Katsunori Inagaki

High-throughput determination of nonsteroidal anti-inflammatory drugs in human plasma by HILIC-MS/MS

A simple and sensitive method was developed and validated here for the analysis of thirteen nonsteroidal anti-inflammatory drugs (NSAIDs) in human plasma samples by hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry (MS/MS). A small volume of plasma (20μL) spiked with compounds was diluted with 80μL of 10-mM ammonium acetate followed by a simple protein precipitation with 400μL of acetonitrile. After centrifugation, the clear supernatant extract was directly injected into the HILIC-MS/MS, without any solvent evaporation and reconstitution steps. The chromatographic separation of the NSAIDs was achieved on a Unison UK-Amino HILIC column (50mm×3mm i.d., particle size 3μm) with a linear gradient elution system composed of 10mM ammonium acetate (pH 6.8) and acetonitrile at a flow rate of 0.4mL/min. The mass spectra obtained by HILIC-MS showed base peak ions due to [M+H](+) for indomethacin, oxaprozin, ketoprofen, alminoprofen, zaltoprofen, tiaprofenic acid, pranoprofen, and ketoprofen-d3 and due to [M-H](-) for etodolac, ibuprofen, diclofenac, fenoprofen, loxoprofen, naproxen, and ibuprofen-d3. Recoveries of these thirteen NSAIDs in plasma were 34.8-113% and the lower limits of quantitation were 0.125-1.25μg/mL. The intra- and interday coefficient of variations for all drugs in plasma were less than 14.6%. The data obtained from actual plasma determinations of zaltoprofen, ibuprofen, and diclofenac are also presented.

Current concepts of elbow-joint disorders and their treatment

BackgroundRecently, many studies have emphasized the importance of the comprehension of detailed functional anatomy and biomechanics of the elbow and its significant contribution in facilitating good functional outcomes of conservative and surgical treatment in the field of elbow disorders.MethodsThe most common disease of elbow disorders and their treatment was reviewed.ResultsLateral epicondylitis of the elbow, is defined as a microscopic tear of extensor carpi radialis brevis tendon, and microscopic findings show immature reparative tissue (angiofibroblastic hyperplasia). The patient needs coordinated rehabilitation, range-of motion-exercise, stretching, and bracing in the second phase. Ninety-five percent of patients with lateral epicondylitis heal spontaneously or conservatively. The medial collateral ligament injury of the elbow is most common in the overhead-throwing athlete. Jobe’s procedure, the original reconstruction technique, and its modifications in bone-tunnel creation, allow a tendon graft to be wound in a figure-eight configuration through the tunnels. Further modification of Jobe’s procedure in bone-tunnel configuration reduced the total number of tunnels and facilitates easier graft tensioning. Outcomes with these reconstruction techniques have proven effective in returning high-level throwing athletes back to their sport. Arthroscopic surgery for the elbow in the throwing athlete has evolved and has proven successful results. Arthroscopic treatment includes debridement of posteromedial synovitis, loose-body removal, and excision of the olecranon spur. Posteromedial elbow impingement is also a source of disability in the overhead-throwing athlete. Twenty-five percent of these patients require a medial collateral ligament reconstruction after removal of a posteromedial bony spur. Linked and unlinked total elbow arthroplasty are successful treatment procedures for patients with rheumatoid arthritis, posttraumatic osteoarthritis, and elderly patients with comminuted distal humeral fractures and the salvage of distal humeral nonunion. Proper selection and implantation of prostheses are also important to achieve good functional outcome and longevity.ConclusionThe success of treatment of elbow disorders depends greatly on surgical design and technique, both of which require comprehensive knowledge of detailed anatomy and biomechanics of the elbow.

A disintegrin and metalloprotease-10 is correlated with disease activity and mediates monocyte migration and adhesion in rheumatoid arthritis.

A disintegrin and metalloproteases (ADAMs) are a family of proteins that have been reported to be involved in several inflammatory conditions. We examined the secretion of ADAM-10 in biological fluids from patients with rheumatoid arthritis (RA) and the role it plays in monocyte migration. ADAM-10 levels were measured using enzyme-linked immunosorbent assays and immunofluorescence. To examine the role of ADAM-10 in RA synovial fluids (SFs), we studied THP-1 (human acute monocyte leukemia cell line) and monocyte chemotaxis. To determine whether ADAM-10 plays a role in cell proliferation in the RA synovium, we assayed the proliferation of ADAM-10 small interfering RNA (siRNA)-transfected RA fibroblast-like synoviocytes (FLSs). The ADAM-10 level in RA serum was significantly higher than that in normal serum and was correlated with a disease activity score of 28. ADAM-10-depleted RA SFs showed a decrease in THP-1 and monocyte migratory activity compared with that of sham-depleted controls. ADAM-10 siRNA inhibited monocyte adhesion to RA FLSs. Finally, blocking ADAM-10 secretion in RA FLSs resulted in decreased production of fractalkine/CX3CL1 and vascular endothelial cell growth factor. These data indicate that ADAM-10 plays a role in monocyte migration in RA and suggest that targeting ADAM-10 may provide a method of decreasing inflammation and potentially treating other inflammatory diseases.

High bone turnover elevates the risk of denosumab-induced hypocalcemia in women with postmenopausal osteoporosis

Hypocalcemia is the most common major adverse event in patients with osteoporosis receiving the bone resorption inhibitor denosumab; however, limited information is available regarding risk factors of hypocalcemia. Therefore, this study aimed to identify the risk factors of hypocalcemia induced by denosumab treatment for osteoporosis. We retrospectively reviewed the records of patients who had received initial denosumab supplemented with activated vitamin D for osteoporosis. Serum levels of the following bone turnover markers (BTMs) were measured at baseline: bone-specific alkaline phosphatase (BAP), total N-terminal propeptide of type 1 procollagen (P1NP), tartrate-resistant acid phosphatase 5b (TRACP-5b), and urinary cross-linked N-telopeptide of type 1 collagen (NTX). Of the 85 denosumab-treated patients with osteoporosis studied, 22 (25.9%) developed hypocalcemia. Baseline serum total P1NP, TRACP-5b, and urinary NTX were significantly higher in patients with hypocalcemia than in those with normocalcemia following denosumab administration (all P<0.01). Multivariate logistic regression analysis revealed that patients with total P1NP >76.5 μg/L, TRACP-5b >474 mU/dL, or urinary NTX >49.5 nmol bone collagen equivalent/mmol creatinine had a higher risk of hypocalcemia (P<0.01). Our study suggests that denosumab may have a greater impact on serum calcium levels in patients with postmenopausal osteoporosis with higher baseline bone turnover than in patients with postmenopausal osteoporosis with normal baseline bone turnover, because maintenance of normal serum calcium in this subgroup is more dependent on bone resorption. Close monitoring of serum calcium levels is strongly recommended for denosumab-treated patients with high bone turnover, despite supplementation with activated vitamin D and oral calcium.

Effects of α2-adorenoceptor agonist dexmedetomidine on respiratory rhythm generation of newborn rats.

Dexmedetomidine, an α2-adrenoceptor agonist which has a slight side effect on breathing, is clinically used as an analgesic and sedative agent. Previous studies have shown depressing or modest effects of α2-adorenoceptor agonists on respiratory rhythm generation in newborn rat preparation in vitro. In contrast, it was recently reported that dexmedetomidine induced long-lasting activation of respiratory rhythm in brainstem-spinal cord preparation isolated from neonatal mice. In the present study, we examined whether dexmedetomidine induces any effects on respiratory rhythm in brainstem-spinal cord preparation isolated from newborn rats. We also examined the effects of dexmedetomidine on reflex response in the spinal cord, which is presumed to be an indication of nociceptive response. We found that the administration of dexmedetomidine, at the range of 0.1-10μM, dose-dependently depressed respiratory rhythm and that the inhibitory effect was reversed by atipamezole, an α2-adorenoceptor antagonist. Spinal cord reflex responses were depressed by the application of dexmedetomidine at the range of 0.1-1nM, a lower concentration than that affecting respiratory rhythm. The inhibitory effect was also reversed by atipamezole. Our findings provide neuronal mechanisms that support the clinical use of dexmedetomidine, which shows sedative and antinociceptive effects with minimal side effects on breathing.

Type 1 diabetes patients have lower strength in femoral bone determined by quantitative computed tomography: A cross-sectional study

Previous studies have reported osteoporosis measured by dual‐energy X‐ray absorptiometry in younger patients with type 1 diabetes. Limitations of 2‐D imaging, however, limit the precision of dual‐energy X‐ray absorptiometry for the measurement of bone mineral density and bone strength.

Teriparatide Administration Increases Periprosthetic Bone Mineral Density After Total Knee Arthroplasty: A Prospective Study

BACKGROUND Teriparatide is a currently available therapeutic agent for osteoporosis. Previous studies have reported that teriparatide affects periprosthetic bone mineral density (BMD) after total knee arthroplasty (TKA). However, little agreement has been reached concerning the treatment of periprosthetic BMD after TKA with teriparatide. Moreover, BMD in the femoral and tibial sides of the joints together has never been examined. We investigated the efficacy of teriparatide to inhibit BMD loss in the femoral and tibial side and considered complications such as migration and periprosthetic fractures after TKA. METHODS Twenty-two knees in 17 patients were included in this study, and a control group of patients who underwent TKA was identified according to their medical records. Dual-energy X-ray absorptiometry was performed for different locations (knee, hip, and lumbar spine), and regions of interest were measured to estimate BMD at initiation of the study as a baseline reference, followed by subsequent measurements at 6 and 12 months. RESULTS As a result of adjusting the difference between the BMDs of the 2 groups at initiation, there was a significant increase in R3 (posterior condyle) and R4 (lateral) at 6 months. Furthermore, there was a significant increase in R2 (anterior condyle), R3 (posterior condyle), and R6 (tibial diaphysis) at 12 months. The study group had a higher adjusted mean BMD in all regions than did the control group at 6 and 12 months. CONCLUSION Teriparatide may be a reasonable treatment option for osteoporotic patients to preserve or improve periprosthetic BMD after TKA.

A disintegrin and metalloproteinase (ADAM)-10 as a predictive factor for tocilizumab effectiveness in rheumatoid arthritis

Abstract Objectives: A disintegrin and metalloproteinase (ADAM)-10 is expressed in rheumatoid arthritis (RA). In this study, we focused on ADAM-10 as a predictive factor for the treatment with biologics in RA. Methods: The levels of ADAM-10 and fractalkine/CX3CL1 in RA and healthy controls serum were measured using enzyme-linked immunosorbent assays. Fifteen patients were treated with adalimumab (ADA), and 20 patients were treated with tocilizumab (TCZ). Results: ADAM-10 positively correlated with fractalkine/CX3CL1 in the sera of RA patients and was presented at a significantly higher level compared to that in normal serum (487 ± 80 pg/ml and 85 ± 33 pg/ml, respectively, p < 0.05). ADAM-10 highly correlates with fractalkine/CX3CL1 in the sera of RA patients. The level of ADAM-10 decreased after the treatment with TCZ but not with ADA. In addition, we found that the level of ADAM-10 in TCZ responders was significantly higher than that of the TCZ nonresponders at 24 weeks (619 ± 134 pg/ml and 109 ± 25 pg/ml, respectively). Multiple regression analysis showed that ADAM-10 was only identified as independent predictive variable for the improvement of DAS28 (ESR) at 24 weeks. Conclusions: ADAM-10 may be a predictor of the effectiveness of TCZ in treating RA.

Microscopic study on resorption of β-tricalcium phosphate materials by osteoclasts.

Abstract Sintered compounds prepared with β-tricalcium phosphate (β-TCP) are commonly used as biocompatible materials for bone regenerative medicine. Although implanted β-TCP is gradually replaced with new bone after resorption by osteoclasts, exactly how osteoclasts resorb β-TCP is not well understood. To elucidate this mechanism, we analyzed the structure of β-TCP discs on which mouse mature osteoclasts were cultured using scanning electron microscopy. We found that β-TCP was resorbed by mature osteoclasts on one side of each disc, as evidenced by the formation of multiple spine-like crystals at the exposed areas. Because osteoclasts secrete acid to resorb bone minerals, we mimicked this acidification by dipping β-TCP slices into HCl solution (pH 2.0). However, no spine-like crystals appeared even though the size of each β-TCP particle was reduced. On dentin slices, osteoclasts formed clear actin rings, which are cytoskeletal structures characteristic of bone-resorbing osteoclasts. No clear actin rings were observed in osteoclasts cultured on β-TCP slices, although small actin dots were observed. Analysis by transmission electron microscopy showed that osteoclasts attached to β-TCP particles. These results suggest that osteoclasts resorb β-TCP particles independently of clear actin ring formation.

Case of femoral diaphyseal stress fracture after long-term risedronate administration diagnosed by iliac bone biopsy

Bisphosphonate excessively inhibits bone resorption and results in pathological fracture of the femur or ilium. The subject of this study was administered risedronate for 7 years; we suspected an easy fracture of the femoral diaphysis. In this study, we report the results of this patient’s bone biopsy and bone morphometric analysis. A 76-year-old female patient presented with right femoral pain. Bone mineral density of the anteroposterior surface of the 2nd to 4th lumbar vertebrae (L2–L4) was decreased and levels of bone turnover markers were high. Therefore, we initiated treatment with risedronate. As she continued the medication, urinary levels of cross-linked N-terminal telopeptides of type I collagen and alkaline phosphatase (bone-type isozyme) were found to be within the normal ranges. After 7 years of administration, the patient experienced pain when she put weight on the right femur and right femoral pain while walking. Plain radiographic examination revealed polypoid stress fracture-like lesions on the right femoral diaphysis and on the slightly distal-lateral cortical bone. Similar lesions were observed on magnetic resonance imaging and bone scintigraphy. We suspected severely suppressed bone turnover. Bone biopsy was obtained after labeling with tetracycline, and bone morphometric analysis was performed. On microscopic examination, slight double tetracycline labeling was observed. The trabeculae were narrow, and the numbers of osteoblasts and osteoclasts were decreased. Further, rates of bone calcification and bone formation were slow. Hence, we diagnosed fracture as a result of low turnover osteopathy. Risedronate was withdrawn, and Vitamin D3 was administered to improve the bone turnover. At 6 months, abnormal signals on magnetic resonance imaging had decreased and her pain while walking or undergoing the stress test disappeared as well. Thus, long-term administration of bisphosphonates may lead to easy fracture, although bone turnover markers were observed to be within the normal range. During bisphosphonate administration, physicians need to monitor closely and treat their patients for any pain experienced in the femoral region while walking or undergoing a stress test.