Determination by Enhanced Luminescence Technique of Liver Antioxidant Capacity

Abstract

A simple approach to quantitative determination of antioxidant capacity of rat liver homogenate is proposed. It consists of measuring chemiluminescence generated by a suitable system “detector” for OH radicals produced from sodium perborate. The system generating the light signal contained luminal and compounds producing enhancement of light emission, such as sodium benzoate and indophenol. Two different methods, utilizing the same technique of enhanced luminescence, were set up.

In a previous work, a parameter b, contained in the equation, which best describes the dependence of the intensity of light emission (E) on liver homogenate concentration (C) (E - a C/exp(bC), was found to be related to the level of antioxidants in the homogenate. Therefore, in the first method, the light emission from several dilutions of both liver homogenates, and homogenate and antioxidant mixtures, stressed with sodium perborate, was detected by a luminometer. The best fitting of data to theoretical equation provided b values, which were introduced in a system of equations relating such values to the antioxidant concentration. The solution of above system supplied the antioxidant concentration in the homogenate in terms of the equivalent concentration of the antioxidant used.

In the other method, evaluations of the antioxidant capacity of liver homogenates were obtained by the determination of the ability of 10% homogenates to quench the light emission induced by either peroxidase or cytochrome c in comparison to the ability of antioxidant solutions.

Both methods are able to evidence the decrease of the antioxidant concentration of liver homogenates after oxidative stress with ter-butylhydroperoxide. The value of both concentration changes and standard errors indicates that the method using a standard curve obtained with peroxidase, such as catalyst of radical reaction, and deferoxamine, such as antioxidant, is to be preferred.

Key Words:

• Enhanced luminescence
• antioxidants
• free radicals
• oxygen radicals
• oxidative stress
• liver
• rat