Assessment of a subcellular metal partitioning protocol for aquatic invertebrates: preservation, homogenization, and subcellular fractionation

Measurements of the subcellular partitioning of trace metals have been used to predict assimilation efficiencies for metals between trophic levels and to evaluate metal detoxification mechanisms in aquatic organisms. Using field-collected larvae of the insect Chaoborus, we assessed the performance of two preservation protocols, three homogenization treatments, and a cell fractionation protocol to determine the best combination of techniques for assessing the subcellular partitioning of Cd, Cu, Ni, and Zn. Preservation of larval samples for either 2 years at -80 degrees C or 1 year at -80 degrees C or -196 degrees C had little influence on the subcellular partitioning of these metals. Using several marker enzymes, i.e., cytochrome C oxidase and citrate synthase for mitochondria, beta-hexosaminidase for lysosomes, and lactate dehydrogenase for the cytosol, we show that optimal and high-yield homogenization is achieved using a combination of mechanical disruption using a micro-pestle followed by vortexing and that the subsequent fractionation procedure employing differential centrifugation/NaOH digestion was effective. We conclude that the preservation and homogenization protocols as well as the fractionation procedure that we tested using Chaoborus larvae can serve as a model approach for improving current protocols and for designing protocols for previously untested biological samples.