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" 1" 1' l 1 \/ ~ 1’47 ‘.~ ‘ ' I ~- ' ’7 "“' 0 9'43"? wt 9 §_U.- Q7 11mm SYATE cause (I “LAND W. screws STUDIES ON HIGH PROTEIN DIETS STUDIES ON HIGH PROTEIN DIETS THESIS Submitted to the Faculty of Michigan State College in partial fulfillment of the re- quirements for the degree of Master of Science. BY MARY HELEN Egacusom . 1930 ACKNOWLEDGMENTS - - The author of this thesis wishes to ac- knowledge the valuable assistance of Dr. C. A. Hoppert, Associate Professor of Chemistry, for his aid in directing this experiment, and his kindly criticism of the manuscript. The author also wishes to express her gratitude to Dr. L. B. Sholl, Assistant Animal Pathologist of the Experimental Station, for his kindness in making histological studies and to .Mr. J. F. Witter, Assistant in Animal Pathology, for his kindness in making the sections. 9ii~96 II III IV TABLE OF CONTENTS Introduction Page I A. Review of Literature 2 Experimental Work 8 A. Table I: Percentage Constituents of Diets 9 Procedure 10 A. Animals Used .10 B. Care of Animals 10 C. Observations 10 Results 12 A. Growth 12 B. Table II: Average Gain of Animals in Series I - IV over a Period of Sixteen Weeks 12 C. Table III: Average Gain of Animals in Series V and VI Over a Period of Twelve Weeks 13 D. Bone development 13 E. Table IV: Percentage Ash in Femurs and Humuri of Rats on Various Rations 13 F. Condition of Teeth lu . Reaction of the Digestive Tract 15 . Table VI: Average Ph Values of Five Parts , of the Digestive Tract 15 I. Bacteria Found in Digestive Tract 16 J. Condition of Kidneys 18 TABLE OF CONTENTS - 2 _ K. Table V: Relationships Between Body Page 19 Weight and Kidney Weight of Rats V Discussion of Results 23 VI Summary . 27 VII Bibliography 30 INTRODUCTION Proteins are a necessary constituent of plant and animal cells. Animals, unlike plants, cannot build their protein from inorganic materials of the air and soil but must obtain them from their food. The cleavage products of proteins, the amino acids, are the "building stones" for construction and maintenance of animal tissue. Normally, so far as we know, the amino acids result- ing from the digestion of protein, pass through the in- testinal wall into the blood stream and are carried by the portal vein to the liver and then passed into the gen— , eral circulation. The amino acids, supplied in the food in excess of those needed for the various functions of the body and those from the normal breaking down of body protein, are deaminized largely in the liver. Most of the ammonia, thus formed, is converted into urea. Also there are other end products of protein metabolism, chiefly uric acid and creatinine. The latter is apparently independent of in- gested protein. The kidneys are the main excretory organs for these products and dispose of a normal amount without suffering any injury. At present there is no direct proof that the kidneys have a limited capacity for eliminating the end products of protein intake. The wide prevalence of kidney diseases, particularly nephritis, has led many investigators to make experimental _ 2 _ study of the effect of high protein diets with Special refer; ence to the condition of the kidneys in the hope that it might throw some light on the cause of human nephritis. In reviewing the experimental results secured by various investigators, the Opinion seems prevalent that with the exception of kidney hypertrOphy, no damage was caused to the kidneys of animals fed with diets rich in protein. (1) Osborne, Mendel, Park, and Darrow reported on the effects of diets, 90 percent or more protein, on various organs of rats grown to a large size. The only striking change found was in the kidneys which were hypertrophied but on micrOSCOpical examination showed no change of degenerative or inflammatory nature. This occurred when the protein was of animal or vegetable origin and was rich or poor in phos- phorus. Diets of 20, 45, and 95 parts protein, used by (2) Reader and Drummond, showed typical growth curves of male and female rats. At the 20 percent level growth was normal, at the 45 percent level growth was normal at first but retarded and at the level of 90 percent growth was subnormal, becoming stationary at about a third of the calculated normal weight. Reproduction was normal in the first group, few litters were produced in group two; and there was no reproduction in group three. In a number of animals, killed at about four months of age, the organs appeared normal with the exception of the kidneys which showed increasing hypertrOphy with increased protein intake. No explanation for growth failure in high - 3 - protein diets was made but Hartwell's theory of a relationship between vitamin B and protein was said to be worthy of study. It was found by (3) Hartwell that the young of female rats fed on diets of 0.9 to 6.0 grams of protein per day had spasms which increased in severity with the increase in pro- tein. Also there was an increasing mortality and on the 6.0 gram protein diet none of the young survived. A second series was carried out similar to the first except that tomato juice was used as a source of Vitamin B in amounts increasing with the increase in protein. The young from females on 1.2 grams protein per day showed normal growth and development with 6 c.c. tomato juice per day. (4) Nelson corroborated Hartwell's theory that there seems to be a quantitative relationship be— tween protein and vitamin B. Rats fed on diets of approximately the same number of calories but of a larger amount of protein in some cases were found by (5) Jackson to grow normally and they showed no ab- hormalities in the urine and no clinical signs of nephritis. 0n autOpsy the kidneys were found to be considerably hyper- tr0phied but showed no lesions in the glomeruli, tubules, or interstitial tissue. (6) Miller fed rats on diets rich in protein and from one animal in each group a kidney was removed to double the load on the remaining one. Kidneys of animals on high pro- tein diets showed hypertrophy amounting in case of the neph— rectomized animals to an increase in weight averaging 0.5% gramsor 85 percent of the average weight of the right kidneys - h _ of the controls. Blood analysis and microscopic examinations of kidneys showed no evidence of kidney damage. The effect of high protein diets fed to white mice was studied by (7) Beard. He found that 75 to 80 percent protein diets gave subnormal growth but no marked differences in weight of kidneys and liver resulted after periods as long as 107 days. Dfiets containing excess protein were used on rate by (8) Addie and E. M. and L. L. MacKay. The period of ob— servation was made sufficiently long to cover what would correspond to thirty years of a man's life. The diets were made adequate for growth and maintenance. The rats on the high protein diet did not reach the weight of the controls. On poet mortem examination the principal cause of this was found to be due to an almost complete absence of fat in the animals. No micrOSCOpic differences were found in the kid- neys of the different groups but the weight of the kidneys was higher and of the liver lower in the high protein diets. This would seem to indicate that rate can live for a third of their life span on a diet very high in protein (supplied as casein) and yet escape any kidney damages. Moreover, this would tend to refute the hypothesis that a large consump- tion of protein is in and by itself necessarily harmful to the kidneys. (9) Osborne, Mendel, Park, and Winternitz reviewed some previous experiments with rats on high protein diets, some of which had two thirds or more of the entire caloric intake _ 5 _ of protein, and found that the rats were capable of growing to adult size. The rats became adjusted to high protein diets and it is thought that the failure of other investi- gators to secure good growth on high protein diets was due to vitamin inadequacy of the rations. There was evidence of kidney disturbances and, as noticed before, hypertrOphy of the kidney was a characteristic outcome of the high protein diet, the change taking place rapidly and with all proteins tested. Contrary to these observations (10) Newburgh, and (11) Maynard and Bender have found actual injury of the kidneys of animals fed on high protein diets. Maynard and Bender made studies on the kidneys of three rats from a 50 percent protein ration, two from an 18 percent protein level and two from stock diet. The kidneys from the high protein group showed marked enlargements and considerable degeneration. A large amount of hyaline material was found distending the convoluting and collecting tubules and also a multiplication of connective tissue with a decreased number of tubules. Coagulum was present in the lumen of the tubules. In contrast the kidneys from the lower protein rations shared little or no hyaline and no connective tissue but did show a degeneration of tubules but of a lesser degree. Whether this is dangerous or not remains to be seen. At the date this was published a rat had been placed on a_high protein diet and after the tenth lactation one kidney was removed. The animal had recovered, was in good health and pregnant thirty days after the operation. - 6 _ According to Newburgh the degree of kidney injury is determined by the type of protein, its concentration, and the length of feeding. The character of the protein is more im— portant than the other two factors. When the diet contained 25 percent protein there was no evidence of injury in the first period of eight months. After more than a year an ab- normally large number of casts were found in the urine. Very little injury was caused in eight months by diets containing as high as 37 percent protein, but, after another eight months there was evidence of marked injury. The differences in the number of casts caused by different kinds of protein, fed at two different time intervals, are very striking. Casein, beef and liver were fed at 75 percent levels for 2H0 and #80 days re- spectively. At the end of 240 days, the number of casts found in the urine from the casein diet was 1100, from the) beef diet were 3200 casts, and that from the liver diet showed 15000 casts. At the end of #80 days there were 2M00 and 16000 casts from the casein and beef diets respectively, and the animal from the liver diet was dead. Diets of 75 percent dried liver caused granular kidneys in less than a year but the same amount of casein, fed 16 months, caused only moderate tubular injury. The effect of a similar amount of beef muscle was intermediate between these two. The kidneys of a rat receiving a diet of about a third of its weight in protein showed injuries of the tubules - everywhere and atr0phy of the tubular membrane but no evidence of injury of the glomerulus. A kidney from a rat on the same _ 7 _ diet but for a longer time showed marked injury of the tubules and glomeruli. It was shown that on a diet of liver protein rats would be killed in less than a year with chronic neph- ritis. The kidneys from a rat receiving 75 percent beef pro- tein suggested an effect of chronic nephritis. MicrOSCOpic sections showed numerous areas in which seriously injured glomeruli and necrotic tubules were imbedded in fibrotic tissue. A normal kidney was shown to be perfectly smooth but the kidney of a rat fed on a liver diet was rough and granular, filled with scars and masses of connective tissue, showing the gross appearance of "chronic nephritis“. -8- EXPERIMENTAL WORK In view of the fact that there is such conflicting evidence on the effects of high protein diets, it seemed desirable to do further work with high protein diets, using diets that simulated practical human diets and special refer- ence made as to their effects on the kidneys. An attempt was made also to answer certain questions which suggested them- selves in reviewing literature on this subject. In the event . of kidney injury, is it simply a strain due to the elimina- tion of large quantities of the normal end products of pro- tein metabolism? Is it due to an abnormal intestinal flora that may be encouraged by a high protein diet or is it due t3 the increased elimination of ammonium salts of the fixed acids that result from the ingestiOn of large amounts of proteins which are acid forming? Experimental work included observations on the kidneys, the intestinal flora, the reaction along the digestive tract, the growth of the animals, their bone development, and the con- dition of their teeth. In this work a high protein diet with various modifica- tions, intended to throw some light on the causes of kidney injury, was compared with a normal diet composed of similar ingredients. In all cases, the proteins used were of poth animal and vegetable origin . As a control animals were fed the regular laboratory stock ration. The make up of these diets is given in Table I. _ 9 _ Table I: Percentgge Constituents of Diets High Protein Diet 1 2 3a 3b Ital-L5 6 1 8a 8239:1910 10a 10b Corn meal 6 60 to 35 25 30 20 5k 3k.5 3h 20 10 30 20 34 24 Whole milk powder 30 l5 l5 l5 Gelatine 10 2o 25 20 25 20 20 20 20 25 20 25 2o 25 Gluten 10 2o 25 2o 25 20 20 2o 20 25 20 25 20 25 Sugar 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Butter 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Alfalfa 355555555555555 Casein 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Lactose 5 5 CaCO; 1 NaHCO3 ' . h .5 1 Yeast 5 5 Codliver oil 1 1 Oil meal 6 NaCl 1 _ 11 _ spleen and digestive tract through the cecum were removed. The femurs and humuri were also removed for ashing and the teeth examined for dental caries. The kidneys were closely examined micrOSOOpically, weighed accurately, and placed in Zenker's solution with the spleen d 'and liver for histological examination. This study was kindly performed by Dr. L. B. Sholl of the Pathology Department. The digestive tract, after removal, was divided into five parts, the stomach, three equal sections of the small intes- tine, and the cecum. The contents of each part were aseptic— ally removed, by stripping, into 10 c.c. of sterile distilled water, thoroughly mixed and allowed to stand for approximate- ly one minute. A sma11.amount of each solution was removed for bacteriological examination; the Ph of the remaining solu~ tion was determined by the Quinhydrone Electrode method. After the first two series, I and II, a bacteriological examination was made only of the fecal material obtained by defaecation of the animal at the time of-etherizing. _ 10 - PROCEDURE Animals‘gged The animals used were of the laboratory albino and piebald stock. Care of Animals The rats were kept in round wire cages with raised wire bottoms. Each cage was placed in a granite pan with paper in the bottom which could easily be removed for cleaning purposes. The cages were cleaned twice a week. The animals were watered daily by means of drinking tubes and fed once a day by placing the ration in feeding cups within the cage. For rations, see Table I. The weights of the animals were taken once a week and re- corded. Six series of animals were placed on experiment. In each series, excepting VI, were fourteen animals, two in each cage on a single diet. As nearly as possible, the rats were dis— tributed equally with respect to weight and sex. In each series two animals were fed stock ration and two were fed the "normal" ration. As indicated the series were numbered I through VI respectively. The animals were termed L1, L2, L3 etc. The animals were placed on experiment at the age of about four weeks with the exception of series IV, in which the animals were about five weeks of age. Observations As stated before, the animals were weighed each week and the weights recorded. They were kept on experiment for thir- teen tc twenty four weeks and were killed by etherizing after _ 11 _ weighing for the last time. When the rat was dead it was cpened and the kidneys, liver, spleen and digestive tract through the cecum were re- moved. The femurs and humuri were also removed for ashing and the teeth examined for dental caries. The kidneys were closely examined micros00pically, weighed accurately, and placed in Zenker's solution with the spleen and liver for histological examination. This study was kindly performed by Dr. L. B. Sholl of the Pathology De- partment. . The digestive tract, after removal, was divided into five parts, the stomach, three equal sections of the small in- testine, and the cecum. The contents of each part were asep— tically removed, by stripping, into 10 c.c. of sterile dis- tilled water, thoroughly mixed and allowed to stand for approximately one minute. A small amount of each solution was removed for bacteriological examination; the Ph of the remain- ing solution was determined by the Quinhydrone Electrode method. After the first two series, I and II, a bacteriolog— ical examination was made only of the fecal material obtained by defaecation of the animal at the time of etherizing. _ 12 _ RESULTS Growth The differences in the rate of growth of animals on these various diets were very striking. The growth of the animals on the "normal" ration was consistently good and compared very favorably with that of the animals on control stock ration. From the first, it was readily noticeable that the rate of growth of the animals on high protein was much less than that of the animals on “normal" and stock rations; this was true of all modifications of the high protein diets. For the first two or three weeks, the animals seemed just able to maintain them- selves. From all indications, there was a period of adjustment to these diets, after which the animals began to grow. The only exceptions were the animals on high protein diets with milk and yeast added reapectively. These animals gained weight from the first week but not to the extent of those of "normal" and stock control animals. I In no case did the final weight of the animals on high pro- tein reach that of the normal and stock ration animals. Table II shows the average gain of the animals in Series I through Series IV over a period of six weeks and Table III shows the average gain of the animals in Series V and VI over a period of twelve weeks. 2g21g_11; Averag@_ggjn of Animals in Series I — 1V over a Period of Sixteen Weeks Diet Males Females 1 231 141 2 227 1A1 3a 142 66 8a 153 131 4a 96 7o 5 23:4: 54 -’ _ 13 _ Table III: Averag e Gain of Animals in Series V and VI Over a Period of Twelve Weeks Diet Males Females 1 219 2 215 5b 65 75 171 81 2b Z9 88 b 1 0 117 10b 119 101 The number of animals on each diet can be found on Table V and the composition of the diets are in Table I. On Diet 8 in Table II there were no male animals and Diets 1 and 2 in Table III had no females. Bone development Table IV shows the averages of the results of ashing of the femurs and humuri of the animals in Series I through Series IV. The bones from the animals in Series V and VI were also ashed and the results, from these animals remain- ing on the diets for thirteen weeks, corresponded very close— ly to those in Table IV. Table IV: Percent Ash in Femurs and Humuri of Rats on Various Rations Diet Average percent Average percent ggh of femggs ash of humuri Males Females Males Females 1 59.53 58. .82 w; 60. 87 2 58.56 57 8 t 5 56 95 5a 55.36 57 5 55 19 56 26 ea 5e.-8 59.05 57.12 61. on ”a 57.“? 55 91 57.22 58 30 5 5A.65 55.72 57 58 7 55 57 56.96 58 6 56 96 9a 53.73 56. 6 60. 76 58. 50 10a 58. 3 60. 51 _ 1h _ There was not a great deal of difference in the ash anal- yses of the bones of these animals kept on the different diets. Bone development apparently was normal in all cases. Condition of the teeth Inasmuch as dental caries seemed prevalent among the lab- oratory animals observations were made on the teeth of all animals used in this experiment. The incisors of all the animals were apparently normal, the enamel was hard and smooth, and there was no evidence of brittleness in the teeth. However, the prevalence of caries in the lower molars of the majority of animals was very astounding. This occurred in the "normal" and stock diets as well as in the high protein diets. No attempt was made to carry out a detailed study of this condition as work is being done in this department on the effects of diet on teeth. Quite a number of skulls of animals from this experiment were removed and turned over to the department for future study. ' One of the most striking results obtained from notations on the appearance of the teeth of the animals used in this ex- periment, was that dental caries seemed to first make their appearance in the teeth of'hormal" and stock rationanimals. Animals on high protein diets, with the addition of lactose, showed very bad cases of caries similar to that found in the cases of Diet 1 and Diet 2. In several cases there were two and three teeth missing from the lower molars of animals on these.diets. Carious teeth were found in animals on high protein diets. _ 15 _ The addition of milk, yeast, and cod liver oil to these high protein diets seemed to have some effect in retarding the appearance of caries in the teeth. However, they eventually became carious although to a somewhat lesser degree than the others. An interesting fact presented itself in the case of four animals in Series III, receiving sodium bicarbonate with their high protein diets. These animals had the best teefi1 of the series although they were kept on the rations for twenty four weeks. Two of the animals had good teeth and two had only small cavities in their teeth. This lends it- self to the suggestion that sodium bicarbonate probably neutral— izes acid which might be instrumental in causing dental caries. Reaction of the digestive tract Table VI shows a comparison of the reactions along the digestive tract of the rats on diets l, 2, 3a, 4a, 5, 6, 6a, 9a and 10a respectively of the first four series of animals. The numbers under Ph represent the following:_l, the stomach, 2, the first section of the small intestine, 3, the second sec— tion of the small intestine, H, the third section of the small intestine and 5, the cecum. Table VI: Average 1_‘Values of Five Parts of the Digestive Tract of Rats on Various Diets Ph Stock High H.P.& H.P.& H.P.& H.P.& H.P.& H.P.& __.Diet Normal Protein Milk Lactose CaCO NaHCO3 Yeast G.L.O 1 3.22 4.445 3.935 4:73 4.12 5.06; 4.51 3.6 3.64 2 6. 8 6.133 6.3 5 6.50 6.49 6.32 6.02 6.46 6.47 K 6.72 6.476 6.503 6.501 6.65 6.70 6.80 6.605 6.56 6.97 6.817 6.80 6.88 7.02 7.24 7.03 6.64 6.86 5 7.00 6.696 6.81 6.98 6.898 7.09 7.11 6.64 6.98 - 16 _ The Ph values of the digestive tract of animals on stock diet increase steadily from acid to the neutral point in the cecum. The last part of the small intestine is nearly neutral. The average Ph value of the digestive tract of animals on normal ration increases through the last section of the small intestines which approaches the neutral point but does not reach it. There is a dr0p in the Ph of the cecum. The values for normal ration were slightly lower than those of the high pro- tein rations. The high protein diet seemed to cause an increase in the Ph of the digestive tract but the neutral point was not reached. The addition of lactose, calcium carbonate, and sodium bicar- bonate caused a greater increase in Ph values. The last part of the small intestines of the animals receiving lactose and sodium bicarbonate was neutral and that of those receiving calcium carbonate was slightly alkaline. The reaction of the cecum drOpped to neutral in the case of animals getting calé cium carbonate and to nearly neutral (but on acid side) in animals receiving lactose; the cecum of the rats receiving sodium bicarbonate was slightly alkaline and of a value higher than that of any in the cecum of animals in these series. The addition of yeast caused a decided drop in the reaction of the digestive tract.‘ The reactions were acid and of the same value in the cecum of the animals. Cod liver oil addition gave an increase in Ph values reaching very nearly the neutral point in the cecum. gagteria;found#;n the digestige tractg One cubic centimeter of the solution from each Ph de- termination was removed, aseptically, and plated on whey agar --17 _ which was found to be a very good medium for the bacterial growth. The plates were incubated at 3700 for #8 hours and ex- amined with respect to the different types of colonies and the relative numbers. The different types were isolated and grown on different laboratory media. Note was made of the morphological and cultural characteristics. In the first two series (Series I and II) it was found that the number of organisms increased along the digestive tract. The largest number of organisms was found in the cecum, in each case and in only one case of fourteen animals were bacteria found in the stomach. This was a single colony found in the stomach of a rat on the'mormal" ration. On examination it was found to be a gram positive coccus and the character- istics of its morphology and growth on media indicate that it was probably a staphylococcus. Coccus and bacillus forms were found in the digestive tract and fecal material of the animals studied. The number of bacillus forms increased and the number of coccus forms de- creased along the digestive tract. In the fecal material bacillus forms were most prevalent. It was interesting to find that gram positive forms predominated in most of the cases. One type alone was gram negative; it was a short, plump rod re- sembling B.Coli in most ways but gave acid with no gas when grown on lactose broth. This organism completely peptonized litmus milk and formed a heavy white pellicle on it, it gave acid and gas when grown on dextrose, maltose and sucrose, and gave a distinct putrefactive odor when grown on whey agar. Other organisms found were staphylococci and strepto— cocci. The predominating forms found were rather large gram positive rods of varying sizes and characteristics, but, in general, resembling each other. A large, bipolarly stained, plump rod which completely peptonized milk, liquified gelatine with a pellicle formation, gave acid on all sugar broths but lactose was found in a number of cases in the cecum. A type of bacillus resembling B Acidophilus was quite prevalent in the digestive tracts of the rats. A smaller type of bacillus, also gram positive,was very common. It was interesting to note that in the high protein diets there were more gram negative organisms than in the other diets but gram positive rods predominated. In cases of animals re- ceiving lactose very few gram n gative organisms were found. In the fecal material studied, large numbers of dead organisms were noticed. Condition of Kidneys One of the outstanding effects of high protein diets was the distinct hypertr0phy of the kidneys. Table V shows the re- lationship between the body and kidney weight of all the rats used in this eXperiment. - 19 _ Table V: Relationship Between Body Weight and Kidney Weight of Rats - Number of Average time Average Wt. Average Wt. Diet Animals on diets per animal of Kidneys Percent 1 3 males 13 weeks 303 gms. 2.0990 .6927 2 females 2 200 1.5604 .7802 2 8 males 20 294 2.7167 .9240 5 females 20 203 1.7757 .8747 3a 3 males 19 246 2.8072 1.1409 females 19 143 1.7079 1.1943 3b 2 males 13 128 2.1657 1.691 2 females 13 130 2.1820 1.678 8a 2 males 22 256 3.2420 1.2664 3 females 22 203 2.3448 1.1550 8b 2 males 13 231 3.1006 1.3422 2 females 13 132 1.6813 1.2737 4a 5 males 21 220 2.7392 1.2450 6 females 21 166 2.0938 1.2613 4b 1 male 13 157 2.6765 1.7047 2 females 13 136 2.4608 1.8094 (Ca003) 4 males 23 178 2.3465 1.3182 5 2 females 23 3132 1.6368 1.2400 (NeH003) 2 males 24 239 2.9251 1.2152 7 2 females 24 195 2.3505 1.2053 9a 1 male 20 288 2.2251 1 0156 Yeast 1 female 20 236 2. 950 1.0572 9b 2 males . 13 195 2.8940 1.4841 Yeast 2 females 13 178 2.4156 1.3570 C.L.O. 2 females 21 181 2.1226 1 1727 10a 0.L.0 2 males 13 198 2.7453 1.3865 10b 2 females 13 151 2.0367 1.3488 _ 20 _ From the results of this eXperiment, as shown in Table V, it is very apparent that an increased intake of protein causes an increase in the size of the kidneys. Using animals on stock ration as a standard it seems that the "normal" ration is slightly high in protein. Upon examination of the external appearance of the kidneys of these animals, it was found that there were distinct and marked differences in their gross appearance. The kidneys from the "normal" and stock ration animals had small, smooth, firm, dark kidneys. Those from the animals on high protein diets varied from large, smooth, dark or slightly discolored kidneys to rough, granular, light colored kidneys. The exterior appearance of the kidneys from animals on high protein diets varied with the diets and the different modifications. Some kidneys were merely enlarged with a few cavity-like holes in them and others were large, dull, porous kidneys with so many perforations that it made them have the exterior appearance of a sponge. The effects of increasing the protein content of the diets is shown in comparing the kidneys of the first four series of animals with those of the last two series. The latter series were on high levels of protein but for a shorter length of time. The size of their kidneys was greater in pr0portion to their weight and the gross appearance was more outstanding than in the first four series. The effect of time in feeding high protein diets was clear- ly shown by animals in the first four series. In cases of the animals killed after being on diets sixteen to twenty weeks, there were no decided changes in the appearance of the kidneys - 21 _ although there was distinct hypertrophy. The kidneys of animals killed at the end of twenty-two to twenty-four weeks on the various high protein diets showed distinct changes in external appearances. In general the kidneys of the animals of the first series (I to IV) may be described as follows: On high protein diet they were slightly granular.with small perforations and several larger cavities, on high protein with milk added they were some- what porous and rough with dark lines; on high protein with lactose they were finely granular with a few holes and perfora- ations; on high protein with Ca003 they were very bad, having a very rough, porous, irregular, granular appearance and in one case part of the kidney appeared infected and eaten away; those on high protein with sodium bicarbonate were slightly granular with a few perforations; those on high protein with yeast were slightly rough but had no holes; and those receiving cod liver oil with high protein were quite smooth but had a few small holes. The kidneys from animals on the higher protein level showed a fine but distinct granular appearance; those from animals receiving milk were rough and granular; those from animals receiving lactose were granular with several perfora- tions; those from animals receiving yeast were porous and granular;and those from animals receiving cod liver oil were merely granular. It was impossible for Dr. Sholl to study the histolog- ical structure of all the kidneys removed for study, but the descriptions of a few studied were given as follows: _ 22 - The kidneys of Leo, an animal receiving diet 3a (high protein at first level with milk) showed “marked congestion and some cloudy swelling. Several small cavities were present in the cortex, appearing as Spaces from which the glomeruli had drOpped oufit L19, an animal on the same diet showed"some dis- tension of the tubules and some cloudy swelling. Considerable granular material was present in the tubules." The kidneys of L21, on Diet Ma.receiving lactose, showed "considerable congestion and slight cloudy swelling. Some granular material was present in the tubules and two or three small hemorrhages in the cortex were found in one sec- tion." L25, an animal on Diet 5, receiving calcium carbonate showed "one small band of cellular infiltration and fibrosis. One rather marked focus of infection with polymorphoneuclear exudate was present. There was some cloudy swelling and pyknosis". L26’ an animal on the same diet showed "congestion and some slightly cloudy swelling of the kidneys. Some tubules contained granular material and cell debris. Several small foci in the cortex showed some endothelial cell and lymphocyt- ic infiltration and some fibrosis tubules in these areas were degenerated and many contained hyaline casts." - 23 - DISCUSSION OF RESULTS High protein diets fed to rats showed no striking changes in their bone development as compared with that of normal ani- mals; the essentials for normal bone develOpment evidently were not lacking in these diets. However, the rate of growth ofthese animals was much less than that of the "normal" and stock ration animals. The addition or milk, yeast, and cod liver oil to these diets caused some increase in the growth rate of animals but it did not approach that of normal animals. The diets were made to contain,as nearly as possible, the essentials for growth and maintenance and the failure of animals to grow was probably not due to a lack of these factors. Two possibili- ties for explanation of the failure of animals to grow normal- ly on these diets might be (1) the large amounts of amino acids and other end products of protein metabolism may have a toxic effect on the tissues of the animals, or, (2) the increased task of the body to eliminate these end products of metabolism may cause the growth to be retarded. Whether or not palata- bility was a factor could not be shown as no effort was made to keep records of the food consumed by the animals. Food was kept before the animals continually. No real explanation is forthcoming for the prevalence of dental caries found in the teeth of the animals on high pro- tein diets. The fact that the teeth of the animals, fed on a high protein diet with the addition of sodium bicarbonate, were not in as bad a condition as those of the animals on the other rations, offers an interesting piece of information as regards the theory that an acid reaction in the mouth may give _ 2h - rise to carious teeth. It is possible that the sodium bicarbonate might have neutralized acid in the mouths of these animals, thus establishing an environment that was unsuitable for the development of carious teeth. A great deal of significance is not attached to the Ph values found in the digestive tracts of the animals used in this experiment. The addition of milk, lactose, calcium car- ‘ "“ f m” "3 bonate, cod liver oil and sodium bicarbonate caused some in- ‘ creases in the Ph values of the digestive tract. No correla- At tion could be formed between the Ph and the bacteria of the digestive tract of these animals. The same types of bacteria were generally found in all the animals on these diets. The fact that proteolytic bacteria were not found in the digestive tracts of animals, fed high protein rations, in as large numbers as might be expected, finds a possible ex— planation in the fact that ten percent of sucrose was included - in all these diets. Bacteria generally prefer a carbohydrate medium and will act on carbohydrate, if it is present, for their food supply before attacking protein. The addition of lactose to high protein diets seemed to give rise to a greater number of gram positive bacilli; this might be due to a great- er increase of carbohydrate for the food supply of the bacter— ia. . The kidneys from rats fed on high protein diets showed distinct macros00pic differences when compared with the kid— neys of normal animals. Hypertrophy was shown in all cases and there were marked differences in the appearances of the kidneys. There was a difference in extent of the changes _ 25 - in the kidneys of the animals kept on high protein diets. They varied from a finely granular state to a rough, irregularly granular condition; the number of cavities varied in number and size. Some kidneys were uniformly porous and others had only a few cavities. It was noticed that the animals fed on high protein with the addition of sodium bicarbonate showed hyper- trOphy of the kidneys but the external appearance was normal excepting for a dark line on one of the kidneys and th ani— mals fed high protein diets with calcium carbonate had by far the worst kidneys. No explanation can be given, to account for these results. The two kidneys of normal rats were found to be very near— ly of the same weight and in the cases of hypertrophy the kid- neys of each animal were still of about the same weight. This indicates that the work of elimination is equally divided be- tween the two kidneys,as might be eXpected. There was no evidence that bacterial infection caused.the macrosCOpic and micrOSCOpic changes in the kidneys of rats fed on high protein diets. In only one case was an infection found This animal received calcium carbonate with the high protein diet. The animal had appeared sick for some time be- fore the conclusion of the eXperiment and an autOpsy showed that the side of one kidney appeared eaten away. This kidney weighed a great deal less than the other one. Upon micro-- scopic examination it was found that there was a marked focus of infection with a polymorphonuclear exudate. The fact that the addition of sodium bicarbonate to a high protein diet seemed to improve the external condition of _ 26 _ the kidneys might find possible eXplanation in that the sodium bicarbonate might assist in the elimination of the acidsthat are formed as a result of the ingestion of large amounts of proteins which are usually acid forming. In a number of kidneys examined microscopically, there was some evidence of degeneration and that the animals were possibly not kept on the diets long enough to show distinct changes. However, in some cases, there were decided micro- scopic changes and differences in the kidneys of animals kept on high protein diets. The results of this eXperiment support the work of New- burgh and others showing that high protein diets cause macro- scopic and micrOSCOpic changes in the kidneys of animals. l. _ 27 _ SUMMARY Studies made on several high protein diets showed that they were not as conducive to growth as were the stock and normal diets. (a) The addition of milk, yeast, and cod liver oil increased the growth of the animals somewhat but the growths never reached those of normal animals. (b) The animals seemed to go through a period of adjustment to high protein diets before they started to gain weight. The bone development of animals on these high protein diets was apparently normal. (a) The addition of milk, yeast, and cod liver oil to the high protein diets had no appreciable effect on the ash con- tent of the bones of these animals. Dental caries were found in the teeth of all animals on high protein diets. (a) Animals on high protein rations with lactose added, had the worst cases of caries. (b) Animals on high protein diets receiving sodium bicarbo- nate had the best teeth. (0) The addition of milk, yeast, and cod liver oil seemed to retard the appearance of caries but did not prevent the ultimate appearance of carious teeth. The Ph values increased along the digestive tract of ani— mals fed on high protein diets and were fairly uniform. ('5 - 20 - (a) The addition of milk, lactose, calcium carbonate, cod liver oil and sodium bicarbonate to the high protein diets increased the Ph values of the digestive tract. (b) The addition of yeast to the high protein diets appar— ently decreased the Ph values to a Certain extent. (0) Since the Ph values are quite uniform in the digestive tract of the rats fed on high protein diets, a great deal of significance cannot be attached to them. The number of bacteria increased along the digestive tract of rats receiving high protein rations. (a) The types of organisms found were gram negative and positive cocci and gram.negative and positive bacilli. (b) Gram positive bacilli predominated in the digestive tract. One type resembled B.Acidophilus. (c) Gram negative bacilli, apparently of only one kind, increased in numbers with an increase in protein ingested, the largest numbers being found in the cecum and fecal material. This type resembled E. Coli. (d) The variety of organisms found in animals on high pro- tein diets was not strikingly different from that found in normal animals, the only difference being in the relative numbers of different types. (e) When lactose was added to the high protein ration the number of gram positive organisms were increased. The ingestion of high protein diets caused distinct changes in the kidneys of animals as compared with the kidneys of normal animals. (a) Distinct hypertrOphy of the kidneys took place when ani- mals were fed on diets rich in protein. (b) The external appearances of kidneys from animals con— suming high protein diets differed from those of normal ani- mals. They varied from fairly smooth kidneys with a few small cavities to irregular granular kidneys with many per— forations. (c) Hicrosc0pic changes found in the kidneys of some of the animals on high protein rations included granular material in some of tubules, degeneration of some of the tubules, hyaline casts in some of the tubules, cell debris in some of the tubules, two or three small hemorrhages in the cortex of one section, some_fibrosis and endothelial cell and. lymphocytic infiltration in several foci in the cortex of one section, some cloudy swellings, and some pyknosis. (d) The amount of protein ingested and the lengths of the feeding periods were found to be important factors in caus— ing changes in the kidneys of rats. (e) There was some evidence that the addition of sodium bicarbonate to a high protein diet might have had some effect in preventing serious changes in the kidneys of ani- mals. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) _ 3o _ BIBLIOGRAPHY Osborne, T. B., Mendel, L. B., Park, E. A., and Darrow, D., Soc. EXpt. Biol. and Ned. Proc. 20, No. 8, pp. 452—u53 (1923) Reader, V. B., and Drummond, J. C., Jour. Physiol. 59, No.6 pp. 1L72-478 (1925) Hartwell, G. A., Biochem. Journ., 18, No.5, pp. 785-794 (192A) Nelson, P. H., Journ. Home Econ., 18, No.7, pp.383- 38 8 (1926) Jackson, H. Jr., Soc. Expt. Biol. and Ned. Proc., 22, pp. #82- %3 (1925) Miller, A. J., Jour. Expt. Med., #2, No.6, pp. 953-957 (1925) Beard, H. H., Amer. Jour. Physiol. 75, No.3, pp. 6&5-695 (1926) Addis, T., MacKay, E.M., and L. L., Jour. Biol. 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