Mathematical treatment of chemiluminescence data allowing an optimised kinetic analysis of vascular NAD(P)H-dependent superoxide anion production

Regulation of vascular redox homeostasis plays a central role in the control of vascular tone (e.g.: redox modulation of endothelial NO synthase and soluble guanylate cyclase activities). Superoxide anion is one of the main reactive oxygen species involved in the modulation of the redox status of the vessel. It is crucial to know if pathological situations or drug treatments are able to modify NAD(P)H oxidase-dependent production of O-2(-). As a steady-state approach is the only way for a valid assessment of these parameters, a mathematical treatment allowing visualisation of the linear portion of the initial velocity of O-2(-) production was developed. Using two vascular preparations (rat aortic rings and mouse thoracic aortae), the chemiluminescence (CL) in the presence of lucigenin was recorded every second in the 15-30 minute period after successive injections of NADPH and superoxide dismutase (SOD). Because, both O-2(-) and photons are labile and evanescent products, visualisation of the steady-state requires calculation of their cumulative production by integral calculus using a first order integral equation. The cumulative production of CL and/or O-2(-) was plotted as a function of the recording time. The initial velocities, as a function of the NADPH concentrations, were determined from the linear portion of these plots. For a precise molar quantification of O-2(-) production, a calibration curve of initial velocities using xanthine oxidase + xanthine as a source of O-2(-) was plotted by comparing CL and cytochrome C reduction. The NADPH-dependent CL production exhibited Michaelian behavior and SOD acted as a noncompetitive inhibitor. For example, the values for NADPH oxidase in rat aorta were: Km = 237 +/- 2 muM; Vmax 500-600 pmol O-2(-)/min/ring.