ENDOCRINE DISORDERS

Mol Diag Ther 2008; 12 (3): 157-170 1177-1062/08/0003-0157/$48.00/0 2008 Adis Data Information BV. All rights reserved.

Biomarkers for Osteoporosis Management

Utility in Diagnosis, Fracture Risk Prediction and Therapy Monitoring
Patrick Garnero1,2
1 INSERM Unit 664, La ennec School of Medicine, Lyon, France 2 Biochemical Marker Division, Synarc, Lyon, France

Contents
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 1. Current Biochemical Markers of Bone Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 2. New Biochemical Markers of Bone Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 2.1 Noncollagenous Bone Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 2.1.1 Bone Sialoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 2.1.2 Urinary Osteocalcin Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 2.2 Osteoclastic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 2.2.1 Tartrate-Resistant Acid Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 2.2.2 Cathepsin K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2.3 Regulators of Osteoclastic and Osteoblastic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2.3.1 RANKL and OPG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2.3.2 Wnt Signaling Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2.4 Post-translational Modifications of Bone Matrix Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2.4.1 Type I Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 2.4.2 Noncollagenous Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 3. Variability of Biochemical Markers of Bone Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 4. Clinical Uses of Bone Markers in Postmenopausal Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 4.1 Markers of Bone Turnover to Predict Individuals at Increased Risk of Fracture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 4.2 Bone Markers to Identify Which Patients Will Benefit from Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 4.3 Bone Markers for Monitoring Treatment of Osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 4.4 Bone Markers to Promote Adherence to Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

Abstract

Osteoporosis is a systemic disease characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in an increased risk of fracture. While the level of bone mass can be estimated by measuring bone mineral density (BMD) using dual X-ray absorptiometry (DXA), its measurement does not capture all the risk factors for fracture. Quantitative changes in skeletal turnover can be assessed easily and noninvasively by the measurement of serum and urinary biochemical markers; the most sensitive markers include serum osteocalcin, bone specific alkaline phosphatase, the N-terminal propeptide of type I collagen for bone formation, and the crosslinked C- (CTX) and N- (NTX) telopeptides of type I collagen for bone resorption. Advances in our knowledge of bone matrix biochemistry, most notably of post-translational modifications in type I collagen, are likely to lead to the development of new biochemical markers that reflect changes in the material property of bone, an important determinant of bone strength. Among those, the measurement of the urinary ratio of native () to isomerized () CTX an index of bone matrix maturation has been shown to be predictive of fracture risk independently of BMD and bone turnover. In postmenopausal osteoporosis, levels of bone resorption markers above the upper limit of the premenopausal range are associated with an increased risk of hip, vertebral, and nonvertebral fracture, independent of

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BMD. Therefore, the combined use of BMD measurement and biochemical markers is helpful in risk assessment, especially in those women who are not identified as at risk by BMD measurement alone. Levels of bone markers decrease rapidly with antiresorptive therapies, and the levels reached after 36 months of therapy have been shown to be more strongly associated with fracture outcome than changes in BMD. Preliminary studies indicate that monitoring changes of bone formation markers could also be useful to monitor anabolic therapies, including intermittent parathyroid hormone administration and, possibly, to improve adherence to treatment. Thus, repeated measurements of bone markers during therapy may help improve the management of osteoporosis in patients.

Osteoporosis is a systemic disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in skeletal fragility and susceptibility to fracture.[1] This definition implies that the diagnosis should be performed before any fragility fracture has occurred, which is a challenge for the clinician. The level of bone mineral density (BMD) can be assessed with adequate precision and accuracy using dual X-ray absorptiometry (DXA) and its measurement forms the basis of the diagnosis of osteoporosis. However, the measurement of BMD does not capture all risk factors for fracture. Bone fragility also depends on the morphology, the architecture of bone as well as on the material properties of the bone matrix, which are all regulated by bone turnover (figure 1). Consequently, it has been suggested that bone strength may be reflected, independently of BMD level, by measuring serum and urinary markers of bone formation and resorption. Bone turnover markers may also be useful for monitoring the efficacy of treatment, especially anticatabolic therapies and, more recently, anabolics including parathyroid hormone (PTH). In this paper, we briefly review the technical aspects of the established and newer biochemical markers of bone turnover and then discuss their use in the management of patients with postmenopausal osteoporosis. 1. Current Biochemical Markers of Bone Turnover Bone remodeling is the result of two opposing activities, the production of new bone matrix by osteoblasts and the destruction of old bone by osteoclasts. The rate of bone production and destruction can be evaluated either by measuring predominantly osteoblastic or osteoclastic enzyme activities or by assaying bone matrix components released into the bloodstream and, eventually, excreted in the urine (table I). They have been separated into markers of formation and resorption, but it should be remembered that in diseases where both events are coupled and change in the same direction, such as osteoporosis, any marker will reflect the overall rate of bone turnover. In postmenopausal osteoporosis the most sensitive markers for bone formation currently available are serum total osteocalcin (also known as bone gamma-carboxyglutamic protein [BGP]),
2008 Adis Data Information BV. All rights reserved.

bone alkaline phosphatase (bone ALP), and the procollagen type I N-terminal propeptide (PINP) [see Garnero adn Delmas[2] for a review]. For the evaluation of bone resorption, most assays are based on the detection of type I collagen fragments in serum or urine. These include the crosslinks pyridinoline and deoxypyridinoline, the telopeptide fragments generated by cathepsin K (CTX, NTX) and matrix-metalloproteases (MMP) [CTX-MMP or ICTP], and fragments of the helical portion of type I collagen molecule (helical peptide).[2] The measurement of most of these biochemical markers can be achieved with high throughput and analytical precision on automated platforms.[3,4] 2. New Biochemical Markers of Bone Metabolism Recently a number of new biochemical markers of bone metabolism have been investigated, although available clinical data in patients with postmenopausal osteoporosis are still limited.
2.1 Noncollagenous Bone Proteins

Although the vast majority of bone matrix is composed of type I collagen molecules, approximately 10% of the organic phase is constituted of noncollagenous proteins, some of which are almost
Bone mass/density

Architecture Bone shape and geometry Microarchitecture

Bone turnover: formation/resorption

Microdamage Osteocyte apoptosis

Bone matrix properties Mineral Collagen/crosslink Noncollagenous proteins

Fig. 1. Determinants of fracture resistance and the pivotal role of bone turnover in their regulation.
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specific for bone tissue. It has been suggested that the measurement of these proteins or fragments thereof could represent specific biochemical markers of bone turnover.
2.1.1 Bone Sialoprotein

Table I. Biochemical markers of bone turnover. Markers with the most significant performance characteristics in postmenopausal osteoporosis are in italics Formation Serum/plasma Total and bone ALP Intact and total osteocalcin Urine PICP, PINP Total and free pyridinoline Total and free deoxypyridinoline NTX CTX Type I collagen helical peptide 620633 ALP = alkaline phosphatase; CTX = C-terminal crosslinked telopeptide of type I collagen; MMP = matrix-metalloprotease; NTX = N-terminal crosslinked telopeptide of type I collagen; PICP = C-propeptide of type I collagen; PINP = N-propeptide of type I collagen. NTX CTX CTX-MMP (ICTP) Resorption

Bone sialoprotein (BSP) is an acidic, phosphorylated glycoprotein of 33 kDa (glycosylated: 7080 kDa) that contains an arginine-glycine-aspartate (RGD) integrin binding site. Although BSP is relatively restricted to bone, it is also expressed by trophoblasts and is strongly upregulated in a variety of human primary cancers, particularly those that metastasize to the skeleton.[5] A small amount of BSP is released in the circulation and, as such, is a potential marker of bone turnover.[6] Serum BSP levels are increased in malignant bone diseases and postmenopausal osteoporosis, and are reduced by anti-resorptive treatments.[6] However, with the currently available immunoassays, accurate measurements of serum BSP remains challenging because of its tight association with circulating factor H.
2.1.2 Urinary Osteocalcin Fragments

Although most newly synthesized osteocalcin is captured within bone matrix, a small fraction is released into the blood where it can be detected by immunoassays. Circulating osteocalcin comprises different immunoreactive forms, including the intact molecule and fragments of different sizes.[7] The majority of these fragments is generated from in vivo degradation of the intact molecule and, thus, also reflects bone formation. However, in vitro studies suggest that some osteocalcin fragments could also be released from osteoclastic degradation of bone matrix[8] and, thus, may reflect bone resorption. Elevated levels of urinary osteocalcin were reported in osteoporotic postmenopausal women;[9] values decreased after 1 month of treatment with the bisphosphonate alendronate. This contrasts with the absence of significant change in serum total osteocalcin levels. A recent study in 601 postmenopausal women age 75 years and older evaluated over 5 years, showed that urinary osteocalcin levels were associated with BMD loss in the spine and hip.[10] In the same population, high levels of urinary but not serum osteocalcin were associated with a greater risk of clinical vertebral fracture independently of BMD.[11] Theoretically, urinary osteocalcin fragments may constitute a specific bone resorption marker, although the value of its measurement in osteoporosis remains to be further evaluated.
2.2 Osteoclastic Enzymes
2.2.1 Tartrate-Resistant Acid Phosphate

Bone acid phosphatase is resistant to L-(+)-tartrate (TRACP), whereas the prostatic isoenzyme is inhibited. Acid phosphatase circulates in blood and shows higher activity in serum than in plasma because of the release of platelet phosphatase activity during the the clotting process. In normal plasma, TRACP corresponds to isoenzyme 5. Isoenzyme 5 is represented by two subforms, 5a and 5b; TRACP5a derives mainly from macrophages and dendritic cells whereas TRACP5b is more specific for osteoclasts.[12,13] These two subforms differ by their carbohydrate content, optimal pH, and specific activity. TRACP5a is a monomeric protein whereas TRACP5b is cleaved in two subunits. Total plasma TRACP activity is measured by colorimetric assays. However, the lack of specificity of plasma TRACP activity for the osteoclast, its instability in frozen samples, and the presence of enzyme inhibitors in serum, are drawbacks which limit the development of clinically useful enzymatic TRACP assays. To overcome these limitations, two different immunoassays for serum tartrate TRACP have been developed that preferentially detect isoenzyme 5b. The first uses antibodies that recognize both intact and fragmented TRACP5a and 5b. The selectivity of this assay for TRACP5b is partly achieved by performing measurements at optimal pH for TRACP5b activity.[14,15] More recently, a new immunoassay using two monoclonal antibodies raised against purified bone TRACP5b with limited crossreactivity for TRACP5a has been described.[16] One of the antibodies captures active intact TRACP5b while the second eliminates interference of inactive fragments. We found that this new ELISA for TRACP5b was highly sensitive in detecting increased bone turnover followMol Diag Ther 2008; 12 (3)

Acid phosphatase is a lysosomal enzyme that is primarily present in the bone, prostate, platelets, erythrocytes, and spleen.
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ing menopause and was also very responsive to alendronate therapy.[17] Serum TRACP5b predominantly reflects the number and activity of osteoclasts. It may, thus, provide information on the bone resorption process that is complementary to that given by collagen-related markers.[15] Another advantage of serum TRACP5b relates to its limited diurnal variation and negligible effect of food intake. These features result in a lower intra-patient variability for TRACP5b than for collagen-based biochemical markers. However, the magnitude of changes observed following bisphosphonate treatment in postmenopausal women is also lower for TRACP5b than for collagen markers.[18]
2.2.2 Cathepsin K

and RANKL with BMD and biochemical markers of bone turnover in postmenopausal women and elderly men.[24]
2.3.2 Wnt Signaling Molecules

The enzyme cathepsin K is a member of the cysteine protease family and, unlike other cathepsins, has the unique ability to cleave both helical and telopeptide regions of type I collagen.[19] The enzyme is produced as a 329-amino-acid precursor procathepsin K, which is cleaved into its active form with a length of 215 amino acids. This cleavage takes place in vivo within the low-PH bone resorption lacunae. Clinical data on serum cathepsin K is still scarce. Increased serum cathepsin K levels have been reported in patients with active rheumatoid arthritis,[20] Paget disease of bone,[21] and in postmenopausal women with fragility fractures.[22] Because circulating concentrations of cathepsin K are very low and the currently available assays lack sensitivity, accurate measurements of this enzyme remain challenging.
2.3 Regulators of Osteoclastic and Osteoblastic Activity
2.3.1 RANKL and OPG

The Wnt signaling pathway plays a pivotal role in the differentiation and activity of osteoblastic cells.[25] There are 19 closely related Wnt genes that have been identified in humans. The primary receptors of Wnt molecules are the seven transmembrane frizzled-related proteins (FRPs; FRZB), each interacting with a single transmembrane low-density lipoprotein (LDL) receptorrelated protein 5/6 (LRP5/6). Different secreted proteins, including soluble FRPs, Wnt inhibitory factor-1 (WIF1), and dickkopf homologs (DKK)14 prevent ligand-receptor interactions and consequently inhibit the Wnt signaling pathway. Alterations in the Wnt signaling pathway and its regulatory molecules, including DKK1 and SFRP1, have been shown to play an important role in bone turnover abnormalities associated with osteoporosis, arthritis, multiple myeloma, and bone metastases from prostate and breast cancer.[26] Immunoassays for circulating DKK1 have recently been developed. Serum DKK1 levels are reportedly increased in clinical situations characterized by depressed bone formation, such as multiple myeloma.[27] Circulating levels are also higher in diseases characterized by focal osteolysis such as multiple myeloma,[27] bone metastases from breast or lung cancer,[28,29] and rheumatoid arthritis.[30] Conversely, in patients with osteoarthritis of the hip, a clinical situation characterized by focal sclerosis of subchondral bone, lower serum DKK1 levels have shown to be associated with a decreased risk of joint destruction.[31] Currently, there are no data on serum DKK1 in postmenopausal osteoporosis. As with the assessment of OPG and RANKL, it is possible that circulating DKK1 adequately reflects local bone contribution.
2.4 Post-translational Modifications of Bone Matrix Proteins
2.4.1 Type I Collagen

The RANKL/RANK/OPG system is one of the main regulators of osteoclast formation and function (see Kearns et al.[23] for a review). RANK (TNFSF11) and OPG (osteoprotegerin, TNFRSF11B) are members of the tumor necrosis factor (TNF) receptor superfamily, and RANKL (TNFRSF11A) is a member of the TNF ligand superfamily. Although the major contribution of this pathway in postmenopausal bone loss has been clearly established in various animal and clinical models, the serum measurement of RANKL and OPG remains difficult. Indeed, at present it remains unclear as to what proportion of circulating OPG is monomeric, dimeric, or bound to RANKL, and which of these forms is the most biologically relevant to measure. The same issues arise for the measurements of circulating RANK-L which, in its free form, is barely detectable in healthy individuals. It is also unlikely that circulating levels of OPG and RANKL adequately reflect local bone marrow production. These limitations probably explain the conflicting data on the association of circulating OPG
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Type I collagen, the main organic component of bone matrix, undergoes a series of enzymatic and nonenzymatic intra- and extracellular post-translational modifications during maturation of bone tissue (figure 2). Among the enzymatic modifications, ex vivo studies performed on human and animal bone specimens have shown that alterations in the hydroxylation of lysine residues, glycosylation of hydroxylysine, and the relative concentration of immature and mature telopeptide crosslinks, are associated with biomechanical properties that are, in part, independent of BMD.[32] Nonenzymatic modifications including the advanced glycation endproducts (AGEs) could also contribute to the mechanical properties of bone tissue. AGEs occur spontaneously in the presence of extracellular sugars (figure 2). In contrast to enzymatic
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Isomerization EKAH D GGR CTX CTX

N Lys Pentosidine PYD DPD N+ AGE e.g. pentosidine a Lys OH-Lys Lysyl oxydase Immature divalent crosslinks DHLNL HLNL Mature trivalent crosslinks PYD DPD b Sugar Arg CTX

Fig. 2. Schematic representation of the extracellular post-translational modifications of type I collagen molecules in bone matrix. A monomer of type I collagen is constituted of 2 alpha 1 chains and one alpha 2 chain. These three chains are associated in a triple helical structure, except at the two ends in the region of the N- and C-telopeptides, where chains remained unwound. During maturation of bone tissue, the type I collagen molecules undergo several post-translational modifications including (a) the telopeptide crosslinks that involved the lysine (Lys) and hydroxylsine (OH-Lys) residues within the N- and C-telopeptides (CTX). These residues are oxydated by the enzyme lysil oxidase, producing in a first step the divalent immature crosslinking molecules (dihydroxylysinornorleucine [DHLNL] and hydroxylysinonorleucine [HLNL]), which upon further maturation are transformed into the mature trivalent crosslinks such as pyridinoline (PYD) and deoxypyridinoline (DPD); (b) the advanced glycation endproducts (AGEs), which are formed by the nonenzymatic glycation of some amino acids such as arginine (Arg) when sugar (e.g. glucose) is in excess within the extracellular matrix. The most evaluated AGE pentosine makes a bridge between an Arg residue of one molecule and a Lys residue of an adjacent collagen molecule; and (c) the non-enzymatic isomerization of the aspartic acid (D) residue in the EKAH D GGR sequence (also known as CTX sequence) of the C-telopeptide. Isomerization converts the native D residue () present in newly synthesized collagen to its isomerized form () in the matured collagen (see text for details on the isomerization process).

telopeptide crosslinks whose concentration in bone tissue plateaus with skeletal maturity, AGEs such as pentosidine accumulate with age in human cortical bone.[33] In biomechanical studies performed on human femur, Wang et al.[33] showed that a higher concentration of pentosidine was associated with a lower bone strength and greater fragility. Thus, high levels of pentosidine, and potentially other AGEs, may alter the capacity of the bone to absorb energy upon loading before it breaks. In the human vertebral bone, increased bone levels of pentosidine have also been shown to be associated with decreased resistance to fracture.[34,35] In 432 elderly Japanese women, increased urinary pentosidine was moderately associated with an increased risk of incident vertebral fracture independent of BMD and bone turnover markers.[36] A clinical situation where AGEs may be particularly relevant is type 2 diabetes mellitus, a disease characterized by altered glucose metabolism and increased bone fragility despite increased BMD. An increased risk of fracture in type 2 diabetes is also related to a higher prevalence of nonskeletal risk factors, including a tendency to fall, poor balance and gait, functional limitations, poor vision, and a history of stroke and peripheral neuropathy. A recent study has shown an association between increased serum pentosidine levels and the presence of prevalent vertebral fracture in postmenopausal women with type 2 diabetes but not in men with type 2
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diabetes independent of the potential confounding factors such as serum glycated albumin, BMD, and renal function.[37] However, pentosidine is only one of several AGEs present in bone matrix and is not specific to this tissue. The identification of the major AGE of bone tissue that directly alters mechanical properties would be extremely useful. -isomerization of the aspartate (D) residue within the sequence (CTX) of the C-telopeptide of type I collagen is another nonenzymatic post-translational modification believed to reflect bone matrix maturation (figure 2). In newly synthesized type I collagen molecules, the peptide bond between the aspartic acid residue and adjacent glycine (G) involves the carboxyl group in position of the aspartic acid (D). Thus, this native sequence is named CTX. With the maturation of type I collagen, there is a translocation of the peptide bond from the carboxyl group in position to the carboxyl group in position of the aspartic acid residue, leading to the so-called CTX-isomerized sequence.[38,39] In contrast to the enzymatic telopeptide crosslinks and some nonenzymatic AGEs (e.g. pentosidine), this spontaneous nonenzymatic modification does not lead to the formation of crosslinks between adjacent collagen molecules. However, isomerization does alter the 3-dimensional conformation of type I collagen by introducing a kink in the peptide backbone.[35,38,39]
1207AHDGGR1214
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The structure of bone matrix can be altered in certain clinical situations (e.g. Paget disease or bone metastases) characterized by a very high bone remodeling in localized area of bone tissue, leading to the so-called woven bone or in genetic disorders caused by mutations in type I collagen genes (e.g. osteogenesis imperfecta). In patients with Paget disease and bone metastases from breast cancer, immunohistologic studies have shown that type I collagen molecules within the disorganized woven bone matrix were mostly on a non-isomerized CTX conformation, contrasting with normal lamellar bone which mainly comprises isomerized collagen.[40,41] It is possible to detect alterations of type I collagen isomerization in vivo by measuring the urinary excretion of native () and isomerized () CTX fragments using specific immunoassays. In patients with Paget disease of bone, we have shown that the urinary excretion of CTX was markedly increased compared to that of CTX, resulting in an abnormally high / CTX ratio.[40] The relationships between these two isoforms could be normalized after bisphosphonate therapy,[42,43] a treatment that has been shown to result in the formation of a bone matrix with normal lamellar structure. More recently, we reported an increased urinary / CTX ratio in adults with osteogenesis imperfecta, suggesting altered bone collagen maturation.[44] The investigation of type I collagen isomerization may also be of clinical relevance in postmenopausal osteoporosis. In the OFELY (Os des Femmes de Lyon) prospective study,[45] it was found that an increased urinary / CTX ratio was significantly associated with increased fracture risk independent of both the level of hip BMD and overall bone turnover evaluated by serum bone ALP. The effects of antiresorptive therapies on the nonenzymatic modifications of collagen have more recently been investigated both in animal models and in clinical studies.[46-48] In vertebral trabecular bone from dogs, treatment with alendronate and risedronate but not raloxifene induced a decrease in the / CTX ratio and a marked increase in pentosidine. This suggests that treatment with the bisphosphonates that have a profound effect in suppressing bone remodeling may increase bone collagen maturation.[46] These animal ex vivo data are consistent with the decrease in urinary / CTX ratio observed in postmenopausal women receiving alendronate 10 or 20 mg/day or oral daily (2.5 mg) or intermittent ibandronate.[47] In agreement with the canine data, there was, however, no significant change in the urinary / CTX ratio after treatment with raloxifene or estradiol.[47] In the postmenopausal women participating in the PaTH (Parathyroid Hormone and Alendronate for Osteoporosis) clinical trial,[48] we were unable to confirm a significant decrease in the urinary / CTX ratio after 1 or 2 years with the lower dose of alendronate (10 mg/day). Thus, it is possible that only profound
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suppression of bone turnover can induce detectable changes in the urinary / CTX ratio. Altogether, the above described data indicate that the extent of post-translational modification of collagen and, more specifically, those related to nonenzymatic processes can contribute to the biomechanical properties of bone tissue. At present, the urinary / CTX ratio appears to be the most promising non-invasive biomarker to assess these changes in bone matrix maturation, although further studies are necessary to confirm its clinical utility in the assessment of postmenopausal osteoporosis.
2.4.2 Noncollagenous Proteins

Bone matrix also contains noncollagenous proteins that can undergo post-translational modifications. Osteocalcin contains three residues of -carboxyglutamic acid (GLA). GLA results from the carboxylation of glutamic acid residues, an intracellular post-translational modification which is vitamin K-dependent. It was postulated that impaired -carboxylation of osteocalcin could be an index of both vitamin D and vitamin K deficiency in elderly populations. In two prospective studies performed in a cohort of elderly institutionalized women followed for 3 years[49] and in a population of healthy elderly women (the Epidemiologic des Ost eoporoses study [EPIDOS]),[50] levels of undercarboxylated osteocalcin which can be evaluated indirectly via the incubation of serum with hydroxyapatite or directly by specific immunoassay above the premenopausal range were associated with a 2- to 3fold increase in the risk of hip fracture, whereas total osteocalcin was not predictive. A decreased ratio between carboxylated and total osteocalcin, an index of increased undercarboxylated osteocalcin, was associated with increased fracture risk in elderly women living independently.[51] The mechanisms relating to increased undercarboxylation of osteocalcin and fracture risk are unclear. Serum undercarboxylated osteocalcin,[52] but not total osteocalcin, has shown to be associated more strongly with ultrasonic transmitted velocity at the os calcis and tibia (which may reflect, in part, changes in bone microarchitecture) than with BMD. Recent studies also suggest that the degree of carboxylation may be an important determinant of the endocrine function of osteocalcin to regulate energy metabolism in mice,[53] although its relevance in humans remains to be explored. 3. Variability of Biochemical Markers of Bone Turnover As discussed above, automated assays with high analytical precision are now available for most established markers.[3,4] In addition to controlling for analytical precision error, the biologic variability of biochemical markers needs to be taken into account when making clinical decision. Indeed, the serum and urinary
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concentration of bone marker is dependent on their production, volume of distribution, half life, and clearance. These parameters, which depend on factors such as age, gender and bodyweight, ethnicity and geography, pregnancy and lactation, diet, exercise and mobility, menstrual and seasonal cycles, previous fracture, the type of metabolic bone disease, and hepatic and kidney function, will impact the overall variability.[54] Some biochemical markers also exhibit a marked circadian variability that does not appear to be altered by gender, menopausal status, postmenopausal osteoporosis, and the presence of bone metastases. Finally, the concentration of some markers, including serum CTX, are markedly affected by food intake,[55] which is mediated in part by the gastrointestinal hormone glucagon-like peptide-2 (GLP2).[55,56] In an individual patient, some of these factors, such as circadian rhythms, fasting and menstrual status, diet, and exercise, are controllable by standardizing the timing and conditions of sample collection (table II). Other factors such as age, ethnicity and geographic location, previous fractures, season, pregnancy and lactation, drugs, immobility, and co-morbidities, cannot be conTable II. Sources of pre-analytical variability of bone turnover markers (ordered by relative importance) Controllable factors Diurnal (circadian) variability Diet Seasonal changes Exercise Uncontrollable factors Age menopausal status Gender Ethnicity Recent fractures (up to 1 year) Pregnancy and lactation Renal and hepatic function Drugs antiresorptives bone anabolics glucocorticoids anticonvulsants gonadotropin-releasing hormone agonists Diseases metabolic bone diseases diabetes mellitus thyroid diseases arthritic diseases (e.g. rheumatoid arthritis, osteoarthritis) Immobility
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trolled, but still need to be considered when interpreting the data of biochemical markers (table II). All together, when sampling is standardized, studies have shown that the biologic intra-patient variability is in the order of 1015% for serum bone formation markers, but higher for type I collagen-related resorption assays, especially when measured in urine. However, this biologic intrapatient variability is often of a lower magnitude than the changes observed during treatment with antiresorptive therapies or PTH (see section 4). Thus, the measurement of bone markers allows the documentation of changes in bone turnover that are related to the pharmacologic intervention (and not only to physiologic variation) in a majority of subjects. 4. Clinical Uses of Bone Markers in Postmenopausal Osteoporosis In women with postmenopausal osteoporosis, biochemical markers of bone turnover may be useful for the following applications: 1. predicting individuals at increased risk of fracture; 2. predicting which patients will benefit from treatment; 3. assessing the efficacy of treatment; 4. promoting adherence to therapy. Clearly, biochemical markers do not have a role in the diagnosis of osteoporosis, and cannot be a substitute for BMD assessment by DXA.
4.1 Markers of Bone Turnover to Predict Individuals at Increased Risk of Fracture

With the emergence of effective, but rather expensive, treatments for osteoporosis, it is essential to detect those women who are at higher risk of fracture and, therefore, would benefit most from therapy. Indeed, although several prospective studies have demonstrated a strong association between BMD measurements and the risk of hip, spine, and forearm fractures,[57] about half of the patients with incident fractures have baseline BMD above the diagnostic threshold of osteoporosis (defined as a T-score of 2.5 SD or more below the average value of young healthy women). Clearly, there is a need to improve the identification of patients at high risk of fracture. Prospective studies investigating the relationships between bone formation markers and fracture risk have yielded conflicting and inconsistent data (see Garnero[58] for a review). Conversely, five prospective, population-based studies consistently reported that a high level of bone resorption, assessed by urinary or serum CTX, urinary-free deoxypyridinoline, serum TRACP5b, or urinary osteocalcin fragments (only one study for these two latter markers), was associated with about a 2-fold higher risk of hip,
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Table III. Relationships between increased baseline levels of bone resorption markers and fracture risk in prospective studies of untreated postmenopausal women Study cohort Epidemiologic des Osteoporoses study (EPIDOS)[60] Age (y) >75 Fracture type Hip Biochemical marker Urinary CTX Urinary-free deoxypyridinoline Rotterdam study[61] OPRA study[11] Malmo Os des Femmes de Lyon (OFELY) study[62] Hawaii Osteoporosis Study a b c d e Upper two tertiles. Upper quartile. Mean. Per SD increase. (HOS)[63] >75 75 64d 69d All All Urinary-free deoxypyridinoline Serum TRACP5b Urinary-OC All All Urinary-CTX Serum-CTX Urinary-CTX Above the upper limit of premenopausal range. Relative risk (95% CI) of incident fracture for high levels of markers 2.2 (1.3, 3.6)a 1.9 (1.1, 3.2)a 1.7 (1.1, 2.6)b 2.2 (1.2, 4.2)c 2.1 (1.1, 4.1)c 2.3 (1.3, 4.1)a 2.1 (1.1, 3.6)a 1.5 (1.2, 2.0)e

CTX = C-terminal crosslinked telopeptide of type I collagen; OC = osteocalcin.

vertebral and nonhip, and nonvertebral fractures over follow-up periods ranging from 1.8 to 5 years[11,58] (table III). A recent analysis of the Malm o cohort in elderly women (all 75 years of age) indicates that some bone resorption markers may still predict fracture risk 9 years after measurement,[59] although the association attenuates with time. In all these analyses, the odds ratios of fracture were not modified after adjustment for potential confounding factors such as mobility status, and were only marginally decreased after adjustment for BMD measured by DXA. The use of an odds ratio is not ideal for clinical decision making, since the risk may decrease or remain stable with age, whereas the absolute risk increases. Thus, estimating the absolute risk of fracture over a 10-year period is probably more appropriate. Based on the data from the EPIDOS[60] and OFELY[62] studies, it was found that combining urinary CTX with BMD or history of previous fracture, results in a 10-year probability of hip fracture that was about 70100% higher than that associated with low BMD alone.[64] The use of bone markers in individual patients may be appropriate in some situations, especially in women who are not identified as being at risk using BMD measurement alone. In the elderly women participating in the EPIDOS study, it was shown that urinary-free deoxypyridinoline was more strongly associated with hip fracture risk in those with normal BMD than in those with osteoporosis.[65] In a study of younger postmenopausal women from the OFELY cohort, half of all incident fractures actually
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occurred in osteopenic women (with a BMD T-score between 1.0 and 2.5). Among these women, high levels of bone ALP were significantly associated with increased fracture risk (table IV). The combination of high bone ALP and a history of prior fracture was very effective in identifying women who would experience a fracture in the following 9.1 years.[66] Women at high risk of fracture may benefit from therapeutic intervention, especially if risk factors are amenable to bonespecific agents. Indeed, some BMD-independent risk factors, such as falls, are particularly important for hip fracture, but may not be modified by pharmacologic interventions. Conversely, some studies have shown that bisphosphonates and raloxifene have a positive effect on fracture risk in osteopenic women,[67] and a greater reduction in fracture risk with alendronate has been shown among postmenopausal women with high pretreatment levels of bone turnover markers.[68] Thus, in selected cases where BMD and clinical risk factors are not sufficient to make a treatment decision, bone turnover markers may be used in the assessment of fracture risk.
4.2 Bone Markers to Identify Which Patients Will Benefit from Treatment

It has long been suggested that pretreatment levels of biochemical markers may be useful in the selection of the most appropriate therapy. Conceptually, there is a belief that patients
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with high bone turnover are more likely to respond to anticatabolic agents, whereas patients presenting with low bone turnover would benefit more from anabolic treatments. Several studies have found that among estrogen-,[69-71] calcitonin-,[72] and bisphosphonate-treated women,[73] higher pretreatment levels of bone turnover were associated with a greater increase in spinal BMD after 13 years. Conversely, little data exist on the relationship between pretreatment levels of bone turnover and the antifracture efficacy of anti-catabolic treatments. In a pooled analysis of several risedronate versus placebo trials, Seibel et al.[74] reported that the spine fracture risk reduction was similar among women with low and high pretreatment bone turnover as assessed by urinary deoxypyridinoline. However, the number of patients needed-totreat (NTT) to avoid one fracture during 1 and 3 years of treatment with risedronate was significantly reduced in patients with high baseline turnover compared with women with low baseline values. More recently, in the Fracture Intervention Trial[68] of alendronate, which included a larger number of incident fractures, it was reported that women with high pretreatment levels of serum PINP or bone ALP had a greater reduction in nonspine fracture than women with lower turnover (see figure 3). Using a Markov predictive model, it was also shown that high levels of bone turnover markers may be useful to identify a subset of osteopenic women who could benefit (cost-effectively) from bisphosphonate therapy to reduce the risk of vertebral or nonvertebral fractures.[75] However, because the association in these studies was modest and may vary according to the drugs, even within the same class, it is still unclear whether the baseline assessment of biochemical markers will be of clinical value to predict which individual patients will benefit more from anticatabolic therapy. Anabolic treatments including PTH have been viewed as a particularly attractive therapeutic agent for patients with low bone turnover. However, recent studies with both teriparatide (rhPTH

134) and recombinant human full length PTH 184 indicate that PTH therapy increases BMD and reduce fracture risk whether or not bone turnover is suppressed before treatment.[76,77] In fact, with both agents the increase in spine BMD was larger in those with higher pretreatment bone turnover. The teriparatide-mediated absolute risk of spine fracture reduction was also greatest in those women with higher pretreatment levels of bone markers, although the relative risk reduction was independent of pretreatment bone turnover.[77]
4.3 Bone Markers for Monitoring Treatment of Osteoporosis

Monitoring the efficacy of osteoporosis treatment is a challenge. The goal of treatment is to reduce the occurrence of fragility fractures, but their incidence is low and the absence of events during the first year(s) of therapy does not necessarily imply that treatment is effective. Thus, the use of surrogate markers with a more rapid response is clearly needed for an efficient monitoring of treatment in osteoporosis. A surrogate marker can be defined as a laboratory measurement or physical sign used as a substitute for a clinically meaningful endpoint that directly measures how a patient feels, functions, or survives. Changes induced by a therapy on a surrogate endpoint are expected to reflect changes in a clinical meaningful endpoint, i.e. incidence of fracture. Measurement of BMD is a surrogate marker of treatment efficacy that has been widely used in clinical trials. Its use in the monitoring of treatment efficacy in the individual patient, however, has not been validated. Several randomized, placebo-controlled studies have shown that treatment of postmenopausal women with antiresorptive therapies, including bisphosphonates, estrogens, selective estrogen receptor modulators (SERMs), the anti-RANKL antibody denosumab, and cathepsin K inhibitors, induces a prompt decrease of

Table IV. Fracture risk in osteopenic postmenopausal women with low bone mineral density (BMD), high bone turnover (bone ALP), or prior fracture. In all 322 osteopenic women (lowest T-score at the spine or hip between 2.5 and 1), the 10-year probability of fracture was 18.6 (reproduced from SornayRendu et al.,[66] with permission from the American Society for Bone and Mineral Research) Predictor 2.5 BMD < T-score 2 Bone ALP (highest quartile) Prior fracture(s) Bone ALP (highest quartile); prior fracturea 2.5 BMD < T-score 2; bone ALP highest quartile, prior fracturea a One or more risk factor(s). ALP = alkaline phosphatase.
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n 102 90 44 122 180

Age-adjusted hazard ratio (95% CI) 2.5 (1.3, 4.6) 2.2 (1.4, 3.8) 2.2 (1.2, 4.3) 2.6 (1.5, 4.5) 5.3 (2.3, 11.8)

10-year probability of fracture (%) 26.7 28.4 38.0 29.4 25.6

Sensitivity (%) 50 43 29 59 85

Specificity (%) 72 75 89 66 50

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a Percentage without nonspine fracture Percentage without nonspine fracture 1.00 0.95 0.90 0.85 0.80 0.75 0 1 2 3 4 Time to first nonspine fracture (y) 5 p = 0.44 1.00 0.95 0.90 0.85 0.80 0.75

b Percentage without nonspine fracture 1.00 0.95 0.90 0.85 0.80 0.75

Alendronate Placebo

p = 0.05

p = 0.0001

1 2 3 4 Time to first nonspine fracture (y)

1 2 3 4 Time to first nonspine fracture (y)

Fig. 3. Increased pretreatment bone turnover and the risk of nonspine fracture in postmenopausal women treated with alendronate (the Fracture Intervention Trial).[68] Women were categorized by tertile of pretreatment procollagen type I N-terminal propeptide (PINP) levels: (a) tertile 1 PINP <41.6 ng/mL; (b) tertile 2 PINP 41.656.8 ng/mL; and (c) tertile 3 PINP >56.8 ng/mL. For each baseline tertile of PINP, the graph shows the percentage of women without facture during follow-up in women receiving alendronate or placebo. p-Values refer to the difference between placebo and alendronate within each tertile of PINP (reproduced from Bauer et al.,[68] with permission from the American Society for Bone and Mineral Research).

bone resorption markers within a few weeks of treatment, with a plateau reached after 36 months (see Cremers and Garnero[78] for a review). The decrease in bone formation markers is delayed following treatment, reflecting the physiologic coupling of formation to resorption; a plateau is usually achieved after 612 months of treatment, as shown for bisphosphonates in figure 4. However, with inhibitors of cathepsin K, current evidence suggests only small changes in bone formation markers during the first year of therapy. The onset and magnitude of the decrease of bone markers with antiresorptive therapies depend on the route of administration (faster for intravenous than oral bisphosphonates), the mechanism of action of the therapy, and the marker investigated. Notably, it has been shown that estrogen induces a significant decrease in the urinary excretion of free, peptide-bound, and total deoxypyridinoline, while bisphosphonate has no significant effect on free deoxypyridinoline.[79] This different response may be related to the differential effects of the bisphosphonates and estrogen on the enzymatic pathways of type I collagen degradation, leading to the generation of the various forms of deoxypyridinoline and/or the renal metabolism of peptide-bound crosslinks that converts part of the peptide bound to free deoxypyridinoline. From a practical point of view, it is not recommended that urinary-free deoxypyridinoline be used to monitor bisphosphonate therapy. The relationship between bone marker changes and fracture risk with antiresorptive therapy has been investigated in several post hoc analyses of randomized clinical studies.[80-85] It was found that the changes in serum osteocalcin, bone ALP, and PINP with raloxifene were associated with the subsequent risk of vertebral fractures in osteoporotic women participating to the Multiple Outcomes of Raloxifen Evaluation (MORE) study, while changes in hip BMD were not predictive.[80-82] In postmenopausal women
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with osteoporosis treated with oral risedronate, changes in urinary CTX and NTX after 36 months predicted the risk of subsequent incident vertebral fractures at 3 years.[83] A significant association between changes in bone ALP and vertebral, hip, and nonspine fracture was also found in women treated with alendronate participating in the Fracture Intervention Trial.[84] Alendronate-treated women with at least a 30% reduction in bone ALP at 1 year had a lower risk of nonspine (hazard ratio [HR] 0.72; 95% CI 0.55, 0.92)
PTH bone formation markers PTH bone resorption markers Oral bisphosphonates bone formation markers Oral bisphosphonates bone resorption markers

100

Change from baseline (%)

0 0 6 12 18 24 30 36

100 Time (mo)

Fig. 4. Schematic representation of the response of biochemical markers of bone formation and resorption with intermittent parathyroid hormone (PTH) treatment and oral bisphosphonate.
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and hip fractures (HR: 0.26; 95% CI 0.08, 0.83) relative to those with reductions lower than 30%. In the risedronate study, it was shown that the relationship between vertebral fracture risk and the levels of CTX reached after 36 months was not linear and that there may be a level of bone resorption reduction below which there is no further fracture benefits.[85] Although optimal cut-offs still need to be validated in prospective studies, it has been proposed that the goal of antiresorptive therapy is to reduce bone turnover markers within the lower half of the reference range for healthy young, premenopausal women.[85] This requires the determination of accurate reference intervals for the lower limit of bone turnover markers in healthy premenopausal women, data which are now becoming available.[86,87] Bone turnover markers may also be useful to monitor the effects of anabolic treatments, including the intermittent administration of PTH. In the PaTH study (with PTH 184) and the Fracture Prevention Trial (with teriparatide),[76,88] it was shown that PTH produces a marked and rapid (within a month) increase in markers of bone formation followed by a delayed increase in bone resorption markers (figures 4 and 5) In this situation, bone formation markers, especially serum PINP,[4,77,88] appear to be the most useful in the prediction of the late grain of BMD. The use of bone markers to predict the antifracture efficacy of PTH remains to be investigated. Strontium ranelate has a unique mechanism of action; it both moderately stimulates bone formation and, at the same time, decreases bone resorption as documented by changes of biochemical markers. However, because the magnitude of these variations is small,[89] bone markers are not useful for monitoring the efficacy of strontium ranelate in individual patients. Conversely, changes in hip BMD have shown to be a strong predictor of its antivertebral facture efficacy.[90] Whether bone turnover markers will be useful in predicting the fracture efficacy of the bisphosphonates zoledronic acid and ibandronate, and other types of antiresorptive therapies currently in development such as denosumab and cathepsin K inhibitors, remains to be investigated.
4.4 Bone Markers to Promote Adherence to Therapy

1500 Change from baseline at 3 months (%) 1000 400 400 350 300 250 200 150 100 50 0 50 100 +136%

Parathyroid hormone Alendronate Median percentage change Variation of PINP

+ LSC LSC 59%

Fig. 5. Serum procollagen type I N-terminal propeptide (PINP) to monitor effects of parathyroid hormone and bisphosphonate in postmenopausal women with osteoporosis. The graph shows the individual percentage changes of serum PINP measured by automated assay in postmenopausal women with osteoporosis participating in the Parathyroid Hormone and Alendronate for Osteoporosis (PaTH) study after 3 months of treatment with subcutaneous parathyroid hormone 1-84 100 g/day (n = 119) or oral alendronate 10 mg/day (n = 60). The median percentage change in each treatment group is shown; the variation of PINP within the least significant change (LSC) range of 20 and +20% is also noted. After 3 months, 83% and 88% of the women had experienced changes in serum PINP that exceeded the LSC after parathyroid hormone (>+20%) and alendronate (<20%) treatment, respectively (reproduced from Garnero et al.,[4] with permission).

tion after 3 months, with a change of treatment if response to the assessed treatment was not satisfactory. From this analysis it has been suggested that monitoring the effectiveness of bisphosphonate treatment with bone markers may improve quality of life. Clowes et al.[92] randomized 65 postmenopausal women to three different groups: no monitoring, monitoring by nurse visit, and monitoring by regular assessment of bone markers. After 1 year of treatment with raloxifene, the monitored groups (nurse or marker) showed a significantly (57%) increased cumulative adherence to therapy compared to no monitoring, although there was no difference between the types of monitoring. In the larger IMPACT study with oral residronate,[93] 2302 postmenopausal women with osteoporosis were randomized to either a reinforcement or noreinforcement group; reinforcement was the expression of feedback by a physician based on urinary NTX bone marker results. Compared with the women in the no-reinforcement group, the subgroup of patients who received a positive message (based on a significant decrease in urinary NTX), showed a significantly higher persistence to therapy. However, persistence was lower in women receiving a negative message (increase in urinary NTX). Although the total number of incident fractures after 1 year was limited, it was significantly lower [8 patients with 9 fractures
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The failure of a patient to respond to osteoporosis treatment may be due to poor adherence (probably the most important single factor), poor intestinal absorption (for oral bisphosphonates), or additional factors contributing to bone loss or other unidentified factors. Using a decision tree and Markov modeling, Chapurlat and Cummings[91] compared two strategies: the treatment of a woman without specific monitoring and the treatment of the same woman with the measurement of a serum marker of bone resorp 2008 Adis Data Information BV. All rights reserved.

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versus 17 subjects with 18 fractures, odds ratio (95% CI): 0.4 (0.2, 1.0)] in the bone marker reinforcement group. Thus, if confirmed by additional studies, monitoring using bone markers may improve adherence to treatment and, ultimately, be beneficial for optimal fracture reduction. 5. Conclusion Reliable assays for assessing bone formation and bone resorption are now available. Data have consistently shown that some of these biochemical markers may be used to improve the prediction of fracture risk, together with BMD and clinical risk factors and monitor efficacy of antiresorptive and PTH therapies in postmenopausal women. New biochemical markers reflecting different biologic pathways of osteoblastic and osteoclastic activity and changes in structural properties of bone matrix have more recently been developed. Their value in the management of patients with osteoporosis alone or in combination with existing markers should be further evaluated. Acknowledgments
No sources of funding were used to assist in the preparation of this review. The author has no conflicts of interest that are directly relevant to the content of this review.

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Correspondence: Dr Patrick Garnero, Synarc, 16 rue Montbrillant, Lyon, 69003, France. E-mail: patrick.garnero@synarc.com

2008 Adis Data Information BV. All rights reserved.

Mol Diag Ther 2008; 12 (3)