Detection of Reactive Oxygen Species in Biological Samples

Aerobic metabolism is constantly subjected to oxidative stress derived from the utilization of oxygen, which generates various reactive oxygen species (ROS). ROS
react with a large variety of oxidizable cellular components, including proteins, nuclei acids, unsaturated fatty acids, NAD(P)H, dopa, ascorbic acid, and glutathione.
This interaction is thought to be critical for a wide spectrum of normal physiological processes. For instance, a growing number of signal transduction pathways
appear to be modulated by reactive oxygen and nitrogen species effects on redox-sensitive regulatory kinases, phosphatases and transcription factors. However,
oxidative damage caused by excess ROS could contribute to the aging process as well as to the development of human cancer and more than 50 diseases. At the
cellular level, the oxidative damage is manifested by genetic mutation, lipid and protein peroxidation, protein crosslinking and inactivation, and changes in
endogenous antioxidant levels. In order to differentiate between physiological and pathological effects of ROS, it becomes necessary to develop analytical methods
to identify and quantify ROS.

The term ROS refers to redox derivatives of molecular oxygen. The parent molecule in the generation of ROS is a free radical, the superoxide anion. Free radicals
are identified as those molecules containing an odd number of electrons. Other major free radicals in this cascade are hydroxyl radical and hydroperoxyl radical.
Hydrogen peroxide, although not a free radical, is also a highly reactive ROS. Aside from these oxygen species, reactive nitrogen species are usually identified with
pathological states and mediators of cellular injury. The reactive nitrogen species cascade includes the parent radical nitric oxide (NO) and its derivatives nitrogen
dioxide and peroxynitrite. Mitochondria are the major source of ROS in eukaryotic cells, and it is estimated that 2-5% of electron flux through the mitochondrial
electron transport chain escapes to produce superoxide anions. Additional enzymatic systems involved in ROS production include xanthine oxidase, NADPH
oxidase, cyclooxygenase, lipoxygenase, phospholipase A2, and cytochrome P450.

Endogenous ROS are difficult to measure because they are produced in minute quantities and are quite unstable due to their interaction with antioxidation
enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and cellular components like thiol residues, molecular oxygen, and metalloproteins. The
currently available methods include assays based on oxidation of chromophores, chemiluminescence techniques, fluorescence-based assays, enzymatic assays,
high performance liquid chromatography (HPLC), and electron spin resonance spectroscopy. It should be noted that each of these methods has its own drawback
and advantage, and some assays could identify specific molecular species while others could be used to estimate total ROS production. The lability and reactivity
nature of ROS is such that simultaneous use of multiple detection/assay methods for each biological system is recommended to verify their involvement and
possible role.

While many methods developed for the assay of ROS require the use of sophisticated and expensive equipment, some assays based on ROS-mediated oxidation
of chromophores are easy to perform, requiring only a spectrophotometer to detect changes in absorption of visible light wavelengths. Our Peroxide Assay Kit is
based on peroxides-mediated oxidation of Fe+2 to Fe+3 in the presence of the xylenol orange dye (Free Radical Res. 37:1209-1213, 2003), which can be
conveniently quantified by spectroscopy. As low as 0.1-5 uM peroxides can be detected using the assay kit in 30 min. Unlike other ROS detection kits, no protein
component is incorporated into our assay kit, thus ensuring reagent stability and assay reproducibility.
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