Measuring antioxidant potential in corals using the FRAP assay

Abstract

In this paper, we standardized a method for determining antioxidant potential in corals. This was determined using a simple, reproducible and inexpensive method: the ferric reducing/antioxidant potential (FRAP) assay. This procedure involves the reduction of Fe III-TPTZ to a blue colored Fe II-TPTZ by biological antioxidants and chemical reductants, some of which might have no antioxidant activity in a sample. The FRAP assay compares the change in absorbance at 600 nm of a sample compared with the change in absorbance of a known standard (FeSO 4·7H 2O) to determine antioxidant levels. This assay was used to determine changes in antioxidant potential in the corals Pocillopora damicornis and Pocillopora meandrina exposed to different temperatures (28, 29, 30 and 31 °C) for 3 h. Corals were also incubated at 31 °C for time intervals of a 0.5, 1 and 3 h. Antioxidant potential in the coral host increased with temperature and time, as indicated by FRAP values, compared to control samples at ambient sea surface temperatures (26.5–27 °C). Lower FRAP values could be a response to lower production of reactive oxygen species (ROS) or the result of an increase in ROS that react with the antioxidants. Because of the complex interactions within cells, one test is normally not enough to understand precisely what is going on within the cell. Rather, a broad array of tests is required to determine the different cellular parameters that are occurring within a biological system. To our knowledge, this is the first time that FRAP has been used to determine antioxidant status in a marine organism. The FRAP technique can potentially be a useful and inexpensive tool for marine biologists engaged in ecotoxicological studies.