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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., (© 2018 Wiley Periodicals, Inc.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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.

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.

Purpose: The purpose of this study was to compare patients with primary hyperparathyroidism with and without nephrolithiasis with regard to (1) biochemical profile, and (2) presence and extent of bone involvement., Patients and Methods: Of 70 unselected patients enrolled in a longitudinal study on the natural history of primary hyperparathyroidism, 62 who underwent complete bone densitometry evaluation were considered. The patients had mild hypercalcemia (2.77 +/- 0.02 mmol/L), as well as elevated parathyroid hormone levels by mid-molecule, N-terminal, and immunoradiometric assays. Bone densitometry was assessed by dual-photon absorptiometry of the lumbar spine and femoral neck, and single-photon absorptiometry of the forearm., Results: Eleven of the 62 patients (18%) had nephrolithiasis. There was no difference in serum parathyroid hormone levels, calcium, phosphorus, serum 25-hydroxyvitamin D, and 1,25-dihydroxyvitamin D3 between those with and without kidney stones. Total daily urinary calcium excretion was higher among those who formed stones (8.2 +/- 1.0 mmol versus 6.1 +/- 0.4 mmol, p less than 0.05), but not when expressed per mmol of creatinine (0.72 +/- 0.07 versus 0.69 +/- 0.04). Urinary hydroxyproline was also higher in patients who formed stones (58 +/- 11 mg/24 hours versus 37 +/- 2 mg/24 hours; p less than 0.05). Hypercalciuria occurred in 39% of the entire cohort (n = 24), and in 33% (n = 17) of those without stones. Only 29% (n = 7) of those with hypercalciuria had nephrolithiasis. Calcium excretion correlated positively with serum 1,25-dihydroxyvitamin D3 (r = +0.32, p less than 0.05), and negatively with forearm bone mineral density (all patients: r = -0.34, p less than 0.05; hypercalciuric patients: r = -0.53, p less than 0.05). Circulating 1,25-dihydroxyvitamin D3 levels were elevated in a similar proportion of (1) all patients (31%, n = 19); (2) those with nephrolithiasis (27%); and (3) those without stones (31%). Bone mineral density was less than 80% of normal in 61% of patients, but forearm, femoral neck, and lumbar spine density were indistinguishable among those with and without stones., Conclusions: Cortical bone demineralization occurs to the same extent and frequency in patients with and without nephrolithiasis, and these two subgroups share similar biochemical and bone densitometric profiles. The pathophysiologic events leading to renal and skeletal involvement in primary hyperparathyroidism may be less selective than previously believed, as evidenced by: (1) increased urinary hydroxyproline in patients with nephrolithiasis, and (2) documentation that urinary calcium excretion reflects not only vitamin D status, but bone resorption was well.

Alkaline phosphatase, osteocalcin and hydroxyproline levels were evaluated in patients with the following conditions: primary hyperparathyroidism, renal dialysis, hyperthyroidism, Cushing's syndrome, long term corticosteroid therapy, Paget's disease, osteoblastic metastases, osteolytic or mixed metastases, and nutritional osteomalacia. In all cases the levels of the three substances were increased, with the following exceptions: a) in endogenous or exogenous hypercortisolism states osteocalcin level was reduced and those of alkaline phosphatase and hydroxyproline were unchanged; and b) in blastic or lytic metastases osteocalcin level was unchanged. In general, alkaline phosphatase and hydroxyproline levels had a higher sensitivity than those of osteocalcin in structural bone disease (Paget's disease, blastic or lytic metastases), whereas the converse was true for endocrine bone disease (the remaining conditions except osteomalacia, which is mixed, both structural and endocrine; in this syndrome, the three substances showed the same sensitivity.

Scintigraphic findings in ten cases of systemic mastocytosis are described. Four radionuclide bone patterns were noted: normal, unifocal, multifocal, and diffuse. Compared with radiographic surveys, bone images were better able to show the widespread skeletal involvement in patients with diffuse disease, and to detect a greater number of focal lesions. Serum calcium, phosphorus, and bone-derived alkaline phosphatase, as well as urinary calcium, phosphorus, and hydroxyproline levels, were usually within normal limits even when the bone scintigrams were clearly abnormal. Plasma and urinary histamine levels were highest in patients whose bone images detected widespread skeletal involvement. In systemic mastocytosis, not only does scintigraphy document active bone disease more effectively than laboratory studies of bone metabolism and radiographs of bone, but it also appears to reflect the general severity of the disease process.

Plasma hydroxyproline fractions were measured in 17 normal subjects and in 54 patients on maintenance haemodialysis therapy (MHT) with various degrees of dialysis osteodystrophy. On the basis of both radiological and histological findings these patients were divided into three groups: radiologically normal, histologically normal and those with osteitis fibrosa. The mean total plasma hydroxyproline concentrations were significantly elevated in all groups of MHT patients. However, these increases were mainly due to peptide-bound and free hydroxyproline fractions. The highest values for these two fractions were found in patients with osteitis fibrosa. The free to peptide-bound hydroxyproline ratio was not significantly altered in the majority of patients on dialysis; the mean ratio was significantly lower in patients with osteitis fibrosa when compared with patients with no histological evidence of bone disease. This finding would suggest that there is no inhibition of hydroxyproline catabolism in patients on haemodialysis and the measurements of both free and peptide-bound hydroxyproline were equally sensitive in identifying patients with osteitis fibrosa.

A study has been made of urinary peptide output in rats before and after production of a Fanconi syndrome induced by a single injection of sodium maleate. There was an unequivocal increase of urinary peptides on the first and second days after the injection, without any detectable change in the concentration of plasma peptides. 2. Similar results were obtained in osteolathyritic rats in which skeletal lesions had been produced by ingestion of beta-aminopropionitrile. 3. The fractional amino acid content of urinary peptides after maleate and beta-aminopropionitrile is shown to be significantly different from that in control animals. 4. Evidence is presented that the increased output of peptides is mainly due to increased renal clearance similar to that previously described for amino acids, glucose and several electrolytes in this type of experimental Fanconi syndrome.

To obtain a clue for the metabolic disorder of glycosaminoglycans (GAG) in orthopedic diseases, a screening study on excretion pattern of urinary GAG from orthopedic patients was performed by the procedures of Nagatsuka et al. (1980). All the urines examined gave three regular GAG-bands. Besides the regular bands, some samples gave irregular bands. Of 123 cases examined in the present study, the numbers of cases with an abnormal excretion pattern of urinary GAG in the regular bands (A) and with the irregular band(s) (B) were as follows: 23(A) and 7(B) in 38 cases of rheumatoid arthritis (RA); 8(A) and 5(B) in 13 cases of malignant bone tumor; 11(A) and 3(B) in 12 cases of benign bone tumor; 16(A) and 3(B) in 25 cases of bone metabolic and connective tissue diseases; 6(A) and 4(B) in 9 cases of osteaorthritis; 6(A) and 3(B) in 8 cases of spine diseases; and 14(A) and 8(B) in 18 cases of other orthopedic diseases. The excretion patterns of urinary GAG suggested that the metabolic rates of chondroitin sulfates tended to elevate in most cases of (A). Also, certain metabolic disorder of dermatan sulfate and heparan sulfate was suggested in several cases of (A). In addition, the abnormal metabolism of acidic glycoprotein(s) was suggested in many cases of (B). Excretion of a large amount of dermatan sulfate was elucidated in one case (Case 20 of bone metabolic and connective tissue diseases) by digestion with chondroitinases after the screening study. Therefore, the metabolic disorder of dermatan sulfate was indicated in this patient. The low activity of alpha-L-iduronidase in her urine supported this indication. This patient was finally diagnosed as mucopolysaccharidosis I-S (Scheie syndrome).

Crude glycosaminoglycan (GAG) fraction was directly precipitated with cetylpyridinium chloride without prior dialysis of urine of orthopedic patients. The crude GAG fraction was then fractionated with trichloroacetic acid (TCA). The TCA-insoluble peptide-bound GAG fraction thus obtained was treated with alkali to eliminate the peptide moiety for enzymatic analysis. The GAG compositions of this fraction and the TCA-soluble fraction were determined by digestion with mucopolysaccharidases (chondroitinase AC, chondroitinase B, chondroitinase C, heparitinase and Streptomyces hyaluronidase). When the amount of the crude GAG fraction was small, no significant amount of the TCA-insoluble peptide-bound GAG fraction was obtained. The GAG composition of this case was also determined by the same procedures after direct alkali-treatment of the crude GAG fraction. The data indicated that the proportion of the TCA-insoluble peptide-bound GAG fraction was very small. The alkali-treated TCA-insoluble peptide-bound GAG fraction contained a larger proportion of heparan sulfate than the TCA-soluble GAG fraction. It was clearly demonstrated that the patients with Werner's syndrome and mucopolysaccharidosis I-S (Scheie) excreted large amounts of hyaluronic acid and dermatan sulfate respectively, into urines. It was indicated in most cases that major urinary GAG were chondroitin 4-sulfate, chondroitin 6-sulfate plus chondroitin and heparan sulfate, while minor ones were dermatan sulfate and hyaluronic acid. In addition, the data suggested a wide range of the degree of desulfation or urinary GAG, and the presence of significant amounts of keratan sulfate plus acidic glycopeptides in the urinary GAG fractions. The present data provided more precise information on urinary GAG from orthopedic patients than those reported previously.

The urinary neopterin index (U-NEOPT-I), expressed as micro-moles of neopterin/moles of creatinine) of 67 patients with definite or classical rheumatoid arthritis was significantly higher than that of sex- and age-matched persons or of 24 non-matched patients suffering from osteoarthrosis or arthralgia (2p less than 0.001). Furthermore, the patients with active disease had statistically significantly increased U-NEOPT-I when compared with patients with a clinically less active disease. In this study, the U-NEOPT-I value was the equal of CRP as a measure of rheumatoid activity. We suggest that neopterin might be involved in the pathogenesis of rheumatoid arthritis.

In patients with multiple myeloma, with moderate and severe bone disease, the urinary hydroxyproline excretion was disproportionately elevated with respect to the activity of bone isoenzyme of alkaline phosphatase when compared with the relationship between the variables observed in 58 age- and sex-matched controls and in 50 healthy young subjects. A significant positive correlation was found between urinary hydroxyproline excretion and the clinical variables related to the extent of bone involvement in multiple myeloma (X-rays, patient's performance status, anaemia). In 9 out of 13 patients with moderate and severe bone disease the chemotherapy-induced remission was associated with a significant (p less than 0.05) rise in the activity of bone isoenzyme of alkaline phosphatase and decrease (p less than 0.005) in urinary hydroxyproline excretion. In successfully treated patients, the relationship between the biochemical variables indicated increased but proportionate extents of whole-body rates of bone formation and resorption. This was not the case in patients in whom no chemotherapy-induced remission was noted. The simultaneous evaluation of the activity of bone isoenzyme of serum alkaline phosphatase and urinary excretion of hydroxyproline improves the assessment of bone involvement in multiple myeloma and the efficacy of treatment.

A simple method for the quantitative determination of glycuronic acid-containing glycosaminoglycans (UA-GAG) is described. Sample solutions of glycosaminoglycans were digested with chondroitinase AC, chondroitinase C, chondroitinase B, heparitinases, and Streptomyces hyaluronidase, respectively, and the absorbance was read at 232 nm after digestion. The contents of 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-4S), 6-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-6S) plus N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-OS), 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-4S) plus N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-OS), heparan sulfate, and hyaluronic acid in the sample solutions were calculated from the absorbance with reference to that of the digestion products of known amounts of standard UA-GAG. The analytical data obtained with the mixtures of authentic UA-GAG were in close agreement with the theoretical values. Application of this procedure to the urinary GAG fractions from orthopedic patients gave satisfactory results.

The response to parathyroid hormone (PTH) and calcitonin (CT) was studied in eight children with various bone diseases by determining the serum calcium (Ca) and phosphate (P) concentration, urinary phosphate excretion rate, renal phosphate clearance, the percentage of filtered phosphate reabsorbed by the renal tubule (%TRP), creatinine clearance (Ccr), urinary cyclic adenosine 3',5' monophosphate excretion rate (UcGMPV). Administration of PTH caused no significant change in serum Ca and P values, whereas CT produced a decrease in Ca (delta Ca, -1.4 +/- 0.1 mg/100 ml) and P (delta P; -1.1 +/- 0.1 mg/100 ml). There was an increase in UcAMP V (delta UcAMP; 437 +/- 74 nmoles/min/100 ml Ccr) without any significant change in UcGMPV after administration of PTH. Phosphaturia was produced by both PTH (delta TRP, -18 +/- 3%) and CT (delta TRP, -13 +/- 2%). However, CT did not elicit any increase in either UcAMPV or UcGMPV.

Simple methods for the detection of keratan sulphate in urine have been applied to over 300 urine samples collected from children and adults with bone and cartilage dysplasias with or without mental retardation. Abnormal keratan sulphate excretion, which is a feature of type IV mucopolysaccharidosis (Morquio syndrome), is found in patients with that condition only during childhood. Abnormal excretion is also a feature of Kniest dysplasia and GM1 gangliosidosis and may be present in a number of other bone and cartilage dysplasias of unknown aetiology.

In a group of hospital patients with various diseases, the urinary hydroxyproline-to-creatinine ratio showed a significant correlation (r = 0.63; p less than 0.001) with the 5-hr bone-to-soft-tissue ratio for 99mTc-pyrophosphate uptake. In patients on chronic hemodialysis, a similar correlation was found between the 5-hr bone-to-soft-tissue ratio and hydroxyproline levels in plasma and serum. The findings suggest that 99mTc-pyrophosphate binding by bone is related to collagen metabolism.

Twenty-four-hour urinary hydroxyproline excretion (HOP) (normal values: 6-22 mg/day/m2) was measured by the Hypronosticon test in 50 untreated patients with plasma cell myeloma. At diagnosis, HOP was elevated in 36 of 50 patients (72%) with a mean value of 35.9 mg/day/m2. Extent of bone lesions and clinical stage were accurately assessed in all patients. Higher HOP values were found in patients with a higher degree of bone lesions (multiple lytic areas and/or destruction of skeletal segments). According to clinical stages, HOP was very elevated only in stage III (mean value: 43.7); in stages I and II the mean value (25.2) was just above the normal range. Our data indicate that HOP in multiple myeloma at diagnosis is closely related to the extension of skeletal lesions and that during the clinical course it may be useful in the follow-up of bone disease.

A significant correlation between the activity of the bone isoenzyme or serum alkaline phosphatase and the urinary hydroxyproline excretion in osteomalacia, osteoporosis, primary hyperparathyroidism with osteodystrophy, Paget's disease, secondary bone tumours, and in a control group was found (P less than 0.001). This close correlation was not observed between these variables in patients with active acromegaly. Diagnosis determined from these indices of formation and turnover of bone matrix agreed with that established by histological and histochemical examination of bone, by X-ray investigation of the skeleton, and by the radionuclear 85Sr test. The relationship between the activity of bone isoenzyme and urinary hydroxyproline excretion differed in metabolic bone diseases with a high bone turnover, in patients with osteoporosis and in patients with early osteoclastic bone metastases.

Young male rats were fed a diet containing cadmium (0, 10, 30, 100, and 300 ppm) so as to morphologically investigate the relationship between bone and kidney lesions caused by experimental cadmium intoxication. In the early stage of the experiment, before the occurrence of kidney lesions, ingeted cadmium caused osteoporotic changes in bone. In later stages, slight pathologic changes in the kdidney occurred in association with urinary excretion of cadmium. However, there was no evidence of osteomalacic change in bone during the 12-week experimental period. These findings suggest that cadmium may act primarily on bone, rather than secondarily through disturbances of the kidneys, which have some protective ability against cadmium intoxication in the early stage of ingestion of the metal.