Quantification of vancomycin and clindamycin in human plasma and synovial fluid applying ultra-performance liquid chromatography tandem mass spectrometry

Periprosthetic joint infection is a challenging infection involving the joint prosthesis and adjacent tissue, such as synovial fluid, synovial tissue, and bone tissue. The current treatment consists of multiple surgical revisions and long-term antibiotic therapy. Treatment failure can cause poor functional outcome and reduced quality of life. Further research on the extent of antibiotic penetration into the infected tissues is of great importance. Our work aimed to develop and validate a novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of the commonly administered antibiotics vancomycin and clindamycin in plasma and synovial fluid. An extraction procedure consisting of zinc sulfate precipitation and dilution with eluent was used for both analytes. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 C18 column (1.8 µm, 2.1 × 100 mm), and quantification was carried out by a Waters Xevo TQ-S micro mass spectrometer. Stable isotope-labeled vancomycin-d10 served as internal standard. The method validation was performed based on the guidelines of the EMA and FDA. The calibration curves were linear over the range of 0.5–50 mg/L, with a coefficient of determination above 0.990. The validation results for precision and accuracy, specificity, matrix effects and stability were all within the acceptance range. An accurate and rapid method for the simultaneous quantification of vancomycin and clindamycin in human plasma and synovial fluid on the UPLC-MS/MS was developed, optimized and validated. The analysis has a run time of 5.2 min and 50 µL sample volume is needed. This developed method was successfully applied in eight patients with PJI and is suitable to determine the exposure of antibiotics in plasma and synovial fluid in patients during current PK/PD studies.</p

Ferritins: furnishing proteins with iron

Ferritins are a superfamily of iron oxidation, storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. The majority of ferritins consist of 24 subunits that individually fold into 4-α-helix bundles and assemble in a highly symmetric manner to form an approximately spherical protein coat around a central cavity into which an iron-containing mineral can be formed. Channels through the coat at inter-subunit contact points facilitate passage of iron ions to and from the central cavity, and intrasubunit catalytic sites, called ferroxidase centers, drive Fe2+ oxidation and O2 reduction. Though the different members of the superfamily share a common structure, there is often little amino acid sequence identity between them. Even where there is a high degree of sequence identity between two ferritins there can be major differences in how the proteins handle iron. In this review we describe some of the important structural features of ferritins and their mineralized iron cores and examine in detail how three selected ferritins oxidise Fe2+ in order to explore the mechanistic variations that exist amongst ferritins. We suggest that the mechanistic differences reflect differing evolutionary pressures on amino acid sequences, and that these differing pressures are a consequence of different primary functions for different ferritins

In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes

Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA-antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA-positive patients and a mouse monoclonal antibody against the ANCA-antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti-myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B-treated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocyte

Iron metabolism in Rhodobacter capsulatus Characterisation of bacterioferritin and formation of non‐haem iron particles in intact cells

The water‐soluble cytochrome b from the photosynthetic bacterium Rhodobacter capsulatus was purified and shown to have the properties of the iron‐storage protein bacterioferritin. The molecular mass of R. capsulatus bacterioferritin is 428 kDa and it is composed of a single type of 18‐kDa subunit. The N‐terminal amino acid sequence of the bacterioferritin subunit shows 70% identity to the sequence of bacterioferritin subunits from Escherichia coli, Nitrobacter winogradskyi, Azotobacter vinelandii and Synechocystis PCC 6803. The absorbance spectrum of reduced bacterioferritin shows absorbance maxima at 557 nm (α band), 526 nm (β band) and 417 nm (Soret band) from the six haem groups/molecule. Antibody assays reveal that bacterioferritin is located in the cytoplasm of R. capsulatus, and its levels stay relatively constant during batch growth under aerobic conditions when the iron concentration in the medium is kept constant. Iron deficiency leads to a decrease in bacterioferritin and iron overload leads to an increase. Bacterioferritin from R. capsulatus has an amorphous iron‐oxide core with a high phosphate content (900–1000 Fe atoms and approximately 600 phosphates/bacterioferritin molecule). Mössbauer spectroscopy indicates that in both aerobically and anaerobically (phototrophically) grown cells bacterioferritin with an Fe core is formed, suggesting that iron‐core formation in vivo may not always require molecular oxygen