Novel fluorometric protocol for assessing myeloperoxidase activity.

Neutrophil myeloperoxidase (MPO) is an essential enzyme for the innate immune system. Measuring MPO activity is vital for understanding neutrophil characteristics and functions in various diseases. MPO activity can be measured using several methods, including spectrophotometric and fluorometric protocols. This paper introduces a fluorometric method for specifically quantifying MPO activity based on the H2O2-dependent oxidation of thiamine. We optimized this new method using the robust statistical approach response surface methodology (RSM) and Box Benken Design (BBD). We extensively examined the effects of several experimental parameters using the RSM methodology and determined the best conditions for accurate and sensitive MPO activity measurement. The optimal conditions were determined using the analysis of variance (ANOVA) for second-order polynomial equations. The resulting F-value (4.86) indicated that the model was significant. However, the lack-of-fitness F-value (1.79) suggested it did not differ significantly from the corresponding p-value. The greatest MPO activity (30 ± 2 U L-1) was obtained under optimum conditions, which were 1000 µM of H2O2, 10 min incubation time, and 1000 µM of thiamine. Our results suggest that this advanced fluorometric method has significant accuracy, sensitivity, and linearity up to 60 IU. The new and standard colorimetric methods also showed a good correlation. These results indicate that the new fluorometric method can be dependable and efficient for assessing MPO activity. The new method is characterized by excellent accuracy, sensitivity, and linearity, making it a valuable protocol for researchers and clinicians interested in assessing MPO activity.

An optimized method for estimating glutaminase activity in biological samples.

Spectrophotometric methodologies have been used to assess glutaminase activity, for which coloured complexes have been developed that measure spectrophotometry across the visible spectrum using different reagents. The present paper describes a precise, simple and reliable procedure for quantifying glutaminase activity, which is a key enzyme in glutamine hydrolysis and also involved in glutamine metabolism regulation. The procedure presented here measures glutaminase activity by incubating glutaminase enzyme at 37 °C for 20 min with a glutamine substrate dissolved in a buffer (pH 8.6). The enzymatic reaction contains suitable activity of glutamate oxidase, which acts to convert glutamate to hydrogen peroxide and 2-oxoglutarate. To terminate the enzymatic activity, a working solution containing pyridine-2,6-dicarboxylic (PDA) acid and ammonium vanadate (AV) was added following incubation. Oxo-peroxo-pyridine-2,6-dicarboxylato-vanadate (OPDV), a stable orange-coloured chelate complex measuring 435 nm spectrophotometrically, was produced by the interaction between the generated hydrogen peroxide and the supplied reagent. Using the response surface methodology (RSM) as an indicator of the assay's accuracy, we employed the Box-Behnken design (BBD) to improve the method's design (the OPDV-Glutaminase assay). Improvement factors were the volume of working reagent solution (PDA/AV), volume of glutamate oxidase solution (GO), and incubation time. In matched samples, this novel method was verified against a Bland-Altman plot assessment of glutaminase activity using the indophenol methodology. A correlation value of 0.99 between the two methods' comparisons showed that the novel protocol was equally applicable to the reference method.