It appeared especially interesting to study the distribution and transport of polyunsaturated fatty acids in the body in view of the fact that parenteral administration of the acid lipidic fraction of various organs to subjects with acute pain had an effect within only a few minutes. The rapid action was independent of the nature of the induced change, that is, decrease of the intensity of pain of acid pattern, and increase of pain of an alkaline pattern. This effect in opposite directions, occurring after the same short interval and seen in hundreds of cases, eliminated the possibility of a psychological factor, as suspected at the beginning.

The fact that the change occurs at the level of the painful lesion itself raised the question of rapid transport between the injection site and the lesion. In order to investigate it, we used two fatty acid preparations containing easily identifiable substances, norbixine and polyconjugated fatty acids. Norbixine could be identified by its characteristic color while the polyconjugated fatty acids were identified by their specific curves in spectral analysis in ultraviolet light.

Adult New Zealand rabbits were injected intraperitoneally with 8 cc. of 0.3% solution of bixine in sesame oil. The injected animals were bled at different intervals by heart puncture. The red cells were separated from the plasma by immediate rapid centrifugation. Each fraction, plasma and red cells, was hydrolyzed separately with 5% KOH. The acid lipidic fractions obtained as a solution in benzene was passed through a chromatographic column with alumina. Bixine was easily recognized because of its red color. After elution with chloroform the amount present was determined photometrically. Similar experiments were made by using a 10% solution of eleostearic acid in oil. The acid lipidic fractions obtained separately from the red cells and the plasma were submitted to spectral analysis and the presence and amount determined by the characteristic peaks. Both norbixine and eleostearic acid were seen to appear in the red cells in less than two minutes, the amount increasing rapidly.

A marked difference was found between the amount of these fatty acids in the red cells and in the plasma of the same blood, for all the samples. The red cells contained 5 to 6 times as much of the injected lipids as the plasma. This unequal distribution, also seen for other highly unsaturated fatty acids, indicates an important physiological role for the red cells which has not been recognized before. Red cells appear to be preferred vehicles for transporting polyunsaturated fatty acids through the blood.

Chapter 12, Note 3. Conjugation Method

Spectral analysis of a mixture of fatty acids such as obtained from cod liver oil has shown that prolonged conjugation sometimes is detrimental for some members. Prolongation of conjugation was found necessary, however, since the members with a lower number of conjugated double bonds needed more time to appear. We investigated the factors which would intervene in these changes. High temperature was seen to affect the polyconjugated formations. With ethylene glycol or glycerol as solvent, conjugation took place rapidly, but the peaks of tetraenes and especially pentaenes and hexaenes were seen to go down rapidly. This was not seen to occur if the temperature of conjugation was lower. In this last case, the conjugation was seen to take much more time. This study led us to use ethyl alcohol as a solvent. Maximum conjugation however, required a longer time, usually around 100 hours. With this method we could obtain from the same preparations much higher amounts not only of conjugated pentane and hexane but also diene and triene. (Fig. 280) We also utilized the same method for analytical purposes with the same good results.