Determination of lipid peroxides in invertebrates tissues using the Fe(III) xylenol orange complex formation

Abstract

Reactive oxygen species (ROS) are subproducts of the oxidative metabolism known to initiate chain reactions with polyunsaturated fatty acids that generate lipid peroxides (LPO). The objective of this work was to adapt the ferrous oxidation/xylenol orange (FOX) assay to measure LPO in invertebrate tissues i.e.: from polychaeta (Laeonereis acuta) and crab (Chasmagnathus granulata) species. Whole polychaetes were homogenized in methanol 100%, being determined the optimal sample volume and the time required for color development. It was tested five sample volumes (8-30 microl), following color development up to 215 min. Absorbance stabilization was observed after 90 min, being linearly related with sample volume. A similar procedure was adopted for crab tissues (anterior gills, posterior gills, and hepatopancreas). Differences between species and between organs of the same species were observed when analyzed nonspecific absorbance increments after adding the standard cumene hydroperoxide (CHP). In polychaeta and crab anterior gills tissue, absorbance increments were lower (21-25%) than samples without tissue extracts (blanks) that received CHP. In crab posterior gills and hepatopancreas, the nonspecific increment was almost negligible. Correction formulae are given to account for these differences and simplified protocols for each tissue and species are also included. Great differences in the lipid peroxides content was detected between worms (127.05 +/- 19.32 nmoles CHP/g of wet tissue) respect to anterior gills, posterior gills, and hepatopancreas from the crab species (52.65 +/- 3.59, 30.54 +/- 4.73, and 48.51 +/- 8.78 nmoles CHP/g of wet tissue, respectively).