H. C. Sherman

A dietetic production of rickets in rats and its prevention by an inorganic salt

The occurrence of rickets in white rats maintained under laboratory conditions has been well known to pathologists since the first publication of Morpurgo in 1900; and the essential identity of the lesions with those of human rickets has been established by the work of Morpurgo himself, of Schmorl, of Weichselbaum, and especially by the detailed histological studies of Erdheim. One of us (A. M. P.) also has had opportunity to become familiar with the disease in rats, in the course of an investigation of the possible influence of the thymus upon the teeth and skeletal system. In none of these investigations, however, were the dietary conditions of the rats standardized and controlled. In continuing by means of feeding experiments upon rats the study of the mineral elements in nutrition which has engaged much of the attention of one of us for the past fifteen years, we have found a relatively simple diet which has led to the development of rickets in every one of the cases thus far examined; while complete protection was afforded by the addition of 0.4 per cent. of potassium phosphate (K2HPO4) to this diet, or (more strictly) by the introduction of this mount of potassium phosphate in place of a part of the calcium lactate which the rickets-producing diet contained. In our experience healthy rats of families on normal diet when separated from their mothers at 28 to 30 days of age average about 40 grams in weight and a calcium content (of about 0.3 gram or 0.7 per cent. If then placed upon good diet the calcium content of the body increases in greater ratio than the body weight so that the percentage of calcium in the body rises continuously until at about four to six months of age the adult percentage of calcium—about I to 1.25 per cent. of the body weight—is reached, after which the weight of the body and the weight of calcium which it contains continues parallel until growth is complete.

Experiments with Reference to the More Heat-Stable Factor of the Vitamin B Group (Factor P-P, Vitamin B2 or G).

Sparing solubility of this substance in alcohol. Extraction experiments with 80% alcohol were deemed of special interest because of the use of such alcohol extracts by Goldberger and associates 1 and because alcohol of almost the same concentration (79%) had been used by Osborne and Wakeman 2 for the precipitation of their “fraction II” which contained a relatively high concentration of both of the recently differentiated water-soluble factors required in the growth of rats. In the present experiments, 400 gm. of air-dry bakers yeast were treated with 1500 cc. of alcohol (80% by weight), thoroughly stirred, and allowed to stand at room temperature (20°-25° C.) for 24 hours; then filtered with suction, and the yeast washed on a Büchner filter with 750 cc. of alcohol of the same strength; then again stirred with 1500 cc. of the alcohol, allowed to stand 24 hours, filtered and washed as before. The residue was dried in the air at room temperature. The extract obtained by combining the 2 filtrates and the washings was a clear yellow solution of pH 6.1. It was concentrated on the steam bath and evaporated at room temperature on cornstarch. Feeding experiments indicated that this extract contained the greater part of the antineuritic vitamin but scarcely measurable amounts of the heat-stable factor. The extracted residue, however, was somewhat less potent than the original yeast as a source of this latter factor. Extraction as above with alcohol 60% (by weight) gave a greenish yellow-brown solution of pH 5.9, which frothed easily. Feeding experiment with the extracted residue indicated that it retained about one-half of the heat-stable factor of the dry yeast. The evaporated extract, while showing decided activity, seemed less potent than the extracted residue.

The balance of acid-forming and base-forming elements in foods, and its relation to ammonia metabolism

In continuation of previous work 1 ash analyses have been made of a number of foods and from the percentages of total sulphur, phosphorus and chlorine on the one hand, and of sodium, potasium, calcium and magnesium on the other, the excess of acid over base or of base over acid which will result from the oxidation of the food has been calculated. Previous ash analyses have also been studied and supplemented by such determinations as were necessary to permit the calculation of this balance for a wide range of food materials. Meats and eggs show a predominance of acid-forming elements; in fruits and vegetables the base-forming elements predominate. From this standpoint the fruits and vegetables tend to balance the meats of the diet. Milk and the cereals contain acid-forming and base-forming elements in more nearly equivalent proportions. Through the kindness of Mr. L. H. Smith, samples of corn which had been bred through ten generations for high and low protein content respectively were obtained from the Illinois Agricultural Experiment Station. The ash-analyses of these were very similar except for the higher sulphur content of the high protein corn, which resulted in this sample showing a slight predominance of acid-forming elements, while in the low protein corn the base-forming elements predominated. In order to determine to what extent the excess of acid brought into metabolism by the oxidation of the food is neutralized by ammonia in man, an experiment was made in which the influence upon ammonia excretion of a known change in the diet was studied quantitatively. The change of food consisted in substituting rice for potatoes in a simple mixed diet and (neglecting the feces but allowing for the unoxidized sulphur excreted during each period) was calculated as equivalent to the introduction of 28.3 C.C. normal acid per day.

Growth and reproduction upon simplified food supply

Rats were fed rations consisting of white bread (made without milk or butter) either alone or with only one other article of food. Later, ground whole wheat was substituted for white bread in several cases. In preliminary experiments with animals placed upon the experimental rations at the time of weaning, bread alone resulted in cessation of growth at once and death after about six weeks. With bread and meat there was some growth at first, but the survival period was only slightly longer than with bread alone; with bread and apple there was no growth, but the survival period was considerably longer; with bread and turnip there was continuous slow growth; with bread and milk there was continuous growth at a normal rate. In this case the bread and milk ration consisted of equal weights of fresh bread and market milk, making a food mixture in which the white bread furnished four fifths and the milk one fifth of the total calories. On this ration young rats of both sexes (taken at weaning time from mothers which were receiving mixed food) made normal growth and the males were capable of normal reproduction but the females usually failed to breed and none of them raised any young. On a ration containing the same proportion of milk (about one fifth of the total calories) but with whole wheat instead of white read or patent flour, young were successfully suckled (though at the cost of considerable loss of weight on the part of the mother) and are growing at somewhat less than the average rate. When about two fifths of the total calories were supplied by milk and the rest by whole wheat, the mother has suckled the young without undue loss of weight and the young have made a fully normal rate of growth.

The Relation of Vitamin D to Deposition of Calcium in Bone.

Quantitative studies in our laboratory 1 have shown that when the food of the growing animal (rat) includes a liberal allowance of vitamin D and a constant supply of phosphorus (0.42%), the calcium content of the bodies as determined at intervals during the period of growth is markedly influenced by the calcium content of the food, as varied in these experiments from 0.16 to 0.50% of calcium in the dry food mixture. In the present experiments we find further, that in rats receiving a basal diet containing a generous allowance of calcium and phosphorus in good proportions (calcium, 0.74%; phosphorus, 0.58%) but with the vitamin D supply restricted practically to a bodily store acquired by 21 to 28 days of age, the growing body has at a given age about the same calcium content as has been acquired by similar animals which received 0.32% calcium, 0.42% phosphorus, and a liberal supply of vitamin D. The calcium content of the femurs of rats which had been kept from the age of 21 or 28 days to the age of 56 days on the vitamin-D-deficient diet which we have previously described 2 is about twice as high as in otherwise similar rats fed the high-calcium rickets-producing diet of Steenbock (No. 2965) for the same length of time. Our rats have not had rickets according to the criterion of the “line” test, yet the deposition of calcium in their growing bones has been influenced by the vitamin D content of the food, even though the percentages of calcium and phosphorus in the dry food mixtures were favorable and uniform. Graded amounts of vitamin D supplied in the form of whole (summer) milk powder induced graded increments in calcification up to what appears to be the maximum potential level.

further experiments upon the purification of pancreatic amylase.

In recent years much light has been thrown upon the chemical nature of some typical enzymes, both by the direct method of purification and analysis and by other methods which, while indirect, have phoven none the less instructive. Pancreatic amylase has been purified by the methods developed in this laboratory 1 to a maximum enzymic activity practically constant in many independent purification experiments. This material in the dry state is an amorphous white powder having the ultimate composition, and showing the color reactions of typical proteins. Moreover, the enzyme material showed, on analysis by the Van Slyke method, 2 the same forms of nitrogen as are found in typical proteins, and in typical quantitative proportions. Like the purified malt amylase previously described by Osborne, 3 this pancreatic amylase on heating in water yielded a coagulated albumin and left in solution a proteose or peptone. Our purified pancreatic amylase preparation has much the highest enzymic activity of any material of which we have found adequate record. In thirty minutes at 40°, this material splits 20,000 times its weight of starch and forms 10,000 times its weight of maltose; and notwithstanding its gradual inactivation in solution, this material has, in sufficiently long experiments, digested as much as 4,000,000 times its weight of starch and formed as much as 2,800,000 times its weight of maltose. 4 This great enzymic activity was shown at a dilution of 1 :100,000,000, whereas the most delicate tests for protein are probably not valid at dilutions greater than about 1:100,000. Thus the failure of protein reactions in solutions enzymically active does not show, as many writers assume, that the enzyme is of other than protein nature, since as in this case, the enzymic activity may constitute a test 1000-fold more delicate than any reaction which can be employed as a test for the presence of protein material.

The Calcium Content of the Body in Relation to That of the Food.

The importance of calcium in normal development accompanying growth, and the purely scientific interest in the so-called law of the minimum, have both emphasized the desirability of more extended investigations of the relation between the calcium intake and the calcium content of the growing body. In the present investigation, chemical analyses have been made of the bodies of large numbers of rats which, starting from comparable heredity and nutritional backgrounds and with all factors other than food maintained strictly uniform, had received food of differing calcium content. The calcium content of the diet was varied in two ways: (1) by varying the ratio of two natural foods of widely different calcium content wheat and milk; (2) by feeding specially designed laboratory diets in which calcium, in the form of calcium lactate, was the sole variable factor. In both cases vitamin D was amply supplied. It was thus found that individuals otherwise well nourished but with low calcium intake do grow up with calcium-poor bodies. It is also evident as a result of these experiments, that the body may be able to retain calcium at a sub-normal rate on a diet relatively poor in calcium and so at an abnormally late age may finally come to approximate a normal percentage of body calcium. Application of these findings to the problem of normal human nutrition should prevent any relaxation of attention to adequate calcium supply in the food. Vitamin D as a “calcium mobilizing” factor is not a substitute for calcium. The body at birth has a low calcium content and its normal development involves a large increase not only in the amount but also in the percentage of calcium which it contains, and so requires a liberal supply of calcium in the food.

A quantitative study of the destruction of vitamin B by heat

Using the rat growth method with the system of controls and precautions developed by Dr. Edgeworth, Dr. Spohn and the authors, studies were made of the percentage destruction of vitamin B at the temperatures 100°, 110°, 120°, 130° C when in each case the heating was continued for 4 hours and the vitamin was contained in a water solution (tomato juice) at the natural acidity of the tomato juice, namely pH = 4.3. The juice of canned tomatoes was chosen as a suitable form in which to study vitamin B and for the further reason that the results here obtained would thus become directly comparable with the results of the studies, previously made in our laboratory, upon the heat destruction of vitamin C. For descriptions of methods and for experimental data and their discussion, reference must be made to the original papers in the Journal of the American Chemical Society. 1 In the paper of which the present communication is an abstract the effects of different amounts of vitamin B upon the weight curve of the young rat are shown quantitatively and the percentage of vitamin destroyed by heat at each temperature is computed from the experimental determination of the quantity of heated juice which shows the same vitamin value as a given standard quantity (usually 4 cc. per rat per day) of the unheated juice. It is also explained that the percentage destruction computed from the experimental data may differ slightly according as we accept as coincident those results which approximate each other within close limits of experimental error (method A) or “correct” such results by computing what change in the quantities of juices fed would have brought the final points of the weight curves into exact theoretical coincidence (method B). These two methods of interpreting the experimental data yield respectively the following results:

The Effect of Temperature and of Hydrogen Ion Concentration upon the Rate of Destruction of Anti-Scorbutic Vitamine.

Experiments were made upon 300 gram guinea pigs fed a new basal diet designed to furnish practically optimum amounts of all nutrients except the antiscorbutic vitamine. The latter was furnished exclusively in the form of filtered canned tomato juice. Relative amounts of this vitamin in the treated and untreated portions of this juice were measured by determining the amounts necessary to prevent scurvy or by a quantitative rating of the severity of the scurvy produced. The technique and the probable degree of precision of the results will be discussed in a later paper. In the case of tomato juice of natural acidity, PH 4.2, it was found that boiling for one hour destroyed practically 50 per cent., and for four hours practically 70 per cent. of the antiscorbutic vitamine present. The time curve of the destructive process is therefore much flatter than that of a unimolecular reaction. The latter finding applies also to similar heating experiments at 60° and at 80°. In such experiments at 60° to 100°, the temperature coefficients are relatively low (Q10 = 1.1 to 1.3). In experiments in which the natural acidity was first neutralized in whole or in part, the juice then boiled for one hour and immediately cooled and reacidified, it was found at PH 5.1 to 4.9 (natural acidity less than half neutralized) the destruction during one hours boiling was increased to 58 per cent. Neutralization of a larger proportion of the natural acidity regularly increased the rate of destruction of the vitamine at 100°. When alkali was added to an initial PH of I I, which fell to about 9 during the hour of heating, the destruction found by feeding of the juice thus treated but immediately cooled and reacidified, was about 65 per cent.

Growth and reproduction upon simplified food supply. II. Influence of food upon mother and young during the lactation period

Breeding rats were fed upon diets containing respectively one sixth whole milk powder to five sixths ground whole wheat or one third whole milk powder to two thirds ground whole wheat. Young were successfully reared on both diets and both would be regarded as adequate for growth, reproduction and successful suckling of the second generation. The larger proportion of milk in the second diet resulted in the following evidences of improved nutrition: (1) Increase in the number of young produced. (2) Increase in the percentage (and therefore also in the number) of young successfully suckled. (3) Better maintenance of the body weight by the mother while suckling the young. (4) Higher average weight of young at a standard weaning age of four weeks. (5) More economical utilization of the calories of food consumed (as well as of the body material of the mother) in the rearing of the young to weaning age.