Tamás Kriska

Quantitative studies on the respiratory burst generated in peritoneal macrophages

The luminol-dependent chemiluminescence of macrophages during the zymosan-stimulated respiratory burst has been studied both in the absence and in the presence of the radical inhibitor 3,5 di-tert-butyl-4-hydroxyphenyl propionic acid. In addition, the consumption of luminol and of the inhibitor has been followed analytically. Based on the rates of the consumption of the inhibitor, an iteration procedure yields a value of 2.2 x 10(-7) M for the steady-state concentration of radicals generated by cells at the maximum of the chemiluminescence in the presence of inhibitor. Approximate calculations have indicated that under the experimental conditions applied, additional formation of superoxide anion radicals by the oxidation of luminol is negligible. By assuming that in an inhibitor-free system the disappearance of radicals takes place via their combination process as well as by their interaction with luminol and/or with luminol-derived species, numerical integration yields a calculated curve of radical concentration versus time in fair agreement with experimental data and a rate-constant value for the combination of radicals of approximately 10(6) M-1 s-1, supporting literature findings according to which primarily superoxide anion radicals are formed.

Quantification of the formation of free radicals in macrophage systems

A mechanism is suggested for the zymosan stimulated, luminol dependent chemiluminescence of macrophage system both in the absence and in the presence of free radical inhibitor. Based on the mechanism and relevant data of elementary rate constants computer simulation has been performed in order to describe the free radical formation and consumption, the kinetics of the chemiluminescence signal as well as the consumption of the inhibitor and luminol molecules in the course of the overall process.

Mechanism of in-vivo photodynamic effects

In order to compare the efficiency of the three different mechanisms suggested for photosensitization in biology and medicine: the sensitizer radical, (Type I), the singlet oxygen (Type II), and the native free radical-triplet sensitizer interaction (MTO mechanism) mediated effects, a possible mechanism is suggested including the primary steps of all three mechanisms. Simulation and sensitivity tests based on this mechanism have revealed: that (i) the main contributing elementary steps to the overall photodynamic effect are the decay of the triplet sensitizer; the interaction of sensitizer radicals with biomolecules and the interactions of native free radicals with biomolecules. (ii) The extent of the contribution of the latter is determined by the concentration of the native free radicals in the tissue preceding illumination. (iii) By changing the assumed rate constants of the triplet-doublet interactions it has been established, that with the decrease of their rate constants the overall photodynamic effect decreases. And (iv) its contribution might become negligible. Kinetic consideration has shown, that while continuous illumination results in steady states, illumination by pulsing light leads to non-steady states of the transient species and the latter enables the distinction between the mechanisms provided one of them is predominant under conditions of the measurements.