Heme octapeptides derived from various cytochromes c (CHP), as represented in TABLE I, have been studied by a number of workers as a model for metal conjugated enzymes. Recently we have reported on some oxidase activities besides those of peroxidase in CHP which original cytochromes c lack. On the other hand, yeast and mammalian cytochromes c have been used clinically as a remedy for cerebral apoplexia, poisoning especially due to carbon monoxide, etc., there is, however, no satisfactory answer as to the mechanism of such effects of cytochromes c on these conditions. The purpose of the present study was to produce some types of experimental hypoxia and to investigate the biological effects of CHP in vivo. The methods of producing some types of experimental hypoxia were as follows.
Exp. 1. Hemorrhage: In order to produce hemic hypoxia, male albino rabbits weighing 2.5-3.0kg were bled from the ear vein daily for 8 consecutive days, the amount of blood withdrawn being in the range from 5 up to 20ml in the course of the study.
Exp. 2. Exposure to low pressure: Five or 10 male Imamichi rats, weighing 210g on an average, were exposed to high altitude in a large well-ventilated glass chamber. The rate of ascent was 30mmHg/min, and when the altitude of 250-300mmHg was reached, the chamber was left standing for 1-3 hrs.
Exp. 3. Histotoxic hypoxia: Racemic isoproterenol hydrochloride, 5mg/kg in the form of saline solution, was injected intraperitoneally into male Imamichi rats weighing approximately 210g.
CHP in 1mg/ml saline solution was injected intravenously in Exp. 1, intraperi-toneally in Exp. 2 and Exp. 3. The usual dose for this agent is 0.1-1.0mg/kg of body weight. Control groups consisting of 5-10 animals were given injections of an equivalent volume of saline solution. Serum lipids were extracted with chloroform-methanol and weighed by a modified method of Folch et al. The serum and tissue enzymes, which were homogenized, were calculated by methods as described elsewhere. Histopathological examinations were performed by using preparation of ordinary paraffin sections succeeded by H-E and other stainings and histochemical ones including succinic dehydrogenase stain.
When the number of erythrocytes decreased to 1/3 or less of the initial level on the 6 th or 7 th day, the sera became milky due to elevation of total lipids contents, mostly triglycerides. CHP-Injections inhibited hemorrhage lipemia caused by anemic anoxia as shown in Fig. 1. The effects of exposure to altitude on serum and liver levels were indicated in Fig. 2. Serum GOT, GPT and acid phosphatase increased according to the duration of exposure, whereas liver acid phosphatase decreased apparently. Histologically, the tissue damages due to high altitude were most apparent in the liver where centrilobular cord cells degeneration as the from of vacuolic degeneration and congestion were observed, which were milder in case of animals given CHP than the controls. The rats receiving injections of racemic isoproterenol hydrochloride developed myocardiac necrosis. The tissue levels of GOT was decreased as shown in Fig. 4. Histologically, the cardiac lesions formed by this compound were characterized by formation of several small necrobiotic foci of muscle fibers accompanied by interstitial edema, congestion and cellular infiltration including neutrophills. These lesions were most significant in the subendocardiac myocard of frontar and apical portions of left ventricles. Intensity of these lesions were rather mild in the cases which were administered with CHP.
The data obtained as described above indicate the therapeutic activities of CHP for various ischemic tissue damages. It should be emphasized that the biological functions of CHP in various types of hypoxia may be connected with its characteristic enzymatic activities, but evidence thereof remains to be obtained in a future study.