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Over the past few decades, scientists have been trying to identify tissue-specific markers that would help to better understand the pathogenesis of bone and cartilage diseases and could be used clinically for the screening, diagnosis and follow-up of bone or joint diseases. Historically, only a few components known to be involved in bone, mineral or cartilage turnover were available for this purpose (e. g., urine hydroxyproline, serum and urine calcium and phosphate levels). However, since most if not all of these substances have wider biological functions beyond bone, mineral and cartilage metabolism, their clinical value as tissue-specific markers was limited. Hence, there was a need to identify more specific indices of bone and cartilage metabolism. Since the 1980s, a number of collagenous and non-collagenous breakdown products as well as cell-specific enzymes have been discovered and developed into markers of musculoskeletal tissue metabolism. This review describes their chemical and biological function, available analytical methods and possible clinical applications., Competing Interests: Conflict of Interest: The authors have no conflict of interest to disclose., (© Georg Thieme Verlag KG Stuttgart · New York.)

The Cre/loxP recombination system can be used to circumvent many of the limitations of generalized gene ablation in mice. Here we present the development and characterization of transgenic mice in which Cre recombinase has been targeted to cells of the osteoblast lineage with 2.3 kb (Col 2.3-Cre) and 3.6 kb (Col 3.6-Cre) fragments of the rat Col1a1 promoter. Cre mRNA was detected in calvaria and long bone of adult Col 2.3-Cre and Col 3.6-Cre mice, as well as in tendon and skin of Col 3.6-Cre mice. To obtain a historical marking of the temporal and spatial pattern of Cre-mediated gene rearrangement, Col-Cre mice were bred with ROSA26 (R26R) mice in which Cre-mediated excision of a floxed cassette results in LacZ expression. In Col 2.3-Cre;R26R and Col 3.6-Cre;R26R progeny, calvarial and long bone osteoblasts showed intense beta-gal staining at embryonic day 18 and postnatal day 5. The spatial pattern of beta-gal staining was more restricted in bone and in bone marrow stromal cultures established from Col 2.3-Cre;R26R mice. Similar differences in the spatial patterns of expression were seen in transgenic bone carrying Col1a1-GFP visual reporters. Our data suggest that Col 2.3-Cre and Col 3.6-Cre transgenic mice may be useful for conditional gene targeting in vivo or for obtaining osteoblast populations for in vitro culture in which a gene of interest has been inactivated.

Unlabelled: Bone metabolism follows a seasonal pattern with high bone turnover and bone loss during the winter. In a randomized, open-label 2-year sequential follow-up study of 55 healthy adults, we found that supplementation with oral vitamin D3 and calcium during winter abolished seasonal changes in calciotropic hormones and markers of bone turnover and led to an increase in BMD. Supplementation with oral vitamin D3 and calcium during the winter months seems to counteract the effects of seasonal changes in vitamin D and thus may be beneficial as a primary prevention strategy for age-related bone loss., Introduction: Bone metabolism follows a seasonal pattern characterized by high bone turnover and bone loss during winter. We investigated whether wintertime supplementation with oral vitamin D3 and calcium had beneficial effects on the circannual changes in bone turnover and bone mass., Materials and Methods: This prospective study comprised an initial observation period of 12 months ("year 1"), followed by an intervention during parts of year 2. Fifty-five healthy subjects living in southwestern Germany (latitude, 49.5 degrees N) were randomized into two groups: 30 subjects were assigned to the treatment group and received oral cholecalciferol (500 IU/day) and calcium (500 mg/day) during the winter months of year 2 (October-April), while 25 subjects assigned to the control group obtained no supplements. Primary endpoints were changes in calciotropic hormones [serum 25(OH)D, 1,25(OH)2D, and parathyroid hormone], markers of bone formation (serum bone-specific alkaline phosphatase) and of bone resorption (urinary pyridinoline and deoxypyridinoline), and changes in lumbar spine and femoral neck BMD., Results: Forty-three subjects completed the study. During year 1, calciotropic hormones, markers of bone turnover, and BMD varied by season in both groups. During the winter months of year 1, bone turnover was significantly accelerated, and lumbar spine and femoral BMD declined by 0.3-0.9%. In year 2, seasonal changes in calciotropic hormones and markers of bone turnover were either reversed or abolished in the intervention group while unchanged in the control cohort. In the subjects receiving oral vitamin D3 and calcium, lumbar and femoral BMD increased significantly (lumbar spine: +0.8%, p = 0.04 versus year 1; femoral neck: +0.1%, p = 0.05 versus year 1), whereas controls continued to lose bone (intervention group versus control group: lumbar spine, p = 0.03; femoral neck, p = 0.05)., Conclusions: Supplementation with oral vitamin D3 and calcium during winter prevents seasonal changes in bone turnover and bone loss in healthy adults. It seems conceivable that annually recurring cycles of low vitamin D and mild secondary hyperparathyroidism during the winter months contributes, at least in part and over many years, to age-related bone loss. Supplementation with low-dose oral vitamin D3 and calcium during winter may be an efficient and inexpensive strategy for the primary prevention of bone loss in northern latitudes.

Glucocorticoid excess leads to bone loss, primarily by decreasing bone formation. However, a variety of in vitro models show that glucocorticoids can promote osteogenesis. To elucidate the role of endogenous glucocorticoids in bone metabolism, we developed transgenic (TG) mice in which a 2.3-kb Col1a1 promoter fragment drives 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) expression in mature osteoblasts. 11beta-HSD2 should metabolically inactivate endogenous glucocorticoids in the targeted cells, thereby reducing glucocorticoid signaling. The inhibitory effect of 300 nm hydrocortisone on percent collagen synthesis was blunted in TG calvariae, demonstrating that the transgene was active. Collagen synthesis rates were lower in TG calvarial organ cultures compared with wild-type. Trabecular bone parameters measured by microcomputed tomography were reduced in L3 vertebrae, but not femurs, of 7- and 24-wk-old TG females. These changes were also not seen in males. In addition, histomorphometry showed that osteoid surface was increased in TG female vertebrae, suggesting that mineralization may be impaired. Our data demonstrate that endogenous glucocorticoid signaling is required for normal vertebral trabecular bone volume and architecture in female mice.

A variety of in vivo and in vitro experimental models have been used to explore the effects of glucococorticoids in bone. Chronically high levels of glucocorticoids typically decrease bone mass in humans and animals and inhibit markers of bone formation in organ and cell cultures. However, under certain experimental conditions, glucocorticoids can stimulate osteoblast differentiation and bone formation in vitro. The relevance of these effects seen in culture models to the role of endogenous glucocorticoids in bone remains unclear. In this article, we briefly review possible pathways for the opposing effects of glucocorticoids on bone formation and propose several genetic loss-of-function mouse models in which disruption of glucocorticoid signaling in cells of the osteoblast lineage would provide a means to determine the role of endogenous glucocorticoids in bone.

Background: Myeloma-induced bone loss is related to an uncoupling of bone formation and bone resorption. The aim of the present study was to assess the potential clinical value of biochemical markers of bone formation in the work up of patients with plasma cell dyscrasias., Methods: Serum total alkaline phosphatase, bone-specific alkaline phosphatase (BAP), and osteocalcin (OC) were measured in 43 patients with newly diagnosed multiple myeloma (MM), in 40 patients with monoclonal gammopathy of undetermined significance (MGUS), in 40 patients with untreated benign vertebral osteoporosis (OPO), and in 48 healthy adults., Results: In MM and MGUS patients, serum BAP, but not serum OC, was lower than in healthy controls (P<0.05). Serum OC was higher in patients with OPO than in healthy controls (P<0.05). The strongest associations between markers were found in OPO patients and in healthy adults. MM patients with early-stage disease or without detectable osteolysis had decreased serum BAP values (P<0.05). Serum OC was higher in MM patients with stage III disease (P<0.05) than in healthy controls. MM patients with OPO-like bone involvement had lower BAP values than sex- and age-matched MGUS patients with OPO-like bone involvement and patients with benign OPO (P<0.05)., Conclusions: In patients with plasma cell dyscrasias, serum BAP, rather than serum OC, appears to reflect a suppressed bone formation rate and may be helpful in the differentiation between benign and myeloma-induced OPO. However, the overall clinical use of biochemical markers of bone formation in patients with plasma cell dyscrasia appears limited.

To test the potential of immunoreactive BSP, a non-collagenous bone matrix component, as a clinical guide in patients with plasma cell dyscrasias, serum BSP concentrations were measured in 62 patients with newly diagnosed multiple myeloma (MM) followed over a period of 4 years, in 46 patients with monoclonal gammopathy of undetermined significance (MGUS), in 71 patients with untreated benign vertebral osteoporosis (OPO), and in 139 healthy adults. Results were compared with clinical and laboratory data, including serum osteocalcin (OC), and urinary pyridinoline (PYD) and deoxypyridinoline (DPD) as markers of bone turnover. In MM, serum BSP, and urinary PYD and DPD were higher than in healthy controls and in MGUS or OPO (P< 0.001). BSP levels correlated with the bone marrow plasma cell content (r = 0.40, P< 0.001), and serum beta2-microglobulin (r = 0.31, P < 0.01). The differentiation of MM from healthy controls and from MGUS or OPO was highest for BSP. After chemotherapy, BSP reflected the response to treatment and correlated with the change in monoclonal protein (r = 0.55, P< 0.001). MM patients with normal baseline BSP levels survived longer than patients with initially elevated BSP values (P< 0.001, log rank test). Only serum monoclonal protein and BSP were independent predictors of survival. We conclude that in MM, BSP levels are associated with skeletal involvement and tumour cell burden. The quantification of serum BSP may be a non-invasive method for the diagnosis and follow-up, and may improve the prognostic value of conventional staging in MM.

Molecular markers of bone turnover have gained increasing relevance in the evaluation of patients with metabolic bone diseases. Their clinical applications include the assessment of future osteoporotic fracture risk, complementation of bone density measurements, diagnosis of certain metabolic osteopathies, therapeutic decision making, and monitoring of therapeutic efficacy and patient compliance. One should be aware, however, that the results from large epidemiologic or clinical trials are sometimes difficult to translate into the everyday clinical situation. The individual patient often has more than one disease that might affect either bone turnover or the handling of the parameters mentioned (or both). Analytic and biologic variability of bone markers can be significant and also needs to be considered when using these indices. In the scientific setting, conventional and new markers of bone turnover can help to elucidate formerly unknown mechanisms and pathways. Because the development of ever more specific and sensitive markers of bone metabolism is progressing rapidly, we are likely to witness new insights into the pathophysiology of bone diseases in the near future.

Recent studies suggest a circannual pattern of bone turnover. To further investigate the underlying mechanisms, 41 healthy subjects (25-80 years old) living in a southwestern German city were studied prospectively over a period of 18 months. Participants were examined every 4 weeks, and blood and urine samples were obtained on each visit. The following parameters were measured: serum 25-hydroxyvitamin D3 [25(OH)D3], 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and parathyroid hormone (PTH), as regulators, and serum total alkaline phosphatase (TAP), bone-specific alkaline phosphatase (BAP), urinary total pyridinoline (PYD), deoxypyridinoline (DPD), and the aminoterminal telopeptide of collagen type I (NTX), as biochemical markers of bone turnover. The presence of significant circannual rhythms for the various markers was tested using the Pharmfit method. In the total group, 25(OH)D3, 1,25(OH)2D3, and PTH as well as BAP, PYD, DPD, and NTX showed a significant seasonal variation. 25(OH)D3 revealed the highest amplitude (38.0%) with an acrophase in August. Levels of the biochemical markers and of PTH were highest in winter with amplitudes of up to 17.7% (DPD). Results were most pronounced in premenopausal women, in subjects <50 years of age, and in subjects who did show a significant individual rhythm in 25(OH)D3 levels. No differences were found regarding other anthropometric or life style factors. Correlation analyses revealed strongest associations between the amplitudes of a vitamin D metabolite and a biochemical marker in premenopausal women. We conclude that specific markers of bone turnover show significant circannual rhythms. These changes are related directly to variations in the hormonal regulation of skeletal homeostasis. In postmenopausal women and in men, other effects may superimpose the circannual variation of biomarkers of bone turnover.

The goals of this study were to examine the role of insulin-like growth factor I (IGF-I) on bone formation and to test the hypothesis that the inhibitory effects of glucocorticoids on bone formation are independent of the IGF-I pathway. In serum-free organ cultures of 18-day fetal mouse calvariae derived from Igf1 null mice (Igf1-/-) and their wild-type (Igf1+/+) and heterozygous (Igf1+/-) littermates, we measured the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), percent collagen synthesis (PCS), the incorporation of [3H]thymidine into DNA, and messenger RNA (mRNA) levels of osteoblast markers in the presence or absence of dexamethasone. After 24 h of culture, calvariae of all genotypes had similar levels of PCS. However, after 48-96 h of culture, PCS was significantly lower in Igf1-/- calvariae compared with Igf1+/+ calvariae. Treatment of calvariae with 100 nM of dexamethasone for 48-96 h decreased PCS in all genotypes. After 72 h of culture, [3H]thymidine incorporation was similar in all genotypes and 100 nM dexamethasone caused a significant reduction in [3H]thymidine incorporation in all genotypes. Dexamethasone at 100 nM decreased alpha1(I)-collagen (Colla1) mRNA and increased alkaline phosphatase, bone sialoprotein, and osteopontin mRNA in all genotypes after 72 h of culture. Type I IGF receptor mRNA levels were highest in Igf1-/- calvarial cultures. Dexamethasone at 100 nM increased Igf2 and type I IGF receptor mRNA levels in all genotypes. We conclude that one intact allele for Igf1 is sufficient to maintain normal rates of collagen synthesis in fetal mouse calvarial cultures. Moreover, the inhibitory effects of glucocorticoids on collagen synthesis and cell replication are at least partially independent of the IGF-I pathway in this model.

This article describes the biochemical background and functional properties of molecular markers derived from osteoblasts, the collagenous, or noncollagenous bone matrix. Clinical applications and the selection, timing, and monitoring of antiosteoporotic treatment are critically discussed. Special emphasis has been put on reviewing the value of bone resorption markers in the prediction of future bone loss and the risk assessment for osteoporotic fractures. The potential of these indices as independent risk factors for future fractures alone or in combination with bone mineral density measurements has recently been demonstrated in large cohort studies. More data are needed, however, to establish biochemical markers of bone resorption as useful tools in the assessment of individual fracture risk.

Advanced tumor osteopathy is characterized by abnormal bone turnover. Using a rat model of parathyroid hormone-related peptide (PTHrP)-mediated tumor osteolysis, the aim of the present study was to define the sequential changes in, and the association between, biochemical and histomorphometric indices of bone metabolism during the early stages of developing tumor osteopathy. Eight-month-old Wistar rats (n = 48) were subcutaneously inoculated with either 2 x 10(6) cells of the Walker carcinosarcoma 256, or saline on day 0, and treated with either saline or the bisphosphonate ibandronate until killing on day 8. Serum calcium (sCa), alkaline phosphatase (sTAP), and osteocalcin (sOC) and urinary calcium (uCa), deoxypyridinoline (uDPD), and pyridinoline (uPYD) were measured daily. In a second semilongitudinal experiment (n = 70), the number of osteoclasts and osteoblasts (N.Oc, N.Ob), trabecular bone volume (BV/TV), and osteoid volume (O.Ar) were assessed by histomorphometry. In untreated tumor-bearing animals, osteoclast numbers increased by 74% on day 3 (5.4 +/- 2.4 vs. 3.1 +/- 1.5/mm(2), p < 0.05), and trabecular bone volume fell by 24% on day 4 (12.5 +/- 2.0 vs. 15.8 +/- 1.2%, p < 0.05). Both time course and magnitude of these changes were closely reflected by an increase in uDPD (0.46 +/- 0.14 vs. 0. 31 +/- 0.15 nmol/12 h, p < 0.05) and uPYD on day 4 (1.44 +/- 0.25 vs. 1.03 +/- 0.3 nmol/12 h, p < 0.05), sCa (3.8 +/- 0.52 vs. 3.0 +/- 0. 13 mmol/L, p < 0.01), and uCa (0.13 +/- 0.08 vs. 0.03 +/- 0.01 mmol/12 h, p < 0.001) on day 6, and sTAP (254 +/- 127 vs. 120 +/- 40 U/L, p < 0.001) on day 7 (mean +/- SD), whereas sOC remained unchanged until day 8. When combining the results of the two experiments, a high correlation was found between the number of osteoclasts and the urinary excretion of PYD (r = 0.91) and DPD (r = 0.89). Treatment with ibandronate delayed hypercalcemia, abolished hypercalciuria, and accelerated bone resorption. We conclude that osteoclast activation is an early event in PTHrP-mediated osteolysis, which is closely reflected by the renal excretion of pyridinium cross-links of type I collagen. Therefore, specific biochemical markers of collagen breakdown may be useful as early indicators of developing tumor osteopathy.

Background: In Paget disease of bone (PD), serum total alkaline phosphatase (TAP) is a valid marker of disease activity. The aim of the present longitudinal study was to compare TAP with new and potentially more specific markers of bone turnover in bisphosphonate-treated patients with PD., Methods: Twenty patients with active PD were studied before and after treatment with 2 mg of intravenous ibandronate over a period of 12 months. TAP (by colorimetry), serum bone-specific alkaline phosphatase (BAP; by enzyme immunoassay), serum osteocalcin (OC; by ELISA), serum bone sialoprotein (BSP; by RIA), and urinary total pyridinoline (PYD; by HPLC) and deoxypyridinoline (DPD; by HPLC) were measured as markers of bone turnover., Results: Before treatment, TAP, BAP, and BSP were increased in all 20 patients, whereas OC was increased in 10, PYD in 13, and DPD in 15 patients. Three months post treatment, nine patients showed normalized TAP values, and a >/=25% re-increase (i.e. , relapse) was observed in all patients after 12 months. A normalization of BAP was achieved in six patients only. No significant changes were found for OC. BSP was decreased significantly at 24 h, and DPD at 48 h post treatment. A normalization of BSP was found in 8, of PYD in 18, and of DPD in 16 cases. Both PYD and DPD increased significantly from 9 months post treatment onward., Conclusions: Most markers of bone turnover show similar long-term changes after treatment of active PD with ibandronate. With regard to cost-effectiveness and assay performance, TAP remains the marker of choice in therapeutic monitoring of PD. However, more specific markers may improve the biochemical assessment of PD in certain situations.

The interest in and the need for effective measures to be used in the screening, diagnosis, and follow-up of disorders of connective tissue, bone, and mineral metabolism has markedly grown. Next to clinical and imaging techniques, indices of bone turnover have come to play an important role in the assessment of metabolic bone disease. In osteoporosis, recent research has shown that bone markers may also be used to predict future bone loss and hip fractures (in larger cohorts of older patients), identify individuals at risk for osteoporosis, select therapy, and predict and monitor the therapeutic response in individual patients. The development of new markers of bone metabolism has greatly enriched the spectrum of serum and urine analytes used in the assessment of skeletal pathologies. Besides total alkaline phosphatase, other markers such as bone-specific alkaline phosphatase, osteocalcin, or the collagen propeptides are being used to measure bone formation. Bone resorption, previously assessed only by the measurement of urinary calcium and hydroxyproline, may now be detected more precisely by a number of new serum and urine markers. Among these, the pyridinium crosslinks and the telopeptides of collagen type I are presently considered the most specific markers of bone resorption. More recently, bone sialoprotein has also been suggested as a marker of bone resorption in serum. Tartrate-resistant acid phosphatase is now measurable by immunoassay. This article surveys the biochemistry and relevant technical aspects of the currently available markers of bone metabolism.

Molecular markers of bone and cartilage metabolism are increasingly used as helpful tools in the diagnosis and early detection of joint disease. Their quantification may help to improve the ability for therapeutic monitoring and follow-up of patients affected with rheumatic or degenerative cartilage disorders. The biochemical background and the functional properties of markers of bone, cartilage, and synovial tissue turnover are described. Current clinical applications and the diagnostic and prognostic validity of these metabolites are presented with special emphasis on recent research activities. Finally, molecular aspects of bone and joint diseases are discussed, highlighting genetic contributions to the development of joint disease.

Although urinary measurements of collagen degradation provide valid estimates of bone resorption, their clinical application is hampered by pronounced analytical and biological variability. Therefore, immunoassays for the determination of such parameters in serum have been developed. In this study, we assessed the performance of three new serum markers of bone turnover, i.e., C-terminal and N-terminal telopeptides of type I collagen (S-CTX and S-NTX) and bone sialoprotein. Results were compared with urinary total pyridinoline, total deoxypyridinoline, and urinary C-terminal telopeptides of type I collagen (U-CTX) and urinary N-terminal telopeptides of type I collagen (U-NTX). The study population included healthy men (n = 27), premenopausal (n = 30) and postmenopausal (n = 31) women, patients with hepatic dysfunction (HF, n = 24), renal failure (RF, n = 30), breast cancer without (BC-, n = 24) and with (BC+, n = 30) bone metastases, primary vertebral osteoporosis (OPO, n = 27), primary hyperparathyroidism (PHPT, n = 16), active Paget's disease of bone (n = 18), multiple myeloma (MM, n = 18), and patients with hypercalcemia of malignancy before and after treatment with pamidronate (HOM, n = 28). Changes in urinary and serum markers were similar in most metabolic bone diseases. However, differentiation between healthy controls and OPO, or PHPT, was improved by the serum markers. In MM, all serum and urinary markers were elevated (p < 0. 05 vs. controls). In BC+, skeletal involvement was reflected by significant increments in all indices (p < 0.01 vs. BC-), except U-CTX and S-CTX. In HOM, pamidronate-induced changes in biomarkers were most pronounced for U-CTX and S-CTX and S-NTX. HF and RF were associated with elevated levels of all serum markers (p < 0.05 vs. controls). In conclusion, measurements in serum reflect bone resorption to the same extent as the urinary indices. Since serum markers circumvent some of the limitations of urinary measurements, their use potentially improves the assessment of skeletal disorders.

To investigate the effect of medrogestone on bone mineral density (BMD) and bone turnover under conditions of estrogen withdrawal, premenopausal women with endometriosis were treated with goserelin (Zoladex), combined with either placebo (group A, n = 12) or 10 mg medrogestone (Prothil, group B, n = 11) for six months, and followed for an additional six months. Lumbar spine BMD was measured at 0 and 6 month. Markers of bone turnover were serum bone alkaline phosphatase (sBAP) and osteocalcin (sOC) by ELISA, and urinary total pyridinoline (uPYD) and deoxypyridinoline crosslinks (uDPD) by HPLC. Patients in both groups had a similar and significant decrease in BMD after 6 months (4%, p < 0.01). The time course of changes in bone turnover, in contrast, was different in both groups. In group A, crosslink excretion increased from one month onwards, while no changes were seen in group B. In group A, sBAP levels rose during treatment, while in group B, this rise was delayed until treatment was terminated. Additionally, group B showed an initial suppression of sBAP and sOC. In both groups, sOC increased after treatment was discontinued. Medrogestone at 10 mg/d does not prevent lumbar bone loss in premenopausal women under estrogen deprivation. In the medrogestone add back group, the changes in bone turnover are compatible with low turnover bone loss,as ooposed to a state of high turnover seen in the unopposed goserelin group. This effect may be due to glucocorticoid receptor mediated actions of medrogestone on bone.

Physical activity is considered an important factor in attaining bone mass. However, the mechanisms by which exercise affects bone metabolism are not completely understood. The present study was performed to investigate the effects of aerobic and anaerobic exercise on bone turnover. Twenty healthy young males (aged 20-29 years) were followed through an 8-week program of aerobic (n = 10) and anaerobic training (n = 10). Ten age-matched individuals served as controls. Serum bone-specific alkaline phosphatase (BAP), serum osteocalcin (OC), and urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd) were determined as indices of bone metabolism. After 4 weeks of aerobic training, serum BAP and OC (p < 0.01), and urinary Pyd (p < 0.001) and Dpd (p < 0.01) were significantly reduced. After 8 weeks, BAP and OC levels had returned to baseline values, whereas the urinary cross-link excretion remained low. In the anaerobic training group, elevated levels of BAP (p < 0.05 vs. week 4), OC (p < 0.05 vs. week 4), and Pyd (p < 0. 01 vs. week 0) were observed after 8 weeks of exercise. Changes in urinary Pyd and Dpd (week 0 vs. week 8) were positively correlated with changes in the mean power level in the Wingate test, a parameter of the anaerobic performance capacity (r = 0.50 and r = 0. 55, p < 0.01, respectively). In the controls, no significant changes in biochemical markers were observed. We conclude that aerobic and anaerobic training excert different effects on bone metabolism. While aerobic training led to changes compatible with reduced bone resorption activity, anaerobic training seems to result in an overall accelerated bone turnover. Therefore, the impact of physical activity on bone turnover may depend on the kind of exercise performed.

We evaluated an automated chemiluminescence immunoassay (CLIA) developed for the measurement of urinary free deoxypyridinoline (DPD). The new DPD method by CLIA is based on the competition of DPD with particle-bound pyridinoline for a limited amount of monoclonal mouse anti-DPD antibody. Total imprecision (CV) was 3.2-9.0% at 30-270 nmol/L. Regression analysis of urinary DPD concentration (second morning-void) measured by CLIA (y) and enzyme immunoassay (EIA) for adult volunteers (n = 449) with and without bone disease revealed a best fit equation of: y = 1.08 +/- 0.03x - 1.15 +/- 0.98 nmol/L (r = 0.964, S(y/x) = 14 nmol/L). CLIA and EIA methods were correlated with HPLC measurement of urinary free DPD (r = 0.846 and 0.871, respectively). For healthy adults, the creatinine-normalized excretion of DPD (mean +/- SD) measured by CLIA for 61 men (4.1 +/- 1.2 micromol DPD/mol creatinine) and 76 premenopausal women (5.3 +/- 1.8 micromol DPD/mol creatinine) did not differ significantly (P >0.05) from DPD excretion measured by EIA, and both immunoassays showed a significant gender difference (P <0.001) in reference intervals. In a clinical trial, DPD excretion (micromol DPD/mol creatinine) measured by CLIA differed substantially from the reference population for 54 untreated pagetic (12.7 +/- 8.0 SD), 255 untreated osteoporotic (7.5 +/- 4.1), 21 osteomalacic (12.4 +/- 8.5), 17 primary hyperparathyroid (9.4 +/- 4.4), and 14 secondary hyperparathyroid (9.2 +/- 5.1) patients. Clinical sensitivities of the CLIA and EIA methods range from 38% to 80% in bone disorders and limit the use of the DPD measurement in disease detection. DPD excretion after pamidronate treatment in a subgroup of the pagetic patients fell dramatically as assessed by CLIA or EIA. We conclude that the automated CLIA method for DPD is a convenient and reliable method that may aid in the evaluation and management of bone disease and is applicable to high volume testing in the routine clinical laboratory.

With the increasing demand for clinically useful biomarkers of bone turnover, a number of assays for the measurement of bone resorption markers have been developed. In the present study, automated (ACS: 180 DPD, Chiron Diagnostics, USA) and manual (DPD-ELISA, Pyrilinks-D, Metra Biosystems, USA) immunoassays for free DPD, and a manual immunoassay for the aminoterminal telopeptide of type I collagen (NTX, Osteomark, Ostex International, USA) were compared to the automated HPLC method for free DPD. Urine samples from a total of 538 healthy and diseased subjects aged 20 to 80 years were analyzed. The age and sex stratified reference ranges were essentially identical for the HPLC, ACS: 180 and the DPD-ELISA, but differed from the NTX assay. Individual values for free DPD as generated by HPLC and immunoassay techniques were highly correlated with each other, whereas correlations between assays measuring free and peptide-bound crosslink components were less pronounced. Precision of the automated techniques (HPLC and ACS: 180) was superior to that of the manual immunoassays. Disease-specific changes in crosslink excretion were similar for all assays and most pronounced in metastatic osteopathy, primary hyperparathyroidism and untreated Paget's disease of bone. We conclude that the automated assays for free DPD in urine, i.e. the HPLC and the ACS: 180 assay, show better analytical performance than the manual immunoassays studied. All techniques used in the present study appear to provide similar or identical clinical information. Therefore, the decision which assay to use largely depends on the laboratory set-up, the number of samples to be analysed, the turn-around time required, and the application for which the test should be used.

Biochemical markers of bone turnover have been shown to provide valuable information for the diagnosis and monitoring of metabolic bone disease. However, these dynamic indexes are influenced by a number of factors that need to be clearly identified to improve their clinical usefulness. To evaluate the contributions of anthropometric, life style, and environmental variables on bone turnover, biochemical markers of bone metabolism were determined in a population-based sample of 580 adults, aged 50-81 yr (297 men and 283 women). Subjects were recruited during 14 consecutive months within the framework of the European Vertebral Osteoporosis Study. Serum total and bone-specific alkaline phosphatase (S-BAP), serum C-terminal propeptide of type I collagen, and serum osteocalcin (S-OC) were measured as bone formation markers. Urinary total pyridinoline and deoxypyridinoline were included as bone resorption indexes. In females, serum levels of 25-hydroxyvitamin D3 were significantly higher (P < 0.01) in summer (May-September) than in winter (October-April), whereas no significant differences were found in males. In both sexes, no seasonal changes were seen in serum PTH. In males, serum total alkaline phosphatase (P < 0.01), S-BAP (P < 0.001), and S-OC (P < 0.05) were significantly higher in winter than in summer. During the same period, females had higher values of S-BAP (P < 0.05), S-OC (P < 0.01), and urinary pyridinoline and deoxypyridinoline (P < 0.001, respectively). Univariate analyses of the effects of life style habits on markers of bone metabolism revealed that in females, regular alcohol consumption and current smoking led to a suppression of markers of bone turnover, whereas in males, only alcohol intake was associated with such changes. In contrast, physical activity was associated with higher levels of bone formation markers and reduced levels of bone resorption indexes in both sexes. As shown by multivariate regression analyses, seasonal variations accounted for more of the variability in most biomarkers (up to 12%) than any of the other anthropometric or life style factors except age. This effect may be attributed to subclinical vitamin D deficiency during the winter period, which is common in countries of the northern hemisphere. We conclude that seasonal variation contributes significantly to the biological variability of bone turnover and needs consideration when interpreting the results of bone marker measurements.

Bone sialoprotein (BSP) is a phosphorylated skeletal glycoprotein. Here we describe a new procedure for the purification of BSP involving wide-pore reversed-phase HPLC, and the development of a homologous RIA for human BSP. The immunoassay showed linearity between 3 and 120 micrograms/L, a lower detection limit of 0.7 micrograms/L, and a mean recovery rate of 99.4%. Intraassay variation was 7.0% (mean = 10.9 micrograms/L) and 6.1% (mean = 38.8 micrograms/L), and interassay variation was 9.2% (mean = 11.1 micrograms/L) and 9.4% (mean = 39.0 micrograms/L). No cross-reactivity was detected with osteocalcin, osteonectin, or bone alkaline phosphatase. Preliminary clinical evaluation in healthy controls (n = 90) showed a mean serum BSP concentration on 12.1 +/- 5.0 micrograms/L (+/- SD). BSP was significantly increased in patients with Paget disease of bone, primary and secondary hyperparathyroidism, and also in subjects with renal failure without skeletal involvement. Impairment of hepatic function did not affect serum BSP concentrations.

To quantify osseous breakdown in multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and benign osteoporosis, we measured urinary levels of pyridinium cross-links of collagen in 50 patients with newly diagnosed and untreated MM, 40 patients with MGUS, 40 untreated patients with osteoporotic vertebral fractures, and 64 healthy adults. Ion-paired, reverse-phase high-performance liquid chromatography (HPLC) was used to measure total urinary excretion of pyridinoline (h-PYD) and deoxypyridinoline (h-DPD). Urinary excretion of free immunoreactive deoxypyridinoline (i-DPD) was determined with an enzyme immunoassay. MM patients had significantly (P < .0001) higher levels of h-PYD, h-DPD, and i-DPD than the healthy adults, patients with MGUS, or patients with osteoporosis. The MGUS and osteoporosis groups presented with elevated (P < .05) levels of urinary pyridinium cross-links when compared with healthy controls. In 20 MM patients who subsequently received chemotherapy, the percent changes in i-DPD did not correlate with the changes in the monoclonal protein. In one of three patients experiencing a transition of initial MGUS into stage I MM, i-DPD increased above the upper limit of the normal range. In 13 patients with stable MGUS, i-DPD remained normal in repeated measurements. Based on the upper limits of the normal range, the sensitivity of urinary pyridinium cross-links in stage I and II MM was low (<50%), but it was between 78% (h-DPD) and 93% (i-DPD) in stage III MM. Specificity in patients with MGUS was between 87% (h-PYD) and 97% (h-DPD). In conclusion, determining the urinary excretion of pyridinium cross-links seems to be a promising noninvasive and thus easily repeatable method for evaluating the actual degree of osseous breakdown. Although measurement of pyridinium cross-link levels is not useful in discriminating patients with MGUS from early-stage myeloma patients, determination of i-DPD levels may contribute importantly to clinical guidance, since increased i-DPD levels seem to identify patients who are particularly likely to benefit from osteoclast-inhibiting drugs such as bisphosphonates. The fact that in a number of patients paraprotein concentrations and i-DPD levels did not change in parallel but instead diverged strongly after chemotherapy might explain the observation that bone lesions sometimes progress even in patients who achieve complete remission.

The collagen cross-linking compound deoxypyridinoline (DPD) has been shown to be a marker of bone resorption and skeletal growth in children. However, the original method for the determination of total (i.e. free and peptide-bound) DPD (tDPD) in urine samples is technically demanding. The recent development of a simple enzyme-linked immunosorbent assay for the quantification of immunogenic DPD (iDPD) in urine samples might be a more convenient technique. Yet, it is unclear at present whether iDPD is equal to tDPD as an index of bone resorption and skeletal growth. Therefore, using 24-h urines from 144 healthy children and adolescents aged 4-19 years, we established reference ranges for the daily urinary excretion of iDPD. A close correlation was found between the daily urinary excretion of iDPD and tDPD related to body weight (r = 0.87, p < 0.001). In 72 subjects aged 4-18 years, the daily excretion of iDPD normalized to body weight was highly significantly correlated with growth velocity (r = 0.70, p < 0.001). We conclude that the enzyme-linked immunosorbent assay for urinary iDPD appears to provide a good index of bone resorption and growth in healthy children.

To evaluate the diagnostic validity of new assays for bone-specific alkaline phosphatase (BAP), we compared measurements of total alkaline phosphatase (TAP) in serum with results for three different assays of serum BAP in healthy adults (n = 119), patients with chronic nonskeletal disorders (n = 123), and patients with metabolic bone diseases (n = 113). Serum TAP was determined by a standard colorimetric assay, BAP by the methods of lectin precipitation (L-BAP), enzyme immunoassay (E-BAP), and immunoradiometric assay (I-BAP). Impairment of liver function resulted in significant increases of all alkaline phosphatase (AP) measurements, with the smallest changes being exhibited by E-BAP. Compared with the results by TAP, diagnostic sensitivity (i.e., of values exceeding the reference interval) was not improved by BAP, but receiver-operating characteristic (ROC) curve analyses revealed improved discrimination for primary hyperparathyroidism by E-BAP. These results indicate that, in the presence of liver disease, the specificity of AP measurements is improved by measuring BAP. In most other clinical situations, serum TAP appears to provide sufficient clinical information; however, the cross-sectional study design used here allows no statement about the usefulness of BAP in serial measurements.

Bone sialoprotein (BSP) is a phosphorylated glycoprotein with a M(r) of 70-80 kDa that accounts for approximately 5-10% of the noncollagenous proteins of bone. Due to its relatively restricted distribution to mineralized tissues, BSP may serve as a potential marker of bone metabolism. Employing a recently developed RIA, serum BSP was measured in 133 healthy subjects, aged 20-80 yr, and in patients with primary hyperparathyroidism (pHPT; n = 26), Paget's disease of bone (PD; n = 14), untreated multiple myeloma (MM; n = 32), and breast cancer with bone metastases (BC; n = 19). Results were compared to clinical and laboratory data, including serum total alkaline phosphatase as a marker of bone formation, and the urinary cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) as markers of bone resorption. In healthy adults, serum BSP values ranged between 5.0-21.6 ng/mL (5-95% interval), with a median of 10.5 ng/mL (total group). In healthy females, a linear correlation was found between serum BSP and age (r = 0.51; P < 0.001), with significantly higher values in postmenopausal than in premenopausal women (13.3 +/- 4.8 vs. 9.0 +/- 3.8; P < 0.01). In the healthy group, BSP values did not change with body mass index, lumbar bone mineral density, serum calcium, serum creatinine, or serum total alkaline phosphatase levels. In contrast, a weak, but significant, correlation was observed between serum BSP and the urinary excretion of PYD and DPD. Compared to those in healthy controls, serum BSP levels were significantly higher in patients with pHPT, PD, MM, or BC (P < 0.01 for all groups). These differences remained after analyses were adjusted for age and sex. In pHPT, serum BSP levels were closely correlated to urinary PYD and DPD (r = 0.87 and 0.83, respectively; P < 0.01), whereas in PD, no correlation was observed between any of the bone markers. Serum BSP levels were highest in patients with MM, and there was a significant difference between early and advanced stages of the disease (30.2 +/- 8.0 vs. 64.3 +/- 6.8; P < 0.01). In a subgroup of 15 patients with metastatic BC, iv bisphosphonate treatment resulted in a rapid reduction of serum BSP levels to 40% of the baseline values within 4 days of treatment. In conclusion, BSP appears to be a sensitive marker of bone turnover, and the present data suggest that its serum levels predominantly reflect processes related to bone resorption.

There is a large body of evidence that points to a systemic link between the somatotropic axis and sex hormones, but epidemiologic data on the interactions between the two hormonal systems are still missing. We examined here the associations between the plasma levels of insulin-like growth factor (IGF) I, IGF-II, IGF-binding protein 3 (IGFBP-3), and sex hormones in a population-based sample of 486 men and women, aged 50-80 yr. The strongest association was an age-independent inverse correlation between all three circulating IGF components and sex hormone-binding globulin (SHBG), the major testosterone-binding protein in plasma. Consistent with this, bio-available (non-SHBG-bound) but not total testosterone levels were positively associated with the IGF system in men, and 3 alpha-androstanediol glucuronide was positively correlated with circulating IGFs in women. Moreover, part of the correlation between the circulating IGF system and bone mineral density at the femur and the calcaneus could be accounted for by SHBG. Our data suggest that sex hormones and the GH/IGF system are significantly interrelated in the elderly population. These hormonal interactions may play an important role in human aging and the pathogenesis of age-related diseases.

Bone metastases strongly affect skeletal metabolism by both their growth and their paracrine activities. However, so far no specific laboratory marker has been found to signal the spread of neoplastic disease to bone. We performed a cross-sectional study of 153 cancer patients and an equal number of healthy controls matched for sex and age, in which we determined serum levels of calcium and total alkaline phosphatase (TAP) as well as the fasting urinary excretion of calcium (uCa) and of the collagen cross-links pyridinoline (uPYD) and deoxypyridinoline (uDPD). The aim of the study was to analyze the diagnostic validity of the biochemical parameters measured with regard to neoplastic bone involvement. In the cancer group, 98 patients had overt bone metastases, as judged from radiographic and radioisotopic bone imaging. The remaining 55 patients were also in an advanced stage of disease, but there was no evidence of malignant bone involvement. In comparison to healthy controls, patients both with and without metastatic bone disease had significantly higher levels of TAP, uPYD, and uDPD (P < 0.0001). Only in cancer patients with bone metastases was the median serum calcium level higher than in the healthy controls (P < 0.02). uCa was the same in cancer patients and the control group. Within the collective of cancer patients, individuals with skeletal metastases had higher levels of serum calcium (P < 0.05), TAP (P < 0.01), and uPYD and uDPD (both P < 0.0001), than patients without evidence of malignant bone disease. uCa did not differ between the 2 groups of cancer patients. The cancer patients were then stratified into 4 subgroups according to the serum calcium level (< or = 2.6 mmol/L >) and the absence or evidence of bone metastases. This stratification revealed that in patients with bone metastases, uPYD and uDPD levels were similar in normocalcemic and hypercalcemic subjects, whereas in hypercalcemic patients, uCa levels significantly exceeded those in normocalcemic patients. When the efficacy of TAP, uCa, uPYD, and uDPD in discriminating between patients with and without bone metastases was evaluated by use of receiver-operating characteristic curves and stepwise multivariate regression analysis, uPYD was found to have the highest diagnostic validity. Using 50 mumol PYD/mol creatinine (i.e. the upper limit of normal range) as the cut-off level, the sensitivity of uPYD was 88.7%, whereas the specificity was only 41.8% (odds ratio, 5.598; 95% confidence interval, 2.547-12.306).(ABSTRACT TRUNCATED AT 400 WORDS)