Office of Technology Development (OTD)

Balaraman Kalyanaraman, PhD

Superoxide has been associated with a variety of pathological conditions ranging from diabetes to atherosclerosis to cancer. As superoxide radical is such an important parameter of oxidative stress, its detection and quantification in vivo is critical. One of the most popular assays for detecting superoxide in cells and tissue involves the florescent-based techniques. Generally, the red fluorescence arising from oxidation of hydroethidine (HE) also called dihydroethidine (DHE) is detected. It was thought that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+) , that binds to DNA and leads to  the enhancement of red fluorescence. However, research at the Medical College at Wisconsin has revealed that the reaction between superoxide and HE results in a completely different fluorescent product, 2-hydroxyethidium. This discovery contradicts the prior art and provides a method for accurately detecting and quantifying superoxide in a sample.     

This invention included both a unique method for synthesizing highly pure amounts of 2-hydroxyethidium and a method for assaying levels of superoxide in a sample. The assay method involves adding HE to a sample, subjecting the sample to conditions under which HE and superoxide react to form 2-hyroxyethidium and detecting specifically the presence of 2-hydroxyethidium in the sample wherein its presence indicates the presence of superoxide in the sample. All known HE-based fluorescence methods for detecting superoxide in biological samples, such as HPLC, can be adapted to this invention by employing the 2-hydroxyethidium specific detection and quantitation methods. These methods can also be used as tools for diagnosing and monitoring the progression of disease and conditions involving abnormal superoxide levels in body tissues. 

• Provides an accurate method for detecting and quantifying superoxide in a sample
• Includes patented method for synthesizing highly pure 2-hydroxyethidium for use as assay controls and standards
• Improves accuracy and sensitivity over current assays
• Useful for research and diagnostic assays

In vivo studies