Abstract
Dipolyunsaturated phosphatidylcholines from bovine retina contain a whole series of unusual fatty acids. Methyl esters from these acids are very strongly retained on polar and nonpolar gas-liquid chromatography stationary phases. On thin layers of silica-AgNO3, they separate as tetra-, penta-, and hexaenoic fatty acid methyl esters. After hydrogenation, the three polyunsaturated fractions give the same series of saturated methyl esters, having 20 (or 22)-36 carbon atoms. High pressure liquid chromatography, as well as gas-liquid chromatography, indicates that the new components of the three fractions are even-carbon homologs of well known polyenoic fatty acids of the n-6 and n-3 families, since they behave as series of 20-36-carbon tetraenoic (n-6), pentaenoic (n-3 and n-6), and hexaenoic (n-3) fatty acids. Their occurrence in phospholipid molecules also having docosahexaenoate (22:6) explains the separation of major dipolyunsaturated phosphatidylcholines from retina into dodecaenoic, undecaenoic, and decaenoic fractions after argentation thin layer chromatography. Using high pressure liquid chromatography, the latter are resolved into individual species having 10-12 double bonds and 42-58 carbon atoms. The unusual PCs are thus endowed not only with the highest degree of unsaturation, but with the longest hydrocarbon chains yet reported for vertebrate glycerophospholipids. It is shown that phosphatidylcholines containing the novel fatty acids are highly concentrated in photoreceptor membranes and that they occur in the retina of vertebrates so distant in evolution as fish, birds, and various mammals.
Highlights
Dipolyunsaturated phosphatidylcholines from bovine retina contain a whole series ofunusual fatty acids
The abbreviations used are: PC, phosphatidylcholine; VLCPUFA, very long chain polyunsaturated fatty acids; Fatty acid methyl esters (FAME), fatty acid methyl esters; fatty acids are abbreviated by the convention, number of carbon atoms:number of double bonds, n - 3 or n - 6 referring to the position of the first double bond counting from the methyl end; GLC, gas-liquid chromatography; HPLC, high pressure liquid chromatography; TLC, thin layer chromatography
The order of elution of compounds present in all seven FAME fractions, and the straight, parallel lines obtained whenlog retention(represented by the parameter k ’ ) was plotted against the number of carbon atoms of the components of each band, were totally consistent with the GLC data indicating that thecompounds of interest, VLCPUFA, belonged to homologous series of tetra,penta,and hexaenoic fatty acids having up to 36 carbon atoms
Summary
Lipids were extracted, isolated, protected from oxygen, and analyzed using simple variations, if any, of established lipid methodological procedures. Several other procedures of FAME preparation were testedon PC (base- or acid-catalyzed methanolysis, sodium methoxide, saponification followedby methylation of the resulting free acids with methanolic HCl orethereal diazomethane), which gave similar yields and recoveries of very long chain FAME This indicated that VLCPUFA 1)were not artifacts (of a given methanolysis procedure) and 2) were bound through ester (not ether or vinyl ether) bonds to the glycerol backbone of PC. Methyl esters were resolved into fractions of similar unsaturation (hexaenoic to saturated FAME) by means of AgN0,-TLC, using the same plates. When saturated FAME obtainedby hydrogenation argentation TLC of acetyldiglyceridederivatives, and fatty of polyenes were subjected to linear temperature programs, acid methylestersfromthesefractions were subjected to they elutedat regular intervals from both polar and nonpolar temperature-programmed GLC on a polar stationary phase phases (Fig. 2)
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