Valer Dzupa

Normalizing Glutamine Concentration Causes Mitochondrial Uncoupling in an In Vitro Model of Human Skeletal Muscle

BACKGROUND Glutamine has been considered essential for rapidly dividing cells, but its effect on mitochondrial function is unknown. MATERIALS AND METHODS Human myoblasts were isolated from skeletal muscle biopsy samples (n = 9) and exposed for 20 days to 6 different glutamine concentrations (0, 100, 200, 300, 500, and 5000 µM). Cells were trypsinized and manually counted every 5 days. Seven days before the end of exposure, half of these cells were allowed to differentiate to myotubes. Afterward, energy metabolism in both myotubes and myoblasts was assessed by extracellular flux analysis (Seahorse Biosciences, Billerica, MA). The protocol for myoblasts was optimized in preliminary experiments. To account for different mitochondrial density or cell count, data were normalized to citrate synthase activity. RESULTS Fastest myoblast proliferation was observed at 300 µM glutamine, with a significant reduction at 0 and 100 µM. Glutamine did not influence basal oxygen consumption, anaerobic glycolysis or respiratory chain capacity. Glutamine significantly (P = .015) influenced the leak through the inner mitochondrial membrane. Efficiency of respiratory chain was highest at 200-300 µM glutamine (~90% of oxygen used for adenosine triphosphate synthesis). Increased glutamine concentration to 500 or 5000 µM caused mitochondrial uncoupling in myoblasts and myotubes, decreasing the efficiency of the respiratory chain to ~70%. CONCLUSION Glutamine concentrations, consistent with moderate clinical hypoglutaminemia (300 µM), bring about an optimal condition of myoblast proliferation and for efficiency of aerobic phosphorylation in an in vitro model of human skeletal muscle. These data support the hypothesis of hypoglutaminemia as an adaptive phenomenon in conditions leading to bioenergetic failure (eg, critical illness).

The correlation between muscles insertions and topography of break lines in pertrochanteric fractures: a comprehensive anatomical approach of complex proximal femur injuries

PurposeThe purpose of our work was to verify the hypothesis that muscle insertions and ligament attachments have an impact on the course of typical break lines in the area of the trochanteric massif, i.e., to provide a more detailed description of the origins and insertions of the musculo-ligamentous apparatus on the surface of the proximal femur, and to find a potential morphological correlate between muscle insertions and ligament attachments to the proximal femur and the course of the break line in a typical pertrochanteric fracture.MethodsA detailed dissection of areas of trochanter major et minor, linea et crista intertrochanterica was performed in 50 anatomical preparations of the proximal femur, and the insertions of the muscular-ligamentous structures were described. The set of 600 radiographs were used to obtain projections of typical break lines on the proximal femur, and corresponding areas of exposed bone surface were identified in the anatomical preparations based on the projections and on 15 real specimens of patients after the pertrochanteric fracture osteosynthesis.Results and conclusionBone covered only with the periosteum, with no reinforcing elements of the origin or insertions of muscles or attachments of ligaments, represents the locus minoris resistentiae for beginning of fractures. Variability in the sizes and shapes of pertrochanteric fracture fragments also depends on variability of the locations and sizes of soft tissue attachment areas at specified sites on the proximal femur.

Distal tibiofibular synostosis after surgically resolved ankle fractures: An epidemiological, clinical and morphological evaluation of a patient sample

INTRODUCTION Ankle fractures comprise a highly morphologically and etiologically diverse group of injuries, which includes various degrees of impairment of bone and ligamentous structures. The complete synostosis and incomplete bony bridging of tibiofibular syndesmosis are among the local late complications after surgically treated ankle fractures. PATIENTS AND METHOD 269 patients were evaluated, including 203 patients with Weber type-B fractures, and 66 patients with Weber type-C fractures. All patients underwent ankle radiography at standard intervals (post-operatively, 6 and 12 weeks, 6 and 12 months). The final assessment one year after osteosynthesis was performed. The study analyzed age, sex, fracture morphology, the location and morphology of ossification, functional outcomes and subjective evaluations of patient status. RESULTS As risk factors there were found male sex, tibiotalar dislocation, syndesmotic screw fixation and Weber type-C fractures. The severity of subjective difficulties and objective status were not dependent on the size of distal tibiofibular synostosis. DISCUSSION AND CONCLUSION Despite relatively extensive imaging findings of complete synostosis or incomplete bony bridging, they only limited functional outcomes to a minimal extent.

Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples

Using skeletal muscle homogenates for respirometry has many advantages, but the main challenge is avoiding the damage to outer mitochondrial membrane (OMM) and complex I. By optimising the amount of muscle and careful titration of substrates and inhibitors we developed a new protocol and compared it to isolated mitochondria. We found acceptable damage to OMM (~10-15% increment of oxygen flux after addition of cytochrome c) and to complex I (~70% of electron flux). Homogenate retained ~90% of phosphorylation capacity of isolated mitochondria. The use of fresh homogenate was crucial as mitochondrial function declined rapidly after 2-3h of cold storage.

The importance of intramedullary hip nail positioning during implantation for stable pertrochanteric fractures: biomechanical analysis.

PurposeProximal femoral fractures are among the most commonly sustained fractures. The current treatment of stable proximal femoral fractures located in trochanteric region primarily involves the use of two systems: extramedullary dynamic hip screws and intramedullary hip nails. Given that these fractures are mainly found in the elderly population, the necessity of a repeat, due to failure of the first, may jeopardize the patient’s life. Decisive factors contributing to the healing of a fracture (or the failure thereof) include fracture pattern, technical implementation of the operation (i.e., position of the implant), implant’s properties and its changes in relation to the surrounding bone tissue during loading. Each screw insertion variant results in damage to various load-bearing bone structures, which can be expected to influence healing quality and stability of newly formed bone.MethodWith the aid of a numerical model and finite element methods, the authors analyzed several different positions of IMHN/PFH-nails in the proximal femur, with the objective of determining positions with an increased risk of failure.Results and conclusionIn model situations, it has been shown that in stable fractures results do not depend on absolutely precise positioning and small deflections in the nails and neck screws positions do not significantly increase the risk of failure for the entire fixation. Damage to load-bearing structures relative to various implant placements does not impact the resultant overall fixation stability. Therefore, it is not necessary to re-introduce implants in the ideal position, which can lead to reduced patient radiation doses during surgery.

Antithrombotic therapy of patients with atrial fibrillation discharged after major non-cardiac surgery. 1-year follow-up. Sub-analysis of PRAGUE 14 study

Background The study investigated the discharge antithrombotic medication in patients with atrial fibrillation (AF) after major non-cardiac surgery and the impact on one-year outcomes. Methods A subgroup of 366 patients (mean age 75.9±10.5 years, women 42.3%, acute surgery 42.9%) undergoing major non-cardiac surgery and having any form of AF (30.6% of the total population enrolled in the PRAGUE-14 study) was followed for 1 year. Results Antithrombotics (interrupted due to surgery) were resumed until discharge in 51.8% of patients; less frequently in men (OR 0.6 (95% CI 0.95 to 0.35); p = 0.029), and in patients undergoing elective surgery (OR 0.6 (95% CI 0.91 to 0.33); p = 0.021). Dual antiplatelet therapy was resumed more often (91.7%) in comparison to aspirin monotherapy (57.3%; p = 0.047), and vitamin K antagonist (56.3%; p = 0.042). Patients with AF had significantly higher one-year mortality (22.1%) than patients without AF (14.1%, p = 0.001). The causes of death were: ischaemic events (32.6% of deaths), bleeding events (8.1%), others (N = 51; 59.3%, 20 of them died due to cancer). Non-reinstitution of aspirin until discharge was associated with higher one-year mortality (17.6% vs. 34.8%; p = 0.018). Conclusion Preoperatively interrupted antithrombotics were re-administrated at discharge only in half of patients with AF, less likely in male patients and those undergoing elective surgery. The presence of AF was recognized as a predictor of one-year mortality, especially if aspirin therapy was not resumed until discharge. Trial registration ClinicalTrials.gov NCT01897220