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Characterization and analysis of the subclasses and molecular species of choline phosphoglycerides from porcine heart by successive chemical hydrolyses and reverse phase high-performance liquid chromatography

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Abstract

Choline phosphoglycerides (CPG) represent the major fraction of heart phospholipids. Since depletion of membrane phospholipids and accumulation of lyso-compounds, particularly lysophosphatidylcholines, have been implicated in arrhythmogenesis, it was of great interest to study the composition of this major phospholipid fraction of the heart at a molecular level in an established animal model. The data presented here describe the first report on the detailed chemical examination of CPG and resolution, characterization and quantitative analysis of the molecular species of this phospholipid fraction from porcine heart by high performance liquid chromatography (HPLC). This fraction constitutes 37.5 ± 0.7% (n = 21) of the total phospholipids and upon successive mild acid and alkaline hydrolyses revealed the presence of essentially three subclasses: diacyl-, alkenylacyl-, and alkylacyl glycerophosphorylcholines, in a relative abundance of 57.7 ± 2.2% (n = 8), 37.3 ± 1.3% (n = 8) and 4.6 ± 0.2% (n = 8), respectively. The fourth subclass, dialkyl CPG was found only in minute amounts (0.43 ± 0.05%, n = 8) and the presence of dialkenyl and alkenylalkyl analogues could not be detected. Alternatively, by converting the CPG fraction to benzoate derivatives after phospholipase C digestion, it was possible to isolate and quantitate subclass composition by TLC/spectroscopy or both subclass compositions and molecular species analysis by HPLC directly by a UV detector online with the column. By these techniques, subclass composition was found to be very similar to that obtained by the chemical hydrolysis technique. By HPLC, up to 25 species can be identified and quantitated in each subclass, their identity being confirmed by gas-liquid chromatography, after their isolation from the column. The analyses showed that up to 74% of the diacyl moiety consisted of 16:0–18:2 (34%), 16:0–18:1 (27%), and 18:0–18:2 (13%) species, while the major species of the alkenylacyl moiety were 16:0–18:2 (44%) 16:0–18:1 (13%), 16:0–20:4 (12%) and 18:1–18:2 (9%) making up more than 75% of the total mass of this subclass. The major molecular species of the alkylacyl moiety was 16:0–18:2, constituting up to 47% of this fraction, while others constituted about 10% (16:0–18:1), 9% (18:1–18:2), 8% (16:0–20:4) and 6% (18:0–18:2), making up 80% of the total mass.

The ether chain composition of alkylacyl CPG whether determined after isolation of this fraction by the chemical hydrolysis technique or by HPLC was indistinguishable. Similarly, the aliphatic moieties of diradylglycerols, and their subclasses, whether analysed directly or reconstituted from the molecular species data, were very similar in composition, confirming the accuracy of the data and the reproducibility of the technique devised. This also suggests that this method is suitable to distinguish minor changes in the molecular species of CPG in the heart during the early phase of ischemia and in arrhythmias, and should facilitate further studies on the metabolism of the individual species in health and disease.

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  1. Departments of Medicine and Clinical Biochemistry, Clinical Science Division, University of Toronto, Medical Sciences Bldg., Rm. 7358, MSS 1A8, Toronto, Ont., Canada

    Nisar A. Shaikh

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Shaikh, N.A. Characterization and analysis of the subclasses and molecular species of choline phosphoglycerides from porcine heart by successive chemical hydrolyses and reverse phase high-performance liquid chromatography. Mol Cell Biochem 96, 43–55 (1990). https://doi.org/10.1007/BF00228452

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