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Superoxide Detection


Superoxide has been implicated in diseases ranging from Alzheimer's to diabetes. The wide range of pathologies associated with superoxide is the result of its ability to react with a variety of cellular targets, including enzyme active sites, nucleic acids and lipids. Cells protect themselves against superoxide damage by producing superoxide dismutase enzymes (SODs). These enzymes accept an electron from a superoxide molecule, reducing the active metal (copper/zinc, iron or manganese) at the core of the enzyme, and releasing oxygen. The reduced enzyme then reacts with a second superoxide, reducing it to produce hydrogen peroxide, and regenerating the enzyme. The high reactivity of superoxide in aqueous solutions almost precludes its being added to a biological test system in a controlled manner. As a result, much of what is known of the involvement of superoxide in disease states comes from studies in which SOD has been inactivated, leaving the cells vulnerable to endogenous superoxide damage.

Detection of Superoxide: The Methods

A number of methods have been developed to detect superoxide. They all employ what has been termed an “indicating scavenger”, that is, a molecule which reacts with superoxide, producing a detectable product. The most commonly used indicating scavengers are cytochrome c, lucigenin and luminol, each of which has its own advantages and pitfalls. Cytochrome c is reduced by reaction with superoxide, producing ferrocytochrome c, which has a detectable absorbance at 550 nm. This assay is relatively insensitive, and is subject to a number of interferences from other chemicals and from enzymes which reduce the cytochrome directly.

Lucigenin is a di-acridinium compound, which emits light on reaction with superoxide. The reaction involves an initial reduction of the lucigenin to a radical. The lucigenin radical can then react with either oxygen, producing superoxide, or with superoxide in an addition reaction, leading to the decomposition of the lucigenin into two acridones, one of which is in an excited state, and decays to produce light. As with most chemiluminescent reactions, lucigenin is more sensitive than colorimetric methods such as cytochrome c reduction. The lucigenin assay has been criticized as a measure of superoxide because lucigenin itself can react to produce superoxide, and because in some cases lucigenin has been observed to stimulate superoxide production by intact cells.

Luminol, unlike cytochrome c and lucigenin, is oxidized by superoxide. This leads into a complex series of reactions between luminol, luminol radicals, oxygen and superoxide.  Ultimately these reactions will produce a luminol endoperoxide, which decomposes with the release of a photon. Again, because superoxide is involved as both initiator and intermediate in the reaction, objections have been raised to the use of luminol as a quantitative measure of superoxide production.

No matter which assay is used, it is important to include the proper controls in the experimental design in order to be certain that any signal detected is due to superoxide. The primary control experiment is to run the reaction in the presence of SOD. If the signal is not abolished by SOD, it does not represent superoxide production. However, the converse is not always true: a signal which is quenched by SOD does not absolutely indicate that superoxide is being produced by the test system.

Both luminol and lucigenin can generate superoxide as they react to produce light. Also, both chemicals can undergo a redox cycle—induced by cellular agents—which produce light by way of a superoxide intermediate. Thus, in special cases SOD will inhibit a signal which did not originate with a superoxide molecule. Therefore, results must be interpreted with caution.

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