Isolation of eicosapentaenoic and docosapentaenoic acids from natural sources as their methyl esters by adsorption and distillation techniques

Abstract

T HE isolation of pure linoleie, linolenic, and arachidonic acids from natural sources by physical rather than by chemical methods has been described in recent publications (1, 2) from this laboratory. The importance of geometrical configuration of the polyunsaturated acids in relation to the alkali-isomerization spectrophotometric method of analysis has also been discussed. New constants determined on these pure "natural" acids were proposed (3) for use in this method.In the course of the work on isolation of araehidonic acid (as its methyl ester) from adrenal lipids, fractions of greater unsaturation than that of methyl arachidonate were obtained (2). Preliminary examination of this material indicated the presence of methyl eicosapentaenoate. After alkali-isomerization the product had characteristic absorption maxima at 346 and 328 m~ in addition to the principal maxima at 315, 301, 268, and 233 millimicrons.This highly unsaturated material has now been subjected to further fractionation on silicic acid adsorption columns and by high-vacuum distillations. Eieosapentaenoic and doeosapentaenoie acids as their methyl esters were isolated and their specific extinction coefficients determined after isomerization with alkali. Further evidence of their identity was obtained by iodine values, refractive indices, and by melting points, saponification equivalents, and X-ray diffraction measurements on hydrogenated portions. The results permit an extension of the spectrophotometric method to the analysis of highly unsaturated lipids, such as are found in nearly all glandular fats and specialized tissues. Experimental and DiscussionStarting Materials. All fractions of methyl esters having iodine values greater than the theoretical value (318.8) for methyl arachidonate, which were obtained in the adsorption fractionation of adrenal lipid methyl esters (2), served as starting material. In all 11.6 g. were obtained from this previous work. The average iodine value was 359.0. The specific extinction 3 coefficients of this concentrate after isomerization in 11% for 45 minutes at 180~ under nitrogen were : k233 = 49.8 ; k26 s -~-35.0 ; ka~ = 21.8 ; k3~6 ~ 8.7. No absorption maximum was found above 346 rag.Adsorption Fractionation on Silicic Acid. Essentially the sama technique as described in previous work (1, 2) was followed except that the glass columns were packed by introducing a pre-heated slurry of the silieic acid-filter aid mixture in petroleum ether.

The alkali isomerization of a methyl docosahexaenoate and the spectral properties of conjugated fatty acids

“…(or one carbon in each of the two end groups), one would expect that the spectra following equal alkaliisomerization treatment would be more nearly alike. A solution effect may also account for the apparent differences mentioned earlier between the eicosapentaenoate isolated by Herb et al (10) and that isolated from fish oil by the present authors (1). However it has not been established whether solubility factors are important in the isomerization of C2o acids with the KOH-glycol reagent.…”

“…A study of possible relationships between diet and the highly unsaturated fatty acids (4, 5, and 6 double bonds) of human blood lipids has been started in this laboratory. The presence of fatty acids with five and six double bonds in fish oils (1,2,3,4,5) and in tile lipids of land animals (6, 7) has been known for some years, but relatively few investigations of their implication in lipid metabolism have been made (8,9,10).…”

South african pilchard oil. 5. The isolation and structure of an eicosapentaenoic acid from South African pilchard oil

“…Tutiya (1940) mentioned the presence of this acid in sardine oil, and Matsuda (1942) found it in bonito oil and supported the 4:8:12:15:18 strUCtUre with oxidative evidence. The presence of eicosapentaenoic acids has been reported by Baldwin & Parks (1943) in menhaden oil; by White & Brown (1948) in ox-adrenal lipids; by Tsuchiya & Kato (1950) in herring oil; by Shorland & Johannesson (1951) in butter fat; by Herb, Witnauer & Riemenschneider (1951) in ox-adrenal lipid; by Tsuchiya & Okubo (1952) in fish-liver oil; by Toyama & Shimooka (1953), who give the alkali isomerization spectrum, in sardine oil; and by Abu-Nasr & Holman (1954), who determined some of the properties of the methyl ester, in cod-liver oil.…”

“…Conjugated diene was formed in greatest amount, followed by triene, tetraene and pentaene in that order. Several authors have obtained much higher proportions of conjugated tetraenes and pentaenes (Herb et al 1951;Toyama & Shimo-oka, 1953;Hammond, 1953) by alkali-isomerizing pentaene acids from other natural sources. This matter obviously needs further investigation.…”

Composition of Edible Fats, Unsaturated Fatty Acids of Butterfat

“…filtrate material, 605 grams, was subjected, in 80to 100-gram batches, to further fractionation by chromatographic adsorption on silicic acid columns. The column and technique for operation under nitrogen were essentially the same as described by Herb and Riemenschneider (22,23,34). The adsorbent mixture, 80% silicic acid-20%, filter aid, was heated to 105°C.…”