Throughout the adult skeleton, bone is continually remodeled by cellular processes of resorption and formation. Teams of osteoclasts are targeted to resorb islands of bone, and osteoblasts follow in a temporally and spatially coupled process to fill in and reshape the region removed. In this process, the role of the osteocyte, the most abundant resident bone cell, is unclear; but osteocytes are a candidate source of the signals that activate a particular bone surface for remodeling. Net loss of bone occurs when the osteoclastic resorption rate exceeds the osteoblastic formation rate; in general, this is the situation in the aging adult skeleton. Particularly in women at or soon after menopause, the higher the bone turnover (remodeling) rate, the more rapid the net loss of bone mass is likely to be. There is growing evidence, therefore, that reliable and convenient biochemical tests for assessing bone turnover can aid in predicting the risk of osteoporosis and in monitoring the patient for a timely and appropriate response to therapy. In this article, current knowledge of molecular markers of bone formation and bone resorption are summarized and their potential use in the clinical management of osteoporosis is discussed.
Several immunoassays have been reported in which results show improved specificity and responsiveness to bone cell activity compared with those in earlier biochemical tests.10,15,23 They and the more traditional assays rely on the measurement of serum or urine enzymes or matrix proteins synthesized by osteoblasts or osteoclasts that spill over into body fluids, or of osteoclast-generated degradation products of the bone matrix itself (Figure 1). To what degree osteocytes release these or other markers of bone metabolic activity is unclear. Serum levels of skeletal alkaline phosphatase or osteocalcin are currently the most convincing formation markers. The most useful resorption markers are products of collagen degradation and currently are best measured in urine.
A biochemical assessment of bone turnover rate provides different information from a bone mineral density (BMD) measurement. In clinical practice, a snapshot of how much bone is present (determined by BMD in the spine, for example) together with a measure of how fast the skeleton is remodeling (a biochemical index) are complementary in helping to predict risk of future fracture in the postmenopausal patient. A specific and responsive biochemical marker can also be used to monitor short-term effectiveness of an antiresorptive therapy in the patient.
Total alkaline phosphatase level in serum is still the most common index of bone formation in clinical use (e.g., to assess and monitor Paget's disease of bone). However, immunoassays to determine osteocalcin and bone-specific alkaline phosphatase levels in serum have now become the preferred tools in clinical research studies when a greater specificity to the osteoblastic process of bone formation is required. The propeptides of Type I collagen can also respond as expected as serum markers of bone formation,40,47 but they represent total Type I collagen synthetic activity in all tissues in the body including skin and soft tissues, not just in bone formation.
The common form of alkaline phosphatase is a cell membrane-associated enzyme expressed by liver, bone, kidney, and placenta from a single gene. Liver and bone are the primary sources of the serum enzymes, and the molecules differ posttranslationally in their glycosylation pattern. Alkaline phosphatase is a prominent product of osteoblasts and osteoblast precursors. The development of monoclonal antibodies that can select for the osteoblast enzyme (bone-specific alkaline phosphatase) by recognizing a posttranslational feature, has provided the basis for a more specific serum index of systemic bone forming activity.39,46 An immunoassay to quantify the enzyme molecule is more attractive than approaches that separate and measure bone-derived and liver enzyme activities in serum.4,9 The measure of bone formation provided by alkaline phosphatase is indirect, depending presumably on a spillover of excess or spent enzyme from active osteoblasts and probably also preosteoblasts, lining cells, and perhaps osteocytes. Therefore, as with all bone-turnover assays, the quantitative correlation between serum levels of bone-specific alkaline phosphatase and the actual rate of bone deposition may not be simple or constant within or between patients.
Human osteocalcin (or bone Gla-protein) is a 49-residue polypeptide expressed under 1,25-dihydroxyvitamin D3 control by osteoblasts as they actively deposit bone. Osteocalcin forms approximately 1% of the organic matrix of bone, where it exists in close association with the surface of the mineral crystallites. Serum osteocalcin34,49 seems to reflect primarily a spillover of osteoblast synthetic activity rather than degradation products of resorbed bone matrix. Osteocalcin in bone is probably degraded on resorption to fragments. These are not recognized by the newer immunoassays, which use specific monoclonal antibodies to measure intact or almost intact molecules that preserve the N-terminal sequence (e.g., by a two-site assay format).14,29
Although immunoassays for serum osteocalcin and bone-specific alkaline phosphatase are emerging as the most responsive and specific indicators of bone formation activity, they do not entirely track each other in all clinical situations. For example, serum osteocalcin is not as elevated as might be expected and correlates poorly with total serum alkaline phosphatases and with the bone-specific enzyme in Paget's disease of bone.16 The relative lack of correlation may reflect the late expression of osteocalcin in the developing osteoblast phenotype compared with alkaline phosphatase, which appears in early progenitors of the marrow stromal cell lineage.13,18 Also, because serum osteocalcin represents only the fraction of the osteoblast's output that escapes incorporation into newly formed bone matrix, this pool may change under different conditions of osteoblast physiology and pathologic activity.
Type I Collagen Propeptides
Collagen Types I, II, and III are synthesized as procollagen monomers from which the N- and C-propeptides are removed extracellularly. The propeptides of all three collagen types appear in serum as by-products of collagen synthesis. Immunoassays for N- and C-propeptides of Type I collagen have been developed as markers of bone formation.10,15,20,23,40,47 Because bone is a major source of collagen Type I turnover, serum levels of these propeptides have been used as an index of total bone-forming activity in the body. Data from clinical studies of various bone diseases show responsiveness when turnover is markedly altered but not in conditions in which bone remodeling changes are known to be subtle.37 In studies that compared serum Type I collagen N- and C-propeptide levels, results showed that the correlation was also poor.20 Therefore, these markers are not in widespread use.
Various biochemical tests have evolved for assessing bone resorption. Most rely on quantifying collagen degradation products in urine. Some of the newer markers that are based on degraded products of cross-linking domains from collagen fibrils offer a higher specificity to osteoclastic resorption activity. Hydroxyproline, which is generic to all collagenous proteins, is a less specific marker than the newer collagen Type I-specific markers.
Total urinary hydroxyproline level (after acid hydrolysis of peptides) is the traditional index, but its usefulness is blunted by contribution from the diet, lack of specificity to bone collagen, degradative losses of the free amino acid in the liver, and the relatively tedious chemical assays used for its determination. More precise assays for hydroxyproline by high-performance liquid chromatography (HPLC) appear to offer greater specificity for bone metabolism than the traditional colorimetric assay.48
All collagenous proteins contain glycosylated hydroxylysine residues. There are two forms, glucosylgalactosylhydroxylysine (GGHyl) and galactosylhydroxylysine (GHyl). They appear to be excreted quantitatively in urine as the free hydroxylysine glycosides, presumably as products of collagen catabolism.44,45,61 Human bone collagen is unusual in containing more GHyl than GGHyl (in contrast with that in skin collagen, for example). Galactosylhydroxylysine also dominates in urine, indicating that bone is the primary source and that GHyl can provide a reasonable index of bone resorption rate. From a clinical perspective, however, the current HPLC assays are not convenient. If a reliable immunoassay for GHyl be developed, it might have clinical potential.
Tartrate-Resistant Acid Phosphatase
Tartrate-resistant acid phosphatase is a prominent product of osteoclasts thought to be active in bone matrix degradation.22 Tartrate-resistant acid phosphatase activity is elevated in serum in clinical conditions that involve increased bone resorption.42 However, certain other cell types can produce tartrate-resistant acid phosphatase.38 Immunoassays for the enzyme molecule,12,41 ideally that are specific to the osteoclast-specific form, might prove useful.
The collagen cross-linking amino acids, pyridinoline and deoxypyridinoline, have been applied extensively in research as bone resorption markers.17,56 These complex amino acids form in the fibrils and have no means of metabolism when collagen is degraded. They are excreted in urine where they can be quantified by HPLC relying on their natural fluorescence for detection.5 Approximately 67% are excreted in the form of small peptides and the remainder as the free amino acids. Two forms exist: hydroxylysylpyridinoline (or simply pyridinoline, HP, or Pyr) and lysylpyridinoline (deoxypyridinoline, LP, or Dpy). They are posttranslational variants that result from incomplete hydroxylation of lysine residues, at sites that will become cross-links, during the synthesis of collagen Types I, II, III, and IX.24
Pyridinolines form mature cross-links in collagens of most connective tissues other than skin (Figure 2). Because bone is a major reservoir of Type I collagen in the body and is believed to remodel much faster than most major connective tissues, bone is likely to be the major source of the pyridinoline pool in urine. This conclusion is supported by the similar ratios of Pyr to Dpy in human bone (3.5:1) and urine (range, 2:1-7:13). Bone is distinguished by its high proportion of Dpy compared with that in most nonbone tissues, in which Dpy is usually less than 10% of the Pyr content.3,24 Urinary Dpy therefore is more specific than Pyr as a marker of bone degradation.64 However, Dpy is not unique to bone,25,62 and the overall Pyr content of bone collagen is actually quite low (0.3 mole/mole) compared with that in other connective tissues (1.5 mole/mole in cartilage; 0.5-1 mole/mole in vascular tissue, tendon, ligament, fascia, lung, intestine, liver, muscle, and so forth.26 Because little is known about collagen degradation rates in tissues other than bone, Pyr and Dpy results should be interpreted cautiously regarding their specificity to bone metabolism.
Collagen Telopeptide Assays
There are two intermolecular sites of Pyr cross-linking in Type I collagen, N-telopeptide-to-helix and C-telopeptide-to-helix. Two pools of Pyr-containing peptides (molecular weight <2 kDa) originating from these sites can be identified in urine. They appear to be discrete, core amino acid sequences attached to the cross-link that resist proteolysis.36 The cross-linked N-telopeptide fraction from urine had a Pyr:Dpy ratio of 2:1, consistent with an origin in bone. Similar domains prepared from human bone collagen showed that 67% of the Dpy in collagen is located at the N-telopeptide site and 33% at the C-telopeptide site.35,36 The α2(I) N-telopeptide also appeared to be a favored site of cross-linking in bone collagen.35 Cross-linked N-telopeptides (NTx) have been targeted, therefore, as a promising marker of bone resorption in urine. A monoclonal antibody, mAb 1H11, provides the basis of the NTx immunoassay.26 It recognizes peptides that contain the α2(I) N-telopeptide sequence QYDGKGVG, where K is involved in a trivalent cross-link. The N-telopeptide antigen is recognized by 1H11 only when proteolytically cleaved to the eight-residue sequence. Osteoclasts generate immunoreactive NTx, but not free pyridinolines (Pyr or Dpy), when cultured on human bone particles in vitro.1 The antigen, therefore, is a proteolytic neoepitope. A microtiter-plate enzyme-linked immunosorbent assay (ELISA) has been developed that can be applied directly to urine.33,36
Two different assays for collagen Type I cross-linked C-telopeptides are also in use. The ICTP (I-C-Telopeptide) assay is based on a polyclonal antiserum raised against the full-length C-telopeptide domain isolated from human bone collagen as a cross-linked peptide.53 It recognizes antigen in serum but not in urine. The CrossLaps assay uses a polyclonal antiserum raised against a synthetic peptide (EKAHDGGR), matching a short segment of the collagen α1(I) C-telopeptide containing the cross-linking lysine residue.7,8 This sequence was selected with the expectation that it would be protected from degradation when embodied in Pyr cross-linked structures and excreted in urine.7,36 CrossLaps is a urine assay, therefore, for cross-linked peptides (CTx) analogous to NTx. ICTP is a serum assay. From the published clinical data, ICTP and CrossLaps are clearly measuring the products of different pathways.30 The ICTP assay is apparently not an index of normal bone resorption and shows no response in patients on antiresorptive therapies.54
Most reports on Pyr levels in urine have measured the total pools of Pyr and Dpy by HPLC after acid hydrolysis to convert peptides to free amino acids.62 The free fraction of Pyr and Dpy has also been targeted. This can be done by HPLC, but immunoassays are more convenient and allow direct analysis without pretreatment steps. A polyclonal antibody-based ELISA that recognizes free Pyr was originally described55 and further developed into polyclonal and monoclonal antibody-based versions.63 More recently, a monoclonal antibody-based ELISA that is specific for free Dpy has also become commercially available.57
Several reports of clinical studies have recently appeared in the literature in which these newer assays of bone formation and resorption were applied and compared. In most reports, mean data from subject groups have been compared to establish the sensitivity of individual markers to reflect altered rates of systemic bone turnover (e.g., in postmenopausal vs. premenopausal women; in Paget's disease patients; in subjects at baseline; and in those receiving estrogen, bisphosphonate, and other therapies). Few reports have presented results in patients to assess the reproducibility and reliability of an assay as a clinical tool in patient evaluation.
Bone formation markers (serum osteocalcin and bone-specific alkaline phosphatase) generally give tighter overall coefficients of variance than urinary assays, which are usually applied to morning urine and normalized to a creatinine determination to correct for differences in dilutions. Diurnal variations in creatinine excretion and bone marker excretion,6 therefore, can add to the longitudinal biological variance in results of urine assays. Collecting 24-hour urine samples can perhaps avoid this but is less ideal in clinical practice.
Serum formation markers respond more slowly to an altered bone remodeling rate that occurs naturally (e.g., at menopause21), after surgical oophorectomy,50 or on intervention with the antiresorptive agents, estrogen51 and bisphosphonates.27 In most clinical situations, resorption and formation are coupled, but changes in formation markers lag several months behind change in resorption markers, whether remodeling activity rises or falls.
Results of several studies comparing the newer formation and resorption markers have been reported.27,30,33,59 Using the percentage increase in bone turnover in postmenopausal women compared with that in premenopausal women as a measure, bone-specific alkaline phosphatase was the most responsive formation marker.30 The level of osteocalcin showed a similar response, but the level of Type I collagen C-propeptide did not change.30 In response to the bisphosphonate alendronate, the concentrations of formation markers bone-specific alkaline phosphatase, osteocalcin, and collagen Type I C-propeptide all dropped 40% to 50%, but did so gradually during a period of 6 to 12 months.30 In this same study, the most responsive resorption marker was the cross-linked N-telopeptide, NTx, in urine. In another study of menopause women,21 this analyte increased the most at menopause and decreased with and the highest percentage from baseline in bisphosphonate intervention trials.30,33,59 NTx detected the response to alendronate after 1 month, declining rapidly into the normal premenopausal range, long before changes in BMD were detectable. Total Pyr, and particularly total Dpy, measured by HPLC after acid hydrolysis, were also responsive, but quantitatively less so than NTx.30,33 In results of a separate study, the cross-linked C-telopeptide (CTx) in urine was also significantly elevated after menopause and suppressed by estrogen and bisphosphonate therapies.27,28
Free pyridinolines measured in urine by immunoassay turn out to be unresponsive or only weakly responsive to bisphosphonate intervention compared with the response of the cross-linked N- or C-telopeptides, total Pyr or total Dpy, when measured by HPLC.27,30,59 The exact biological reason has not been defined, although it is notable that osteoclasts resorbing bone in vitro do not release free pyridinolines but do generate the immunoreactive telopeptide epitope NTx.1 Most recently, it was shown that the osteoclast-specific protease, cathepsin K, is responsible for this release of NTx at the correct cleavage site from bone collagen.2 Therefore, free pyridinolines must be generated by peptide breakdown after the osteoclast, probably in the liver, kidneys, or both. Tissue origins for free pyridinolines other than bone collagen should also be considered25 (Figure 2). Regarding nontissue origins, it is notable that free pyridinolines in urine, as well as total pyridinolines and the telopeptide markers,27 were suppressed during long-term estrogen therapy, perhaps by systemic (i.e., nonbone) effects of estrogen in contrast with effects of bisphosphonates, which target bone cells selectively.
Further studies are needed to understand the cellular and molecular origins of the various markers better. Findings should include determining from which tissues the individual markers can originate and which organs are the principal sites of any secondary catabolism needed to generate each collagen analyte. A desirable resorption marker in urine would be a unique product of osteoclastic resorption that escapes significant further metabolism in the liver and kidneys and is rapidly cleared. It is notable that cathepsin K, considered to be a specific and major protease of the osteoclast,19 turns out to be highly active in quantitatively releasing the telopeptide neoepitope NTx from bone collagen.2
Correlations Between Biomarkers, Bone Mineral Density and Fracture Risk
In results of several recent studies, markers have been correlated with BMD measurements and fracture incidence in postmenopausal and osteoporotic women. Garnero et al31 in a cross-sectional study of women in France found that with increasing time after menopause, the telopeptide markers of resorption, NTx, and CTx, correlated negatively and increasingly strongly with BMD of the spine, hip, forearm, and total body. In findings in a study of women in early menopause who were treated with hormone replacement therapy or calcium supplement, high bone turnover was a predictor of rapid bone loss in the group receiving calcium alone11,58 (Figure 3). In the hormone replacement therapy-treated group, high bone turnover before treatment predicted a greater gain in bone mass. Schneider and Barrett-Connor60 reported results from the Rancho Bernardo community-based study indicating that NTx levels in older men and women correlated inversely with BMD of the hip and spine. There was a significant decrease in BMD in subgroups stratified by increasing quintiles of NTx.
Riggs et al52 reported data from a fluoride trial showing that high bone turnover was a predictor of fracture independent of BMD. From the results of the French EPIDOS study of elderly women, Garnero et al32 also reported that free Dpy in urine was elevated in a subgroup in which subjects eventually had fractures compared with free Dpy levels in an age-matched control group. In a recent comparison of bone markers, BMD, and fracture incidence in a Minnesota community-based population, a negative correlation was found between certain of the bone turnover indexes and BMD at the spine, hip, and forearm.43 Even after adjusting for BMD, elevated levels of bone resorption markers and reduced levels of bone formation markers were independently associated with the risk of osteoporotic fractures.
Biochemical markers that can reliably index systemic bone turnover activity have a place in the prediction of risk for osteoporosis (i.e., in planning a prevention strategy) and in monitoring therapy in the patient (appropriate drug taken correctly at the optimum dose and bone turnover decreased into premenopause range). Barriers to rapid adoption have been confusion about which marker to use, cost (who will pay) and lack of clear guidelines on the use of bone biomarkers in clinical practice.
The author thanks Kae Pierce for preparing the manuscript.
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