Biochemical markers of bone turnover in postmenopausal osteoporosis

Introduction

Bone turnover markers (BTMs) allow assessment of bone resorption and formation by measuring their concentration in blood and urine ( Box 201.1 ). BTM assays are inexpensive; for example, in the European Union they cost around £20 per assay. The assays are noninvasive, and markers can be measured several times in one individual. The term BTM is used for markers that reflect the activity and number of osteoclasts (bone-resorbing cells) and osteoblasts (bone-forming cells). Therefore these markers are distinct from the locally acting regulators of bone cell activity (e.g., osteoprotegerin and sclerostin) and hormones (e.g., parathyroid hormone [PTH]). In this chapter we will focus on the use of BTMs in postmenopausal osteoporosis, but these markers are also useful in other forms of osteoporosis and other bone diseases (e.g., Paget’s disease), which are less common than postmenopausal osteoporosis.

Bone Turnover

Regardless of age and health status, bone turnover comprises two processes in bone tissue: bone resorption and bone formation. During resorption, osteoclasts remove small amounts of bone at distinct sites of the skeleton, thus digging resorption cavities known as resorption pits. This process lasts 4 to 6 weeks, during which time bone tissue components are catabolized and released into the bone microenvironment and subsequently transferred into the bloodstream and partly excreted in the urine. The concentration of bone components or products of their catabolism in blood and urine reflects bone resorption activity. Subsequently, osteoblasts arrive at the newly formed pit to start bone formation. Osteoblasts secrete molecules that fill in the cavity with osteoid, a connective tissue rich in collagen, and these molecules are released into the circulation. Blood concentrations of the molecules released by osteoblasts (bone formation markers) reflect bone formation activity. Finally, osteoid is mineralized. Jointly, this entire bone formation process lasts 4 to 5 months. The orderly sequence of these events in one location is called coupling, and the site at which these events occur is called the bone remodelling unit (BRU). Bone turnover permits removal and replacement of old and damaged bone and thereby preserves bone strength. Bone turnover also supports mineral and acid–base homoeostasis.

Bone Remodeling

In the mature adult skeleton (>30 years), the predominant process is bone remodeling. This process takes place in trabecular and cortical bone. The amount of resorbed bone equals that which is subsequently formed at every BRU (remodeling balance). This process allows for constant bone renewal, associated with relatively constant bone mass and strength.

After menopause, however, the rate of formation of BRUs doubles. Moreover, at every BRU the amount of the new bone is reduced compared with that which was newly resorbed in the same remodeling cycle. This represents a negative remodeling imbalance. These two factors, the higher number of BRUs and remodeling imbalance at each BRU, underlie the accelerated bone loss and osteoporosis that are common in postmenopausal women. Increased BTM concentrations coupled with negative remodeling imbalance can be associated with increased rates of bone loss and fracture risk.

Antiresorptive drugs inhibit osteoclasts, reduce bone resorption, and might restore remodeling balance. A decrease in bone resorption results in a rapid reduction in bone formation because of reduced release of coupling factors. Subsequently, bone formation marker concentrations are low because bone formation is operating in a reduced number of BRUs. The balance between resorption and formation markers can be described mathematically in a bone balance index, and the changes from bisphosphonates have been described.

Bone Turnover Assessment

Bone histomorphometry is the gold standard technique for assessment of bone turnover. Bone biopsies taken from the iliac crest allow assessment of the amount of bone, bone turnover, and bone mineralization. When tetracycline is administered twice before a bone biopsy is performed, it is incorporated in the mineralization front and appears bright yellow under fluorescent light. The distance between the two labels divided by the time interval introduces a time component and reflects the mineralization rate. This rate, jointly with other histomorphometric parameters, permits calculation of the bone formation rate and of other dynamic parameters of bone turnover.

However, a bone biopsy can only be done on a few occasions in the same individual, as the procedure itself induces high bone turnover locally (rapid acceleratory phenomenon) of unpredictable duration. Thus it is unclear when a repeat sample can be taken from the same biopsy location without interference. Bone biopsy analysis is laborious, expensive, and limited to a small bone sample. Histomorphometric measures of bone formation are more reliable than are measures of bone resorption.

Bone turnover can also be assessed by use of calcium balance and tracer kinetic techniques. This approach is expensive and might involve long-term hospitalization. Bone turnover can also be assessed after administration of radiolabeled bisphosphonate or 18F-fluoride-PET scanning; the PET scanning method allows the study of local bone turnover.

BTMs have been validated against quantitative bone histomorphometry. The largest BTM study, done in 370 postmenopausal women with osteoporosis, showed a weak correlation between serum levels of N-terminal propeptide of type I procollagen (PINP) or bone ALP and histomorphometric bone formation estimates, and between serum C-telopeptide of type I collagen (CTX-I) and histomorphometric bone resorption estimates, with most correlation coefficients between 0.21 and 0.36. However, BTMs indicate the status of the entire skeleton, and bone biopsy represents a small part of this whole. The precision error of bone histomorphometry is large. BTM concentrations are determined both by the rate of their production and by the rate of their degradation and excretion.

A combined calcium balance and tracer study in women with osteoporosis showed positive correlation between estimated resorption and concentration of bone resorption markers, with the highest correlation coefficient for deoxypyridinoline, which is highly specific for bone. Thus the available studies of BTM validation are not perfect, and the reference methods used have major limitations, including availability, cost, and small sample size. To our knowledge, no study has been done to correlate changes in biochemical BTMs with changes in bone turnover assessed using other approaches in the setting of treatment with antiresorptive drugs.

Bone Cells

Osteoblasts develop from mesenchymal stem cells and produce osteoid. After their work is complete, osteoblasts undergo apoptosis, become lining cells, or are embedded in bone as osteocytes. Type I collagen is the most abundant bone protein and comprises 90% of total bone protein. It is secreted as procollagen, and subsequently, the N-propeptides and C-propeptides are cleaved enzymatically. Type I collagen is deposited in a quarter stagger array held together by pyridinium crosslinks: deoxypyridinoline and pyridinoline. Osteoblasts express their highest concentration of collagen during the proliferative phase, their highest concentration of bone ALP during matrix maturation, and of osteocalcin during matrix mineralization. The production of distinct markers during various phases of osteoblast development can help explain why their circulating levels do not always change in parallel.

Osteoclasts develop from monocyte-macrophage lineage cells. These cells attach to bone with a sealing zone and secrete acid, which dissolves the bone mineral. Osteoclasts release enzymes that are active at a low pH (e.g., cathepsin K) and that digest proteins and release their fragments, e.g., N-telopeptide of type I collagen (NTX-I) or CTX-I. Assays are available for measurement of these protein fragments in serum, plasma, or urine. Tartrate-resistant acid phosphatase isoform 5b (TRAP5b, also known as ACP5) is one of the enzymes produced by osteoclasts. It is fairly specific to bone and appears to reflect the number of osteoclasts. This is in contrast to NTX-I and CTX-I, which are indicative of osteoclast activity. After osteoclasts complete resorption, they undergo apoptosis.

Osteocytes are derived from osteoblasts. They are numerous and sense the mechanical forces applied to bone. Osteocytes regulate bone resorption and formation by production of locally active factors (e.g., RANKL and sclerostin) and can remove bone by the process of osteocytic osteolysis. It is unknown how much this removal contributes to the release of bone turnover markers from bone.

Practical Aspects

Bone resorption markers show a circadian rhythm and decrease by 20% to 40% shortly after ingestion of a meal. The blood sample for CTX-I measurement should be drawn after an overnight fast, between 7:30 am and 10:00 am. The sample can be collected as serum or EDTA plasma, and the latter is preferable if the sample cannot be processed within 2 hours. The sample can be frozen, preferably at −80°C, although TRACP5b is unstable during long-term storage. For urinary markers (deoxypyridinoline, NTX-I, and CTX-I) the sample should be taken as the second morning void (SMV) after breakfast. Excessive fluid intake should be avoided the day before. No preservative for the SMV is required if the urine sample is processed within several hours. The results of urinary marker assays should be expressed as a ratio to urinary creatinine. Assessment of 24-hour urine output eliminates reliance on a urinary creatinine comparison, which might be helpful in patients with sarcopenia who have low urinary creatinine.

Bone formation markers have little circadian rhythm (<10%), so the sample can be drawn at any time of day, the blood sample centrifuged, and the serum or plasma measured immediately or frozen. For determination of osteocalcin levels, hemolysis should be noted, as this can give anomalous low results.

Choice of Bone Turnover Markers

Usual practice is to measure one marker (either for bone formation or bone resorption) when investigating bone turnover. The International Osteoporosis Foundation (IOF) and International Federation of Clinical Chemistry recommend serum CTX-I (bone resorption) and PINP (bone formation) as two reference markers. All studies should include these two markers at a minimum. PINP is derived from posttranslational cleavage of type I procollagen. Serum PINP primarily originates from bone, shows weak circadian variation, and increases during bone formation-stimulating therapy. The values from the two auto analyzer methods give similar results. CTX-I is a product of breakdown of type I collagen. It is specific for bone and decreases during antiresorptive treatment. CTX-I occurs in its native α and β isomerized forms, which undergo racemization (D-forms and L-forms). In practice, the choice of BTM is determined by local availability and cost. BTM selection also depends on the clinical context. For example, bone ALP, intact PINP, and TRAP5b would be appropriate for assessing a mineral and bone disorder associated with chronic kidney disease, and osteocalcin or CTX-I would be appropriate for assessing glucocorticoid-induced osteoporosis.