, as reduced mobility accelerates the bone loss due to aging and increase CTX and P1NP levels in elderly subjects (n=111) [21,22]. Moreover, BTMs are increased following a fracture, and subjects with increased frailty risk had significantly high levels of PINP, β-CTX and PTH as well as low levels of 25(OH)D [23,24].

Increasing data underlie the relevance of bone metabolism and turnover biomarker concentration on type 2 diabetes mellitus (T2DM) pathophysiology [25-27]. In particular, OC has been significantly related to glucose metabolism, through the increase of insulin release and sensitivity and energy expenditure, and reduction of visceral fat [24-28,29]. In a general population, an inverse relationship was found between OC and fasting plasma glucose, fasting insulin, and homeostasis model assessment-estimated insulin resistance (HOMAIR) [30]. In patients with T2DM, serum OC levels resulted negatively related to glucose and fat mass, and atherosclerosis surrogates (brachial-ankle pulse wave velocity and carotid intima-media thickness, IMT), and positively to total adiponectin levels [30,31]. In this context, two very recent meta-analyses confirm that OC results significantly lower in T2DM patients than controls, and it is inversely associated with the development of T2DM [32,33].

Other sources of variability are related to sample handling. In particular, OC decrease with hemolysis and if left to room temperature even for few hours or underwent successive freeze-thaw cycles, subjected to rapid degradation in sample often characterized by presence of heterogeneous OC fragments, and can be present in undercarboxylated form [4,6]. This form results mainly associated with enhanced cell function, while the carboxylated form appears more involved in improved insulin sensitivity [34]. Renal function can also affect OC levels, although OC evaluation retains important advantages as biomarker, as it is widely used, retains high tissue specificity, and relatively low within-subject variation [4].

The measurements of BALP still retain cross-reactivity with the liver form, which can be significant in liver disease patients [4]. Moreover, BALP half-life is long (1-2 days), which renders this biomarker less dependent to circadian rhythm than other BTMs [4]. In any case, samples must be stored at -20°C, because the activity of the enzyme increased at room temperature [4].

Serum CTX and P1NP retain high specificity, because mostly derived from bone, but CTX may vary in patients with liver or renal abnormality, whereas P1NP presents significant biological variability [4,35].

Analytical variability

The lack of standardization is still a problem, and betweenlaboratory variability may be significant, rising question on the validity of comparing results from different laboratories [36]. For osteocalcin, for which several immunoassays are available, the measurement is complicated by the presence in variable amount of several fragments, which may negatively influence the reproducibility of repeated sampling (Table 2). However, the difference between BTM results may be significant even if laboratories used the same method (Table 2). For this reason, one reference laboratory to monitor serial samples for the same patients is advocated.

Scientific Society recommendation and guidelines proposed by IOF, IFCC, and NBHA, have recommended the use of serum P1NP and CTX as the reference standard for bone formation and resorption, respectively, and advocate efforts for sample collection, standardization, establishment of reference intervals, and need for External Quality Assurance Schemes (EQAS) [1-6]. Both biomarkers retain high specificity, and for CTX the standard is well characterized as an eight-aminoacid peptide that permits the development of reference standard [4-6]. Sources of variability are well known, as well as procedures for sampling, and storage [4-6]. For it concerns method, s-P1NP is available as RIA (Orion) or automated immunoassay (Total P1NP, Roche Diagnostics, Germany; Intact P1NP, IDS-iSYS, UK), and s-CTX as ELISA (IDS, UK) or automated immunoassay (Roche Diagnostics, IDS-iSYS) [1-6].

Result reporting and interpretation

An example of available data on reference values for main BTMs is reported in (Table 3) [37-50]. BTM reference ranges are not established and may vary according to general population or patient cohort tested (Table 3). In particular, there is a great variability between individual subjects, and these values are more variable in postmenopausal women, and clearly reference ranges differ according to the method used (Table 3). Moreover, there is heterogeneity in reporting age intervals, biomarker name or abbreviation, and measurements units, which render more complex the interpretation and comparison of results.

Table 3: Reference Intervals for serum BTM in adults.

Guidelines suggested the use of “least significant changes” in the follow-up under anti-osteoporotic treatment to render more meaningful the clinical decision [2]. In particular, reduction of at least 30% for serum bone turnover biomarkers has been recommended, even in OP postmenopausal women with BTM levels in the premenopausal range at the start of treatment [2].

Scientific organizations have recently recommended the measurements of sPINP and sCTX as markers of bone formation and bone resorption, respectively [4-6]. In any case, this advice do not exclude the use of other BTM, as OC or BALP, but possibly used in parallel with P1NP and CTX, when clinicians are more familiar with the other BTM or if there are previous patient data obtained by using the other BTM. Moreover, these four biomarkers have effectively different characteristics, as BALP presents cross-reactivity with the liver form and so it is not advised in patients with liver disease, but it can be preferred in patients with renal function because less influenced by this factor respect to the other biomarkers (Table 2).

Standardization on patient condition (fasting status, etc.) and control of sample timing, handling and storage are important aspects to decrease controllable variability and increase the accuracy by which BTM may reflect the rate of bone remodelling (Table 2). Significant intra-method as well as inter-assay differences exists for BTM (Table 2). This analytical variability can be controlled referring to the same laboratory for serial assays, and standardization of methods for the reference BTM is advocated. Also a need for international reference standard, as well as the identification of reference intervals for the general population or relevant patient cohort remains a high priority target (Tables 2 and 3).

In this scenario, new possible additive biomarkers are continuously proposed, including periostin, a matricellular protein preferentially localized in the periosteal tissue, sphingosine 1-phosphate, a lipid mediator mainly involved in osteoclastogenesis, and sclerostin, an osteocyte factor, because both P1NP and CTX retain limitations related to lack of absolute specificity for bone tissue, and incapacity to reflect osteocyte activity or periosteal apposition [51].

Moreover, genome-wide studies identified genetic variants associated with bone mineral density and fracture risk, that could identify new additive biological pathways underlying bone metabolism, and provide new possibility of OP intervention and treatment [52-57].

Traceability, validation, harmonization, standardization, external quality assurance programs need to be improved to expand BTM clinical applicability. In any case, despite high variability, BTM changes are greater enough to identify subjects at high risk for bone loss and subsequent fracture, or to monitor the efficacy of OP therapies, due to BTM capacity to rapidly respond to treatments. In particular, reduction in BTMs after 3 months- 6 months of anti-resorptive therapy predict successive reduction in fracture risk, whereas the changes BMD in patients receiving therapy, particularly under anti-resorptive therapy, are not closely related to the fracture risk reduction [58]. It has been suggested that the increase in bone strength following antiresorptive treatment may be partly explained by a reduction in trabecular perforations that might be captured in the measurement of BTMs, but not by BMD [59]. However, only clinicians aware of BTMs benefits and limitations may find in these biomarkers a useful tool in association with other clinical, instrumental and laboratory parameters for the management of their OP patients.