THE BIOLOGICAL VALUE OF THE NITROGEN OF MIX- TURES OF PATENT WHITE FLOUR AND ANIMAL FOODS. (Received for publication, January 21, 1926.

Transcription

1 THE BIOLOGICAL VALUE OF THE NITROGEN OF MIX TURES OF PATENT WHITE FLOUR AND ANIMAL FOODS. BY H. H. MITCHELL AND G. G. CARMAN. (From the Division of Animal Nutrition, Department of Animal Husbandry, University of Illinois, Urbana.) (Received for publication, January 21, 1926.) The peculiar function of protein and protein derivatives in the body, which, in practical nutrition, cannot be served by any other nutrient, is to provide structural units (amino acids) for the synthesis of the nitrogenous constituents, protein and nonprotein in character, of the tissues and secretions of the body. These constituents are extremely varied in character, but it is fair to assume, since the assumption does not contradict any of the known facts or the accepted theories of physiology, that the proportions of these constituents that are catabolieed endogenously and that must be replaced in order to maintain the integrity of the tissues, are fairly constant for different conditions of the environment or of the animal itself. The known constancy of the creatinine excretion in the urine speaks for the correctness of this assumption. Furthermore, it is known that the proportions of the different nitrogenous constituents needed in the formation of new tissue in growth and reproduction, and in the elaboration of milk during lactation, are very little affected by conditions of feeding, activity, or variable environmental factors. Hence, though the percentage of nitrogen in the different nitrogenous compounds of the body varies great.ly, the need for them, or for their precursors (dietary amino acids), may fairly be measured by the total nitrogen content of the tissue constituents catabolixed endogenously (maintainance), or by the total nitrogen content of the new tissues formed in growth and reproduction, or by the total nitrogen content of the milk produced in lactation, A complete determination of the protein value of a food or of a mixture of foods in covering these nitrogen requirements of ani 183

2 184 Biological Values of Foods mals must consider (1) the total nitrogen content of the food or ration, (2) the loss or wastage of nitrogen in digestion, and (3) the loss or wastage of nitrogen in the process of its conversion into tissue constituents or body secretions. It is possible to determine the true protein content of foods and rations by laborious chemical methods, but in the routine analysis of foods the determination of protein is based solely upon the nitrogen content, which when multiplied by a conventional factor (6.25) is called the crude protein content. This procedure was originally introduced as a purely chemical expedient, but after all it would appear to possess more significance from a biological standpoint than the true protein content itself. The nonprotein nitrogenous constituents of foods are, in the main, amino acids or simple derivatives of amino acids, which may, in all probability, participate to a significant extent in the synthesis of the nitrogenous constituents of animal tissues and secretions, either individually, or in conjunction with the endproducts of protein digestion, They should not, therefore, be excluded from consideration in the determination of the protein value of foods and rations. For this reason, the total nitrogen content of a food or mixture of foods is a better starting point in this determination than the total content of true protein. Another argument for this conception is the fact that the losses of protein, or, more appropriately, amino acids, in digestion and metabolism, can only be measured in terms of nitrogen. Coefficients of digestibility of proteins and biological values of proteins are necessarily based solely upon nitrogen determinations, and refer solely to the utilization of food nitrogen in the animal body. They are therefore of significance only with reference to the total nitrogen content of the food or ration with which they have been obtained. While, therefore, in the chemical characterization of a food or ration, the true protein content, referring to a definite class of chemical compounds, possesses a much greater significance than the content of crude protein, in the biological evaluation of the food or ration, the total nitrogen content, converted into a purely conventional protein term, i.e., crude protein, must be given the preference. Therefore, both in the determination of the protein values of foods and in the determination of the so called protein requirements of animals, it seems advisable to speak in terms of nitrogen,

3 H. H. Mitchell and G. G. Carman According to numerous dietary studies made by the U. S. Department of Agriculture and summarized by Langworthy in Circular 110 of the Office of Experiment Stations, white flour contributes almost 30 per cent of the protein (nitrogen) in the average American dietary. Other cereal preparations contribute about 13 per cent, while the legumes contribute about 3 per cent of the dietary protein. The nitrogen of these foods possesses relatively low biological values, much lower than the nitrogen in staple animal foods. The addition of animal foods to such staple plant foods as those just mentioned, would conceivably correct the protein value of the diet in two distinct ways; first, by providing additional nitrogen of high biological value, and second, by supplementing the plant nitrogen and increasing in this way its value to the body. In so far as this supplementing action is based upon the amino acid makeup of the different classes of foods, it would be expected to appear whenever the amino acids constituting the limiting factors in the utilization of the two sources of nitrogen to be mixed, are not the same. If the indispensable amino acid limiting the utilization of the nitrogen of meat, for example, is not the limiting factor for the nitrogen of white flour, it is evident that if the two foods are fed together, the nitrogenous compounds of meat will enhance the biological value of the nitrogenous compounds of white flour, and also that the nitrogenous compounds of white flour will enhance the biological value of the nitrogenous compounds of meat. Only when the limiting amino acid is the same for both flour and meat will no supplementing relation exist. On the other hand, the supplementing effect may not be great, even if present, since, when the first limiting deficiencies of the two sources of nitrogen are mutually satisfied, the utilization of the mixture may still be seriously limited by the relative deficiency in each individual food of some common indispensable amino acid. In determining the value in the human diet of food products of animal origin, we have been interested not only in the inherent biological value of the nitrogen of such foods, but also in the extent to which their nitrogenous constituents are capable of supplementing the nitrogenous constituents of staple cereal foods, themselves possessing low biological values. Among these foods, patent white flour is the most important quantitatively and, therefore, has received our first attention. The plan of the experi

4 186 Biological Values of Foods ments to be reported in this paper has been to determine in the same experiment and with the same experimental animals, the biological value of the nitrogen of the animal food alone, of white flour alone, and of a mixture of the two, always in the proportion of one part of nitrogen from the animal food to two parts of nitrogen from white flour. Each test has generally been conducted upon ten rats handled in two groups of five. The nitrogen balance determinations on these two groups of animals were made by different persons and the order in which the foods to be tested were fed was different for the two groups. The details of the experiments relative to the preparation of rations, the method of feeding, and the methods of collecting the Ingredients. Wheat flour.... Dried egg... Butter fat... Cod liver oil... Salt mixture... Starch... Sucrose... Agar... TABLE I. Composition of Rations. White flour ration Egg ration. Low N ration. excreta were similar to those already deskribed in earlier reports of similar experiments (1) from this laboratory. I. White J lour and Whole Egg. The first combination of foods tested in this study was white flour and whole egg. The composition of the rations used in this test is given in Table I. The white flour was a standard brand in the midwest. The dried egg was prepared in the laboratory from eggs obtained from the Poultry Division of the Agricultural Experiment Station. The butter fat was prepared from University butter by melting and centrifuging to remove the casein. The cod liver oil was the product put out for animal feeding by the E. L. Patch Co. The salt mixture was prepared according to the directions of Osborne and Mendel (2). In this, as in all later

5 H. H. Mitchell and G. G. Carman 187 experiments, each rat received daily, besides its allotment of these rations, 25 mg. of yeast vitamin (Harris) as a source of vitamin B. This amount of vitamin concentrate contained from 2 to 3 mg. of nitrogen, which has been entirely neglected in the calculations based on the nitrogen metabolism data. The mixed ration used in this experiment was prepared by combining the white flour and the egg rations in the ratio of 2 to 1. The complete data of the nitrogen metabolism periods with the intermediate calculations leading to the final biological values for the first five rats are contained in Table II. The experimental data for the other five rats are given in Table III. In this and all subsequent tables, the intermediate calculations leading to the final biological value will be omitted. The biological values calculated for these different rations presumably represent the percentage of the absorbed nitrogen used by growing rats for both maintenance and growth. Their calculation involves the assumption that the excretion of fecal nitrogen per gm. of food consumed on the low nitrogen ration in the first and final periods measures the excretion of body nitrogen in the feces in the intervening periods. The change in the excretion of the fecal nitrogen per gm. of low nitrogen ration from the first to the sixth period is assumed to occur in a linear fashion with respect to time. The second assumption involved in these calculations is that the excretion of nitrogen in the urine per 100 gm. of body weight in the first and final periods of low nitrogen feeding is a measure of the excretion of body nitrogen in the urine in the intervening periods, the change in these values from the first to the sixth period being also assumed to be linear. If the estimated daily quantity of body nitrogen in the feces exceeds the total fecal nitrogen actually found, as frequently occurred in many of these experiments, it has been assumed that all of the food nitrogen is digestible. Also, with Rats 226 to 230, inclusive, on the whole egg ration, the estimated quantity of body nitrogen in the urine generally exceeded the actual total urinary nitrogen. This is interpreted to mean that no food nitrogen in these periods was wasted in the urine, so that the absorbed nitrogen was utilized to the extent of 100 per cent. This is a very rare occurrence, however, which has not been shown in any experiment but the one mentioned above.

6 TABLE II. Nitrogen Metabolism Data and the Calculation of Biological Values for the First Group of Rats. Period 1. Low nitrogen ration (0.016 per cent N). g?n. gnz. gm. mg. nag. mg. mc7. nw. n&l. mg. mg. ml. Per cent * r * 42 23t * 42 25t * 39 22t *, 38 23t Period 2. Egg ration (1.27 per cent N) Period 3. Flour, egg ration (1.28 per cent N) Period 4. Flour ration (1.20 per cent N) Period 5. Flour ration (1.29 per cent N)

7 H. H. Mitchell and G. G. Carman 189 TABLE IIConcluded. Period 6. Low nitrogen ration (0.025 per cent N). grn. gm. gm.?ng. mg.?ng.??zg. nzg. nzg.?w. 77x7. mg. Per cent * 22 9t * 30 13t * * 25 11t * * These figures represent the metabolic fecal nitrogen per gm. of food consumed and are used in estimating the body nitrogen in the feces in the intermediate periods from the amount of food consumed. t These figures represent the endogenous urinary nitrogen per 100 gm. of body weight and are used in estimating the body nitrogen in the urine in the intermediate periods from the average body weight. With each of the two groups of rats in this experiment, either the white flour ration or the whole egg ration was fed in two consecutive periods to see how close a check could be obtained in the estimated biological value of the food nitrogen. With Rats 221 to 225, inclusive, very close checks were obtained between these two periods of like feeding, the average biological value for the two periods being identical. With Rats 226 to 230, inclusive, a good check was also obtained with reference to the whole egg ration, in spite of the fact that with four of the five rats, the amount of food consumed was approximately 1 gm. more per day in the second period than in the first. The duplicate biological values for each of the rats are as follows: White flour nitrogen. Whole egg nitrogen. Rat No. Period 4. Period 5. Rat No. Period Average

8 Biological Values of Foods A summary of the biological values obtained for the ten rats in this test is given in Table IV. It is evident that the second group of rats utilized their dietary nitrogen consistently better than the first group of rats. The biological value for egg nitrogen pre TABLE Nitrogen Metabolism Data for the Second Group of Rats. III. g Body weights. 4 d. d c j s z Fr z F G Period 1. Low nitrogen ration (0.016 per cent N). Qm. Qrn. gm. mg. mo. mg Period 2. Flour ration (1.29 per cent N) Period 3. T Flour, egg ration (1.28 per cent N). 1 Peri{ 3C 1 6. Low nitrogen ration (0.025 per cent N). Body weights. d d 4 d.$ 2.$ r; d * G z g td d 8 Is F a 2 G Period 4. Egg ration (1.27 per cent N). on. Qnz. 0m. mo. mo. mo Period 5. Egg ration (1.27 per cent N) viously reported (3) was 93, very close to the average for the ten rats in this test In the last column of Table IV, the biological va,lue of the mixed ration has been estimated from the biological values found for the

9 H. H. Mitchell and G. G. Carman two rations separately and from the proportions in which they were combined. These estimated values may be taken as the values that would have been obtained for the mixed ration if no supplementary relation existed between the nitrogen compounds in white flour and the nitrogen compounds in whole egg. However, for each of the ten rats the estimated bio!opical value of the mixed ration is less than the value actually obtained, indicating that an appreciable supplementary relation exists between these two sources of nitrogen. As an average for the first group of rats the actual biological value obtained for the mixture was 7 per cent Rat No. TABLE Summary of Biological Values Obtained. 1 wtzp 1 Egg ration Average Average I 99 IV. Mixture. 7 Estimated values II no supplementing effect existed greater than the value to be expected if no supplementary action occurred. With the second group of rats the average increase was 11 per cent. II. Flour and Egg Albumin. The next experiments were designed to obtain information concerning the biological value of the nitrogen of egg albumin and egg yolk, separately, and each combined with white flour nitrogen in the proportion of 1 to 2. Although very constant values were obtained for egg albumin among the different groups of rats experi

10 192 Biological Values of Foods mented upon, an unaccountably wide variability existed among the values obtained for the egg yolk fed under analogous conditions. For a number of groups of rats giving consistent values for egg albumin, the values obtained for egg yolk varied all the way from 75 to 100 per cent, although the ration was con sumed readily and in adequate amounts and no evidence was apparent during the course of the experiments for the presence of any abnormal conditions. It was hardly thought worth while, therefore, to present such a series of discordant values for this fraction of the hen s egg. We hope to continue experimental work on egg yolk nitrogen. Ingredients. Dried egg albumin... L< whole egg... White flour... Cellu *... Sucrose... Cornstarch... Butter fat... Salt mixture... TABLE V. Composition of Rations White flour ration * A product obtained from the Chicago Dietetic Supply House, containing, by actual analysis, 37.8 per cent crude fiber and only per cent of nitrogen. The composition of the rat,ions used in these experiments is given in Table V. It will be noted that in place of a ration containing no nitrogenous nutrients, we have used in these experiments a ration containing a small amount of dried whole egg, the concentration being equivalent to about 4 per cent of crude protein. There has existed in this type of work some objection to the use of a ration conta,ining no nitrogenous materials in the initial and final periods. In such periods of nitrogenfree feeding, the animals invariably lose in weight, and, in the last days of experimental feeding, frequently leave considerable food residues, so that the average consumption of food per day for these periods has generally been distinctly less than for the intervening test periods. A

11 H. H. Mitchell and G. G. Carman 193 subnormal food consumption in these periods, in which the rat is standardized relative to the nitrogen of body origin in urine and feces, throws some doubt on the applicability of the results obtained to other periods in which the food consumption is distinctly larger. The nitrogen of dried whole egg is apparently completely digestible, and, in the low concentration used in this low eggnitrogen ration, is probably completely utilized in metabolism. When fed at twice the concentration used in this ration, its biological value has been found to average approximately 94 per cent. For a lower concentration, this value would presumably increase (4). Direct testimony on these points will be presented in the following section of this paper. Another slight departure in the makeup of these rations as compared with the rations heretofore used, is the inclusion in all rations of equal amounts of a source of roughage. We have used for this purpose a commercial product known as Cellu Flour, which is practically nitrogenfree and which contains about 38 per cent of crude fiber. The results of the nitrogen metabolism periods are given in Table VI, and the biological values domputed from them are summarized in Table VII. The biological values obtained for egg albumin nitrogen are very concordant with the exception of that for one rat, No. 263, for which an abnormally low value was obtained; the average values obtained for the four groups of rats fed the egg albumin ration are 86, 81, 81, and 82. The biological values for white flour nitrogen for the two groups of rats fed upon the white flour ration are not as concordant as the values obtained with egg albumin, the averages being 48 and 59. For the mixed ration, the individual values agreed satisfactorily, averaging 65 for the first group of rats, and 67 for the second group of rats. The computed biological values for the mixed ration, given in the last column of Table VII, were, in general, slightly lower than the values found experimentally, but in several cases the computed and found values were either the same, or the computed values were larger than the found values, this being especially true in the second group of rats, Nos. 260 to 264 inclusive. Possibly with this group of rats the high values obtained with the white flour ration were abnormally high, so that actually a greater supplementary

12 194 Biological Values of Foods TABLE VI. Nitrogen Metabolism Data. Body weights. 4 i 4 aa g j.$ ~ il Fr 8 g jj 2 Period 1. Low eggnitrogen ration (0.732 per cent N). Period 4. Flour ration (1.53 per cent N). ; Qrn. Qm. I Qrn. mg. mg. WZQ Period 2. Egg albumin ration Period 7. Low eggnitrogen (1.43 per cent N). ration (0.732 per cent N) l! ( ( ( * ( * 40 Period 3. Egg albumin, flour ration (1% per cent N) Period 1. Low eggnitrogen ration Period 6. Egg albumin ration (0.732 per cent N). (1.43 per cent N) * Because the fecal nitrogen determinations.. in these cases were lost, the average amount of fecal nitrogen per gm. of food consumed for the other three rats in the group was assumed to apply to these rats.

13 H. H. Mitchell and G. G. Carman 195 TABLE VIcontinued. Body weights. 4 d Body weights. d Period 4. Flour ration (1.53 per cent N). gm. Pm. gm. ml. ml?. nw. Qnz t t Period 5. Flour, egg albumin ration (1.48 per cent N.) Period 1. Low eggnitrogen ration (0.679 per cent N) Period ration gm I I I Low eggnitrogen (0.732 per cent N). mn. mg. mg. 9.0( 9 9.0( ( ( ; 15 I mo Period 5. Low eggnitrogen ration (0.679 per cent N) Period 3. Egg albumin ration (1.37 per cent N). t Since the fecal nitrogen determinations were lost for these rats, an average digestibility of the dietary nitrogen was assumed.

14 196 Biological Values of Foods TABLE VIConcluded Period 1. Low eggnitrogen ration (0.679 per cent N). vm. mn. om. 7nQ. m!j Period 2. Egg albumin ration (1.37 per cent N) Rat No Eggr;tliynmin m Qm Period 5. Low eggnitrogen ration (0.679 per cent N). om TABLE VII. Summary of Biological Values. Rat No. Zgg albumi ration. OnZ. 7.0! ( 8.9! 8.9: T mu. Mixed no ration ?w Average I Average

15 H. H. Mitchell and G. G. Carman 197 effect between the nitrogen of white flour and the nitrogen of egg albumin existed than the computed values for the mixed ration would indicate. In any case, however, the supplementing effect is comparatively slight between these two sources of nitrogen, quite distinctly smaller than the supplementing relation between whole egg nitrogen and white flour nitrogen. III. Flour and Milk. Combinations of milk and white flour are frequent in the preparation of pastries in human dietetics. Although it is known that marked supplementary relations exist between the nitrogenous compounds of milk and those of whole cereals, we have been Ingredients. White flour... Skim milk powder... Sucrose... Starch... Butter fat... Cod liver oil... Salt mixture... Agar... Dried whole egg... TABLE Composition VIII. of Rations. V b hite flour Milk ration. ration. Low nitrogen ration. Egg ration unable to find any experimental results demonstrating such a relation between the nitrogen of milk and the nitrogen of patent white flour. The composition of the rations in this experiment is given in Table VIII. The test rations were all designed to contain the usual percentage of nitrogen, namely from 1.2 to 1.3 per cent. In Period 1, a ration practically free of nitrogen was fed. In the final periods of the experiment, however, the urinary and fecal excretion on this nearly nitrogenfree ration was compared with the excretions on a ration containing a small amount of whole dried egg. The purpose of this test was to see whether, for all practical purposes, such a low eggnitrogen ration might not be used in place of the ration containing no known source of nitro

16 198 Biological Values of Foods TABLE IX. Nitrogen Metabolism Data. Period 1. Low nitrogen ration (0.022 per centn). g?n. gnz. om. ml. mg nw Period 2. Flour ration (1.29 per cent N) _ Pm _ Period 6. Egg ration (0.645 per cent N). gm Period 3. Flour, milk ration (1.29 per cent N) Period 1. Low nitrogen ration Period 4. Flour ration (1.29 (0.022 per cent N). per cent N) LO LOO * These figures are taken from the preceding period because, for Rat 241, the fecal nitrogen determination was lost, and for Rat 242, the food intake record was unsatisfactory. mg Period 7. Low nitrogen ration (0.013 per cent N) : : * : 9* ! 11 13

17 H. H. Mitchell and G. G. Carman TABLE IXConcluded. Period 2. Milk ration (1. 31 per Period 5. Egg ration (0.747 cent N). per cent N). gm. vm. gm. mg. ww. ml. gm. Pm. wk. mg. ml. WI Period 3. Milk, flour ration (1.29 Period 6. Low nitrogen ration per cent N). (0.013 per cent N) t This was a 6 day period. Rat No. Milk ration. Flour ration. Mixed ration TABLE X. Summary of Biological Values t Computed for mixed ration Average Average

18 200 Biological Values of Foods gen. This substitution was considered advisable, since such an egg ration would be consumed much more readily than the low nitrogen ration. The data of the nitrogen metabolism periods in this test are given in Table IX. For Rats 231 to 235, inclusive, the experiment was highly successful in every respect. For Rats 240 to 244, inclusive, however, in Period 4 in which the milk ration was fed, such an inadequate consumption of food resulted, leading to abnormally low biological values for the milk nitrogen, that the milk periods have not been reported with these rats. Even in a second period of milk feeding, the food consumption was not what it should have been, and abnormal biological values still were obtained. With these rats, therefore, the results of Periods 4 and 5 have been omitted from the table. A summary of the biological values computed from the metabolism data is given in Table X. For the first group of rats, milk nitrogen possessed an average biological value of 84, practically identical with that previously reported (4). In view of the abnormally low values obtained with Rats 240 to 244, inclusive, for milk nitrogen, another group of rats was given a low eggnitrogen ration immediately followed by a milk ration similar to the one used in the present experiment. The biological values calculated for these rats were 87, 87, 79, 88, and 86, averaging 85. It is not considered necessary to report the detailed nitrogen balance data on this test. The biological values for white flour nitrogen are somewhat higher for the first group of rats than for the second, the respective averages being 54 and 48. Similarly for the mixed ration, the values for the first group of rats averaged 73 and for the second group of rats, 69. The biological values to be expected for this mixed ration if no supplementary relation existed, averaging 64, are all distinctly lower for the first group of rats than the values actually found. If it may be assumed that for Rats 240 to 244, inclusive, the biological value of milk proteins was actually 84, the computed biological value for the mixture would average 60, as compared with an average value of 69 found by direct experiment. A comparison of the period in which a low eggnitrogen ration was fed, with the period immediately following, in which a ration

19 H. H. Mitchell and G. G. Carman practically free of nitrogen was fed, is of interest in determining whether the former ration can legitimately be substituted for the latter in standardizing rats in these protein studies. For both groups of rats the average excretion of fecal nitrogen was closely the same in the two periods; i.e., 1.61 mg. and 1.58 mg. per gm. of food. For Rats 231 to 235, the average excretion of urinary nitrogen on the egg ration was slightly lower than that on the nitrogenfree ration (12.7 mg; per 100 gm. of body weight as compared with 15.5 mg.), while for Rats 240 to 244 the reverse was true (12.7 mg. as compared with 11.1 mg.). It seems fair to conclude from this test, bearing in mind the extent of the biological variations involved, that the substitution of the egg ration for the nitrogenfree ration will give practically the same standardization of the rats. IV. Flour and Meat. The importance of the ordinary cuts of meat in the human diet depends to a large extent upon their high content of protein of high digestibility and superior biological value. However, it should be realized that different kinds of meat and even different cuts of the same meat may vary in this respect. The ordinary assumption that the protein in cheap cuts of meat is equal in nutritive value to the protein in expensive cuts of meat can only have reference to digestibility. Any assumption that they are equal with respect to the biological value of their nitrogen is quite unfounded. It is a matter of common observation that different cuts of meat differ in texture and particularly in tenderness. These differences presumably depend upon differences in the proportion of connective to muscle tissue. In all probability the nitrogen of connective tissue would possess a lower biological value than the nitrogen of muscle tissue. The extent of the chemical differences existing among the nitrogen compounds in different cuts of meat is illustrated by the following data obtained in this laboratory.1 Two cuts of meat were obtained from the same hog carcass, one a shoulder cut (picnic) and the other a tenderloin. From inspection it was evident that the shoulder cut contained more connective t.issue than the tender 1 These results were obtained by Mr. J. H. Longwell.

20 202 Biological Values of Foods loin. IJpon analysis the shoulder cut was found to contain 2.89 per cent nitrogen and the tenderloin 2.94 per cent. Of the total nitrogen, 23.5 per cent was watersoluble in the case of the shoulder cut, and 28.2 per cent in the case of the tenderloin, while of the soluble nitrogen 16.3 per cent was coagulable by heat in the case of the shoulder cut, and 21.8 per cent in the tenderloin cut. In another investigation of the same question, two different cuts of beef from two different animals were compared. The first cut was the tenderloin muscle from a young heifer in good condition. The second cut was the lower round (heel) cut from a bull. These two cuts probably showed as great a difference in content of connective tissue as can be found. The tenderloin showed no visible connective tissue, while the round cut from the bull was Ingredients. Patent white flour..... Dried veal... Dextrinized starch... Sucrose... Butter fat... Cod liver oil... Salt mixture... TABLE Composition I XI. of Rations. Low nitrogen ration Veal ration I White flour ration. evidently extremely fibrous. The tenderloin cut contained 3.41 per cent nitrogen, while the round cut contained 3.06 per cent. Of the total nitrogen, 23.2 per cent was watersoluble in the tenderloin cut, and 16.9 per cent in the round cut. Of the soluble nitrogen, 49.9 per cent was coagulable by heat in the tenderloin cut and 47.8 in the round cut. The first experiment dealing with meat was concerned with the biological value of the nitrogen of veal alone and with the supplementary relation existing between veal nitrogen and white flour nitrogen. The veal was obtained at a local market and contained no visible fat. It was ground, dried, reground, and passed through a sieve. During these operations it was evident that this particular cut, the location of which in the carcass was not known, contained a good deal of connective tissue

21 H. H. Mitchell and G. G. Carman 203 TABLE XII. Nitrogen Metabolism Data. Period 1. Low nitrogen ration (0.055 per cent N). ma gm gm. mg. mg. nzg Period 2. Veal ration (1.20 per Period 5. Low nitrogen ration cent N). (0.016 per cent N) Period 3. Flour ration (1.29 per cent N) Period 1. Low nitrogen ration (0.055 per cent N). _ Period 4. Flour, veal ration (1.30per cent N) gm. wk. mg. mg. ng , * 9 * Period 4. Flour, veal ration (1.30 per cent N)

22 204 Biological Values of Foods TABLE XIIConchded. Period 2. Flour ration (1.29 per cent N). om. Qm. QWL. mq. m0. 172Q. om , Period 3. Veal ration (1.20 per cent N). Period 5. Low nitrogen ration _ (0.016 per cent N). om. om. 7 nzq. nz : t ! t * The average value for the other three rats was assumed in these cases, since the fecal nitrogen determinations were lost. The composition of the rations used is given in Table XI. A ration practically free of nitrogen, instead of one containing small amounts of dried egg, was used in this particular test. The complete results of the nitrogen metabolism are given in Table XII. A summary of the biological values will be found in Table XIII. The biological values of white flour nitrogen were very similar for the two groups of rats in this test, averaging 49. The values for veal nitrogen, averaging 64 and 59, were much lower than those previously obtained for this meat. The relatively poor biological value of the nitrogen of this particular sample of veal was in all probability related to its high content of connective tissue. The nitrogen of the mixed ration, containing two parts of white flour nitrogen to one part of veal nitrogen, possessed practically the same biological value as was obtained for veal alone, the averages being 63 and 60. The biological values to be expected if no supplementary relation existed between the two sources of nitro 3 3

23 H. H. Mitchell and G. G. Carman 205 gen, are in all cases less than the values actually found, averaging 9 per cent less for one group of rats and 8 per cent less for the other. Rat No Average Average.... Biological of flour TABLE XIII. Summary of Biological value nitrogen. Biological value of veal nitrogen TABLE XIV. Composition of Rations. Dried egg* White flour... 0 Dried lean beef*... 0 Cornstarch Salts... 4 Butterfat Sucrose Cellu Flour... 4 * Etherextracted. Values. Biological value of nitrogen of mixed ration. Computed biological value of nitrogen of mixed ration White 5our ration. Beef ration The second experiment was concerned with beef.2 The rations used are described in Table XIV. The beef was prepared in the same way as the veal in the preceding test, except that it was f The metabolism data in these experiments were secured by Miss J. R. Beadles and Mr. P. M. Baldwin.

24 206 Biological Values of Foods TABLE XV. Nitrogen Metabolism Data. Body weights. 4 6 d.f: gd+zj;,; c) g g 2 ig Period 1. Low eggnitrogen ration (0.745 per cent N). gm om gm. ml. mg. mg Period 2. Beef, flour ration (1.41 eriod 5. Low eggnitrogen ration per cent N). (0.745 per cent N) , Period 3. Flour ration (1.35 per cent N) Period 1. Low eggnitrogen ration (0.745 per cent N). om Period 4. Beef ration (1.51 per cent N) Period 4. Beef ration (1.51 per cent N)

25 H. H. Mitchell and G. G. Carman TABLE xvconcluded. Body weights. 4 d i.c 2 $.; 3 ; z i;;!2 z 2 z Period 2. Flour ration (1.29 per cent N). gn *m. mg. rng. w&g Period 3. Flour, beef ration (1.41 per cent N) Period 5. Low eggnitrogen ration (0.745 per cent N). Qm Qm z mo. VW mo extracted with ether after drying and grinding, since it contained. considerable intramuscular fat. In this test the ra.ts received separately not only their daily allotment of 25 mg. of yeast vitamin (Harris), but also a drop of cod liver oil. The results of the nitrogen metabolism periods will be found in Table XV. The biological values are summarized and averaged in Table XVI. The white flour nitrogen in this test had an average biological value of 55, the two groups of rats giving very constant values in this respect. The beef nitrogen possessed an average biological value of 69 for both groups of rats, the individual biological values again being quite constant. The mixed ration, containing one part of beef nitrogen to two parts of white flour nitrogen, possessed an average biological value of 70 with one group of rats, and 76 with the other. Seven of the ten rats gave higher biological values for the mixed ration than for the beef ration itself. For both groups of rats in the beef test the estimated biological value of the mixed ration, on the assumption of

26 208 Biological Values of Foods no supplementing effect, was 60, so that the supplementing effect is represented by average increases in the expected biological value of 10 and 16. Although the biological value of beef nitrogen is less than that of whole egg nitrogen, egg albumin nitrogen, or milk nitrogen, the biological value of a mixture of two parts of flour nitrogen to one part of beef nitrogen possesses at least as high a biological value.as similar mixtures of white Aour and whole egg, egg albumin or milk. From the experiments reported in preceding sections of.&his paper, such a mixture of flour and whole egg was found to Rat No Average Average TABLE XVI. Summary of Biological Values. Beef ration. I Mixed ration. Found values. :omputed valuea. have nitrogen with an average biological value of 75; for flour and egg albumin, mixed in a similar fashion, the average biological value was 66; while for flour and milk the value found was 71. It appears that beef nitrogen, of all the kinds of food nitrogen tested in these experiments, has the most marked supplementing effect on the nitrogen of white flour. In experiments on the growth of rats on rations containing diff erent sources of nitrogen, Osborne and Mendel (5) conclude that egg or milk proteins supplement flour proteins more efficiently than do meat (beef) proteins. Their results are expressed in terms

27 H. H. Mitchell and G. G. Carman of gain in weight per gm. of protein consumed in a period of 4 weeks. With wheat flour contributing twothirds of the protein, and at a protein level of 10.3 per cent, the average result for wheat flour and egg powder was 1.80, for wheat flour and milk powder 1.90, and for wheat flour and meat powder 1.66, when an added source of vitamin B was included in the ration. However, the gain in weight per gm. of protein consumed is influenced by the amount of food consumed, since the greater this amount in proportion to the weight of the rat, the greater will be the amount of protein consumed above the requirements for maintenance and, therefore, available for growth. In the particular experiment under discussion, the food intake of the rats on the flour and egg, and flour and milk mixtures, was quite distinctly higher than the food intake on the flour and meat mixture. Also the variation among the four rats constituting any one of the four groups of rats on experiment was such that it is extremely doubtful that the differences between the averages possess any significance. In connection with the beef and flour experiment, some attempt was made to investigate the accuracy of the experimental technique employed in these protein studies, and to substantiate the assumptions upon which the methods of calculation are based. Some preliminary experiments performed several years ago inclicatecl that the rat rapidly adjusts itself to changes in the quantity or character of its nitrogen intake. In order to redetermine whether the usual period of preliminary feeding of 3 days duration was adequate to permit the establishment of equilibrium with the ration, Particularly with the low eggnitrogen ration used in the standardizing of all experimental rats, the following experiment was undertaken. Four rats were taken from the ordinary stock ration and put upon the low eggnitrogen ration used in this experiment. The urine was collected for each of these four rats anclitsnitrogencontent was determined daily. The results are shown in Table XVII. For two of these rats, Nos. 3 and 4, the endogenous level of nitrogen excretion was reached on the 2nd day. The adjustment was slower with Rats 1 and 2, but on the 4th or 5th clay the level can be assumed to have been attained. On the basis of these results, it was considered expedient to increase the period of preliminary feeding from 3 to 4 days. This was clorn in the experiment on

28 210 Biological Values of Foods white flour and beef, and will be adopted as a routine procedure in all future work from this laboratory. In the calculation of the biological value of dietary protein throughout these studies, the assumption is made that if the urinary nitrogen per 100 gm. of body weight on the standardizing ration varies from the first to the final period, the change really indicates a change in endogenous catabolism rather than an error in experimental technique. It was thought that some light could be thrown upon this question by determining the creatinine nitrogen in the composite collections of the rat urine in the initial and final periods on the standardizing ration. The method used TABLE Adjustment of the Urinary Excretion of Nitrogen to the Endogenous Level I Rat 1. mo XVII. Daily excretion of urinary nitmgen. Rat 2. Rat 3. Rat 4. m0. mo. mng for the determination of creatinine in the weekly composite samples of rat urine was an adaptation of Folin s well known method. As used, it gives values for total creatinine, inclusive of any creatine that might be present. The details of the method are as follows : Triplicate samples of 20 or 25 cc. each (depending upon the quantity of creatinine present) are measured out into 250 cc. weighed Erlenmeyer flasks. To one of these portions two drops of methyl red indicator are added and it is then titrated exactly to the neutral point with 5 per cent sodium hydroxide solution. The other two portions of urine are then neutralized by adding

29 H. H. Mitchell and G. G. Carman 211 the same quantity of NaOH. 20 cc. of saturated picric acid solution are added to these samples, which are then made up to a volume of 150 cc. with distilled water. After the addition of two or three small pieces of porous clay plate, the solutions are boiled gently over an electric hot plate, until the volume is slightly less than 20 cc. The flasks and contents are then cooled, placed on the balance, and distilled water added until the weight of the contents is 21 gm. In preparing the standard creatinine solution, 1 cc. of creatinine zinc chloride solution (containing 0.5 mg. of creatinine per cc.) is placed in a 100 cc. volumetric flask. 20 cc. of saturated picric acid solution and 1.5 cc. of 10 per cent NaOH are then added, and at intervals of 1 minute the same quantity of alkali is added to each of the urine solutions as prepared according to the procedure described in the preceding paragraph. After standing 10 minutes, the standard solution is made up to 100 cc., and, at intervals of 1 minute, the urine samples are transferred from the Erlenmeyer flasks into 100 cc. volumetric flasks and made up to the mark. The samples are then compared in the calorimeter with the standard creatinine solution, setting the standard at the 20 mm. mark. If the reading of the unknown is less than 13 mm. or greater than 27 mm., the determination is repeated using a smaller or a larger sample as necessary. The results of these creatinine determinations3 in the first and final standardizing periods are given in Table XVIII in mg. of creatinine nitrogen daily, and in percentage of creatinine nitrogen on the total urinary nitrogen. The average percentage of creatinine nitrogen in the first period was 8.55 for Rats 311 to 315, inclusive, and 8.09 for Rats 321 to 325. For Period 5 the averages were, respectively, 7.89 and The similarity in these percentages indicates that the creatinine nitrogen excretion hasvaried from Period 1 to Period 5 to much the same extent as the total urinary nitrogen on the low eggnitrogen ration. This fact may be taken to confirm the assumption that the urinary excretion on this ration, after a preliminary feeding of 4 days, constitutes a good determination of the endogenous nitrogen catabolism of the rats. For example, it will be seen from Table XV that the * The ereatinine determinations were made by Mr. R. L. Zimmerman.