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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.

Biomedical Research Service