EFFECT OF DIETARY PROTEINS AND AMINO ACIDS ON THE SUSCEPTIBILITY OF MICE TO BACTERIAL INFECTIONS

Young mice were maintained for periods of 1 to 6 weeks on experimental diets containing all known growth factors, but differing in their protein and amino acid contents. All diets were supplemented with L-cystine. The effect of the nutritional regimen on infection was tested by inoculating the animals with either one of four pathogens (Mycobacterium tuberculosis var. bovis, Mycobacterium fortuitum, Staphylococcus aureus, Klebsiella pneumoniae type C), and by observing the survival time. The infective dose was administered by either one of three routes: intravenous, intraperitoneal, or air-borne (aerosol). In some experiments, the animals were maintained in groups of five throughout the tests. In other experiments they were housed in individual cages. This difference in housing did not affect the results in a detectable manner. Mice fed diets containing 5 or 8 per cent casein as sole source of amino acid (except for cystine supplementation) proved more susceptible to the experimental diseases than did mice fed diets containing 15 or 20 per cent of the same protein. Susceptibility to infection developed when wheat gluten, or soybean α-protein, was substituted for casein—even in high concentrations (15 or 20 per cent). In one experiment, mice were fed a diet containing as sole source of amino acids a mixture of soybean and rice flour, so designed as to provide a protein concentration of 15 per cent, with an amino acid pattern similar to that of casein. These animals gained weight at the same rate as those fed a diet containing 15 per cent casein and they exhibited a satisfactory level of resistance to bacterial infection. The infection-enhancing effect of low casein concentration (5 and 8 per cent) could be corrected by supplementing the diet with the proper mixture of amino acids. This could be done using either synthetic or natural amino acids. In contrast, susceptibility to infection developed when low casein diets were supplemented with unbalanced mixtures of amino acids. The infection-enhancing effect of gluten diets could not be corrected by supplementing the latter with lysine even though this supplementation markedly improved weight gains in uninfected animals. It appears in conclusion that the relative proportion of the various amino acids in the diet is as important a factor as their total amount in conditioning resistance to bacterial infections. This effect of nutrition on resistance can be detected irrespective of the route of infection: intravenous, intraperitoneal, or air-borne. Moreover, the effect has been observed with two strains of mice differing markedly in their natural resistance to bacterial infection.     

THE INFLUENCE OF HIGH PROTEIN DIET ON THE KIDNEYS

Seven groups of rats were fed on diets containing protein varying in amount from 1.36 to 40.13 per cent derived mainly from grain, casein, meat, and milk with carbohydrate, fat, and vitamines. From a number of animals one kidney was removed to double the load on the remaining one. The time of feeding was from 9 weeks to 6 months. Blood uric acid, blood urea nitrogen determinations, and microscopic examinations of the kidneys revealed no evidence of kidney damage. There was evidence of kidney hypertrophy consisting of increased weight of the kidney, large diameters of the capillary tufts, convoluted tubules, and kidneys in the animals receiving high protein diet. The nephrectomized animals that ate high protein had no kidney changes save hypertrophy and this amounted to an increase in weight of an average of 0.54 gm. or 85 per cent of the average weight of the right kidneys of the controls.     

THE EFFECT OF VARIOUS PROTEIN RATIONS ON THE SERUM PROTEIN CONCENTRATION OF THE RAT

1. The effect of various percentages of protein (casein) in the diet on the serum protein concentration of the rat was studied. 2. With high protein rations (50 per cent or over) there is no drop at all of serum protein concentration, even though the diet is otherwise poorly balanced and inadequate as evidenced by weight loss. 3. With protein rations under 20 per cent the serum protein concentration falls promptly to a level of about 5.25–5.50 gm. per cent, where it is maintained regardless of whether the diet contains 1, 5, or 10 per cent of protein. Further depletion of the blood colloid concentration seems to meet with resistance on the part of the body.     

The relative effects of protein,choline, and methionine in the treatment of experimental dietary cirrhosis in the rat

Cirrhosis of the liver was produced in rats by feeding a diet low in protein (4 per cent casein) and deficient in lipotropic factors. The degree of liver cirrhosis was determined from specimens obtained at biopsy. Comparable groups of animals then were treated with diets containing 4 per cent casein and 30 per cent casein. The 4 per cent casein diets were supplemented with choline and methionine; the 30 per cent casein diets were fed with and without added choline. On supplementing the low protein diet with choline and methionine the animals remained feeble, their growth remained stunted, and their livers showed signs of progressive cirrhosis. In contrast, animals fed the higher protein diet (with or without added choline) grew normally, and their livers showed signs indicating arrest and regression of the disease process. These studies suggest that the feeding of high protein (30 per cent casein) diets to rats with nutritional cirrhosis produces reparative effects greater than those attributable to the supplements choline and methionine.     

ALTERATION OF RESISTANCE OF THE RAT TO TUBERCULOSIS WHEN MAINTAINED ON AN ATHEROGENIC DIET

Albino rats maintained for 50 to 60 days on rations containing cholesterol, sodium cholate, and thiouracil were found to be highly susceptible to the lethal effects of infection with Mycobacterium tuberculosis. Maximum loss of resistance occurred only when all three components were present in a single ration. A lesser degree of susceptibility resulted from the deletion of one or more of the factors from the diet. Animals maintained on control rations never died of the infection. Histopathological studies of stained sections of tissues of infected animals revealed that the reaction of the host to the bacillus was more extensive and less well contained in animals fed the complete atherogenic ration than in the controls.     

THE EFFECT OF VARIOUS DIETS ON THE LIVER DAMAGE CAUSED BY EXCESS CYSTINE

1. The effect of 9 different diets on the liver lesions resulting from excess dietary cystine has been studied in 130 rats. 2. The incidence and severity of each of the following liver lesions were varied by changes in the composition of diets containing 5 or 10 per cent cystine: (a) Hemorrhage was least severe with low fat diets. (b) Necrosis was most severe with synthetic diets. (c) Cirrhosis was delayed by a diet high in lard, 20 per cent, and cod liver oil, 5 per cent, but not by a diet high in butter, 25 per cent. (d) Fatty infiltration was found consistently only with low protein, high fat diets. In other words, the pathogenesis of the liver lesion due to excess dietary cystine can be modified by diet. 3. In the presence of cystine as 5 per cent of a low protein, high fat diet, 1 per cent choline inhibited fatty infiltration but did not protect the liver against damage by cystine. 4. In these experiments there was no apparent correlation between fatty infiltration of the liver and the incidence or degree of cirrhosis.     

Influence of dietary magnesium on cardiac and renal lesions of young rats fed an atherogenic diet

Young male rats fed an atherogenic diet for 24 to 26 days developed magnesium deficiency. The renal lesions, calcium deposition in tubular lumens in the outer zona intermedia, are morphologically consistent with those seen in animals fed magnesium-free diets. It was necessary to feed 8 to 16 times the normal requirement of magnesium to prevent completely these lesions. The limitations in the pathogenesis of these renal lesions are discussed. The addition of 2 mg. per cent of thyroxine to the diet markedly lowered or abolished deposition of calcium in the kidney; it also lowered atherogenesis to a minimum but did not abolish it. Early lesions of atherosclerosis of the cardiac valves and of the aorta were observed. These were more prominent in animals on a 10 per cent than in those on a 20 per cent protein diet. The atherosclerosis was diminished but not abolished by exceedingly high dietary levels of magnesium in the 10 per cent protein group. Such a decrease was not noted in the 20 per cent protein group. There did not appear to be significant variation in the morphology of the fatty livers in and between the various groups and subgroups as judged by staining with Sudan IV.     

Chronic sodium chloride toxicity in the albino rat. II. Occurrence of hypertension and of a syndrome of edema and renal failure

Sustained arterial hypertension developed in male, albino rats chronically fed diets rich in sodium chloride with demineralized drinking water available ad libitum. After 12 months of the experimental regimen a positive, linear correlation (r = 0.91) was found between the systolic blood pressure and the concentration of sodium chloride in the diet. A syndrome of edema and renal failure was observed in 18 per cent of the group fed at the level of 7.0 to 9.8 per cent of sodium chloride. Significant histologic changes occurred in the kidneys and certain other organs in rats consuming rations containing these levels of NaCl. The relative volume of the radiosodium space was increased in the rat by high dietary sodium chloride.     

DIET AND TISSUE GROWTH : I. THE REGENERATION OF LIVER TISSUE ON VARIOUS ADEQUATE DIETS.

1. The toxicity of chloroform varies according to the diets used in these experiments in the following order of decreasing susceptibility of the animals: high fat > standard > high carbohydrate > high protein diets. 2. On the high fat and high carbohydrate diets there may be a more or less marked proliferation of the endothelium and the connective tissue stroma in the necrotic area producing in some instances scars resembling the picture of an early cirrhosis. 3. On the diets studied, standard, high carbohydrate, high protein, and high fat, the most active and rapid repair is observed on the standard balanced diet. On the high fat diet the reparative process is definitely delayed in comparison with the others. There are only slight differences between the high carbohydrate and high protein diets which suggest but do not conclusively show a more rapid repair with the latter diet.     

DIET AND TISSUE GROWTH : II. THE REGENERATION OF LIVER TISSUE DURING NUTRITION ON INADEQUATE DIETS AND FASTING.

The relative toxicity of chloroform given subcutaneously to rats fed on two diets deficient in respect to their nitrogenous components, the sole sources of protein in which were, respectively, gliadin and gelatin and to rats without food, is as follows: gelatin diet > fasting > gliadin diet. The rate of regeneration of liver cells after chloroform poisoning in rats fed a diet in which gliadin is the only protein is about as rapid as that in animals fed a diet containing casein as the source of nitrogen. The rate of regeneration on a food containing its nitrogen as gelatin and also during fasting is definitely slower than that in animals fed the gliadin food. The results of these experiments indicate that the requirement for the essential amino acids for growth of individual organs of the body is less than that for correlated growth of the whole body.     

HEMOGLOBIN REGENERATION IN THE CHRONIC HEMORRHAGIC ANEMIA OF DOGS (WHIPPLE) : I. THE EFFECT OF IRON AND PROTEIN FEEDING

1. A group of dogs on a standard salmon bread diet with a slowly regenerating anemia were studied. The addition of liver to this diet during a 2 week period promoted a definitely greater regeneration of hemoglobin than did the addition of an amount of inorganic iron which was equivalent to that contained in the added liver. The more effective result attained with liver cannot, therefore, be attributed solely to the iron intake. 2. The greater response to liver is not due to its content of amino acids which are present in casein, since a diet containing an exactly similar amount of calories, iron and protein nitrogen, made up of inorganic iron and casein does not cause a greater response than that obtained by the addition of that amount of inorganic iron alone to the standard basal diet. 3. Furthermore, the salmon bread diet does not produce a deficiency of the amino acids represented in casein, since dogs eating the high protein (casein) Cowgill dog ration show the same basal hemoglobin regeneration rate and a similar greater response to liver than to inorganic iron. The Cowgill ration, however, supplies some non-ferrous factor involved in hemoglobin regeneration which is not contained, to as great a degree at least, in the salmon bread. 4. Whipple''s chronic hemorrhagic anemia of dogs serves as an accurate assay method for measuring the hemoglobin producing power of a substance. Quantitatively reproducible responses can be obtained.     

Supplemented low-protein diets protect the rat kidney without causing undernutrition.

Low-protein diets supplemented with keto-analogues and essential amino acid (KA-EAA) mixtures or with EAA have been widely used to retard renal deterioration without affecting nutrition. These assumptions have recently been challenged in clinical studies and rest on little or no experimental data. The effects of EAA and KA-EAA supplementations have not been compared. We compared three groups of rats with subtotal nephrectomy that were fed (1) a 16% casein reference (R) diet, (2) a 6% casein plus EAA (A) diet, or (3) a 6% casein plus KA-EAA (K) diet with KA as amino acid salts. The three diets had the same energy and mineral contents, and they induced comparable growth. The two supplements had the same nitrogen content. The only difference found until month 3 was higher proteinuria and plasma urea levels in group R rats. Renal biopsies performed at month 3 showed more severe glomerular sclerosis and tubular changes in R rats than in A and K rats. From months 3 through 7, R rats developed higher plasma creatinine levels than did A and K rats (final median values: 167, 106, and 83 mumol/L; p < 0.04), had more proteinuria (232, 56, and 84 mg/day), and showed greater mortality rates. At the time the rats were killed, 2 R, 6 A, and 5 K rats had survived while receiving the diets. Examination of the remnant kidneys, regardless of time of death, showed that renal lesions were significantly worse in R than in A and K rats, with sclerosis affecting more than 50% of the glomeruli in 7 of 13 R, 4 of 14 A, and 4 of 15 K rats, and less than 25% glomeruli in 2 of 13 R, 10 of 14 A, and 10 of 15 K rats (A and K vs R: p < 0.03). In conclusion, restriction of nonessential amino acids compensated by EAA or by KA-EAA mixtures retards renal damage without affecting growth, but no real benefit of KA or EAA has been observed.     

THE EFFECT OF DIET ON THE FECAL BACTERIAL FLORA OF MICE AND ON THEIR RESISTANCE TO INFECTION

A study was made of the effect of certain dietary regimens on the lactobacillus flora in the stools of mice and on their resistance to infection. Semi-synthetic diets with purified casein or wheat gluten as sole source of protein, gave rise to much smaller numbers of viable lactobacilli in the stools than did other diets containing unidentified natural products—as present for example in mixtures of whole wheat and whole milk, or in certain commercial pellets. Furthermore, one of the lactobacillus types with rhizoid morphology disappeared completely from the stools of animals fed the semi-synthetic diet. The change in the lactobacillus flora became apparent within a very few days after the animals had been shifted from the complex to the synthetic diet Moreover, this change was not completely reversible. Whereas the total numbers of lactobacilli increased when the animals were shifted back from the synthetic to the complex diets, the rhizoid lactobacilli which had disappeared completely from the stools reappeared only slowly or not at all. In twelve consecutive experiments the three diets which gave rise to the large numbers of lactobacilli in the stools also conferred on the mice a much higher resistance to experimental infection with Staphylococcus aureus and Klebsiella pneumoniae, than did the semi-synthetic diets. However, direct evidence has not yet been obtained that the two kinds of phenomena were causally related. Following administration of endotoxin there was a rapid and very large increase in the numbers of enterococci and coliform bacilli in mice fed the semi-synthetic casein diet, but not in those fed the pellets. In two preliminary experiments carried out with another colony of mice, not pathogen-free, it was also found that the rhizoid type of lactobacilli disappeared from animals fed the semi-synthetic casein diet while enterococci and coliform bacilli progressively increased in numbers under the same conditions. The dietary effects on the lactobacillus flora, and on resistance to experimental infection, were equally pronounced whether the mice were housed in individual cages on wire grids, or grouped in larger cages with wood shavings as litter. This was true even if the bedding was changed only once weekly and became therefore grossly soiled.     

EXPERIMENTAL RICKETS IN RATS : IV. THE EFFECT OF VARYING THE INORGANIC CONSTITUENTS OF A RICKETS-PRODUCING DIET.

1. Rachitic bone lesions may be produced in rats by a diet containing an excess of calcium, but deficient in phosphates. 2. Similar lesions follow a diet deficient in calcium, but containing an excess of phosphates. The endochondral lesions, however, are less pronounced. 3. A diet deficient in both calcium and phosphate induces atypical rickets. 4. Inorganic salts other than calcium or phosphate seem to be without influence upon the development or prevention of rachitic lesions. 5. The minimal amount of phosphate (calculated as P) required for normal calcification in young growing rats (30 to 50 gm.) on the diets used in these experiments is approximately 160 mg. per 100 gm. of diet. At 135 mg. individual variations come into play; at 110 mg., or less, rickets invariably follows. 6. An excess of calcium is not necessary to the production of the rachitic lesions. A reduction of the calcium lactate of Diet 84 to 0.6 per cent (111 mg. of Ca per 100 gm. of diet) still results in the development of typical rickets. 7. Adult rats react to a deficiency of phosphate in the diet by the production of calcium-free osteoid in abnormal amount about the spongiosa and cortex. There is not produced any alteration of the epiphyseal cartilage, as in growing animals.     

THE EFFECT OF ANTIBACTERIAL DRUGS ON THE FECAL FLORA OF MICE

Oral administration of penicillin, terramycin, or chloramphenicol to NCS mice rapidly brought about profound changes in their fecal flora. The lactobacilli disappeared completely, whereas the numbers of enterococci and Gram-negative bacilli reached very high levels. In contrast, no effect on the fecal flora could be detected following administration of isoniazid in any amount. The intensity and duration of the effects on the fecal flora were related to the type of drug and to the amount of it administered. Chloramphenicol produced disturbances which were less profound and of shorter duration than those produced by penicillin or terramycin. The duration of the disturbances in the fecal flora produced by antibacterial drugs was markedly conditioned by the nutritional regimen. The fecal flora returned to its pretreatment state (large numbers of lactobacilli, few enterococci, and few Gram-negative bacilli) within less than 4 weeks after discontinuing the drug when the mice were fed a complex diet of ill defined composition (commercial pellets). Contrariwise, the fecal flora remained markedly different from that of control mice when the animals were fed semisynthetic diets containing as source of protein either 15 per cent casein or 15 per cent wheat gluten (both supplemented with cystine); or 15 per cent wheat gluten supplemented with lysine, threonine, and cystine. The fecal flora of mice treated with penicillin contained large numbers of lactose-fermenting Gram-negative bacilli, not found in the untreated animals. These lactose fermenters persisted for several months after discontinuance of the drug in mice fed either the casein or gluten diets, but they disappeared rapidly from mice fed pellets. Similar results, although less striking, were obtained with Swiss mice from colonies maintained under usual conditions, and therefore having a fecal flora more complex than that of NCS mice.     

Effect of dietary proteins and amino acids on the susceptibility of mice to bacterial infections

Groups of young albino mice were fed continuously four different types of diets and were compared with regard to (1) rate of weight gain; (2) resistance to experimental bacterial infections. The protein content of the four diets was as follows: (a) pellets: a minimum of 21 per cent "crude" protein (according to the manufacturer); (b) diet 20 C: 20 per cent casein; (c) diet 8 C: 8 per cent casein; (d) diet 8 C + AA: 8 per cent casein supplemented with 12 per cent of a mixture of essential amino acids. All diets provided an adequate supply of minerals and vitamins. They were administered ad lib. Three strains of pathogens virulent for mice were used for the infection tests, namely: Staphylococcus aureus, Mycobacterium fortuitum, and Mycobacterium tuberculosis bovis. The bacteria were injected by the intravenous route. The experimental regimens were begun at different times before infection, and were continued until death of the animal, or until termination of the experiment. It was found that mice on the 8 C diet exhibited much greater susceptibility to infection than did mice on the 20 C diet; mice receiving pellets were intermediate between these two groups. The infection-enhancing effect of the 8 C diet could be entirely corrected by amino acid supplementation (diet 8 C + AA). Indeed, mice fed diet 8 C + AA proved the most resistant to infection. The fact that animals fed pellets (which contain a minimum of 21 per cent protein) consistently died faster following infection than did animals fed diets 20 C or 8 C + AA suggests that qualitative characteristics of the protein in the regimen are as important as the quantity of protein fed in determining susceptibility to infection. The differences in susceptibility exhibited by the mice on the four experimental diets were the same whatever the species of bacterial pathogen used for the infection test, the size of the infective dose, and the duration of the disease. There was no apparent relation between the effects of the diets on the weight curves of the animals, and on resistance to infection. Mice on diet 8 C (which were most susceptible) gained weight as rapidly as those on 20 C and more rapidly than those fed 8 C + AA (which were most resistant). All the tests reported in the present paper were carried out with young mice, which were placed on experimental diets within 1 to 2 weeks after weaning. Preliminary experiments suggest that the relation between dietary factors and susceptibility to infection was more difficult to bring out in older animals. There was evidence also that this relation was most apparent during the first weeks that the animals were fed the experimental diets, and became less striking after several weeks.     

PROTEIN CONSUMPTION AND THE RESTORATION OF LOST ORGAN TISSUE

1. At all levels of immediate protein consumption, from almost none to excessive amounts, the degree of growth of the single kidney, suprarenal, and ovary left after removal of one of each of these organs is the same up to 5 days after the operation. 2. On a 1 per cent protein diet there is no growth of the remaining organs after the first 5 days. 3. On diets containing from 10 per cent to 57 per cent of protein there is no definite relation between the level of protein consumption and the rate and degree of growth from 5 to 40 days after the operation. 4. When rats are given a 1 per cent protein diet for 10 days before the operation a continuation of this diet reduces the initial growth of the remaining organs and leads to a cessation of growth after the first 2 days.     

INFLUENCE OF DIETARY FACTORS AND SEX ON THE TOXICITY OF CARBON TETRACHLORIDE IN RATS

Six groups of rats on different diets were exposed to the inhalation of carbon tetrachloride (about 300 p. p. m.) for 150 days. Food intake and changes in weight were followed throughout the experiment. Animals fed a diet low in protein showed greater susceptibility than rats on a diet high in protein. Methionine was a good substitute for protein (casein) in the diet. Increase in fat intake with correspondingly lower carbohydrate intake exerted a harmful effect, especially evident in combination with a low protein diet. In this change of the fat: carbohydrate ratio, whether the increased fat or the lowered carbohydrate is the specific factor must remain unanswered at the present time. Necrotizing nephrosis was the presenting sign of the intoxication caused by carbon tetrachloride, in addition to hepatic changes, such as hydropic degeneration, necrosis, and cirrhosis. Dietary factors (methionine and methionine-containing protein, as well as low fat intake) more consistently prevented renal injury than cirrhosis of the liver. Under identical dietary conditions, especially with higher fat intake, male rats appeared to evince greater susceptibility to carbon tetrachloride than female rats. The significance of this observation and its wider applicability has been discussed.     

CIRRHOSIS OF THE LIVER CAUSED BY EXCESS DIETARY CYSTINE

1. Cystine fed to young albino rats as 10 per cent of the diet resulted in: (a) Portal hemorrhagic necrosis, resembling eclampsia, within 3 or 4 days. (b) A high mortality rate. (c) Fatty infiltration of hepatic cells in all rats surviving the initial acute lesion. (d) Cirrhosis of the liver in rats surviving more than 2 weeks. 2. 5 per cent dietary cystine produced marked fatty infiltration of the liver, followed by portal hemorrhagic necrosis. Cirrhosis was present in one of the two rats on the diet for 6 weeks. 3. The livers of rats fed 5 or 10 per cent cystine diets followed by the McCollum stock diet, showed evidence of residual cellular damage, and of regeneration as shown by mitotic figures. 4. In this series of 30 rats on excess dietary cystine, a renal lesion was found in only one case.     

Anemia plus hypoproteinemia in dogs; various proteins in diet show various patterns in blood protein production; beef muscle,. egg,lactalbumin, fibrin,viscera, and supplements

Dogs with sustained anemia plus hypoproteinemia due to bleeding and a continuing low protein or protein-free diet containing abundant iron have been used in the present work to test food proteins and supplements as to their See PDF for Structure capacity to produce new hemoglobin and plasma proteins. The reserve stores of blood protein-producing materials are thus largely depleted in such animals and sustained levels of 6 to 8 gm. per cent hemoglobin and 4 to 5 gm. per cent plasma protein can be maintained for considerable periods of time. The stimulus of double depletion drives the body to use all protein building materials with the utmost conservation. This represents a severe biological test for food and body proteins and its assay value must have significance. Measured by this biological test in these experiments, casein stands well up among the best food proteins. The ratio of plasma protein to hemoglobin is about 40 to 50 per cent, which emphasizes the fact that these dogs produce on most diets about 2 gm. hemoglobin to 1 gm. plasma protein. The reason for this preference for hemoglobin production is obscure. The mass of circulating hemoglobin is greater even in this degree of anemia and the life cycle of hemoglobin is much longer than that of the plasma protein. Egg protein, egg albumin, and lactalbumin all favor the production of more plasma protein and less hemoglobin as compared with casein. The plasma protein to hemoglobin ratio is increased, sometimes above 100 per cent. Supplements to the above proteins of casein digests or several amino acids may return the response toward that which is standard for casein. Histidine as a supplement to egg protein increases the total blood protein output and brings the ratio of plasma protein to hemoglobin toward that of casein. Beef muscle goes to the other extreme and favors new hemoglobin production up to 4 gm. hemoglobin to 1 gm. plasma protein—a ratio of 25 per cent. The total amounts of new blood proteins are high. Lactalbumin as compared with casein shows a lower total blood protein output and a plasma protein to hemoglobin ratio of 70 to 90 per cent. Amino acid supplements are less effective. See PDF for Structure Fibrin is a good food protein in these experiments—much like casein. When fed over these 5 week periods it causes a sustained increase in blood fibrinogen. Folic acid in the doses given has no effect on the expected response to various diets. Peanut flour is a very poor diet for the production of new hemoglobin and plasma proteins. Small supplements of casein and beef show a significant response with improved output of blood proteins. Soy bean flour gives a poor response and wheat gluten a good response with adequate output of blood proteins. Visceral products show some variety. Beef heart is not as effective as beef muscle. Beef spleen, kidney, and pancreas give good responses but not up to casein. Pig stomach, beef brain, and calf thymus are below average. The plasma protein to hemoglobin ratio shows a narrow range (40 to 60 per cent) in experiments with visceral products.