The Lipidomics Analytical Core (Core 2) will monitor changes in glycerophospholipid patterns and quantities in native cells as well as cells enriched with alkynyl-derivatized fatty acids or alkynyl-derivatized GPCho lipids following oxidative stress. RAW 264.7 and THP1 cell extracts (in conjunction with projects 1, 3, and 4) will be analyzed by a variety of mass spectral techniques. Liquid chromatography /Mass spectrometry (LC/MS) analysis of cellular extracts will be utilized for both the detection and quantitation of naturally occurring as well as alkynyl-derivatized glycerophospholipids using a large array of odd-carbon internal standards and in-house developed data analysis software. A complimentary high-throughput technique, direct infusion mass spectrometry (DI-MS), will be used to rapidly study changes in lipid patterns within glycerophospholipid classes. In the instances where alkynyl-derivatized lipids co-elute with naturally occurring isobaric peaks, two pathways will be available for the quantitation of these species: 1) Phospholipid levels in control and alkynyl-lipid treated cell extracts can both be quantitated. The difference between these spectra would be a good semi-quantitative estimate for the amount of derivatized lipid species present. 2) Samples will be divided into two aliquots with one used as a control and the second derivatized using a known cobalt/alkyne reaction. This reaction forms a dicobalt hexacarbonyl adduct with any alkyne-containing compound that shifts both the retention time as well as the molecular weight of the compounds. The differences between these sets of spectra yield a more reliable value for the amount of alkyne-containing phospholipid present in the cellular extracts. The detailed procedures are described in the Core section. The second role for the Lipidomics Analytical Core will be to support the EPA (20:5 fatty acid) studies undertaken by project 2. In this project, we will analyze differences in rodent and human glycerophospholipid levels in subjects fed normal or EPA-containing diets. In the rodent experiments, we will study heart and liver extracts from animals on both diets. Additionally we will also examine animal tissue extracts from subjects which have undergone oxidative stress following infusion with CCI4. For the human studies, glycerophospholipid levels will be determined in plasma samples. In both model types, LC/MS quantitation and DI-MS techniques will be utilized to characterize phospholipid changes due to diet and/or oxidative stress.
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