Howard H. Beard

Blood Regeneration in Nutritional Anemia. Influence of Iron, Iron and Copper, Nickel, Cobalt, Germanium, or Sodium Germanate.

Young rats were made anemic by feeding whole milk for a period of 6 weeks after weaning. When the erythrocyte count was about 3-4 million per cu. mm., and the Hb content 4-6 gm. per 100 cc., additions of Fe, Fe + Cu, Fe + Ni, Fe + Co, and Fe + Ge, Fe + Na2GeO3 were made. Erythrocyte counts and Hb estimations were made weekly on a few drops of blood obtained by clipping the end of the tail. A Levy counting chamber with double Neubauer ruling was used. Hb determinations were made by the acid hematin method, color comparison being made with a calibrated Newcomber disc. The results are summarized in the table. When 0.5 mg. Fe was given daily to anemic rats recovery of both erythrocytes and Hb was complete in 4.3 and 6 weeks; with 1 mg. the time for recovery was 3.5 and 4.8 weeks; and with 1.5 mg. it was 3.5 and 3.7 weeks, respectively. Since this iron solution was prepared from standard iron wire using hydrochloric and nitric acids, it would hardly seem that the effect of iron salts, as FeCl3, in blood regeneration could be due to the impurities these salts might contain, as Hart and coworkers suggest. 1 Mitchell and coworkers 2 had previously shown that some iron salts, e. g., FeCl3, were effective in blood regeneration in rats fed whole milk diets. Our results seem to confirm those of Mitchell in respect to this one iron salt. The experiments also appear to confirm those of Hart and his coworkers 3 on the supplementing action of copper when added to iron, but do not indicate that copper is unique in this respect. The data in the table show that traces of copper, nickel, cobalt, germanium and sodium germanate plus 0.5 mg. Fe all brought about regeneration of erythrocytes and hemoglobin in less time than did 0.5 mg. iron alone. A number of other elements have been studied.

Protein and amino acid feeding upon creatine formation in muscle, and creatinine elimination in urine.

It is generally believed that creatine is a tissue constituent with a special function and that it arises in the body as a result of a specific cellular demand for it. There is also much evidence to show that it may also be derived from certain precursors, e. g., arginine, 1 glycine, 2 cystine, 3 and histidine, 4 in the diet. About 3 years ago, using young rats and mice, it was observed in this laboratory that the feeding of d-arginine monohydrochloride gave slightly larger increases in muscle creatine than creatine itself, when each of these substances formed 5% of the standard casein diet. The possibility that this amino acid was one of the precursors of creatine in the animal body was suggested. With positive evidence for the other amino acids mentioned above, a systematic study was begun to determine the influence of feeding proteins, amino acids, and related substances upon creatine formation in the muscles, and creatinine elimination in the urine, the results of which are presented below. Young rats were placed on Shermans Diet B for a period of 10 days after weaning. Amounts of purified amino acids up to 1.5 gm. were fed either as such or mixed with a small amount of the stock diet. In other experiments casein or edestin were fed. At the end of 17 to 48 hours the animals were killed and the muscle creatine determined by the method of Rose, Helmer and Chanutin. 5 The litter mate control animals received no amino acid or protein supplement. The average creatine content of the muscles of 118 control rats was 0.40%. The average results obtained are given in Table I. The effect of amino acid feeding upon creatinine elimination in the urine was next studied with the hope that further light might be thrown upon the origin and metabolism of creatine.

Effect of Radiant Energy With and Without Iron Upon Nutritional Anemia in the Rat

The effect of radiant energy upon the metabolism of small doses of Fe in the nutritional anemia of the rat seems to have been little studied. Foster 1 produced anemia in rats by milk feeding and observed the effect of radiant energy in preventing and curing this type of anemia. There was a slight but definite effect in increasing the hemoglobin and the number, size and saturation of the red cells. We have made the following studies upon the effect of radiant energy upon both the prevention and cure of nutritional anemia in the rat. Preventive Studies. Young rats at weaning were fed upon whole milk. Daily doses of pure Fe from 0.05 to 0.30 mg. were given, with and without 3/4 hour ultraviolet irradiation of the rat daily, with the General Electric Sunlight Mazda Lamp, Type S-I, at a distance of 4 feet. This lamp has recently been described by Carter. 2 Weekly estimations of hemoglobin and red cell counts were made by the technique described by Beard and Myers.8 With milk alone, milk plus irradiation, irradiated milk, and milk plus 0.05 mg. Fe daily the anemia became progressively worse. There was a striking effect of irradiation on red cell recovery with both 0.05 and 0.10 mg. Fe daily, namely, a drop of 41 and 0.6% on these doses of iron may be compared to an increase of 33 and 50% when 3/4 hour irradiation was given. The anemia was prevented by daily doses of Fe from 0.15 to 0.30 mg. These doses with irradiation were also much more effective in preventing the anemia than were these doses of Fe alone, with the exception of the 0.25 mg. dose. Irradiated milk with 0.30 mg. Fe daily gave a much more striking increase in both cells and hemoglobin than any other type of therapy used in this study. These results are given in Table I. A polycythemia occurred in 4 animals on doses of Fe alone and in 12 animals on Fe and irradiation.

Effect of Hydroquinone in Vitamin A Deficiency.

Since the discovery about 1913 that a lack of vitamin A in the diet would cause ophthalmia in rats, attempts have been made to show that this pathological condition might also be due to other factors than the absence of this vitamin. The so-called “salt ophthalmia” of McCullom, Simmonds and Becker 1 , 2 has been shown by McCullom, Simmonds and Becker 3 and Jones 4 to be due to an oxidative destruction of vitamin A in the diet by ferrous sulfate if the diets are not made up frequently. However, when the diet is made up daily, or every few days, no ophthalmia develops. Recently Huston and Lightbody 5 have presented evidence to show that hydroquinone had a definite beneficial effect when used in a diet low or lacking in vitamin A. Huston, Lightbody and Ball6 also concluded that hydroquinone had a definite anti-oxygenic effect on the vitamin A of milk fat and cod liver oil. If these views are correct, a very important physiological rôle must be attributed to this phenol. In order to test out the hypothesis of Huston and Lightbody, we fed 2 groups of rats on a vitamin A free diet consisting of extracted and heated casein, 18 gm., corn starch, 51 gm., crisco, 23 gm., salt mixture (Osborne and Mendel), 5 p., dried yeast, 3 gm., with 2 drops daily of a 5% solution of Vigantol to supply vitamin D. 0.05 and 0.1 gm. of hydroquinone per 100 gm. of the above diet were fed to the 2 groups of animals from the time of weaning. This diet was made up weekly. The 11 animals of these 2 groups all lost weight, developed ophthalmia and died. When 1 part of a substance is included in 1000 parts of food the chances are very good that each animal will not receive a given amount of this small part each day. Hence a third group of rats were fed the above diet from the time of weaning and were given daily by mouth 5 drops of a solution of hydroquinone equivalent to 7 mg. of the phenol. The same results were obtained as described above. 3 animals of this group were cured of ophthalmia by cod liver oil.

Cytological Studies on Experimental Rat Tumors

Conclusions Study of experimental tumors produced by ultraviolet rays in the albino rat revealed low average values for the nuclear-nucleolar ratio of groups of 100 cells. The nucleolus from experimentally produced malignant tumor cells was found to vary greatly in size and number as it does in malignant cells from human lesions.

Pathological Changes in Liver and Spleen in Nutritional Anemia in Rat

Pure milk fed to young rats produces severe anemia which finally results in death in the majority of cases. In our studies of the pathological changes which take place during the course of nutritional anemia in the albino rat, the findings in the liver and spleen seem to have considerable value in the differential diagnosis of this type of anemia as contrasted with the infectious type caused by Bartonella muris. All our examined animals showed a severe degree of anemia with an erythrocyte count of less than 3 million per c.mm. and a Hb content of less than 4 gm. per 100 cc. Most of the animals died spontaneously, while 5 were killed during the course of the disease. The spontaneous death of the anemic animals was consistently preceded by a rapid loss of weight during the last days of life. The liver and spleen were weighed and fixed with the other organs in Zenker, alcohol and formalin. Slides for microscopic study were stained with hematoxylin-eosin, van Gieson, Mallory, Bielschovsky, Sudan III and Turnbulls Iron stain. Changes in the Liver. Macroscopically, the changes were not marked. The size and weight were slightly decreased, the surface smooth and pale, the consistency soft. The cut surface showed a slight yellowish brown mottling and occasional small petechial hemorrhages. The microscopic changes were most pronounced in those animals which died spontaneously from anemia. The central veins and the capillaries in the center of the lobule were greatly distended and filled with an edematous fluid. In the center of the liver lobule the cells were atrophied; those near the center were swollen and showed a marked vacuolization of the plasma. The majority of these vacuoles proved to be fat droplets when stained with Sudan III. They completely filled the cells surrounding the atrophic areas in the center of the lobule and were present even in livers in which atrophy of the central part of the lobules was just in the beginning stage. The periportal zones showed marked hypertrophy of the cells, with no fat, and an increase in the size of the cell and of the nucleus. Most of the cells contained 2 or more nuclei.