Metabolic reduction assay

The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure-activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Exploration of these SAR studies were undertaken to optimize inhibitory activity and to improve the physicochemical properties of the sialic acid-based influenza virus sialidase inhibitor. A few in vitro assays are commonly employed to measure the effectiveness of influenza virus sialidase inhibitors. The first involves a fluorometric assay that measures release of a synthetic fluorophore following its cleavage from Neu5Ac by sialidase. Dye-uptake assay, such as the Neutral Red uptake assay, measures the uptake of a vital stain, Neutral Red in cell culture. The process requires intact membranes and active metabolism in the cell, and is expressed as percent protective rate against virus infection. The plaque-reduction assay is used to measure sialidase inhibition indirectly in cell culture, and provides some measure of the inhibitor s effect on the viability of the influenza virus. In vitro and in vivo systems for analysis of inhibitors of influenza virus enzymes have been reviewed.71... 

One example is the known interference by reducing compounds that affect the chemical conversion of substrate to a colored indicator. This is especially true for the tetrazolium assays (Ulukaya, Colakogullari, and Wood 2004 Chakrabarti et al. 2000 Pagliacci et al. 1993 Collier and Pritsos 2003). The growing list of interfering compounds includes ascorbic acid and sulfhydryl reagents such as glutathione, coenzyme A, dithiothreitol, etc. Similar interferences by compounds that affect the oxidation and reduction chemistry of cells are likely to cause artifacts with the resazurin reduction assay. Assays that measure markers of metabolism also can be influenced by the pH of the culture medium and other factors that may stimulate or stress the metabolic rates of cells. 

In vitro quantitative antibacterial activity was evaluated using Alamar Blue Assay [56]. It is a colorimetric oxidation-reduction assay which involves the addition of Alamar blue dye as an indicator. It evaluates the metabolic activity of the microorganisms. The activity of the evaluated drug is expressed as minimum inhibitory concentration ( 0,g/ml). Sampangine, SAMM2 and SAM MMl were tested for their activity against Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 15442, Bacillus subtilus ATCC 6633, and E. coli ATCC 10536. Streptomycin was used as a positive control. 

Animal assays. When assessing the nature of a protein quality reduction, the conventional methods of protein quality measurement have certain limitations. For example, a reduced TD in an NPU assay is not necessarily the result of a reduced protein digestion. Obviously the same result will be obtained if the increase of fecal nitrogen is caused by an enhanced excretion of endogenous protein or if there is a fixation of metabolic nitrogen by the colonic micro-flora. 

MTT assay is a standard colorimetric assay used to determine cytotoxicity of potential medicinal agents and other toxic materials. It is based on the reduction of the tetrazolium salt MTT by viable cells. A mitochondrial dehydrogenase enzyme is able to cleave the tetrazolium rings of the pale yellow MTT and form dark purple formazan crystals, which are largely impermeable to cell membranes resulting in the accumulation of these crystals within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of crystals, which are solubilized. The metabolic activity of cells is directly proportional to the concentration of the created formazan product (22), whose color is quantified in a colorimetric assay. 

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