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 luminol 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(b.C), 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.

Oxidative Stress and Antioxidant Status in Hypo- and Hyperthyroidism

“…Another explanation could be that at cellular level, there are other antioxidant systems [201], whose activity has not been evaluated by the mentioned investigations.…”

Antioxidant-Sensitive Triiodothyronine Effects on Characteristics of Rat Liver Mitochondrial Population

“…In this method the ability of the 10% homogenates (w/v) in 0.1 Lubrol to quench the light emission from a peroxidase-induced luminescent reaction was compared with that of antioxidant solutions. Because the more reproducible results were obtained using desferrioxamine [24], in this study the activity scale was obtained with such a substance and the overall antioxidant capacity of tissue homogenate was expressed as an equivalent concentration of desferrioxamine.…”

“…The plates were incubated at 37°C for 30 s under shaking and, then, transferred to a luminescence analyzer (Amerlite Analyser), which supplied the emission values as percentages of the emission of a standard constituted by 25 Ìl of a solution of 22 ng/ml horseradish peroxidase. Such values were used to fit dose-response curves using [24]. In this method the ability of the 10% homogenates (w/v) in 0.1 Lubrol to quench the light emission from a peroxidase-induced luminescent reaction was compared with that of antioxidant solutions.…”

Effects of Prolonged Aerobic Exercise on Myocardial Responses to Ischaemia‐Reperfusion in the Rat

“…The plates were incubated at 37°C for 30 s under continuous shaking and then transferred to a luminescence analyser (Amerlite Analiser). The emission values were fitted to dose-response curves using the statistical facilities of the Antioxidant capacity (C A ) was determined according to Venditti et al (1995). Briefly, 250 µl of the above reaction mixture was added to 10 µl of 110 ng ml _1 peroxidase plus 15 µl of either desferrioxamine, at concentrations ranging from 0.01 to 3 mM, in 15 mM Tris (pH 8.5) or buffer alone.…”