Morphological Changes—The same level separation was used in the study of the effects of lipids on complex organisms. Acting at chromosomal levels, lipids led to the appearance of monstrosities. Various lipids, especially insaponifiable fractions of organs, were injected into larvae of flies. While an immediate change in the cells of the larvae could be traced to the subnuclear level as seen in chromosomes, monstrosities were seen to be induced in the resulting flies. A similar effect became evident when lipids were injected into hens' eggs before or during incubation. Especially with cholesterol but also with insaponifiable fractions of organs, a high proportion of chickens were hatched with spastic paraplegia.

The same problem is being studied, in collaboration with P. Fluss, in Drosophila melanogaster, grown for many generations in media to which an entire series of different lipids from one or the other group are added. This study is in progress and the results will be published later.

We have seen that the antagonistic effects induced by the two groups of lipids could be related ultimately to opposite changes in the fundamental biological process of aging. This appeared clear for lower morphological levels of organization and was especially evident for cells. While anti fatty acids induce changes which can be regarded as corresponding to prolonged youth, the polyunsaturated fatty acids induce rapid aging with pyknosis and karyorrhexis and death of the cells as old entities. This could be seen clearly in tumors, in which cells with youthful character lead to non necrotic tumoral masses, while cells that age rapidly produce necrosis in the tumors followed by ulceration if the tumor is superficial.

The effect of conjugated fatty acids was somewhat more complex, indicating an abnormality in the induced processes. Their administration was followed by the appearance of cytoplasmic and even nuclear vacuoles, corresponding ultimately to an anomaly of water metabolism.

The effect of lipids upon adipous cells appeared to be of special interest. The anti fatty acids, especially the sterols, when injected subcutane ously in animals, induced a characteristic process in the adipous cells near the injection she. These cells became very enlarged and highly irregular, with their content changed into an emulsion only slightly stained with sudan. The fatty acids, on the contrary, imparted to adipous cells an abnormal resistance to destruction. They remained persistently unchanged even in the midst of very active processes which usually cause them to disappear. Unchanged adipous cells were found encircled by the invading cancerous cells, deep within tumors in animals treated with fatty acids.

On Pain—From the start of this research, the opposing effects of the two groups of lipids upon pain has been most impressive. For the fatty acids the degree of saturation is important. The saturated members of the fatty acid series and even oleic acid are entirely without effect. Linoleic and linolenic acids show a slight influence, while the polyunsaturated members show a marked effect. Administration of highly unsaturated fatty acids and of acid lipidic fractions of certain organs, such as placenta, liver, spleen or blood, uniformly decreased pain of an acid pattern and increased pain of an alkaline pattern. These opposite effects have, from the beginning of our study, contradicted the idea that this influence upon pain was the result of the direct action of these agents upon the nervous system. Furthermore, the opposite effects exerted upon the same pain by the other group of lipids have confirmed the hypothesis that the action takes place at the level of the painful lesion, where the differences between the two pains was found to correspond to two opposite acid base offbalances.

In the study of the effects of lipids on the pH of the second day wound crust, made in collaboration with Carlos Huesca, we have demonstrated that lipids influence pain through changes induced on the acid base balance present at the tissue level. The positive lipids constantly lowered this pH while the negative lipids elevated it. (Note I Chapter V) Even more import ant than this temporary pH effect in establishing the mechanism of lipid action in pain was the change in the actual pattern of an existing pain after administration of these agents. Polyunsaturated fatty acids in sufficient amount were found to convert an acid pain pattern to an alkaline pattern, while sterols changed an alkaline pattern into an acid one. We will return to this important fact later.

A pathogenic role for lipids becomes evident too, when pain can be induced through the administration of lipids in previously painless lesions. Such lesions treated with large amounts of lipidic preparations became painful. An alkaline pattern of pain was seen to appear after fatty acid administration, while an acid pattern followed use of the insaponifiable fraction. (Note 36)

At the tissue level, lipids also affect such acid base symptoms as vertigo, itching, dyspnea, tremor, and even mental diseases. In these conditions the same antagonism between the two groups of lipids—and the same opposite effects upon the acid and the alkaline pattern—can be noted along with the same possibility for changing the pattern to the opposite type if big doses of lipids are administered.

Wound Healing—The same manifest antagonism between the two groups of lipids was also noted in their influence upon the evolution of wounds. Changes in the sloughing and healing process were followed by measuring the size of wounds (Note 37) as well as by serial histological examinations. The lipids with a negative polar group were seen to retard the evolution of the processes by prolonging the first catabolic phase. Positive lipids generally had an opposite effect. However, here too it could be observed that sterols have relatively little effect on the healing of connective tissue, but manifestly favor proliferation of the epithelia. This was especially evident in the changes in scar formation of the skin of treated animals. In rabbits treated with cholesterol, the epithelial scars were found to have 8-10 layers instead of the 2-3 characteristic for the rest of the skin and for the scars in control animals.

Regeneration—In collaboration with E. F. Taskier we studied the effect of lipids upon the regeneration of liver in rats, after the resection of almost 3/4 of this organ. The rate of regeneration could be estimated by correlating it to the time of appearance of fatty droplets filling up almost all the cells, as a first step in the regenerative process. In very young animals, this change in fatty liver cells was seen to take place even within the first 24 hours after resection. The change was progressively delayed as the age of operated animals increased. In old animals the change in fatty liver cells appeared only after the fourth day.

The administration of lipids had a marked effect on appearance time of fatty cells. Sterols induced precocious appearance in old animals. From this point of view, sterol treated animals appeared to react as young individuals, with fatty cells evident even on the second day. The fatty acids and acid lipidic fractions of organs showed an opposite effect, delaying the time of appearance of the fatty cells. Young animals treated with polyunsaturated or conjugated fatty acids showed no fatty droplets in the liver cells for as long as three to four days. With higher doses of the same agents, the fatty infiltration did not occur at all.

It is interesting to note a parallelism between fatty infiltration of liver cells and the richness of adrenals in sudanophil substances. An almost complete lipid depletion of the adrenals was seen after high doses of fatty acids and coincided with a total lack of fatty cells in liver regeneration. (Note 38)

Organic Level—Effects of the two antagonistic groups of lipids at the organic level have been studied in terms of manifestations clearly associated with various organs. We will review these effects briefly here, with more details to come when the therapeutic use of lipoids is discussed.