Novel fluorometric protocol for assessing myeloperoxidase activity

• Published in: Enzyme and microbial technology 2023-12, Vol.171, p.110320-110320, Article 110320
• Main Authors: Majid, Rawa, Al Talebi, Zainab Abbas, Al-Kawaz, Hawraa Saad, Hassan Alta'ee, Abdulsamie, Alsalman, Abdul Razzaq S., Hadwan, Asad M., Hadwan, Muntadhar M., Hadwan, Mahmoud Hussein
• Format: Article
• Language: English
• Subjects: Bland-Altman plot; Myeloperoxidase; Response surface methodology; Spectrofluorometric assessment; Thiamine
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2023.110320

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. [Display omitted] •Myeloperoxidase enzyme activity in biological samples was studied using a novel fluorometric method.•The diagnostic method has been validated on biological samples as part of the validation process.•The method may be utilized professionally and in research investigations.