The effect of liquid-liquid extraction on metabolite detection and analysis using NMR spectroscopy in human synovial fluid.

The evaluation of joint disease using synovial fluid is an emerging field of metabolic profiling. The analysis is challenged by multiple macromolecules which can obscure the small molecule chemistry. The use of protein precipitation and extraction has been evaluated previously, but not in synovial fluid. We systematically review the published NMR spectroscopy methods of synovial fluid analysis and investigated the efficacy of three different protein precipitation techniques: methanol, acetonitrile and trichloroacetic acid. The trichloroacetic wash removed the most protein. However, metabolite recoveries were universally very poor. Acetonitrile liquid/liquid extraction gave metabolite gains from four unknown compounds with spectral peaks at δ = 1.91 ppm, 3.64 ppm, 3.95 ppm & 4.05 ppm. The metabolite recoveries for acetonitrile were between 1.5 and 7 times higher than the methanol method, across all classes of metabolite. The methanol method was more effective in removing protein as reported by the free GAG undefined peak (44 % vs 125 %). However, qualitative evaluation showed that acetonitrile and methanol provided good restoration of the spectra to baseline. The methanol extraction has issues of a gelatinous substrate in the samples. All metabolite recoveries had a CV of > 15 %. A recommendation of acetonitrile liquid/liquid extraction was made for human synovial fluid (HSF) analysis. This is due to consistency, effective protein precipitation, recovery of metabolites and additional compounds not previously visible.

The influence of sample collection, handling and low temperature storage upon NMR metabolic profiling analysis in human synovial fluid.

The impact of metabolism upon the altered pathology of joint disease is rapidly becoming recognized as an important area of study. Synovial joint fluid is an attractive and representative biofluid of joint disease. A systemic review revealed little evidence of the metabolic stability of synovial joint fluid collection, handling or storage, despite recent reports characterizing the metabolic phenotype in joint disease. We aim to report the changes in small molecule detection within human synovial fluid (HSF) using nuclear magnetic resonance (NMR) spectroscopy at varying storage temperatures, durations and conditions. HSF was harvested by arthrocentesis from patients with isolated monoarthropathy or undergoing joint replacement (n = 30). Short-term storage (0-12 h, 4°C & 18°C) and the effect of repeated freeze-thaw cycles (-80°C to 18°C) was assessed. Long-term storage was evaluated by early (-80°C, <21days) and late analysis (-80°C, 10-12 months). 1D NMR spectroscopy experiments, NOESYGPPR1D and CPMG identified metabolites and semi-quantification was performed. Samples demonstrated broad stability to freeze-thaw cycling and refrigeration of <4 h. Short-term room temperature or refrigerated storage showed significant variation in 2-ketoisovalerate, valine, dimethylamine, succinate, 2-hydroxybutyrate, and acetaminophen glucuronide. Lipid and macromolecule detection was variable. Long-term storage demonstrated significant changes in: acetate, acetoacetate, creatine, N,N-dimethylglycine, dimethylsulfone, 3-hydroxybutyrate and succinate. Changeable metabolites during short-term storage appeared to be energy-synthesis intermediates. Most metabolites were stable for the first four hours at room temperature or refrigeration, with notable exceptions. We therefore recommend that HSF samples should be kept refrigerated for no more than 4 hours prior to freezing at -80°C. Furthermore, storage of HSF samples for 10-12 months before analysis can affect the detection of selected metabolites.

Can joint fluid metabolic profiling (or "metabonomics") reveal biomarkers for osteoarthritis and inflammatory joint disease?: A systematic review.

AIMS: Metabolic profiling is a top-down method of analysis looking at metabolites, which are the intermediate or end products of various cellular pathways. Our primary objective was to perform a systematic review of the published literature to identify metabolites in human synovial fluid (HSF), which have been categorized by metabolic profiling techniques. A secondary objective was to identify any metabolites that may represent potential biomarkers of orthopaedic disease processes. METHODS: A systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines using the MEDLINE, Embase, PubMed, and Cochrane databases. Studies included were case series, case control series, and cohort studies looking specifically at HSF. RESULTS: The primary analysis, which pooled the results from 17 published studies and four meeting abstracts, identified over 200 metabolites. Seven of these studies (six published studies, one meeting abstract) had asymptomatic control groups and collectively suggested 26 putative biomarkers in osteoarthritis, inflammatory arthropathies, and trauma. These can broadly be categorized into amino acids plus related metabolites, fatty acids, ketones, and sugars. CONCLUSION: The role of metabolic profiling in orthopaedics is fast evolving with many metabolites already identified in a variety of pathologies. However, these results need to be interpreted with caution due to the presence of multiple confounding factors in many of the studies. Future research should include largescale epidemiological metabolic profiling studies incorporating various confounding factors with appropriate statistical analysis to account for multiple testing of the data.Cite this article: Bone Joint Res. 2020;9(3):108-119.

The Use of Sonographically Guided Botulinum Toxin Type A (Dysport) Injections Into the Tensor Fasciae Latae for the Treatment of Lateral Patellofemoral Overload Syndrome.

BACKGROUND: Pain in the anterior and lateral parts of the knee during exercise is a common clinical problem for which current management strategies are often unsuccessful. PURPOSE: To investigate the effect of an ultrasound-guided botulinum toxin (BT) injection into the tensor fasciae latae (TFL), followed by physical therapy, in patients classified with lateral patellofemoral overload syndrome (LPOS) who failed to respond to conventional treatment. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 45 patients (mean ± SD age, 32.4 ± 8.6 years) who met the inclusion criteria of (1) activity-related anterolateral knee symptoms, (2) symptoms lasting longer than 3 months, (3) a pathological abnormality confirmed by magnetic resonance imaging, and (4) previous failed physical therapy received an ultrasound-guided injection of BT into the TFL followed by physical therapy. Patient-reported outcomes were collected at 5 intervals: before the injection; at 1, 4, and 12 weeks after the injection; and at a mean 5 years after the injection. In 42 patients, relative iliotibial band (ITB) length changes were assessed using the modified Ober test at the first 4 time points. A computational model was run to simulate the effect of TFL weakening on gluteus medius (GMed) activity. Statistical analysis was undertaken using 1-way analysis of variance and paired t tests with Bonferroni post hoc correction. RESULTS: There was a significant improvement in Anterior Knee Pain Scale scores from before the injection (61 ± 15) to 1 (67 ± 15), 4 (70 ± 16), and 12 weeks (76 ± 16) after the injection and in 87% of patients (39/45 patients available for follow-up) at approximately 5 years (from 62.9 ± 15.4 to 87.0 ± 12.5) after the injection (all P < .010). A significant effect on the modified Ober test was identified as a result of the intervention, with an increase in leg drop found at 1 (3° ± 5°), 4 (4° ± 5°), and 12 (7° ± 6°) weeks after the injection compared with before the injection (all P < .010). Simulating a progressive reduction in TFL strength resulted in corresponding increases in GMed activity during gait. CONCLUSION: An injection of BT into the TFL, combined with physical therapy, resulted in a significant improvement of symptoms in patients with LPOS, which was maintained at 5-year follow-up. This may result from reduced lateral TFL/ITB tension or to an increase in GMed activity in response to inhibition of the TFL.

Isolated gallbladder perforation following blunt abdominal trauma in a six-year-old child.

Isolated perforation of the gallbladder secondary to blunt trauma is rare. Furthermore, only a few cases exist in the infant age group. It has vague symptoms and interpretation of the radiology imaging is challenging. Diagnosis is usually made at operation. We report the case of a six-year-old boy who fell on to the handlebars of his scooter, sustaining an isolated gallbladder perforation. The authors highlight the importance of interpreting the volume of intraperitoneal fluid and early diagnostic laparoscopy.

Blunt abdominal trauma resulting in gallbladder injury: a review with emphasis on pediatrics.

BACKGROUND: Gallbladder injury in blunt abdominal trauma is a rare and difficult diagnosis. Gallbladder injury is reported to be between 1.9% and 2.1% of all abdominal traumas. It has vague symptoms usually with inconclusive investigation results; hence, it is often diagnosed at laparotomy. The patient typically has vague abdominal pain and occasionally a period of remission depending on the type of gallbladder injury. In pediatrics, blunt abdominal trauma presents additional challenges of difficult historians and compensating physiology. Any delay in diagnosis and definitive management will worsen the prognosis. Making the diagnosis requires astute clinical acumen and radiologic interpretation. The classification system of Losanoff has merit in guiding treatment. While cholecystectomy is the preferred treatment, there are occasions when the gallbladder may be left in situ and these are discussed. METHODS: Literature searches were performed using Pubmed and Medline with keywords "abdominal trauma," "gallbladder injury," and "gallbladder perforation." DISCUSSION: The authors highlight the incidence of associated visceral injuries in gallbladder trauma (>90%). Gallbladder perforation is more likely in cases when the gallbladder is distended and thin-walled at the time of injury. Therefore, we recommend that gallbladder perforation is suspected in those patients who have drunk alcohol or eaten recently. Despite the developments in modern computed tomography, identifying gallbladder perforation is difficult because of the subtlety and rarity of the condition. We draw attention to the proposed anatomic classification systems because they are of some use in guiding treatment. In the absence of a diagnosis after blunt abdominal trauma and with intra-abdominal free fluid, the clinician faces the difficult decision of whether surgery is indicated for a potential visceral injury. After discussing the available evidence, the authors advocate a low index of suspicion for performing diagnostic laparoscopy.

A comprehensive review of trauma and disruption to the sternoclavicular joint with the proposal of a new classification system.

BACKGROUND: The sternoclavicular joint (SCJ) is rarely injured but should not be overlooked in cases of high-energy trauma. Stability is reliant on the ligamentous attachments. The methods of injury and the clinical presentations are examined. Obtaining informative plain radiology of the SCJ is challenging and the best methods to achieve this are discussed. METHODS: The Pubmed and Medline databases were searched for all literature relating to the keywords of "sternoclavicular" or "SCJ." CONCLUSIONS: Early closed reduction in acute injury is advisable. Complications of posterior dislocation to the SCJ are potentially severe and occasionally life threatening. Long-term stability is often difficult to achieve and can be significantly debilitating. Operative methods to restore joint stability are examined and the evidence to support them is presented. We propose a simple classification system to aid in making management decisions.