Abstract
Silica and C18 reverse phase high performance liquid chromatography (HPLC) were used to fractionate synthetic molecular species of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and semi-synthetic platelet-activating factor (PAF) synthesized from beef heart plasmalogens. A single coincident peak from silica HPLC was observed for either a mixture of synthetic AGEPC's with alkyl chain lengths from C12 to C18 or for beef heart-derived PAF. This peak was well separated from other classes of phospholipid standards including 2-lysophosphatidylcholine and 3H-labeled lyso-PAF. Subsequently, the synthetic AGEPC mixture or beef heart PAF was separated into individual species on a C18 reverse phase column. Beef heart-derived PAF was fractionated into at least four molecular species of PAF activity which had similar retention times as the radioactivity of 3H-labeled beef heart PAF. Approximately 56% of the radioactivity of 3H-labeled PAF was found in the fraction with a similar retention time as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 10% as 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, 11% as 1-O-pentadecyl-2-acetyl-sn-glycero-3-phosphocholine, and 13% in an unidentified fraction which eluted after C-16-AGEPC. The unidentified fraction did not correspond to any of the homologous series of synthetic AGEPCs with saturated alkyl chain lengths from C12 to C18. Recoveries of radioactive phospholipids from silica or reverse phase columns were greater than 95%.
Highlights
MethodsMaterialsAll solvents and phosphoric acid were high performance liquid chromatography (HPLC) grade and were purchased from Fisher Scientific Co. (Pittsburgh, PA)
I To whom reprint requests should be addressed at Department of Pathology, University of Texas Health Science Center, San Antonio, T X 78284
Several procedures [7, 8] have been used to isolate platelet-activating factor (PAF) from other phospholipid classes. We describe in this communication the separation of synthetic AGEPCs and semisynthetic PAFs from other major phospholipid classes by silica high performance liquid chromatography (HPLC) and the subsequent fractionation of PAF activity into several molecular species by reverse phase HPLC
Summary
MaterialsAll solvents and phosphoric acid were HPLC grade and were purchased from Fisher Scientific Co. (Pittsburgh, PA). Tritium-labeled beef heart-derived PAF (30-60 Ci/mmol) and lyso-PAF, 1-0-alkyl-sn-glycero-3-phosphocholine, (30-60 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Both of these radiolabeled compounds were purified immediately prior to use by thin-layer chromatography (TLC) on 250 p Silica Gel G plates (Analtech Inc., Newark, DE) using a solvent system of chloroform-methanol-water 65:35:6 (v/v/v), as described [10]. Such purification was necessary since 10-20% of the 'H was associated with degradation products that migrated with the solvent front. We refer to the semisyntheticcompounds prepared from bovine heart as PAF inasmuch as they have not been fully characterized
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