Chemiluminescent determination of leukocyte alkaline phosphatase: An advantageous alternative to the cytochemical assay

The determination of leukocyte alkaline phosphatasd (LAP) is used as an aid to diagnose many diseases in the laboratory. For example, it can be used to distinguish chronic myeloid leukemia (CML) from other myeloproliferative disorders (particularly myelofibrosis and polycythemia) and leukemoid reactions (LR). Traditionally, this test is performed with the use of subjective cytochemical assays that assign a score to the level of LAP. Here we present a nonsubjective, quantitative, sensitive, and inexpensive chemiluminescent technique that determines LAP based on the commercial reagent Immulite (R) (AMPPD). To validate this methodology, intact leukocytes obtained from 32 healthy subjects, nine CML patients, and nine LR patients were submitted to the optimized protocol. By measuring the light emission elicited by four concentrations of neutrophils, we were able to estimate the activity of LAP per cell (the slope of the curve obtained by linear regression). A high linear correlation was found between the chemiluminescent result (slope) and the cytochemical score. The slope for healthy individuals ranged between 0.61 and 8.49 (10(-5) mV.s/cell), with a median of 2.04 (10(-5) mV.s/cell). These results were statistically different from those of CML patients (range = 0.07-1.75, median = 0.79) and LR patients (range = 3.84-47.24, median 9.58; P < 0.05).

Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay

In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H2O2 system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-scruin-HRP-H2O2 System consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at less than or equal to4 degreesC. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O-2 uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta -methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyryleholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme. Copyright (C) 2001 John Wiley & Sons, Ltd.

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.

Disfunção sexual feminina e níveis dos hormônios esteroidais em mulheres obesas atendidas no ambulatório de cirurgia bariátrica do hospital universitário Onofre Lopes em Natal/RN

A disfunção sexual corresponde a alterações em uma ou mais fases da resposta sexual humana e apresenta maior prevalência na população feminina. Ademais, a participação de alguns fatores como obesidade e níveis dos hormônios esteroidais na disfunção sexual feminina (DSF) permanece incerta. O presente estudo deteve-se na análise da ocorrência de DSF numa população de mulheres portadoras de obesidade, cadastradas no Ambulatório de Cirurgia Bariátrica do Hospital Universitário Onofre Lopes, da Universidade Federal do Rio Grande do Norte, no município de Natal, RN. O estudo foi realizado em uma amostra composta por trinta e uma mulheres, com idade entre 20 e 50 anos, com índice de massa corpórea (IMC) > 30 Kg/m2. A todas as pacientes foi aplicado um questionário composto por uma seção com dados socio-econômicos, e outra abordando a saúde sexual feminina, sendo esta última correspondente ao Female Sexual Function Index (FSFI), para diagnóstico de DSF. A partir dessa caracterização, as pacientes foram reunidas nos grupos CD (pacientes com disfunção, n= 9) e SD (sem disfunção, n= 22). Para a análise do efeito da obesidade na DSF, as pacientes foram reunidas nos grupos 1 (6 pacientes com IMC grau I e II: entre 30 e 40 Kg/m2) e 2 (25 com IMC grau III: acima de 40). Para o estudo da participação dos hormônios esteroidais foram determinadas as concentrações séricas de cortisol, estradiol e dehidroepiandrosterona (DHEA) pelo método de quimiluminescência. A análise estatística dos dados foi realizada usando os testes ANOVA, MANOVA (Pillai), além de análise de Cluster. Para identificar as diferenças entre os domínios do FSFI, foi usado o teste T de Student. A significância considerada para todos os testes foi para p< 0,01. Das pacientes estudadas, 25,8% apresentaram DSF de acordo com o escore total do FSFI. A análise estatística posterior evidenciou que as diferenças ocorreram para os domínios desejo, excitação e orgasmo. Não foi encontrada relação da presença de DSF com os diferentes graus de obesidade ou com os níveis hormonais dos esteróides cortisol, estradiol ou DHEA. Contudo, foi encontrado aumento significativo nos níveis séricos de estradiol para o grupo 1, que corresponde ao de menor índice de IMC. Estes resultados mostram que a prevalência de DSF não diferiu entre os graus I,II e III de obesidade das pacientes deste estudo mas, quando presente, a disfunção ocorre nos domínios desejo, excitação e orgasmo. A maior concentração de estradiol encontrada nas pacientes de menor índice de IMC sugere uma possível relação entre as duas variáveis que precisa ser investigada em estudos futuros.

Decreased oxidative stress in patients with ulcerative colitis supplemented with fish oil omega-3 fatty acids

OBJECTIVE: the potential pathogenicity of free radicals may have a pivotal role in ulcerative colitis. Fish oil omega-3 fatty acids exert anti-inflammatory effects on patients with ulcerative colitis (UC), but the precise mechanism of the action of fish oil on oxidative stress is still controversial. The aim of the present work was to verify the blood oxidative stress in patients with UC and determine whether the association of sulfasalazine to fish oil omega-3 fatty acids is more effective than isolated use of sulfasalazine to reduce the oxidative stress.METHODS:, Nine patients (seven female and two male; me. an age = 40 +/- 11 y) with mild or moderate active UC were studied in a randomized crossover design. In addition to their usual medication (2 g/d of sulfasalazine), they received fish oil omega-3 fatty acids (4.5 g/d) or placebo for 2-mo treatment periods that were separated by 2 mo, when they only received sulfasalazine. Nine healthy individuals served as control subjects to study the oxidative stress status. Disease activity was assessed by laboratory indicators (C-reactive protein, alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, erythrocyte sedimentation rate, albumin, hemoglobin, and platelet count), sigmoidoscopy, and histology scores. Analysis of oxidative stress was assessed by plasma chemiluminescence and erythrocyte lipid peroxidation, both induced by tert butyl hydroperoxide (t-BuOOH) and by plasma malondialdehyde. Antioxidant status was assayed by total plasma antioxidant capacity (TRAP) and microsomal lipid peroxidation inhibition (LPI). Superoxide dismutase (SOD) and catalase erythrocyte enzymatic activities were also determined.RESULTS: No significant changes were observed in any laboratory indicator or in the sigmoidoscopy or histology scores, with the exception of erythrocyte sedimentation rate, which decreased with both treatments. Oxidative stress was demonstrated by significant decreases in TRAP and LPI levels, increased chemiluminescence induced by t-BuOOH, and higher SOD activity in patients with UC. Treatment with fish oil omega-3 fatty acids reverted the chemiluminescence induced by t-BuOOH and LPI to baseline levels but that did not occur when patients received only sulfasalazine. Levels of plasma malondialdehyde, erythrocyte lipid peroxidation, and catalase were not different from those in the control group.CONCLUSIONS: the results indicated that plasma oxidative stress occurs in patients with UC, and there was a significant decrease when the patients used sulfasalazine plus fish oil omega-3 fatty acids. However, there was no improvement in most laboratory indicators, sigmoidoscopy, and histology scores. The results suggested that omega-3 fatty acids may act as free radical scavengers protecting the patients against the overall effect of oxidative stress. (C)Elsevier B.V. 2003.

Imunossensor amperométrico

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

Microscopia de força atômica aplicada em imunoensaios

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

Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au-cystamine-glutaraldehyde-lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 +/- 0.2) X 10(4) M-1 for Jacalin-Fetuin and (4.5 +/- 0.1) x 10(2) M-1 for ConA-maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin-carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides. Published by Elsevier B.V.

Biossensores baseados no processo de inibição enzimática

Fast, selective, reproducible and reliable detections have been carried out by using enzymatic biosensors in several areas. The enzymatic biosensors based on the inhibition represent an important role in analytical chemistry. Enzymes like cholinesterases, peroxidases, tyrosinases, etc. have been immobilized on electrochemical and optical transducers and the enzymatic activity decreasing in the presence of the inhibitor is related with its concentrations. This article presents a review on the enzymes used on the construction of these sensors, emphasizing the respective applications.

Study of the electrochemical reduction of amoebicide Teclozan and its amperometric determination in pharmaceutical formulations

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Flow injection analysis of paracetamol using a biomimetic sensor as a sensitive and selective amperometric detector

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

Aplicação e avanços da espectroscopia de luminescência em análises farmacêuticas

This paper provides a review on the latest advances and applications of the luminescence spectroscopy for the development of pharmaceuticals analyses methods, basically based on the photo- and chemiluminescence. The different forms of the drugs determination on pharmaceuticals through the fluorescence and chemiluminescence are discussed. The analyses include the drugs native fluorescence (liquid and solid-phases); the fluorescence from the oxidizing or reducing forms of the drug; the fluorescence from the chemical derivatization and their photochemistry and hydrolysis reactions. The quenching of luminescence and chemiluminescence generation for the pharmaceutical quantification are also shown. Finally, the trends and future perspectives of the luminescence spectroscopy in the field of the pharmaceutical research are discussed.

Desenvolvimento e avaliação de eletrodos de difusão gasosa (EDG) para geração de H2O2 in situ e sua aplicação na degradação do corante reativo azul 19

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

Harmonical oscillator and electro-mechanical analogy: an interdiscinary experiment to high precision mass variation measurements

De forma geral, os cursos de física clássica oferecidos nas universidades carecem de exemplos de aplicações nas áreas de química e biologia, o que por vezes desmotivam os alunos de graduação destas áreas a estudarem os conceitos físicos desenvolvidos em sala de aula. Neste texto, a analogia entre os osciladores elétrico e mecânico é explorada visando possívies aplicações em química e biologia, mostrando-se de grande valia devido ao seu uso em técnicas de medição de variação de massa com alta precisão, tanto de forma direta como indireta. Estas técnicas são conhecidas como técnicas eletrogravimétricas e são de especial importância em aplicações que envolvem biossensores. Desta forma, o texto explora o estudo da analogia eletromecânica de forma interdisciplinar envolvendo as áreas de física, química e biologia. Baseado nessa analogia é proposto um experimento que permite a sua aplicação em diferentes níveis conceituais dessas disciplinas, tanto em abordagem básica como mais profunda.

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

Objective-To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils.Sample Population-Blood collected from 5 healthy adult Holstein cows.Procedure-Isolated neutrophils or whole brood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured.Results-incubation with TNF-alpha, IL-8; PAF, and IFS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils.Conclusions and Clinical Relevance-Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and setectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.