BIOORGANIC CONCEPTS INVOLVED IN THE DETERMINATION OF GLUCOSE, CHOLESTEROL AND TRIGLYCERIDES IN PLASMA USING THE ENZYMATIC COLORIMETRIC METHOD

Bioorganic and biological chemistry have been found to be highly motivating to undergraduate students and in this context, biochemical blood parameter analysis emerges as highly attractive content. In this proposal, several aspects related to analyses of glucose, cholesterol and triglycerides using the enzymatic colorimetric method were involved, and the findings have at least two relevant implications: i) introducing students to connections between organic chemistry and biology based on enzymatic processes, including reactivity and mechanistic aspects; ii) performing a micro scale bioassay analysis. The proposal requires two theoretical classes (2 h per class) and one practical class (4 h).

The azomethine-H modified colorimetric method of boron determination at fertilizers.

The present work studied the azomethine-H colorimetric method for boron determination to fertilizers analysis applications. The reagent azomethine-H needs lights conditions of reaction that, jointed to the big sensibility and specificity, diffused its use in boron's dosage in many materials. The most suitable experimental parameters were established for such colorimetry, and analysis conditions: standard curve between 0,200 e 2,25 mg. L(-1) boron, in maximum absorption at 415nm, I cm cell and reading between 30 and 90 minutes standing after the final homogenization of the boron's solution in analyse and with dye reactive (azomethine If 0,90 % m/v solution at buffer ammonium acetate 1,82 mol . L(-1) - potassium acetate 0,10 mol . L(-1) - acetic acid 1,67 mol . L(-1)-EDTA 2,7 . 10(-2) mol . L(-1)-NTA 2,1.10(-2) mol . L(-1) pH 5,5). Statistics analysis of the results didn't present big differences when the same results were got by the colorimetric method offered and by the potentiometric, from AOAG, whatever in manufactured fertilizers or in lab sintetized fertilizers.

Adapted colorimetric method for measurement of feline urinary glycosaminoglycans

Several methods have been employed to quantify urinary glycosaminoglycans (GAGs), such as chromatography associated with electrophoresis and colorimetric methods, cheaper and faster ones, which employ mainly azure A and B, alcian blue, and dimethylmethylene blue (DMB). The purpose of this study was to standardize a reproducible and cheap method to measure total urinary GAGs in feline urine. Two colorimetric methods based on DMB were tested with chondroitin sulfate C as standard. Urine samples were obtained from 12 healthy cats and some modifications were made for the chosen method to be adequate. The modified technique using DMB acetate buffer carried out in this study can be used to measure feline urinary GAGs. © 2012 Springer-Verlag London.

Screening the toxicity and biodegradability of petroleum hydrocarbons by a rapid colorimetric method

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Field evaluation of a novel colorimetric method - Double-site enzyme-linked lactate dehydrogenase immunodetection assay - To determine drug susceptibilities of Plasmodium falciparum clinical isolates from northwestern Thailand

A double-site enzyme-linked lactate dehydrogenase enzyme inummodetection assay was tested against field isolates of Plasmodium falciparum for assessing in vitro drug susceptibilities to a wide range of antimalarial drugs. Its sensitivity allowed the use of parasite densities as low as 200 parasites/mul of blood. Being a nonisotopic, colorimetric assay, it lies within the capabilities of a modest laboratory at the district level.

A simplified, sequential, phosphorus fractionation method

Hedley er al. (1982) developed what has become the most widely used land modified), phosphorus (P) fractionation technique. It consists of sequential extraction of increasingly less phytoavailable P pools. Extracts are centrifuged at up to 25000 g (RCF) and filtered to 0.45 mu m to ensure that soil is not lost between extractions. In attempting to transfer this method to laboratories with limited facilities, it was considered that access to high-speed centrifuges, and the cost of frequent filtration may prevent adoption of this P fractionation technique. The modified method presented here was developed to simplify methodology, reduce cost, and therefore increase accessibility of P fractionation technology. It provides quantitative recovery of soil between extractions, using low speed centrifugation without filtration. This is achieved by increasing the ionic strength of dilute extracts, through the addition of NaCl, to flocculate clay particles. Addition of NaCl does not change the amount of P extracted. Flocculation with low speed centrifugation produced extracts comparable with those having undergone filtration (0.025 mu m). A malachite green colorimetric method was adopted for inorganic P determination, as this simple manual method provides high sensitivity with negligible interference from other anions. This approach can also be used for total P following digestion, alternatively non-discriminatory methods, such as inductively coupled plasma atomic emission spectroscopy, may be employed.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

Colorimetric enzymatic assay of L-malic acid using dehydrogenase from baker's yeast

A colorimetric method has been developed and optimized to measure L-malic acid in samples of fruit juices and wine. This method is based on oxidation of the analyte, catalyzed by malate dehydrogenase (MDH) from dry baker's yeast, and in combination with the reduction of a tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). In the present study, the method exhibited sensitivity in the range of 500-4000 mu M of L-malic acid in the reaction cuvette, with the lower detection limit of 6.7-10(-2) g/L, the upper limit of 53.6.10(-2) g/L and a maximum standard deviation of only 2.5 % for the analyzed samples. The MDH activity from baker's yeast was also optimized, the enzyme showed a high stability at pH=8.0-9.0 and the activity was maintained completely at temperatures up to 40 degrees C for 1 hour. The results show that the colorimetric method using enzymatic preparations from dry baker's yeast is a simple and low-cost method with possibility of wide application.

A Method for Determination of Ammonia in Air using Oxalic Acid-Impregnated Cellulose Filters and Fluorimetric Detection

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Colorimetric Assessment of BCR-ABL1 Transcripts in Clinical Samples Via Gold Nanoprobes

Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

Masticatory afficiency in denture wearers with bilateral balanced occlusion and canine guidance

The maintenance of masticatory function is especially important for patients wearing complete dentures due to their limitations. Thus, the bilateral balanced occlusal concept is used to achieve greater masticatory efficiency. However, a critical review of the literature reveals that there is not sufficient scientific evidence to support bilateral balanced occlusion as the most appropriate occlusal concept in complete dentures. Therefore, the aim of this study was to evaluate the masticatory efficiency in complete dentures wearers with bilateral balanced occlusion and canine guidance. A double-blinded controlled crossover clinical trial was conducted. The sample was composed by 24 edentulous patients who wore sets of complete dentures with both occlusal concepts during equal periods of 3 months. Objective data were collected through the masticatory efficiency test performed by the colorimetric method with the beads, in which capsules of a synthetic material enclosing fuchsine-containing granules were used. Subjective data were recorded by patient's ratings of their chewing function. No significant statistical difference was found for masticatory efficiency (p=0.095) between the two occlusal concepts studied. The results suggest that bilateral balanced occlusion does not improve the masticatory efficiency in complete denture wearers.

Confiabilidade da eficiência mastigatória com beads e correlação com a atividade muscular

TEMA: a avaliação da eficiência mastigatória pela análise colorimétrica com beads, pode ser um método promissor, mas não há relatos sobre a sua confiabilidade. OBJETIVO: investigar a confiabiabilidade das beads para teste de eficiência mastigatória e a correlação com a atividade eletromiográfica dos músculos masseter e temporal anterior. MÉTODO: participaram dezenove sujeitos adultos jovens, nove do gênero masculino e dez do feminino com idades entre dezoito e vinte-oito anos, com dentição completa, sem histórico de desordem temporomandibular, trauma, cirurgia na região de cabeça e pescoço, tratamento ortodôntico ou fonoaudiológico. O teste de eficiência mastigatória foi realizado com beads nas condições: mastigação habitual, mastigação unilateral direita e esquerda, com duração de 20 segundos. Simultaneamente, foi realizada a eletromiografia. A atividade em máxima intercuspidação habitual dos dentes também foi registrada. A quantidade de fucsina liberada após a mastigação foi medida usando o espectrofotômetro Beckman DU-7 UV-Visible (Beckman Inc., Palo Alto, CA, USA). RESULTADOS: houve alta confiabilidade do teste de eficiência mastigatória (r = 0,86, p < 0,01) e correlação significante com a atividade eletromiográfica (r = 0,76, p < 0,01). Também houve correlações positivas quando as provas foram analisadas separadamente. CONCLUSÃO: o teste de eficiência mastigatória realizado com beads mostrou-se um método confiável e correlacionado positivamente à atividade eletromiográfica dos músculos temporal anterior e músculos masseter.

Influence of alpha-tocopherol on the levels of serum anti-oxLDL antibodies

Purpose - This paper aims to evaluate the association between the a-tocopherol with the levels of serum anti-oxLDL autoantibodies and the risk markers for cardiovascular disease. Design/methodology/approach - A normolipidemic control group (n=30) and a hypercholesterolemic group (n=33) were used. Plasma lipid profile (colorimetric method), anti-oxLDL autoantibodies (ELISA) and a-tocopherol (HPLC) were analysed. Findings - The a-tocopherol (ß=-0.714; p=0.001) is negatively associated with anti-oxLDL autoantibodies in serum and with other risk markers for cardiovascular disease (BMI, WC, total cholesterol, LDL-c) and positively associated with HDL-c. Originality/value - Oxidized low density lipoprotein (oxLDL) and their autoantibodies are increased in subjects with hypercholesterolemia. The a-tocopherol can influence the levels of serum anti-oxLDL autoantibodies

Capacity of the extracellular matrix of the bone marrow to induce proliferation of myeloid cells in vitro in model of protein malnutrition in mice

The aim of this study was to verify the capacity of the extracellular matrix (ECM) obtained from bone marrow of malnourished mice to sustain survival and to induce the proliferation of myeloid cells. We also verified the capacity of the tests to interact with in vitro hematopoietic cytokines. Male ""Swiss"" mice were submitted to protein malnutrition with a diet contents of 4% casein until they lost 20% of the original weight, while the group-control was kept with a diet content of 14% of casein. The bone marrow was extracted with 1.0 mg of aprotinin/mL in PBS. The proliferation tests were carried out with myeloid cell line FDCP-1, by the colorimetric method of reduction of the MTT. The obtained ECM from nourished and undernourished mice induced cellular proliferation in vitro. Tests performed with Il-3 and GM-CSF cytokines in a concentration of 10 and 500 rho g/mL displayed synergic and regulatory effects respectively. The ECM obtained from the malnourished group submitted to the binding to GM-CSF demonstrated higher cellular proliferation than the ECM obtained from the control group (p<0.05). The results suggest that the alterations in the composition of ECM of bone marrow caused by malnutrition might lead to modification of the GM-CSF activity modulation.