In collaboration with Carlos Huesca Mejia and Priscilla Teitelbaum, we studied several thousand samples of lipid preparations through spectral analysis, in ultraviolet and first portion of visible light, and more rarely in infrared, using the Beckman spectrophotometer. We will limit ourselves to note here only some of the principal conclusions reached.

1) Concerning the chemical isomerization procedures, we could show the importance of the temperature used when a mixture of fatty acids is treated. The conjugation in vitro as it is usually carried out, with ethylene glycol or glycerol as solvents, was seen to result in preparations with too low amounts of tetra-, penta- and hexaenic conjugated members. A relatively rapid disappearance of the conjugated formations with 4, 5 and 6 double bonds was seen to be induced by the high temperature used. This led us to utilize a new method of conjugation, at lower temperatures. Using ethyl alcohol as solvent, preparations with high conjugated formations were obtained.

2) We utilized the spectral analysis for quantitative determination not only for di-, tri- and tetraenes as usually employed, but also for pentaenes and hexaenes. For this purpose we determined the extinction coefficient corresponding to these pentaene and hexaene formations. This was made possible by isolating the respective pentaenic and hexaenic conjugated members through appropriate solvents.

3) We studied various materials and especially different organs in order to correlate their richness in different fatty acids to their biological activity, by using the spectral analysis of the in vitro conjugated fatty acids, as mentioned above.

4) Similarly, we tried to correlate the existence of characteristic peaks in the spectral analysis curve of unsaponifiable fractions of organs to their biological activity.

5) We utilized spectral analysis for the study of the effects of various agents such as chlorine, sulfur, sulfuric acid or oxygen upon the conjugated fatty acids.

6) We showed that minimal changes are induced in the nonpolar groups of conjugated fatty acids by changing their polar group from carboxyl into a primary alcohol, by treatment with lithium aluminum hydride.

7) We studied the influence exerted by conjugated fatty acids upon carcinogens. This can be partially revealed by the quenching action induced upon the fluorescence of these latter agents.

8) In an extensive study we investigated the influence exerted by radiation upon fatty acids in vitro and in vivo. This influence was characterized by the appearance of conjugated trienes, and is presented in Chapter "Radiation," and in other Notes.

Chapter 6, Note 8c. Vapor Fractionation Of Fatty Acids

In a group of experiments we applied the gas chromatography method to the study of fatty acids. The principal aim was to investigate the value of the information furnished by this method concerning the presence of conjugated fatty acids. This study was made in collaboration with Ivan Bier and with Winston Dindial who prepared the samples.

Methyl esters of eleostearic acid, linoleic acid and its conjugated isomers; of linseed oil fatty acids and the conjugated preparation; of cod liver oil fatty acids and the conjugated preparations; and of samples of fatty acids obtained from animals and tissues under normal and abnormal conditions were obtained. We analyzed through vapor fractionation, these different preparations as such, the preparations obtained through condensation on cold fingers during distillation in vacuum at different temperatures, and the different fractions obtained through distillation in vacuum. For all these tests we used the Perkin Elmer vapor fractioner with a column of succinyl polymers heated up to 235°C. Under these conditions, no differences could be seen between the respective conjugated and nonconjugated samples. Figs. 250 and 251 show examples of such analyses of a cod liver oil fatty acid preparation and of a preparation obtained after treatment with KOH in butyl alcohol. Fig. 252 shows the spectral analysis of this last product.

Under the condition of analysis used, the gas chromotography method does not permit the identification of the conjugated isomers present. This is the reason why the analytical method could not indicate the presence of such members in materials obtained during abnormal conditions. The conjugated isomers can be identified by other methods—such as spectral analysis and especially oxalic index—after oxidative fission.

We are now trying to obtain columns that would permit working at much higher temperatures and would permit us to identify these conjugated fatty acids. In view of the minimal amount of material needed for analyses and the precision of the results usually obtained, an adaptation of this method for the identification of conjugated members would be of especial great value.