Aberrant methylation-induced dysfunction of p16 is associated with osteoblast activation caused by fluoride.

Chronic exposure to fluoride continues to be a public health problem worldwide, affecting thousands of people. Fluoride can cause abnormal proliferation and activation of osteoblast and osteoclast, leading to skeletal fluorosis that can cause pain and harm to joints and bones and even lead to permanent disability. Nevertheless, there is no recognized mechanism to explain the bone lesions of fluorosis. In this work, we performed a population study and in vitro experiments to investigate the pathogenic mechanism of skeletal fluorosis in relation to methylation of the promoter of p16. The protein coded by the p16 gene inhibits cdk (cyclin-dependent kinase) 4/cdk6-mediated phosphorylation4 of retinoblastoma gene product and induces cell cycle arrest. The results showed that hypermethylation of p16 and reduced gene expression was evident in peripheral blood mononuclear cells of patients with fluorosis and correlated with the level of fluoride exposure. Studies with cell cultures of osteoblasts revealed in response to sodium fluoride (NaF) treatment, there was an induction of p16 hypermethylation and decreased expression, leading to increased cell proliferation, a longer S-phase of the cell cycle, and development of skeletal fluorosis. Further, the methylation inhibitor, 5-aza-2-deoxycytidine, reversed the p16 hypermethylation and expression in response to NaF. These results reveal a regulatory role of p16 gene methylation on osteoblasts activation during the development of skeletal fluorosis.

Clinical utility of bone markers in various diseases.

Measurements of bone markers (BMs) in peripheral blood or urine are a pivotal part of bone research within modern clinical medicine. In recent years the use of BMs increased substantially as they can be useful either to diagnose bone (related) disease and to follow its natural history, but also to monitor the effects of interventions. However, the use of BMs is still complicated mainly due to (pre)analytical variability of these substances, limited accessibility of assays, variable cut-off values in different countries and laboratories and heterogeneous results with regard to clinical implications of measuring BMs in several studies. This review will provide the clinician with a practical guide, based on current evidence, in which circumstances to test which bone markers for optimal diagnostic purposes, in order to improve patient care in different areas of bone diseases including Paget's disease, primary osteoporosis, tumor induced osteomalacia, hypophosphatemic rickets, van Buchem disease, chronic kidney disease, rheumatoid arthritis, neoplasma/multiple myeloma, type 2 diabetes mellitus and primary hyperparathyroidism. The clinician should consider fasting state, recent fractures, aging, menopausal status, concomitant liver and kidney disease when ordering and interpreting BM measurements as these factors might result in misleading BM concentrations. We found that BMs are clearly useful in the current diagnosis of tumor induced osteomalacia, van Buchem disease, Paget's disease and hypophosphatemic rickets. In addition, BMs are useful to monitor disease activity in chronic kidney disease, Paget's disease and are useful to monitor treatment adherence in osteoporosis.

Markers of Bone and Cartilage Turnover.

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.

Unexpected widespread hypophosphatemia and bone disease associated with elemental formula use in infants and children.

OBJECTIVE: Hypophosphatemia occurs with inadequate dietary intake, malabsorption, increased renal excretion, or shifts between intracellular and extracellular compartments. We noticed the common finding of amino-acid based elemental formula [EF] use in an unexpected number of cases of idiopathic hypophosphatemia occurring in infants and children evaluated for skeletal disease. We aimed to fully characterize the clinical profiles in these cases. METHODS: A retrospective chart review of children with unexplained hypophosphatemia was performed as cases accumulated from various centres in North America and Ireland. Data were analyzed to explore any relationships between feeding and biochemical or clinical features, effects of treatment, and to identify a potential mechanism. RESULTS: Fifty-one children were identified at 17 institutions with EF-associated hypophosphatemia. Most children had complex illnesses and had been solely fed Neocate® formula products for variable periods of time prior to presentation. Feeding methods varied. Hypophosphatemia was detected during evaluation of fractures or rickets. Increased alkaline phosphatase activity and appropriate renal conservation of phosphate were documented in nearly all cases. Skeletal radiographs demonstrated fractures, undermineralization, or rickets in 94% of the cases. Although the skeletal disease had often been attributed to underlying disease, most all improved with addition of supplemental phosphate or change to a different formula product. CONCLUSION: The observed biochemical profiles indicated a deficient dietary supply or severe malabsorption of phosphate, despite adequate formula composition. When transition to an alternate formula was possible, biochemical status improved shortly after introduction to the alternate formula, with eventual improvement of skeletal abnormalities. These observations strongly implicate that bioavailability of formula phosphorus may be impaired in certain clinical settings. The widespread nature of the findings lead us to strongly recommend careful monitoring of mineral metabolism in children fed EF. Transition to alternative formula use or implementation of phosphate supplementation should be performed cautiously with as severe hypocalcemia may develop.

[Comparison of the therapeutic effect on skeletal fluorosis and impact on urine fluoride value among fire needle therapy, electroacupuncture and calcium carbonate D3].

OBJECTIVE: To observe the impacts on skeletal fluorosis pain, joint motor dysfunction and urine fluoride excretion in the treatment with fire needle therapy, electroacupuncture and calcium carbonate D3. METHODS: The randomized controlled trial was adopted. Ninety-five patients were randomized into a fire needle group (31 cases), an electroacupuncture group (33 cases) and a calcium carbonate D3 group (31 cases). In the fire needle group and the electroacupuncture group, Ashi points, Dazhui (GV 14), Geshu (BL 17), Quchi (LI 11), Hegu (LI 4), Xuehai (SP 10) points were selected and stimulated with fire needle and electroacupuncture separately, three times a week. In the calcium carbonate D3 group, calcium carbonate D3 tablets was prescribed for oral administration, 600 mg each time, twice a day. The duration of treatment was 2 months in the electroacupuncture group and calcium carbonate D3 group and 1 month in the fire needle group. VAS score, the range of motion (ROM) and urine fluoride value were compared before and after treatment in the patients of the three groups. RESULTS: After treatment, VAS value and ROM were improved significantly in the patients of the three groups (all P < 0.05), the difference was not significant in comparison of the three groups (all P > 0.05). After treatment, the urine fluoride value was increased significantly in the fire needle group [(7.89 +/- 3.61) mg/L vs (9.81 +/- 4.17) mg/L, P < 0.01] and was increased in the electroacupuncture group [(7.53 +/- 3.46) mg/L vs (8.97 +/- 4.21) mg/L, P < 0.05]. The difference was not significant in comparison before and after treatment in the calcium carbonate D3 group (P > 0.05). CONCLUSION: The fire needle therapy, electroacupuncture and calcium carbonate D3 all have the clinical value in the prevention and treatment of skeletal fluorosis and the difference in the therapeutic effect has not been discovered among them yet at present. But it has been found that the fire needle therapy and electroacupuncture display the active significance in the promotion of urine fluoride excretion.

[Excretion of urinary metallothionein and osteal damage induced by cadmium in an environmentally cadmium exposed population].

OBJECTIVE: To observe the relationship between urinary metallothionein excretion and osteal damage induced by cadmium in a general population. METHODS: The inhabitants living in both cadmium polluted and non-polluted areas were asked to participate in this study. Urinary cadmium (UCd) and blood cadmium (BCd) were measured by GF-AAS. Total cadmium(TCd)was evaluated with environmental cadmium exposure. URBP, UB2M, UALB and UMT were measured by ELISA method. UNAG, UNAGB were measured by fluorescence analysis method. Forearm bone mineral density in human were mensurated by SPA. RESULTS: UMT can reflect the change of cadmium body burden. Renal dysfunction and osteoporosis would appear successively after high level of cadmium exposure. UMT had a complex relationship with bone mineral density which related to the amount of UMT excretion. The BMDLs of UCd were calculated using software of BMDS Versionl. 3.2 for these biomarkers. The values of BMDL of these biomarkers were arranged: UNAGB < UNAG < UB2M < UMT < URBP < Tscore < UALB. CONCLUSION: Cadmium exposure could induce bone damage which occurred later than renal dysfunction related to cadmium exposure. UMT could be not only a specific and sensitive biological indicator of cadmium-induced renal dysfunction but also could reflect the damage on bone induced by cadmium.

Biphasic anti-osteoclastic action of intravenous alendronate therapy in multiple myeloma bone disease.

Multiple myeloma is a malignancy of plasma cells with osteolytic bone destruction. Bisphosphonates inhibit osteoclast activity and are widely used for the treatment of myeloma bone disease. We analyzed the changes in urinary cross-linked N-telopeptides of collagen (u-NTx) and urinary calcium (u-Ca) after bisphosphonate alendronate therapy in ten patients with myeloma bone disease. In all patients, the levels of u-Ca and u-NTx decreased within a week. After the maximum decrease of u-NTx, u-NTx started increasing in half of the patients. However, this further increase in u-NTx decreased again without any additional therapy. Disease severity and pretreatment u-NTx concentrations did not differ between patients with and without the rebound. Patients who did not have rebound had decreased bone marrow monocytes and decreased serum concentrations of interleukin 18, which is produced by monocytes. Our results suggest that impaired activity of monocytes, which are possible osteoclast precursors, is related to reduced bone destruction in multiple myeloma.

Investigation of bone disease using isomerized and racemized fragments of type I collagen.

In the collagen type I C-telopeptide an aspartyl-glycine site within the sequence AHDGGR is susceptible to molecular rearrangement. In newly synthesized collagen this site is in the native form, denoted alpha L. During aging a spontaneous reaction occurs resulting in three age-modified forms: an isomerized form (beta L) a racemized form (alpha D), and an isomerized/racemized form (beta D). In this study, we measured the urinary excretion of the four forms of C-telopeptides (CTX) in healthy adults and in patients with bone diseases. Levels of all CTX forms were higher in healthy postmenopausal women (P<0.001) compared with premenopausal controls. Levels decreased within 3 days of bisphosphonate treatment indicating that all CTX forms reflect bone resorption. In hyperthyroidism, characterized by a generalized increased bone turnover, native (alpha L) and age-modified (beta L, alpha D and beta D) forms increased to a similar extent compared to controls, resulting in normal ratios between the alpha L and age-modified forms of CTX. Conversely, in Paget's disease and prostate cancer-induced bone metastases, conditions characterized by focal increased bone turnover, alpha L CTX levels were more elevated than those of age-related CTX forms, resulting in increased ratios between native and age-modified CTX. For example, the ratio alpha L/alpha D was increased 7-fold in Paget's disease (P<0.001) and 2-fold in prostate cancer-induced bone metastases (P<0.002). In conclusion, the study suggests that in conditions with a localized alteration in bone turnover the ratio between alpha L CTX and the age-modified forms is significantly elevated. This may provide a new diagnostic and monitoring tool for diseases such as metastatic bone cancer and Paget's disease.

Clinical utility of biochemical markers of bone remodeling.

Remodeling is essential for bone health. It begins with resorption of old bone by osteoclasts, followed by the formation of new bone by osteoblasts. Remodeling is coupled (formation is linked to resorption). After middle age or perhaps beginning earlier, bone loss occurs because resorption exceeds formation. This imbalance is accentuated by estrogen deficiency as well as by many diseases and conditions. Biochemical markers that reflect remodeling and can be measured in blood or urine include resorption markers (e.g., collagen cross-links) and formation markers (e.g., alkaline phosphatase). Bone markers exhibit substantial short-term and long-term fluctuations related to time of day, phase of the menstrual cycle, and season of the year, as well as diet, exercise, and anything else that alters bone remodeling. These biological factors, in addition to assay imprecision, produce significant intra- and interindividual variability in markers. Bone marker measurements are noninvasive, inexpensive, and can be repeated often. Unfortunately, most of the studies that provided insight on clinical situations did not focus on markers as a primary endpoint. Bone markers have been useful in clinical practice and have been helpful in understanding the pathogenesis of osteoporosis and the mechanism of action of therapies. In clinical trials, markers aid in selecting optimal dose and in understanding the time course of onset and resolution of treatment effect. Clinical questions that might be answered by bone markers include diagnosing osteoporosis, identifying "fast bone losers" and patients at high risk of fracture, selecting the best treatment for osteoporosis, and providing an early indication of the response to treatment. Additional information is needed to define specific situations and cut points to allow marker results to be used with confidence in making decisions about individual patients.

Evaluation of bone disease in multiple myeloma: a comparison between the resorption markers urinary deoxypyridinoline/creatinine (DPD) and serum ICTP, and an evaluation of the DPD/osteocalcin and ICTP/osteocalcin ratios.

Markers of bone metabolism were measured in 73 newly diagnosed myeloma patients and in age-matched controls. Correlations to bone disease on X-rays and survival were performed. In urine deoxypyridinoline/creatinine (DPD) and in serum carboxyterminal pyridinoline cross-linked telopeptide of type I collagen (ICTP), procollagen type I carboxy-terminal extension peptide (PICP) and osteocalcin were analyzed. The ratios DPD/osteocalcin and ICTP/osteocalcin were calculated. Skeletal X-ray findings were divided into no, limited and extensive bone involvement. DPD and ICTP levels were significantly elevated in patients compared to controls. Levels increased with advancing skeletal involvement. Serum osteocalcin was elevated in patients without visible bone disease. The level decreased with more advanced bone involvement. The finding of significantly elevated osteocalcin and ICTP levels in patients without bone involvement on X-rays indicates that bone markers might reflect bone disease better than X-rays in untreated myeloma patients. Ratios between bone resorption and bone formation markers added no further information on bone disease or survival. Only ICTP had prognostic value with an inverse correlation between serum levels and survival.

Non-invasive assessment of bone density in primary biliary cirrhosis.

BACKGROUND/AIMS: Low bone mass is an important complication of primary biliary cirrhosis (PBC), resulting in an increased risk of fractures and reduced mobility. In the present study, we sought to determine the frequency of low bone mass in PBC, and its relationship to disease severity and non-invasive markers of bone turnover. METHODS: In 36 women with PBC, bone mineral density of the lumbar spine and hip was assessed by dual emission X-ray absorptiometry. Serum and urinary markers of bone turnover were compared with those from age- and sex-matched controls. RESULTS: Spinal osteopenia (T score, -1.5 to -2.5) was present in 15 of the 36 patients (42%), while six others (16%) had established osteoporosis (T < -2.5). Osteopenia of the femoral neck was found in 17 patients (47%), and osteoporosis in five (14%). The severity of liver disease, as determined by Mayo Clinic R score and histological stage, correlated negatively with both regional bone mineral density and total bone mineral content expressed as a ratio to lean body mass. There was a strong positive correlation between serum levels of the procollagen degradation peptides, PICP and PIIINP (r = 0.65, P < 0.001), and both peptides correlated significantly (P < 0.001) with histological stage and Mayo Clinic R score. Fasting urinary pyridinoline and deoxypyridinoline to creatinine ratios were also significantly raised. CONCLUSIONS: Low bone mass in PBC correlates positively with disease severity, and is associated with a net increase in bone resorption, as assessed by urinary collagen cross-link excretion. These markers of bone turnover may be of value in controlled clinical trials aimed at improving bone mass in PBC.

Novel serum markers of bone resorption: clinical assessment and comparison with established urinary indices.

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.

Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation.

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.

Automated and manual assays for urinary crosslinks of collagen: which assay to use?

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 in the assessment of bone disease.

As the mean age of our population increases, increasing attention has been paid to the diseases associated with aging, including diseases of the skeleton such as osteoporosis. Effective means of treating and possibly preventing such skeletal disorders are emerging, making their early recognition an important goal for the primary care physician. Although bone density measurements and skeletal imaging studies remain of primary diagnostic importance in this regard, a large number of assays for biochemical markers of bone formation and resorption are being developed that promise to complement the densitometry measurements and imaging studies, providing an assessment of the rates of bone turnover and an earlier evaluation of the effects of therapy. In this review, emphasizing the recent literature, the major biochemical markers currently in use or under active investigation are described, and their application in a number of diseases of the skeleton including osteoporosis is evaluated.

Marked increase in urinary excretion of nitrate and N-nitrosothioproline in the osteogenic disordered syndrome rats, lacking ascorbic acid biosynthesis, by administration of lipopolysaccharide and thioproline.

Urinary excretions of nitrate and N-nitrosothiazolidine-4-carboxylic acid (N-nitrosothioproline; NTPRO) were determined in rats with osteogenic disordered syndrome (ODS, od/od), lacking L-ascorbic acid (ASC) biosynthesis, after i.p. administration of Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) followed by thiazolidine-4-carboxylic acid (thioproline, 20 mg/rat). L-Ascorbic acid-sufficient ODS rats showed the excretion of nitrate and NTPRO at the levels of 20.3 +/- 7.9 mumol/24h and 369 +/- 111 pmol/24 h respectively, whereas the levels of nitrate and NTPRO in ASC-deficient (scorbutic) rats increased to 54.7 +/- 5.6 mumol/24 h (P < 0.01) and 796 +/- 367 pmol/24 h (P < 0.05) respectively. Administration of L-arginine further increased urinary excretion of nitrate and NTPRO while D-arginine showed no effect. NG-Monomethyl-L-arginine, a specific inhibitor of nitric oxide synthase (NOS), strongly inhibited endogenous formation of both nitrate and NTPRO. These results indicate that increased excretion of NTPRO in ODS rats stimulated by LPS involves induction of NOS leading to an increase in endogenous formation of reactive nitrogen oxides such as N2O3, a potent nitrosating agent at physiological pH conditions. Increased NOS activities in the plasma and various tissues of ODS rats were observed 5 h after treatment with LPS. The possibility of extragastric N-nitroso compound formation in inflammation sites is discussed.

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.

Automated high-performance liquid chromatographic determination of hydroxylysylpyridinoline and lysylpyridinoline in urine using a column-switching method.

An on-line urine clean-up system was developed for the simultaneous determination of free and total pyridinoline, hydroxylysyl-pyridinoline (HP) and lysylpyridinoline (LP) by high-performance liquid chromatography (HPLC) using a column-switching technique. The method is based on a combination of gel permeation chromatography (GPC) and ion-pair reversed-phase HPLC. In the GPC column, pyridinoline is preseparated from endogenous urinary substances with 0.03 M heptafluorobutyric acid (HFBA) as the mobile phase. After column switching, the eluate fraction containing pyridinoline is further separated by ion-pair chromatography using an octadecylsilica (ODS) column with 0.03 M HFBA-acetonitrile (81:19) as the mobile phase. The detection limits were 36 and 44 pmol/ml for free and total HP, respectively, and 44 pmol/ml for both free and total LP at a signal-to-noise ratio of 3. The coefficients of variation for free and total pyridinoline were 1.5 and 3.5%, respectively. The determination of one sample including the clean-up is completed within 25 min. This system is precise and is useful for the determination of pyridinoline in large amounts of urine. The usefulness of pyridinoline as a biomedical marker for bone resorption was also examined.