Glycerides and Cholesterol Esters

Szonyi et al. (1962) have described a differential infrared spectrophotometric method for the determination of trans unsaturation in fats. The method utiUzes absorption at 965 cm which is due to C—H out-of-plane deformation vibrations of trans unsaturated compounds. The method is rapid, accurate, and directly appUc-able to the determination of trans unsaturation in triglycerides. It is applicable to samples which contain low concentrations of trans acids (down to 2%) and also to samples with fatty acids of mixed chain length. The absorptivities used in the calculations for this method are those established by Shreve et al. (19506). 

Freeman (1964) has analyzed mixtures of cholesterol esters (e.g., cholesteryl oleate) and triglycerides (e.g., triolein) by using the difference in the positions of the ester carbonyl bands. To analyze serum lipids he first removed phospholipids (1735-cm ester groups) by adsorbing them on silicic acid. Measurements were then made on carbon tetrachloride solutions of the unadsorbed lipids at 1745 and 1730 cm the approximate band positions of triglycerides and cholesterol esters, respectively. Minor lipid constituents produced only negligibly small errors and the over-all accuracy was about 5%. Freeman said that with minor modifications in procedure as little as 0.4 ml of serum could be analyzed and with microcells as little as 0.05 ml. 

Freeman et al. (1967) have developed an instrument which uses infrared absorption for the semiautomatic analysis of mixtures of triglycerides and cholesteryl esters. The method of analysis is based on the carbonyl absorption bands of triolein and cholesteryl oleate at 1745 and 1730 cm , respectively. Sample preparation consists of an extraction of lipids from serum in such a way as to exclude phospholipids. With adequate resolution and good precision of frequency setting, measurements at the two positions can be used successfully in a standard two-component spectrophoto-metric analysis. The nonautomatic part of the system is the handling of samples. 

Substitution of a methyl group at the a-methylene carbon of methyl esters of laurate, myristate, and palmitate results in characteristic infrared spectra that readily distinguish them from the straight-chain homologs and facilitate their unequivocal identification when separated from complex lipid mixtures by gas-liquid partition chromatography (Napier, 1966). Spectral changes observed with the methyl esters are also evident in the cholesterol esters of 2-methylalkanoates and also serve in their identification. 

Characteristic spectra of methyl and cholesterol esters of 2-methyllauric, 2-methylmyristic, and 2-methylpalmitic acids have been given by Napier (1966). Alkyl substitution at the 2-position is readily identified (Freeman, 1952) by a reversal 

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