High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]+ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MSn analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well

Influence of 'Trichobilharzia regenti' (Digenea: Schistosomatidae) on the defence activity of 'Radix lagotis' (Lymnaeidae) haemocytes

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2–36 h post exposure (p.e.) to the parasite. At later time points, 44–92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae

VÝVOJOVÉ PORUCHY ZUBŮ A JEJICH DIAGNOSTIKA POMOCÍ RENTGENOVÝCH SNÍMKŮ

Introduction: The Development of detention begins in sixth week of intrauterine life with making the dentogingival lamina, that gives the origin to tooth germs. If the insult operates in these early stages of development, it can result in termination of the development of tooth, the disorder in later development manifests as a malformation of tooth - the anomaly of shape, size, defects of hard dental tissues, variations in eruption, etc. The development of tooth is related to the development of the whole organism, therefore the failure of the overall development interferes with the development of teeth. Except for systemic disorders, factors as trauma and inflammation may play a significant role. The differentiation between local and systemic causes of the anomaly can be very difficult. Methods: An indispensable component in diagnostic and as a tool for screening of disorders of impacted teeth is the radiographic examination. In dentistry we use intraoral RVG images, OPG panoramic image and for 3D imaging CBCT and CT. Results: Atomic force microscopy(AFM) was used to study the enamel surface of extracted teeth in order to compare the structure of malformed and healthy enamel. Conclusion: Developmental disorders of the teeth and dental hard tissues are relatively common clinical practice and their diagnostics and therapy is usually not an easy task. It is often found as an incidental finding on chest radiograph. Atomic force microscopy provides high-resolution images and is an important source of information about the structure of human enamel

Effects of yeast hydrolysate supplementation in low-fish meal diets for pikeperch

Plant proteins have been increasingly used as sustainable substitutes for fish meal (FM) in aquafeeds; however, their high inclusion level compromises fish performance. The objective of this study was to examine whether yeast hydrolysate (YH) supplementation can improve the utilisation of high soybean meal (SM) diet and ameliorate its potential deteriorating impacts in pikeperch (Sander lucioperca). A basal diet was formulated using 44% FM, and four additional diets were produced by replacing 30 or 60% of FM with SM with or without the addition of 2% YH (FM, SM30, SM60, SM30 + YH, and SM60 + YH diets). Each diet was fed to three groups of fish (35.3 ± 0.10 g, 150 fish per group) to visual satiety four times daily for 70 days. Fish growth was not impacted by FM replacement level or YH application. However, SM60 group exhibited markedly higher feed conversion ratio and lower survival rate than those fed the FM- and YH-supplemented diets (P < 0.05). The highest and the lowest protein efficiency ratio values were obtained for the SM30 + YH and SM60 groups, respectively. Whole-body lipid content decreased in SM60 and SM60 + YH groups, and muscle lipid decreased in all the replacement groups. Serum triglyceride and glucose concentrations tended to decrease as FM replacement level increased. The highest alanine aminotransferase, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities were detected in the SM60 group, and YH addition significantly decreased the AST and LDH activities. Serum lysozyme activity decreased in SM30, SM60 and SM60 + YH groups. Serum myeloperoxidase and antiprotease activities decreased in SM60 group, and YH supplementation improved their activities. No effects of diets were observed on serum antioxidant parameters such as catalase activity and malondialdehyde concentration, and gut morphological indices. Number of goblet cells in midgut decreased by increasing the SM inclusion level and a slight improvement was observed by YH application. These findings suggest that YH supplementation has the potential to support the replacement of up to 60% FM with defatted SM in pikeperch feed without deteriorating growth, feed utilisation, and survival rate. Further, YH incorporation mitigated the damaging impacts of high SM diet on liver function and non-specific immune response

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination.

Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination.

Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses