Nina Simmonds

A delicate biological test for calcium-depositing substances

Some time ago we described two diets, Nos. 2638 and 2677, which we used with a view to the development of a biological test which would show the calcium-depositing power of any given substance. 1 The test was carried out as follows: The faulty diet was first fed to a group of young rats for the purpose of making the epiphyseal cartilage free from calcium, and producing a rachitic metaphysis. After a sufficiently long period had elapsed, the test substance was added to the diet of those animals which were to serve as test subjects, while the faulty diet without the test substance was continued in the case of the control rats. Substances, which when added to the faulty diets enabled the organism to deposit lime salts, caused the reappearance of the provisional zone of calcification in the bones. This biological test we called the “line test,” because the new provisional zone of calcification appeared as a line of calcium salts extending transversely across the bone with a limeless cartilage on one side of it and a limeless metaphysis on the other. The success of this test depends on the use of a diet which uniformly causes the epiphyseal cartilage and the metaphysis to be free from calcium salts. It is not sufficient that a diet should merely produce rickets. The rickets which it produces must be of so severe a type that no vestige of calcium remains in the cartilage, and a wide metaphysis is formed. Moreover, the diet must be so constituted that the animals restricted to it will grow and maintain a fair state of general health and nutrition. The diets which we earlier described were not satisfactory, since they did not invariably produce typical rickets.

II. The prevention of the development of rickets in rats by sunlight

In June, 1919, Huldschinsky 1 reported that the ultraviolet ray exerted a curative action in rickets. The criterion on which he relied was the evidences furnished by the X-ray of calcium deposition at the ends of the long bones. He found that there were definite signs of calcium deposition after four weeks of treatment and that at the end of eight weeks healing was almost complete. In May, 1920, Huldschinsky 2 again reported the curative effects of treatment with the ultraviolet ray in rickets in a series of thirty children, aged between one and one half and six and one half years, who exhibited all clinical manifestations of the disease. In all, healing was accomplished after twenty-two to twenty-six treatments covering a period of two months. In April, 1920, Putzig 3 corroborated the findings of Huldschinsky. He obtained cures by means of the quartz lamp in premature infants suffering from rickets. In July, 1920, Riedel 1 further confirmed Huldschinskys findings in a series of one hundred children suffering from rickets. In June, 1921, Hess 2 confirmed Huldschinskys findings in a series of six cases. The favorable influence of sunlight in rickets has been recognized by some students of the disease for a long time, notably by Palm 3 (1890), and experimental evidence of its beneficial effect on mineral metabolism in the puppy has been furnished by Raczynsky 4 in 1912. Huldschinsky made use of sunlight together with the ultraviolet ray in two cases of his series and Riedel relied on treatment with sunlight in some of his cases, supplementing with the quartz lamp ray only on sunless days. Hess 5 was the first, so far as we are aware, to demonstrate, by means of the radiograph, that sunlight alone exerts the same curative action as the ultraviolet ray.

The prevention of rickets in the rat by means of radiation with the mercury vapor quartz lamp

All the evidence as to the preventive and curative effects in the rickets of human beings of the radiations from the mercury vapor quartz lamp has been furnished by the X-ray. In order to determine the protective action of these radiations in experimental rickets in rats and also to examine the bones themselves we performed the following experiments. Nineteen rats, mostly mixed black and white and about seven weeks old, were placed on diet 3143 which, as previous experience has shown, produces rickets comparable in every respect to the rickets manifesting itself in human beings. Nine rats were kept as control animals under ordinary laboratory conditions in a room completely screened with windows of ordinary glass (cage “R” animals). Ten rats were exposed to the radiation from a Hanovia mercury vapor quartz lamp (Alpine type) (cage “U-V” animals). One animal (16Y) in cage “R” was found paralyzed thirty-eight days after being placed on diet (age about eighty-eight days) and was killed. We have previously pointed out that the development of paralysis of the posterior extremities not infrequently occurs in rats fed on diet 3143. Another animal (26Y) was killed fifty-eight days after being placed on the diet (age about one hundred and eight days); and the other seven animals were killed sixty-four days after being placed on the diet (age about one hundred and fourteen days). The animals in cage “U-V” (rayed animals) were exposed to the radiations at a distance of three feet for varying periods of time daily for sixty-four days and were then killed. The rayed animals as contrasted with the control animals showed marked physical vigor as evidenced by growth, activity, good appetite, thick smooth coats and reproductive power.

Studies on experimental rickets, IV. Cod liver oil as contrasted with butter fat in the protection against the effects of insufficient calcium in the diet

In our experimental work we have made observations which demonstrate in a striking way the differences in the effectiveness of cod liver oil as contrasted with butter fat in influencing the rate and extent of growth, and their effects on the histological structure of the bones. This is well illustrated by the results of restricting young rats to the following diet: This diet induces fairly good growth and fertility during at least 8 to 10 months, notwithstanding its deficiency in calcium. The calcium content of this food mixture is 0.059 gms. per 100. The optimum calcium content of this diet is reached when about 1.5 per cent. of calcium carbonate is added. The phosphorus content of this mixture is 0.3546 gms. per 100, and is not far from the optimum content. This food mixture is slightly alkaline owing to its content of sodium bicarbonate. This food mixture with butter fat to the extent of 3, 10, or 20 per cent. of the food mixture fails to induce an appreciable amount of growth. With 2 0 per cent. of butter fat the animals gain but a few grams in weight, look very inferior, become short and stocky, and are rough coated. This diet when 10 per cent. of casein is added, but without the addition of either butter fat or cod liver oil, causes pathological changes characteristic of rickep. With small amounts of cod liver oil (3 per cent.) no rachitic changes are seen in the bones. Even 20 per cent. of butter fat fails to effectively direct the growth processes in the bones toward the normal condition. They usually die within a few months. This food mixture containing 3 per cent. of butter fat and 1.5 per cent. of calcium carbonate is a highly satisfactory diet for the promotion of growth to the full adult size, the maintenance of high fertility throughout the breeding period in the females and the successful nutrition of the young by the mothers. Even a fifth

The influence of light and darkness upon the development of xerophthalmia in the rat

The preventive influence of direct sunlight and of radiation with the mercury vapor quartz lamp upon the development of experimental rickets in rats has been demonstrated in experiments reported in previous studies. A logical further step was to determine whether or not direct sunlight and radiation with the mercury vapor quartz lamp would also prevent the development of xerophthalmia in rats fed diets which, under ordinary conditions of roomlight, would lead to the development of both rickets and xerophthalmia and of xerophthalmia alone. If this information could be ascertained it would be a valuable contribution to the study of xerophthalmia and by analogy would suggest either the unity or the divisibility of the factors contained in cod liver oil, which prevent and cure both xerophthalmia and rickets. It was desired also to determine whether or not different combinations or groups of light rays—as for example, direct sunlight, quartz lamp radiations and roomlight—have the same or similar influence upon rats fed xerophthalmia producing diets. It was conceivable also that complete absence of light rays might have a different effect upon experimental animals than that produced by radiations showing either a complete solar spectrum (direct sunlight) or spectra considerably different from sunlight (quartz lamp radiation and roomlight). On October 22, 1921, fifteen young albino rats were placed on a diet low in fat-soluble A and phosphorus (3127). Previous experience had shown that young rats on this diet would develop rickets and xerophthalmia. The animals were divided into three groups of five each. One group was to be kept in a laboratory room screened with ordinary window glass; a second group was to be kept in total darkness, excepting for the rays from a red electric light bulb such as is used in photographic dark rooms 1 ; the third group was to be kept in ordinary room-light, but was to be radiated with a mercury vapor quartz lamp.

Is there more than one kind of rickets?1

I. CLINICAL OBSERVATIONS SUGGESTING THE EXISTENCE OF MORE THAN ONE KIND OF RICKETS. For at least two years our attention has been attracted to the possibility that there might be more than one kind of rickets. We were led to think of this possibility as the result of the consideration of certain peculiar manifestations of the disease and associations with other diseases. The facts are as follows: Rickets occurs with great frequency in premature children, even when breast fed. It seems to affect the head more than the extremities or ribs. In a group of cases of rickets the disease shows an especial tendency to involve the shafts of the long bones declaring itself clinically by the occurrence of multiple fractures from trivial causes. There is a curious association between rickets and certain forms of secondary anemia (the so-called alimentary anemias and the anemias of the von Jaksch type). The rachitic involvement of the head in the children suffering from the combined conditions appears to be out of all proportion to the involvement of the extremities. Rickets is associated with the most severe forms of chronic interstitial nephritis or developmental defects of the kidney in which there is the most extreme degree of disturbance in renal function. Curiously, tetany sometimes occurs with rickets and sometimes not. Since the conception that there might be more than one kind of rickets was supported solely by clinical observations of at most a suggestive character, we did not feel at liberty to express it as a definite hypothesis. Recently, however, our experiments have yielded results which indicate that there may be two distinct forms of the disease. II. THE EXPERIMENTAL EVIDENCE. In previous publications 1 we have described certain defective diets which, when fed to the young rat, produced marked disturbances in the growth and calcification of the skeleton.

The distribution of the fat soluble A, the growth-promoting substance of butter fat, in the naturally occurring foodstuffs.

That butter fat and egg yolk fats contain a substance whose chemical nature is unknown, which is indispensable for growth or prolonged maintenance of health was first pointed out by McCollum and Davis. Later they showed the presence of this substance in the maize kernel and in wheat embryo, and presented some evidence that if it is found in the oat kernel it is in very small amount. 2 Our further studies have confirmed these observations. McCollum and Kennedy 3 have discussed the desirability of employing the term “fat-soluble A” for this, to distinguish it from the “water-soluble B,” a substance which is widely distributed in the natural foodstuffs of both animal and vegetable origin and is likewise indispensable for growth or prolonged maintenance. The water-soluble B only is concerned with the production and cure of polyneuritis in pigeons. Our experimental work with the grains has shown that the content of the fat-soluble A is greater in the maize kernel than in wheat, and greater in wheat than in the oat kernel. In all three the content is too low to induce growth at the maximum rate even though all other factors in the diet be near the optimum. We have much experimental evidence indicating that the unknown A is principally confined to the germ of the seed. Sunflower seed appears to be fairly rich in this substance. We have also found that the leaves of certain plants, especially alfalfa and cabbage are very rich in the fat-soluble A as compared with the grains. It is probable therefore that it is universally associated with metabolizing plant cells. We have rats in our colony which have grown to very near the normal adult size at slightly below the normal rate on a simple mixture of polished rice sixty and powdered alfalfa leaves forty per cent.

Studies on experimental rickets, V. The production of rickets by means of a diet faulty in only two respects

The following diet, when fed to the young rat, in a comparatively short time (three to five weeks) produces rickets. The diet is composed of: This diet is extremely poor in fat-soluble A, the anti-xerophthalmic substance. Young rats develop xerophthalmia when placed upon it in from four to five weeks. Its proteins are of good quality and are supplied in abundance (21 per cent.). The phosphorus content is relatively low (0.209 gms. per 100 gms. of the food mixture). The calcium content is approximately the optimum. The gross evidences of rickets in the rats eating the faulty ration were briefly these: deformities of the thorax consisting in flattening or hollowing along the line of costochondral junctions; pigeon breast; enlargement and distortion of the costochondral junctions; fractures of the ribs; enlargements of the ends of all the long bones; diminished resistance of the bone to cutting; great diminution in the tensile strength; and finally, the presence of a zone of a white or pale yellow color, between the cartilage and the shaft, visible to the naked eye, the rachitic metaphysis. The microscopic evidences of rickets in the skeleton of the rats receiving the diet were convincing. There was increased thickness and great irregularity of the proliferative cartilage which extended in irregular prolongations toward the shaft, complete absence of calcium deposition in the cartilage or great defects in calcification, the presence of an intermediate zone between cartilage and shaft, the zone already alluded to as the rachitic metaphysis, composed of cartilage in all stages in the process of metaplasia into osteoid, osteoid trabeculz, blood vessels accompanied by marrow elements, irregular deposits of calcified intercellular substance encased in osteoid and, finally, by broad osteoid investments of the trabeculz of the shaft.