ABSTRACT EFFECT OF DIFFERENT SOURCES OF PROTEIN ON SERUM UREA NITROGEN AND ACID-BASE HOMEOSTASIS OF HUMAN SUBJECTS By Mushtaq Ahmad The entire study was divided into two parts, one being the effect of ingestion of sodium bicarbonate and the other being the effect of different sources of protein, i.e.. animal, wheat and potato, fed isonitrogenously to human subjects and rats on the serum urea nitrogen (SUN) and acid- base homeostasis of human subjects in particular, and blood and urine composition in general. P rt I Two experiments were carried out. In the first of these, three healthy, adult male human subjects consumed typical American diets of the same kind and same amount for five days. They then ingested l5 9 NaHCO3 per day with the same diet for an additional five days. There was no variable except ingestion of bicarbonate between the two phases. During the bicarbonate phase, the urine pH became alkaline Mushtaq Ahmad and the total titratable acidity of the urine dropped from 22.6 t 2.5 to l7.4 t 4.5 mEq per day. The serum urea nitrogen also dropped from l6.7 t l.3 mg/dl to l2.7 t 2.3 mg/dl (P < .05). There was no change in serum creatinine concen- tration. This suggests that the changes observed in acid- base balance have an effect on SUN concentration. Serum glucose and cholesterol levels were not changed by the in- gestion of bicarbonate. In the second experiment, during the control phase, four healthy adult male human subjects drank, for seven days, a constant volume of liquid diet in which wheat gluten was the only source of protein (Taylor gt a1. Brit. J. Nutr. 32: 407, l974). After the seven day control phase, 10 g NaHCO3 was ingested daily in three doses with the same diet. When the control liquid diet was consumed by these subjects, they excreted acid urine with a pH of 5.6 and titratable acidity of 95.2 t 30.3 mEq/day. When bicarbonate was ingested with the same diet, the urine pH increased to 7.3 and the titratable acidity dropped to 24.2 t 5.3 mEq/day. At the same time, a possible reduction in SUN was observed in three of the four subjects, while an increase in SUN occurred in one subject. No change was observed in serum creatinine, sodium and potassium in either phase. This supports the observations made in the first experiment that the change in acid-base balance of human subjects, by in- gesting NaHCO3 has an effect on SUN. Mushtaq Ahmad early. To examine the effect of various kinds of proteins, two different plant protein sources, i.e., potato and wheat, were studied in three experiments with humans and one with rats. The first study was designed to evaluate the effect of potato protein when it isonitrogenously replaced mutton. For that study 3 normal young men consumed typical American diets except that the protein intake was restricted to 46 g per day. During the experimental phase of the study, potato protein was incorporated into the menus so it isonitrogenously replaced the protein in the control diet. The blood samples drawn at the end of the 7 days of the control period had SUN values of l3.0 mg/dl; the blood taken. at the end of the 7 dawsof the experimental period had 7.0 mg/dl. This represented a significant reduction (P < 0.05) in sun. In the second experiment 6 healthy college students consumed diets in which practically all the protein came from mutton. After 7 days on that diet, potato protein replaced that in the mutton on an isonitrogenous basis. The blood drawn at the end of the control period contained l4.8 mg/dl of SUN which decreased to l0.3 mg/dl when the potato diet was eaten for 7 days. A slight increase in plasma CO2 and HCOS was also observed in the second experi- ment at the end of the experimental phase. The titratable acidity decreased from ZOJ 2 7A to H35 t'lB mEq/day. Mushtaq Ahmad In the third experiment, two isonitrogenous rations containing l0% protein of either wheat or casein, were fed to two different groups of adult, male Sprague-Dawley rats. The rats were fed for four weeks ad libitum when blood samples were secured by cardiac puncture. Then the rats were pair-fed for another four weeks and blood samples were drawn at the end of that period by cardiac puncture. Serum analyses showed significantly (P < 0.05) lower urea nitrogen levels in the rats fed wheat rations than in those fed the casein ration, both during the ad libitum and pair-fed periods. The SUN values of l0% casein fed group were l3.9 mg/dl and 13.8 mg/dl on ad libitum and pair feeding respectively, while the 10% wheat protein fed group of rats had SUN values of ll.6 mg/dl and ll.5 mg/dl on ad libitum and pair feeding respectively. Finally, in the fourth experiment, a group of eight healthy, aduTt,male, college students were selected for the experiment to demonstrate whether or not diets high in wheat (90% of the total daily protein intake) had an effect on the serum urea level as compared to a typical American diet which is omnivorous in nature. In order to check the con- sistency of the effect produced during the wheat diet on various parameters of fasting blood, period I and IV were repeated. For this purpose, four of the eight subjects agreed to continue for seven additional days when the "control" diet was repeated and for another six days when the strictly "bread" diet was served. The first control Mushtaq Ahmad period lasted 15 days. Thereafter, for six days all eight subjects ingested 10 grams of sodium bicarbonate with their control diet to examine the alkalinizing effect on the serum urea nitrogen. Then for another two weeks. diets high in wheat were served. During this experiment, a gradual reduction of serum urea nitrogen was observed. The SUN decreased in subjects when the bicarbonate was ingested together with the control diets. There was a further, but slight reduction in SUN when a rigidly monitored bread diet was served. There was no essential change in creatinine and uric acid concentra- tions of the serum. Throughout the entire period of study, the hemoglobin, total protein and albumin concentrations in the serum remained constant. Also, no change in the level of serum glucose was observed. Although urinary pH on control and bread diets was on the acidic side. the titrata- ble acidity during the bread diet was significantly lower. The titratable acidity which was 340 t 7.0 mEq/day on con- trol diet dropped to 318 t 29 mEq/day on wheat diet. This change in acid-base balance would suggest that the reduction in SUN in the subjects consuming wheat diets may be due to the change in acid-base balance as reflected by titratable acidity of the urine. EFFECT OF DIFFERENT SOURCES OF PROTEIN 0N SERUM UREA NITROGEN AND ACID-BASE HOMEOSTASIS OF HUMAN SUBJECTS By Mushtaq Ahmad A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition I976 In Honor of My Parents Mr. and Mrs. Ghulam Isa Khan ACKNOWLEDGMENTS I wish to express my deepest appreciation to my advisor, Dr. Olaf Mickelsen, for direction, encouragement, support and insight throughout my graduate program; to my committee members, Dr. R.W. Luecke, Dr. B.H. Selleck, Dr. R. Schemmel, and Dr. H.A. Lillevik, for their helpful suggestions in pre- paring this dissertation. Warmest gratitude is extended to Mrs. Claire Mickelsen, Dr. and Mrs. J.L. Gill for their encouragement, concern, and thoughtfulness, expressed in so many ways. A special acknowledgment to Dr. B.H. Selleck, Depart- ment of Physiology, who worked many hours in the preparation of the dissertation. I would also like to acknowledge the enthusiastic support given by the late Dr. J.S. Feurig, (then Director General of Olin Health Center) who served as a member of the guidance committee. Special thanks are expressed to Dr. J.T. Bond for technical advice and encouragement. I am also thankful to Dr. D.D. Makdani and Dr. E.D. Schlenker for their help in performing laboratory work. This project would have not been possible without the assistance of my friends. at Michigan State University. including Dr. N.W. Shier, M.S. Bari (Statistics). K. Kari- mullah (Engineering), M. Mittermair (Food Manager, Owen Hall), ii C. Geiger, N. Beck, C. Fairchild, and Joe Marshman. I am very thankful to my friend and teacher, Mr. Tajjamal Hussain, Chairman, Department of Agricultural Chem- istry, University of Peshawar for his concern and encourage- ment throughout my graduate program. Special gratitudes to all of the twenty individuals who have participated in various experiments. I wish to acknowledge the constant support and encour- agement from my learned friend, Dr. F.B. Shorland (0.B.E.) from whom I have learned many invaluable things. Finally, I am very grateful for the financial support given to me by the Fulbright Commission for the fellowship which helped to make this program possible. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES. INTRODUCTION Part II. REVIEW OF LITERATURE The Influence of Diet on Renal Function in Health and Disease Dietary Protein and Renal Function Blood Urea Nitrogen as an Index of Renal Function Blood Urea as Influenced by Protein Intake in Normal Subjects a. Quantity of Proteins. l. Humans. . . 2. Animal Studies. b. Nature of Dietary Protein as it Affects Renal Function Effect of Age on Blood Urea Levels and Glomerular Filtration Rate . . Acid-Base Disorders, Renal Function, and Blood Urea Nitrogen. Effect of Diet on Acid-Base Homeostasis. Renal Function and Blood Urea Nitrogen EFFECT OF SODIUM BICARBONATE INGESTION 0N SUN. . . . . . . . . . . . . . . . . . . . Chapter I. Effect Of Ingestion 0f Sodium Bicarbonate 0n SUN 0f Normal Individuals iv Page vii xiv )2 12 I3 18 22 24 24 26 29 37 43 46 57 III. Introduction Experimental Procedure Analytical Procedures. Statistical Analysis Results. Discussion Chapter II. Effect Of Ingestion 0f Sodium Bicarbonate 0n SUN 0f Individuals Consuming A Liquid Wheat Gluten Diet. Introduction Experimental Procedure Analytical Procedure Results. Discussion EFFECT OF THE NATURE OF DIETARY PROTEIN FED ISONITRDGENOUSLY ON THE ACID-BASE HOMEOSTASIS AND ON VARIOUS PARAMETERS OF BLOOD AND URINE. . . . . . . . . . . . . . . . . Chapter I. The Comparative Effects Of Iso- nitrogenous And Isocaloric Diets On The Acid-Base Balance And Various Parameters Of The Blood And Urine 0f Normal Human Subjects . . . . Introduction Experimental Procedure Analytical Procedure Results. Discussion Chapter 11. Serum Urea Levels In Adult Rats Fed Isonitrogenous Rations Which Pro- vided l0% Protein From Casein 0r Wheat Introduction 57 57 6O 6T 6T 65 7O 71 72 73 77 86 87 91 92 TO] 110 lIO Experimental Procedure . . . . . . . . . . . . ‘11 Analytical Procedure . . . . . . . . . . . . . 114 Results. . . . . . . . . . . . . . . . . . . . 115 Discussion . . . . . . . . . . . . . . . . . . 118 Chapter III. The Effect Of Diet High In Wheat Bread On Various Parameters Of The Blood And Urine In Human Subjects Introduction . . . . . . . . . . . . . . . . . ‘23 Experimental Procedure . . . . . . . . . . . . 124 Analysis . . . . . . . . . . . . . . . . . . . 127 Results. . . . . . . . . . . . . . . . . . . . 13‘ Discussion . . . . . . . . . . . . . . . . . . I53 SUMMARY AND CONCLUSION................ ‘65 BIBLIOGRAPHY.....................I73 APPENDICES......................190 vi Table l. 2. 3. 4. 5. 6. LIST OF TABLES Glomerular filtration rate and renal plasma flow in malnourished subjects Distribution of urea concentration for boys and girls one to 20 years of age. Effect of the ingestion of 15 grams NaHC03 per day in three equal doses on serum level of urea and creatinine in three normal subjects who were (1) on typical American diet for five days as control and (2) same amount and same kind of diet + 15 gms of NaHC03 per day in three equal doses for f1ve days. Blood samples were taken at the end of each dietary period. Effect of the ingestion of 15 grams NaHC03 per day in three equal doses on urinary acidity in three normal subjects who were (1) on a typical American diet for five days as control and (2) same amount and same kind of diet + 15 grams of NaHCO per day in three equal doses for five ays. . . . . . . . . . Effect of the ingestion of 15 grams NaHC03 per day in three equal doses on serum level of glucose and cholesterol in three normal subjects who were (1) on typical American diet for five days as control and (2) same amount and same kind of diet + 15 gms of NaHCO3 per day in three equal doses for five days Serum urea nitrogen (SUN) and urinary pH values of four normal adults who were on (1) liquid formula diet, according to Young gt 31., Am. J. Clin. Nutr. 26, 967, l973, as control diet and (2) same liquid formula diet + 10 grams of NaHCO3 in three doses per day . vii Page 16 4O 62 63 64 74 9a. 9b. 10. II. I2. I3. Urinary volume and acidity of four normal adults who were on (1) liquid formula diet, according to Young gt gt., Am. J. Clin. Nutr. 26:967, 1973, as control diet and (2) same liquid formula diet + 10 grams of NaHCO3 in three doses per day. Each dietary period lasted for a week. Values shown are average of last two days of each period. Serum sodium and potassium values of four normal subjects who were fed (1) liquid formula diets, according to Young et al., Am. J. Clin. Nutr. 26, 967, 1973, a: _" control diet and (2) same liquid formula diet + 10 grams of NaHC03 in three doses. Each dietary period lasted 7 days. Blood samples were drawn at the end of each period. Composition of meat diet (control) Composition of potato diet (experimental). Serum values for urea nitrogen and urinary pH of normal adult males. Three subjects were fed iso-nitrogenous diets (l) mutton (control) for 7 days, followed by (2) potato (experimental) for 7 days . . . . . . . . . . . Effects of isonitrogenous and isocaloric diets based on (1) meat and (2) potato diets, respectively on serum levels of urea and creatinine and carbon dioxide and uric acid of the human subjects. Each dietary period lasted for 7 days. Effect on urinary acidity of individuals fed isonitrogenous and isocaloric diets based respectively on (1) meat and (2) potatoes. Each dietary period lasted 7 days . . . . . . . . . . . Effect of urinary volume of individuals fed isonitrogenous and isocaloric diets based respectively on meat and potato. Values shown are the averages of the 3 day urine collection of each dietary period viii 75 76 89 90 94 95 96 97 I4. 15. I6. I7. I8. I9. 20. 2]. 22. Effect on serum electrolytes of indivi- duals fed isonitrogenous and isocaloric diets based on (1) meat and (2) potato diets. Each dietary period lasted for 7 days in each case. . . . Effect on serum levels of proteins of human subjects fed isonitrogenous and isocaloric diets based respectively on (1) meat and (2) potato diets. Each dietary period lasted 7 days. Effects of isonitrogenous diets based respectively on (1) meat and (2) potato + bread on blood and urine composition of human subjects Proximate composition of the casein and grain rations The composition of isonitrogenous casein and wheat rations (10% protein level) used in the rat studies . Weight in grams of rats fed rations con- taining 10% protein from casein or wheat rations. The animals were fed ad libitum or pair fed for four weeks. Serum urea nitrogen (SUN) and creatinine in two groups of rats fed isonitro- genous casein or wheat ration (l0% protein) both ad lib and pair-fed for a period of four weeks. All values are given in mg/dl. Daily protein, fat and calories in diets used for human subjects throughout the entire study. . . . Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum urea nitrogen levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study ix 9B 99 100 I12 II3 II6 lI7 I28 I34 23. 24. 25. 26. 27. Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum creatinine levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. . . . . . . . . . Effect of typical omnivorous American diet. the same type of diet + 10 g sodium bicarbonate per person per day. and 'high bread' diet on serum uric acid levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum protein levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum albumin levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum hemo- globin levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. I35 I36 I37 I38 I39 28. 29. 30. 3T. 32. Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on hematocrit levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. All the values are expressed as volume %. Blood pH of the individuals fed isonitro- genous the same type of diet, typical omnivorous American diet + 10 g sodium bicarbonate per person per day and 'high bread' diet. The last values listed under each dietary period repre— sent the sample collected at the end of that phase of the study Effect of typical American diet, the same type of diet + 10 g NaHC03 per person per day, and 'high bread' diet on plasma bicarbonate of the individuals. The last values listed under each dietary period represent the samples collected at the end of that phase of the study Effect of typical American diet, the same type of diet + 10 g NaCH03 per person per day, and 'high bread' diet on plasma carbon dioxide of the individuals. The last values listed under each dietary period represent the samples collected at the end of that phase of the study Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum sodium levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. xi I40 I4I I42 I43 144 33. 34. 35. 36. 37. 38. Effect of tYDical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and ’high bread' diet on serum potas— sium levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum calcium levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum phos- phorus levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on the excretion of titratable acid of the individuals as reflected by urinary acidity. The values given are the averages of the last 3 days of each dietary period Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread' diet on serum choles- terol levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study. Effect of typical omnivorous American diet, the same type of diet + 10 g sodium bicarbonate per person per day, and 'high bread’ diet on serum glucose levels of human subjects. The last values listed under each dietary period represent the samples collected at the end of that phase of the study xii I45 I46 I47 I48 I49 I50 39. Average body weight of the individuals fed various isonitrogenous diets. . . . 151 40. Urinary volume in milliliters of the individuals. Average of the last 3 days of each dietary period . . . . . . 152 xiii LIST OF FIGURES Figure l. The three types of reactions giving rise to endogenous acid production. (Adapted from, Relman, A.S., ggg. tgt. Mgg. 12: 295, 1964) . . . . . . . . 2. Average titratable acid excretion and urine pH of individuals fed typical American diet alone and with 15 g sodium bicar- bonate 3. Average titratable acid excretion and urine pH of individuals consuming Taylor's wheat gluten liquid diet, and the same diet plus 10 g sodium bicarbonate. 4. Average titratable acid excretion and urine pH of 6 human subjects who were fed iso- nitrogenous and isocaloric (1) meat and (2) potato diets respectively. 5. Average titratable acid excretion and urine pH of 8 subjects fed isocaloric and iso- nitrogenous (l) typical American diet; (2) same diet plus 10 g sodium bicarbo- nate, and (3) diet high in wheat bread xiv Page 42 68 83 I08 I72 INTRODUCTION The prominent role played by plant proteins in the diets of a large segment of the world's population has led to increasing concern that these plant proteins, especially in wheat, are not of sufficient nutritional value for normal growth and development. This has been assumed true for both animals and human subjects. This question has been thoroughly reviewed by Vaghefi gt gt. (1 ) and Mickelsen (2 ). These authors conclude that the proteins in whole wheat and 70% extraction flour contain an adequate quota of all essential amino acids to maintain nitrogen equilibrium, if a suffi- ciently long adjustment period is used and if enough wheat product is consumed to meet the protein requirements of the subject. A great deal of attention has been given to the nutri- tional quality of the protein in bread. The results of studies aimed at evaluating the protein in American white bread have been almost unanimous in concluding that it would not support growth when it was the only source of protein in a diet. There is no doubt about the low PER of bread as measured by the accepted rat bioassay. The primary reason for suspecting a disparity in results secured with the weanling rat and human subject; is the relative rates of growth. 2 The pioneer work of Bolourchi gt__l, ( 3) left no doubt that protein in 72% extraction flour contains an adequate amount of essential amino acids to produce nitrogen equili- brium in healthy adult subjects. In that study which lasted 70 days, a full academic term, 12 young men were fed a typical American diet for three weeks. The protein in that diet was restricted to 70 grams per day. For the next 50 days, the subjects received a diet which also provided 70 grams of protein per day with 90 to 95% of it from 72% extraction flour. During that 50-day "bread" period, there was no animal protein in the diet. Nitrogen balances were performed using daily urine and stool samples. These results indicated that during the first 10 days of the bread diet, all the subjects were in negative nitrogen balance. However, in the remaining 40 days of the bread diet period, the subjects were in positive nitrogen balance. The initial negative nitrogen balance during the first ten days of the bread diet has not been explained. It was probably due to a number of factors. The most obvious explanation is that during the negative nitrogen period, the requisite enzymes were being formed in concentrations ade- quate to cope with the new amino acid mixture presented by the bread diet. That this is of minor importance has been suggested by Bolourchi, gt gt. (1938) who pointed out that the activity of most enzymes in the body exceeds, by a number of fold, the daily needs. The maximum increase in any one amino acid during the bread diet was only two fold that in 3 the control period. To accomodate such a change should re- quire but a day or so for the induction of the necessary enzyme activity even if its activity were inadequate ini- tially. Other possible explanations may involve the effect that acid base changes in the body may have on enzymatic and physiological reactions. The over-all effects of the change in diet may also be important. It has been pointed out (Mickelsen, 1976) that the presence of large amounts of white bread in the diet produces alternations similar to those attributed to fiber. There are several factors which limit the translation of animal protein bioassay results to human subjects. One of these involves the nutrient requirements. This is especially important for rapidly growing rats which require a high con- centration of all nutrients in their ration. This require- ment, which is far greater than that of human infants, is associated with the fact that the young rat grows relatively at a much faster rate than human infants (4 ). At birth, the rat weighs 5 to 6 grams; it is usually weaned on the let day, when it weighs 35 to 40 grams. That represents a seven-fold increase in body weight in three weeks. Under ordinary circumstances, the laboratory rat is started on nutritional experiments at weaning. Thereafter, it frequent- ly increases its weight at the rate of 6 grams per day providing its ration is adequate. This means, it doubles its weaning weight in 6 days. On the other hand, the human baby gains weight at a much slower rate. Consequently, the dietary requirements, from a quantitative standpoint. of these two species are entirely different. By limiting the protein level in a ration to 10%, many plant proteins produce poor growth in weanling rats, whereas animal proteins, so called "superior proteins," produce better growth per gram of protein and thus receive a high PER score. This was evident in Bolourchi gt gl.'s (3 ) studies where her weanling rats grew very poorly when fed a diet in which bread provided the only source of protein. Another factor which has become more important as a result of the MSU bread study, is the time required to establish an equilibrium state in nitrogen balance studies. For nitrogen balance studies, many investigators working with adult human subjects have assumed and some still believe that three, five, or at the most, seven days are adequate for the development of an equilibrium state. Most workers claim that in such a period of time, it should be possible to determine the efficiency of a given protein; this is not true as has been shown in the Michigan State University bread study (1 ). During that experiment, one of the unexpected observa- tions made when the subjects ate the bread diet, was a marked reduction in blood urea levels (5 ). The first blood sample secured after the start of the bread diet was on the 25th day and the next on the 50th day. The average value at the latter time was 6.4 mg /100 ml , while at the end of the control period it had been 16.9 mg /100 ml. There have been suggestions that the vegetarian diet and the acid-base equilibrium of the individual may have some impact on kidney function in general and reduction in blood urea levels, in particular. Most reports in the literature are either of a preliminary nature or limited in scope. Therefore, on the basis of the data available, it has been considered important to further investigate the effect of different dietary sources of protein, i.e., animal vs. vegetable, on renal function as well as blood and urine composition. The nutritional status of patients with renal malfunc- tions have been the subject of study almost from the time of the first description of the uremic syndrome. The unique problem of deranged metabolism, precarious nitrogen balance. low protein intake, and states of malnutrition, have attrac- ted nutritionists to the clinical environments of nephrology (6,7). The uremic syndrome is a constellation of clinical and physiological abnormalities resulting from extensive deterioration of renal function. It is believed that many of the manifestations of uremia, such as disturbed functions of the peripheral and central nervous systems, bleeding abnormalities, pericarditis, and gastrointestinal distur- bances, are the results of retained toxic metabolites. The end-product of exogenous and endogenous nitrogen metabolism have long been suspected as the toxicants responsible for the uremic syndrome (8 ). Urea, the major end-product of mammalian protein metabo- lism, when retained in the body. has been exonerated as the primary etiologic agent in the uremic syndrome. During the past decade, advances in nutritional therapy along with the development of long-term hemodialysis and renal transplantion, have added immensely to the possibili- ties for treating chronic uremic patients. Anderson gt_gl. ( 9) reported that of the three methods only nutritional therapy is applicable in every case. The magnitude of the problem posed by chronic uremic patients is evident from the mortality statistics. Basing calculations on the report that only about one in five patients who die from this condition could benefit from dialysis (10). This represents 1500 patients per year in the United Kingdom and about 7500 patients per year in the United States. These figures are based on the reported yearly mortality of 7000 for Britain and 35,000 in the United States. In the United Kingdom, the estimated cost of treating one patient in a dialysis center is.¥2500 per year; in the United States, the cost is more than $3000. To implement the recommendation of the Committee on Chronic Kidney Diseases in the U.S.A. for a national dialysis and transplantion program would involve the expenditure of one billion dollars from 1970-1975. The annual mortality rate in the United States is in excess of 30% for patients undergoing dialysis (10,11). Also, dialysis patients must spend many hours each week attached to a kidney machine. These patients are under a severe dietary restriction and suffer important side effects that may include impotency, depletion of calcium in the bones. and neurological and psychiatric changes. 0n the other hand, yearly costs for transplant patients are close to $3,000 (12). From these figures, it may be seen that few countries have the wealth or medical resources to cope with all financial and logisti- cal implications of dialysis and/or transplants. Clements (13) suggested that the most important thing to remember about kidney transplants is not that they are marvels of surgical techniques, but that they are the outward and visible manifestations of the failure of medicine to preserve the integrity of this organ. It is for this reason that every effort should be made to determine how renal failure can be prevented or the patient maintained in reason- ably good health for as long as possible before having to be subjected to either dialysis or a transplant. The level of urea in the blood is used as an index of kidney function. Any unexplainable increase in the blood urea level represents a potential derangement in kidney activity (14). One of the dietary factors that is associated with an increase in blood urea is the level of protein in the diet. The more protein that is consumed, the higher the level of urea in the blood. The concept that the only dietary factor affecting the blood urea level is the amount of protein in the diet was suggested by the work of a number of scientists (15,16). Those investigators provided no basis for a con- clusion other than that the level of protein in the diet was the only factor appearing to influence the blood urea level. Furthermore, earlier investigators (15,16) apparently had established the concept that the only dietary factor affec- ting the blood urea is the amount of protein consumed, since there was no theoretical reason for believing otherwise. Furthermore, no thought was given to the possibility that in normal individuals, the functioning of the kidney could be influenced by the nature of dietary protein. That factors other than dietary protein may influence the blood urea level was demonstrated by some Edinburgh pediatricians(l7,l8). They reported that children who devel- oped renal disturbances secondary to scarlet fever, showed a reduction in their high blood urea levels when they were given sodium bicarbonate. With that, most of the symptoms associated with renal disturbances disappeared. The present concept of protein excretory products basi- cally, is a confirmation of Folin's observations (19). He showed that in mammals, urea excretion is related to the dietary protein intake. That assumes that all proteins have the same effect on urinary urea excretion, and by indirec- tion, the same effect on the blood urea level. This concept dominated the attempts to provide dietary care for those patients required to undergo routine renal dialysis as a consequence of poor kidney function. To mini- mize the load imposed on the kidneys of these patients, their diets have been designed to provide as little protein as possible. By thus reducing the load on their kidneys, the patients should be enabled to extend the time between dialysis. Admittedly, renal dialysis is only a temporary solution to the ultimate problem faced by these patients. Today, the long-term prognosis for these patients depends on the main- tenance of a successful kidney transplant. Another potential approach, based only on theoretical consideration is, as soon as the abnormality has been discovered, to delay the development of the syndrome. The rationale for this suggestion comes from the classi- cal M.S.U. Bread Study. Additional support for this propo- sition is indirectly provided by the composition of the initial diets developed for renal dialysis patients. The Giovannetti and Giordano diets were based on wheat products. It is likely that wheat was the primary ingredient in these diets, since the Italians for whom they were developed, use a number of wheat products that are low in protein. This may have been the primary reason for the nature of these original diets. One of the primary goals of the present study is to evaluate the effect of different proteins on the blood urea concentration of both human subjects and rats. The concept that is proposed as a justification for this project is that different proteins are not alike in-so-far as their effect on blood urea levels is involved. The results of this work 10 should provide a firm basis for indicating the mechanism and possible implications of reduced blood urea levels brought about by a high bread diet. For this purpose, the experiments were designed to test human subjects and rats to see whether blood urea levels can be changed either by (l) changing the nature of dietary pro- tein, (2) changing the acid-base state of the individual, or (3) whether both factors work together to bring about this change. PART I REVIEW OF LITERATURE The IIIEIUQPFF-IXE.DIPI FUL-R9PPI Function ifl-fl?fiIIh.fiUd Disease The kidneys are paired on either side of the spine; they are flattened, bean shaped, and located retroperitoneally in the lombar region. In a 70 Kg. man, together they weigh about 300 grams and thus constitute 0.4% of the body weight. In spite of their small size, the kidneys hold the key posi- tion in controlling the homeostasis of the internal environ- ment (20). The kidneys act as an important metabolic and endocrine organ with profound effects on homeostasis. But, occasionally one of their vital function goes away. The structures. especially the proximal convoluted tubules, are also the guardians of the body's nutritional wealth. Nutritional homeostasis can often be achieved by sound dietary and nutritional principles and practice even in advanced renal-urologic disorders. The effect of diet on kidney function, size and compo- sition, has thoroughly been reviewed in the Ph.D. thesis of Dr. Jenny T. Johnson, 1972 (21). This review will be concerned primarily with the influ- ence of quantity and quality of protein on a number of phys- iological effects-~i.e.. nature of protein on renal function, blood urea levels and acid-base homeostasis of the indivi- dual. Furthermore, the effect of change in the acid-base state of the individual on the blood urea nitrogen also will be reviewed. I2 I3 Dietary Protein and Renal Function Changes in the amount of dietary protein consumed sub- stantially affect the function, metabolism and structure of mammalian kidneys. Several well documented and significant changes in renal concentrating ability (22-25) have appeared. These cover such topics as urea reabsorption, (26-30) glomerular filtration rate, renal plasma flow and maximal rate of tubular secretion of p—amino hippuric acid (31-34), activity of enzymes related to the intermediary metabolism of amino acids and carbohydrates (35-39) and growth and weight of the kidneys, and tubular structure (40,41). Dicker gt gt. (42) have shown that in rats, a protein deficient diet led to decreased ability of the kidney to concentrate the urine. McCance and co-workers (43) evaluated the ability of the kidney to concentrate urine in Ugandan children on protein-deficient diets. When severely malnour- ished children were admitted to the hospital, the maximal urine concentration was less than half that attainable on discharge. The parents of these children also ate a low protein diet and could not produce a normally concentrated urine. When their diet was improved. they achieved the same concentration as their European counterparts. Also when Europeans ate the local protein-poor diet, their urinary concentrating ability decreased. I4 Likewise, semi-starved individuals have been reported to have an increased urine volume (44). In the Minnesota study, as an example, the urine volume was 3 to 4 liters in a 24 hour period, while the young men were starving. Although the subjects in the Minnesota starvation study had polyuria, no specific determination of renal function in these men was actually made. Pullman gt g1. (45) described experiments in which they fed normal adults diets high in protein (2.3 to 3 g protein per kg of body weight), medium and low in pro- tein (0.1 to 0.4 g of protein per kg of body weight). Glomerular filtration rate and renal plasma flow dropped while the low protein diets were fed and increased with the high protein diets. Nielson and Bang (46) studied normal subjects fed varying amounts of protein and found that the glomerular filtration rate was relatively unaffected by changes in the protein content of the diet. Sargent and Johnson (47) reported that when normal subjects were fed a caloric-deficient diet, irrespective of the percentage of calories from protein, fat or carbohydrates, there was a constant reduction on glomerular filtration rate as measured by creatinine clearance. Over a period of four years, these investigators have studied a total of 211 subjects under strict control of diet, fluid intake and daily activity. They concluded that many alterations can be provoked by nutritional imbalance. caloric deficiency. dehydrateion, physical work, and extremes of temperature. 15 Table 1 summarizes data for renal plasma flow and glomerular filtration rate in children and adults with pro— tein calorie malnutrition. Alleyne (48) found that the glomerular filtration rate (GFR) was markedly reduced in malnourished children and improved as they recovered. He could not find any consistent difference in inulin or PAH clearances between edematous and non-edamatous children. Gordillo ”t gt. (49) measured inulin and PAH clearances in ten children who were at least 40% below average weights for their age. Seven of these children were well hydrated and three were severely dehydrated. In well-hydrated, malnour- ished children, the clearance rates were about half the normal values while in the dehydrated,malnourished they were about one-fifth normal. Working with adults, Klahr gt gt. (50) demonstrated a marked decrease in both GFR and the renal plasma flow (RPF) in ten adults with protein malnutrition. Values for these measurements increased following protein repletion. GFR increased somewhat during repletion leading to an increase in filtration fraction. On the other hand, no decreases were observed in the glomerular filtration rate and the renal plasma flow in five cases of severe malnutri- tion with protein "deficiency edema" (51). These subjects differed from others reported in the literature in that they were oliguric at the time of the experiment. It seems that in children, protein calorie malnutrition leads to a decrease in both renal plasma flow and glomerular filtration rate (47,48). 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R :e L I I I K e: er .m000> mm0em>0 cu0m eoe cm>0m me0 0cowp0w>m0 0e00c0pmx «“000 0000 004000 xmhmw mm0em>< 0_N 00 00_ 00 .<.z 0e_ 00 00, e0 .z.0 000 00 000 N0 .z.0 _0\0e 00\0e _0\0e 00\0e 000000wm FOmemwpor—u mmouzpw POqumm—oco wmouapw . 0002020000x0 0000200 .0000 m>we eoe 0m0o0 _0uam mmecp :0 000 emu moux0z 00 050 00 + pm00 eo 0:03 ms00 000 000020 0500 ANV 0:0 Poepcou 00 0000 m>me eow pm00 =000eme< _0uwu0u co 00v memz on: 0pum0000 P0seoc 0meeu :0 Foemp0mpoeu 0:0 m0ouuFm $0 Fm>m_ Eaem0 :0 0m0o0 00uam mmeeu :0 000 emu mouz0z 0E0em 00 mo cowp0mmcw meg mo Homewm--.m mum<0 65 Utinary RH and Titrgtable Acid Excretion There was an increase in the alkalinity of the 24 hour urine sample when sodium bicarbonate was ingested since the pH of the urine increased markedly from 6.0 to 7.7 (Table 4). The titratable acidity dropped significantly (Table 4), from 22.6 t 26 mEq/day to 17.4 14.5 mEq/day (Fig. 2). Although the fluid intake was kept as constant as pos- sible, the urinary output increased in the experimental phase. Serum Glucose and Cholestergt These two parameters of the blood were practically the same at the end of the experimental period as at the end of the control period (Table 5). iscussion These studies confirmed those of Lyon gt gt.(l7) des- cribed in the introduction to this chapter and thus the same conclusion reached by Lyon gt _t. also apply to this study. In our studies with normal subjects the addition of sodium bicarbonate to the typical American diet resulted in a low SUN. The cause of this decrease in SUN was either (1) decreased urea production, (2) increased urea clearance by increased GFR and/or increased urine flow, or (3) dilu- tion of the total body urea by the addition of extracellular fluid to body with ingestion of sodium bicarbonate. Although Lyon gt gt. stated that sodium bicarbonate was useful in treating patients with renal failure by 66 compensating for the metabolic acidosis of renal failure. the studies in this thesis were performed on normal indi- viduals and do not necessarily prove that sodium bicarbonate treatment would be useful for lowering SUN in uremics. Indeed the addition of sodium would be detrimental to those uremics who have difficulty in excreting sodium. The overall concept that acid-base alteration can affect the SUN is substantiated by the experiments described in this chapter. Plasma creatinine showed no change during the ingestion of sodium bicarbonate. This observation seems to suggest that the GFR increase which occurs with the ingestion of sodium bicarbonate was not large enough to lower the serum creatinine concentration. No changes were observed in the levels of serum glucose and cholesterol. This suggests but by no means proves that sodium bicarbo- nate ingestion has apparently no effect on the metabolism of these two parameters of the serum. The urinary pH (Table 4) was raised by the ingestion of sodium bicarbonate, while titratable acid was lowered. In conclusion, the administration or ingestion of sodium bicarbonate has an effect on the lowering of SUN concentration in normal individuals. According to the ob- servations made in this study, it is more likely that the effect may be either upon the urea production and/or the urea clearance. 67 Figure 2.--Average titratable acid excretion and urine pH of individuals fed typical American diet alone and with 15 g sodium bicarbonate. 68 . 0000>a>mrm >000 mxnmmquoz g 00 .. 90230.. * 90023022... II, 1? I No 1 ll MEo/DAY 001 0.0 0 00020 0: PH \ m . .. \ UT 0 3.11151111011qu o MumL. I. _ . _ F . a w m w m 2 0w Chapter II Effect Of Ingestion Of Sodium Bicarbonate 0n SUN 0f Individuals Consuming A Liquid Wheat Gluten Diet Intr _ggtjgn _————.-—~—- The purpose of this experiment was to repeat the studies performed by Taylor, gt gt, (114), paying special attention to the changes produced in the acid-base balance of the human subjects consuming the liquid wheat gluten diet. That group reported that there is an inverse rela- tionship between the biological value of a protein and the level of blood urea. Hence, when wheat gluten was the only source of nitrogen, the blood urea increased in the human subjects. From the composition of the liquid gluten diet used in Taylor's study, it appears that it would produce an acidosis in the body - especially since cola drinks were consumed. If previous work of Lyon gt gt. (17) is valid, then possibly the acidic condition produced by Taylor's diet would counteract the reduction in urea levels that we have so frequently observed in subjects fed diets in which the only source of protein was from wheat. This work involved a study to observe if the ingestion of sodium bicarbonate would lower SUN of normal individuals consuming the Taylor's liquid wheat gluten diet. This study would therefore determine (1) if acid-base changes were a possible cause of the alterations in SUN of individuals consuming wheat gluten as their source of dietary protein and (2) whether or not the SUN changes with changes in the acid-base state of individuals on an entirely different dietary regimen from the study with typical American diets 70 71 described in the previous chapter. ttperimental Procedure a. Control Period: Four normal, healthy, adult male subjects were fed a constant volume of the fluid diet des- cribed by Taylor's associates; Young gt gt. (”H.182), Scrimshaw gt _t. (183). This was consumed in four equal doses at 8:00, 12:00, 17:00, and 21:00. This diet provided 2110 kilocalories. . .the remaining kilo calories needed to maintain body weight came from crackers containing flour, shortening and sucrose. Since Taylor gt gt. (114) indicated that soft drinks (mostly colas) were permitted gg libitum with no definite indication of amount and brand, the sub- jects in our study were provided with 16 oz. of a specific soft drink (sprite, uncola) every day. By that means, the acidity attributable to the soft drink was the same in all subjects. Water was permitted, but the volume consumed was kept constant throughout the study for each subject. Each subject had in the refrigerator, a bottle containing a measured amount of water. All ingredients of the diet except for the soft drink were obtained from the same sources mentioned by Young gt gt. (181). The composition of the liquid diet is given in Appendix A. Body weights were recorded every other day throughout the entire study period (Appendix E). The control period was 7 days. 72 b. Experimental Period: The procedures carried out in the control period were also performed during the experimen- tal period. The only difference was that a total of 10 grams of NaHCO3 in 3 doses per day was consumed by each individual with water. This phase was extended for 7 days. c. Sample Collection: 1. Urine Every day during the study, 24-hour urine samples were collected by each subject. They were provided half-gallon, wide mouth, plastic bottles, which contained 10 m1. of toluene as a preservative. The urines were ana- lyzed immediately after they were brought to the laboratory for total volume, pH and titratable acidity, as described in Chapter I of Part II of this thesis. 2. Blood Each subject, before breakfast, reported to the 01in Health Center on the first and last days of the control and experimental periods. All blood samples were withdrawn from anticubital veins. After allowing the blood to stand for 4-6 hours in a refrigerator, serum was then isolated. Analytical Procedure A. Urine The urine samples were analyzed in a similar manner as described in Part 11, Chapter I. 73 B. Serum The serum was obtained from the whole blood as described above and was preserved in the freezer until analyzed. Serum urea nitrogen for each subject was determined by the diacetyl monoxine method and creatinine by Folin's picric acid method (184). Serum sodium and potassium con- centrations were analyzed by an atomic absorption spectro- photometer, Perkin-Elmer 303 Model. Rgsults ggrum Urea Nitrogen (SUN) SUN values in three individuals seemed to drop slightly when NaHCO3 was taken with the liquid formula diets, despite the maintenance of a constant protein intake. One of the individuals whose control urine pH was high had an increase in his serum urea nitrogen levels when he ingested sodium bicarbonate (Table 6 ). The serum urea nitrogen levels were not significantly different at the end of the experimental phase as compared to those at the end of the control phase. Serum Creatinine The average concentration in the four subjects was the same at the end of the experimental (1.53 i 0.8 mg/dl) as it was at the end of the control period (1.53 i 0.10 mg/dl). gtinary pH and Titratable Acid As postulated, the liquid diet even without cola con- sumptionseemed UJproduce an acid condition in the body; because urinary pH was on the acidic side when this diet 74 .0000:u _0p:mswemuxm 0:0 _oep:00 mo 0m0_0> zam m:0 :mmzpmn m0:memee00 Amo.ovuv 0:0000wcmw0 0000000000000 0: 00 memepxxx .em:mwc 000.0vuv 000:00000:000 00 0owemu F0p:mswemuxm mo 0:m me“ 00 :u 0e0:0e:xx .m=_0> mm0em>0 :00m eom :m>0m me0 0:00000>m0 0e00:0pmx xxee..00.e 00.0.00.0 0.0.0.00 0e..00.0 x0e.0.00.e xxx0.0.0.00 000e0>< 00.e 00.0 0.0: 00.0 00.0 0.0_ .<.: 0_.e 00.0 0.0: 00.0 00._ 0.0: .0.0 00.e 00.0 0.00 00.0 e0._ 0.e_ .0.0.e _0.e 00._ 0.0 00.0 00._ 0.0_ .3.0 :0 e0\0e _0\0e :0 _0\0e e0\0e 0e000e0 00000000e0 Z00 0e000e0 0000000000 200 00000000 0 Iu 0e0:0e: 0:0 Azzmv :mmoepw: 0me0 Euemm--. 0 m0m<0 75 vav .emzoe e00 .0.» xxx 000:00000:m00 0F 000emu 000:050emuxm 00 0:0 mew 00 000 emu :000me0xm 0000 m_0000 .o 00 00 00:0 e0000: Amo.ovuv 000000000cmw0 me0 000emu P0p:mswemuxm e0 0:0 mcp 00 02000> 0:0e0xx .0m:_0> mm0em>0 000 e00 :m>0m me0 0:00000>m0 0e00:0umx m.mflxxxm.0m Ppmflxxmomp m.om0N.mm xoou—mNF m00em>< 0.0m owmp 0.000 ommp .<.z 0.NN m_mp . 0.000 0000 .m.o 0.00 0000 ~.Nm mNNF .m.m.u 0.0N mpmp m.mm_ OFNP .3.0 000\cms PE 000\cme PE 11 cowpmeuxm 000< 0e0:0e: m5000> m:0e: :0wpme0xm 000< 0e0:0e: me:~0> m:0e: pumwnum e00: 0N e00: 0N e00: 0N e00: 0N 00 me0 :30:0 0mup0> .xmmz 0 e00 00000P 000emu 0e00000 :000 .000 emu 00000 mme:u :0 000:02 00 0E0em 00 + 0000 00=Ee00 00:00, 0500 Amy 0:0 0000 _0e0:00 00 .0000 .momnmm .euuz .:000 .0 .E< ..Hm MM 0:000 00 0:00e0000 .0000 00:5eoe 000000 00v :0 memz 0:: 000000 00Ee0: e000 00 0000000 0:0 manpo> 0e0:0e:--.e m0m mm0em>0 FF0 eoe :m>wm me0 m:owu0w>m0 0e00:0umx m.e.~.m PHQQF 0.0.e.¢ xo._.¢¢e mmmem>< 0.: gee e.¢ 00, .<.: 0.0 ewe 0.0 00. .m.u m.m :ve e.¢ wqe .m.m.e 0.0 eve 0.0 m0, .3.o mecm: +¥ m\:m: +02 4\:m: +¥ m\:m: +mm 4 op m:w0eouu0 .mgmw0 0pseeoe 0030?, A_v 0m» memz 0:3 mpumnaam P0Eeo: eaoe we mm=F0> 53000000: 0:0 520000 Enemm--. m mmma>mrm >000 mxnmmqeoz .Vo . z 8 8239. Q 90%/«APE? ‘ All I MEo/DAY no. / i /. No1 3. _ _ e . _ _ . cmezm 0: lllml PART III EFFECT OF THE NATURE OF DIETARY PROTEIN FED ISONITRDGENOUSLY ON THE ACID-BASE HOMEOSTASIS AND ON VARIOUS PARAMETERS OF BLOOD AND URINE Chapter I The Comparative Effects Of Isonitrogenous And Isocaloric ‘Diets On The Acid-Base Balance And Various Parameters Of The Blood And Urine Of Normal Human Subjects Introduction There is sufficient evidence both from our preliminary work and from reports in the literature to suggest that the acid-base condition in the body has a marked influence on a number of physiological and biochemical reactions. It should be emphasized that ingestion of meat because of its relati- vely high concentration of sulfur and phosphorus, tends to produce an acidic condition in the body; whereas vegetarian diets usually produce a more alkaline condition. These acid- base changes produced by different diets are reflected by changes in urinary pH and titratable acidity. The acid or base forming properties of food depend on the inorganic con- stituents or ash residue remaining after the food has been metabolized in the body; and, according to the type of the food eaten, the ash is neutral, acid, or alkaline. On the basis of the preceeding studies, the experiments in this chapter were designed to explore (l) whether change from meat to potato diet alters serum urea nitrogen, creat- inine and uric acid in particular, and other blood parameters which are determined by SMAC (186) automatically; and (2) whether change from meat to potato diet alters the urinary pH and titratable acidity. This study was initiated because it was suspected that shifting from meat to potato diet would make more alkaline metabolic products. By making the body more alkaline, it was suspected that serum urea nitro- gen would decrease on the potato diet. If this assumption were to be validated by these experiments, this finding 86 87 would substantiate the proposal that acid-base changes in the body affect the SUN and also would suggest that the decreased BUN observed in individuals on wheat diet (5 ) may also be caused by alkaline dietary metabolic products. Experimental Procedure To determine that a vegetable source of dietary protein can change the SUN, two experiments were performed as fol- lows. The protocol and diets were the same; in that they were isonitrogenous and isocaloric. The major source Of protein during the control period, in both cases. was meat and during the experimental period, it was potato. The purpose of the first experiment was to explore whether a vegetarian diet has an effect on blood urea nitrogen and acid-base balance as reflected by the urinary pH. In this first study with three subjects, only a few blood parameters were examined; and only the pH of the urine was determined. In the follow—up experiment, about six months after the first,six college students were chosen for the study, Numerous serum parameters were measured. Also in this second study. urinary titratable acid excretion and urine flow rate were measured along with the urine pH. Subjects In all, there were ten volunteers who participated in these two experiments. One of the subjects in the first experiment caught flu and was dropped. The rest of the subjects remained healthy and had no complaints 88 throughout the entire study. The subjects had no signifi- cant change in body weight (Appendix F). §ghedule Both studies consisted of a control period of seven days and an experimental period of seven days. Twenty-four hour urines were collected every other day. Blood samples were drawn at the beginning and end of the control period and then at the end of the experimental period. All meals were provided to the volunteers at no cost. 9.19.2: During the control period, the subjects received a variety of foods which provided 46 g of protein per day per person regardless of the subject's size. The major source of the protein was meat. The amount of protein given during the control period was chosen so that it would be isonitrogenous with that in the experimental diet. The caloric intake in the control period was raised by providing extra carbohydrates (Table 9a), to make both the diets approximately isocaloric. The subjects were required to consume all of the food provided. The diet of the experimental period also contained 46 g of protein per day per person. However, the major source of protein on this experimental diet was potatoes. The sub- jects were required to consume all the food provided in this experimental diet including the drippings of mutton tallow, which were a major source of calories. 89 TABLE 9a.--Composition of meat diet (control) Menu: This was based on Mrs. Mickelsen's Formula II dated Feb. 7, l975. The object of the menu was to provide 123 g of lamb fat or llO7 kcal of fat in the diet containing 2800 kcal total. Each person received Formula II in the following amount: kcal Protein (g) 200 g lean meat containing: l7% protein lZO 34 5% fat 90 - ll4 g mutton fat l026 - Ordinary bread (with calcium) 6 slices 420 12 Jam, maple syrup, honey, hard candy 300 - Dripping, mutton tallow (on bread) l5 g 135 - 2 oranges or apples 8O - lettuce or other greens 4O - 22ll 46 2 cups coffee or tea per day (no sugar or cream) Honey (lst exp., 3 subjects) 2 table- spoons per day Honey (2nd exp., 6 subjects) l table- spoon per day Sprite 12 oz per day Carbohydrates added specifically to increase caloric intake of meat diet lst exp weighed amounts of candy and sugar 2nd exp weighed amounts of chocolate fudge 6T7 Total 2828 Kcal 90 TABLE 9b.--Composition of potato diet (experimental) _-—.., _q. ~- -1 . --—- —-_- ”.— gggal Protein (9) l032 g potato (boiled and peeled) 785 21.7 l23 g mutton fat llO7 - l2? 9 potato concentrate 446 lO.6 7 slices bread 490 l4.0 Total 2828 46.2 2 oranges or apples, honey, Sprite, lettuce, coffee or tea was consumed in same amount as given during the control diet. _—_ 91 Both diets were cooked in the laboratory by a volunteer. The complete dietary intakes for the control and experimental periods are shown in Tables 9a and 9b. The same daily menu was followed throughout the entire study. Sample Collection Twenty-four hour urines were collected by each indivi- dual every other day. They were provided with wide mouth, plastic bottles containing toluene, as a preservative. A plastic bag was provided so that the subjects could carry their bottles. Blood samples were drawn in the Olin Health Center and after four-six hours standing in a refrigerator; they were centrifuged for serum collection. All blood samples were collected from the anti-cubital vein before breakfast. Serum samples were preserved by freezing. Analytical Procedure A. Urine As the urine samples were brought to the laboratory, they were examined for the pH and titratable acidity as described previously on page 60. 8. Serum The frozen serum samples were sent to the Laboratory of Clinical Medicine, where they were analyzed by SMAC (l86) for SUN, creatinine, uric acid, Na+, K+, Ca++, P+++, total protein, total C02, glucose, cholesterol, and albumin. Since the serum was exposed to the atmosphere, the values for total 92 CO2 are not valid. Also since the blood pH was allowed to change after it had been collected, serum potassium level may also not be valid. Since nonetabolicinhibitor was added to the blood to prevent glucose metabolism, the serum glucose analysis is probably also not valid. C. Diet The protein and caloric contents of the diet were cal- culated from the U.S. Agriculture Handbook, No. 8. Results Effect of Potato Diet on Serum Urea Nitrogen The average SUN changed slightly from 14.2 mg/dl at the start of the control diet to l3.3 i 4.7 mg/dl at the end of the control period. The change is not statistically signif- icant. But, when the subjects consumed the potato diet which was isonitrogenous and isocaloric to the control diets, the serum urea level in each subject decreased (Table l0). The decrease was about 50% on an average. At the end of the seven day experimental period, the average SUN values were 7.0 i l.3 mg/dl as compared to 13.3 mg/dl at the end of the control (meat) diet. In the follow-up experiment with six college students, when the approximately same dietary regimen was followed, the SUN values (l3.0 t 4.0 mg/dl) at the beginning of control increased slightly toward the end of the control diet to l4.8 t 3.0 mg/dl. There is nothing to suggest that this slight change was of any physiological significance or that 93 it is statistically significant. At the end of the experi- mental period, the SUN was observed to be lower than the respective control SUN in each of the six individuals. The reduction amounted to about a 30% decrease in average values for the serum urea nitrogen. The average value dropped from 14.8 t 3.0 mg/dl at the end of the control diet to 10.3 t 1.6 mg/dl of serum at the end of seven days on the potato diet (Table 11). Serum Creatinine and Uric Acid In the second experiment (Table 11), there was no change in the concentration of creatinine between the end of the control phase, 1.0 t 0.1 mg/dl and the end of the experi- mental phase, 1.0 z 0.1 mg/dl. On the other hand, uric acid concentration may have slightly decreased on the potato diet as compared to the meat diet; however, the observed change is not statistically significant. Serum uric acid which was 6.9 t 1.4 mg/dl at the end of the meat diet decreased to 5.7 t 0.8 mg/dl at the end of the potato diet. Urinary pH and Titratable Acidity In both experiments, the urinary pH changed signifi- cantly when the experimental diet was consumed by the sub- jects (Tables 10 and 12). The urinary pH in the first experiment in all three subjects changed from acid to alka- line. At the end of the control phase, the urinary pH was 5.5 t 0.3 and at the end of the experimental phase, it increased to 7.3 t 0.1 (Table 10). In the second study, the pH of the urine which was 5.8 t 0.2 at the end of the .0m000> mm0em>0 000 e00 :m>0m me0 0:00000>m0 0e00:00m0 .Amo.ovav 0000 000:050em0xm 0:0 :0:0 0m00 00e0:00 :0 emzop 000:00000cm00 00 :0 m:0e0 0:0 em:m0: 000:00000:m00 00 200 00.owom.0 m._Ho.0 0000~ umfimmp om.0000.m 0.00m.m_ 0N000~ ONHNFF mm0em>< _0.e 0.0 000 00 00.0 0.00 000 000 .0.0.: 00.0 0.0 000 00 00.0 0.00 000 000 .<.z 00.0 0.0 000 000 00.0 0.0 000 000 .3.0 _0\0e 00\00 _0\00 00\00 _0\00 00\00 :0 :mmoe00z 00em0 m00em0 x0 :m00e00z 00em0 m00emu 0e0:0e0 0me0 -0m00:u -0Pm0e0 0e0:0e0 0me0 -0m00:0 -0Pm0e0 0<0zmszmaxm 0000200 .0000 0 e00 0000:me0em0xm~ 000000 ANV 00 0m300000 .0000 0 e00 000e0:0uv :00005 000 00000 000:000e00:-000 0m0 mem: 00000000 mme:0 .0m00e 00:00 00sec: 00 :0 0e0:0e0 0:0 :mmoe00: 0me0 e00 0m000> E:emm--.o_ 000<0 C‘s-03: 0.x.» 0...!» «(02: AF» CC tCVQC Mada—C “0.0.0301UCUV tCN U..C(.a\p\r\va+.rillw i1 lilllieie .0 .0 0000\0. 95 .0000> 00: me0 m0000 00:0 :0 0m000> N00 00000 0:00 .0m000> mm0em>0 000 e00 :m>0m me0 0:00000>m0 0e00:00m« .0m000> 50em0 0m00 00m: Eoe0 0:00000:m00 0000000000000 00: memz m:0:000me0 0:0 .0000 00e0 .Noo 20em0 0m00 000000 .000.0v0v 0m00 000000 m:0 :0 em300 000:00000cm00 me0 0m000> zsm _.0.0._ 0.0.0.0 0.0.0.0_ 0.00 0.0.0._ 0.000.0 0.0.0.00 00.00 000e0>< 0.0 0.0 0.0 00 0.0 0.0 0.00 00 .3.0 0.0 0.0 0.00 00 0.0 0.0 0.00 00 .3.0 0.0 0.0 0.00 00 0.. 0.0 0.00 00 .0.0 0.0 0.0 0.00 00 0.0 0.0 0.00 00 .0.0 0.0 0.0 0.00 00 0.0 0.0 0.00 00 .0.0 0.0 0.0 0.00 00 0.0 0.0 0.00 00 .0.0 _0\00 _0\0: 00\00 _0\00 000:0 0000 00\02 0\:e 0:000 000: 00\0s 0 :0 00000000 -000e0 00e0 2-00e0 0000 -000e0 00e0 2-00e0 0 00 0000 000000 0000 000: .0000 0 e00 0m0000 000em0 0e00m00 :000 .000m0000 :050: 00 0000 00e0 0:0 m00x000 :00e00 0:0 m:0:000me0 0:0 0me= 00 00m>m0 50em0 :0 00m>000m00me .00m00 000000 000 0:0 00mE A00 :0 0m000 00m00 00e0000000 0:0 000:m00e00:000 00 000m000--.00 000<0 96 .000.0v00 000_0> 0000 0000 :0e0 0:000000000 00000000000000 .0m000> mm0em>0 000 e00 :m>0m me0 0:00000>m0 0e00:00m« 0 0m0000 000em0 0e00m00 :000 .0m000000 0N0 0:0 00mg 00 00000 00e0000000 0:0 000:m00e00:000 0m0 000000>0000 00 00 00.0.0.00 0.0.0.0 00,000.00 0.0.0.0 000e0>< 0.00 0.0 0.00 0.0 .3.0 0.0 0.0 0.00 0.0 .3.0 0.00 0.0 . 0.00 0.0 .0.0 0.0 0.0 0.00 0.0 .0.0 0.00 0.0 0.00 0.0 .0.0 0.00 0.0 0.00 0.0 .0.0 000\cms 000\cms :000me0xm :000me0xu 000: 000000e000 :0 0:0:0e0 000: 000000e000 :0 0e000e0 0000000 00000 0:000 + 000000 0000 0002 .0000 v :0 00m>000m00me 0m000 00000 0e000e0 :0 000000--.0_ 000:0 97 .Amo.0v0v 0000 0002 00 00000500 00 00:00; 0000000000000 000 0000 000000 :0 manpo> 00.00500r .00000> 00000>0 _F0 00» 00>0m 000 0000000>00 00000005..a oom000mmm ommfioom FmN0000mm mwpnmmm x—omnom0 ma000>< comp mmop 0000 0000 0000 .3.o mum 000 mmm owe omm .3.m omo 000 000 com ofim .o.H mmop ooFP mum 000 omm .0.o OFQ 000 000 emu omm .¥.o 00m coop owe omw 000 .H.0 000 00000: 00m 00000: ug0um mpomnnam huHo o00 0 0;» $0 000000>0 0;» 000 03000 00=F0> .000000 000 0005 :0 >00>00000000 00000 00000 0000000000 000 00000000000000 000 000000>0000 00 we:_0> 000000: 00 000000--.m0 000<0 98 .00000> 0m000>0 000 00>0m 000 0000000>00 00000000 00. 0.0“ 0.00 0.00 00 00 0.0 0.00 0.00 00 000 0.0 0.00 0.0 000 000 0.0 0.00 0.0 000 000 0000000 0.0 0.00 0.0 000 000 0.0 0.00 0.0 000 000 .3.0 0.0 0.0 0.0 000 000 0.0 0.00 0.0 000 000 .3.0 0.0 0.00 0.0 000 000 0.0 0.00 0.0 000 000 .0.0 0.0 0.00 0.0 000 000 0.0 0.00 0.0 000 000 .0.0 0.0 0.00 0.0 000 000 0.0 0.00 0.0 000 000 .0.0 0.0 0.0 0.0 000 000 0.0 0.0 0.0 000 000 .0.0 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 II 1+0 ++00 +0. +02 -00 $0 ++00 +0. +02 -00 0000.200 0000 00000 + 000000 0000 0000 000 000000 000000 0000000 0000 .0000 0000 :0 0000 0 .00000 000000 Amy 000 0005 00v :0 00000 00000 0000000000 000 00000000000000 000 000000>0000 00 000000000000 50000 :0 000000--.00 00000 99 .00000> 00000>0 000 000 00>00 000 0000000>00 0.000000?0 N.000.0 0.000.0 0.000.0 00.000.0 00000>0 0.0 0.0 0.0 0.0 .3.0 0.0 0.0 0.0 0.0 .0.0 0.0 0.0 0.0 0.0 .0.0 0.0 0.0 0.0 0.0 .0.0 0.0 0.0 0.0 0.0 .0.0 0.0 0.0 0.0 0.0 .0.0 0000 0000 0000 , 0000 0000000 0000000 00000 0000000 0000000 00000 00000000 0000 00000 + 000000 0000 0000 .0000 0 000000 000000 0000000 0000 .00000 000000 ANV 000 0000 000 00 >00>00000000 00000 00000 0000000000 000 00000000000000 000 00000000 00000 00 00000000 00 000>00 0:000 00 000000--.m0 00000 100 000000 0000000 000000 000000. 00000>0 000 000 000 000 .0.0 000 000 000 000 .3.0 000 000 000 000 .0.0 000 000 000 000 .0.0 000 00 N00 00 .0.0 000 00 000 000 .0.0 00000 00000 .0000 00000 I 00000000000 000000000000 00000000000 000000000000 00000000 0000m 50000 0:000 0:000 00000 00 0000 00000 + 000000 00000 00 0000 0000 .00000000 00000 00 00000000000 00000 000 00000 00 00000 + 000000 0N0 000 0000 00V 00 000>00000000 00000 00000 00000000000000 00 0000000--.00 00m<0 101 control period rose to 7.2 2 0.5 at the end of the experi- mental period (Table 12). This shows the alkalinizing effect of the potato diet. Similarly. total titratable acidity of the 24 hour urine which was 20.7 t 7.4 mEq/day during control dropped to 10.6 t l.7 mEq/day as the urine became more alkaline at the end of the experimental potato diet. The reduction in acidity was accompanied by a slight increase in the average daily urinary volume (Table 13). The time course of the change in urine pH and titratable excretion is shown in Figure 4. Electrolytes and Protein There was no change observed in the serum electrolyte concentration throughout the entire study (Table l4). Fur- thermore, the total serum protein and serum albumin remained constant (Table l5). Discussion In two different experiments with human subjects, diets high in potatoes and free from animal protein produced a reduction in serum urea levels and an increase in urinary pH. This was true when the subjects were fed diets, in which the sole source of protein was potatoes and bread as compared to an isonitrogenous, typical American, omnivorous diet. The influence of different sources of dietary protein on the blood urea level was reported in l93l by Lyon in Edinburgh (l8). They clearly demonstrated the reduction in blood urea nitrogen when their patients were shifted to 102 diets wherein the protein was derived from vegetables. These authors interpreted the decreased BUN as being caused by change in the protein from animal products to vegetable products. They also suggested that body acid-base changes with different diets may also influence the BUN. The results of the experiments described in this chapter support the latter (acid-base) proposal of Lyon. In the study (Table 10) with three subjects, there is a reduction of almost 50% in the serum urea nitrogen after 7 days on the isocaloric, isonitrogenous potato diet. At the same time, urinary pH on potato diet increased to the point where the urine became alkaline. The potato diet was free from meat which is rich in sulfur and phosphorus. Thus, the potato diet produced alkalinity in the body, as reflected by increased urine pH. In a follow-up study, with six normal, young college students, the dietary regimens, shown in Table 9a and 9b, were followed strictly. In this second study, a similar reduction (30%) in serum urea levels was observed when the experimental diet was fed (Table 11). This SUN reduction was, again, accompanied by increased urinary pH when the experimental potato diet was fed. These observations further substantiate results of other investigators, who have pre- sented evidence that alkaline diets reduce SUN (l8). In the studies described in this thesis, the cause of the decrease in urea nitrogen seems to be extra-renal rather than due to changes in the renal function because serum creatinine and 103 uric acid were not altered. It should be emphasized how- ever, that changes in renal function can take place without significantly altering serum creatinine and uric acid con- centrations. When the diet was changed from meat to potato, there seemed to be an effect on the rate of urine flow. The 24 hour urine volume was 40% higher on potato diet as compared to meat diet (Table l3). From the increase in urine flow on potato diet, it can be suggested that the reduction of SUN on potato diet might, in the past, be due to an increased urea clearance, brought about by increased flow rate in the nephron. Previous reports from this laboratory have indicated that there is no change in the fecal nitrogen, or urinary urea nitrogen, when the subjects were changed from a typical American diet to diets high in wheat (5 ). Thus, the change brought about by the nature of dietary sources of protein seems to have no influence upon the production rate of urea. 0n the other hand, others have observed a decrease in liver arginase activity when rats ingest protein from wheat as compared to milk protein, casein (ll3). This latter observa- tion suggests that urea production in the rat is lower when wheat is the source of dietary protein. Thus, there is con- flicting evidence concerning the rate of urea production by the liver when animals have different dietary sources of protein. Since the studies performed in this thesis had no urea analysis performed on the urine, no further information 104 regarding rate of urea production by the body can be given. Although there seemed to be a decrease in the concen- tration of uric acid in the serum when the subjects consumed potato diets (Table ll), this change was not statistically significant. It is possible that, due to the impaired uric acid excretion in acidosis (186-19l), there was less uric acid excretion on the control diet. No changes in serum electrolyte concentration occurred between the end of the control and the end of the experi- mental periods (Table l4). These observations indicate that calcium, phosphorous, Na and Cl control was maintained during the dietary changes. The lack of change in the serum potassium concentration, suggests that only small acid-base changes occurred as a result of the shift in meat to potato diet. Acidosis usually increases and alkalosis decreases serum potassium; however, in this study, the subjects con— suming the potato diet had approximately seven times the potassium intake of those on the meat regimen. Therefore, it is possible that the high potassium intake of the subjects on the potato diet raised their serum potassium levels in spite of their alkalotic condition. The obviously higher intracellular potassium levels of the individuals consuming the potato diet would probably have a profound effect on their body chemistry. Whether it can alter SUN is unknown. Total protein and albumin levels in the subjects' blood on the two different diets remained constant. Since changes in serum protein levels usually occur rather slowly. the 105 constancy of the total protein and albumin levels during the course of the study is not surprising. This observation also suggests that the proteins from the potato used in this study were of such biologic value that no drastic changes in protein synthesis resulted. Although the only statistically significant changes observed in this study were decreases in SUN, decreased urine titratable acid excretion, and perhaps increased urine flow rate, it is possible that an infinite number of other changes also took place. When the body becomes more alkaline: intracellular potassium increases, plasma bicarbo- nate increases, renal bicarbonate excretion increases, ionic calcium decreases, urinary ammonia excretion decreases, hepatic glutamine synthesis decreases, etc. Therefore, the possibility exists that any one of an infinite number of alterations in the body can be the cause of the decreased SUN. Thus, it appears that the reduction in serum urea level ~associated with the potato diet may be due to either a de- crease in its production or increased clearance. Although there is still confusion as to the effect of the nature of dietary protein on SUN, there appears to be agreement that a vegatarian diet which produces an alkaline-type reaction in the body, if consumed for some time, results in a reduction in serum urea levels (l8). Such a relationship was suggested by several investiga- tors (l8). Although the change observed in this study seems 106 to show a relationship between the alkalinization of the body as reflected by a decreased titratable acidity of the urine and reduction in serum urea levels, the physiological and biochemical significance thereof is still unknown. In conclusion, the data obtained as a result of these studies suggest that the changes of acid-base balance in human subjects has an effect on SUN concentration. If the alkalinizing diets of plant origin are consumed, they lower the serum urea concentration significantly. Figure 4.--Average titratable acid excretion and urine pH of 6 human subjects who were fed isonitrogenous and isocaloric (l) meat and (2) potato diets respectively. 108 am a~4x>a>wrm >nHo mxnmmasoz so am 9232. f mxvmfzmzfir MEo/DAY :xuzm a: Chapter 11 Serum Urea Levels In Adult Rats Fed Isonitrogenous Rations Which Provided 10% Protein From Casein 0r Wheat Introduction Urea metabolism under normal conditions appears suscep- tible to a variety of factors other than changes in dietary protein level. The production and excretion of protein metabolites reflects the sum of many metabolic processes in the body. Nitrogen end-products, of which urea is the major constituent, added to the blood and excreted through the kidneys are, to a large extent, the result of an interaction between the diet and the organism. Normally, the blood urea level in healthy individuals is controlled by several factors. So far, the quantity of protein has been the main aspect of this problem that has received any attention by investigators interested in nutri- tion. However, there are very few instances in which the correlation between urea level and the quality of dietary protein was intentionally studied separately from dietary protein level. The present experiment was undertaken to examine whether or not there is an effect on the serum urea levels when two different kinds of protein are fed at the same level of dietary nitrogen. For that, healthy, adult male rats were fed isonitrogenous rations which contained l0% protein either from casein or wheat. 111 Experimental Procedure An Mel}. Adult, Sprague-Dawley male rats were used for this study. The rats weighed between 250 and 300 g. The reason for selecting adult rats was that earlier workers using weanling rats fed different kinds of protein secured results which werenot directly applicable to human subjects. The nutrient requirements of growing rats are entirely different from those of the adult, especially in quantitative terms. The rats were divided into two groups with six rats in each group. In order to give them an adjustment period, these rats were put on two different diets for four weeks to establish the same sort of nutriture prior to the actual experiment. One of these was the grain ration which contained 23% protein and the other was the casein ration which had 20% protein. At the end of the four weeks, those rats fed the grain ration were transfered to the whole wheat ration which had 10% protein (Table l8) while those fed the casein ration were given the casein ration which contained l0% protein (Table 18). Both of the l0% rations were fed for four weeks. 3.231%: The composition of the diets are shown in Tables 17,18 The whole wheat grain was ground to medium fine flour. The other ingredients were added, in amounts shown in Table 18 The rations were prepared by thorough mixing in an electric mixer. Before the rats were caged separately, they were 112 TABLE 17.--Proximate composition of the casein and grain rations. Casein Ration Grain Ration1 % % Protein 20% (from casein) 23.4 Fat 5% (corn oil) 3.0 Carbohydrates (added or by difference) 66.0 (cornstarch) 53.5 Fiber or cellulose 2.0 3.8 Moisture - 10.0 Ash 5.0 6.3 Vitamin mixture 2.0 - 100% 100% 1The ingredients in the grain ration are listed by Schemmel, et al., (192) 113 TABLE 18.--The composition of isonitrogenous casein and wheat rations (10% protein level) used in the rat studies. Casein Ration Wheat Ration % % Casein1 1] - Whole wheat (ground)2 - 80 Corn starch 75 11 Vitamin mixture3 2 2 Mineral mixture4 5 4 Corn oil 5 3 Cellulose5 2 - 766? “163%” 1Casein was purchased from General Biochemicals, Chagrin Falls, Ohio. The casein contained 91.25% protein. 2Wheat was obtained from the Department of Crop Science. Michigan State University (Genesee variety of wheat) 3Vitamin mixture purchasef from Nutritional Biochemicals Corporation, Cleveland, Ohio. 4Mineral mixture was prepared according to A.O.A.C. (193). 5Cellulose, microcrystaline, from Nutritional Biochemicals Corporation, Cleveland, Ohio. 114 weighed and paired on the basis of their weights. After four weeks, the rats getting the 20% casein rations were put on the ration containing 10% protein from casein and the other group fed the grain ration was then fed the ration containing 10% protein from wheat. The composition of these rations is shown in Tables 17 & 18. These rats were fed their proper rations, ad libitum, for four weeks; water was available all the time. At the end of the experiment, blood samples were secured by cardiac puncture under light ether anesthesia. The blood samples were drawn five hours after the feeding cups were removed from the cage, so that they were in a fasting state. During the initial 4 week period, daily food consumption was recorded. After four weeks of ad libitum feeding, the rats were continued on the same protein rations but were pair-fed for an additional four weeks; water was available all the time. At the end of the period, blood samples were drawn. Serum was obtained from the blood samples and the sera analyzed for urea nitrogen and creatinine concentration. Analytical Procedure A. Blood The serum was preserved in a freezer until analyzed. The sera were analyzed for urea nitrogen concentration and creatinine as described in previous experiments. 115 Results Weight Gain_ While the rats were fed the 20% protein rations con- taining either casein or grain, they gained weight quite rapidly. After four weeks on the 20% protein rations, when the rats were fed isonitrogenous casein and wheat rations at a 10% protein level, they also gained weight, but at a slower rate. The rats either maintained or gained a little weight in both groups when they were pair-fed. The results are shown in Table 19. Serum Urea Nitrogen When the rats were fed ad libitum, the serum urea nitro- gen (Table 20) was lower in the group consuming the wheat ration as compared to those fed the casein ration (P<.05). A similar difference in SUN levels occurred during the paired feeding period (P<.05). There was essentially no change in the serum creatinine values both during ad libitum and paired feeding periods. That was in contrast to the urea levels which were signifi- cantly lower when the animals were fed the wheat ration despite the fact that both it and the casein ration had the same level of protein. The SUN concentrations of the rats fed the wheat ration were significantly lower than those fed the casein ration in both cases, i.e., ad lib and paired feeding. The SUN values of 10% casein fed group were 14.0 mg/dl and 14.2 mg/dl on ad libitum and pair feeding periods, while the rats fed the 10% wheat protein ration .mapm> mmmgw>m comm com cm>wm mew meowumF>wn ucmucmpmx Amy mp:m_mz 116 “_hmee Nmneme mmhmee unhmee omnome kmhm_e m_He¢m xepueem amatm>< ooa - mn¢ mme mow mme mm¢ mmm mmm o umv mo¢ one mme m~¢ mpa mam 0mm m oqv mmv oev Nov me m¢v mum omm v Fnc mmv Fme «we moc «me mom nmm m owe «me mmq mme Nae Nov mmm mcm N mmv mow ope ooe ope onm mpm opm F paws: cwmmmo pawn: c_mmmu :ngu =_mmmu :wmgw :wmmmu mama oN\~P\m mm\mp\¢ om\vm\m ou\m\m mmumo op mm\m_\¢ on oN\¢N\m :wmuoga xom mepwcH :o mxmmz N no» swam omm nWP c< cum QPF c< scrum; :_ Pm>m_ :wmaogm &op cowumc cw Pm>mp :wmpogm xom .xoega< .mxmmz gnaw com com swan so sapwnwp um um» mew: mpaswco one .mcowpmg “was: co :vmmmu soc» cwmpoga go? newcvaucou mcompmc awe mum; mo mamcm cw pcmwmz--.m_ m4m mmmsm>m PFm com cm>mm wen mcowpm_>mu ccmucmpmx .Amo.vmv mpmswcm www-cmwmmu mg» cw can» vmmuummgz mg» C? Luzop xpucmquwcmwm mm: :mmoguw: owe: Esgmm mg» .ume even use nw— cm .mmmmu :pon :H No.0umo.p m.um.pp ~o.emm.o N.H¢F Fo.nmo.p N.oum.P~ _o.H¢o.~ xF.OH¢F mmmgm>< mo.P m.—F po.P m.mp mo.P o.pp mo.P _.¢_ No.p o.NF o.p P.¢_ vo._ v.__ mo.F m.mP no.F o.FF mm.o m.mp mo.P w.pp mo.~ m.m~ mew: 23m m:_: 22m mew: zzm mew: 23m -wpmmcu -wuawcu -wpmmgu -Pumwgu azogu pawn: aaocu cwmmmu aaogo poms: asogw :mmmmu omu-m~_m mew mmspm> Fp< .mxmmz Lao» we nowcma m com ummugwma new awp um :uon Acvmuoga gopv meowpm; poms: so crummu maocmm -ocuwcomw com mum; mo maaogm ozu cw mcwcwumocu can Azzmv ammocgwc awe: E:cmm--.o~ m4m< _mom om mo Fm Lmnouuo _Nom cop Pu om Lmnouoo _mom NN— Pm mm Lmnouuo meM mop mm mu Lwnouuo mwmm cm om mm sweepuo wmmm eop on om Lwnouuo mwnm mop —N mm Lmnouuo NONN Fmp on em Lwnouuo mmmm mop Fm mm Lm20poo wpxm mPF mm mm LwnOpuo mmmm oNF mm Fm Lmnouuo munm mm mm om Lwnouuo mmkm ¢op pm mp LmnOHUO mpmm mm on mp LmQOpuo Nmnm ¢FP mm up Lmnouuo MNNN wm cu mp Lmnouuo m¢¢m em mm mp LmQOHUO mm¢~ mm mm «P Lmaouuo mmmm mm Fm mp Lmnouuo AH nostmav _Ochoo Anapmewpmmv Nompmeepmmv .dmwwx_mc< awe ma_co_mo Amv you Amy cwmuoca mung .xnapm mgwpcm as» “nonmaocgp muumnnsm cuss; Low umm: mpmwu cw mmPLOqu new um» .cwmpoca prmouu.FN m4m<» mF—Hmmom mmmgm>< 129 mmom Noom mmom m—Pm Noom mmmm vam cvom evmm wmmm Neom mmom m—mm ommm opmm ommm mmmm mm cmngm>oz mm Lmnsm>oz _m cmnEm>oz om Lmnem>oz empem>oz cmnsm>oz Lmnsm>oz Lonem>oz Lmaew>oz Lmnem>oz gmnEm>oz cmnem>oz cwnsm>oz gmaem>oz gmnem>oz Lmn5m>oz gmnem>oz Or-NM'U'LDLONCDOS A>H vowswav cmmgm mmwoFom +1 F mumsm>< mmFM «mom ~¢om mumm momm ommm SONOF-r-N r-I—OMr—O r—F-v—F-r—F- AHH vowgmav gmaam>oz LmnEm>oz LmnEm>oz Lmnem>oz Lmnsm>oz LmnEm>oz FNMQ‘LDK) mouxaz .A.u.p=ouv--.PN usmqk 130 NNFHmmpm whom “mo ammcm>< momm me Q“ m caneauao mopm 4“ mm q Lansaumo comm Na mm m cansmuao oo_m en mm N Language oowm mm mm _ taQEmumo om_m em om om tansm>oz AH> newtmav uamcm kmhm_om __H0_F “on amacm>< Pmmu QNF mm mm tansm>oz Pmmm cup Pu mm conem>oz oeom Fo_ NR RN Lansa>oz hmom mop mm mm Lanem>oz _mmm mm, mm mN Lansm>oz nmom mo, mm 4N taasmsoz A> nowgmav Pocpcou .A.c.o=ouv--._mmsm<» 131 (anticubital vein) were drawn at the Student Health Center where the blood pH, hemoglobin, hematocrit, plasma HCOS and total CO2 were determined. The sera were sent to the commercial laboratory for analysis of the other blood con- stituents, as described in Chapters 1, Part III. .Lrifls. Urines were analyzed for titratable acidity and pH as soon as they were brought to the laboratory. The 24-hour urine volumes were recorded every time urine was collected. The procedures are described in Chapter I, Part II. Results Serum Urea Nitrogen (SUN) The SUN values may have slightly decreased when the subjects were given 10 g/day of NaHCO3 along with their con- trol diet; however this change was not statistically signifi- cant. When the subjects were transferred to the wheat bread diet (Table22), at first the SUN returned to the control values, but after 4 days the SUN may have decreased; but the change is not statistically significant. Four of eight subjects who continued for a longer period (Period V and VI, which were repetitions of Periods I and IV) showed a possible increase in the SUN levels 6 days after their diets were changed to the typical American pattern; however, this change was not statistically significant. When those subjects were again fed the high wheat diet, the SUN seemed to decrease after three days and remained there throughout the remainder of 132 the period; however these changes were not statistically significant. It should be pointed out that even though the above changes were not statistically significant, the observation that the changes were repeatable adds to the credibility. Serum Creatining and Serum Uric Acid The serum creatinine (Table 23) of the subjects showed very slight fluctuations during the entire period of the study, but there was no definite change during any part of the dietary regimen. At all times, these values remained within normal limits (Table 23). Similarly, uric acid (Table 24) fluctuated to a very small extent which was of no apparent physiological or biochemical significance, since the variations were within normal limits. Sgrum Protein and Albumig Serum protein and albumin values remained relatively constant within the normal range over the entire course of the study (Tables 25,26). Hemoglobin and Hematocrit No alterations were observed throughout the entire period of study as far as these parameters were concerned. They remained well within normal limits (Tables 27,28). gfl, Bicarbonate, Carbon Dioxide and Serum Electrolytes Similarly, blood pH, plasma HCOS and CO2 remained essentially unchanged on all the dietary regimens (Tables + ++ 29-31). The serum electrolytes, Na+, K , Ca and phos- phorus remained constant (Tables 32-35). The constancy of 133 these parameters occurred despite the large amount of NaHCO3 given the subjects for six days during Period 11. Urinary Acidity The pH of the urine (Table 36) increased from 6.1 1 0.2 on the control diet to 7.5 i 0.2 when the subjects consumed 10 g of NaHCO3 with the control diet. The pH of the urine drOPPEd to 6.0 t 0.2 when the subjects were fed the high wheat diets. On the other hand, total titratable acidity 0f the urine was 34.0 t 2.5 mEq/day when the subjects were fed the control diet. When the subjects were on the wheat diet, the titratable acidity statistically significantly decreased, indicating that their body fluids became more alkaline as evidenced by the fact that the urine titratable acid excretion dropped to 13.8 i 2.9 mEq/day. Cholesterol and Glucose The serum cholesterol value decreased slightly in each individual while he ate the bread diet. There was an in- crease in serum cholesterol when four out of the eight subjects were fed the control diet (Period V) but it dropped from 181 to 154 mg/dl during the bread diet which is not significant statistically (Table 37). Similar trends in serum glucose concentration which were not statistically significant were observed (Table 38). Since no inhibitor of glucose metabolism was added to the blood when it was collected, the serum glucose analysis was probably underestimated. 134 m.Nnfl ¢.PMH w. 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emegm>e Hwo gew ce>wm ewe mcewpew>me eweeceumx HatHHE mHMHmHmH HemawmmH xmewfimeNH memgm>< mmm mHeH mum .>.m HemH emmH eNmH .=.e mmmH HeHH mcHH .m.¥ mmmH mHHH emu .H.H emmH HmmH mmeH .H.H emeH MHHN HmmH .¥.u mam meeH emNH .e.m eHm eeHH ewe .m.w mmmum mammmm Hogecoe meemfingm >H HH H mane m peep use we mmmge>< .eewgme xwmpmwe gene we .mwmeew>wecw esp we mgopwpwwpws cw msepe> xweewgzuu.oe memm 16.7 mg/dl in the first phase to 12.7 mg/dl when NaHCO3 was. given. No changes were observed in creatinine, glucose or cholesterol concentrations of the serum throughout the Study. 165 166 From these observations, it was concluded that NaHCO3, which is an alkalinizing agent lowers the SUN in human sub- jects. This does not effect the creatinine, glucose or cholesterol levels of the serum. These data support the observations made by earlier workers. Experiment 2: During this experiment, four, healthy male, adult human subjects consumed a wheat gluten liquid formula diet for seven days in the control phase. In the experimental phase the subjects consumed the same daily amount of this liquid diet plus 10 g of NaHCO3 in three doses every day for seven days. During the control phase, the urine of these subjects was highly acid with a titratable acidity of 95.2 mEq/day. The urine in the experimental phase became more alkaline with a titratable acidity of 24.2 mqu day. The serum urea nitrogen in three of the four subjects decreased when the bicarbonate was given while in one case, there was an increase. There was no essential change in serum creatinine, sodium and potassium. In conclusion, it may be suggested that the wheat glu- ten, liquid diet used in the control phase of the study, was an acidifying diet. When the acidotic condition was corrected with the ingestion of NaHCO3, the SUN dropped but not sig- nificantly. It can be concluded from these data that any changes in acid-base homeostasis of the human subject has an effect on SUN. The decrease in SUN may possibly be due to (1) increased urea excretion and/or (2) less urea production. 167 Part II To examine the effect of various kinds of protein fed isonitrogenously on the acid-base balance and SUN, four ex- periments were conducted. Three experiments were carried out with human subjects and one with rats. Experiment 1 and 2: The first two experiments were exactly the same, but performed at two different times. first in February, 1975, and the second in October, 1975. In both experiments, nine (three in the first and six in the second) healthy male, adult individuals were fed a typical omnivorous American diet during a seven day control period. In the following seven day experimental period, the subjects con- sumed isonitrogenous and isocaloric diets, in which the primary sources of protein was from potatoes. Blood and urine samples were collected for the analyses of various con- stituents. The titratable acidity of the 24 hour urine was 20.7 mEq/day at the end of the control period and it dropped to 10.6 mEq/day at the end of the experimental period. Associated with that reduction in acidosis, the SUN also decreased from 13.3 mg/dl to 7.0 mg/dl in the first experi- ment and from 14.8 mg/dl to 10.3 mg/dl in the second experi- ment. The above data suggest that the potato diet was an alkalinizing agent which lowered the SUN significantly. Thus, vegetarian dietswhich aretmsic in nature lower the SUN. Experiment 3: This experiment was carried out with two groups of male, adult Sprague-Dawley rats. One group was 168 fed a casein ration and the other, a wheat ration. Both rations were isonitrogenous and contained 10% protein. The rats were fed these rations ad lib for four weeks. At the end of the four weeks, blood samples were secured by cardiac puncture. Thereafter, the rats were pair-fed and after four weeks of such feeding, blood samples were again drawn by cardiac puncture. The SUN of the rats fed the casein diets at the end of the ad libitum feeding was 13.9 mg/dl and 13.8 mg/dl during the paired feeding, while the 10% wheat protein fed group of rats had SUNlevels 11.6 mg/dl and 11.5 mg/dl on ad libitum and pair feeding which was significantly (P<0.05) lower than that of the casein-fed rats. The wheat ration lowered the SUN in the adult rats when compared with the values in the casein-fed rations. This may be due to increased urea excretion, as reported by other workers. Experiment 4: In this experiment, eight healthy, adult male college students were the subjects. Prior to the study, each subject received a physical examination with special attention to the condition of the kidney as determined by their serum creatinine and SUN. The entire 54-day study was composed of six metabolic periods of varying length. During this study, the daily protein intake of the subjects was limited to 70 g. The subjects were permitted to adjust their caloric intake with protein-free foods to maintain body weight. The foods were served in the Olin Health Center cafeteria at Michigan State University. Periods I and IV 169 during which typical American diets and the bread diet res- pectively were consumed, were repeated as Period V and VI. This was done to examine the consistency of the reduction in SUN observed during Period IV. Fasting blood samples were secured at the 01in Health Center. 24-hour urines were collected by each individual on almost every day of the study. Titratable acidity of the urine indicated that the diet high in wheat fed to human subjects reduced the acidity of the urine. The average titratable acidity of the urine which was 34.0 mEq/day dropped to 13.8 mEq/day. Also a trend in the reduction of SUN was observed. There was no essential change in other constituents of the blood. When four of the eight subjects again consumed control diets high in animal protein for six days, their SUN increased. Following that, when they consumed the bread diet for another six days, their SUN was reduced. Unfortunately, the titratable acidity of the urine was not determined during these periods. As an overall conclusion, it is suggested, on the basis of analytical data obtained from various experiments, that a change in acid—base balance of human subjects has a definite effect on SUN. Also proteins of plant origin, like potatoes and wheat flour. result in the formation of urine more alka- line than that produced when a diet high in animal protein is consumed. This is associated with a reduction in the SUN in humans. Furthermore, the SUN is lowered on high bread diets, if sufficient time is given to the experiment. Similar 170 responses in SUN levels are seen in adult rats fed isonitro- genous diets in which the protein source is either casein or wheat. This reduction in SUN does not occur when weanling rats are used since wheat protein is not an adequate source of essential amino acids to support normal growth in these animals. Another factor which has become important is the age of the rats. If weanling rats are used for this type of experi- ment, no reduction in SUN is observed, because of the greater nutritional requirements of the growing rats as compared with adult rats. It can be suggested that extensive work is needed to examine the urea clearance, insulinlevels of the plasma, glutamine stores, and degradation of urea in the gut. 171 Figure 5.--Average titratable acid excretion and urine pH of 8 subjects fed isocaloric and isonitrogenous (1) typical American diet; (2) same diet plus 10 g sodium bicarbonate, and (3) diet high in wheat bread. 172 3 + e ‘ 8 3228 H $218 2 32:8 3 mm ‘1‘; . 1111 II we // . :Sepgwrm >222 / // mm / I / H m ... fie \e/I 92% Ha: BIBLIOGRAPHY 10. 11. 12. BIBLIOGRAPHY Vaghefi, 5.8., Makdani, D.D., and Mickelsen, 0., Lysine supplementation of wheat proteins, A review., Am. J. Clin. Nutr., 27: 1231, 1974. Mickelsen, 0., The nutritional value of bread, Cereal Foods World, 20: 308, 1975. Bolourchi, S., Friedemann, C.M., and Mickelsen, 0., Wheat flour as a source of protein for adult human subjects, Am. J. Clin. Nutr., 21: 827, 1968. Bernhart, F.W., Comparison of essential amino acids, nitrogen and caloric requirements of the weanling rats and breast-fed infants, J. Nutr., 100: 461, 1970. Bolourchi, S., Feurig, J.S., and Mickelsen, 0., Wheat flour, blood urea concentration, and urea metabolism in adult human subjects, Am. J. Clin. Nutr., 21: 836, 1968. Hegsted, D.M., Minimum protein requirements of adults, Am. J. Clin. Nutr., 21: 352, 1968. Swendseid, M.E., Amino acid requirements in uremia, Am. J. Clin. Nutr., 21: 382, 1968. Lewis, E., Magill, J.W., Nutritional aspects of uremia, Am. J. Clin. Nutr., 21: 349, 1968. 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Scott, 0., and Mason, G.D., Renal tubular reabsorption of urea in sheep, Quart. J. Exp. Physiol., 55: 275, 1970. Shannon, J.A., Jolliffe, N., and Smith, H.W., The excretion of urine in the dog: IV, The effect of maintenance diet, feeding, etc., upon the quantity of glomerular filtrate, Am. J. Physiol., 101: 625, 1932. Dicker, S.E., Effect of the protein content of the diet on the glomerular filtration rate of young and adult rats, J. Physiol., 108: 197, 1949. Pullman, T.N., Alving, A.S., Dern. R.J., and Landowne, M., The influence of dietary protein intake on specific renal functions in normal men, J. Lab. Clin. Med. 44: 320, 1954. Smith, H.W., The Kidney, structure, and Function in Health and Disease, Oxford Univ. Press, New York, p. 93, 473, 1958. Konsishi, F., and Brauer, R.W., Renal hypertrophy and function in irradiated and nephrectomized rats fed a high protein diet, Am. J. Physiol. , 202: 88. 1962. 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Bagdade, J.D., Disordered carbohydrate and lipid meta- bolism in uremia, Nephron, 14: 153, 1975. Bagdade, J.D., Uremic lipemia, Arch. Int. Med., 126: 875, 1970. Kiehm, T.G., Anderson. J.W., and Ward, K., Beneficial effects of high carbohydrate. high fiber diet on hyperglycemic diabetic men, Am. J. Clin. Nutr., 29: 895, 1976. Jenkin, D.J.A., Goff, D.V., Leeds, A.R., Alberti, K.G.M., Wolever, T.M.S., and Gassul, M.A., Unab- sorbable carbohydrate and diabetes: Decreased post- prandial hyperglycemia, Lancet, 2: 172, 1976. APPENDICES 190 Appendix A COMPOSITION OF THE LIQUID FORMULA DIET USED BY MIT WORKERS Amount/day in gms for a Ingredient 70 kg man Kcal Liquid Formula? Wheat Gluten 66.7 264 Maltose-Dextrin Mixture 170 680 Corn oil 130 1170 Lemon Juice 7.0 - Vanilla 7.0 - NaCl 1.0 — Cellulose 2.0 - K2HPO4 5.6 - Water 550 - Non Protein Energy Source, Composition Ingredient Beverages Corn-Starch Dessert Protein-Free Cookies Composition/100 gm solution w/w Soft drink powder (0.3) Sucrose (4.2) Carbohydrate(19.0) Dessert (21.3) Sugar (3.9) Carbohydrate(3.95) Corn oil (2.6) Corn Starch (47) Sucrose (13.0) Salt (1.0) Carbohydrate (3.0) Amount for 70 kg man/day 1300 gm, supply- ing 1200 Kcal 456 gm, supplying 630 Kcal Total of 8 cook- ies, supplying 1000 Kcal *All the ingredients were obtained from the same source as reported by these investigators. 191 Appendix 8 CONSENT FOR PARTICIPATION IN A STUDY TO EVALUATE THE EFFECT OF WHEAT FOODS ON BLOOD UREA LEVELS I agree to serve as subject for a study to evaluate the effect of wheat foods on blood urea levels. I recognize that this will involve consuming a regular American type diet for 26 days and a vegetarian diet for 18 days consisting primar- ily of white bread, wheat products plus vegetables and fruits. I understand that the diets served me will provide 70 g protein and approximately 2,600 Kcal per day. If the caloric content of the diet is not sufficient to maintain my body weight, I will consume extra protein-free foods like purified butter, jam jelly, hard candies, etc. which will be made available to me. During the study, I will refrain from the consumption of any alcoholic beverages and eat only what is made available to me through the study dietitians in the Olin Health Center Cafeteria. This study requires that I eat three meals each day for 44 days in the dining room of the Olin Health Center Cafe- teria. I will report for these meals at the scheduled hours. In exchange for these meals, which will be at no cost to me, I will eat no food or drink any fluids (water excepted) other than that which is provided at the Olin Health Center Cafe- teria. I also permit the execution of the tests and measure— ments as shown in the attached Calendar of Events. The blood samples will be drawn by personnel in the laboratory of the Olin Health Center. Containers for the collection of urine and feces will be provided by the Food Science Department. In giving my consent to participate in this study, I agree that all the following statements are true: (1) I understand that the blood, urine and stool samples are to be used for scientific research. This study has been explained to me and I am aware of any risk involved therein, including the normal risks incident to the withdrawal of blood by needle and syringe. (2) (3) (4) (5) 192 I have not been coerced in any way to participate in this study, and my consent to participate in it has been given voluntarily. I understand that I am free to discontinue participation in the experiment at any time. If I do so, there will be no recrimi- nation; however, I will not be eligible for the payment which is to be made to the subjects on completion of the study. I understand that all results of the study will be held in strict confidence and that I will remain completely anonymous in any reports relating to the study. I understand that, if I so desire, I will be given a summary of the results obtained during this study. Such summary will be available sometime after the completion of the study. I understand that if I adhere to the schedule of eating three meals each day at the scheduled hours in the Olin Health Center Cafeteria, if I eat or drink (water excepted) nothing other than that available to me through the dining room,if I allow my blood sample to be drawn, and collect urine and stool samples according to the schedule for tests and measurement mentioned above, I will be paid shortly after the completion of the study. Signature of subject Project Leader Date 193 Appendix C QUESTIONNAIRE FOR WHEAT DIET AND BLOOD UREA LEVEL STUDY Name Address Phone Number(s) 1. Are you on any diet which has been prescribed for health reasons? Yes____No 2. Are you currently on a weight reducing program? Yes No___ 3. Are you now on any prescribed medication? Yes No 4. Have you even been told that you have a kidney problem? Yes No 5. Will you be able to eat 3 meals a day in Olin Health Center Cafeteria for 44 days? Yes No 6. Is your schedule during Fall Quarter such that you can report to the Olin Health Center Cafeteria for breakfast between 7:00 and 8:00 a.m. (Saturdays and Sundays 7:30 to 8:30 a.m.), for lunch between 11:30 a.m. and 1:00 p.m., and dinner between 5:00 and 6:00 p.m.? Yes___ No___ 7. Will you, throughout the entire period of study, be able to refrain from strenuous physical activity that might result in sweating? Yes___ No___ 8. What is your: Age _; Weight_______; Height__”____ 9. Are you married? Yes No 194 Appendix 0 SCHEDULE FOR MSU BREAD STUDY Fall 1975 Stool Date Diet Blood Urine (Complete (Fasting) (24—hr.) 24-hr.) PERIOD I Oct. 13-14 Regular Diet --1 -- -- 15 " X -- ~- 16-22 " -- -- -- 23-27 " -- X -- 28-30 " -- X X 31 " X X -- PERIOD II Nov. 1-2 Regular Diet + NaHCO3 -- X -- 3-5 " -- X X PERIOD III Nov. 6 Regular Diet + Bread X X -- 7 ll ___ x _- PERIOD IV Nov. 8-9 Bread Diet -- X -- 10 " X X -- 11-13 " -- X -- 14 " X X X 15-17 " -- X X 18 " X X X 19 " -- X X 20 " X X X 21-22 " -- X X PERIOD V Nov. 23 Regular Diet -- -- -- 24 " -- -- -- 25 " X X -- 26 " -- -- -- 27 " -- -- -- 28 " -- -- -- 29 " x x -- Nov. Dec. PERIOD VI 0 Bread Diet II mman—Iw 195 2. -- No sample collected. X Sample collected. 196 >vvmchx m week zmHoznm H: eeceem em HeeH. Hm» Hm; Hm» >vvmonx m week zeeoyam H: eeceem em nzm Heee4 ona uoa>qo onq mceemefl\em