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Measurement of ionized calcium is more important than measurement of total calcium in serum samples. In the present study, equations were derived from complexation and acid dissociation equilibrium equations, and were used to determine the concentration of ionized calcium from the observed serum concentrations of total calcium, albumin, total protein, and inorganic phosphate. The ionized calcium concentration was calculated in 67 serum samples from healthy subjects and 34 outpatients previously identified as having abnormal serum calcium levels. The correlation coefficient between our method (y) and the calcium-ion-selective electrode method (x) was 0.953 and the linear regression equation was y = 0.97x at pH 7.4 with a factor of α = 0.21, which was based on the differences between the concentrations of calcium phosphorus compounds obtained by the electrode method and by calculation. The developed calculation is as useful and accurate as the electrode method, and therefore extremely useful for clinical diagnoses. [ABSTRACT FROM AUTHOR]

Abstract  Bisphosphonate is an effective drug to reduce fracture risk in osteoporotic patients; however, factors affecting the efficacy of bisphosphonate treatment are not fully known, especially in Japanese patients. In the present study, we examined the relationships between an increase in lumbar spine bone mineral density (BMD) by bisphosphonates and several pretreatment parameters, including biochemical, bone/mineral, and body composition indices, in 85 postmenopausal osteoporotic patients treated with alendronate or risedronate. BMD increase was measured by dual-energy X-ray absorptiometry at the lumbar spine before and 2 years after treatment. BMD increase at the lumbar spine was observed as independent of age, height, weight, body mass index, and fat mass, although lean body mass seemed slightly related. On the other hand, fasting plasma glucose (FPG) levels were significantly and positively related to BMD increase at the lumbar spine. In multiple regression analysis, FPG levels were not significantly related to BMD increase at the lumbar spine when lean body mass was considered. As for bone/mineral parameters, BMD increase at the lumbar spine was not significantly related to serum levels of calcium, parathyroid hormone (PTH), and alkaline phosphatase or urinary levels of deoxypiridinoline and calcium excretion. As for BMD parameters, Z-scores of BMD at any site and bone geometry parameters obtained by forearm peripheral quantitative computed tomography were not significantly related to BMD increase at the lumbar spine. BMD increases at the lumbar spine were similar between groups with or without vertebral fractures. In conclusion, BMD increase at the lumbar spine by bisphosphonate treatment was not related to any pretreatment parameters, including body size, body composition, and bone/mineral metabolism in postmenopausal Japanese women with primary osteoporosis, although FPG correlated partly to BMD through lean body mass. [ABSTRACT FROM AUTHOR]

Abstract  Glucocorticoid (GC) therapy induces rapid bone loss, but the early changes in calcium and bone metabolism in patients treated with GC have not been clarified. To investigate the changes in calcium and bone metabolism during the early stage of GC therapy, we analyzed various biochemical markers of bone metabolism. The serum levels of calcium (Ca), phosphorus, parathyroid hormone (PTH), osteocalcin (OC), bone alkaline phosphatase (BAP), and type I collagen cross-linked N-telopeptide (NTx), as well as the urinary levels of Ca, creatinine, and NTx, were measured on days 0, 3, 7, and 28 of GC therapy. The subjects were divided into the following four groups: 9 patients receiving pulse therapy (P), 18 patients receiving prednisolone (PSL) at doses ≥40 mg/day (H), 9 patients receiving PSL at doses ≥20 mg/day (M), and 11 patients receiving PSL at doses ≤10 mg/day (S). The serum OC level showed a marked decrease on day 3 of GC therapy (−41.2% ± 6.6%, P P P [ABSTRACT FROM AUTHOR]

Objectives: Interleukin (IL)-1b and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis. On the other hand, plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, exerts functions in the pathogenesis of various diseases. However, the functional roles of PAI-1 in the chondrocytes have been still remained unknown. Methods: In the present study, we investigated the roles of PAI-1 in the effects of IL-1b on the chondrocytes using wild-type and PAI-1-deficient mice. Results: IL-1b significantly elevated PAI-1 mRNA levels in the chondrocytes from wild-type mice. PAI-1 deficiency significantly blunted the mRNA levels of TGF-b and IL-6 enhanced by IL-1b in murine chondrocytes. Moreover, PAI-1 deficiency significantly decreased the mRNA levels of MMP-13, -3 and -9 as well as MMP-13 activity enhanced by IL-1b in the chondrocytes. In addition, PAI-1 deficiency significantly reversed type II collagen mRNA levels suppressed by IL-1b in the chondrocytes. On the other hand, active PAI-1 treatment significantly enhanced the mRNA levels of MMP-13, -3 and -9 as well as decreased type II collagen mRNA levels in the chondrocytes from wild-type mice. Conclusion: We first demonstrated that PAI-1 is involved in MMP expression enhanced by IL-1b in murine chondrocytes. PAI-1 might be crucial for the cartilage matrix degradation and the impaired chondrogenesis by IL-1b in mice. [ABSTRACT FROM AUTHOR]

Background: Subchondral osteopenia is important for the pathophysiology of osteoarthritis (OA). Although previous studies suggest that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is related to bone metabolism, its role in OA remains unknown. We therefore investigated the roles of PAI-1 in the subchondral bone in OA model mice.Methods: Wild type (WT) and PAI-1-deficient (KO) mice were ovariectomized (OVX), and then destabilization of the medial meniscus (DMM) surgery was performed.Results: DMM and OVX significantly decreased the trabecular bone mineral density of the subchondral bone evaluated by quantitative computed tomography in PAI-1 KO mice. The effects of OVX and/or PAI-1 deficiency on the OARSI score for the evaluation of the progression of knee degeneration were not significant. PAI-1 deficiency significantly augmented receptor activator nuclear factor κB ligand mRNA levels enhanced by IL-1β in mouse primary osteoblasts, although it did not affect osteoblast differentiation. Moreover, PAI-1 deficiency significantly increased osteoclast formation from mouse bone marrow cells.Conclusion: We showed that PAI-1 deficiency accelerates the subchondral osteopenia after induction of OA in mice. PAI-1 might suppress an enhancement of bone resorption and subsequent subchondral osteopenia after induction of OA in mice. [ABSTRACT FROM AUTHOR]

Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/ CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury. [ABSTRACT FROM AUTHOR]

Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC. [ABSTRACT FROM AUTHOR]

Further development in research of bone regeneration is necessary to meet the clinical demand for bone reconstruction. Recently, we reported that plasminogen is crucial for bone repair through enhancement of vessel formation. However, the details of the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) in the bone repair process still remain unknown. Herein, we examined the effects of plasminogen activators on bone repair after a femoral bone defect using tPA-deficient (tPA-/-) and uPA-deficient (uPA-/-) mice. Bone repair of the femur was delayed in tPA-/- mice, unlike that in wild-type (tPA+/+) mice. Conversely, the bone repair was comparable between wild-type (uPA+/+) and uPA-/- mice. The number of proliferative osteoblasts was decreased at the site of bone damage in tPA-/- mice. Moreover, the proliferation of primary calvarial osteoblasts was reduced in tPA-/- mice. Recombinant tPA facilitated the proliferation of mouse osteoblastic MC3T3-E1 cells. The proliferation enhanced by tPA was antagonized by the inhibition of endogenous annexin 2 by siRNA and by the inhibition of extracellular signalregulated kinase (ERK)1/2 phosphorylation in MC3T3-E1 cells. Vessel formation as well as the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were decreased at the damaged site in tPA-/- mice. Our results provide novel evidence that tPA is crucial for bone repair through the facilitation of osteoblast proliferation related to annexin 2 and ERK1/2 as well as enhancement of vessel formation related to VEGF and HIF-1α at the site of bone damage. [ABSTRACT FROM AUTHOR]

Vitamin D deficiency and advanced glycation end products (AGEs) are suggested to be involved in the pathogenesis of osteoporosis and sarcopenia. However, the effects of vitamin D and AGEs on myogenesis and the interaction between muscle and bone remains still unclear. We previously showed that osteoglycin (OGN) is secreted from myoblasts and stimulates osteoblastic differentiation, suggesting that it plays important roles in the interaction between muscle and bone. The aim of this study is thus to examine the effects of vitamin D and AGEs on myoblastic differentiation of C2C12 cells and osteoblastic differentiation of osteoblastic MC3T3-E1 cells through OGN expression. 1α,25-dihydroxyvitamin D3 (1,25D) and eldecalcitol, an active vitamin D analog, induced the expression of MyoD, myogenin and OGN, and these effects were abolished by vitamin D receptor (VDR) suppression by siRNA in C2C12 cells. Moreover, conditioned medium from 1,25D-pretreated C2C12 cells stimulated the expression of type 1 collagen and alkaline phosphatase in MC3T3-E1 cells, compared to control medium from 1,25D-untreated C2C12 cells. In contrast, conditioned medium from VDR-suppressed and 1,25D-pretreated C2C12 cells showed no effects. AGE2 and AGE3 suppressed the expression of MyoD, myogenin and OGN in C2C12 cells. Moreover, 1,25D blunted the AGEs' effects. In conclusion, these findings showed for the first time that active vitamin D plays important roles in myogenesis and muscle-induced osteoblastogenesis through OGN expression. Active vitamin D treatment may rescue the AGEs-induced sarcopenia as well as - suppressed osteoblastic differentiation via OGN expression in myoblasts. [ABSTRACT FROM AUTHOR]

Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vectortransfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues. [ABSTRACT FROM AUTHOR]

Background Deep venous thrombosis (DVT), which is often associated with pulmonary embolism (PE), is a serious complication after total knee arthroplasty (TKA). In the present study, we examined the overall thrombotic and thrombolytic status using Global Thrombosis Test (GTT) in non-anticoagulated blood of patients undergoing TKA to develop the predictable marker for the incidence of DVT. Methods DVT was diagnosed using doppler ultrasonography a day after the surgery in 31 patients with osteoarthritis (n = 24), rheumatoid arthritis (n = 6) and ankylosing spondylitis (n = 1) by the well-trained operator. We measured overall thrombotic and thrombolytic status using GTT and other biomarkers, which is associated with blood coagulation and fibrinolysis, before and immediately after the surgery. Results Newly-generated DVT during the operation was detected in 11 of 31 patients (35.4%) 1 day after TKA. There were no differences in markers of coagulation (PT and APTT), platelet activity (platelet aggregation-induced by ADP and collagen) and fibrinolysis (FDP and D- dimer) between non-DVT and DVT group both before and after the surgery. Both Pre- and Post-operative GTT-occlusion times (OT), an index of platelet reactivity, were tended to be shorter, but not significant, in DVT group compared with non-DVT group. Pre-operative GTT-lysis time (LT), an index of thrombolytic activity, was significantly shorter in DVT group compared with non-DVT group, while there were no differences in post-operative value of this index between DVT group and non-DVT group, suggesting overall thrombolytic activity was enhanced in DVT group before surgery. Conclusions Our data suggest that enhancement of pre-operative thrombolytic activity assessed by GTT may be a predictable marker for the incidence of DVT after TKA. [ABSTRACT FROM AUTHOR]

In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1--deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism. [ABSTRACT FROM AUTHOR]

The effects of Enzamin on obesity-related metabolic disorders in obese db/db mice were examined to explore a novel agent for the prevention of insulin resistance. Db/db mice were treated with water containing Enzamin (0·1 and 1·0%) for 8 weeks from 6 weeks of age. Enzamin treatment at 1·0%, but not at 0·1%, significantly decreased the fasting plasma glucose, serum total cholesterol and TAG levels in db/db mice, without affecting body weight gain and body fat composition. Furthermore, insulin sensitivity and glucose tolerance were improved by the treatment of db/db mice with 1·0% Enzamin. Immunohistochemical studies and gene expression analysis showed that 1·0% Enzamin treatment suppressed macrophage accumulation and inflammation in the adipose tissue. In addition, 1·0% Enzamin treatment increased serum adiponectin in db/db mice. Treatment with 1·0% Enzamin also significantly suppressed the expression of NADPH oxidase subunits, suggesting an antioxidative effect for Enzamin in the adipose tissue. Furthermore, in vitro experiments demonstrated that the lipopolysaccharide-induced inflammatory reaction was significantly suppressed by Enzamin treatment in macrophages. Enzamin treatment increased the expression of GLUT4 mRNA in muscle, but not GLUT2 mRNA in the liver of db/db mice. Enzamin also increased the mRNA expression of carnitine palmitoyltransferase 1a (CPT1a, muscle isoform) in db/db mice, whereas Enzamin treatment did not affect the mRNA expression of CPT1b (liver isoform) in db/db mice. In conclusion, our data indicate that Enzamin can improve insulin resistance by ameliorating impaired adipocytokine expression, presumably through its anti-inflammatory action, and that Enzamin possesses a potential for preventing the metabolic syndrome. [ABSTRACT FROM AUTHOR]