A third group of natural constituents, which act as anti fatty acid agents is composed of sterols, which are absorbed and circulated bound to polyunsaturated fatty acids. Except in brain and red cells where only free sterols are encountered, the sterols are found both as esters and free substances in all cells, tissues and organs.

Cholesterol, phytosterol and a few sterols were utilized in pure form in our research. In addition, sterols were obtained and used as mixtures, as in the insaponifiable fractions of tissues, organs, organisms and biological products. In some studies, these fractions were further separated into constituents, largely through column chromatography. We used cholesterol in different preparations. (Note 4) Watery or gum cellulose suspensions were used for in vitro and in vivo studies.

Cholesterol was seen to induce a change in the shape of some bacilli, such as B. subtillis, B. megatherium and B. anthracis, turning them into irregular round formations. At the same time, their Gram positive staining became abnormally intense. The agar cultures had a creamy aspect. The influence upon Gram positivity explains the fact that Gram positive individuals could be obtained in cultures of various Gram negative microbes such as Esch. coli or Eb. typhy after repeated treatment with colloidal cholesterol preparations added to broth. The Gram positive forms, however, could not be isolated.

Cholesterol was seen to influence red cells in form, shape, volume, sedimentation, velocity, and oxygen combining capacity. A vermillion color, which persisted for a long time, was obtained through in vitro treatment of blood with cholesterol or through intravenous injections in animals. Although such injections were lethal, they induced the abnormal vermillion color. Cholesterol produced a manifest change toward less alkaline values in the second day wound crust pH. A favorable effect upon rabbit skin wounds was obtained, with abnormally intensive proliferation of the epithelium, this healing effect, however, was less manifest for irradiated wounds. In subjects with ulcerated lesions, prolonged administration of cholesterol was frequently observed to induce hemorrhages, especially of an arterial character. However, in patients with coronary occlusion or endarterial obliterations, the administration of cholesterol was followed by an increase of symptoms apparently related to exaggeration of the degree of occlusion. This effect upon blood vessels also could be seen in animal tumors. Administration of cholesterol induced zones of necrosis in the tumors which could be related to proliferation of the endarterial cells leading to thrombosis and ischemic infarct. The portions of tumor corresponding to these ischemic infarcts showed characteristic necrosis with unaltered structure but without the normal staining.

In animals injected with cholesterol and then submitted to trauma in the Noble Collip drum, shock was prevented. Injection of cholesterol prior to the experiment reduced mortality to zero while in untreated controls mortality was high. A similar but less constant effect was obtained when cholesterol was administered immediately after trauma. It is noteworthy that in animals injected with cholesterol before being placed in the drum, the blood not only did not become abnormally black but the usual bleeding from the nose, mouth and paws (if not taped during the trauma) was abnormally bright red.

The effect upon experimental tumors was investigated through the dipping technique repeated in successive generations. Changes in the evolution of certain tumors, such as mammary adenocarcinoma in mice, were induced. The effect of cholesterol was similar to that of insaponifiable fraction but was less manifest and will be discussed in more detail later.

Cholesterol's effect upon the central nervous system was interesting. Often, immediately after administration, both in animals and humans, transitory somnolence was observed. However, repeated administration induced convulsions. (Note 5) Exophthalmia was seen in mice after injection of cholesterol and was most manifest 24 hours thereafter. It contrasted with normals and especially with animals injected with fatty acids or similar lipoids, who showed enophthalmia.

An ether oil cholesterol solution induced paraplegia with early foot ulcerations in rats and rabbits. This occurred particularly in females and was related to a predominance of sterols in this sex. Castration or administration of sex hormones did not change this special susceptibility of the nervous system of females to cholesterol. However, the administration of fatty acids or of acid lipid preparations of organs or tissues did suppress it. (Note 21, Chapter VI) Changes in systemic analyses were generally not obvious and, when present, were slow in appearing. They corresponded to changes toward offbalance A.

Unsaponifiable Fractions (Insaponifiable or Non saponifiable)

When insaponifiable fractions were prepared from various tissues and organs, big differences could be seen in the quantity and the number of sterol compounds naturally present. However, a certain specificity related to the origin of these insaponifiable fractions appeared most interesting. The insaponifiable fractions of various materials were prepared by the usual methods. Most of the fractions are soluble in oil in higher proportion than cholesterol, with some of them even miscible with oil. More concentrated solutions in sesame oil could be prepared than for cholesterol. In most of the experiments, 5 or 10% solutions were used. Colloidal suspensions also were prepared in the same manner as for cholesterol.

In spite of the extreme variations in sources of the insaponifiable fractions, almost all have some properties in common. Some are similar to cholesterol in their effects particularly at the lower levels. The marked differences appear at higher levels. They induce hemorrhages in the adrenals between the fascicular and reticular zones.

On the healing process, especially of radiation wounds, insaponifiable fractions of placenta, embryos and butter—materials related to growth— show impressively greater activity than cholesterol or preparations of insaponifiable fraction of other origin. They induce healing processes even in standardized radiation lesions where cholesterol has a weak effect.

In their influence upon tumors, the preparations of insaponifiable fractions of different organs differ markedly. No changes at all were obtained with some preparations such as from pig intestine, for example, while interesting results were obtained with others. The differences appeared especially evident in experiments in which a direct influence upon the tumor was exerted. Transplants of Ehrlich mammary adenocarcinoma in mice were dipped in insaponifiable preparations and grafted. In general, no immediate visible effects were seen with this technique for the first transplant generation. By repeating the same procedure for following transplant generations, changes were obtained which varied with the preparations used. The insaponifiable fraction of human placenta, for instance, produced a marked increase in malignancy, together with morphological changes, the tumor changing from an adenocarcinoma to an encephaloid. Further treatment of the transplants led to still greater malignancy with a sarcoma toid transformation. Thereafter, negative results were obtained with new treatment of the transplants. (Note 6) With this procedure, plancenta preparations showed a manifest influence even at the third transplant generation, and they were negative passages for the fifth to sixth transplant generations. With pig intestine preparations, even after ten successive passages, malignancy was unchanged.

The specificity according to origin was also seen in other experiments, such as in the influence exerted by these preparations upon the development of specific lesions produced by smallpox virus in low reacting species such as mice or rats. Preparations from receptive animals, and especially from organs sensitive to the virus, were more capable of inducing local receptivity than were preparations from refractory animals. For instance, positive effects were obtained with vaccinia virus in mice and rats previously injected subcutaneously with the insaponifiable fraction of rabbit skin or brain, while no such effects were seen when the insaponifiable fractions of pig or hen intestines were used.

Differences were observed between the insaponifiable fractions of different organs for conditions principally manifested at the organ level. Conditions affecting mainly one organ were treated with the insaponifiable fraction corresponding to that organ. These preparations often appeared much more active than those from other organs. We investigated the effects of a heart insaponifiable fraction on patients with myocardial insufficiency, especially when responses to other therapeutic agents could no longer be obtained. In like manner, we used the insaponifiable fraction of liver for manifest liver insufficiency. The rate of liver regeneration in rats after subtotal resection was found most accelerated by liver insaponifiable fractions. The good effects obtained in the treatment of intractable diarrhea with insaponifiable fractions from pig and hen intestines will be discussed below. We investigated preparations obtained from lymph nodes and spleen for the treatment of shock, particularly in its acute form. Similarly we also used adrenal insaponifiable fraction to influence induced adrenal insufficiency, and brain insaponifiable fraction in an attempt to influence insomnia. The results of these studies will be discussed in the section dealing with therapy. The changes obtained with the respective preparations indicate that they have a specificity which represents an important factor in normal and abnormal physiology.

In a second group of researches, constituents of the rough preparations of insaponifiable fractions from various sources were separated by different methods. The ketonic and nonketonic constituents were obtained and, when tested in animals, showed several differences in biological properties.

Further research of specificity was made using separations through the chromatographic column method. Most of these preparations are still under laboratory investigation. Experiments are being conducted with different organ preparations, some fractions obtained being identified as common to all organs, while others are specific to one organ or to a group of organs. These experiments already have revealed a marked plurality of constituents for the insaponifiable fractions of organs which has to be related to the plurality of constituents found in the acid lipid fractions of the same organs and which was discussed above. The specificity seen for organs would thus greatly concern their lipidic constituents which form the acid and the insaponifiable fractions. It is especially in terms of specificity that the acid lipidic and insaponifiable fractions of various organs are being investigated in research now in progress. (Note 7)