Novel Extraction Method for Combined Lipid and Metal Speciation From Caenorhabditis elegans With Focus on Iron Redox Status and Lipid Profiling

Parkinson ' s disease progression is linked to iron redox status homeostasis via reactive oxygen species (ROS) formation, and lipids are the primary targets of ROS. The determination of iron redox status in vivo is challenging and requires specific extraction methods, which are so far tedious and very time-consuming. We demonstrated a novel, faster, and less laborious extraction method using the chelator ethylene glycol l-bis(beta-aminoethyl ether)-N,N,N ',N '-tetra acetic acid (EGTA) as a stabilizing agent and synthetic quartz beads for homogenization under an argon atmosphere. Additionally, we combined the metal extraction with a well-established lipid extraction protocol using methyl-tert-butyl ether (MTBE) to avoid the problems of lipid precipitation in frozen samples and to determine lipid profiles and metal species from the same batch. The nonextractable matrix, such as the debris, is removed by centrifugation and digested to determine the total metal content of the sample as well. Lipid profiling using RP-LC-MS demonstrated high accordance of the modified extraction method to the reference method, and the organic solvent does not affect the iron redox status equilibrium. Furthermore, rigorous testing demonstrated the stability of the iron redox status equilibrium during the extraction process, secured by complexation, inert atmosphere, fast preparation, and immediately deep frozen extracts.

Automated summary

The progression of Parkinson's disease is linked to iron redox status homeostasis and ROS formation, with lipids being the primary targets of ROS. A novel extraction method using EGTA as a stabilizing agent and synthetic quartz beads has been demonstrated to be faster, easier, and more reliable for determining iron redox status in vivo. Combining metal extraction with lipid extraction protocols using MTBE allows for the simultaneous determination of lipid profiles and metal species, effectively addressing the challenges of lipid precipitation and total metal content determination in frozen samples.