46] Oxystat technique in study of reactive oxygen species

Abstract

Publisher Summary This chapter discusses the oxystat technique in study of reactive oxygen species. The oxystat system allows samples to be collected from the filtrate as well as directly from the incubation chamber. Thus, the formation of products of oxidative reactions in biological material, such as malon- dialdehyde, can easily be determined during the course of incubation. Furthermore, by putting the incubation chamber directly in front of the photomultiplier of a single-photon counting apparatus, low-level chemiluminescence emitted by electronically excited species such as singlet molecular oxygen ( 1 O 2 ) and triplet carbonyls ( 3 RO) can be detected. Finally, the stimulation of O 2 uptake may serve as an additional indicator for the formation of reactive oxygen species. The oxystat system consists of an incubation chamber, an O 2 sensor, a pump, and a computer. The water-jacketed incubation chamber is made of plexiglass. It is equipped with a magnetic stirrer and a port for the O 2 sensor. At the top two stainless steel needles serve as entrances. The inlet for the O 2 -saturated infusion medium ends directly above the magnetic stirring bar. The second needle, which does not project into the chamber, is used as an injection port for the addition of reagents and for taking samples of the incubation mixture. The bottom of the incubation chamber is equipped with a membrane holder covered by a filter membrane. The actual PO 2 in the incubation chamber is determined by a polarographic O 2 sensor.