Modification of the oxygen radical absorbance capacity assay and its application in evaluating the total antioxidative state in fish

The oxygen radical absorbance capacity (ORAC) assay is widely accepted as a common method for evaluating the total antioxidative activity in animals and food. The ORAC assay can simultaneously detect both hydrophilic (H-) and lipophilic (L-) antioxidants in animal tissues. In the present study, we modified the ORAC assay and estimated the total antioxidative state in two important fish species. The liver was initially extracted using a hexane and dichloromethane (Hex/Dc) mixture to prepare L-antioxidants associated with an L-ORAC value followed by the use of an acetone, water, and acetic acid (A/W/AA) mixture to prepare H-antioxidants as associated with an H-ORAC value, according to the original ORAC procedure. We altered the extraction order and reagents used in the modified ORAC assay. The H-antioxidants were initially extracted using 75 mM phosphate buffer (pH 7.4) instead of A/W/AA followed by Hex/Dc. Surprisingly, the H-ORAC value of the liver in rainbow trout (Oncorhynchus mykiss) measured using our modified ORAC assay was five-fold higher than that measured using the original procedure. In contrast, there were no significant differences in the liver L-ORAC values between the modified and original procedures. In addition, the plasma total antioxidative activity measured using the modified ORAC procedure was ten-fold higher than that measured using the original procedure. Analysis of the modified ORAC assay method revealed that high doses of dietary oxytetracycline, a widely used antibiotic, can affect the total antioxidative state and induce oxidative stress in coho salmon (Oncorhynchus kisutch). The present study suggests that total antioxidative activity, particularly H-antioxidative activity, in animal tissues and food based on the original ORAC assay might be underestimated in the literature. The modified procedure of the ORAC assay was simply and effectively improved to evaluate the total antioxidative state in animal tissues.