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Journal Article 2016; 37(Suppl 1): 139-143 PubMed PMID: 28263542 Citation  Keywords:  Antioxidants:analysis, Biological Assay:methods, Biomarkers, Colorimetry, Dithionitrobenzoic Acid, Glutathione:analysis, Humans, Smartphone:utilization,.   

OBJECTIVES: Glutathione belongs to the family of small-molecular weight antioxidants like ascorbic acid, cysteine, α-tocopherol, uric acid, etc. These molecules play important role in the neutralization of free radicals and reactive oxygen species (ROS). Oxidative stress may lead to ageing and the development of large scale of pathological states of organism. This low molecular weight antioxidant´s level can alter under pathological conditions from reduced (GSH, thiols) to oxidized (oxidized glutathione -GSSG, disulfides) form. A GSSG-to-GSH ratio is indicative marker of oxidative stress. There is a large scale of methods how to determine this biomarker. The trend of the analysis is to minimalize the instrument equipment, sample application volume and analysis cost.

DESIGN: Reduced glutathione (GSH) solutions were prepared in water in the concentration 0-16 mmol/L. Other small-molecular weight antioxidants like 0.25 mmol/L ascorbic acid, 0.15 mmol/L TROLOX and 0.02 mmol/L N-acetyl-cysteine (NAcCys) were studied as possible interferents. The samples were mixed with 5,5´-dithiobis-(2-nitrobenzoic) acid (DTNB) resulting in yellow colored drops forming. Coloration was assayed using camera integrated in a smartphone and color channels analysis. The total volume of 10 µl of sample was applied for one analysis. The smartphone-based data were compared with the reference Ellman assay.

RESULTS: The calibration of glutathione was evaluated. The blue channel intensity data were decreasing according to the increasing glutathione concentration. Red and green channel intensities were stagnating during the whole concentration scale of glutathione. Limits of detection were 0.4 mmol/l for glutathione. Addition of 0.25 mmol/L of ascorbic acid, 0.15 mmol/L of TROLOX and 0.02mmol/L of N-acetylcysteine to GSH in final concentration 0-16 mmol/L had minimal influence on the assay. The results from smartphone-based analysis correlate with the standard Ellman method. The detection limit for GSH was 0.03 mmol/L.

CONCLUSION: The smartphone-based assay seems to be promising because of simplicity, reliability, robustness and low cost. In spite of the fact that there is a large scale for approaches for the glutathione determination, the main advantage of our colorimetric method is portability and easibility to perform the assay in the field and publically availability of smartphones for home applications.

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Journal Article 2016; 37(Suppl 1): 132-138 PubMed PMID: 28263541 Citation  Keywords:  Biological Assay:methods, Biosensing Techniques:methods, Blood Glucose:metabolism, Enzymes, Immobilized:metabolism, Glucose Oxidase:metabolism, Horseradish Peroxidase:metabolism, Humans, Smartphone:utilization,.   

OBJECTIVES: Glucose is an important diagnostic biochemical marker of diabetes but also for organophosphates, carbamates, acetaminophens or salicylates poisoning. Hence, innovation of accurate and fast detection assay is still one of priorities in biomedical research.

METHODS: Glucose sensor based on magnetic particles (MPs) with immobilized enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) was developed and the GOx catalyzed reaction was visualized by a smart-phone-integrated camera.

RESULTS: Exponential decay concentration curve with correlation coefficient 0.997 and with limit of detection 0.4 mmol/l was achieved. Interfering and matrix substances were measured due to possibility of assay influencing and no effect of the tested substances was observed. Spiked plasma samples were also measured and no influence of plasma matrix on the assay was proved.

CONCLUSIONS: The presented assay showed complying results with reference method (standard spectrophotometry based on enzymes glucose oxidase and peroxidase inside plastic cuvettes) with linear dependence and correlation coefficient 0.999 in concentration range between 0 and 4 mmol/l. On the grounds of measured results, method was considered as highly specific, accurate and fast assay for detection of glucose.

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Journal Article 2016; 37(Suppl 1): 17-24 PubMed PMID: 28263526 Citation  Keywords:  Animals, Biological Assay:methods, Environmental Monitoring, Fishes, Toxicity Tests:methods, Waste Water:toxicity, Water Pollutants, Chemical:toxicity,.   

: Wastewater, especially containing hospital effluents, exhibits high chemical complexity and specificity since it includes various chemicals, biocides, pharmaceuticals, surfactants, radionuclides, disinfectants and pathogens. Biological tests provide true evidence of the wastewater quality and unlike chemical analytical tests show comprehensive pollution effects on the environment and human health. Normalized conventional bioassays are not sensitive enough for ecotoxicological evaluation of wastewater and there is a great need for the development of suitable sensitive bioassays in order to characterize properly the residual toxicity of treated effluents. Provisions of binding EU legislation regarding protection of animals used for scientific purposes and legislation dealing with test methods for identification and classification of health hazard of chemicals, pharmaceuticals, biocides, medical devices and consumer products such as cosmetics for environmental ecosystems and for man require to employ alternative toxicological methods respecting the 3Rs concept with priority given to methods in vitro. The Fish Embryo Test (FET) is identified as a relevant, reliable and efficient alternative test method in vitro for determination of acute toxicity for fish. Using the FET, additional toxicological endpoints may be investigated to assess organ specific bioaccumulation, genotoxicity and mutagenicity, developmental toxicity, teratogenicity, various forms of neurotoxicity or endocrine disruptivity. The addition of multiparametric sensitive endpoints makes the FET a true alternative in vitro assay and a powerful tool in toxicology.

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Journal Article 2007; 28(4): 382-385 PubMed PMID: 17693986 Citation  Keywords:  Animals, Biological Assay:methods, Cells, Cultured, Dose-Response Relationship, Drug, Enzyme Stability:physiology, Growth Hormone:metabolism, Growth Hormone-Releasing Hormone:analogs & derivatives, Male, Pituitary Gland:cytology, Rats, Rats, Inbred WKY,.   

OBJECTIVE: It has been found that hGH-RH analogues with increased resistance to enzymatic degradation have a much higher potency than the native hGH-RH (1-29)-NH2 and have an ability to partially reverse growth hormone deficiencies.

THE AIM: The aim of these studies was to elaborate a method which can be used for preliminary evaluation of new GH-RH analogues both from the point of view of their potency to release GH and enzymatic stability.

METHOD: Two highly active GH-RH analogues with increased resistance to trypsin-like enzymes, and hGH-RH(1-29)-NH2 used as a standard, in doses 1 nM, 10 nM, and 100 nM were added to pituitary rat cell culture, and medium was collected after 30, 60, 120 and 240 min. GH concentration was measured by RIA kit.

RESULTS: It was observed that the potency of these two GH-RH analogues was several times higher than that of native compound. Moreover, the stimulation was much longer. This suggests that high activity of these analogues in vivo could be the result of increased enzymatic stability.

CONCLUSION: This method can be used for selecting more potent and more stable releasing peptides before in vivo evaluation....

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