Abstract
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(β-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.
Highlights
Parkinsons disease is the second most common neurodegenerative disorder, which affects about 1 percent of the population above 65 years
The glycerophospholipids are the main components of biological membranes and of high interest for determining potential cell damages caused by reactive oxygen species (ROS), whose alteration is typical for Parkinson’s disease progression (Kolodkin, Sharma et al, 2020)
The results showed similar extraction efficiencies compared to the reference method for the important lipid classes
Summary
Parkinsons disease is the second most common neurodegenerative disorder, which affects about 1 percent of the population above 65 years. The underlying mechanisms are not fully understood, but an imbalance in forming and scavenging of reactive oxygen species (ROS) is accepted to play a crucial role in Parkinsons disease progression The iron redox state and the possibly caused lipid peroxidation in biological systems are of high interest for enlightening cellular mechanisms in Parkinsons disease progression. Biological variability regarding iron redox and lipid metabolisms lead to relatively high batch to batch variances, which worsens the significance of the results. Yields and recoveries were similar to the lipid extraction protocols by Bligh and Dyer (Bligh and Dyer 1959) and Folch (Folch, Lees et al, 1957). Use of this extraction protocol over the last decade has increased
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