Because of the role of abnormal fatty acids in the pathogenesis of the offbalance type D, particularly related to radiation, a study was made of preparations of acid lipids obtained from abnormal tissues, organs and organisms. In an initial group of researches, animals that had died of radiation sickness, shock, acute infections or after adrenalectomy were used. Guinea pigs infected with B. anthracis and mice infected with strep hemo lyticus were used as sources of abnormal lipids in a large number of experiments. Acid lipids obtained from autolysates of tissues were employed. We also used the fatty acids of cod and sardine oil, which may be considered to correspond not to natural but to altered fatty acids since they were obtained after autolysis of cod liver and whole sardine bodies.

After having determined that conjugation of double bonds is the basis f abnormality in pathogenic fatty acids, conjugated isomers of different fatty acids were prepared and studied. Eleostearic acid obtained from tung oil was used extensively in animal and clinical research. Parinaric acid was obtained from nuts of parinarium laurinum and was used to a lesser degree. Various conjugated fatty acid isomers, recognized through spectral analysis and oxalic indices, were obtained in a higher proportion from different lipoacidic preparations by using a modification of classical methods of conjugation. (Note 3) Many mixtures of naturally occurring fatty acids found in saponifiable fractions were conjugated by the same method. Conjugated di, tri, tetra, penta—and hexaenic fatty acids were further separated from these mixtures and studied. The unexpected relationship of these conjugated fatty acids to oxygen was of interest. While the treatment of linoleic acid at 37°C with oxygen leads to progressive increase in the amount of peroxides present, this does not occur for the conjugated isomer (Fig. 123) apparently because of repeated destructions of the peroxides formed.

Spectral analysis of the fatty acids of the entire body of rats

Fig. 122. Spectral analysis of the fatty acids of the entire body of rats, after chemical isomerization shows the presence of di- and triethenic members, and little of members with more double bonds. (0.002% in ethyl alcohol).

Curves of peroxides of samples of linoleic acid

Fig. 123. Curves of peroxides of samples of linoleic acid (a) and its conjugated isomer (b) induced through the passage of oxygen (100 ml per minute for 30 cc of sample) at 37°C. While peroxides are progressively increasing in the linoleic acid preparation, they do not change in the conjugated isomer.

In general, the effects produced by conjugated fatty acids at different levels of the organization are more intensive and persist longer than those of the nonconjugated isomers. It is interesting to mention here the influence exerted by these preparations upon viruses. Although there are no changes for bacteriophages, a marked influence is seen in vivo upon receptivity of the organism to viruses. Subcutaneous injection of conjugated fatty acids in rabbits establishes a zone of refractivity toward inoculation of smallpox vaccine in the skin, which is greater and more persistent than that observed for nonconjugated isomers. The effect upon microbes also is clear. Gram negative strains with manifest morphological changes were obtained from B. anthracis, and persisted as such for 6-15 passages before the old morphological and tinctorial characters returned. The effect upon red cells and leucocytes in vitro is more apparent than for corresponding nonconjugated isomers. This is also true for the influence upon the respiration in vitro of tissues or ascites cells.

The difference in the effects of conjugated and nonconjugated fatty acids is particularly striking in certain manifestations. For example, pain with an acid pattern is more easily changed to alkaline by treatment with conjugated fatty acids than with nonconjugated isomers. Once the alkaline pattern appears, it persists for a long time. A manifest effect is seen upon lesions induced by radiation. Standardized radiated wounds in rats, which heal in about four weeks in control animals and require six to eight weeks to heal when treated with nonconjugated fatty acid preparations, fail to heal at all when treated with corresponding doses of conjugated isomers of the same fatty acids.

The isomers of fatty acids also differ in their effect upon animals in shock. When anaesthetized animals are scalded by immersion up to the level of the xiphoid in water at 90°C, immediate fatal shock occurs if the immersion lasts for more than four seconds. With a three second scalding the animals die after several hours. The administration of fatty acid preparations markedly reduces the survival time, and this effect is more manifest for conjugated members, especially for eleostearic acid. In general, animals in shock induced by any means, such as by the Noble Collip drum, show a special sensitivity toward conjugated fatty acid preparations.

The effect of conjugated fatty acids at the systemic level, as recognized by blood and urine analyses, is in the same direction as that for the non conjugated but is more manifest.

There are significant differences between the effects of conjugated and nonconjugated fatty acids upon the evolution of transplanted and spontaneous tumors. In a small proportion of mice, a mixture of nonconjugated fatty acids prepared from cod liver oil or sardine oil prevents the appearance of methylcholanthrene induced tumors. The conjugated fatty acids obtained through the treatment of these preparations show this preventive effect in a large proportion of mice. These experiments are interesting from several standpoints and are presented in some detail in Note 4.

Conjugated fatty acids produce an increase in the chloride content of wounds in animals. Values are 40% higher than in untreated lesions. The same increase of chlorides occurs in tumors. When Dba mice with adenocarcinoma were treated for ten days with conjugated fatty acid preparations, and tumors were removed and chloride content determined, values were up to 180% higher than for controls with untreated tumors. (Note 5) An interesting effect was noted in two rat tumors. For years, passages of Guerin's rat tumor and of sarcoma induced in our laboratory by the injection of benzpyrene have shown a peculiar character. Although they grow to large size, the tumors have no necrotic zones. After treatment with conjugated fatty acids, large necrotic zones appeared, leading to ulceration and death. The appearance of these zones of necrosis corresponds to changes in the fundamental character of the tumor. Transplants of fragments of these treated tumors, although taken from nonulcerated regions, or young tumors, resulted in tumors showing early central ulceration. This character persisted without further treatment in succeeding generations. The ulcerating tumors appear to be a mutant of the original and the mutative change can be related to intervention of conjugated fatty acids. This was confirmed by the fact that similar changes were constantly obtained in the same tumors with the same agents. A similar but less manifest and less constant effect is obtained with preparations of cod liver oil fatty acids administered repeatedly in large amounts. The same effect was obtained with the injection of the fatty acids directly in the tumor itself.

The second day wound crust pH shows marked changes toward alkalinity under the influence of conjugated fatty acids. The effect upon regeneration time of liver is also manifest; cells full of fatty droplets do not appear at all or appear much later than in untreated controls. In rats weighing 200 grams, with a large enough dose, such as 2 cc. of 10% solution of polyconjugated fatty acids obtained from cod liver oil and repeated for two days, the adrenals show complete depletion of fats. They become small and red in color and contain no sudanophil material. The liver regenerates with small cells with compact cytoplasm and almost entirely bare of fatty droplets.

The effect of conjugated fatty acids upon the lymphatic system is manifest. A marked involution of thymus, spleen and lymphatic gland follows the injection of conjugated fatty acid preparations, particularly of a mixture of conjugated cod liver oil fatty acids. The effect upon tumors is irregular. In some animals a marked retardation occurs, in others no effect can be observed. In convulsions, no greater effect is seen than that produced by nonconjugated isomers. Blood in vitro assumes a color darker than when other fatty acids are used. Eosinophiles are markedly reduced after administration of conjugated fatty acid preparations. Important changes in organs were obtained with repeated injections of the respective cells treated in vitro with suspensions of these acids. Changes in the analytical values of urine, however, are not greatly different from those obtained with nonconjugated isomers. With sufficient amounts of conjugated fatty acids, a frank hypothermia is obtained.