Thomas B. Osborne

Experimental production of rickets with diets of purified food substances.

The possibility of producing experimentally in animals symptoms essentially identical with those associated with rickets in human beings promises to advance the study of this disease greatly. The efforts in this direction have hitherto been concerned with the effects of diets of varied composition—usually for the most part mixtures of natural foods or materials derived therefrom without much manipulation. This has made comparisons between different rations somewhat difficult and often unconvincing because with a change in the natural foods several chemical ingredients are altered at the same time and consequently the cause of any marked change induced thereby in the animal usually cannot be charged directly to changes in any one chemical factor. For example, Sherman and Pappenheimer have demonstrated that rickets is brought about in a few weeks in rats by a diet of patent flour 95 per cent plus a mixture of three inorganic salts (Ca lactate 2.9 per cent, NaCl 2.0 per cent, Fe citrate 0.1 per cent). In experimental feeding tests under otherwise comparable conditions we found that the introduction of 10 per cent of a protein (lactalbumin was used) to replace an equal weight of flour in the Sherman-Pappenheimer ration increased the severity of the symptoms. The calorie value of the two foods was essentially alike. One diet was made far richer than the other in protein of good biological quality; but this result was attended with “dilution” of the flour so that the various food factors which it specifically introduced into the diet, viz., phosphates and certain other inorganic ingredients, vitamines, and carbohydrates, were decreased. Were the gains or the losses responsible for the increased severity of the rickets? Obviously the physiological “analysis” or interpretation becomes complicated where several food factors are altered by an even seemingly simple change in the diet.

The influence of butter-fat on growth

When young rats are fed on mixtures of isolated food substances and inorganic salts such as the “protein-free milk” foods earlier described by the authors, 1 they cease sooner or later to grow and they then decline upon these diets. Milk food speedily brings restoration of growth; and it has been shown that the “essential” accessory factor responsible for this effect is a component of the cream which is present in butter. 1 Further experiments now indicate that the butter-fat separated by centrifugal methods from unsalted butter contains the substance which averts the cessation of growth and possible nutritive decline noted when lard is used instead of milk-fat. Butter-fat thus prepared is free from nitrogen, phosphorus and ash-yielding constituents. The growth-promoting substance therefore is not a phosphatide (lecithin) or an inorganic compound.

Kidney hypertrophy produced by diets unusually rich in protein

In preliminary reports 1 Osborne and Mendel demonstrated that rats can grow to considerable size on diets consisting of nine-tenths or more of protein, provided that they receive a suitable supply of vitamins A and B as well as of inorganic salts. Both casein and washed meat were used as the sources of the protein. Similar tests have since been made with rations containing about 75 per cent. of protein in the diet. It seemed unlikely that rations on which young rats grew from 60 to 260 grams could be extremely harmful to the organism. However Squier and Newburgh 2 have concluded, in harmony with a widespread popular belief, that “a high protein diet in man is a renal irritant” and Newburgh and Clarkson 3 have described the production of arteriosclerosis in rabbits on “diets containing 27 and 36 per cent. of protein derived chiefly from beef.” For this reason it seems worth while to give a preliminary account of our observations on some of the organs of rats growing on our diets very high in protein. The only striking change was found in the kidneys, which in the animals on the high protein diets were greatly hypertrophied. The average weight of the kidneys was almost twice that of the kidneys of control animals and their size about one-third greater. Microscopic examination showed no changes of an inflammatory or degenerative nature. The exact histological condition of the kidneys and of the other organs will be reported in full in asubsequent paper. Hypertrophy of the kidneys existed without hypertrophy of the heart. The ratio of the weight of the heart as well as of the liver to the body weight was about the same in the animals fed the high protein diets as in the control animals. The ratio of the weight of the kidneys to the body weight in the animals on the high protein diets was, on the average, almost double that of the control animals.

Nutritive factors in plant tissues. V. Further observations on the occurrence of vitamin-B

In the course of our studies of the distribution of vitamins in plant products we have collected data regarding a number of important edible foods for which no information in this respect seems to be available at present, with the possible exception of indirect suggestions obtained by other than animal feeding trials. Our experiments, made with rats, supplement numerous earlier ones 1 conducted by the same technique and indicate that asparagus, celery, dandelion, lettuce, and parsley all contain noteworthy amounts of vitamin-B. The details of the investigation will be published elsewhere.

Does growth require preformed carbohydrate in the diet

Carbohydrates are ordinarily regarded as indispensable components of the food intake. This belief is based on the presence of more or less carbohydrate in the food mixtures consumed by man and the higher animals, and the fact that sugar is a constant constituent of the blood. Furthermore, it has been concluded that carbohydrates are essential for the proper metabolism of the fats because ketone substances may be excreted in diabetes when sugar fails to be burned up in the normal manner in the organism. We have found that rats receiving a diet in which the amount of digestible carbohydrate was at most exceedingly small can grow from an early age to adult size. The rations which we fed included protein—casein, edestin, or lean beef which had been thoroughly extracted with boiling water—inorganic salts, agar-agar, lard, butter fat and 0.4 gm. daily of dried brewery yeast furnishing vitamin B. The yeast can scarcely be regarded as a significant source of available carbohydrate. Success was likewise attained in experiments in which no agar-agar was introduced. In the latter case the only obvious sources of preformed carbohydrate were the yeast employed and such carbohydrate impurities as might still adhere to the protein preparation fed.

Growth on diets containing more than ninety per cent. of protein

Although it has been demonstrated that a carnivorous animal can be kept alive and maintained in activity for considerable periods on an exclusive diet of meat it is not known whether growth as well as maintenance can proceed on a regimen entirely free from both fats and carbohydrates. Hammarsten has stated that omnivora and herbivora cannot survive on such a ration. The few experiments on record in relation to this problem have without exception been conducted on a wrong plan, the food mixtures being inadequare in respect to one or more essential factors. Our successful experiences in growing rats on foods extremely poor in fats 1 and in carbohydrates 2 respectively encouraged us to test diets containing only minimal quantities of both. The mixtures included protein 95 per cent., inorganic salts 5 per cent., along with a supply of vitamins A and B in the form of tablets of alfalfa (0.4 gm.) and dried brewery yeast (0.2 gm.) daily. On such diets, when casein furnished the protein component, animals have already grown to three times their weight at the beginning of the trial. The vitamin-bearing substances were the only noteworthy sources of either fat or carbohydrate, and supplied 4-8 per cent. of the food eaten. Whether rats will attain adult size and normal function on such diets, furnishing protein as the almost exclusive source of energy and tissue substance, remains to be determined further. If future experiments prove as successful as those here described various problems of nutrition and physiological function can be approached from new experimental standpoints.

Do fruits contain water-soluble vitamine?

Although fresh fruits have long been classed as valuable antiscorbutic foods there are comparatively few recorded scientifically planned tests of their potency aside from the familiar studies of the juice of oranges and lemons. With respect to the possible presence, in fruit, of water-soluble vitamine (water-soluble B) comparable to this essential factor in yeast, scarcely anything has been published. We have begun experiments on rats in the otherwise adequate diet of which fruits and fruit juices furnish the the sole source of the water-soluble vitamine. When larger portions (more than 5 grams per day) of fresh apples and pears are fed the characteristic decline in weight observed where vitamine-free diets devoid of water-soluble vitamine are used, is averted. The bulky character of such fruits has made it impracticable to feed more than 10 grams per day without decreasing too greatly the intake of other essential nutrients. Ten c.c. of orange juice per day suffice to promote considerable growth. The inner peel of the orange (which Hess has found to be antiscorbutic) seems also to contain some of the other water-soluble vitamine. It is already evident that the proportions of the latter in the fruits tested is not large in relation to the quantities edible.

Vitamines in green leaves

In view of the very scanty data recorded respecting the relative content of vitamine in green foods compared with other plant and animal products which have been studied more in detail, we have fed albino rats on diets containing, as the source of water-soluble vitamine, spinach, cabbage, clover, timothy and alfalfa, dried in their immature state. Far less dried spinach supplies sufficient water-soluble vitamine to promote normal growth than do whole wheat, soy beans, dried egg, meat, milk or potatoes. Spinach leaves are much richer in the fat-soluble vitamine than are most of the products used in our ordinary rations. Thus rats fed for over 160 days, during which they consumed only 25 to 34 grams of spinach, have grown from 60 to 250 grams at a nearly normal rate and have as yet shown no evidence of deficient nutrition. Such quantities are not much larger than have heretofore been considered to be necessary when butter fat supplied the fat-soluble vitamine. A somewhat larger quantity of cabbage than of spinach leaves is needed to promote normal growth. It probably also contains the fat-soluble vitamine. Timothy, clover and alfalfa contain both vitamines, but further experiments are needed to establish the relative amounts of these. Tests of a large variety of tubers, stems, leaves and fruits are now in progress. If one may draw conclusions from the limited data at present available, it seems that the green vegetables and fodders are richer in vitamines than most of the staples like meats, potatoes, cereals, fats and sugar products, in the diet of man. Therefore they unquestionably contribute largely to the dietary need of the average person.

The nutritive value of some cotton-seed products in growth

When certain animals are fed on a ration containing an abundance of cotton-seed meal they frequently give evidence of so-called cotton-seed injury. This has been attributed to irritation from the indigestible husks, the oil, harmful microorganisms, and specifically toxic chemical compounds. The possibility suggests itself that the rations are frequently far from ideal or adequate in respect to the various essential nutrients, inorganic salts and “accessories.” Richardson and Green 1 have found that when the ration of rats is otherwise suitable, toxic symptoms do not follow the use of cotton-seed meal. With their approval we refer to our own experiments, which are still in progress. To ascertain whether the cotton-seed proteins are notably deficient for the purposes of nutrition, we have conducted feeding experiments on rats in which these proteins furnished practically all of the food nitrogen and in which the other essential dietary components were supplied by adding to the products to be tested a suitable mixture of “protein-free milk,” butter fat and starch which, with the addition of adequate protein, has been shown in hundreds of experiments to be sufficient for perfect growth. In this way we have found that satisfactory growth can be made by rats when either cotton-seed globulin or the total cotton-seed protein precipitated from alkali extracts of cotton-seed meal is employed without other significant protein sources in the mixture. No toxic symptoms have appeared, even when the supposedly harmful meal also was used, during a period in which the animals reached a large size. In experiments in which the inorganic components were furnished by our “artificial protein-free milk” there was no failure of growth when the cotton-seed meal was used, thus suggesting that the latter contains the equivalent of the “determinant,” “food accessory,” or “vitamin” deemed essential for nutrition and furnished in fat-free milk.

The influence of beef fat on growth

The inability of young albino rats to complete their growth on a diet consisting of isolated proteins, starch, “protein-free milk” and lard has directed attention to the need of some substance to supplement the ordinary nutrients so that the characteristic increment in body weight may proceed to its normal limits. 1 Among naturally occurring fats, butter fat, egg yolk fat, and cod liver oil have been shown to be effective as adjuvants to the above artificial dietary in order to promote growth; whereas lard, almond oil, and olive oil behave otherwise. We have now found that beef fat is likewise capable of promoting renewal of growth when it has been checked on the lard diets; or if beef fat is incorporated with the food at an early period there is no cessation of growth until long after the time at which nutritive failures on the inadequate diets usually occur. The content of the growth-promoting substance appears to be smaller in beef fat than in butter fat. By fractional separations it can be obtained in the more liquid portions of the fat—the “beef oil.” Beef oil and beef fat, like butter fat and cod liver oil, seem to exert a curative effect in certain affections of the eyes which the rats experience as the result of malnutrition. A more detailed account of the work will appear in the Journal of Biological Chemistry.