Analysis of Myocardial Plasmalogen and Diacyl Phospholipids and Their Arachidonic Acid Content Using High-Performance Liquid Chromatography
| Autor: | Peter M. Olley, Donna Panas, Richard Schulz, Gary D. Lopaschuk, Ken Strynadka |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Male
Plasmalogen Plasmalogens Biophysics Phospholipid Biochemistry Rats Sprague-Dawley chemistry.chemical_compound Phospholipase A2 Phosphatidylcholine Animals Molecular Biology Chromatography High Pressure Liquid Phospholipids chemistry.chemical_classification Phosphatidylethanolamine Arachidonic Acid Chromatography Phospholipase C biology Myocardium Fatty Acids Fatty acid Heart Cell Biology Rats Perfusion chemistry biology.protein Spectrophotometry Ultraviolet lipids (amino acids peptides and proteins) Arachidonic acid |
| Zdroj: | Analytical Biochemistry. 213:140-146 |
| ISSN: | 0003-2697 |
| Popis: | A high-performance liquid chromatographic method for the assay of diacyl and plasmalogen (alk-1-enyl) phospholipid content and the determination of their fatty acid content from tissue homogenates is described. Myocardial phospholipids are rich in plasmalogens and have a high content of unsaturated fatty acids, including arachidonic acid, esterified in the sn-2 position. Using a three-stage HPLC assay we have analyzed the phospholipid subclass content and the amount of arachidonic acid esterified to these fractions extracted from isolated perfused rat hearts. After HPLC separation of total myocardial phospholipids, the phosphatidylcholine and phosphatidylethanolamine peak fractions are treated with phospholipase C to remove polar head groups and ultraviolet-absorbing benzoate derivatives are made. Separation and quantification of diacyl and plasmalogen content of the total phospholipids with nanomolar sensitivity is then achieved using isocratic elution with a silicic acid HPLC column. The separated plasmalogen and diacyl glycerobenzoates are then subjected to alkaline hydrolysis to remove fatty acids from the sn-2 position. The 2-(2,3-napthalimino)ethyltrifluoromethanesulfonate esters of the free fatty acids are then prepared and analyzed with subnanomolar sensitivity using reverse-phase chromatography with gradient elution. As plasmalogen-specific phospholipase A2 is activated during myocardial ischemia and comprises the majority of total phospholipase A2 activity in the heart, this methodology allows for a sensitive and complete determination of the changes in the mass of these phospholipids and their arachidonic acid content. |
| Databáze: | OpenAIRE |
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