The conditions established for obtaining in vitro pepsin digests were determined from feeding experiments with rats which indicated that approximately 30% of ingested egg protein nitrogen is absorbed before the chyme has reached the area of the intestine where tryptic activity is significant. Consequently, the quantity of pepsin used and the dura-tion of incubadura-tion in the in vitro procedure were adjusted to produce approximately 30% release of microbiologically available essential amino acids from egg protein. The quantity of amino nitrogen released when 1 gm of egg protein is digested with 25 mg of pepsin for 24 hours is almost 90 % of that which occurs when either the amount of pepsin or the incuba-tion time is doubled. With pepsin of the proper activity the condiincuba-tions are such that small variations in time, temperature, and quantity of enzyme will not cause significant variation in the PDR index. The activ-ity of the pepsin used in these experiments was U.S.P. 1-10,000. Use of 3 X crystallized pepsin resulted in a more rapid release of amino acids but did not change the pattern of amino acids made available.
Following the proposal of Mitchell and Block (9), whole egg protein was used as the reference protein for the PDR index of net protein utilization. These investigators reported that whole egg protein was almost perfectly utilized in digestion and metabolism for the growing rat. It was demonstrated in the present work that when the well-utilized standard egg protein was treated with digestive enzymes in a manner simulating the physiological sequence, then the pattern of amino acids changed as digestion proceeded. These^results suggest that the optimal pattern varies with the stage of digestion or position in the digestive tract.
In this respect, many investigators have*jfound that whole protein is better utilized in the diet than protein* jhydrolyzates or amino acid mixtures containing essentially equal quantities of amino acids (97-100).
Therefore, it appears that the most efficiently utilized pattern of amino acids may vary with the stage of digestion.
The patterns of microbiologically available essential amino acids present in enzymatic digests and in complete hydrolyzates of whole egg and casein are given in Table X. It can be seen that there are large differences in the proportions of amino acids liberated from the two proteins by pepsin digestion. These differences are considerably reduced when digestion is continued by treatment with trypsin, or trypsin and erepsin. For example, isoleucine represented 24.3% of the 9 amino acids measured in the pepsin digest of whole egg, whereas it was only 8.8% in
TABLE Χ COMPARISON OF THE PATTERNS" OF THE MICROBIOLOGIC ALLY AVAILABLE AMINO ACIDS IN ENZYMATIC DIGESTS AND IN COMPLETELY HYDROLYZED WHOLE EGG PROTEIN AND CASEIN Whole egg Casein Pepsin + Pepsin + Pepsin + trypsin + Pepsin + trypsin + Pepsin trypsin erepsin Total Pepsin trypsin erepsin Total Amino acid (%) (%) (%) (%) (%) (%) (%) (%) Leucine 33.4 24.6 21.2 17.5 57.0 28.4 25.2 20.5 Isoleucine 24.3 13.6 12.1 12.0 8.8 11.2 10.1 12.1 Threonine 16.5 14.4 13.0 10.6 18.6 13.6 12.8 8.6 Valine 7.4 12.9 14.7 14.4 1.6 8.2 11.5 15.1 Phenylalanine 7.3 11.6 11.3 12.0 7.2 9.1 10.8 11.5 Methionine 4.6 5.6 7.0 6.8 0.2 2.9 3.0 6.3 Tryptophan 4.3 3.9 3.5 2.9 6.0 3.1 3.2 2.4 Lysine 1.8 10.4 13.6 17.9 0.5 21.1 20.9 16.8 Histidine 0.3 3.0 3.6 5.8 0.1 2.3 2.5 6.6 ° Each amino acid value was calculated as the psrcentage of the sum of the 9 amino acids indicated.
the pepsin digest of casein. This ratio of 3:1 was reduced to almost 1:1 following treatment with trypsin. Similarly, the proportion of methionine in the pepsin digest of whole egg was 23 times as great as in the pepsin digest of casein. After tryptic action the ratio is diminished to only about 2:1. Leucine is present in much smaller proportion in the pepsin digest of whole egg than of casein; it is present in almost equal propor-tions in the tryptic digests of both proteins. When the proteins are hy-drolyzed to completion the proportions of the 9 amino acids in the two hydrolyzates are almost identical.
Melnick et al. (70) have reported that similar differences in the rate of release of individual amino acids occur after treatment of proteins with pancreatin; however, the results presented here demonstrate that when proteins are first treated with pepsin, as occurs under physiological conditions, the differences between proteins upon subsequent treatment with trypsin are considerably reduced. These data prompted the hypoth-esis that if appreciable absorption of the products of peptic digestion occurred, some of the differences in biological value between proteins of comparable total amino acid content could be accounted for by the diversity in pattern of amino acids released by pepsin digestion.
Using whole egg protein as the standard, the total essential amino acid patterns in proteins can be compared by computing "egg ratios" (11).
The integrated essential amino acid egg ratios of a variety of proteins are presented in Table XI. These values are calculated according to the method of Oser (11), as modified by Mitchell (42) to include tyrosine but omit arginine in computing the revised index (modified essential amino acid index, MEAA). In general, the MEAA index overestimates the biological value. This fact lends support to the hypothesis that much of the potential nutritional value of many proteins is lost by the release and absorption of disproportionate amounts of essential amino acids at an early stage in digestion. In the new index the pattern of essential amino acids obtained from analysis of the pepsin digest are combined with the amino acid pattern of the remainder of the protein to produce an inte-grated index—the pepsin digest-residue (PDR) amino acid index.
Comparison of the PDR amino acid index with the net protein utiliza-tion and the biological value of food proteins for the growing rat and for man is presented in Tables X I and XII. Values for the chemical score and the modified essential amino acid index of Mitchell (MEAA) are also given for comparison. It will be noted that the chemical score, based upon the limiting amino acid, always underestimates the biological value.
While the MEAA index gives values in closer agreement with the biolog-ical value, there are many cases where the correlation for the MEAA index is also poor. The PDR indices are found to be in close agreement
T A B L E X I
COMPARISON OF THE P D R INDEX OF FOOD PROTEINS WITH THEIR BIOLOGICAL VALUE AND N E T UTILIZATION FOR THE GROWING R A T
Net P D R / Chemi
Biological Digesti utiliza PDR digesti MEAA cal Protein0 value bility tion index bility index score
Whole egg 98* 99 97 100 101 100 100
Egg albumin 97b 100 97 95 95 96 84
Defatted egg 87b 97 84 83 86 93 75
(conVl)
Lactalbumin 84' 98 82 82 84 92 70
Soy flour 96 72 71 74 82 44
Casein, Labco 97 66 65 67 92 64
Brewers' yeast 6 6c' 93 61 61 66 72 36
White flour 52c 100 52 51 51 65 26
α The test proteins used in this study were obtained from the following sources:
dried whole egg, Blue Star Foods Co.; egg albumin, Emulsol Corp.; defatted egg, Viobin Corp.; lactalbumin (Labco), Borden Co.; soy flour (Nutri Soy), Archer-Daniels-Midland Co.; casein (Labco, vitamin free), Borden Co.; brewers' yeast, (U.S.P. XII), Nutritional Biochemicals Corp.; and white flour (Pillsbury's Best) Pillsbury Mills, Inc.
b Mitchell and Beadles (101).
c Mitchell and Block (9).
d Greaves et al. (102).
e Chick et al. (103).
' Mitchell (104).
T A B L E X I I
COMPARISON OF THE P D R INDEX OF FOOD PROTEINS WITH THEIR BIOLOGICAL VALUE AND N E T UTILIZATION FOR ADULT M A N
Net P D R / Chemi
Biological Digesti utiliza P D R digesti MEAA cal Protein value bility tion index bility index score
Whole egg 100··6 96 96 100 104 100 100
Egg albumin 91c 101 92 95 94 96 84
Soy flour 7 3d 90 66 69 77 81 44
Casein, Labco 68c 96 65 63 66 90 64
White flour 4 1d 97 40 49 51 64 26
"Murlin et al. (105).
6Murlin et al. (106).
'Hawley et al. (107).
<*Bricker et al. (108).
with the net utilization values of the respective proteins for the growing rat and for man. Therefore, the values obtained by dividing t h e ' P D R index by digestibility are closely correlated with the biological values
(net utilization/digestibility).
PDR Index/Digestibilit y MEAA Inde x
FIG. 1. A comparison of the biological values of selected food proteins with their respective P D R index/digestibility and M E A A index values.
The regression lines correlating the biological values with the MEAA indices and the PDR/digestibility values are shown in Fig. 1. The corre-lation coefficient, r, of the regression line for the PDR index/digestibility is only slightly better than that for the MEAA index since the latter index does, in general, show the proper order of value. However, the regression line for the PDR index/digestibility passes closer to the origin and shows a much smaller standard error of estimate, SE, indicating that
this index more accurately reflects the quantitative differences in biolog
ical value between the various proteins.