2.4.2. Coumarin Boronic Acid (CBA) Assay

To assess the oxidative potential of proteins in serum, we applied the real-time monitoring of HP formation, using the CBA-based assay, as previously described [21,26]. Briefly, 50 μL of serum sample was transferred to 96-microwell black plates and mixed with 150 μL phosphate buffer (50 mM, pH 7.4) containing catalase (100 units/mL), DTPA (diethylenetriaminepentaacetic acid; 0.1 mM), and non-fluorescent CBA as a substrate (0.8 mM). The fluorescence intensity of the reaction product, that is, the 4-hyroxycoumarin (COH), was measured (Ex/Em: 360 nm/465 nm) at 10 min intervals, using a plate reader (ClarioStar, BMG Labtech, Ortenberg, Germany), for 20 h. Each serum sample was analyzed in duplicates, and the arithmetic means presented the experiment's outcome. In each case, the proper background fluorescence was subtracted, and the sample fluorescence was adjusted to the total protein concentration in the serum.

The intensity of fluorescent product generation demonstrated exponential growth that was fitted to the logistic growth model. The resulting sigmoidal logistic growth curve and growth dynamics were described by the three parameters: (1) saturation level, 'K concentration', representing a numerical upper limit of growth; (2) rate factor, 'R', which describes the velocity of the fluorescent product growth; and (3) the area under the curve until saturation level, representing a cumulative generation of the CBA fluorescent product over time.
