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.

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.

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.

Immunoassay of pyridinoline crosslink excretion in normal adults and in Paget's disease.

A new immunoassay (ELISA) based on an antiserum that preferentially recognizes the free form of the pyridinoline (Pyr) crosslink was used to assess the urinary excretion of Pyr in a large normal sample of the population comprising 236 men and 193 women 30-90 years of age. Urinary Pyr increased significantly with age in both sexes. In women Pyr was higher than in men (57 +/- 21 versus 46 +/- 17 nmol/mmol creatinine, p < 0.001), and the menopause was reflected by a mean 37% increase, from an average 43 to 59 nmol/mm creatinine (p < 0.001). In 52 patients with active Paget's disease of bone, Pyr excretion was markedly increased (206 +/- 160 nmol/mmol creatinine, p < 0.001 versus controls) and decreased by 71% after 3 days of IV treatment with the bisphosphonate pamidronate. Free Pyr measured by the ELISA and the total urinary excretion measured by HPLC were highly correlated both in normals (n = 74, r = 0.83, p < 0.001) and in Pagetic patients (n = 20, r = 0.93, p < 0.001). The mean intra- and interassay coefficients of variation of the ELISA were below 9 and 15%, respectively. It is concluded that this new convenient immunoassay of Pyr should be valuable for the clinical investigation of patients with osteoporosis and other metabolic bone diseases.

Urinary excretion of pyridinoline crosslinks correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis.

Vertebral osteoporosis, a common disorder in elderly women, is characterized by a wide spectrum of bone turnover abnormalities on iliac crest biopsy. The level of bone formation can be assessed noninvasively by measuring serum osteocalcin, whereas conventional biochemical markers of bone resorption lack specificity and do not reflect bone resorption assessed from histology. We measured the urinary excretion of pyridinoline crosslinks Pyr and D-Pyr, a specific marker of bone and cartilage collagen degradation, along with serum osteocalcin and urinary hydroxyproline, in 36 elderly women with vertebral osteoporosis who had a simultaneous iliac crest biopsy. Urinary pyridinoline crosslinks, but not hydroxyproline, correlated significantly with histologic resorption, assessed by the osteoclast surface (r = 0.35, p less than 0.05 for Pyr; r = 0.46, p less than 0.01 for D-Pyr). In addition, Pyr and D-Pyr were correlated with the bone formation rate as well as serum osteocalcin, with correlation coefficients ranging from 0.69 to 0.80, p less than 0.0001. These data indicate that Pyr and D-Pyr are sensitive markers of bone turnover in elderly women with vertebral osteoporosis. The poor correlation between the level of urinary collagen crosslinks and histological assessment of bone resorption indicates the low sensitivity of iliac crest histomorphometry in the measurement of resorption rate of the skeleton.

Different effects of bisphosphonate and estrogen therapy on free and peptide-bound bone cross-links excretion.

We have measured the free and peptide-bound type I collagen cross-link excretions in normal women and in patients with metabolic bone disease using the HPLC technique and immunoassays recognizing specifically the free or peptide-bound forms of pyridinoline (Pyr). After menopause, free deoxypyridinoline (free D-Pyr) excretion measured by HPLC without urine hydrolysis and expressed as a fraction of the total excretion was lower than in premenopausal women (45 +/- 15% vs. 59 +/- 12%, p < 0.005), whereas the fraction of free Pyr was not changed. In normal pre- and postmenopausal women (n = 43), the fraction of free D-Pyr was negatively correlated with bone turnover rate as assessed by the total urinary excretion of Pyr (r = -0.64, p < 0.001). In patients with a variety of metabolic bone diseases characterized by increased bone turnover (osteoporosis, Paget's disease, and hyperthyroidism), the fractions of free Pyr and free D-Pyr were significantly lower than in premenopausal controls (p < 0.001 for all diseases). After 3 days of intravenous (iv) treatment with the bisphosphonate pamidronate in patients with Paget's disease and osteoporosis, the urinary excretion of cross-linked peptides measured by high performance liquid chromatography (HPLC) or enzyme-linked immunoassay (ELISA) (NTX and CrossLaps) was markedly decreased (-52% and -85% for NTX, -71% and -93% for CrossLaps in Paget's disease and osteoporosis, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Assessment of bone resorption with a new marker of collagen degradation in patients with metabolic bone disease.

We have used a new enzyme-linked immunoassay (ELISA) to measure the urinary excretion of type I collagen peptides (CrossLaps) released during bone matrix degradation in a sample of healthy adults comprising 146 women and 60 men, aged 31-89 yr, and in patients with metabolic bone disease. The intra- and interassay coefficients of variation were less than 10% and 13%, respectively. The recovery of CrossLaps antigen from urine samples ranged from 92-115%, and the ELISA was linear for serial sample dilutions. The CrossLaps assay does not cross-react with either free pyridinoline (Pyr) or free deoxypyridinoline (D-Pyr). CrossLaps measured by ELISA and the total excretion of Pyr measured by high performance liquid chromatography were highly correlated in normal women (n = 91; r = 0.73; P < 0.001). Urinary CrossLaps excretion increased with age in women, but not in men. In women, the menopause was reflected by a mean 141% increase in CrossLaps excretion [from an average 217 to 524 micrograms/mmol creatinine (Cr)] that was higher than the mean increase in total D-Pyr (+91%) and total Pyr (+47%) measured by HPLC and the mean increase in bone alkaline phophatase (+48%) and osteocalcin (+41%). Urinary CrossLaps excretion was increased from control values in Paget's disease (n = 32; mean, 1810 +/- 2300 micrograms/mmol Cr; P < 0.001), in patients with primary hyperparathyroidism (n = 10; mean, 780 +/- 380 micrograms/mmol Cr; P < 0.001), and in patients with hyperthyroidism (n = 27; mean, 1280 +/- 970 micrograms/mmol Cr; P < 0.001), with Z-scores (number of SD from the mean of sex- and age-matched controls) of 4.4 +/- 6.6, 1.5 +/- 1.2, and 6.7 +/- 6.5, respectively. In patients with Paget's disease, CrossLaps values were highly correlated with urinary hydroxyproline levels (r = 0.91; P < 0.001), and the decrease in urinary CrossLaps excretion was greater than that in urinary hydroxyproline (-71% vs. -17%; P < 0.001) after 3 days of i.v. treatment with the bisphosphonate pamidronate. In patients with hyperthyroidism, CrossLaps excretion was elevated above the normal range in most patients (78%) and returned to normal within 1 month of treatment for hyperthyroidism. It is concluded that this new convenient assay represents a sensitive and specific index of the bone resorption rate, and that it should be useful for the clinical investigation and therapeutic monitoring of patients with osteoporosis and other metabolic bone diseases.

Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment.

To evaluate the clinical utility of recently developed biochemical markers of bone turnover to monitor the response of osteoporotic patients to antiresorptive therapy, we compared the results of three advanced assays for markers of bone resorption and four of bone formation to high pressure liquid chromatography (HPLC)-fluorometric assays for urinary pyridinoline and deoxypyridinoline. These assays were also used to resolve the uncertainties concerning the rate of bone turnover in late postmenopausal (late-PMP) osteoporotic women. The rate of bone turnover in 85 women (mean +/- SD age, 63 +/- 6 yr) with low bone mass and all more than 5 yr postmenopausal (mean +/- SD yr PMP, 16 +/- 7 yr) was compared to that in 46 premenopausal women (mean +/- SD age, 40 +/- 5 yr) randomly selected from a large cohort and all having a normal spine bone mineral density (BMD). The late-PMP osteoporotic patients were a subset of patients enrolled in a double blind, placebo-controlled, randomized study comparing the effects of several doses of oral alendronate, a potent and specific inhibitor of bone resorption. Periodically during the 2-yr study, the women's spinal BMD and the level of several markers of bone turnover were measured. Serum total and intact osteocalcin, bone-specific alkaline phosphatase, and carboxy-terminal propeptide of type I collagen measured by RIA were used to assess bone formation. To assess bone resorption, we measured the urinary excretion of total pyridinoline (HPLC Pyr) and deoxypyridinoline (HPLC D-Pyr) by HPLC, type I collagen cross-linked N-telopeptide and urinary free PYR (F-Pyr) by enzyme-linked immunosorbent assay, and the serum concentration of type I collagen cross-linked C-telopeptide (ICTP) by RIA. All bone formation markers, except carboxy-terminal propeptide of type I collagen, and all bone resorption markers, except ICTP, were significantly increased above normal (33-171%; P < 0.001) in late-PMP osteoporotic women. The long term within-patient variability assessed over a 15-month period in the placebo group was low and was somewhat lower for serum markers (12.5-17.4%) than for urinary markers (24-29%). Under treatment with alendronate, resorption markers decreased earlier than markers of bone formation, consistent with a direct action of the drug to inhibit osteoclastic bone resorption. With the exception of F-Pyr and ICTP, the levels of bone markers were reduced to the normal premenopausal range, and this steady state was maintained from 6-15 months.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of menopause and hormone replacement therapy on the urinary excretion of pyridinium cross-links.

Pyridinoline (Pyr) and deoxypyridinoline (D-Pyr) are two cross-links of collagen molecules that are present in the extracellular matrix and released during its degradation. In contrast to the wide distribution of collagen, Pyr is present in bone and cartilage, but not in significant amounts in other connective tissues, and D-Pyr appears to be specific for bone tissue. Therefore, the urinary excretion of Pyr and D-Pyr might be a sensitive marker of bone matrix degradation. Using a specific high pressure liquid chromatography assay we have measured Pyr and D-Pyr cross-links in a 24-h and a fasting urine sample in 60 early postmenopausal women and 19 premenopausal women matched for age. Menopause induced a 62% increase in Fu Pyr (49.8 +/- 18.7 vs. 30.8 +/- 8.0 pmol/mumol creatinine; P less than 0.001) and an 82% increase in Fu D-Pyr (8.2 +/- 3.4 vs. 4.5 +/- 1.4 pmol/mumol creatinine; P less than 0.001). In 20 postmenopausal women on hormone replacement therapy, urinary Pyr and D-Pyr returned to premenopausal levels within 6 months, contrasting with unchanged levels during placebo treatment. The 24-h excretion of Pyr and D-Pyr was significantly lower than the fasting excretion, but was similarly decreased after hormone replacement therapy. Pyr and D-Pyr excretion measured in the same urinary sample were highly correlated (r = 0.85 for fasting and 0.83 for 24-h sampling), but correlations between fasting and 24-h values were weak (D-Pyr, r = 0.30; Pyr, r = 0.29; P less than 0.05 for both). Correlations between urinary cross-links and other markers of bone turnover (Fu hydroxyproline/creatinine and plasma osteocalcin) were significant but low (Pyr vs. osteocalcin, r = 0.29, P less than 0.05; Pyr vs. hydroxyproline, r = 0/.34; P less than 0.01; D-Pyr vs. osteocalcin, r = 0.39; P less than 0.01), except for D-Pyr vs. hydroxyproline (r = 0.24; P = 0.07), suggesting that these markers reflect different events of bone metabolism. Finally, a single measurement of the fasting excretion, but not of the 24-h excretion, of cross-links was significantly correlated (Pyr, r = 0.34; P less than 0.05; D-Pyr, r = -0.46; P less than 0.01), with the subsequent spontaneous rate of bone loss assessed by repeated measurements of the radial bone mineral content in 37 postmenopausal women.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum-free gamma carboxyglutamic acid (free Gla) is a poor biochemical marker of bone resorption in early postmenopausal women.

In this study, we assessed the potential value of serum-free Gla to reflect changes of bone turnover at menopause. Serum-free Gla was measured in 49 healthy postmenopausal women and 19 healthy age-matched premenopausal women. No difference in serum-free Gla concentrations was observed between the two groups. In a double-blind longitudinal study of 36 postmenopausal women (a subgroup of the above 49 women) treated with either oral oestradiol valerate in a cyclical combination with cyproterone acetate (n = 19) or placebo (n = 17) for nine months, the serum-free Gla concentration was unchanged with placebo and decreased by only 10% after hormonal replacement therapy. Initial serum-free Gla concentrations in postmenopausal women did not correlate with other biochemical markers of bone turnover. The predictive value of serum-free Gla concentration for the spontaneous rate of bone loss in early postmenopause was also assessed, and the marker showed no correlation to bone loss. These data indicate that the serum concentration of free Gla is not a useful marker of the bone turnover rate in the early postmenopause.

Automatic-interactive measurement of resorption cavities in transiliac bone biopsies and correlation with deoxypyridinoline.

Measuring bone resorption accurately by histomorphometry of bone biopsies is a challenge. Several techniques have been proposed including the measurement of eroded surfaces and resorption depth, but they have not been compared between themselves nor with biochemical assessment of bone resorption. In addition, there is a need for a rapid method that could be used more routinely. We describe here an automatic interactive method using a color analyzer (Visiolab, BIOCOM, France) with a specific software for the evaluation of erosion depth, eroded volume, eroded surface, osteoclast number, and surface. Thirty transiliac undecalcified bone biopsies stained with Goldner's trichrome were used in this study, taken from subjects suffering from osteoporosis or primary hyperparathyroidism. At the time of the biopsy a 2 h fasting morning urine sample was collected for measurement by HPLC of total deoxypyridinoline, the most sensitive marker of bone resorption. There was a highly significant correlation between maximum erosion depth measured directly and the one calculated according to the count of eroded lamellae (E. F. Eriksen, et al. Metab Bone Dis Relat Res 5:243-252; 1984) (r = 0.76; p = 0.0001). A significant correlation was found between urinary deoxypyridinoline and eroded volume/bone volume in cancellous and endocortical bone measured with the automatic interactive technique (r = 0.48; p = 0.007). In contrast, other histological indexes of bone resorption did not correlate with urinary deoxypyridinoline. The volume of resorption cavities appears to be the most valid index of bone resorption rate as it was correlated with the urinary excretion of total deoxypyridinoline.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Increased plasma free gamma carboxyglutamic acid levels during deep vein thrombosis and intravascular disseminated coagulation.

Gammacarboxyglutamic acid (gla) is a non essential amino acid synthesized in presence of vitamin K, predominantly found in coagulation and bone proteins. In 14 cases of deep vein thrombosis and in 11 cases of disseminated intravascular coagulation, compared to 19 normal subjects and 9 patients hospitalized for leg pain, free plasma gla levels were found significantly elevated (respectively 372 +/- 244 and 559 +/- 361 versus 146 +/- 34 and 120 +/- 40 pmol/mL). In six paired plasma and serum, gla levels were similar. These results suggest an involvement of blood coagulation in gla generation with need of a catabolism of the activated factors. A significant decrease was noticed during vitamin K antagonist therapy and liver disease, both instances in which the synthesis of gla containing coagulation factors is affected. During hepatocellular carcinoma with elevated desgamma carboxyprothrombin, gla was found normal, denying an global impairement of the vitamin K metabolism.

Evidence that free gamma carboxyglutamic acid circulates in serum.

We report a rapid, sensitive and reproducible method to measure free gamma-carboxyglutamic acid (GLA) in serum using precolumn derivatization with O-phthalaldehyde, reversed-phase chromatography and deproteinization of serum by ultrafiltration. Serum free GLA level in 62 healthy adults was 167 +/- 46 pmol/ml and was increased (302 +/- 195 pmol/ml) in 14 patients with primary hyper-parathyroidism, a disease characterized by an increased bone turnover. Peptide bound GLA averaged 413 pmol/ml. In rabbits receiving massive doses of warfarin during 6 days there was a time- and dose-dependent decrease of serum free GLA but a significant fraction was still measurable. These data indicate that free GLA circulates and originates both from the metabolism of the vitamin K-dependent clotting factors and from bone metabolism.

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.