Serum total periostin is an independent marker of overall survival in bone metastases of lung adenocarcinoma.

More than 35% of lung adenocarcinoma patients have bone metastases at diagnosis and have a poor survival. Periostin, a carboxylated matrix protein, mediates lung cancer cell dissemination by promoting epithelial-mesenchymal transition, and is involved in bone response to mechanical stress and bone formation regulation. This suggests that periostin may be used as a biomarker to predict survival in lung cancer patients. Serum periostin was assessed at diagnosis in a prospective cohort of 133 patients with lung adenocarcinoma of all stages. Patients were divided into localized and bone metastatic groups. Both groups were matched to healthy controls. Survival analysis and Cox proportional hazards models were conducted in the total population and in bone metastatic group. The median serum periostin level was higher in bone metastatic (n = 67; median: 1752 pmol/L) than in the localized group (n = 66; 861 pmol/L; p < 0.0001). Patients with high periostin (>median) had a poorer overall survival in the whole population (33.3 weeks vs. NR; p < 0.0001) and the bone metastatic group (24.4 vs. 66.1 weeks; p < 0.001). In multivariate analysis, patients with high periostin had increased risk of death (HR = 2.09, 95%CI [1.06-4.13]; p = 0.03). This was also found in the bone metastatic group (HR = 3.62, 95%CI [1.74-7.52]; p = 0.0005). Immunohistochemistry on bone metastasis biopsies showed periostin expression in the bone matrix and nuclear and cytoplasmic staining in cancer cells. Serum periostin was an independent survival biomarker in all-stage and in bone metastatic lung adenocarcinoma patients. IHC data suggest that periostin might be induced in cancer cells in bone metastatic niche in addition to bone microenvironment expression.

Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

Early effects of zoledronic acid and teriparatide on bone microarchitecture, remodeling and collagen crosslinks: comparison between iliac crest and lumbar vertebra in ewes.

Iliac crest bone biopsies are used to assess the mechanism of action of drug treatments, yet there are little data comparing this site to sites prone to fracture. The purpose of this study was to compare the delay and the amplitude of responses to treatment in two different bone sites. The short-term effects of zoledronic acid and teriparatide on microarchitecture, collagen crosslinks and bone remodeling were evaluated in iliac crest and lumbar vertebrae. Aged ewes (n=8/gr) received either vehicle (CTRL) or a single injection of zoledronic acid (ZOL, 10mg) or daily injections of teriparatide (TPTD, 20 µg/d) for 3 months. Blood samples were collected monthly for assessing bone turnover markers. At the end of the study, a transiliac bone biopsy (IC) and L1 lumbar vertebrae (LV1) were collected to assess bone microarchitecture; pyridinoline (PYD), deoxypyridinoline (DPD), pentosidine (PEN) content, static and dynamic parameters of bone remodeling. In CTRL, Tb-BV/TV was significantly higher in LV1 than IC (p<0.0001). This was associated with a trend of higher Tb.N, Tb.Th, DA, an inferior Conn.D and a lower bone turnover as shown by the decreases of osteoid parameters, MS/BS, Ac.f in LV1 when compared to IC. In addition, the ratio PYD/DPD was 4 times higher in LV1 than IC. After 3 months, significant decreases of sALP (p<0.001) and sCTX (p<0.001) were observed in the ZOL-group whereas in TPTD-group, after transient increases, they returned to baseline values. When compared to their respective CTRL, ZOL induced significant increases in Tb.BV/TV, Conn.D, Tb.N and Tb.Sp, in IC but not in LV1. Regardless of the site, ZOL markedly depressed the bone turnover: The static parameters of bone formation significantly decreased and the diminution of MS/BS, BFR/BS and Ac.f varied from -94 to -98% vs CTRL (p<0.01 to 0.001). It was associated with a diminution of the DPD content and the PYD/DPD ratio mainly in IC cortices. In contrast, after 3 months, TPTD did not modify the 3D structure and microarchitecture in IC and LV1, except a trend of higher Conn.D in IC, compared to IC-CTRL. TPTD treatment induced a significant increase in cortical porosity in LV1 (p<0.05) when compared to LV1-CTRL. Static parameters of bone formation and resorption were augmented in both sites, significantly only in LV1 (p<0.05) with a trend of increases in MS/BS and BFR/BS, compared to LV1-CTRL. In conclusion, in adult ewes, the bone mass, microarchitecture, remodeling and collagen crosslink content differ according to the bone site (iliac crest and vertebra). Furthermore, after 3 months, the responses to ZOL and TPTD were of different magnitude and delay between the two bone sites. The distinction of bone sites to study the early effects of anti-osteoporotic therapies appears meaningful in order to approach their site-specific anti-fracture efficacy.

Quantification of immature and mature collagen crosslinks by liquid chromatography-electrospray ionization mass spectrometry in connective tissues.

We describe a novel high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method for the simultaneous quantification of enzymatic immature (dihydroxylysinonorleucine DHLNL, hydroxylysinonorleucine HLNL) and mature (pyridinoline PYD, deoxypyridinoline DPD) collagen crosslinks in connective tissues. The crosslinks were separated on a C18 Atlantis T3 reversed-phase column with heptafluorobutyric acid (HFBA) as volatile ion-pairing reagent in an acetonitrile-water mobile phase. Detection was carried out by electrospray ionization mass spectrometry in a positive ion mode with selected ion recording (SIR). This method is more sensitive and selective than ion exchange chromatography with post-column ninhydrin detection which is the reference method used for the simultaneous quantification of collagen enzymatic divalent and trivalent crosslinks. The intra and inter-day precision errors were less than 3.4 and 7.7%, respectively for DHLNL, 3.5 and 5.9%, respectively for HLNL, 4.0 and 5.2%, respectively for PYD, 8.2 and 10.7%, respectively for DPD. This novel technique should be useful to quantify simultaneously DHLNL, HLNL, PYD and DPD in connective tissues and to evaluate the maturation of collagen by determination of the ratio between immature and mature enzymatic crosslinks.

Association between collagen cross-links and trabecular microarchitecture properties of human vertebral bone.

UNLABELLED: It has been suggested that age-related deterioration in trabecular microarchitecture and changes in collagen cross-link concentrations may contribute to skeletal fragility. To further explore this hypothesis, we determined the relationships among trabecular bone volume fraction (BV/TV), microarchitecture, collagen cross-link content, and bone turnover in human vertebral trabecular bone. Trabecular bone specimens from L2 vertebrae were collected from 51 recently deceased donors (54-95 years of age; 20 men and 30 women). Trabecular bone volume and microarchitecture was assessed by microCT and bone formation, reflected by osteoid surface (OS/BS, %), was measured by 2D histomorphometry. Pyridinoline (PYD), deoxypyridinoline (DPD), pentosidine (PEN) and collagen content in the cancellous bone were analysed by high-performance liquid chromatography. Associations between variables were investigated by Pearson correlations and multiple regression models, which were constructed with BV/TV and collagen cross-links as explanatory variables and microarchitecture parameters as the dependent variables. RESULTS: Microarchitecture parameters were modestly to strongly correlated with BV/TV (r(2)=0.10-0.71). The amount of mature enzymatic PYD and DPD cross-links were not associated with the microarchitecture, either before or after adjustment for BV/TV. However, there was a positive correlation between PEN content and trabecular number (r=0.45, p=0.001) and connectivity density (r=0.40, p=0.004), and a negative correlation between PEN content and trabecular separation (r=-0.29, p=0.04). In the multiple regression models including BV/TV, age and PEN content was still significantly associated with several of the microarchitecture variables. In summary, this study suggests a link between trabecular microarchitecture and the collagen cross-link profile. As PEN reflects non-enzymatic glycation of collagen and generally increases with bone age, the association between PEN and trabecular architecture suggests that the preserved trabeculae may contain mainly old bone and have undergone little remodeling. Thus, vertebral fragility may not only be due to alterations in bone architecture but also to modification of collagen cross-link patterns thereby influencing bone's mechanical behavior.

Urinary levels of pentosidine and the risk of fracture in postmenopausal women: the OFELY study.

UNLABELLED: The aim of the study was to investigate prospectively whether the levels of urinary pentosidine could predict fractures in postmenopausal women from the OFELY cohort. The results of the study suggest that urine pentosidine concentration is not an independent risk factor for fractures in postmenopausal women from a French cohort. INTRODUCTION: Pentosidine has been described as an independent risk factor for hip and vertebral fracture in postmenopausal Japanese women. We investigated the prediction of urinary pentosidine on all fragility fracture risk in healthy untreated postmenopausal women from the OFELY cohort. METHODS: Urinary pentosidine was assessed at baseline in 396 healthy untreated postmenopausal women aged 63.3 +/- 8.4 years from the OFELY cohort using high-performance liquid chromatography method. Incident clinical fractures were recorded during annual follow-up and confirmed by radiographs, and vertebral fractures were assessed on radiographs performed every 4 years. Multivariate Cox's regression analysis was used to calculate the risk of urinary pentosidine levels after adjustment for age, prevalent fractures, and total hip bone mineral density (BMD). RESULTS: During a mean follow-up of 10 years, 88 of the 396 postmenopausal women have undergone incident vertebral (n = 28) and peripheral (n = 60) fractures. Fracture risk was higher in postmenopausal women with pentosidine in the highest quartile (p = 0.02), but it did not remain significant after adjustment for age, BMD, and prevalent fracture. CONCLUSIONS: Urine pentosidine concentration is not an independent risk factor of osteoporotic fracture in healthy postmenopausal women from the OFELY cohort.

Effects of infliximab therapy on biological markers of synovium activity and cartilage breakdown in patients with rheumatoid arthritis.

BACKGROUND: Defining the remission criteria of rheumatoid arthritis (RA) remains a critical issue. Markers of synovium activity, urinary glucosyl-galactosyl-pyridinoline (Glc-Gal-PYD) and of cartilage destruction, urinary C-terminal crosslinking telopeptide of type II collagen (CTX-II) have been shown to reflect disease activity and joint damage progression in RA. METHODS: The prospective study cohort comprised 66 RA patients treated with infliximab and methotrexate and 76 healthy controls. Measurements of urinary Glc-Gal-PYD and CTX-II were performed at baseline and at 1 year of infliximab therapy. RESULTS: At baseline, urinary Glc-Gal-PYD and CTX-II levels were increased in patients with RA and correlated with modified Sharp scores and progression of joint damage. Patients with more progressive joint destruction had higher Glc-Gly-PYD and CTX-II baseline levels. CONCLUSION: These markers reflected bone erosion evolution and might be useful for treatment monitoring and evaluation of RA. Markers remained high even in clinical responders after infliximab, suggesting persistence of synovitis.

Simple and sensitive method for quantification of fluorescent enzymatic mature and senescent crosslinks of collagen in bone hydrolysate using single-column high performance liquid chromatography.

A rapid high performance liquid chromatographic method was developed including an internal standard for the measurement of mature and senescent crosslinks concentration in non-demineralized bone hydrolysates. To avoid the demineralization which is a tedious step, we developed a method based on the use of a solid-phase extraction procedure to clean-up the samples. It resulted in sensitive and accurate measurements: the detection limits as low as 0.2 pmol for the pyridimium crosslinks and 0.02 pmol for the pentosidine. The inter- and intra-assay coefficients of variation were as low as 5% and 2%, respectively, for all crosslinks.

Bisphosphonates alter trabecular bone collagen cross-linking and isomerization in beagle dog vertebra.

UNLABELLED: Changes in organic matrix may contribute to the anti-fracture efficacy of anti-remodeling agents. Following one year of treatment in beagle dogs, bisphosphonates alter the organic matrix of vertebral trabecular bone, while raloxifene had no effect. These results show that pharmacological suppression of turnover alters the organic matrix component of bone. INTRODUCTION: The collagen matrix contributes significantly to a bone's fracture resistance yet the effects of anti-remodeling agents on collagen properties are unclear. The goal of this study was to assess changes in collagen cross-linking and isomerization following anti-remodeling treatment. METHODS: Skeletally mature female beagles were treated for one year with oral doses of vehicle (VEH), risedronate (RIS; 3 doses), alendronate (ALN; 3 doses), or raloxifene (RAL; 2 doses). The middle dose of RIS and ALN and the lower dose of RAL approximate doses used for treatment of post menopausal osteoporosis. Vertebral trabecular bone matrix was assessed for collagen isomerization (ratio of alpha/beta C-telopeptide [CTX]), enzymatic (pyridinoline [PYD] and deoxypyridinoline [DPD]), and non-enzymatic (pentosidine [PEN]) cross-links. RESULTS: All doses of both RIS and ALN increased PEN (+34-58%) and the ratio of PYD/DPD (+14-26%), and decreased the ratio of alpha/beta CTX (-29-56%) compared to VEH. RAL did not alter any collagen parameters. Bone turnover rate was significantly correlated to PEN (R = -0.664), alpha/beta CTX (R = 0.586), and PYD/DPD (R = -0.470). CONCLUSIONS: Bisphosphonate treatment significantly alters properties of bone collagen suggesting a contribution of the organic matrix to the anti-fracture efficacy of this drug class.

Homocysteine and fracture risk in postmenopausal women: the OFELY study.

UNLABELLED: Homocysteine has recently been described as an independent risk factor for osteoporotic fractures in the elderly. We prospectively followed 671 postmenopausal women belonging to the OFELY study, mean age 62 years, during a mean follow-up of 10 years. After adjustment for age, there was no significant relation between the plasma level of homocysteine and the subsequent risk of fracture. INTRODUCTION: Plasma homocysteine increases with age. Recent studies have described homocysteine as an independent risk factor for osteoporotic fractures in elderly. We investigated the role of plasma homocysteine in the subsequent risk of fractures in healthy ambulatory postmenopausal women. METHODS: Homocysteine was measured at baseline in 671 postmenopausal women from the OFELY cohort (mean age 62.2 +/- 9 years). Incident clinical fractures were recorded during annual follow-up and vertebral fractures were evaluated with radiographs every four years. A cox proportional hazards model based on time to first fracture was used to calculate hazard ratios for quartiles of homocysteine values. RESULTS: Mean homocysteine was 10.6 +/- 3.4 mumol/l, increasing with age. After adjustment for age, homocysteine was significantly associated with physical activity, calcium intake, serum albumin and serum creatinine but not with bone turnover markers and bone mineral density. During a mean follow-up of 10 years, 183 fractures occurred among 134 women. After adjustment for age, the overall relative risk of fracture for each 1 SD increment of homocysteine was 1.03 (95%CI 0.87-1.31). Fracture risk was higher in women with homocysteine in the highest quartile without adjustment but no longer after adjustment for age. CONCLUSIONS: Homocysteine is not an independent risk factor of osteoporotic fractures in healthy postmenopausal women from the OFELY cohort with a broad age range.

Urinary CTX-II and glucosyl-galactosyl-pyridinoline are associated with the presence and severity of radiographic knee osteoarthritis in men.

OBJECTIVE: To investigate the association between biochemical markers of bone, cartilage, and synovial turnover with the presence and severity of knee osteoarthritis (OA) in men. METHODS: 176 men aged 59-70 years from the MRC Hertfordshire Cohort were studied. Weightbearing anteroposterior and lateral semiflexed radiographs were taken of both knees. A lifestyle questionnaire including basic demographic details and a questionnaire detailing knee pain was completed. This random sample was stratified based on the Kellgren and Lawrence (K&L) score, and the following biochemical markers were analysed: serum osteocalcin, serum C-terminal crosslinked telopeptide of type I collagen (CTX-I), urinary C-terminal crosslinked telopeptide of type II collagen (CTX-II), and urinary glucosyl-galactosyl-pyridinoline (Glc-Gal-Pyd). RESULTS: Age, body mass index (BMI), social class, smoking, and alcohol consumption were similar across K&L grades. Only one subject had a grade 4 K&L score, and was amalgamated with grade 3 subjects. A strong significant association was found between the presence of knee OA and urinary CTX-II and urinary Glc-Gal-Pyd (p=0.0001 and p=0.009), which persisted after adjustment for age and BMI. A significant positive association was also found between urinary CTX-II and urinary Glc-Gal-Pyd and the severity of K&L grade, joint space narrowing, and osteophytes scores, which persisted after adjustment for age and BMI. No associations between the presence and severity of knee OA were found for serum CTX-I or serum osteocalcin. CONCLUSIONS: Urinary CTX-II and Glc-Gal-Pyd, but not systemic markers of bone turnover, are strongly associated with disease severity and the presence of OA at the tibiofemoral and patellofemoral joints in men.

Effects of ibuprofen on molecular markers of cartilage and synovium turnover in patients with knee osteoarthritis.

OBJECTIVE: The aim of this study was to evaluate the effect of ibuprofen on the urinary excretion of C-terminal crosslinking telopeptide of type II collagen (CTX-II) and urinary glucosyl galactosyl pyridinoline (Glc-Gal-PYD), two new molecular markers of cartilage and synovial tissue metabolism, respectively, in patients with knee osteoarthritis (OA). METHODS: We studied 201 patients with knee pain and radiographic evidence of knee OA who were on treatment with non-steroidal anti-inflammatory drugs (NSAIDs) prior to study initiation. After an initial screening visit, patients were withdrawn from their pre-study NSAID and, following a flare of their OA symptoms, were randomised to ibuprofen (2400 mg/day) or placebo. Urinary CTX-II and Glc-Gal-PYD levels were measured at time of randomisation (baseline) and after 4-6 weeks of treatment. RESULTS: After 4 to 6 weeks, urinary CTX-II (+17%, p = 0.023) and Glc-Gal-PYD (+10%, p = 0.020) increased significantly from baseline in the placebo group whereas marginal or no increase was observed in the ibuprofen group (CTX-II +2%, NS and Glc-Gal-PYD +4%, p = 0.045). For urinary CTX-II, the difference in the change from baseline between placebo and ibuprofen treated groups was significant (13%, p = 0.017). At baseline, urinary levels of CTX-II and Glc-Gal-PYD were higher in patients with knee swelling (n = 127) than in those without (n = 74) (p<0.02 for both markers). When patients were stratified according to presence or absence of knee swelling at baseline, the increases over 4-6 weeks of urinary CTX-II and Glc-Gal-PYD in the placebo group were restricted to patients with knee swelling (+22% from baseline, p = 0.001 and +12%, p = 0.011, for urinary CTX-II and Glc-Gal-PYD respectively). In patients with knee swelling who were treated with ibuprofen this increase was not observed and the difference from placebo was significant for urinary CTX-II (p = 0.014). CONCLUSION: In patients with a flare of knee OA, specifically in patients with evidence of joint inflammation documented by knee swelling, there was a significant increase in markers reflecting cartilage and synovium metabolism that could partly be prevented by high doses of ibuprofen. These data suggest that patients with a flare of knee OA are characterised by increased cartilage and synovial tissue degradation, which may be partly prevented by high doses of NSAIDs.

Collagen type II C-telopeptide fragments as an index of cartilage degradation.

We report the development of an assay for measurement of the urinary concentration of collagen type II C-telopeptide fragments. This assay was developed for providing a specific marker of joint metabolism. A monoclonal antibody, recognizing a linear six amino acid epitope from the middle region of the collagen type II C-telopeptide was used in a competitive enzyme-linked immunoassay (ELISA) format for measurement of urine samples. The technical performance and specificity of the assay was evaluated and a panel of samples from patients with rheumatoid arthritis (RA) (n = 27), osteoarthritis (OA) (n = 29), Paget's disease (n = 9), and healthy controls (n = 428) was measured in the assay. The ELISA was specific for the peptide EKGPDP derived from collagen type II C-telopeptide: it did not recognize peptides from the N-telopeptide of the molecule or from other collagen types. Collagen type II C-telopeptide fragments measured in the assay resisted seven freeze-thaw cycles and >20 h of storage at room temperature. RA and OA patients showed significant 2.33-fold (95% confidence interval [CI] 1.50-3.16) and 1.53-fold (CI 1.24-1.82) elevations in CartiLaps concentration, respectively. Paget's disease patients did not have elevated CartiLaps levels. RA patients with radiological evidence of cartilage damage had significantly higher (1.79-fold, CI 1.04-2.54) CartiLaps levels than RA patients without radiological evidence of cartilage destruction. The Cartilaps assay showed high technical precision and an ability to differentiate populations with an elevated joint metabolism from normal controls. This suggests that the assay may have clinical value in assisting in the diagnosis of joint diseases and in monitoring progression and therapy in RA and OA.

Cross sectional evaluation of biochemical markers of bone, cartilage, and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage.

OBJECTIVE: To analyse the relations between the urinary levels of type II collagen C-telopeptide (CTX-II) and glucosyl-galactosyl pyridinoline (Glc-Gal-PYD)-two newly developed biochemical markers of type II collagen and synovial tissue destruction respectively-disease activity and the severity of joint destruction in patients with knee osteoarthritis (OA). The clinical performance of these two new markers was compared with that of a panel of other established biochemical markers of connective tissue metabolism. METHODS: The following biochemical markers were measured in a group of 67 patients with knee OA (mean age 64 years, median disease duration eight years ) and in 67 healthy controls: for bone, serum osteocalcin, serum and urinary C-telopeptide of type I collagen (CTX-I); for cartilage, urinary CTX-II, serum cartilage oligomeric matrix protein (COMP), and serum human cartilage glycoprotein 39 (YKL-40); for synovium, urinary Glc-Gal-PYD, serum type III collagen N-propeptide (PIIINP), serum hyaluronic acid (HA); and for inflammation, serum C reactive protein. Biochemical markers were correlated with pain and physical function (WOMAC index) and with quantitative radiographic evaluation of the joint space using the posteroanterior view of the knees flexed at 30 degrees. RESULTS: All bone turnover markers were decreased in patients with knee OA compared with controls (-36%, -38%, and -52%, p<0.0001 for serum osteocalcin, serum CTX-I and urinary CTX-I, respectively). Serum COMP (+16%, p=0.0004), urinary CTX-II (+25%, p=0.0009), urinary Glc-Gal-PYD (+18%, p=0.028), serum PIIINP (+33%, p<0.0001), and serum HA (+ 233%, p<0.0001) were increased. By univariate analyses, increased urinary Glc-Gal-PYD (r=0.41, p=0.002) and decreased serum osteocalcin (r=-0.30, p=0.025) were associated with a higher total WOMAC index. Increased urinary CTX-II (r=-0.40, p=0.0002) and Glc-Gal-PYD (r=-0.30, p=0.0046) and serum PIIINP (r=-0.29, p=0.0034) were the only markers which correlated with joint surface area. By multivariate analyses, urinary Glc-Gal-PYD and CTX-II were the most important predictors of the WOMAC index and joint damage, respectively. CONCLUSION: Knee OA appears to be characterised by a systemic decrease of bone turnover and increased cartilage and synovial tissue turnover. CTX-II, Glc-Gal-PYD, and PIIINP may be useful markers of disease severity in patients with knee OA.

Urinary excretion of glucosyl-galactosyl pyridinoline: a specific biochemical marker of synovium degradation.

OBJECTIVE: Glucosyl-galactosyl pyridinoline (Glc-Gal-PYD), which has been identified in urine, is a glycosylated analogue of pyridinoline. The tissue distribution of this molecule has not been yet determined and its utility as a potential biochemical marker of joint degradation in patients with joint diseases has not been investigated. METHODS AND RESULTS: In this study, we demonstrate that Glc-Gal-PYD is abundant in human synovium tissue, absent from bone and present in minute amounts in cartilage and other soft tissues, such as muscle and liver. Using an ex vivo model of human joint tissue degradation, we found that Glc-Gal-PYD is released from synovium tissue, but not from bone and cartilage. The urinary level of Glc-Gal-PYD was increased by 109% in patients with rheumatoid arthritis (RA) compared with healthy adults, but was normal in patients with Paget's disease of bone. In addition, Glc-Gal-PYD was higher in those patients with destructive disease, as assessed by X-rays of the joints, than in those with non-destructive RA. CONCLUSION: Glc-Gal-PYD may be useful for the clinical investigation of patients with joint disease.

Racemization and isomerization of type I collagen C-telopeptides in human bone and soft tissues: assessment of tissue turnover.

Urinary excretion of the type I collagen C-telopeptide (CTx) has been shown to be a sensitive index of the rate of bone resorption. The human type I collagen sequence A(1209)HDGGR(1214) of CTx can undergo racemization of the aspartic acid residue Asp(1211) and isomerization of the bond between this residue and Gly(1212). These spontaneous non-enzymic chemical reactions takes place in vivo in bone, and the degree of racemization and isomerization of CTx molecules may be an index of the biological age and the remodelling of bone. The aim of the present study was to investigate the degree of racemization and isomerization of type I collagen in human connective soft tissues, in order to estimate the rate of collagen turnover in adult tissues and compare it with that of bone. We also performed a systematic evaluation of the pyridinium cross-link content in adult human tissues. Using antibodies raised against the different CTx forms, we found that bone and dermis are the tissues that show most racemization and isomerization. The type I collagen of arteries, lung, intestine, kidney, skeletal muscle and heart shows significantly less racemization and isomerization than that of bone, suggesting that these soft tissues have a faster turnover than bone. We also found that pyridinoline and, to a lesser degree, deoxypyridinoline are distributed throughout the different tissues investigated. Because bone type I collagen is characterized by a high degree of both racemization/isomerization and deoxypyridinoline cross-linking, the concomitant assessment of these two post-translational modifications is likely to result in a highly specific marker of bone resorption.

Measurement of urinary excretion of nonisomerized and beta-isomerized forms of type I collagen breakdown products to monitor the effects of the bisphosphonate zoledronate in Paget's disease.

OBJECTIVE: We have previously shown that the woven pagetic bone in patients with Paget's disease is characterized by an impaired degree of beta-isomerization of C-telopeptides of type I collagen molecules, which results in a preferential urinary excretion of nonisomerized type I collagen C-telopeptide breakdown products (CTX). The aim of this study was to measure the urinary excretion of nonisomerized (alpha) and beta-isomerized (beta) CTX in patients with Paget's disease treated with a bisphosphonate. METHODS: We studied 28 patients with active Paget's disease of bone who were a part of a randomized, double-blind, placebo-controlled study comparing the effects of several doses of a single injection of zoledronate, a new potent bisphosphonate. Serum bone alkaline phosphatase (BAP) and type I collagen C-terminal extension propeptide (PICP) and urinary excretion of free deoxypyridinoline (free D-Pyr), N-telopeptide breakdown products (NTX), alphaCTX, and betaCTX were measured at baseline and 5, 10, 30, and 60 days after injection. RESULTS: At baseline, all markers were significantly increased in the patients compared with a group of 97 sex- and age-matched controls, with a greater increase in BAP (12-fold), NTX (19-fold), and alphaCTX (10-fold) compared with PICP (2.2-fold), free D-Pyr (2.5-fold), and betaCTX (3-fold). The ratio of alphaCTX to betaCTX was about 3-fold higher than in controls (2.1 versus 0.76; P < 0.001). After a single intravenous injection of zoledronate (200 or 400 microg), all markers decreased within 5 days, except for BAP and free D-Pyr, which decreased on day 10. The maximum decrease was greater and occurred faster for NTX, alphaCTX (-55% after 10 days), and betaCTX (-42% after 10 days) than for free D-Pyr (-25% after 30 days). After the initial decrease, the urinary excretion of betaCTX increased between days 10 and 30 and returned to pretreatment levels after 2 months, in contrast to the sustained decrease in alphaCTX and NTX that was maintained up to 60 days. The urinary ratio of alphaCTX to betaCTX decreased significantly between days 10 and 60, and returned to within the normal range in most patients after 2 months of treatment, probably reflecting the progressive replacement of woven bone by a lamellar bone with a higher and normal degree of beta-isomerization of type I collagen, as previously documented by histology. CONCLUSION: The determination of the urinary ratio of alphaCTX to betaCTX could be useful for monitoring the effect of bisphosphonate treatment in restoring bone quality in patients with Paget's disease.

Decreased beta-isomerization of the C-terminal telopeptide of type I collagen alpha 1 chain in Paget's disease of bone.

In Paget's disease of bone, the normal lamellar bone is replaced by a woven structure with an irregular arrangement of collagen fibers. In this study, we investigated whether the degree of beta-isomerization within C-telopeptide of alpha 1 chain of type I collagen was altered in Paget's disease compared with other bone diseases with no alteration of bone structure. In Paget's disease (n = 26), but not in patients with primary hyperparathyroidism (n = 6) or hyperthyroidism (n = 17), the urinary excretion of nonisomerized (alpha) fragments derived from degradation of type I collagen C-telopeptide (CTX) was markedly increased compared with beta-isomerized CTX (+ 13-fold vs. + 3.5-fold over controls) resulting in an urinary alpha CTX/beta CTX ratio 3-fold higher than in controls (2.6 +/- 1.0 vs. 0.8 +/- 0.3, p < 0.001). In five pagetic patients in complete remission, as demonstrated by normal total alkaline phosphatase activity, the alpha CTX/beta CTX ratio was normal. The immunohistochemistry of normal and pagetic human bone sections showed a preferential distribution of alpha CTX within woven structure, while lamellar bone was intensely stained with an anti-beta CTX antibody, suggesting a lower degree of beta-isomerization of type I collagen in the woven pagetic bone. In collagenase digest of human bone specimens, we found a lower proportion of beta-isomerized type I collagen molecules in pagetic bone (40% of beta CTX) than in normal bone taken from trabecular (68%) and cortical compartments (71%). In conclusion, we found that in Paget's disease the alpha CTX/beta CTX ratio in bone and in urine is markedly increased. This altered beta isomerization can be accurately detected in vivo by measuring urinary degradation products arising from bone resorption.

Comparative evaluation of markers of bone resorption in patients with breast cancer-induced osteolysis before and after bisphosphonate therapy.

The understanding of the pathophysiology and the monitoring of metastatic bone disease remains unsatisfactory. We compared several new markers of bone turnover in normocalcaemic patients with breast cancer-induced osteolysis before and after a single infusion of the bisphosphonate pamidronate. We studied 19 ambulatory patients with advanced breast cancer and extensive bone metastases who did not receive any systemic antineoplastic therapy. Pamidronate was administered at doses of 30, 60, 90 or 120 mg and the patients were followed weekly during a mean of 8 (range 4-10) weeks. Compared with healthy premenopausal women, the percentage of elevated values at baseline was 47% for fasting urinary calcium (uCa), 74% for hydroxyproline, 83% for CrossLaps (a new marker of type I collagen degradation) and 100% for the collagen cross-links (measured by high performance liquid chromatography), namely pyridinoline (Pyr) and deoxyPyr (D-Pyr). Pretreatment levels of uCa did not correlate significantly with any of the four markers of bone matrix resorption, whereas the correlations between these four markers were generally significant (r(s)=0.43-0.71). Alkaline phosphatase correlated significantly with markers of bone matrix resorption (r(s)=0.54-0.74). All parameters, except phosphaturia (uPi) and the bone formation markers (osteocalcin and alkaline phosphatase), fell significantly after pamidronate therapy, up to day 42 for hydroxyproline, D-Pyr and CrossLaps and day 56 for uCa. This longer lasting effect was probably due to the parathyroid hormone (PTH) surge following the decrease in serum calcium, implying that the decrease in uCa can overestimate the effects of bisphophonates on bone resorption. The decrease in bone turnover parameters was most marked for CrossLaps, indicating the potential of this new marker for monitoring therapy. Sequential determinations of markers of bone matrix resorption should be useful in delineating the optimal therapeutic schemes of bisphosphonates and for evaluating treatment effects on bone in cancer patients.