mt summon» VALUE or TWO summsmsb BREADS COMPARED wum THAT or A STANDARD wan: BREAD Micrfhobqanool M. S. wane“ STATE UNNERSiTY Dorothy Juno Walwodh 1-960 llflllfll I IHRIIMIWIHWITHWIWFWW ¢ L» l THE NUTRITIONAL VALUE OF TWO SUPPLEMENTED BREADS COMPARED WITH THAT OF A STANDARD WHITE BREAD By Dorothy June Walworth AN ABSTRACT Submitted to the College of Home Economics Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition 1960 Approved Dorothy June Walworth ABSTRACT The nutritional value of the protein in each of 2 breads, advertised as containing more protein per unit weight than the average bread, was compared with a standard white bread. One bread contained Roman meal. The other bread contained a blend of wheat flours supplemented with lysine. Each bread was analyzed for nitrogen. Thirty three weanling, male, albino rats were divided into 3 equal groups and fed 90 per cent bread diets. Food and water were allowed ag_libitum throughout the 2 week ex- perimental period. Food intake and weight records were kept. At the close of the experimental period, animals were decapitated, and livers were removed and analyzed for xanthine oxidase activity, nitrogen, fat, and moisture. Carcasses were analyzed for nitrogen, fat, and moisture. The bread containing Roman meal provided approxi- mately 1/3 more protein (dry weight) than did the standard white bread. However, rats fed the bread containing Roman meal did not grow at a rate greater than the control, thus, had lower protein efficiency ratios than did rats fed the standard white bread. The xanthine oxidase system in the livers from rats fed bread containing Roman meal was not significantly more active per unit weight of liver nitrogen than this enzyme Dorothy June Walworth system in the livers from rats fed the standard white bread. No significant differences were observed between these 2 groups with respect to the composition of the carcass, or the liver with one exception. Livers from rats fed bread supplemented with Roman meal contained a greater per cent of nitrogen than did the livers from rats fed the standard white bread. This difference was small, but significant. Therefore, while the bread containing Roman meal provided more protein than did the standard white bread, as determined by chemical analysis, the quality of this protein was not improved; the proportions of amino acids supplied by the protein of Roman meal did not complement those of white flour. The additional cost of Roman meal bread (10 cents more per pound) was not warranted in terms of nutritional value. The second bread studied presented an entirely different picture. The amount of protein provided by the lysine supplemented bread was approximately twice that provided by the standard white bread as determined by chemical analysis. Significantly more growth and signif- icantly higher protein efficiency ratios were observed in rats fed the lysine supplemented bread than in rats fed the standard white bread. The xanthine oxidase system was significantly more active in livers from rats fed the lysine supplemented bread than in livers from rats fed the standard white Dorothy June Walworth bread. Rats fed the lysine supplemented bread had signifi- cantly larger livers, which contained a greater per cent of nitrogen and a smaller per cent of fat, than did rats fed the standard white bread. The only significantdiffer- ence observed between these 2 groups, with respect to car- cass composition, was a greater per cent of nitrogen in the carcasses of rats fed the lysine supplemented bread. In addition to providing more protein, the lysine supplemented bread provided a better balanced amino acid pattern, thus, the protein was more efficiently utilized than that of the standard white bread. As a result, the nutritional value of the protein in the lysine supplemented bread was markedly superior to that of the standard white bread. The superior nutritional value of the protein in the lysine supplemented bread may be worth the additional cost of 37 cents per pound for individuals consuming sub- optimum protein. However, it is recognized that the majority of people in the United States consume adequate protein. Liver xanthine oxidase activity was an excellent measure of the nutritional value of the protein in bread. Significant differences in the activity of this enzyme in the livers from young rats were observed after an experi- mental feeding period of only 14 days, therefore, it is a sensitive, fast, and economical assay method. THE NUTRITIONAL VALUE OF TWO SUPPLEMENTED BREADS COMPARED WITH THAT OF A STANDARD WHITE BREAD By Dorothy June Walworth A THESIS Submitted to the College of Home Economics Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MA STER OF SC IENCE Department of Foods and Nutrition 1960 ACKNOWLEDGMENTS The author gratefully acknowledges the encourage- ment and guidance given her throughout this project by Dr. Dorothy Arata. Thanks are also extended to Dr. Catherine Carroll for her interest and help. TABLE OF CONTENTS INTRODUCTION 0.0000000000IO0.0000000000000000.0.0000... REVIETV‘J OF LITERATLIRE 00......0.0.0.0000...0.00.00.00.00 The Protein Ofy‘lheat 0.0.00.0...OOOOOOOOOOOOOOOOOOOOO CDUUUJl—J Protein Supplements to Wheat Diets .................. Crystalline Amino Acid Supplementsto Wheat Diets .... 11 The Relationship of Liver Xanthine Oxidase to Dietary Protein ................................... l9 EXPERIMENTAL PROCEDURE ................................ 21 .RESULTS ............................................... 26 DISCUSSION AND CONCLUSIONS ............................ 30 SUMMARY ............................................... 34 LITERATURE CITED ...................................... Al APPENDIX .............................................. i A Summary of the Information Available from the Manufacturer of Each Bread ........................ 1 Data for Individual Rats (Group I) .................ii—v Data for Individual Rats (Group II) ...............vi-ix Data for Individual Rats (Group III) .............x-xiii TABLE II. III. ITJ 0 II. III. IV. VI. VII. VIII. IX. LIST OF TABLES PAGE Food Consumption and Growth Data ............. 37 .......................... 38 Liver Analysis Data Liver Xanthine Oxidase Data .................. 39 ........................ AO Carcass Analysis Data A PPENDIX Group I Food Consumption and Growth Data for. IndiVj—dual Rats 0.......0..0.....000.0.0 11 Group I Liver Analysis Data for IndiVidual Rats ....0.00.0.0..0..0...0000..0 iii Group I Liver Xanthine Oxidase Data for Individual Rats . . . . . 0 0 0 0 0 . 0 0 . 0 0 0 . . . 0 . 0 . . . . 0 iv Group I Carcass Analysis Data for IndiVidual Rats .0........00........00..0..0 v Group II Food Consumption and Growth Data for IndiVj-dual Rats . . 0 . . . 0 . . . 0 0 . 0 . 0 . . . vi Group II Liver Analysis Data for IndiVidual Rats 0....00..........0.....0.... Vii Group II Liver Xanthine Oxidase Data for Individual Rats . . 0 O . . . . 0 0 I . O . . . . O 0 0 0 . 0 .viii Group II Carcass Analysis Data for IndiVidual Rats 0 O . . . O . O . . . . . . . . 0 0 O 0 0 0 O . 0 . . 0 ix Group III Food Consumption and Growth Data for Individual Rats ..... x Group III Liver Analysis Data for Individual Rats ....0..0........0.0.0.000... Xi TABLE I. II. III. IV. II. III. IV. VI. VII. VIII. LIST OF TABLES PAGE Food Consumption and Growth Data ............. 37 .......................... 38 Liver Analysis Data Data .................. 39 Liver Xanthine Oxidase ........................ 40 Carcass Analysis Data APPENDIX Group I Food Consumption and Growth Data for IndiVidual Rats ...0.............0.0.... 11 Group I Liver Analysis Data for IndiVj-dual Rats O....0...0.........0...0.... iii Group I Liver Xanthine Oxidase Data for IndiVidual Rats ...0.0......0...0..........0 iv Group I Carcass Analysis Data for IndiVj—dual Rats 0.....0...00.....0.0....0... V Group II Food Consumption and Growth Data for IndiVj—dual Rats . . 0 0 . 0 0 . . 0 0 . 0 0 . . . . . Vi Group II Liver Analysis Data for IndiVidual Rats 0......00...0.....0.......0. Vij— Group II Liver Xanthine Oxidase Data for Indj—Vidual Rats 0 . . O . . . 0 . .. 0 . . . 0 . . . . 0 0 0 .Viii Group II Carcass Analysis Data for Individual Rats ......... ix Group III Food Consumption and Growth Data for Individual Rats .......0.0..0...000 X Group III Liver Analysis Data for Individual Rats ..........00...0.00...0.0... Xi TABLE PAGE XI. Group III Liver Xanthine Oxidase Data for IndiVj—dual Rats .....0......0...0.0....0 xii XII. Group III Carcass Analysis Data for IndiVj—dual Rats 0...0....0....000.00.0..0...Xiii INTRODUCTION INTRODUCTION In recent years, the emphasis on protein and on weight reduction has increased the popularity of ”high rotein—-low calorie” foods. Manufacturers have supple- mented bread with a variety of foods, and emphasized the protein content of the bread in their labeling. These breads constitute approximately 2 per cent of the total bread market (Friedman, 1959). When applied to a popula- tion of approximately 170 million people, who consume approximately 120 pounds of bread per person per year (Block and Mandl, 1958), this represents a considerable volume of business. Advertising claims concerning ”high protein” breads present vague information, which implied a need for increasing dietary protein, and emphasizes the value of a particular product for meeting this need. Labeling claims refer to ”more,” ”better,” or ”improved” protein in the bread. A few breads are advertised as containing ” When a protein, ”equivalent to meat, milk, and eggs. variety of these special breads were examined, the quan— tity of protein ranged from approximately equal to twice that of a standard bread, while the quality of protein varied from B/A to twice that of a standard bread (Friedman, 1959). Thus, claims and label declarations, with respect to protein, are often misleading. The majority of these special breads, advertised as containing more or better quality protein than the standard bread, are supplemented with natural foods. How- ever, recently bread has been supplemented with a chemical compound, lysine, the amino acid most deficient in wheat. Although lysine supplements have improved the nutritional value of the protein of wheat, most investigators question the necessity of supplementing cereal products with lysine in the United States, where the majority of the people con- sume an adequate intake of high quality protein. This experiment was undertaken to study the nutri- tional value of the protein contained in 2 commercially produced breads, advertised as ”high protein” breads, as compared to that of a standard white bread. One bread contained a supplement composed of cereal grains. The other bread contained a supplement of lysine. The cost of these breads was also considered. Measurements used in this study included growth, protein efficiency ratio, per cent of nitrogen, fat, and moisture in the livers, and per cent of nitrogen, fat, and moisture in the carcasses of young rats. Since the activ- ity of the liver enzyme, xanthine oxidase, is affected by dietary protein, and has been used successfully in measur- ing the nutritional value of other proteins (Litwack et_al,, 1952; DJu et al., 1957), this criterion was also used in this study. REVIEW OF LITERATURE REVIEW OF LITERATURE In the later half of the nineteenth century experimenters believed the value of a protein food depended upon the amount of nitrogen it contained. After the dis- covery of some of the amino acids, variations in the nutri- tive value of protein foods were explained in terms of differences in their amino acid composition. The present concept of the nutritive value of protein indicates that dietary protein must supply eight ”essential” amino acids, which can not be synthesized by man in adequate amounts, in addition to supplying nitrogen for body synthesis of other compounds. The amount of protein nitrogen, and the proportion of the essential amino acids supplied by a food determine the nutritional value of the protein it contains. THE PROTEIN OF WHEAT Quantity 9£_Protein: The quantity of protein in whole wheat is not absolute, but depends upon environment and variety of wheat (Harris et_§l,, 1945), and on soil conditions and fertilizers (El Gindy et_al,, 1957). These experimenters found the protein content of whole wheat ranged between 10 per cent and 17 per cent. On an equal weight basis, the protein content of fractions of the wheat grain varies in the following order, approximately 26 per cent of the germ, 14 per cent of the bran, and 11 per cent of the endosperm, while the intact grain contains approximately 12 per cent protein (Morris 23.313: 1946). Although the germ and bran contain more protein than does the endosperm on an equal weight basis, they constitute only a small portion of the total wheat kernel. The total wheat kernel contains approximately 2 per cent germ, 14 per cent bran, and 84 per cent endosperm (Lowe, 1950). The protein content of flour depends upon the protein content of the whole grain, and the extraction of the bran and germ during milling. Whole wheat flour, which contains the germ and bran, provides more protein than does patent flour, which contains only the heart of the endo— sperm. Generally, white flour represents between 72 per cent and 75 per cent extraction. The protein content of "standard grade” flour (the flour most widely used) varies from 9 per cent to 12 per cent (Morris et al., 1946). The protein content of bread depends on the amount of protein present in the flour and in the other ingred- ients used in making the bread. Kulp et_al, (1956) anal- yzed 255 samples of enriched white bread from various loca- tions throughout the United States, and noted an average protein content of 8.6 per cent. This agrees with the figure released by the United States Department of Agri- culture (1950), i.e. 8.5 per cent protein in white bread containing 4 per cent nonfat milk solids. Commercial bakers use between 3 and 4 per cent nonfat milk solids in the preparation of the standard white bread. ”Although bread flour contains sufficient total protein (13 per cent of the calories), less than one-half of this protein is available to the animal for the forma- tion of new tissue. The recognized protein inadequacy of wheat flour is therefore not the result of a lack of total protein per calorie but due solely to the low biological value of the wheat proteins.” (Howard et_al,,_l958) Quality 2£_Protein: During the early part of the twentieth century, mxapoor quality of wheat protein was demonstrated by Osborne and Mendel (1912, 1914, 1920). Other investigators substantiated their results. When compared with other foods, wheat grains contain a poorer quality protein than do oats (Jones et_§1,, 1948), rye Ocon and Markuze, 1931; Sure, 1954, 1955), meat, eggs, or milk (Mitchell and Carman, 1924; Mitchell and Block, 1946; Mitchell, 1947). The poor quality of the protein in wheat is attributed to its amino acid composition. When the amino acids present in the protein in whole wheat were compared with those in egg, which supplies an amino acid mixture almost completely utilizable in animal metabolism, the protein in whole wheat was approximately 63 per cent de- ficient in lysine (Mitchell and Block, 1946). In order for a protein to supply the Optimal proportion of amino acids for utilization by the animal, it must contain 5.3 grams of lysine per 16 grams of nitrogen (Howard gt_al,, 1958; Block and Mandl, 1958). Since the lysine content of the protein in whole wheat ranges between 2.5 and 2.7 grams per 16 grams of nitrogen (Block and Weiss, 1956; Hepburn et_al,, 1957), the deficiency of this amino acid limits the utilization of the protein in whole wheat. The proteins contained in various fractions of the whole wheat grain differ in quality as well as quantity. Wheat germ and wheat bran promoted higher protein efficien- cy ratios in young rats than did whole wheat. 0f the three, wheat germ contained the highest quality protein (Hove et_ a1}, 1945). The better quality of the protein in wheat germ and wheat bran as compared with whole wheat is due, in part, to a difference in lysine content. The protein of wheat germ contains approximately 5.5 grams of lysine per 16 grams of nitrogen (Block and Weiss, 1956), which is equivalent to the lysine content of an ideally balanced protein mentioned previously. The protein of wheat bran contains approximately 3.8 grams of lysine per 16 grams of nitrogen (Block and Weiss, 1956), which is more than the lysine content of the protein in whole wheat. Standard white flour, which consists primarily of wheat endosperm, contains a poorer quality protein than does whole wheat. The protein in white flour contains approximately 2.0 to 2.2 grams of lysine per 16 grams of nitrogen (Block and Mandl, 1958). Of the essential amino acids contained in whole wheat, only lysine is decreased "*3 as a result of milling (Hepburn E19. 31» 1957). On the other hand, milling increases the digestibility of white flour as compared with whole wheat, thus, more of the protein contained in white flour is available to the animal for growth and metabolism. The increase in digest- ibility partially compensates the loss of lysine during milling. The quality of protein in bread depends on the proportions of amino acids supplied by the protein in flour and other ingredients. Ingredients, which contain a well-balanced protein or supply an extra quantity of lysine, improve the protein quality of the bread. Al- though a small loss of lysine occurs during the baking process, this does not significantly affect the protein quality of bread (Rosenberg and Rohdenburg, 1951). The protein of standard white bread contains approximately 2.5 to 2.8 grams of lysine per 16 grams of nitrogen (Block and Weiss, 1956; Block and Mandl, 1958). Thus the ingredients used in making bread have added 0.5 to 0.6 grams of lysine per 16 grams of nitrogen above that supplied by white flour. The evidence presented supports the fact that whole wheat bread contains a higher quality protein that does standard white bread. The protein of whole wheat bread produced higher biological values in both young and adult rats (Mitchell, 1947), and in adult human subjects (Murlin §£_gl., 1941) than did the protein of white bread. Analysis of the amino acid composition (Block and Mandl, 1958) and growth in young rats (Block et_§13, 1959) indi- cated the protein of whole wheat bread was better balanced and more efficiently utilized than the protein of white bread. In conflict with these data, French and Mattill (1935) found the biological value of white bread equal to that of whole wheat bread. The difference in their results and those of other investigators may be due to the fact that 10 per cent of the nitrogen in their experimental diets came from sources other than bread. PROTEIN SUPPLEMENTS TO WHEAT DIETS Supplements g£_Nonfat Milk Solids: Application of the supplementary relationship between the proteins of wheat and milk resulted in the improvement of the protein of bread. Hove et_a1.(l945) added 3, 6, or 20 per cent nonfat milk solids to a diet of white flour, which supplied 10 per cent protein. They observed an increase in the growth and protein efficiency ratios of young rats as the amount of milk solids in the diet increased. In a similar study, Carlson et_al. (1946) noted an increase in the biological value and protein stored in the bodies of young rats, as the amount of milk solids in the dietincreased from 3 to 6 per cent. Corresponding results were obtained when a diet of whole wheat bread, which was supplemented with either 3, 6, or 12 per cent nonfat milk solids, was fed to young rats at the 11.5 per cent protein level (Sabiston and Kennedy, 1957). Jahnke and Schuck (1957) suggested that the addi- tion of more than 6 per cent nonfat milk solids adversely affects the taste and texture of bread. However, Welton et_al, (1959) have added 25 per cent nonfat milk solids to bread, which increased the nutritional value of the protein to approximately 2.5 time that of the standard white bread, as measured by rat growth. This bread was similar in appearance to standard white bread, and had excellent flavor. Supplements g£_Yeast: The protein of yeast may effectively supplement the protein of wheat, because the concentration of lysine in yeast protein is comparable to that: of whole egg (Block and Weiss, 1956). The addition of 5 per cent dry yeast to white bread, at the 12.5 per cent protein level, increased the growth of young rats (Light and Frey, 1943). Similarly, when young rats were fed a diet of enriched flour, which contained 1, 3, or 5 per cent yeast and supplied 9 per cent protein, growth increased with increased amounts of yeast in the diet (Sure, 1948). Sure stated, ”The increased biological value of the proteins of milled enriched wheat flour with dried food yeast is due to the yeasts' provision of lysine and possi- bly other dietary essentials.” IO The addition of 3 per cent yeast to a white bread diet promoted more growth in young rats than did the addi- tion of 3 per cent nonfat milk solids. However, the addi- tion of both yeast and nonfat milk solids to a white bread diet, at the 12 per cent protein level, promoted better growth than did the addition of either alone (Seeley et_al,, 1950). V Supplements 2£_Wheat Germ: Improvement of the pro- tein of white flour with the addition of wheat germ was suggested by the previous discussion relative to the lysine contained in wheat germ protein (p. 6). Hove et_a1, (1943) noted that wheat germ, skim milk, or beef muscle promoted equal growth and protein efficiency in young rats when fed at the 10 per cent protein level. The addition of either 4 or 6 per cent wheat germ to a mixed diet, which contained 48 per cent white flour, significantly increased the growth rate of young rats (Westerman §t_al,, 1952). When either 3, 6, or 20 per cent wheat germ was added to a white flour diet, which supplied 10 per cent protein, the growth and protein efficiency ratios of young rats increased with in- creased amounts of wheat germ in the diet (Hove et_al,, 1945). Equal amounts of either wheat germ or nonfat milk solids added to the diet promoted equal results. They concluded wheat germ and nonfat milk solids are of equal value for improving the nutritive value of flour. Supplements of Wheat Gluten: The value of wheat gluten for supplementing the protein of bread is dubious, 11 since it contains less lysine than whole wheat, wheat germ, or wheat bran (Block and Weiss, 1956). The addition of 2 per cent wheat gluten to a white flour diet at either the 10 per cent or 15 per cent protein level did not affect the growth rate or the per cent of nitrogen depoSited in the carcasses of young rats (Howard et_§1,, 1958). They con- cluded, ”The futility of adding wheat gluten to bread from the point of view of protein nutrition is apparent.” CRYSTALLINE AMINO ACID SUPPLEMENTS TO WHEAT DIETS Supplements of Lysine: Small supplements of the amino acid, lysine, improved the protein of wheat. The amount of supplemental lysine necessary for optimum nutri- tional value depended on the amount of lysine supplied by the protein of the wheat, and other ingredients in the product. Hutchinson et_al, (1956) observed that a supple- ment of 0.25 per cent L-lysine was better than lower quan- tities of this amino acid, and equal to greater quantities for promoting growth in young rats fed a white flour diet which supplied 12.5 per cent protein. At this level of supplementation the total lysine in the diet was 0.5 per cent. When the total lysine in the diet was more than 0.5 per cent, the nitrogen efficiency was reduced. Rosenberg and Rohdenburg (1952) supplemented a commercial white bread, which contained 3 per cent nonfat milk solids, with various levels of lysine. The experimen- tal bread diets supplied 12.5 per cent protein. A l2 supplement of 0.2 per cent L-lysine supported better growth in young rats than did smaller amounts of this amino acid. At the 0.2 per cent L-lysine level of supplementation the total lysine content of the diet was 0.5 per cent, a quan- tity identical with that used by Hutchinson 22 _a_:_L_. (1956). Rats fed diets containing larger amounts of lysine grew faster initially but at maturity were equal in weight to animals fed the diet supplemented with 0.2 per cent L-lysine. They suggested fortifying bread with 0.2 parts lysine per 100 parts of flour. Jahnke and Schuck (1956, 1957) noted a supplement of 3 per cent nonfat milk solids and 0.25 per cent L-lysine equaled a supplement of 12 per cent nonfat milk solids for promoting growth in young rats fed a diet equivalent to un— baked white bread, which supplied approximately 12 per cent protein. They suggested that commercial bakers are not likely to use 12 per cent nonfat milk solids in bread, therefore, the possibility of supplementing bread with 0.25 per cent L-lysine should be given consideration for raising the lysine content of diets where this appears to be desirable. These experimenters (Jahnke and Schuck, 1957) also measured the rate at which fat was deposited in the livers of the animals, since this is affected by the amino acid balance of the diet when the protein content of the diet is low. The 0.25 per cent L-lysine supplement had no affect on the amount of fat deposited in the livers of the 13 animals when the diet contained either 3, 6, or 12 per cent nonfat milk solids. The total lysine content of the diets ranged from 0.44 to 0.63 per cent. Culik and Rosenburg (1958) observed five generations of rats fed a commercial white bread which contained 6 per cent nonfat milk solids, and supplied 12 per cent protein, either with or without a supplement of 0.25 per cent L-lysine. The total lysine content of the supplemented 'bread diet was 0.51 per cent. The reproduction and lacta- tion performance of rats fed the lysine supplemented bread was superior to that of rats fed the unsupplemented bread, and equal to that of rats fed a stock ration of mixed foods, which supplied 22 per cent protein. The lysine supplemented bread supported normal reproduction and lactation of rats when fed as the sole source of dietary protein. Experimenters diasgree concerning the extent to which lysine supplementation improves the protein of wheat. .Flodin (1956) suggested that the nutritional value of the gurotein in lysine supplemented wheat products is comparable fix: that of proteins from animal sources. 0n the other hand, jBender'(1958) suggested that lysine supplementation of wheat products increases the utilization of the protein only by approximately 10 per cent. I Supplements 2£_Lysine Plus Other Amino Acids: The possilujity'of further improving the nutritional value of the Iarotein of wheat with supplements of other amino acids in.achdition to lysine has been studied. 14 Desphande et_al, (1957) observed the growth and amount of fat deposited in the livers of young rats fed a white flourdiet, which supplied 9.5 per cent protein. The diet was supplemented with various amounts of lysine and/or threonine, ranging from 0.25 to 0.90 per cent and 0.20 to 0.60 per cent respectively. A supplement of 0.25 per cent L—lysine promoted more growth than did the unsupplemented diet. The addition of either 0.20 per cent DL-threonine or larger amounts of lysine did not further improve the growth. However, maximum growth was attained when the diet was supplemented with 0.50 per cent L-lysine plus 0.40 per cent DL-threonine. Sure, (1952, 1954a, 1955a, 1957) conducted a series of experiments to determine the value of adding lysine, threonine, valine, or methionine to diets of whole wheat flour, or white flour, which supplied approximately 8 per cent protein. In every study, the diets supplemented with amino acids produced more growth and higher protein effi- ciency ratios in young rats than did the unsupplemented diets. A supplement of 0.25 per cent L-lysine and 0.20 per cent DL-threonine was better than a supplement of 0.25 per cent L-lysine only (Sure, 1952, 1954a), and equivalent to a supplement of 0.25 per cent L-lysine, 0.20 per cent DL-threonine, and 0.50 per cent DL-valine for promoting growth and protein efficiency in young rats fed a whole wheat flour diet (Sure, 1954). However, a supplement of l4 Desphande et_gl, (1957) observed the growth and amount of fat deposited in the livers of young rats fed a white flourdiet, which supplied 9.5 per cent protein. The diet was supplemented with various amounts of lysine and/or threonine, ranging from 0.25 to 0.90 per cent and 0.20 to 0.60 per cent respectively. A supplement of 0.25 per cent L-lysine promoted more growth than did the unsupplemented diet. The addition of either 0.20 per cent DL-threonine or larger amounts of lysine did not further improve the growth. However, maximum growth was attained when the diet was supplemented with 0.50 per cent L-lysine plus 0.40 per cent DL-threonine. Sure, (1952, 1954a, 1955a, 1957) conducted a series of experiments to determine the value of adding lysine, threonine, valine, or methionine to diets of whole wheat flour, or white flour, which supplied approximately 8 per cent protein. In every study, the diets supplemented with amino acids produced more growth and higher protein effi- ciency ratios in young rats than did the unsupplemented diets. A supplement of 0.25 per cent L-lysine and 0.20 per cent DL—threonine was better than a supplement of 0.25 per cent L-lysine only (Sure, 1952, 1954a), and equivalent to a supplement of 0.25 per cent L-lysine, 0.20 per cent DL-threonine, and 0.50 per cent DL-valine for promoting growth and protein efficiency in young rats fed a whole wheat flour diet (Sure, 1954). However, a supplement of 15 0.40 per cent L-lysine combined with 0.20 per cent DL- threonine, and 0.50 per cent DL-valine produced the most growth and highest protein efficiency ratios in young rats fed a whole wheat flour diet (Sure, 1954). The nutritional adequacy of this diet was further improved, as measured by growth and protein efficiency ratio, by substituting 0.40 per cent DL-methionine for the 0.50 per cent DL-valine used in the previous study (Sure, 1957). The inclusion of methionine in the amino acid supplement significantly re- duced the amount of fat in the livers of the rats as com- pared with those of rats fed the unsupplemented white flour diet. Supplements of lysine alone, or in combination with threonine, and/or valine did not appreciably affect the amount of fat deposited in the livers of the rats (Sure, 1957). A bread diet identical to one described previously (Rosenberg and Rohdenburg, 1952) was supplemented with various amounts and combinations of lysine, valine, threo— nine, and methionine (Rosenberg et_al,, 1954). Lysine alone was equivalent to any combination of lysine, valine, threo- nine, or methionine for promoting growth and protein efficiency in young rats. Thus, they concluded that the only amino acid deficiency in commercial white bread is lysine. The difference between their results and those of other investigators may be due to the 12.5 per cent protein level of their experimental diet, or the 3 per cent nonfat milk solids contained in the bread. 16 It is difficult to compare the studies available because of the variations in experimental procedures. Perhaps further investigation will clarify the details that at present are obscure. The Practical Aspect§_9f_Supplementing Foods with Amino Acids: Any commercial supplementation of foods with amino acids should be contingent on the cost, safety, effectiveness, and necessity of the program. The economic aspect of supplementing bread with lysine has been discussed extensively by Flodin (1953, 1958). He suggests that lysine can be made available at a cost of l to 3 cents per gram (1953). ”The cost of amino acid fortification, at least in the case of lysine, is potentially in the range of very low-cost protein addi- tives,” (Flodin, 1958). However, the economic interests held by DuPont in this program should not be ignored. It appears doubtful that adverse effects would result from the supplementation of bread with small amounts of lysine under normal conditions. Adverse effects result- ing from lysine supplementation of wheat have been observed only when an excessive amount of the supplementary amino acid was added to the diet (Hutchinson et_al,, 1956). For economic reasons, a food manufacturer would not add an excess of lysine to bread, thus the possibility of an excess quantity of lysine in bread is slim. The effectiveness of supplementing bread with lysine is questionable, and difficult to determine, when 17 considered in relation to a diet of mixed foods, because of the supplementary relationship of various proteins, and the variability of individuals in their choice of foods. Westerman et_al, (1957) noted that rats fed diets contain- ing 37 per cent white flour and 12 per cent meat, milk, and eggs, grew as well as rats fed this diet supplemented with 0.3 per cent L-lysine. When the meat was omitted, lysine supplementation improved growth, however, when meat and milk were both omitted the flour equaled 49 per cent of the diet, growth was slow even with a supplement of lysine. The value of any amino acid supplementation program will not be realized unless the diet is adequate with respect to other nutrients, particularly the vitamins involved in the metabolism of amino acids. In the United States there is no need to improve the protein in the average diet. In 1955 the average diet provided 103 grams of protein per person per day. In this country, 92 per cent of the population met the National Research Council recommended allowances for protein. (United States Department of Agriculture, 1957). Of the total protein consumed in 1957, 65 per cent was from animal sources and 20 per cent from flour and cereal products (United States Department of Agriculture, 1958). The present information available indicates that the daily lysine requirement of young men (Rose et_al,, 1955) and young women (Jones §t_al,, 1956) is low and is 18 more than adequately met in the average American diet. Clark et_§1, (1957) expressed the opinion, ”The possibility of a general inadequacy of lysine in diets consumed by the adult population in this country seems remote.” Nevertheless, it should be acknowledged that a small percentage of people in the United States are consum- ing a diet suboptimum in protein. In two independent sur- veys Williams (1945) and Jean et a1. (1952) observed the average quantity of protein consumed by pregnant women was below that recommended by the National Research Council. It has also been suggested that the protein nutrition of children between the ages of 2 and 6 should be improved (Jeans, 1950; Lynch and Snively, 1955). Morgan (1959) compiled recent surveys of the nutritional status of the United States population. According to this report,the protein consumed by adolescent girls and women over the age of 70 was significantly lower than the National Re- search Council recommended allowances. One way the protein requirements of these individuals may be more adequately met is by commercially supplementing bread with lysine. The Food and Nutrition Board of the National Research Council has examined the possibility of supple- menting bread with lysine. Their conclusions are summarized in the following statements: The committee recognizes the potential offered by amino acid supplementation in human nutrition, but no convincing evidence of a need for such supplementa- tion for the individual eating an average mixed diet in the United States has as yet been presented. The 19 possibility exists, of course, that certain segments of the population could benefit by supplementation of certain food items in the diet. (National Re- search Council, 1959) In other countries, however, severe protein mal- nutrition is prevalent. Sufficient protein is not avail- able, and sources of high quality protein are limited. The grams of animal protein available per person per day in China, India and Indonesia were 4.9, 5.8, and 4.7 respectively (Phillips, 1951). The possibility of improv- ing the dietary protein with supplements of mixed foods locally available, and with supplements of amino acids, is being examined in Mexico (Gomez et_§l,, 1958), Central American and Panama (Behar et_al,, 1958), and French West Africa (Senecal, 1958). Each of these investigators suggested the present knowledge of amino acid supplementa- tion is inadequate, and recommended further investigation. THE RELATIONSHIP OF LIVER XANTHINE OXIDASE T0 DIETARY PROTEIN The liver xanthine oxidase enzyme system is very sensitive to the quantity and quality of protein present in the diet. The activity of this system is affected by the availability of the essential amino acids (Williams and Elvehjem, 1949), thus, it is a suitable criterix1for judging the protein value of the diet. An advantage of using this criteria for measuring protein quality is the speed with which this enzyme system responds to the protein 20 adequacy of the diet. Since the xanthine oxidase system responds maximally to dietary conditions in approximately ten days (Litwack et al., 1952), it provides a more econom- ical method of measuring the value of dietary protein than does growth studies. The results obtained by using the activity of this enzyme as a measure of protein quality agree with the results obtained by long-term growth studies (Litwack SE 31°: 1953). EX PER IMEN TA L PR OCEDURE EXPERIMENTAL PROCEDURE The diets used in this experiment were of the following composition: 90 per cent dried, ground bread; 5 per cent corn oill; 4 per cent mineral saltsg; 0.25 per cent vitamin mixture3; 0.15 per cent choline; and 0.60 per cent sucrose. The bread used in the preparation of diet I was a standard white bread and served as the control. In diet II, the bread contained 5/6 white flour and 1/6 Roman meal (a blend of whole wheat, whole rye, bran, and flax- meal). In diet III, the bread contained a blend of gluten, whole wheat, and unbleached flours supplemented with 1.7 grams of L-lysine monohydrochloride per pound. Those specifications, which were available from the manufacturer of each bread may be found in Appendix, page i. The breads were purchased at local markets. They were oven dried at 50 degrees centigrade for 3 hours, lContaining 7.5 mg cA-tocopherol acetate and 0.38 mg menadione. 2Salts IV obtained from Nutritional Biochemicals, Inc. 3Containing 0.5 mg thiamine, 0.5 mg riboflavin, 1.0 mg niacin, 0.25 mg pyridoxine HCl, 2.0 mg calcium pantothenate, 10 mg inositol, 0.02 mg folic acid, 0.002 mg vitamin B12, 0.01 mg biotin, 10 mg vitamin A, 0.18 mg vitamin D, and 0.225 g sucrose. finely ground in the Hobart grinder, and stored in covered metal containers. Nitrogen was determined in duplicate on 1.0 gram samples of the dried, ground bread by the Macro- Kjeldahl procedure. Thirty three weanling, male, albino rats of the Sprague-Dawley strain were divided into 3 groups of 11 each. The average weight per group was 43 grams. The rats were housed in separate, wire mesh cages and allowed food and water ad_libitum for a period of 14 days. Food intake and weight records were kept. Between 14 and 19 days the animals were decapitated (2 animals from each group per day), the livers removed, weighed, and homogenized and the activity of the xanthine oxidase system determined. The remainder of the liver homogenates were stored frozen for the determination of moisture, nitrogen, and fat. The contents of the gastrointestinal tract were washed out, and the carcasses were weighed and frozen for the determination of carcass moisture, nitrogen, and fat. Liver xanthine oxidase activity was determined by the use of manometric procedures. A modification of the method of Axelrod and Elvehjem (1941) was used in this study. For each determination, the substrate (0.2 milli- liters of 0.038 molar xanthine in 0.05 molar sodium hydrox- ide) was pipetted into the side arm of a Warburg flask. The center well contained 0.2 milliliters of 10 per cent potassium hydroxide. A folded piece of filter paper was inserted into the well to provide a greater surface area 23 for the adsorption of carbon dioxide produced. The flask was chilled in crushed ice. After the flask was prepared, the rat was stunned by a sharp blow on the head and decapitated. The liver was quickly removed from the decapitated animal and immed- iately chilled in ice. The excess moisture was removed by blotting with filter paper. A portion of the liver was weighed and homogenized with five volumes of cold, distilled water, in a Potter-Elvehjem homogenizer. One milliliter of this homogenate, which contained 0.167 gram of tissue, was pipetted into the main compartment of the chilled Warburg flask. The total volume contained in the flask was brought to 2.2 milliliters by adding chilled, distilled water. The flask was seated on the manometer, placed in a water bath maintained at 37 degrees centigrade, and equilibrated for a period of 10 minutes. The substrate was tipped into the main compartment from the side arm, and the stopcock was closed. The manometer reading was recorded every 10 min— utes for a period of 200 minutes.1 Duplicate determinations were run for each rat. Endogenous activity was determined similarly except for the omission of the xanthine substrate from the flask. Data were corrected for endogenous activity and changes in atmospheric pressure. A 30 minute period, within the log phase of the enzyme activity curve, was chosen to calculate 1For the first rat in each group readings were re- corded only 120 minutes. 24 the activity of the xanthine oxidase system. Enzyme data were calculated and reported per 10 milligrams of nitrogen, per gram of fresh weight liver, and per total weight of the liver. When the enzyme determination was completed, the remaining liver, which had been weighed, homogenized, and stored in a frozen state, was thawed, placed into a weighed evaporating dish, and dried at 90 degrees centigrade for 12 hours. The dry liver homogenate was cooled to room temperature in a dessicator, weighed, finely ground, and stored in a covered bottle. The per cent of moisture in the liver was calculated. The amount of fat in the liver was determined by continuous ether extraction in the Goldfisch apparatus. The sample of dried, ground liver was weighed to the near- est milligram and consisted of the entire amount available, approximately 0.5 gram per rat in groups I and II, and approximately 1.0 gram per rat in Group III. The per cent of fat in the liver was calculated on a dry weight basis. The nitrogen content of a 0.25 gram sample of dried, ground, fat free liver was determined by the Macro— Kjeldahl method. A single determination was run for each rat in group I, because of the small amount of sample available. Duplicate determinations were run for each rat in groups II and III. The per cent of nitrogen in the total liver, which included the fat, was calculated. The frozen carcass was sawed into chunks and ground three times to obtain a homogeneous mixture. A 20 gram sample from each carcass was oven dried at 90 degrees centigrade to a constant weight, then pulverized in a mortar. Atmospheric moisture collected during pulverizing was removed by oven drying. The sample was then placed in a covered container and stored in a dessicator. The per cent of moisture in the total carcass, including the liver, was calculated. The amount of fat present in each carcass was deter- mined in duplicate on 1.0 gram samples of dried, pulver- ized carcass by continuous ether extraction in the Goldfisch apparatus. The per cent of fatin the total carcass, which included the liver, was calculated on a dry weight basis. The amount of nitrogen present in each carcass was determined in duplicate on 0.5 gram samples of dried, pulverized carcass by the Macro-Kjeldahl method. The per cent of nitrogen in the total carcass, including the liver, was calculated. All individual data reported was averaged per group, and the standard error ofthe mean was calculated. ”Student's” t test was used as a measure of significance. RESULTS RESULTS The results Of this study are summarized in Tables I, II, III, and IV; data for individual animals are presented in the Appendix, p. ii through p. xiii. PROTEIN CONTENT OF BREAD The protein content (N x 5.7)1 of the breads used in this experiment, reported on a dry weight basis, was 12.5 per cent in the standard white bread (diet I), 16.3 per cent in the bread containing Roman meal (diet II), and 25.0 per cent in the lysine supplemented bread (diet III). FOOD CONSUMPTION AND GROWTH DATA (Table I) No significant differences were observed in the food intake and growth data of rats fed the bread contain- ing Roman meal (group II) as compared with rats fed the standard white bread (group I). Since the bread containing Roman meal supplied 1/3 more protein than did the standard white bread on a dry weight basis, the animals in group II had lower protein efficiency ratios than did the control rats. This difference was small, but significant at the 5 per cent level (P<0.05). 1Association of Official Agricultural Chemists, 1950. 27 The rats fed the lysine supplemented bread (group III) consumed approximately 1 1/2 times the food and grew approximately 5 times as fast as rats fed the standard white bread (group I). As a result, the protein efficiency ratios of the rats fed the lysine supplemented bread were approximately twice those Of the rats fed the standard white bread (P( 0.01). LIVER ANALYSIS DATA (Table II) The per cent of nitrogen stored in the livers from rats fed the bread containing Roman meal (group II) was greater than that in the livers from rats fed the standard white bread (group I). This difference was significant at the l per cent level (P<_0.01). NO significant differ- ences were noted in the per cent of fat, per cent of mois- ture, or dry weight of the livers of rats fed the bread containing Roman meal (group II) as compared with rats fed the standard white bread (group I). Livers from rats fed the lysine supplemented bread (group III) contained a significantly greater per cent of nitrogen and a significantly smaller per cent of fat than did the livers from rats fed the standard white bread (group I). These differences were significant at the l per cent level (P< 0.01). The dry weight of the livers from rats fed the lysine supplemented bread (group III) was approximately 2 1/2 times that of the livers from rats fed 28 the standard white bread (group I). No significant dif- ference in the per cent of moisture was Observed between these 2 groups. LIVER XANTHINE OXIDASE DATA (Table III) When the xanthine oxidase activity was calculated per unit weight Of nitrogen, no significant difference was observed between rats fed bread containing Roman meal (group II) and rats fed the standard white bread (group I). When the xanthine oxidase activity was calculated per gram Of fresh weight liver, or per total weight Of the liver, this enzyme system in the livers from ratsin group II was more active than in the livers from rats in Group I. These differences were small, but significant at the 1 per cent level (P<0.0l). The xanthine oxidase system was significantly more active in livers from rats fed the lysine supplemented bread (group III) than in livers from rats fed the standard white bread (group I), regardless of the units used in calculating. These differences were significant at the l per cent level (P(0.01). Since rats fed bread containing Roman meal and rats fed lysine supplemented bread deposited significantly more nitrogen in the liver than did rats fed the standard white bread (table II), the xanthine oxidase activity expressed in terms of unit weight of liver nitrogen is probably more appropriate. With this unit of measurement, 29 only the rats fed the lysine supplemented bread had significantly more active xanthine oxidase systems in the livers than did rats fed the standard white bread. CARCASS ANALYSIS DATA (Table Iv) There were no significant differences in the per cent of nitrogen, per cent of fat, per cent of moisture, or dry weight Of the carcasses Of rats fed the bread con- taining Roman meal (group II) as compared with those Of rats fed the standard white bread (group I). However, the carcasses of rats fed the lysine supplemented bread (group III) contained a greater per cent of nitrogen than did the carcasses of rats fed the standard white bread (group I). This difference was significant at the l per cent level (P<.0.01). The dry weight of the carcasses of rats fed the lysine supplemented bread was twice that of the rats fed the standard white bread (P<:0.0l). There were no significant differences between these 2 groups in the per cent of carcass fat or moisture. DISCUSSION AND CONCLUSIONS DISCUSSION AND CONCLUSIONS Data presented in this paper emphasize the impor- tance Of quality as well as quantity of protein supplied by a food. Chemical analysis of the bread containing Roman meal supported the advertising claim, ”contains about 25 per cent more protein than most white breads.” The small but sig- nificant increase in the per cent of nitrogen contained in the livers from rats fed the bread containing Roman meal as compared with those of rats fed the standard white bread, was probably due to the greater quantity Of pro- tein supplied by the Roman meal bread. Although the bread containing Roman meal supplied approximately 1/3 more protein, the quality of this protein was no better than that Of the standard white bread when measured by biological asSay. No significant difference was observed in the growth of rats fed the bread contain- ing Roman meal and that of rats fed the standard white bread, thus rats fed the bread containing Roman meal had lower protein efficiency ratios. Since growth studies are usually of longer duration, it is possible that these data were influenced by the experimental period of 14 to 19 days. Measurement Of the activity of the liver xanthine oxidase system, which responds maximally to dietary conditions in approximately 10 days (Litwack et_§l,, 1952), indicated that the quality Of the protein provided by bread containing Roman meal was not improved as compared to that of the standard white bread. The activity of the liver xanthine oxidase system in rats fed bread containing Roman meal was not significantly different than the activ- ity of this enzyme system in livers from rats fed the standard white bread, when measured per unit weight Of liver nitrogen. Therefore, it is suggested that Roman meal, composed of whole wheat, whole rye, bran, and flax- meal, did not provide an amino acid pattern that comple- mented that of white flour. For individuals consuming diets restricted with respect to the quantity and quality of protein, substi- tuting a bread containing a supplement Of Roman meal for a standard white bread is not warranted. This is especially true in View of the increased cost of this supplemented bread; approximately 10 cents more per pound. The nutritional value of the protein in a lysine supplemented bread, which was advertised as containing, ”.3 per cent more protein than the same weight of regular L". ./ ' was also studied. Chemical analysis performed white bread,‘ in this laboratory indicated that the lysine supplemented bread provided approximately twice the quantity Of protein, which was provided by the standard white bread. Biological assay demonstrated a marked improvement in the quality as well as the quantity of the protein provided by the lysine supplemented bread. lore growth, higher protein efficiency ratios, and a greater per cent of nitrogen in the livers and carcasses, were Observed in rats fed the lysine supplemented bread as compared with rats fed the standard white bread. The activity of the liver xanthine oxidase system was in agreement with this data. Livers from rats fed the lysine supplemented bread had significantly more xanthine oxidase activity than did livers from rats fed the standard white bread. It is sug— gested that the blend of wheat flours and the lysine supplement improved the amino acid balance of the protein in this bread as compared with that of the standard white bread. This study did not clarify the metabolic processes involved, which resulted in the significantly smaller per cent of fat in the livers of rats fed the lysine supple- mented bread. Nevertheless, the relationship of amino acid balance in dietary protein to the deposition Of fat in the liver Of experimental animals supports the conclusion that the amino acid balance Of the protein in the lysine supple- mented bread was improved as compared with that of the standard white bread. For individuals consuming suboptimum protein the superior nutritional value of the lysine supplemented bread may be worth the additional cost of 7 cents per pound. However, it is recognized that in the United States there LJ L0 is no need for improving protein consumption by the average person. This study indicated that the use of liver xanthine oxidase activity provided an excellent technique for deter- mining the protein value of bread diets. Significant changes were noted after an experimental feeding period Of only 14 days. Thus, the use of this enzyme system as a criterion Of protein quality is both fast and economical. The assay methods used in this study made necessary the use of weanling rats as experimental subjects. How- ever, the results may contribute additional information concerning the nutritional value of protein in supple- mented breads for human consumption. S UMMA RY S UMMA R Y The nutritional value of the protein in each of 2 breads, advertised as ”high protein” breads, was compared with that of a standard white bread. One bread contained Roman meal. The other bread contained a blend of wheat flours supplemented with lysine. Thirty three weanling, male, albino rats (3 groups of 11) were fed 90 per cent bread diets. Rats in group I were fed a standard white bread. Rats in group II were fed a bread containing Roman meal. Rats in group III were fed a lysine supplemented bread. Food and water were allowed ag_libitum for a period Of 2 weeks. Records of food in— take and weight gain were kept. At the end of the experi— mental period the rats were killed, and the livers were analyzed for xanthine oxidase activity, nitrogen, fat, and moisture. The carcasses were analyzed for nitrogen, fat, and moisture. Each bread was analyzed for nitrogen. The bread containing Roman meal provided approxi- mately 1/3 more protein (dry weight) by chemical analysis than did the standard white bread. However, no significant difference was Observed in the growth of young rats fed the supplemented bread as compared with those fed the standard white bread. As a result, rats fed the bread containing Roman meal had significantly lower protein efficiency ratios. NO significant difference was noted in the xanthine oxidase activity per unit weight Of liver nitrogen between the livers from rats fed the bread containing Roman meal and those from rats fed the standard white bread. The only significant difference observed between these 2 groups, with respect to the composition of the carcass or the liver, was a greater per cent of nitrogen in the livers from rats fed the bread containing Roman meal. This difference was small, but significant, and in View Of other data, probably reflects the greater quantity of protein supplied by the Roman meal supplemented bread. While the bread containing Roman meal provided more protein, the quality of this protein was not improved as compared with that of the standard white bread. Therefore, the amino acid composition Of Roman meal did not complement that of white flour. The additional cost of Roman meal bread (10 cents more per pound),combined with a biological value for protein which is no better than the standard white bread, makes this an unsatisfactory product. The second bread studied was supplemented with lysine and provided approximately twice the amount of pro- tein, by chemical analysis, than was provided by the standard white bread. Rats fed the lysine supplemented bread had significantly greater growth rates and signifi- cantly higher protein efficiency ratios than did rats fed the standard white bread. 36 The xanthine oxidase system was significantly more active in livers from rats fed the lysine supplemented bread than in livers from rats fed the standard white bread. Rats fed the lysine supplemented bread had larger livers, which contained a higher per cent of nitrogen and a lower per cent of fat, than did rats fed the standard white brew. The only difference Observed between these 2 groups, with respect to carcass composition, was a greater per cent of nitrogen in the carcasses of the rats fed the lysine supple- mented bread. This difference was small, but significant. The lysine supplemented bread provided protein which was superior in both quantity and quality as compared with that of the standard white bread. The blend of wheat flours and the lysine supplement improved the amino acid balance Of this protein as compared to that Of the standard white bread. The superior nutritional value Of the protein in the lysine supplemented bread may be worth the addition- al cost of 17 cents per pound to individuals who are con- suming suboptimum protein. However, it is recognized that the majority of people in the United States do not need improved protein nutrition. Liver xanthine oxidase activity was an excellent criterion of the nutritional value Of the protein in bread. Significant differences in the activity of this enzyme in the livers from young rats were Observed after an experimental feeding period of only 14 days, thus, it is a sensitive, fast, and economical method for determining protein nutri- tional value. TABLE I FOOD CONSUMPTION AND GROWTH DATA GROUPl FOOD WEIGHT INTAKE GAIN (s wk) (s/Wk) I 46 :_23 7 :_03 II 46 :.2 8 i.1 III 75 + l 34 + l 1Each group contained 11 rats. 37 PROTEIN2 EFFICIENCY (s/WK) 1.31 + 0.063 1.12 + 0.06 Group I 90 per cent standard white bread. Group II 90 per cent bread containing Roman meal. Group III 90 per cent lysine supplemented bread. 2V 3Standard error of the mean. Ieight gain per gram Of protein eaten. 39 TABLE III LIVER XANTH INE OXIDA SE DATA GROUP1 UL Og/HR/IO MG UL o /HR/GM UL Og/HR/TOTAL NITROGEN LIVER LIVER I 63 i 32 95 i 42 237 : 82 I: 73.: 4 118 :_A 303 :_17 III 106 :_6 214 :_7 1248 :_79 1Group I 9 rats fed 90 per cent standard white bread. Group II 10 rats fed 90 per cent bread containing Roman meal. Group III 10 rats fed 90 per cent lysine supple- mented bread. 2Standard error of the mean. 40 TABLE IV CARCASS ANALYSIS DATAl GROUP2 CARCASS WEIGHT NITROGEN FAT MOISTURE g dry wt) % dry wt) (% dry wt) % I 17.0 i 0.33 8.4 i 0.13 29.1 30.93 67 :_13 II 16.7 :_0.3 8.4 i.0°1 27.6 :_0.9 69 :_1 III 34.2 + 1.0 8.9 + 0.1 28.9 + 1.1 69 + l lData include liver analysis. 2Each group contained 11 rats. Group I 90 per cent standard white bread. Group II 90 per cent bread containing Roman meal. Group III 90 per cent lysine supplemented bread. 3 Standard error of the mean. L ITERA TURE C ITED LITERATURE CITED Association of Official Agricultural Chemists. 1950 Official Methods of Analysis. 7th edition. Association of Official Agricultural Chemists. Washington, D. C. p. 13. Axelrod, A. E. and Elvehjem, C. A. 1941 Xanthine oxidase .content of rat liver in riboflavin deficiency. Journal of Biological Chemistry 140:725. Behar, M., Viteri, F., Bressani, R., Arroyaue, G., Squibb, R., and Scrimshaw, N. 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APPENDIX A SUI~11‘-'IARY OF THE IIxIFORI‘v'IATION AVA ILABLE FROM THE MANUFACTURER OF EACH BREAD Composition gf_the Breads: Bread I Bread II Bread III (diet I) (diet II) (diet III) Protein (fl) 8.0 10.0 14.5 Fat (fl) 3.1 3.1 2.0 Carbohydrate (fl) 50.5 47.3 41.0 Moisture (%) 38.0 38.0 38.0 Calories per pound 1227 1172 1035 All breads contained nonfat milk solids and yeast, and were enriched according to government standards with thiamine, riboflavin, niacin, and iron. The bread used in diet II contained Roman meal, which replaced 1/6 of the white flour. Roman meal is a blend of whole wheat, whole rye, bran, and flaxmeal. The manufacturer stated, ”Contains about 25% more protein than do most white breads.” The bread used in diet III was made of a blend of gluten, whole wheat (part bran removed), and unbleached flour. Eacn pound of bread was supplemented with 1.7 grams of L-lysine monohydrochloride, and 5.4 micrograms of vita- min B12. The manufacturer stated, ”53% more protein than the same weight of regular white bread.” ii TABLE I GROUP Il FOOD CONSUMPTION AND GROWTH DATA FOR INDIVIDUAL RATS RAT # FOOD WEIGHT PROTEIN2 INTAKE GAIN EFFICIENCY (s/Wk) (s/Wk) (s/Wk) 11 45 6 1.20 12 61 8 1.30 I3 49 7 1.16 I4 46 8 1.60 15 47 9 1.80 I6 5? 7 1.16 I7 46 6 1.20 I8 40 6 1.20 19 42 7 1.40 110 42 7 1.40 Ill 38 4 1.00 AVERAGE 46 + 23 7 :_03 1.31 + 0.063 19095 standard white bread. 2Weight gain per gram of protein intake. 3 Standard error of the mean. GROUP AVERAGE 1 WEIGHT (g dry wt) 0.44 0.62 0.61 0.74 0.63 0.71 0.66 0.59 0.47 0.77 0.57 19075 standard white bread. 2 TABLE II I LIVER ANALYSIS DATA FOR NITROGEN (% dry wt) 6.2 6.0 6.0 5.7 6.5 5.7 5.8 6.3 5.9 5.4 5.7 Standard error of the mean. FAT (% dry wt) 9.0 15.7 15.5 15.7 10.0 18.3 14.5 11.9 13.4 17.7 20.6 iii INDIVIDUAL RATS MOISTURE 7.5 75 74 0.62 :0.032 5.9 :0.12 14.8 :_1.12 75 :_19 iv TABLE III GROUP Il LIVER XANTHINE OXIDASE DATA FOR INDIVIDUAL RATS RAT # UL Og/HR/lO MG UL 0 /RR/G UL Og/RR/TOTAL NITROGEN LI ER LIVER 112 -- -- -- I2 64 102 245 3 __ __ -_ I3 I4 47 66 206 I5 56 96 227 I6 64 90 257 I7 64 102 245 lg 68 108 259 19 86 120 240 110 60 84 260 Ill 60 90 194 AVERAGE 63 + 3LL 95 :_4“ 237 + 84 190% standard white bread. 2Data for this animal recorded for 120 minutes was inconclusive. 3No results were obtained for this animal because of defective experimental procedure. 4Standard error of the mean. TABLE IV GROUP Il CARCASS ANALYSIS DATA FOR INDIVIDUAL RATS RAT # (gEgggth) (AlgigGfig) (% 8?; wt) MOIgTURE 11 16.3 9.2 24.1 69 I2 18.6 8.5 29.1 67 I3 16.2 8.4 25.2 69 In 17.5 8.5 30.3 67 I5 18.3 7.8 31.3 66 I6 17.7 8.4 31.2 67 I7 17.2 8.6 24.4 68 18 16.8 8.4 29.4 65 19 16.3 7.8 31.8 67 I10 16.4 8.1 31.1 67 Ill 15.3 8.3 31.9 66 AVERAGE 17.0 I 0.32 8.4 I 0.12 29.1 + 0.92 67 + 1 190% standard white bread. 2Standard error of the mean. vi TABLE V ' GROUP IIl FOOD CONSUMPTION AND GROWTH DATA FOR INDIVIDUAL RATS RAT # FOOD NEIGRT PROTEIN2 INTAKE GAIN EFFICIENCY (S/Wk) (S/Nk) (g wk) IIl 45 8 1.14 112 42 7 1.16 II3 46 8 1 .14 114 66 10 1.00 II5 40 7 1.16 II6 43 7 1.16 II7 47 7 1.00 118 48 5 0.71 119 44 11 1.57 1110 46 9 1.28 IIll 38 6 1.00 AVERAGE 46 + 23 8 + 13 1.12 + 0.063 l90% bread containing Roman meal. 2Weight gain per gram of protein intake. 3Standard error of the mean. RAT # IIl 112 113 IILL II5 II6 II7 118 119 II 10 II11 AVERAGE vi TABLE V ' GROUP IIl FOOD CONSUMPTION AND GROWTH DATA FOR INDIVIDUAL RATS FOOD INTAKE E/Wk) 45 42 46 66 4O 43 47 48 44 46 38 46 :_23 NEIGRT PROTEIN2 GAIN EFFICIENCY (S/Wk) (S/Nk) 8 1.14 7 1.16 8 1.14 10 1.00 7 1.16 7 1.16 7 1.00 5 0.71 11 1.57 1.28 1.00 8 + 13 1.12 :_0.063 190% bread containing Roman meal. 2 Weight gain per gram of protein intake. 3Standard error of the mean. RAT-# IIl 112 II3 II“ 115 II6 II7 118 119 II10 AVERAGE TABLE V ' GROUP IIl FOOD CONSUMPTION AND GROWTH DATA FOR INDIVIDUAL RATS vi FOOD WEIGHT PROTEIN2 INTAKE GAIN EFFICIENCY (S/Wk) (g/wk) (g/wk) 45 8 1.14 42 1.16 46 8 1.14 66 10 1.00 40 7 1.16 43 7 1.16 47 7 1.00 48 5 0.71 44 11 1.57 46 9 1.28 38 6 1.00 46 + 23 8 + 13 1.12 i 0.063 l90% bread containing Roman meal. 2Weight gain per gram of protein intake. 3Standard error of the mean. vii TABLE VI 1 GROUP II LIVER ANALYSIS DATA FOR INDIVIDUAL RATS RAT # WEIGHT NITROGEN FAT MOISTURE (s dry wt) (% dry wt) (% dry wt) % IIl 0.66 6.4 14.2 74 112 0.77 6.8 11.1 72 II3 0.76 6.1 14.5 76 Ila 0.78 6.5 14.7 75 115 0.74 6.2 14.0 75 116 0.76 6.2 14.1 74 117 0.70 6.6 9.0 74 118 0.66 6.6 12.6 76 119 0.72 6.1 14.8 74 IIlO 0.76 6.5 14.1 80 IIll 0.79 6.3 11.7 73 AVERAGE 0.74 $0.042 6.4 :0.12 13.2 30.62 75 + 12 l90:6 bread containing Roman meal. 2Standard error of the mean. viii TABLE VII GROUP IIl LIVER XANTHINE OXIDASE DATA FOR INDIVIDUAL RATS RAT # UL Og/HR/lo MG - UL 0 /RR/G UL O /HR/TOTAL NITROGEN LIFER EIVER IIl 64 108 270 112 66 126 350 1132 -- -- -- 114 90 144 454 115 64 102 296 116 71 114 331 II7 71 120 324 118 71 114 310 119 94 150 420 1110 78 102 296 IIll 56 96 278 AVERAGE 73 :.43 ‘118 I 43 303 3:173 l9056 bread containing Roman meal. 2No results were obtained for this animal because of defective experimental procedure. 3Standard error of the mean. ix TABLE VIII GROUP IIl CARCASS ANALYSIS DATA FOR INDIVIDUAL RATS RAT # WEIGHT NITROGEN FAT MOISTURE (6 dry wt) (% dry wt) (% dry wt) % 111 18.1 8.7 26.7 70 112 16.8 8.4 31.4 ‘ 68 113 16.5 8.2 26.9 70 114 17.5 8.1 28.9 68 115 16.6 8.4 27.6 69 116 16.3 8.5 27.9 69 117 16.7 8.8 22.4 70 118 15.1 8.1 28.0 68 II9 17.5 8.8 24.6 69 IIlo 17.2 7.7 33.0 66 1111 15.0 8.6 26.4 69 2 AVERAGE 16.7 :_0.32 8.4 :_0.1 27.6 I 0.92 69 :_12 l9056 bread containing Roman meal. 2Standard error of the mean. X TABLE IX 1 GROUP III FOOD CONSUMPTION AND GROWTH DATA FOR INDIVIDUAL RATS RAT # FOOD WEIGHT PROTEIN2 ' INTAKE GAIN EFFICIENCY (S/wk) (E/wk) . (Ia/wk) IIIl 70 36 2.25 III2 73 33 1.94 I113 66 32 2.13 1114 77 39 2.29 III5 72 32 2.00 1116 75 32 1.88 III7 75 32 1.88 1118 74 32 1.88 III9 79 35 1.94 IIIlo 82 36 1.89 IIIll 80 34 1.88 AVERAGE 75 I.23 34 + 13 1.99 :_0.043 l90% lysine supplemented bread. 2Weight gain per gram of protein intake. 3Standard error of the mean. xi TABLE X GROUP IIIl LIVER ANALYSIS DATA FOR INDIVIDUAL RATS RAT # WEIGHT NITROGEN FAT MOISTURE (g dry wt) (% dry wt) (5 dry wt) % IIIl 1.40 7.4 6.2 73 III2 1.06 8.7 12.1 73 III3 1.38 8.1 8.0 73 1114 1.54 7.6 8.6 76 1115 1.42 8.2 10.6 72 1116 1.67 7.6 8.4 73 III7 1.68 7.7 7.8 73 I118 1.74 7.0 7.7 75 III9 1.78 7.5 10.3 73 11110 1.43 7.8 12.0 74 IIIll 1.76 6.4 9.5 73 AVERAGE 1.53 :_0.072 7.6 + 0.22 9 2 :_0 62 73 :.12 l90% lysine supplemented bread. 2Standard error of the mean. TABLE XI GROUP IIIl LIVER XANTHINE OXIDASE DATA FOR INDIVIDUAL RATS RAT # UL Og/RR/TO MG UL 0 /RR/G UL 0 /RR/T0TAL NITROGEN LIVER DIVER IIIl 96 192 998 1112 73 174 679 1113 -- -- ~- IIILl 120 216 1385 III5 112 258 1316 1116 111 234 1463 1117 109 228 1423 1118 109 186 1302 1119 80 168 1089 IIIlO 129 258 1406 IIIll 123 222 1421 AVERAGE 106 i_63 214 :_73 1248 :793 1 90% lysine supplemented bread. 2No results were obtained for this animal because of defective experimental procedure. 3 Standard error of the mean. xiii TABLE XII GROUP IIIl CARCASS ANALYSIS DATA FOR INDIVIDUAL RATS RAT # (gEgggth) (gIgigGgN) (%F§:y wt) MOIgTURE IIIl 31.0 9.3 25.0 70 III2 30.9 9.4 27.1 70 III3 32.0 8.9 30.1 69 III4 34.2 8.8 30.0 68 III5 34.6 8.6 32.1 67 1116 31.5 9.4 26.1 70 III7 35.8 8.4 29.5 68 1118 31.1 8.5 21.6 72 III9 36.6 9.1 29.6 69 111lo 37.9 8.8 32.8 68 IIIll 41.1 8.2 33.4 66 AVERAGE 34.2 :1.02 8.9 :_0.12 28.9 :_1.12 69 :_12 l90% lysine supplemented bread. 2Standard error of the mean. It; xii TABLE XI GROUP IIIl LIVER XANTHINE OXIDASE DATA FOR INDIVIDUAL RATS RAT # UL 02/HR/10 MG UL 0 /RR/G UL O /HR/TOTAL NITROGEN L fER LIVER I111 96 192 998 1112 73 174 679 III3 —- -- -- IIIAL 120 216 1385 III5 112 258 1316 1116 111 234 1463 1117 109 228 1423 1118 109 186 1302 1119 80 168 1089 IIIlO 129 258 1406 IIIll 123 222 1421 AVERAGE 106 i_63 214 :_73 1248 :_793 1 90% lysine supplemented bread. 2No results were obtained for this animal because of defective experimental procedure. 3 Standard error of the mean. xiii TABLE XII GROUP IIIl CARCASS ANALYSIS DATA FOR INDIVIDUAL RATS RAT # (gEgggth) (£IgigGgN) (%Fé$y wt) MOIgTURE 1111 31.0 9.3 25.0 70 III2 30.9 9.4 27.1 70 III3 32.0 8.9 30.1 69 IIILL 34.2 8.8 30.0 68 III5 34.6 8.6 32.1 67 1116 31.5 9.4 26.1 70 III7 35.8 8.4 29.5 68 1118 31.1 8.5 21.6 72 1119 36.6 9.1 29.6 69 IIIlo 37.9 ’ 8.8 32.8 68 11111 41.1 8.2 33.4 66 AVERAGE 34.2 :_1.02 8.9 :0.12 28.9 :_1.12 69 + 12 l90% lysine supplemented bread. 2Standard error of the mean. N‘T’l U?“ M‘ RUB!“ IQIE. “Hm. ) ’ ' t -- ' :1 'If {‘1 7371728.) 961 m... I. .5 HICHIGQN STATE UNIV. LIBRQRIES 31293010635104