ABSTRACT WHEAT AS THE SOURCE OF PROTEIN FOR HUMAN ADULTS EFFECT OF WHEAT PROTEIN ON NITROGEN BALANCE, UREA, AND AMINO ACID METABOLISM by Simin D.Bolourchi-Vaghefi Normal young men were maintained in nitrogen equilibrium and good health when they were fed a diet in which white flour provided most of the protein with the remainder coming from fruits and vegetables. This was shown by a balance study carried out with 12 normal young men who were fed a control diet for 20 days. During that time, the diets pro- vided 12.2 g of nitrogen per day from both plant and animal sources. For the next 50 days, they were fed a diet free of animal protein with 90 - 95% of the 11.8 g of nitrogen supplied by white flour. Throughout the study, the subjects maintained their body weights by consuming extra protein-free foods. Nitrogen equilibrium was maintained throughout the control phase. For the first 10 days of the wheat diet, the subjects were in negative nitrogen balance but for the remainder of the 50-day experimental phase, the subjects retained small amounts of nitrogen. The level of physical activity increased throughout the study with the result that 3300 calories were consumed in the control phase and 3800 in the experimental phase. Although fecal nitrogen values were constant throughout the study, the wet weights of the 24/hour fecal samples increased during the first part of the experimental phase to about twice that of the control value; thereafter, the weights decreased but did Simin D. Bolourchi-Vaghefi not reach the control value. When subjects were fed a diet providing 90 to 95% of the protein intake from wheat, their blood urea levels were half what they had been in the preceding control period. During the control period, the sub- jects consumed a mixed diet which was isonitrogenous with that for the experimental period. Practically all the protein in the wheat diets came from commercial white flour. The reduction in blood urea occurred in both men and women. In one study involving 12 men, the low blood urea levels continued for the 50 days the wheat diets were fed. The reduction in blood urea was practically completed within the first 48 hours after the initiation of the wheat diet. It was accompanied by a slower but equally pronounced reduction in the blood non-protein nitrogen level. No alteration in protein metabolism as evidenced by urinary urea, creatinine or uric acid excretion accompanied the reduction in blood urea. The results of this study indicate that the level of blood urea may be influenced to a marked extent by the nature of the dietary protein. The reduction in blood urea level seen when normal subjects consume a wheat diet is as dramatic as the change associated with a drastic alteration in the level of dietary protein. Analysis of the diets served in both periods indicated that percentage-wise, the greatest reduction in intake during the wheat period involved threonine and lysine, but even for these, the wheat diet provided more than the daily requirement. Urinary excretion of the essential amino acids in both control and experimental periods was closely related to dietary intakes. For such amino acids as isoleucine, leucine and valine, one percent or less of the intake was excreted, while for lysine and cystine, Simin D. Bolourchi-Vaghefi about 10% was excreted. Fasting plasma free amino acid levels were within normal limits throughout the study. There was a reduction in the levels of lysine and valine during the experimental phase. The reduction in valine level occurred despite the consistancy of its intake in both the control and experimental periods. WHEAT AS THE SOURCE OF PROTEIN FOR HUMAN ADULTS EFFECT OF WHEAT PROTEIN ON NITROGEN BALANCE, UREA, AND AMINO ACID METABOLISM BY Simin D. Bolourchi-Vaghefi A THESIS Submitted to Michigan State UniVersity in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Foods and Nutrition 1967 This work is dedicated to the people of Iran ACKNOWIEDGMENTS The author gratefully acknowledges the guidance and help given her throughout this study by Dr. Olaf Mickelsen. The interest and encouragement of Mrs. Mickelsen and fellow graduate students in the Department of Foods and Nutrition is appreciated. Warmest gratitude is extended to Dr. Dorothy Arata for her sincere understanding and inspiring lead throughout the author's graduate studies. Special thanks are due to Dr. Dena C. Cederquist, Dr. W. Doyne Collings, and Dr. Richard W. Iuecke for their interest and critical comments. The author wishes to especially thank the government of Iran for their financial support, their faith and encouragement in recognition of her efforts during these years at Michigan State University. The continuous support and encouragement the author received from her husband, especially towards the end of her studies, made the completion of this dissertation possible. ii TABLE OF CONTENTS INTRODUCTION Chapter 1 REVIEW OF LITERATURE . . . . . A. Bread as a Constituent of Human Diets B. Proteins and Amino Acids in Wheat and Bread I. Introduction . a. Biological value of wheat and its products b. Relative concentration of protein in different cereals . . . . c. Comparison of biological value of wheat with other cereals . d. Major amino acid deficiences . e. Loss of lysine due to baking . II. Supplementation of bread with protein and amino acids a. Animal studies b. Human studies 1. Adults . . . . . . . 2. Studies with infants and children . Chapter 2 WHEAT FLOUR AS A SOURCE OF PROTEIN FOR ADULT HUMAN SUBJECTS Nitrogen Balances B. Experimental Procedures I. Subjects II. Schedule III. Diets IV. Sample collections V. Analytical procedures a. Urine b. Feces c. Diets C. Results and DisCussion . . I. Nitrogen balance . . . . . . . . . . II. Body weights . . . . . . . . . III. Protein digestibility . . IV Urine volume . . . . . iii Page U1 \D\D\OU1 ll 12 13 14 14 l7 l7 19 23 23 25 25 25 26 28 28 28 29 29 30 31 34 35 38 Page Chapter 3 INFLUENCE OF WHEAT FLOUR ON BLOOD UREA CONCENTRATION AND UREA METABOLISM OF ADULT HUMAN SUBJECTS . . . . . . . . . 47 A. Experimental . . . . . . . . . . 49 I. Effect of a high bread diet on BUN . . . . . 49 II. Reduction in BUN levels after initiating a high bread diet . . . . . . . . . . . . . 50 B. Results . . . . . . . . . 51 I. Effect of high bread diet on BUN . . . . 51 II. Reduction in BUN levels after initiating a high bread diet . . . . . . . . . . . . . . . 53 C. Discussion . . . . . .. . . . . . . . . . 56 Chapter 4 EFFECT OF HIGH WHEAT DIET ON AMINO ACID METABOLISM . . . . 71 A. Experimental. . . . . . . . . . . . . . . 73 BO Results 0 O O O O O O U C O C O O O O 74 C Discussion . . . . . . . . . . . . . . . 76 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . 84 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . 89 APPENDIX . . . . . .. . . . . . . . . . . . 108 iv TABLE 10 ll 12 13 LIST OF TABLES Caloric intake per capita and percent of calories derived from selected food groups . . . . . . . . The extent to which wheat flour (white or patent) provides the amino acids required by adult human beings and by growing rats . . . . . . . . . Composition of the "core" diets . . . . . . . . Nitrogen retained or lost per day by the individual subjects 0 O I O O O O O O O O O O O O O O O O The average amino acid intake (in g) of subjects during control and experimental phases. . . . . . . . . . Serum urea levels in the subjects, expressed as mg N per 100 m1 0 O O O O O O O C O O O O O O O O O O O O O O O 0 Urinary urea excretion of the individual subjects expressed as g of nitrogen per 24 hours . . . . . . . . . . .. Total serum proteins and albumin to globulin ratios in blood samples . . . . . . . . .. . . . . . . . . . Serum values for 5 normal adults before, during and after a high bre‘d diet 0 O I O C O O O O O O O O 0 Urinary nitrogen constituents of the 5 subjects before, during and after a high bread diet . . . . . Essential amino acid intakes and urinary excretions compared with the requirements of normal male adults . Typical foods served on two of every seven days of the contr01 phase 0 O O O O O O O O O O O O O O O O O 0 Plasma amino acid levels in blood samples secured from the subjects 14 hours after the last meal . . . . . . Page 39 40 41 42 64 65 66 67 68 81 82 83 TABLE ii iii iv vi vii viii ix xi xii xiii xiv Calendar of events for bread study Description of subjects with result of their physical examinations made at the end of the study Height and body weights of the subjects at the start of study with deviations from standard . . . . . Average body weights (in kg.) for each subject for each metabolic period. These values are for the control phase when the "normal diet" was served . Average body weights (in kg) for each subject for each metabolic period. These values are for the experi- mental phase when the "Bread Diet” was served Total serum protein . Serum protein components Level of two enzymes in serum of subjects . Hemoglobin and hemotocrit of the subjects Serum "free" iron Daily urinary creatinine excretion as measured on 5-day pooled urine samples . . . . . . . . . . . . . .. Twenty-four xanthurenic acid excretion before and after feeding 2 gms of tryptophane Determined amino acids in diet samples (mg/day) Amino acid level of blood plasma Urinary amino acid excretion of subjects during control and experimental phases vi Page 109 111 112 113 114 115 116 117 118 119 120’ 121 122 123 128 FIGURE LIST OF FIGURES Page Average body weights for each 5-day metabolic period (in kg) for the subjects. . . . . . . . . . . . . . . . 43 Average heart rates of the subjects during a 10 minute run on a treadmill and for 5 minutes of recovery period. 44 Average fecal weight and fecal nitrogen content expressed as grams per day . . . . . . . . . . . . . . . . . . . . 45 Average retention or loss of nitrogen by the subjects thréughout the study 0 O O O I I O O O O O O O O O O O O 46 Plasma urea nitrogen in blood samples secured at the beginning and end of the control phase and midpoint and end of the experimental phase . . . . . . . . . . . . . 69 Total and urea nitrogen excreted in the urine . . . . . 70 vii INTRODUCTION In many developing countries, the existing food supplies are not adequate to meet the nutritional requirements of the people. _Estimates of per capita calories and available protein made by W.H.cx (1953) indi- cated that in such countries the average diets are insufficient in quantity and defective in quality. According to Borgstrom (1965), the average per capita caloric intake of the world pOpulation is 2200 and .only 1/10 of that is of animal protein. World population, according to Borgstrom, consumes about 85 million metric tons of protein annually. Of this 2/3 is provided by plant products (about 61 million metric tons) such as grains, beans and pulses. Cereals provide #0 million metric tons of the protein while only 24 million metric tons of the protein are supplied from animal sources. (In terms of calories, however, nine-tenths of the human protein intake is derived from plant protein.) Browne et al.(1961) stated that people of wheat growing countries in the Near and the Middle East receive 70% of their daily caloric intake in the form of bread and other wheat products. The average Iranian peasant consumes 1500-2500 calories per day. In some parts of this coun- try, cereal and pulses provide anywhere from S2~to 98.7% of the protein intake and for the majority of the population, cereal and pulses provide between 80 and 90% of the protein intake. (Sen Gupta and Hedayat 1966) As a low cost more readily available food, wheat, bread and flour occupy an important place in the diet of the Iranian peasants. With world food needs increasing and the adequacy of the available food supply already a serious problem in many areas of the world (Borgstrom l965), it would appear justifiable to give more attention to the foods that might be ecomnical‘ly feasible. Plants may be consumed as such or converted into animal protein prior to ingestion by the human. The conversion to animal protein would serve to elevate the cost, since the process is only about 20% efficient. Borgstrom (l965) has calculated that 5-8 calories of primary plant products are required to produce one calorie of animal protein. According to Iepkovsky (l91+2+) only 5 to 10% of the feed fed to animals is recovered as meat and only 15 to 20% as milk or eggs. Thus, in view of the crucial aspects of the world feeding problem, it would be more appropriate to consider plants per se_as protein sources in the diet. Animal studies on the biological value of bread proteinssuggest that amino acidstherein are not adequate for maintaining nitrogen equilibrium in adult rats and are grossly deficient for normal growth of the young. (Mitchell and Carmen 1921+ and 1926). Data from numerous animal studies suggest the proteins of bread are deficient in essential amino acids (for more complete discussion of these studies see the review of the literature). Most of the studies in the literature center on the adequacy of bread proteins for growth or maintenance of rats. Studies in which the primary purpose was to evaluate bread alone as the source of protein for human subjects are very scarce. The closest approach to this was the study of Widdowson and McCance (1954) with German orphans. In that study, girls and boys 8 to 13 years old received 51-73 grams of protein per day for a year. Of this 8-11 grams were of animal origin; the rest came from bread. The purpose of the study was to evaluate the nutritional quality of various types of flour that were used in the baking of bread. This study showed that there were no differences in growth or health among the groups of children who received bread made of flours of various extractions (including enriched). "The addition of 500 ml of reconstituted dried whole milk per day over the period of 6 months caused no apparent improvement in the growth or the health of the children." Since most of the previous work emphasized the inadequacy of the proteins in bread, the first approach to the improvement of bread in this laboratory was to supplement its protein. For a review of those studies please refer to Bolourchi,(1963). In order to determine how valuable animal datazxmain predicting the adequacy of bread for humans, we attempted to mathematically correlate the amino acid content of bread and the requirement. The amino acid intake of adult human beings, who might receive a diet of 2500 calories of which 70% of the calories are obtained from bread, was calculated. These calculations showed that all the essential amino acids would be h provided in more than adequate amounts, regardless of the remaining 30% of caloric intake. However, even when weanling rats were fed a ration containing 90% dried bread, they would not secure an adequate intake of the essential amino acids. On the basis of these calculations, bread made with wheat flour should not require any fortification with proteins or amino acids to maintain nitrogen equilibrium in adult human subjects. A comparison of the requirements of the growing rat for amino acids, and those supplied by bread, indicated lysine to be the most limiting amino acid . Chapter One REVIEW OF IITERATURE A. Bread as a Constituent of Human Diets Cereal products occupy a primary position in meeting the demand of a large and ever increasing group of the world pOpulation. According to Wirths (1966) cereals provide 53% of the total protein,15% of the fats, 70% of all carbohydrates, and 55% of the total calories of the world pop- ulation. He indicated that many civilized nations meet two-thirds of their carbohydrate intake and one—third of their protein needs from cereal, while much higher figures are estimated for peoples of developing countries. The highest consumption of wheat and bread is among Eastern Europe, Near and Middle Eastern countries. The following is a list of annual per capita wheat consumption in some of these countries: Iran 110.9 kg, Israel 118.3 kg, Jordan 112.9 kg, Iebanon 150.5 kg, Turkey 155.9 kg, India 22.4 kg, Japan 25.5 kg, Thailand 1.2 kg, Burma 1.4 kg (Band 1962). Guggenheim (1957) showed that the new settlers of Israel consume about 380 grams of bread and cereal per day. According to Bennett (1942) the per capita consumption of bread in USSR was 123.0 kg per year. In some countries, while the per capita con- sumption is still high, the intake of this foodstuff is in a decline. The consumption of wheat and bread dropped from 150 kg per year per man in 1934-38 to 92 kg in 1945 in Switzerland (Rosen 1947). In Germany at the beginning of the 20th century cereal products supplied 35% of the 5 total calories. By 1962-63 this percentage had dropped to 26%. In that year per capita consumption in Germany was 53 kg of wheat flour, 20 kg rye flour, 5 kg of other cereals and grains supplying 25% of their protein intake (Wirths 1966). According to Mitchell and Carman (1926), in the United States 30% of the protein (nitrogen) in the average American diet was provided by white flour. Friend and Clark (1959) report that 20% of the total protein intake of the average American came from flour and cereal, which would be approximately 70 kg/year/man. Wheat consumption in the United States was about 73 kg/year/man in 1966 (The World Food Budget 1970). Hodges (1966) indicated that the consumption of bread and cereal and other high carbohydrate foods increases with the decrease or scarcity of the total calories. In other words, the percentage of cereal or bread in the diet has an inverse relationship with the degree of affluence of the society. Westerman e: al.(l949) consider a high cereal diet, one consumed by people with "poor food habits" or with low income. They believe that these groups are most likely to include considerable quantities of cereals in their diets, either from habit or by economic necessity, since such foods represent cheaper sources of energy. According to Parpia and Bains (1966) a major portion of the diet in India is composed of cereal grains and legumes. The per capita con- sumption of cereal in India averages 375 g/day (which amounts to 137 kg/ year). This level of cereal in the diet would provide 86% of the protein intake of people which for a reported intake of 2060 calories per day (World Food Budget 1970) would amount to 50.4 grams of wheat. Sen Gupta and Hedayat (1966) give average figures of 600 to 700 grams of cereal and bread consumption in the villages of Iran. For a daily caloric intake of 1500—2500, cereal would provide 66-98% of the calories. Tekeli (1966) states that the principal cereal used in Turkey for human consumption is wheat, which is prepared and consumed in various ways. Iapman (1966) agrees with Hodges (1966) that the nations develop- mental state influences cereal consumption. He emphasizes the contribution of wheat as a source of protein for countries struggling for survival, and the low fat, cholesterol inhibiting effects of diet high in wheat foods. Hegsted (1962) points out that low fat diets are automatically high cereal, high carbohydrate diets. The U.S. Department of Agriculture in their reports on the Worl 1?ood Edeet, and the projection of the food budget for 1970, based on the data collected in the preceding decade, present the data from which Table 1 is taken. This table shows the percentage of caloric intake of people of the sub-regions of the world from selected food groups. These are averages for 1959—61. Table 1. Caloric intake per capita and percent of calories derived from selected food groups. No. of CHO Wheat Other Grain Animal.Pro&nn .l__21 ‘_fi Calories %_of Cal. % of Cal. % of Cal. % of Cal. United States 3,190 40 17.4 3.4 30.4 Canada 3,100 42 18.8 2.5 36.1 Oceania 3,260 43 25.2 1.9 36.5 Northern Europe 3,060 48 23.4 4.6 27.7 Southern Europe 2,720 60 40.1 6.2 12.7 Eastern Europe 3,000 66 32.1 17.5 18.5 West Asia 2,350 72 48.0 13.0 8.2 North Africa 2,210 73 26.4 34.3 9.1 south Africa 2,670 72 14.0 42.7 18.8 India 2,060 74 11.3 52.1 6.4 Japan 2,360 78 11.7 51.5 7.3 USSR 3,040 73 35.7 17.7 14.7 Communist Asia 1,790 87 12.2 62.4 2.4 B. Proteins and Amino Acids in Wheat, Flour and Bread 1. Introduction a. Biological value of Wheat and its Products Osbornaand Mendel (1914) showed that wheat proteins are generally of poor biological value as compared with animal proteins. The studies of Munaver and Harper (1959), Howard et al. (1958), Block and Mandl (1958) and Bender (1958) suggest that the protein content of wheat flour is inferior to animal protein for growth and maintenance of rats. There are numerous reports of animal studies on the biological value of wheat, flour and bread proteins which support the above facts. These studies have shown that lysine is the primary amino acid deficiency as far as growth of rats is concerned (Mitchell and Block 1946, Block and Weiss 1956, Hepburn et a1. 1957, Kon and Markuze 1931, Mitchell and Smuts 1932). Jonick and Kawalizyk (1965) showed in several samples of wheat and rye bread that lysine and methionine levels control the biological value of wheat while methionine and isoleucine limit that of rye. They also showed that although the protein content of rye was less than that of wheat, its biological value was higher probably because of its lysine content. Csonka (1937) analyzed whole wheat flour and patent flour for their amino acid content. He found that cystine and tryptophane were higher 10 in patent flour, while tyrosine and dibasic amino acids were higher in whole wheat flour. Simmonds (1962), on the other hand, found that amino acids were parallel in every extraction that he examined. Guggenheim and Freedmann (1960) showed that the nutritional value of bread protein diets increased as the percentage of extraction of flour arose. They attributed this to an increased lysine content, since the lysine content increased per gram of nitrogen by increase in extraction rate. b. Relative Concentration of Protein in Different Cereals According to Jacobs (1951) the protein concentration of corn is between 8 to 11% varying among products of this grain. Corn meal is said to contain 9.1% protein. Rice contains between 6.5 to 10.5% protein. Whole oat varies in protein content from 9 to 20%. Its protein content averages between 11.5 to 14.0%. Proteins of rye com- prise 9.0% of its weight. Barley has a protein content of 8-20% in different samples. Wheat and wheat products contain 8-12% protein with an average of 10.5%. With the development of the new technique of "air classification" which separates the protein particles by an air current, yielding a higher protein flour, wheat flours with protein content of as high as 20% are obtained (Wrigley 1963). 11 c. Comparison of Biological Value of Wheat with other Cereals Mitchell and Block (1946) found a biological value of 70 for whole wheat bread. They showed that soybean (heated) had a bio- logical value of 75, rolled oats and white rice, a biological value of 66, and corn one of 60. The biological value of white flour was 52. They showed that the greater the deficiency of the amino acid in a pro- tein, the lower the biological value would be. Sure (1947) showed that rats gained 2.06 g per gram of protein intake from rolled oats while an only 0.80 g gain was obtained from each gram of protein from wheat flour. He found that the proteins in polished rice were three times as efficient biologically as those in white flour, when fed on the same level of protein intake. Prior to this Ken and Markuze (1931) obtained results indicating the superiority cd‘rye bread with respect to the biological value of the protein. (0.61 g gain per gram of protein from white wheat bread and 1.14 g gain per g protein from rye bread) In general rice proteins have a higher biological value among the cereals (with the exception of whole wheat grain and soybean) and they appear to be fairly well balanced. The most limiting amino acid in rice is lysine (Mitchell and Block 1946). Jones 33 a1. (1948) showed that rice with a 2.22 protein utilization ratio was superior to wheat (1.72 PUR) and barley (1.55 PUR) and corn (1.42 PUB) in the biological 12 value of its proteins. They consider oats (2.23 FUR) similar to rice as far as biological value of protein is concerned. Rye has a higher lysine content than most of the cereals. However, its protein utili- zation ratio is 1.83 which is lower than that of rice and oats. Proteins of corn are deficient in lysine, cystine and tryptophane. The biologi- cal value of corn is similar to that of wheat. Barley also resembles wheat in its biological value (Jones 933. a_1_.1948 ). lamb Si“. a__1_. (1966) showed that sorghtm proteins were nutritionally inferior to wheat pro- teins in rat growth and reproduction assays. d. Major Amino Acid Deficiencies Wheat, flour and bread are shown to be deficient in lysine. When lysine was added to the wheat diet, there was a large increase in the growth promoting value of wheat (Mitchell and Smuts 1932, Hutchinson et a1. 1958, Rosenberg and Rohdenburg 1952, Jahnke and Schuck 1957, Flodin 1956). While lysine is the most limiting amino acid in wheat and wheat products, additional amino acids have been shown to be limiting. The addition of threonine along with lysine further increased the quality of wheat protein when the level of protein in the rat ration was 9.5% or less. Above this level, the addition of threonine had no effect (Desphande et a1. 1957, Sure 1954, Rosenberg et a1. 1954). According to the studies of Sure (1952, 1954) the most deficient amino acid in 13 whole wheat is lysine, followed by valine and threonine. He showed that in milled wheat flour, however, the sequence of most limiting amino acids varies as follows: lysine, threonine, and valine (Sure 1953 and 1955). Bender (1957 and 1958) showed that when a diet of bread fed to the rats fin‘lO days at 1.5% nitrogen level, the first and second limiting amino acids were lysine and threonine, but methionine proved to be the third limiting amino acid. There is general agree- ment between investigators that the most limiting amino acid in wheat and wheat products is lysine. However, depending on the type of pro- duct; whole grain, wheat flour, or bread, the order of the second and the third limiting amino acid will vary between valine, methionine and threonine. e. losses of lysine Due to Baking Horn at El. (1958) accounted quantitatively for 11 amino acids of whole wheat, when the extraction products; flour, bran, and shorts were analyzed microbiologically. No destruction of amino acids happened during fermentation. Iosses of cystine, lysine, and methionine during baking were significant. Most of the loss occurred in the crust (browning reaction). Jansen st 31. (1964) measured the nutritional losses of added lysine during baking, by rat assays. They showed that 30% of lysine became unavailable nutritionally when the time of baking was increased 14 from zero to 50 minutes. At 20 minutes baking time no loss was observed at 4250F. This was confirmed by rat assay but not by micro- biological assay in which l8% loss was detected at 20 minutes baking time. McGarr gt a1.(1964), Ericson and Iarson (1961) have shown that lysine is lost in bread baking, therefore the biological value of bread is lower than its unbaked ingredients. Hepburn it al. (1957) showed that in the process of milling and baking, lysine is the first amino acid that has an appreciable loss. Then arginine and aspartic acid are also lost to a large extent. Iarson (1966) suggests that protein bound and free lysine can be made nutritionally unavailable by the browning reaction which is a reaction between the 8 group of lysine and carbohydrates or other compounds containing aldehyde groups. II. Supplementation of Bread with Protein and Amino Acids a. Animal Studies Many attempts have been made to improve the nutritional value of bread by different supplements. lysine addition to wheat flour proteins was first shown by Osborn and Mendel (1914) to improve the growth of the rats. Mitchell and Smuts (1932) showed that addition of lysine to 8 and 10% wheat protein diet increased the growth of rats. Hutchinson e__t_ a__]_._. (1958), Rosenberg and Rohdenburg (1952), Jahnke and Shuck (1957) and Flodin (1956) have all confirmed the beneficial 15 effects of fortification of bread with amino acids and protein. Banks at al. (1964) fed rats diets containing, as dietary protein, wheat gluten in agar gel, wheat gluten supplemented with lysine (4.7 g/lOOgg protein) and wheat gluten plus casein (2.1: l) or egg albumin, and also a protein free diet as control. Nitrogen growth index for wheat gluten was 8.6, casein 20.2 and egg albumin 27.0. Supplementing wheat gluten with either lysine or casein improved the nitrogen growth index to a value of 16.7. Thus they showed that the value of wheat gluten as a dietary protein source for weight gain of rats can be improved equally by either type of supplementation. ,Gates and Kennedy (1964) showed that supplementation of bread with 0.25% lysine H01 or 3, 6 and 12% dried milk solids improved growth protein efficiency ratio in rats. This has been repeatedly shown by different investigators. .Ehle and Jansen (1965) measured growth and PER in rats for several wheat products when sup- plemented with lysine and threonine. The PER was proportional to the amount of lysine supplement up to 4.7 or 5.3 grams lysine per 16 grams nitrogen, when 85% and 60% of the protein, respectively, was supplied by wheat, flour, white bread and wheat gluten. 'ins at El. (1964) used lysine (0.25 grams per 10 grams protein), whole milk (7%) or non- fat dry milk (3%) to supplement the basal diet of wheat macaroni for weanling rats. They demonstrated that the nutritive value of wheat macaroni was improved by the addition of lysine, as was shown by an 16 increase weight gain of the rats. Milk protein increased the protein content of the product, the PER and the body weight gain of the rats. McCollum et_al.(192l) showed that milk is an effective supplement to wheat, with respect to protein, calcium and vitamin A. Fairbanks (1938 ) reported that addition of 6% dried milk solids to bread formula increased the nutritive value of the bread. Addition of 12% dried milk solids produced a better gain in weight and animals with longer bones, even when the rats were pair fed. The weight gains were the same when rats were fed diets containing 6% dried milk solids, but the rats receiving 12% supplement had more ash in their carcasses. Mitchell and co-workers (l9h3) showed that enrichment of bread with dried milk solids promoted a better growth and bone calcification than the bread that was enriched with equal quantities of calcium and thiamin present in dried milk solids. Parks E3.Ei- (195A) showed that addition of milk improves the amino acid composition of white bread. (Dried milk solids contain 7.5% lysine and u.;% valine.) light EE.§$f (19u3) stated that addition of 6% dried milk solids to the bread formula produces a bread which is equal to whole wheat bread in so far as growth of rat is concerned. Commercial white bread is baked with H% dried milk solids added to improve the baking quality. The amino acid deficiency of bread as shown by animal bioassays has been the basis of many studies undertaken by investigators to show 17 the necessity of supplementation of bread for human consumption. b. Human Studies 1. Adults As early as 1907 Chittenden showed that nitrogen equi- librium could be maintained in human adult subjects when the protein in the diet (a mixture of plant and animal protein) was as low as 35 to 40 grams per day, the exact amount varying among individuals. He con- cluded that the dietary standards existing at that time were one to two times higher than the actual requirements of adults for protein depend- ing on the source of protein used. Almost a century ago Karl Thomas in Berlin showed that he could maintain nitrogen equilibrium when he consumed sufficient bread to provide, in his diet, 13.1 grams of nitrogen per day from a bread diet. (ahyun 19u8) Hegsted 22.31-(1946) postulated that if healthy adults consume diets "relatively high" in cereal, they should remain in protein equilibrium. Clark gt al.(l963) (personal communications) showed that a woman could maintain nitrogen equilibrium consuming 242 grams of wheat flour without any supplementary amino acids, as the sole source of protein. Hoffman and McNeil (lgug) demonstrated that supplementation of wheat gluten with h% lysine improved the nitrogen balance index for 18 adult human subjects suffering from protein malnutrition. However in this study it was shown that in patients with chronic protein deficiency, even unenriched gluten was capable of producing positive nitrogen bal- ance with the intake of 35 g gluten per day. The authors of this paper expressed their surprise at the ability of "such a poor protein" to maintain nitrogen balance in adults but they attributed it to the fact that the subjects were suffering from undernutrition thus their require- ments for protein were lower. In most of the studies in which the bread proteins were supplemented with amino acids for human consumption, the emphasis has been on the "improved" nitrogen retention of adult subjects due to supplementation of bread with lysine. One of these studies is; that of Rice et a1. (1960). In this study bread was the primary source of the protein for adults. They reported, in an abstract, that supplemen- tation of bread with lysine increased nitrogen retention, not emphasizing the fact that subjects were in nitrogen equilibrium when they consumed only unsupplemented bread. This bread was made with 4% dried milk solids and provided the subjects with 95% of their protein intake. Watts at al.(196h) studied the nitrogen balance of 6 men who received the FAO amino acid reference pattern as compared to the wheat flour pattern, a diet made of amino acidswith the same prOportion in wheat flour. The wheat flour pattern was fed at a level so that it provided an equal amount of lysine as that in the FAO reference pattern. 19 The mean nitrogen retention for each diet was equal;.L0.h2 and +-0.41 g per day for FAQ and wheat pattern respectively. Thus they concluded that the level of lysine in bread is the crucial factor in nitrogen bal- ance studies using wheat bread as the sole source of protein for adults. Clark SE 5341963) showed that only 2 of the 4 adult sub- jects consuming h.5 grams of nitrogen 3.27 g of which came from wheat maintained nitrogen balance. The other 2 subjects remained in negative nitrogen balance even when as much as 1500 mg lysine were given, show- ing that individuals differ in their minimal total protein requirements. The intake of calories tvas constant for each subject and adequate to maintain weight. 2. Studies with infants and children Bressani SE a_l_.. (1960) fed wheat flour diets to l to 5 year old children to study the effects on growth of supplementation of wheat flour with amino acids. These children were just recovering from severe protein malnutrition. A 2-day adaptation period was used before each three 3-day balance period for each diet combination. ‘The basal diet used contained wheat flour 85%, wheat gluten 7%, glycine 3%, and cornstarch 5%. Vitamin and mineral capsules were given daily. The amino acids were substituted for corn starch and when this happened the glycine was reduced so that all diets remained iso-caloric and iso- nitrogenous. These diets provided 2 grams of protein per kg per day, with a calorie intake of 80-100 calories per kg per day. When lysine was added to the wheat diet, the nitrogen retention of the children was increased to that obtained with a milk diet. The basal diet usually had l/3 to 1/2 the value of nitrogen retention obtained on the milk diet. They showed that the addition to the basal diet of other amino acids to bring their level up to those of the FAQ pattern produced more consistent results. Albanese et al.(1949) showed that a lysine supplemented wheat gluten diet, used as the sole source of protein supported a nutri- tional state in infants comparable to that afforded by an evaporated milk formula. Krut it. 53.1.- (1961) in South Africa fed a diet in which bread provided 89% of the protein and 5h% of the calories to children 2-h years old. One group received bread that was supplemented with lysine. The control group received the same bread but an isonitrogenous amount of glycine was used in place of lysine. Children receiving lysine supplemented bread showed a "better growth", (P<:0.02). Serum albumin of all the children drOpped as a result of a change to the bread diet, but the children who were receiving lysine supplemented bread had a lesser drop in their serum albumin . The magnitude of changes in the body weight of the children, although considered by the authors significant, is very little, less than 1/2 kg. There are no data on nitrogen retention or loss by the children. In this paper the beneficial effects of lysine supplementation is also emphasized; 21 although many of the children had to be withdrawn from both the diets since they were adversely affected by the diet. They concluded that bread of low protein content (6.h%) was unsuitable as the principal source of protein for children of 2—h years old unless wheat of suffi- ciently high protein content is used. King at al. (1963) showed that addition of lysine to the bread eaten by "undernourished" Haitian school children improved their growth and weight gain "not impressively but significantly." These children were from rural villages who received most of their protein from vegetable sources. These children were given the bread in addi- tion to their daily meal that they received at their homes. No data on the daily total nutrient intake of children we given. The investigators of this study, however found marked weight gain in children in which supplementary bread without added lysine was given. It appears that the extra calories and protein in the form of 150 grams of bread with 9 grams of jam daily was what caused the latter weight gain in both groups of school children vs the third school group who did not receive any bread. The authors again emphasized the ”not impressive but signifi- cant" increase in weight gain in the group receiving lysine supplemented bread. No reports could be found in the literature of a study carried out to assay the adequacy of the proteins in flour or bread prepared only with white flour, a leavening agent, salt, sugar and fat and water for 22 human adults. According to the calculations made in this laboratory, a diet in which 70% of 2500 calories and 95% of protein are provided by flour and bread, all the essential amino acids are available to the subjects well above the requirements suggested by Rose et a1. (1955). Similar calculations were made by Hegsted (1962).0n the basis of these, he suggested that if all the calories in the diet came from 80% extraction wheat flour, the protein therein would provide all the amino acids required by both adults and growing children. Chapter 2. WHEAT FIOUR.AS A SOURCE OF PROTEIN.FOR ADULT HUMAN SUBJECTS A. Nitrogen Balances Estimates of the nutritional value of wheat flour and bread are based largely on animal experiments. One of the earliest of these studies was that of Osborne and Mendel (191%). This and other work with rats (Howard et a1. 1958, Bender 1958) suggested that the protein in wheat is inferior to animal protein for both growth and maintenance. These studies showed that lysine is the primary amino acid deficiency in wheat (Mitchell and Block l9u6, Hepburn §£.§i' 1957) followed by threonine and then either valine or methionine (Deshpande at El. 1957, Sure 1952). On the basis of such work, many animal studies were carried out to determine the effect of fortifying bread with amino acids and/or proteins.(Csborn and Mendle 1914, MbCollum 23.2}: 1931) The spate of reports describing the deficiencies of wheat protein almost obliterated a paper by Thomas which appeared in 1911. Therein he showed that he could maintain nitrogen equilibrium when he consumed sufficient bread to provide, in his diet, 13.1 g of nitrogen per day. Since then a number of studies with infants and children (Bressani §£.§$f 1960, King El.§lr1963) and adults (Hoffman and McNeil 19u9) have demonstrated the beneficial effects of fortifying bread with proteins and amino acids. Rice_et al. (1960) found that college students showed an increased nitrogen retention when lysine was added to the bread which provided 95% of their protein intake. The bread in that 23 study was made with h% skim milk powder. There are indications that the unsupplemented bread maintained nitrogen balance in both normal subjects and those recovering from malnutrition. The studies of Widdowson and McCance (195h) showed that children grew at a normal rate when the diet provided 50 to 70 g of protein from bread plus 8 to 11 g from animal sources. Since many people in Iran consume diets composed primarily of bread, it appeared desirable to determine how the biological value of this food could be improved. To this end, weanling rats were fed a ration containing 90% dried bread purchased from a bakery in Tehran.. This was supplemented so as to provide an adequate intake of minerals and vitamins. The addition of 7 or 15% dried lentils or up skim milk powder to the bread increased the weight gains of the rats by as much as two- to three-fold. Calculations indicated that the basal bread diet did not contain an adequate concentration of essential amino acids for the growing rat (Table 23. Similar calculations showed that a 2500 kcalorie diet high in bread would provide human adults with more than an adequate amount of all essential amino acids. This would be true if 70% of the calories came from white flour and was independent of the composition of the other 30% of the diet (Table 2). Similar calculations were made by Hegsted in 1962. On the basis of these, he suggested that if 25 all the calories in the diet came from 80% extraction wheat flour, the protein therein would provide all the amino acids required by both adults and growing children. However, to our knowledge, this was not experimentally tested. No nitrogen equilibrium data could be located which involved diets with 90% or more of the protein from wheat with the other 10% secured from plant sources. For this reason, a study was undertaken to deter- mine the effect produced by feeding young men a diet in which 90 to 95% of the protein came from bread with the remainder from other plant sources. B. Experimental Procedures I. Subjects Twelve male college students, 19 to 27 years of age, free of gross signs of thyroid, chest, cardiac, neurological and muscular diseases, were chosen as subjects for the study. Besides passing the physical examination, each subject had to be within 10% of his standard weight (Society of Actuaries 1959). The Minnesota Multiphasic Personality Inventory Test was used as an aid in choosing the subjects. II. Schedule The study consisted of a control phase of 20 days which began on Monday, March 30, 1964. This was followed by the experimental phase of 50 days, which ended Sunday, June 7. Twenty—four hour urine and stool samples were collected throughout the study. Blood samples (about 50 m1. on each occasion) were drawn at the beginning and end of the control phase and, at the midpoint and end of the experimental phase. Both the control and experimental phases were divided into 5-day balance periods. Seven daily menus were used in both the control and experimental phases. This permitted a bag lunch for Sunday evenings whereby both the subjects and cook-dietitians had a little freedom. Apart from Sunday evenings, all meals were served in the diet kitchen. III. Diets During the control phase, the subjects received a normal-type diet which provided an average of 72 g protein (12.2 g nitrogen) per day. From #3 to k5% of the protein in those diets were of animal sources. These diets contained small amounts of milk, eggs and meat. Although the amount of protein in these rations is smaller than the 97 g intake of the average adult in the United States (Leverton 1959), it is approximately the same as that recommended by the Food and Nutrition Board of NRC (196A). The level of protein in the diets served during the control phase was designed to be isonitrogenous with that of the diets in the experimental phase. The diets served during the experimental phase provided an average of 11.8 g of nitrogen per day. Of this, 90 to 95% was derived from 27 commercial wheat flour used largely in preparing bread and rolls. These were baked in the laboratory to insure that the same formula was followed throughout the study. The bread and rolls for both the control and experimental phases were made from: #790 g white flour, 200 g vegetable shortening, 150 g sucrose, 85 g salt and #9 g dry yeast. The diets supplied the recommended nutritive allowances with the exception of calcium. Calcium was sufficient in the contrOl diet, how- ever during the experimental regimen the diets supplied an average of 250 mg of calcium per day. In order to compensate for this deficiency, calcium lactate pills which supplied 650 mg of calcium were given to the subjects daily. Throughout the study the same brands of foods were used. The butter for the experimental phase had been melted and washed free of whey. The resulting oil was reconstituted with sodium chloride and water to a consistency similar to that of butter. Kjeldahl analysis of a number of samples of the whey-free butter indicated the absence of any detectable nitrogen. For this reason, the subjects were permitted enough butter to maintain their body weights. Throughout the study, the subjects were required to consume the "core" diet which was composed of the protein-containing foods. ther foods, such as butter, jam, tea and coffee were permitted in unrestric- ted amounts but the quantities consumed were recorded for each subject. Known amounts of "Instant” coffee and tea were provided at regular 28 intervals. The nitrogen content of the tea and coffee consumed were used in correcting the nitrogen balance figures. IV. Sample Collections Twentyefour hour urine samples were collected in polyethylene bottles containing toluene as a preservative. Stools were collected directly in one quart, wide-mouth cylindrical cartons which had snugly— fitting covers. Each container had some dry ice in it and additional dry ice was available at all times. A plastic carrying case was pro- vided in which the subject could keep his urine and stool containers. Blood was withdrawn from the antecubital vein using Vacutainers, some of which contained heparin and others nothing. On the day the blood sample was collected, the subjects reported to the laboratory where a small amount of blood was secured by a finger prick for hemo- globin and hematocrit determinations. At every meal, an extra serving was prepared and weighed in the same way as that for the subjects. At the end of the day, the combined extra servings were weighed. When water had to be added to the latter to facilitate hemogenization, the volume thereof was recorded and the weight of the sample corrected accordingly. V. Analytical Procedures a. Urine As soon as the urine samples were brought into the laboratory, 29 the volume, specific gravity and pH were determined. Approximately 100 m1 of each sample was saved for urea nitrogen determination. Exactly one-half the urine volume was used in preparing the 5-day composite sample for that metabolic period. These composite samples were saved for determination of total nitrogen (digestion with sulfuric acid in a heating block, followed by Nesslerization) and creatinine (by the Folin picric acid method). b. Feces The stools for each metabolic period were weighed and placed in a covered jar containing a measured volume of 10% hydrochloric acid. As soon as the last sample was received, the entire mass was homogenized. in a blender. Approximately 50 ml was saved for'the determination of nitrogen by the macro-Kjeldahl method. c. Diets The nitrogen content of the diet samples was determined by the macro-Kjeldahl method. Fat was extracted in the Goldfisch apparatus using diethyl ether as the solvent. The calorie content of the diets was measured by means of the Parr Bomb Calorimeter. Carbohydrates were calculated using the USDA Handbook No. 8 (1963).. 30 C. Results and Discussion The "core" diets, which each subject had to consume, provided comparable amounts of nitrogen in both the control and experimental phases (Table 3). The protein values for the two phases differ to a greater extent than the nitrogen values. This difference arises from the use of the factors 6.25 and 5.70 for animal and plant proteins respectively. The increased calorie content of the experimental diets was necessitated by the increased physical activity of the subjects. The control diet initially was designed on the assumption that 3000 kcalories per day should maintain college men in weight equilibrium. During the control phase, the "core" diet had to be supplemented with extra protein-free calories to provide an average intake of 33h6 kcalories. For this reason, the experimental "core” diets were designed to provide approximately 3300 kcalories per day. During the experimental phase the subjects continued to increase their activity. To maintain body weight, extra protein-free calories raised the intake to 3835 kcalories. At first glance, one might conclude that the very high calorie intake of the subjects resulted in a high intake of all essential nutri- ents. The experimental diets contained the same amount of white flour as the diet for which the calculations in Table 1 were made. That 31 2500 kcalorie diet with 70% of the calories from white flour required an intake of 500 g of flour per day. The diets served during the experimental phase provided 515 g of flour per day. The diet used in the experimental phase provides no more protein than that in diets used by large numbers of peOple throughout much of the Middle East (Browne et_al.1961). For this reason, the data collec- ted in the present study should have some relevance in evaluating the contribution that might be made by wheat in the diets of large groups of human adults. I. Nitrogen Balance A week before the control phase started, the subjects restricted their intake of such foods as milk, cheese, meat and poultry. This was done since the ordinary eating habits of the subjects indicated a daily protein intake of 90 to 120 g. During the first metabolic period of the control phase, the sube jects showed an average loss of 0.23 g nitrogen per day (Tablell). The rate of loss varied slightly for the remainder of the control phase, but essentially, the subjects were in nitrogen equilibrium. This is especially true if the criteria proposed by Leverton and Steel (1962) are accepted. They stated that a daily variation of2t0.5 g in the nitrogen balance was compatible with an equilibrium state. The first two metabolic periods of the experimental phase resulted in a definite negative nitrogen balance (Table A). The subjects lost 32 an average of almost 2 g of nitrogen per day during that 10—day period. This was followed by a retention of nitrogen which continued for the remainder of the study. The overall change in body nitrogen content for th experimental phase was a retention of 20.5 g of nitrogen equiva- lent to about 512 g of lean body tissue. The latter would be too small to detect as a change in body weight. It was so small that other losses (e.g. hair, skin, saliva, etc.) probably accounted for most of this apparent nitrogen retention (Mitchell and Edman 1962). The data in Table A show that these normal young men maintained nitrogen balance when fed a diet in which white flour provided 90 to 95% of the daily protein intake. The remaining protein in the diets were of plant origin. Consequently, for norma1.young men, the amino acids in wheat provide adequate amounts of all essential amino acids and, when the calorie intake is adequate, these amino acids can be utilized effectively by the body. If the experimental phase had been limited to the first two bal- ance periods, which covered 10 days, the conclusion might be that wheat protein could not maintain nitrogen equilibrium. The negative balance during those two periods was so great as to justify the conclusion that the protein was inadequate. However, after that, the nitrogen balance became positive and remained so throughout the rest of the study. The initial negative nitrogen balance during the experimental phase probably resulted from an inadequacy of body enzymes required for 33 the proper utilization of the amino acid mixture present in white flour. Validation of such a suggestion is hard to come by. Actual analyses of the experimental and control rations for the essential amino acids show some variations. However, the differences between the two sets of diets are smaller than one might have assumed (Table 5). Threonine showed the greatest difference, being present in the control diets to an extent which was 2.2 times that in the experimental diets. At the other extreme, the amount of methionine in the control diets was only 0.68 times that in the experimental diets. It seems reasonable to assume that most enzymes involved in normal metabolic reactions should be capable of a two« to three-fold range in activity. Under such circumstances, it becomes difficult to visualize how the difference in the amounts of the essential amino acids between the control and experi- mental diets could require drastic alterations in the enzyme patterns of the body. The metabolic adjustment to a high wheat diet exhibited by human subjects is similar to that shown by rats. When adult rats that had been reared on a natural grain (stock) ration were transferred to a ration containing 90% of a high protein, white flour, they were in negative first nitrogen balance for at least the’three days. After that they started to retain nitrogen. The rats that were transferred from the grain to a semi-purified ration containing casein as the protein, did not show this initial loss of nitrogen (Nitsan _e_t_ a__1_. 1967). 3h II. Body Weights The body weights of the subjects showed some fluctuations throughout the study (Fig. 1) but for the group as a whole, there was no great difference between the initial and final weights. The differ- ences in body weights become even less when the weight at the end of the control phase (71.3 kg) is compared with that at the end of the experimental phase (71.0 kg). The slight reduction in body weights of the subjects during the study may reflect their improved physical condition. At the beginning of the study, each subject ran for 10 minutes, on a motor—driven tread- mill at a rate of 6 miles per hour. The pulse rates for this run were lower at the end of the experimental phase than at the beginning of the control phase (Fig. 2). The improvement in physical condition represen- ted by the lower pulse rates during and following the run on the treadmill are not attributable to the wheat diet. However, if the wheat diet could not support normal.body functions, such an improvement in physical performance would not have occurred over such an extended period as 50 days. Since physical activity varied throughout the study and consequently the intake of non-protein foods, no evaluation can be made of the caloric efficiency of the control and wheat diets. The closest approach to this is a comparison of changes in the lean body mass of individual subjects (based on changes in nitrogen metabolism) and their changes in body 35 weights. The control phase was treated as a unit since no prominent changes occurred at that time. The first two metabolic periods during the eXperimental phase were separated out since the average nitrogen balances at that time were negative. The changes in body weight were plotted against the changes in lean body mass. When this was done, the points were so randomly scattered that nothing could be concluded there- from. The absence of any correlation between these two parameters probably stems from the fact that in most cases the changes along both axes of the figure were small. Under such circumstances, an accumulation of small errors could produce a marked deviation from a straight line relationship. The loss of nitrogen through hair, skin, nails, etc., might be a major factor responsible for an "accumulated error." Furthermore, the increased activity of the subjects as the study pro- gressed could have resulted in body fat changes which might account for some of the apparent increases in lean body mass associated with a loss 0f body weight. III- Protein Digestibility IThe digestibility of the protein in the control and experimental diEtS was the same as evidenced by fecal nitrogen excretion. During the Control phase, the fecal nitrogen excretion averaged l.35:l;0.23 g per day and during the experimental phase, it averaged 1.30 £13.17 g. After the first two metabolic periods in the control phase, the fecal ‘ :9 1|;- AC . \ 'Y 1. «s... .A K r i' ~ F» | ~ ‘ 36 nitrogen excretion was very constant (Fig. 3). The slight increase in fecal nitrogen during the control period occurred despite the absence of any significant change in nitrogen balance values (Table d). An unexpected finding was the marked increase in weight of the stools during the first half of the experimental phase (Fig. 3). In the control phase, the average stool weight was SheiflJB g while in the experimental phase, it was l£5:t6.2 (P<0.00l). In the experimental phase, the weight of the stools increased progressively until the eighth metabolic period, when the average was 155 g. From then on, the weight of feces began to return to the values observed during the control phase. Since the stools were analyzed only for nitrogen, it is difficult to identify the substance responsible for their increased weight. Had the increase in weight of the stools during the experimental phase been erbtributable to fat, the percentage thereof would have approached 70%. SUClléi level of fat would have changed the nature of the stools in a VEIUV obvious way. Since there was no change in the appearance of the Stcxol.samples throughout the study, it would seem that fat, per i3! CCNIJJi not account for the increased weight. Minerals and water either tcDg‘e‘ther or individually could not account for the marked change in stx3c>l.weight. On this basis, carbohydrate would appear to be the pxflinuiry stool component that increased during the experimental phase. T?m3 <1arbohydrate might have come from the mucopolysaccharides in the 37 intestinal tract. Until this suggestion can be checked by actual analyses, it must remain in the realm of theory. Relatively little has been done on the variation in weight or compo- sition of stools from normal individuals. The work of Toscani and Whedon (1951) indicated a variation in stool nitro- gen from 0.91 to 2.22 g per day when an intake of 90 g protein was maintained for 18-20 weeks. No satisfactory explanation was available for these fluctuations in stool nitrogens. Analyses of stools indi- cated the absence of glucose, galactose and lactose, with the occasional presence of some pentoses and hexoses from fruits and plants (Gryboski €3.10. 2391961»). Further evidence that very little carbohydrate is normally present in feces comes from the observation that its total caloric content can be accounted for on the basis of fat and protein (Watts at i]: 1963). The dry matter in each day's stool sample can be influenced by the type of diet even when the change appears to be as minor as that represented by the isocaloric substitution of a liter of WhOle milk in a diet devoid of dairy products. This and other studies (wallaeger at a__l_. 191W) indicate that under normal circumstances, the wet weight of the stools parallels their dry matter content. The initial increase in the stool weight during the experimental phase followed by a return toward normal is somewhat analogous to the Ch’a‘nges in the fecal flora of animals whose ration is supplemented with antibiotics. The addition of antibiotics to the rations of a number of “h“.- v._ u ”up. _‘ \‘.; u , ~v ‘4‘ v ‘o.' “-5 W. "at. >4 y,_ 'vnm . : ‘ *0.-c.4 38 animal species frequently produces a marked initial alteration in the microflora of the intestinal tract. If the antibiotic-supplemented ration is continued over a number of weeks, the microflora gradually returns to what it had been before antibiotic feeding was instituted (Gant gt a__l_. 19u3) . IV. Urine Volume The urine volume of the subjects was the same in both control and experimental phases. 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This test of physical fitness was made at the beginning of the control and at the end of the experimental phase. 0| Zl {I VI 1+5 L. FECAL N FRESH WEIGHT OF STOOLS (G/DAY) (G/DAY) .- .- f” - - - - - - c> \n <3 :3 Eg :8 5: ES 23 E5 is :3 r 1 *F1 FAQ? I I I r' 1’ l r I “ s —1 p- a ,_ § - :E L b I 1 L . BSVHd 1V1N3Wl83dx3 Fig. 3 Average fecal weights and fecal nitrogen content expressed as grams per day, for four metabolic periods of the control and ten metabolic periods for the experimental phase. Each metabolic period represents five days. The fecal weights represent the fresh weight of the stools as they were received in the laboratory. #6 AVERAGE NITROGEN RETAINED 0R LOST PER DAY (G) ‘l +N T T8239 pxamlllv xi thmpEng. tram ,J +_ II 2m4>00r.0 tmm_000 WHW. b. >5. 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Dev. Subj. from No. Ht. Wt. "Std." Cm. Kg. % ‘ 1 165 70.45 + 8 2 170 ' 65.91 + 1 3 I70 74.14 0 4 173 75.00 -r 5 5 175 72.73 + 3 6 178 77.27 0 7 167 68.82 '+ 6 8 179 71.45 + 1 9 180 71.45 -5 10 183 79.54 0 11 178 70.00 0 12 173 68 . 18 + 7 113 Table iii Average body weights (in kg.) for each subject for each metabolic period. These values are for the control phase when the "normal diet” was served. Subjects gr 2 ‘__ 3 4 1 70.45 70.22 70.16 69.78 2 65.91 67.04 67.14 66.94 3 74.14 74.46 74.29 74.08 4 75.00 74.91 74.37 74.26 5' 72.73 69.81 69.88 69.92 6 77.27 76.61 76.74 76.60 7 68.82 68.35 67.62 67.36 8 71.45 68.96 66.96 69.14 9 71.45 71.29 ' 71.59 71.36 10 7951+ 77-72 76-97 76-97 11 70.00 70.44 70.43 69.98 12 68.18 68.10 68.27 68.80 Average 71-07 71.57 71.20 71.26 Stand. Dev. 1.5.4? 1- 3.58 3.60 3.41 Hmowm H< >aHoo mnHa HmHH 3. Se.» .34; .24.» in.» re... 4.84.4 Page.» 6.34.» 6.34.4 zonamH wmomm .mH n H.bm zonamH wmomm .u u H.w 124 >BHoo mnHa HmHH aHoo moHa HmHH HH mmw. 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