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16 مارس 10

ANTIOXIDANT ASSAYS

From Oxford Biomedical Research: http://www.oxfordbiomed.com/commerce/info/showpage.jsp?page_id=1042&gclid=CL603tWq15kCFRpN5QodpDEpVw/

Background
It is now well established that oxidative stress is a major risk factor for the development of several diseases including atherosclerosis, cardiovascular disease, and cancer. Oxidative stress is the condition in which there is an imbalance between the concentrations of reactive oxygen species (ROS) and physiological antioxidants, resulting in oxidative damage to many biomolecules within the cell. Products of ROS-mediated oxidation are widely used to monitor oxidative stress. However, it is also important to assess the antioxidant capacity of cells and biological fluids, as well as putative “functional foods” to assess their antioxidant capacity. Organisms possess multiple antioxidant systems to help regulate ROS and prevent oxidative stress. In vertebrates, these include enzymes that metabolize ROS, antioxidant proteins, and smaller molecules that are important antioxidants. These antioxidants include hydrophilic as well as lipid-soluble molecules that are localized throughout various tissues and cell types.

Given the multiplicity of antioxidant pathways, their centrality in the prevention of oxidative stress, and the influences of lifestyle and nutritional supplements on an individual’s antioxidant capacity, it is important to be able to quantitatively measure the total antioxidant capacity or antioxidant power in a biological specimen or in nutrients.

ORAC Assay

Assay Principle
The ORAC assay is based on the oxidation of fluorescein by peroxyl radicals via a classic hydrogen atom transfer (HAT) mechanism. Free radicals are generated by the water soluble compound 2,2’-azobis-2-methyl-propanimidamide (AAPH). The peroxyl radicals thus generated quench the fluorescence of fluorescein over time. The antioxidants block the peroxyl radical mediated oxidation of fluorescein until all of the antioxidant activity in the sample is exhausted, after which the AAPH-generated peroxyl radicals react with and quench the fluorescence of fluorescein. The area under the fluorescence decay curve (AUC) is used to quantify the total peroxyl radical antioxidant activity in a sample and is compared to a standard curve obtained using various concentrations of the water soluble vitamin E analog Trolox. Unlike other antioxidant activity assays, the fluorescent ORAC assay provides a direct measurement of antioxidant capacity against hydrophilic chain-breaking peroxyl radicals.

HORAC Assay

Assay Principle
The HORAC assay is based on the oxidation of fluorescein by hydroxyl radicals via a classic hydrogen atom transfer (HAT) mechanism. Free radicals are generated by hydrogen peroxide (H2O2). The hydroxyl radicals thus generated quench the fluorescence of fluorescein over time. The antioxidants block the hydroxyl radical mediated oxidation of fluorescein until all of the antioxidant activity in the sample is exhausted, after which the H2O2 radicals react with and quench the fluorescence of fluorescein. The area under the fluorescence decay curve (AUC) is used to quantify the total hydroxyl radical antioxidant activity in a sample and is compared to a standard curve obtained using various concentrations of gallic acid. Unlike other antioxidant activity assays, the fluorescent HORAC assay provides a direct measurement of antioxidant capacity against hydrophilic chain-breaking hydroxyl radicals.

Total Antioxidant Power

Background
Oxidative stress has been implicated in a number of diseases such as atherosclerosis, chronic inflammatory disease, chronic renal failure, and cancer.  It is a condition where an imbalance exists between the production of reactive oxidizing species and the body’s ability to neutralize these intermediates, resulting in cellular damage. The body has designed several physiological responses to oxidative stress including counterbalances such as enzymes and variously functionalized molecules (see examples below) that effectively neutralize these damaging species. These antioxidants can be either water or lipid soluble, and are localized transiently throughout various tissues, cells and cell types.

Given the multiplicity of antioxidant pathways, their centrality in the prevention of oxidant stress, and the influences of lifestyle and nutritional supplements on an individual’s antioxidant capacity, it is important to be able to quantitatively measure the total antioxidant capacity or antioxidant power with biological specimens.

Assay Principles
The reduction potential of the sample or standard effectively converts Cu+2 to Cu+1, thus changing the ion’s absorption characteristics. This reduced form of copper will selectively form a stable 2:1 complex with the chromogenic reagent with an absorption maximum at ca. 450 nm. A known concentration of uric acid is used to create a calibration curve, with the data being expressed as mM uric acid equivalents or in μM copper reducing equivalents

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