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

Abstract

T HE preparation of a quite pure methyl docosahexaenoate from hog brain lipids is reported in 'another paper from this laboratory (1). In the present paper the ultraviolet spectral characteristics of this compound, following alkali-isomerization under various conditions, will be considered in relation to alkali-eonjugation-spectrophotometric techniques of analysis of polyunsaturated fatty acids and esters (2,3,4).Also to be described are some general relationships in the ultraviolet spectral characteristics of fatty acids with conjugated double bond systems, whether natural or alkali-induced, which were developed on the basis of previous work by Lewis and Calvin (5). ec 5C 40 o ~_ 3r Fx w r ft. m : t , .,.a \ t-,..r\v,t,..; 350 WAVELENGTH, m~ ! ( t! |1 t# I i 375 400 Fro. 1. Specific extinction coefficient vs. wavelength for small samples of methyl docosahexaenoate isomerized in 21% KOH-glycol at 180~ for 4 rain. (solid line) and 15 rain. (broken line) by tho micro procedure. Ultraviolet Spectral Absorption Following Alkali IsomerizationSmall samples of the methyl docosahexaenoate isomerized at 180~ in 21% KOH-ethylene glycol for 4 and 15 minutes by the micro procedure of Herb and Riemenschneider (6) gave the absorption spectra represented in Figure 1. The data were obtained with a Beckman DU speetrophotometer. Measurements at wavelengths above 320 m/~ were made with an incandescent light source. A slit width of 0.15 ram. was 1Paper No. 3042, Scientific Journal Series, Minnesota Agricultural Experimental S~ation. This work was supported by a grant-in-aid from the Nutrition ]Foundation Inc. The findings reported in this paper a.e to be included in a thesis to be submitted to the University of Minnesota by Earl G. Hammond in p.artia! falfillment of the requirements for ~he Ph.D. degree. 433 used for readings made at 374 m~. (The importance of specifying slit width at this wavelength will be discussed below.) Absorption peaks are found at 233, 268, 279, 301, 315, 333, 352.5, and 374 m~.As with the fatty acids of lower unsaturation, the spectral properties following isomerizati0n depend markedly on time of heating, alkali concentration, and temperature. In addition, two other factors, the speetrophotometer slit width and the size of the sample isomerized, are important in working with hexaenoate.Effect of time of heating and alkali concentration. Figure 2 illustrates the effects of time of heating in alkali isomerization. The fixed conditions were the same as those used in obtaining the data for Figure 1. Evidently the maximal concentrations of conjugated materials are attained very rapidly in the case of hexaenoate. The heating times to reach a maximum for the various conjugated forms of alkali isomerized hexaenoate were found to increase in the following order as might be expected : a) di-, tri-, and tetraene, b) peutaene, and c) hexaene. Presumably if times shorter than 2 minutes were used , the maxima for the first three would be reached in the order listed.After 5 minutes of heating the various conjugated forms...

Application of ultraviolet spectrophotometry to the analysis of fats and derivatives

“…For maximum absorptivity when analyzing docosahexaenoic acid with the Beckman spectrophotometer, a slit width of 0.15 ram. at 374 m~ has been recommended, and to obtain this slit width a tungsten lamp was used as the light source (18). In an investigation at this laboratory a relatively pure hexaenoic acid was isolated, and absorptivities were obtained that were in reasonably good agreement with those published by Hammond and Lundberg (18).…”

“…They also extended the investigation and determined the absorptivity constants with this reagent for linoleic, linolenie, arachidonic, and pentaenoic acids of natural configuration. Absorptivity constants for docosahexaenoic acid have recently been determined with the 21% KOH-glycol reagent (18).…”

“…The data in Figure 2 were obtained from one of four fractions of methyl hexaenoate that appeared to be rather pure. In Table I the average absorptivities of these four fractions, after alkali treatment, are compared at the analytical wavelengths with those reported previously (18). Both samples were isolated from hog brain lipids.…”

“…The importance of this factor in fat and oil analysis was noted by Brice et al (8) in a discussion of the determinations of linolenie acid. Recently Hammond and Lundberg (18) found that slit width was of great importance in the analysis of docosahexaenoie acid. In an analysis it would seem only necessary to use the slit width employed in the determination of the absorptivity 3 constants, but an example of the error that may occur by not employing optimum slit widths may be found from inspection of the curve of a pentaene-hexaene mixture.…”

Highly unsaturated fatty acids. I. A survey of possible animal sources

“…It should be pointed out that some of the constants used in the calculation were determined upon acids derived from fish oil, and these acids probably are not identical to the polyunsaturated acids found in mammalian tissue. For further discussion of the calculation and interpretation of spectrophotometrie data, the reader is referred to current papers dealing with the limitations of the method (12,13).…”