Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

Characterization of some strains from human clinical sources which resemble “Leptotrichia sanguinegens”: description of Sneathia sanguinegens sp. nov., gen. nov.

Three strains of a gram-negative, blood or serum requiring, rod-shaped bacterium recovered from human clinical specimens were characterised by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed the unknown rod-shaped strains are members of the same species as some fastidious isolates recovered from human blood specimens and previously designated "Leptotrichia sanguinegens". Based on phylogenetic and phenotypic evidence, it is proposed that the isolates from human sources be classified in a new genus Sneathia, as Sneathia sanguinegens gen. nov., sp. nov. The type strain of Sneathia sanguinegens is CCUG 41628T.

Effect of blood contamination on the shear bond strength at resin/dentin interface in primary teeth

Purpose: To assess in vitro the shear bond strength at the resin/dentin interface in primary teeth after contamination with fresh human blood. Methods: 75 crowns of primary molars were embedded in acrylic resin and mechanically ground to expose a flat dentin surface. The specimens were randomly assigned to five groups (n=15), according to the surface treatment. Group I (control) had no blood contamination. The other groups were blood-contaminated and subjected to different post-contamination protocols: in Group 2, the surfaces were rinsed with water; in Group 3, the surfaces were air-dried; in Group 4, the surfaces were rinsed and air-dried; and in Group 5, no post-contamination treatment was done. In all groups, a 3-mm dentin bonding site was demarcated, Single Bond adhesive system was applied and resin composite cylinders were bonded. After 24 hours in distilled water, shear bond strength was tested at a crosshead speed of 0.5 mm/minute. Results: Means (in MPa) were: Group 1: 7.1 (+/- 4.2); Group 2: 4.0 (+/- 1.8); Group 3: 0.9 (+/- 0.7); Group 4: 3.9 (+/- 2.2) and Group 5: 1.3 (+/- 1.5). Data were analyzed statistically by the Kruskal-Wallis test at 5% significance level. Groups 2 and 4 were similar to each other (P > 0.05) and both ware similar to Group 1 (P > 0.05). These groups (2, 3 and 4) had statistically significantly higher bond strengths than Groups 3 and 5 (P < 0.05). Blood contamination negatively affected the shear bond strength to primary tooth dentin. Among the blood-contaminated groups, water-rinsed specimens had higher bond strengths than those that were exclusively air-dried or not submitted to any post-contamination protocol before adhesive application.

Validation of a novel ELISA for measurement of electronegative low-density lipoprotein

Background: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. LDL(-) is present in blood plasma of healthy subjects and at higher concentrations in diseases with high cardiovascular risk, such as familial hypercholesterolemia or diabetes. Methods: We developed and validated a sandwich ELISA for LDL(-) in human plasma using two monoclonal antibodies against LDL(-) that do not bind to native LDL, extensively copper-oxidized LDL or malondialdehyde-modified LDL. The characteristics of assay performance, such as limits of detection and quantification, accuracy, inter- and intra-assay precision were evaluated. The linearity, interferences and stability tests were also performed. Results: The calibration range of the assay is 0.625-20.0 mU/L at 1: 2000 sample dilution. ELISA validation showed intra- and inter- assay precision and recovery within the required limits for immunoassays. The limits of detection and quantification were 0.423 mU/L and 0.517 mU/L LDL(-), respectively. The intra- and inter- assay coefficient of variation ranged from 9.5% to 11.5% and from 11.3% to 18.9%, respectively. Recovery of LDL(-) ranged from 92.8% to 105.1%. Conclusions: This ELISA represents a very practical tool for measuring LDL(-) in human blood for widespread research and clinical sample use. Clin Chem Lab Med 2008; 46: 1769-75.

Investigation of whole blood of SJL/J mice using neutron activation analysis

The Br (0.0022 +/- A 0.0006 gL(-1)), Ca (0.113 +/- A 0.012 gL(-1)), Cl (3.07 +/- A 0.36 gL(-1)), K (2.63 +/- A 0.14 gL(-1)), Mg (0.045 +/- A 0.002 gL(-1)) and Na (2.09 +/- A 0.10 gL(-1)) concentrations were determined in whole blood of SJL/J mice using the Neutron Activation Analysis (NAA) technique. Eleven whole blood samples were analyzed in the IEA-R1 nuclear reactor at IPEN (So Paulo, Brazil). These data contribute for applications in veterinary medicine related to biochemistry analyses using whole blood. Moreover, the correlation with human blood estimation allows to checking the similarities for studying muscular dystrophy using this model animal.

Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C 18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 mu m of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 103 less sample than using conventional methods). (C) 2008 Elsevier B.V. All rights reserved.

Estudo in silico da interação da albumina de soro humano com o ibuprofeno

Currently, computational methods have been increasingly used to aid in the characterization of molecular biological systems, especially when they relevant to human health. Ibuprofen is a nonsteroidal antiinflammatory or broadband use in the clinic. Once in the bloodstream, most of ibuprofen is linked to human serum albumin, the major protein of blood plasma, decreasing its bioavailability and requiring larger doses to produce its antiinflamatory action. This study aimes to characterize, through the interaction energy, how is the binding of ibuprofen to albumin and to establish what are the main amino acids and molecular interactions involved in the process. For this purpouse, it was conducted an in silico study, by using quantum mechanical calculations based on Density Functional Theory (DFT), with Generalized Gradient approximation (GGA) to describe the effects of exchange and correlation. The interaction energy of each amino acid belonging to the binding site to the ligand was calculated the using the method of molecular fragmentation with conjugated caps (MFCC). Besides energy, we calculated the distances, types of molecular interactions and atomic groups involved. The theoretical models used were satisfactory and show a more accurate description when the dielectric constant ε = 40 was used. The findings corroborate the literature in which the Sudlow site I (I-FA3) is the primary binding site and the site I-FA6 as secondary site. However, it differs in identifying the most important amino acids, which by interaction energy, in order of decreasing energy, are: Arg410, Lys414, Ser 489, Leu453 and Tyr411 to the I-Site FA3 and Leu481, Ser480, Lys351, Val482 and Arg209 to the site I-FA6. The quantification of interaction energy and description of the most important amino acids opens new avenues for studies aiming at manipulating the structure of ibuprofen, in order to decrease its interaction with albumin, and consequently increase its distribution

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Levels of selected persistent organic pollutants in blood from delivering women in seven selected areas of São Paulo State, Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ex vivo evaluation of the effectiveness of bleaching agents on the shade alteration of blood-stained teeth

Aim To evaluate ex vivo effectiveness of the three formulations of bleaching materials for intracoronal bleaching of root filled teeth using the walking bleach technique.Methodology Extracted premolar teeth were stained artificially with human blood. After biomechanical preparation, the root canals were filled and a 3-mm thick intermediate base of zinc phosphate cement was placed at the level of the cementoenamel junction. The teeth were divided into four groups (n = 12): C (control, without bleaching material), A1 (sodium perborate + distilled water), A2 (sodium perborate + 10% carbamide peroxide) and A3 (sodium perborate + 35% carbamide peroxide). The bleaching materials were changed at 7 and 14 days. Evaluation of shade was undertaken with aid of the VITA Easyshade (TM) (Delta E*ab) and was performed after tooth staining and at 7, 14 and 21 days after bleaching, based on the CIELAB system. Data were analysed by ANOVA for repeated measurements, Tukey and Dunnett tests (alpha = 0.05).Results The Tukey test revealed that group A1 (10.58 +/- 4.83 Delta E*ab) was statistically different from the others (A2, 19.57 +/- 4.72 Delta E*ab and A3, 17.58 +/- 3.33 Delta E*ab), which were not different from each other. At 7 days: A1 was significantly different from A2; at 14 and 21 days: A2 and A3 were significantly better than A1; the Dunnett test revealed that the control group was different from A1, A2 and A3 at all periods (P < 0.05).Conclusion Sodium perborate associated with both 10% and 35% carbamide peroxide was more effective than when associated with distilled water.

Potentiometric Sensor for Furosemide Determination in Pharmaceuticals, Urine, Blood Serum and Bovine Milk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

Background: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse. © 2013 Pereira et al.

Identificação e análise de polimorfismos no gene que codifica a Lectina ligante de Manose (MBL) na espécie Canis lúpus familiaris

Pós-graduação em Biologia Geral e Aplicada - IBB

Enxertos venosos ao avesso e normal, preenchidos com plasma rico em plaquetas em nervo misto de rato

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)