Biological Evaluation of Proteins

Biological Evaluation of Proteins

BY JAMES B. ALLISON Rutgers University, New Brunswick, New Jersey

CONTENTS I. Introduction, . . . . . . ............................................ 11. Evaluation through Nitrogen Balance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Protein Minima for Nitrogen Equilibrium.. . . . . . . . . . . . . . . . . . . . . . . . 2. Nitrogen Balance Index of Nitrogen Intakes.. . . . . . . . . . . . . . . . . . .

Page 155 157 158 . 161

1. Growth and Nitrogen Retention.. . . . . . . . . . . . 2. Protein Efficiency Ratios.. . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Protein Efficiency and Nitrogen Retention. . . . . . . . . . . . . 1. Depletion in Protein

....................................

Potency Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasma Albumin Regeneration.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repletion Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Repletion in Liver Protein.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Repletion in Body Weight ............................... V. Evaluation through Amino Acid Analysis.. . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Correlation between Amino Acid Composition and Biological Tests. . . 2. Some Causes of Disagreement.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . .......................................... 4. 5. 6. 7.

181 185 186 186 187 .189 192 192 193 195 196

I. INTRODUCTION Growt,h, reproduction, repair, maintenance, resistance to disease, and all of the faculties of the living system are correlated with the intake and utilization of foods. The foods are the raw materials which are needed to supply energy to the living machine and to construct and t o repair it, Dietary proteins, through digestion in the gastrointestinal 155

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tract, furnish amino acids, possibly polypeptides too, which are the raw materials needed t o build the body proteins of animals. These body proteins form the matrix of the living system; they are the catalysts, the centers around which the dynamic equilibria of life develop; they are the protein stores of the body. These stores are the proteins of the gut, the liver, the plasma, and other tissues of the body. There are no protein reserves in the sense that there are f a t or carbohydrate reserves but the body utilizes tissue proteins to maintain the nitrogen integrity of essential structures. The studies of Whipple and associates (1946) and Schoenheimer and Rittenberg (1940) emphasize the dynamic equilibria which exist within these stores so that one type of tissue protein contributes to the construction of others and food nitrogen entering into these equilibria loses its identity and becomes part of body nitrogen. The primary purpose, therefore, of dietary.proteins is to fill the protein stores of the body and their biological evaluation is the study of this purpose. Indeed, the biological value of a protein was defined by Thomas (1907) as the amount of nitrogen retained in the body of an animal, a concept which has been so ably developed by Mitchell (1944) and associates in their many excellent papers and reviews. These reviews form a background for this presentation of the biological evaluation of proteins, a presentation which will attempt to expand and supplement the previous surveys. A new phase of research on the nutritive value of proteins began when mixtures of amino acids replaced proteins in the diets of animals. Such studies were inaugurated by Rose in 1930, who demonstrated th a t a mixture of ten of the amino acids, namely valine, methionine, threonine, leucine, isoleucine, tryptophan, lysine, histidine, and arginine, must be included in optimum quantities in the diet for normal growth in the rat. An essential amino acid was defined, therefore, as one which could not be synthesized by the animal in sufficient quantities to meet the needs for growth (Rose, 1938). An amino acid may be essential for growth, however, but not be essential for all the faculties of the living system. It is now clear that the pattern of essential amino acids varies with the physiological state of the animal and with the species. The retention of nitrogen in the animal is a function of this pattern. Consideration will be given in this review, therefore, to the correlation between the amino acid composition of a protein and its nutritive value. The purposes of protein nutrition are filled, however, only if all the raw materials are present in optimum amounts. If carbohydrate and fat are missing, for example, amino acids of the diet are utiliqed to supply energy so t ha t the construction and repair of the living machine is reduced or interrupted entirely. Certain of the vitamins are active spots in the protein catalysts of intermediary metabolism. Absence of these vitamins

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157

interrupts the formation of the catalysts and the machine cannot function, Thus the biological evaluation of any food such as proteins must be done in the presence of all other foods, with the full realization of their interdependence. Unless otherwise mentioned attempts have been made to make protein the only dietary variable in the researches which will be discussed in the following pages. The simplest method used to evaluate nitrogen retention in a n animal is to determine the difference between nitrogen intake and nitrogen excreted. This difference, called nitrogen balance, shows whether a n animal is maintaining, losing, or gaining nitrogen. Evaluation through nitrogen balance will be elaborated in this review into its quantitative forms. These forms become most complicated when applied to the growing animal so that the concept of growth as a function of nitrogen retention will be examined. Growth of tissues can take place also in a n adult animal depleted in protein stores. These stores are reduced in malnutrition, and in their reduction the body loses the capacity to repair damage, to maintain barriers to destructive forces. Repleting the protein stores of the body is a form of therapy very important to medical practice. Dietary proteins may replete tissues differently than they support growth in young animals or maintain the nitrogen integrity of a n adult. This review will consider, therefore, the evaluation of dietary proteins through tissue regeneration.

11. EVALUATION THROUGH NITROGEN BALANCE Nitrogen balance is the difference between dietary nitrogen intake and nitrogen excreted in the urine and in the feces. If the nitrogen intake equals the total nitrogen excreted, nitrogen balance is zero and the animal is said to be in nitrogen equilibrium. If the nitrogen intake is greater than the nitrogen excreted, the animal has gained in nitrogen and is said to be in positive nitrogen balance. If the nitrogen intake is less than the nitrogen output, the animal is losing nitrogen from the body stores and is said to be in negative balance. Thus the term nitrogen balance ( B ) is used for the over-all accounting of nitrogen assimilation. This term is defined mathematically by the following equation:

B=I-(F+U)

(1)

where Z is nitrogen intake, F is fecal nitrogen, and U is urinary nitrogen. Positive nitrogen balances can be maintained on a mixture of so-called essential amino acids, demonstrating that the amino acids are the sources for proteins and other nitrogenous constituents in the body of an animal. If one of the amino acids, essential to maintenance of nitrogen balance, is eliminated from the diet the animal will go into negative balance and

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lose nitrogen from body protein stores. The expression “essential for maintenance l 1 is used advisedly because the terms “essential ” or “indispensable” were applied originally to amino acids not synthesized by the animal organism at a speed necessary to meet the demands for normal growth. Rose and coworkers (Borman et al., 1946) and Albanese (1947) point out, however, that this concept of an essential amino acid need not apply to other functions such as reproduction, detoxication, or even over-all maintenance of nitrogen balance in an adult organism. Lysine, for example, is an indispensable amino acid for growth in the young rat, but Mitchell (1947) has demonstrated that this acid is not essential for the maintenance of nitrogen equilibrium in the normal mature rat. Lysine, on the other hand, is required for maintenance of nitrogen equilibrium in the dog (Rose and Rice, 1939; Allison, Anderson and White, 1949) and in protein nutrition in adult man (Rose, 1938, 1947; Albanese, 1947). Mitchell suggests that synthetic reactions leading to the formation of lysine in a rapidly growing animal such as the rat may be inadequate for growth but adequate for maintenance. Frazier et al. (1947) have demonstrated lysine to be essential t o the proteindepleted rat when regeneration of new tissue takes place. The nitrogen balance method has been used recently with particular success by Rose (1947) in the determination of the amino acids essential for maintenance of nitrogen equilibrium in man. His results demonstrated that valine, methionine, threonine, leucine, isoleucine, phenylalanine, tryptophan, and lysine are essential to this maintenance. The exclusion of any one of these amino acids from the food was followed by It a pronounced negative nitrogen balance, a profound failure in appetite, a sensation of extreme fatigue, and amarked increase in nervousinstability.” The effect of omission of one of these acids is so immediate that there can be little, if any, stores of free amino acids in the body to overcome dietary deficiencies. Dietary deficiencies in essential amino acids are reflected rapidly, therefore, in the reduction of the nitrogen balance. The nitrogen balance method of evaluating amino acid mixtures has been developed by many workers into a measure of the minimum amount of protein necessary to maintain nitrogen equilibrium in animals or in man. 1. Protein Minima for Nitrogen Equilibrium

Melnick and Cowgill (1937) gave careful consideration t o the quantitative determination of the minimum amount of dietary protein nitrogen necessary t o maintain nitrogen equilibrium. They determined this amount of nitrogen in the dog by plotting nitrogen intake against nitrogen balance in the region of nitrogen equilibrium, interpolating to the point of exact equilibrium. Their determinations demonstrated quantitative

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159

variations in the ability of dietary proteins to maintain nitrogen equilibrium, variations associated with the presence and availability of essential amino acids. An acid hydrolyzate of casein, for example, in which the tryptophan was destroyed by the acid was shown to be incapable of maintaining nitrogen equilibrium in the dog no mattcr how much hydrolyzate nitrogen was included in the diet. When an optimum amount of tryptophan was added to the hydrolyzate, nitrogen equilibrium was maintained by feeding 150 mg. hydrolyzate nitrogen/day/kg. body weight. When an insufficient amount of tryptophan was added, 300 mg. nitrogen/day/kg. body weight was required to maintain the nitrogen equilibrium in the same dog. The minimum amount of nitrogen necessary to maintain equilibrium increases, therefore, as the content of an essential amino acid or acids decreases below optimum quantities. It is possible that the protein minima for nitrogen equilibrium arc always determined by the essential amino acid present in least quantity in the protein or made available in the smallest amounts to the animal organism. Risser (1946) , using Melnick’s and Cowgill’s techniques, measured the minimum amount of casein and fibrin required to maintain nitrogen balance in dogs, an amount which was labeled MPN. He found that fortification of casein with cystine reduced the M P N value and that addition of both cystine and methionine reduced the M P N value still further. Frost and Rivser (1946) have applied these nitrogen balance techniques in dogs for the evaluation of casein and fibrin hydrolyzates, variously supplemented by essential amino acids. These authors and others have checked their determinations of protein minima for nitrogen equilibria by feeding this quantity of nitrogen t o animals for many days. Kade et al. (1946) maintained nitrogen equilibrium for 21 to 25 days in dogs fed an acid hydrolyzate of casein fortified with tryptophan. Their data emphasizes the fact that dogs can be standnrdized to retain larger or smaller amounts of nitrogen. Cox and Mueller (1946) compared the relative efficiency of an enzymatic casein hydrolyzate and two mixtures of crystal amino acids in maintaining nitrogen equilibrium in protein-depleted dogs. At levels just sufficient to maintain nitrogen equilibrium, a mixture of ten essential amino acids and glycine promoted nitrogen retention somewhat more effectively than did the hydrolyzate. At a higher level the hydrolyzate was much more effective than the dmino acid mixture. The objection to the determination of protein minima for nitrogen equilibrium as a method of evaluating proteins is the great variation in minima from animal to animal. Melnick and Cowgill (1937) pointed to this variation, emphasizing the need to select animals of similar nitrogen needs if comparisons between one dietary protein and another are to be

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made. Their work was extended b y Allison and Anderson (1945) and Allison, Anderson, and Seeley (1946) to demonstrate that a linear relationship between nitrogen balance and nitrogen intake in the dog exists over the whole range of negative nitrogen balance, extending into the region of positive balance but eventually becoming curvilinear in that region. This relationship is idealized in Fig. 1 for data obtained by

NiTROGEN INTAKE

g./day/m2

FIG.1. Curves illustrating the relationshipbetween nitrogenbalance and nitrogen intake in the diet. They are produced from data obtained by feeding dogs one type of protein such as defatted, dried, whole egg. Curve a represents data obtained on a protein-depleted dog while curves b, c, and d illustrate data obtained on normal dogs with differing protein stores.

feeding one type of protein to dogs. Curve d illustrates data secured on a dog with saturated protein stores, the excretion of nitrogen on a protein-free diet being high, 4 g./day/m.2 body surface area. Approximately 5 g. dietary nitrogen/day/m.2 was required to maintain nitrogen equilibrium in this dog. Such an animal, with high protein stores, can be prepared by feeding a high-protein diet, the proteins being of good quality such as those found in whole egg or milk. Curves b and c represent data obtained on normal animals in which the protein stores are not quite so full, less nitrogen being required t o maintain equilibrium. These data could be obtained from experiments on dogs which had been prepared by feeding ordinary mixed diets. Curve a illustrates data obtained on a dog which had been depleted markedly in proteins by feeding the

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161

animal a protein-free diet for a period of time. This depleted animal required only 1 g. dietary nitrogen/day/m.2 body surface area to maintain equilibrium. The magnitude of the protein stores is reflected, therefore, by the amount of nitrogen excreted from the body tissues when the animal is receiving a nitrogen-free diet, being high when the stores are high and low when the stores are low. The excretion of nitrogen drops when an animal is placed on a protein-free diet, rapidly at first and then more slowly, reaching a relatively constant value in the depleted state. Thus the passage of an animal from a state of high protein stores t o low can be followed roughly by the decrease in excretion in nitrogen. The excretion of nitrogen on a protein-free diet reflects more strictly, however, the metabolism within the protein stores of the body and a change in the excretion may be correlated with a shift in this metabolism, which may or may not in turn be associated with markedly altered stores. Under any circumstances, however, the nutritive value of one protein cannot be compared to another by determining protein minima for nitrogen equilibrium unless the physiological states of the animals are known. The linear relationship illustrated in Fig. 1 suggests that there is a minimum excretion of nitrogen which must be met by dietary nitrogen to maintain nitrogen equilibria. These curves are described by the following equation:

B

=

K'I - Eo

(2)

where B is nitrogen balance, I is dietary nitrogen intake, and E e is the excretion of nitrogen during zero nitrogen intake. E,, the sum of the excretion of feces nitrogen, F,, and of urine nitrogen, U,, may represent a form of minimum nitrogen excretion. The slopes ( K ' ) are identical for lines b, c, and d, in the region of negative nitrogen balance, and are independent of the magnitude of nitrogen excretion or intake. These curves represent data obtained on so-called normal dogs. Under these conditions protein minima may vary but the slopes remain constant. 2. Nitrogen Balance Index of Nitrogen Intake It has been shown by Allison, Anderson, and Seeley (1946) that the slopes of lines such as those plotted in Fig. 1 decrease as the nutritive value of the dietary nitrogen decreases, being a function of the amount of nitrogen retained in the body of the animal. The slopes ( K 1 )have been called the nitrogen balance indexes of dietary nitrogen intake by these authors, since they measure the rate at which dietary nitrogen fills the protein stores of the body. These indexes do not vary in normal dogs even though there may be differences for nitrogen equilibria. The

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JAMES B. ALLISON

indexes do, however, increase in the depleted dog as illustrated by the increased slope of curve c (see Allison, Seeley, Brown, and Ferguson, 1947). As the protein stores of the animal are reduced the position of the curve becomes higher on the y axis and the linear relationship between nitrogen balance and nitrogen intake extends further and further into the region of positive balance. Thus the capacity to grow in nitrogen increases as the stores are depleted (Allison, 1948).

3. Digestibility and Absorbed Nitrogen It is, in many respects, more meaningful to plot nitrogen absorbed from the intestinal tract instead of dietary nitrogen intake so that the nitrogen balance produced is correlated with the dietary nitrogen entering the blood stream rather than the gut. The fraction of dietary nitrogen absorbed from the intestinal tract into the blood stream is called the true digestibility. Thus digestibility (D)is defined as:

D = -A

(3)

I

where A is absorbed nitrogen and Z is dietary nitrogen intake. The nitrogen absorbed from the intestinal tract ( A ) is calculated according t o the following equation:

A

=

Z

-

(F - F,)

=

N intake

-

fecal food N

(4)

where Z is nitrogen intake, F is total feces nitrogen, and F , is the excretion of nitrogen in the feces which comes from body, not food, sources, so-called “metabolic fecal nitrogen.” The quantity F , is usually determined by measuring the amount of nitrogen excreted on a nitrogen-free diet. The assumption is made that the excretion of body nitrogen is constant and does not vary with nitrogen intake. This assumption has been tested in the mouse by Bosshardt and Barnes (1946)) who found that there is a linear relationship between fecal nitrogen and nitrogen intake. Body fecal nitrogen (F,) could be determined, therefore, by extrapolation to zero nitrogen intake. Blaxter and Mitchell (1948) have determined metabolic fecal nitrogen in a similar way in sheep and other animals. The data of Bosshardt and Barnes indicate that F , is different on protein-free or low-nitrogen diets than under conditions of protein feeding. Thus incorrect values for absorbed nitrogen would be calculated from equation 4 if F , was measured on a protein-free diet. The excretion of fecal nitrogen on a protein-free diet is designated F , since it may differ from F,. The value F o is a variable, reflecting the composition of the

BIOLOGICAL EVALUATION O F P R O T E I N S

163

diet and physiological state of the animal. Depletion in proteins reduces, for example, the excretion of feces nitrogen, a reduction which is associated with decreasing protein stores. The value for F , is quite constant, however, under constant experimental conditions and for a given physiological state, and it is approximately equal to F , a t low nitrogen intakes in the dog. It is believed, therefore, that absorbed nitrogen can be calculated with some accuracy from equation 4 in the dog. This has been done and absorbed nitrogen instead of nitrogen intake has been plotted against nitrogen balance. A linear relationship between nitrogen balance and absorbed nitrogen was found in the region of negative nitrogen bal-

0

5 10 ABSORBED NITROGEN g./day/rn?

FIG.2. Curves illustrating the relationship between nitrogen balance and absorbed nitrogen in protein depleted dogs.

ance becoming curvilinear in the region of positive balance, curves such as those shown in Fig. 1 being obtained (Allison and Anderson, 1945). Similar curves were presented by Bricker, Mitchell, and Kinsman (1945) for data obtained in man and by Bricker and Mitchell (1947) for the rat. The data plotted in Fig. 2 illustrates the linear relationship between nitrogen balance and absorbed nitrogen in a group of protein-depleted dogs. Data from depleted animals were selected to illustrate the marked extension of the linear relationship into the region of positive nitrogen balance. The greater the degree of depletion the greater the positive nitrogen balance which can be produced by any one kind of protein. Theoretically, if all the protein stores of the body were filled, the animal

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J A M E S B . ALLISON

could not be put into positive nitrogen balance no matter how much protein was included in the diet. The circles in Fig. 2 describe data obtained while feeding wheat gluten t o the dogs and the angular symbols illustrate similar data obtained while feeding defatted, dried, whole egg protein. These data demonstrate th at less whole egg than wheat gluten nitrogen is required t o produco nitrogen equilibrium or fill the protein stores of the body. The slope of the line which is a measure of the rate a t which the protein stores are being filled is greater for whole egg than for wheat gluten, illustrating the correlation between the slope and biological utilization of the protein. Furthermore, each dietary protein has a n upper limit for utilization. Feeding more than th a t amount is inefficient and may put an excess burden on the organism to form and excrete organic compounds containing waste nitrogen. The mathematical expressions for the linear portion of the curve in Fig. 2 is:

B

=

K A - Eo

(5)

where B is nitrogen balance, A is absorbed nitrogen, and E , is the excretion of nitrogen when the nitrogen intake is zero. E o is the y intercept and K is the slope of the line. 4. Nitrogen Balance Index of Absorbed Nitrogen

The slope ( K ) of the,lines in Fig. 2 has been called the nitrogen balance index of absorbed nitrogen by Allison, Anderson, and Seeley (1915), because this slope is the rate of change of nitrogen balance with respect to absorbed nitrogen, a measure of the rate a t which the protein stores of the body are being filled. These authors defined the index as the tangent to any point on the curve giving the rate a t th a t point of absorbed nitrogen. I n the regions where the curve is linear the index measures t,he rate of greatest change in protein stores and, unless otherwise specified, will be calculated only for these regions. This calculation of the index for absorbed nitrogen, however, is not new. All determinations for biological values, where the fraction of nitrogen rctained in the body of the animal was being sought, have assumed 3 linear relationship between nitrogen balance and absorbed nitrogen. It will be shown later in this review that, if the amount of so-called body nitrogen excreted in the feces and in the urine is constant and separate from food nitrogen, the slopes of these lines are the fractions of nitrogen rrt:iined in the body of the animal; they are the biological values of the protcins as defined by Thomas and developed by Mitchell. It will be demonstrated, however, that K may be a function of rather than equal to the biological value.

165

BIOLOGICAL EVALUATION OF PROTEINS

For that reason the term nitrogen balance index of absorbed nitrogen is retained. The data recorded in Table I summarizes some of the digestibilities and nitrogen balance indexes for absorbed nitrogen which have been obtained for protein sources fed to adult dogs. The “net protein values” recorded in the last column were calculated by multiplying nitrogen intake (I) by digestibility ( D ) , by nitrogen balance index of absorbed nitrogen ( K ) and by the per cent protein in the source. Net protein TABLE I Digeatibilitiea, Nitrogen Balance Indezes of Absorbed Nitrogen, and Net Protein Values Obtained by Feeding Diflerent Proteins to Normal Adult Dogs --

Protein source

+

Casein methionine Fibrin hydrolyzate methionine Egg white Lactalbumin Lactalbumin hydrolyzate Bovine round roast Bovine rib roast Casein Casein hydrolyzate Wheat gluten lysine Chicken entrails Flounder entrails Flounder heads Wheat gluten a-Protein

+

+

--

Digestibility 0.95 0.97 0.95 0.97 0.98 0.99 0.99 0.95 0.99 0.95 0.95 0.96 0.83 0.95 0.82

Nitrogen balance index

Net protein valuc?

1.5

1.1 1. o 0.86 0.74 0.80 0.14 0.14 0.65 0.64 0.64 0.09 0.11 0.06 0.31 0.28

I .2

1.1 1 .o 1 .o 0.85 0.83 0.80 0.80 0.82 0.77 0.77 0.52 0.40 0.39

d

values, the use of which was suggested by Mitchell and Carman (1924), are functions of the retention of nitrogen by the animal, digestibility, and the concentration of protein in the source. These net values measure the rate of filling of the protein stores of the animal by the protein source; they do not evaluate the protein as such in the source. The low net protein value of bovine roast, compared to casein, results from the fact that the meat contains more water and other nonprotein constituents than the casein. Actually the meat protein is a bit superior t o the casein, as indicated by its higher nitrogen balance index for absorbed nitrogen. The effects on the index of improving the pattern of amino acid by supplementation is illustrated by its increase when lysine is added to wheat gluten or methionine to fibrin or casein. Nitrogen balance indexes increase as the animal is depleted in proteins.

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J A M E S B . ALLISON

This increase is illustrated by the data recorded in Table 11, the effect of depletion being more marked on the indexes of the poorer proteins. Thus, when there is greater need, more nitrogen is retained and conserved in the body of the animal. I n this connection, Silber, Seeler, and Howe TABLE I1 Nitrogen Balance Indexes i n Normal and Protein-Depleted Dogs

-

-

Nitrogen balance index

Protein source

Normal Egg white Casein Casein hydrolyzate Wheat gluten a-Protein

-

0.96 0.80 0.80 0.44 0.39

~

Depleted 1.2

0.93 0.92

0.70 0.73

-

(1946) found that depletion in proteins reduced the excretion of amino nitrogen following intravenous administration of amino acid mixtures. The variable nitrogen balance (B) has in it the variable absorbed nitrogen ( A ) , so th at a less complicated equation can be obtained by simplifying equation 5 to the following:

u = (1 - K ) A + u,

(6)

where U is urinary nitrogen excretion, K is the nitrogen balance index of absorbed nitrogen, and U o is the excretion of urinary nitrogen on a protein-free diet. This form of the equation is most useful in calculation of the indexes. It has been used, too, for the calculation of indexes of hydrolyzates fed intravenously into dogs (Allison, Seeley, and Ferguson, 1947). The value C', can be determined by feeding orally a source of protein with a nitrogen balance index of 1. Under these conditions, if the amount of protein nitrogen fed is sufficient to bring the dog into nitrogen equilibrium, the urinary nitrogen excreted will be equal to U , and the animal will not be depleted in proteins. Proteins, with indexes of 1, which have been used for this purpose are found in whole egg, lactalbumin, and egg white. Whole egg was used with particularly good success, the dog being fed this protein source once a week to determine U,, the remainder of the week being devoted to the determination of indexes for hydrolyzates fed intravenously. If a t any time U , dropped below 60 mg./day/kg. body weight the index tended to increase, becoming larger as U , decreased, reflecting the depletion in the protein stores of the animal (Allison, Seeley, and Ferguson, 1947).

BIOLOGICAL EVALUATION O F P R O T E I N S

167

5 . Nitrogen Balance Index a Function of Nitrogen Retention

The nitrogen balance index of a dietary protein has been defined as a function of the rate of filling of the protein stores of a n animal at any given nitrogen intake. This index can be expressed also a s a function of the amount of nitrogen retained in the body of the animal-nitrogen which is used t o construct and repair the living system. The following derivation illustrates the correlation between the index and nitrogen retention. The excretion of nitrogen on a nitrogen-free diet (En) is expressed as follows:

Eo = (Fo

+ Uo)

(7)

where F o is the excretion in the feces and U o the excretion in the urine. Equation 5 can be written, therefore, as:

KA = B

Since by definition: then :

+ F , + Un

(9)

B=I-F-U

KA

=

I

-

F

-

U

(8)

+ Fo + U o

(10)

Absorbed nitrogen is calculated as follows: A = I - ( F - Fo) (11) By substituting equation 11 in equation 10 and solving for K , we obtain the following relationship:

K =

A

-

(I'

-

Uo)

A

Equation 12 states that K is a function of the fraction of nitrogen retained in the body of the animal. K is equal to this fraction when U o represents the excretion of body nitrogen a t all levels of absorbed nitrogen. Under these conditions, U - U o would be equal to the excretion of urinary nitrogen derived from the food. Data are accumulating to demonstrate, however, that body nitrogen excretion is not the same at all levels of nitrogen intake. The conservation of body nitrogen through feeding nitrogen, for example, is illustrated by curve I11 plotted in Fig. 3 (Allison, Seeley, Brown, and Anderson, 1946). The open circles record data obtained by feeding the protein-free diet, the black circles, by feeding protein nitrogen. Cuscin was fed to secure the data plotted in curve I. Since the nitrogen balance index ( K ) for casein is less than 1 (0.8), the excretion of urinary nitrogen increased during the nitrogen-feeding period. Curve I1 illustrates data obtained by feeding a dog egg white protein. The index for

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JAMES B. ALLISON

this protein was 1, so that the excretion of nitrogen was not altered by feeding nitrogen. Curve I11 resulted from feeding egg white to a protein-depleted dog. Under these conditions the nitrogen balance was greater than 1, the urinary nitrogen excreted during the nitrogenfeeding period being less than during the protein-free feeding period. Thus, in the depleted dog, the excretion of nitrogen from the protein stores was less while the animals were receiving nitrogen than during the protein-free feeding period. It will be demonstrated later that proteindepleted animals are not always repleted uniformly in their protein stores, I

z

g 0

1.0

-c--LLQ* 0-* 0- -0ea-

a

I

t 2 >-

1.5

- (=Q,

a 4

z_

a:

3

1.0-

I

I

'*** I

P

1

m

I

FIG.3. Urinary nitrogen (g./day/m.* body surface area) excreted by dogs on a protein-free diet ( 0 )or a diet containing protein nitrogen (0). (Allison, Seeley, Brown, and Anderson, 1946.)

some stores being repleted more rapidly than others. It is not unreasonable, therefore, to find that during the feeding of protein the stores are filled in such a way that body nitrogen is conserved. Some of the protein stores, for example, which contribute to the excretion of nitrogen on a protein-free diet could mix with dietary nitrogen to build protein tissues of the body, thus reducing the excretion of body nitrogen, a form of internal supplementatiop. Dietary proteins such as casein to which an optimum amount of methionine has been added build u p protein stores in the normal dog in such a way that nitrogen balance indexes ( K ) greater than 1 are obtained. The only way indexes greater than 1 can be obtained is for dietary nitrogen to reduce the excretion of body nitrogen. When dogs

BIOLOGICAL EVALUATION OF PROTEINS

169

are receiving a protein-free diet they may be forced to draw upon tissue proteins to supply sufficient methionine for metabolic needs. The breakdown of tissue to supply the methionine on a protein-free diet could be reflected by a high excretion of urinary body nitrogen. If sufficient methionine is supplied in the diet this drain on the tissue may be relieved reducing the excretion of nitrogen. Much larger than optimum amounts of methionine, however, cause marked reduction in certain body protein stores in animals. Brown and Allison (1948), for example, fed a relatively large excess of methionine in a diet containing casein to rats. The animals lost nitrogen from their bodies but the proteins of the liver, kidneys, and plasma globulins were increased. Thus some tissues were being built up while others were torn down under the influence of the abnormal pattern of amino acids in the diet, the abnormal pattern increasing the excretion of body nitrogen. Addition of arginine with the methionine to the casein corrected, in whole or in part, these abnormal shifts of nitrogen in stores of the rat. These experiments and others suggest that the separation of food from body nitrogen is a difficult task. 6. Biological Value

The separation of food from body nitrogen is a necessary task, however, to calculate “ biological values ’’ of dietary protein as defined by Thomas and Mitchell. These values have been defined by them as the fraction of absorbed food nitrogen retained in the body of the animal, a fraction which is often calculated using a relationship such as equation 12. To make this calculation it is assumed that U , is equal to the excretion of body nitrogen a t all nitrogen intakes as well as at zero levels. The reality of the separation of body from food nitrogen, has been re-emphasized by Block and Mitchell (1946-1947). It is possible, as they point out, that certain reactions are irreversible and free from disturbances of dietary amino acids. Indeed, if all the protein stores of the body are filled or repleted uniformly during protein feeding it seems logical to expect a relatively constant minimum body nitrogen excretion. This excretion may shift, however, during experimental periods because of alterations in the equilibria between the protein stores of the body. The ideal experiment to determine “biological value” would be set up so that the physiological state of the animal is not altered. Various methods for measuring the excretion of body nitrogen without altering the physiological state have been discussed by Mitchell. Most of these methods involve a short-time measurement of nitrogen excretion on a protein-free or low-nitrogen diet containing a protein of good quality.

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JAMES B. ALLISON

The low-protein diet often used to measure the excretion of body nitrogen contains proteins of excellent quality and digestibility such as those in whole egg. If the nitrogen of the whole egg is completely retained to maintain the nitrogen integrity of the animal, the excretion of urinary nitrogen represents the minimum protein requirement for maintenance, a requirement that is taken as a measure of the excretion of body nitrogen. Recently, Murlin et al. (1946a) have measured the excretion of nitrogen in man fed a protein-free diet with the aim to calculate “biological values” for dietary proteins. They found that urinary nitrogen U , varied in man with: “(a) level of protein in the pre-experimental diets; (b) the position of the no-protein period in the series of periods; (c) the nature of the protein, called here ‘supporting protein,’ immediately preceding the no-protein, and its level of intake; and (d) conditions antecedent t o the supporting proteins which could affect the accrued nitrogen deficit to the beginning of the no-protein period,” the same factors which cause variations in experimental animals a n d emphasize the susceptibility of this measurement to changing nutrition and physiological state of the animal. Murlin and coworkers, however, were able to measure a value for U,in man by using short-term feeding experiments. They demonstrated a mathematical relationship between body weight and urinary nitrogen ( U o ) . Thus even though the excretions of nitrogen on a protein-free diet are variable, values which satisfy equation 12 for a given physiological state were determined. Whether or not the ‘‘ biological values” so calculated are the actual amounts of nitrogen retained in the body they are functions of th at amount; they are the rate a t which the protein stores are filled. These authors emphasized the necessity for performing the experiments in a definite order if comparisons were to be made between nutritive values of different proteins and amino acid mixtures. They pointed out, for example, th a t the depleting effects of a protein-free period promoted retention of a protein consumed immediately afterward, results similar to those found in animals (Allison ct al., 1946). They emphasized too, that values calculated from data obtained in the region of positive nitrogen balance may be low because of the curvilinear nature of the relationship between nitrogen intake and nitrogen balance in this region. Murlin and associates found that the “biological values’’ of mixtures of amino acids were always lower than the natural protein by 10 to 40%. They explained this lower value as due, in whole or in part, to the presence of unnatural isomers in the mixture of amino acids. Their data support the conclusion that “ th e unnatural isomers in these experiments, insofar as they escape deamination, belong t o this class of dispensable compounds; while insofar a s they are deaminated and can be recognized as contributingextra nitrogen to the

BIOLOGICAL EVALUATION O F PROTEINS

171

urea and ammonia fractions of the urine, they are in the same class as non-essential amino acids” (Murlin et al., 194613).

7. Biological Eficiency Swanson and coworkers (Willman e l al., 1947; Brush et al., 1947) used a standardized procedure for the determination of ‘(biological value” in rats, a procedure based on the classical nitrogen balance test as developed by Mitchell (1924) and which was designed to satisfy the requirements of equation 12. They obtained values, however, greater than 1, which they also recognized could not, by definition, be “biological values.” Thus they called their determination a measure of biological “efficiency ” rather than of biological “value.” They recognized that using these procedures the feeding of nitrogen reduced the excretion of nitrogen from the protein stores of the animal. They measured body nitrogenexcretion dy placing the rats on a nitrogen-low diet until they were somewhat depleted in proteins. This was done so that a constant value for excretion of nitrogen on a protein-free diet could be obtained for substitution in equation 12. It has been pointed out previously that this excretion decreases rapidly a t first and then more gradually until the excretion is essentially constant in a protein-depleted animal. This procedure undoubtedly depleted their rats in proteins so that during repletion the protein stores were shifted so as to reduce the excretion of nitrogen from the animal. Their methods of calculating and illustrating their results are shown graphically in Fig. 4. The body nitrogen spared by methionine, illustrated in Fig. 4, demonstrates that conservation can result from feeding single amino acids. Miller (1944) and Allison, Anderson, and Seeley (1946, 1947) observed a reduction in the excretion of urinary nitrogen when methionine was added to a protein-free diet fed to dogs, a reduction due primarily to decreased excretion of urea nitrogen. Johnson, Deuel, Morehouse, and Mehl (1947), on the other hand, found that the addition of DL-methionhe to low-protein diets of man did not decrease the excretion of urinary nitrogen. The addition of methionine provided a n excess amount in intermediary metabolism, the excess appearing in the urine as sulfate or as unchanged methionine. Similarly, Schwimmer and McGavack (1948) have reported that methionine did not conserve body nitrogen in man receiving a protein-free diet. The work of Cox et al. (1947) supplements these experiments demonstrating that the addition of methionine to casein hydrolyzate did not increase nitrogen retention in man while it did so in the rat and in the dog. Excess methionine, however, reduced the utilization of nitrogen in the rat (Swanson et al., 1946) and in the dog (Brown and Allison, 1947). These

.

172

J A M E S B . ALLISON

and other unpublished data indicate that the rat and the dog are more easily depleted in methionine than is man. Brush et al. (1947) demonstrated further, in the rat, that cystine, choline, and all the essential amino acids, except phenylalanine, valine, and tryptophan, exert some nitrogen-sparing action. The fact that single amino acids can reduce the 3. THE I0 ESSENTIAL l Y l N O ACIDS

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excretion of nitrogen supports the belief that these acids alter the metabolism of the protein stores. These acids may be in such deficiency while the animals are on a protein-free diet that protein reserves are utilized to supply them, the excess catabolism thereby contributing to the excretion of nitrogen. Conservation of this nitrogen could then take place when these acids are included in the diet.

8. Replacement Value Murlin and coworkers (1938) have introduced a method of interpreting nitrogen balances that utilizes the concept of a reference protein of

BIOLOGICAL EVALUATION O F PROTEINS

173

high nutritive value. Such a method is particularly useful in human nutrition where low-protein diets are unpalatable. In its original form this technique was simply a comparison of nitrogen balances of adults fed a given quantity of reference protein nitrogen of high nutritive value, such as the proteins of milk or egg, with the balances produced while receiving the same intake of nitrogen from the food under test. Mitchell (1944) has applied this replacement test successfully to rats using the following formula for calculation of the value ( V R ) . v n = 100

- Bz - Bi ___ I

x

100

where B1 is the nitrogen balance of the rat subsisting on the reference protein diet, Bz is the nitrogen balance of its pair mate subsisting on the protein under test, and I is the average of the nitrogen intakes of the two rats in the pair, intakes which should be nearly the same. Mitchell used this technique to study the effect of food processing on the nutritive value of the protein. In these studies B1 was the unprocessed while B* was the processed proteins. Any change from 100% indicated, therefore, the effect of processing on the protein.

111. EVALUATION THROUGH GROWTH Growth has in it most of the phases of metabolism which contribute toward the retention of nitrogen by the animal. Evaluation of proteins or amino acids through growth is, therefore, one of the most rigorous of all methods, integrating most of the functions of proteins into one measurement. The very breadth of this approach to evaluation, however, makes it difficult to standardize and to interpret (see reviews by Mitchell, 1944, and Barnes and Bosshardt, 1946). 1. Growth and Nitrogen Retention

The concept of nitrogen retention (biological value) was applied originally to the protein requirements for maintenance in adult animals but Mitchell and coworkers (Mitchell (1944) have extended this measurement to growing animals. The nitrogen retained in growing animals is the sum of the fractions of nitrogen retained for growth and for maintenance. Barnes, Bates, and Maack (1946) have shown that the relative proportion of dietary nitrogen entering into growth and maintenance varies markedly depending upon the amount and the nutritive quality of dietary proteins. They found that there was an increase in consumption of dietary proteins by the young rats as the amount of protein source in the diet was raised. The increased consumption of protein increased the rate of growth until a maximum was obtained. They used four

174

JAMES I). ALLISON

protein sources in their study, whole egg, soy flour No. I, soy flour No. 2, and wheat gluten. Soy flour No. 1 was processed to develop a highquality protein; soy flour No. 2 was processed to produce a n intermediatequality protein. T o determine the minimum maintenance requirements of young 60-g. rats, grams of protein gained was plotted against grams of protein absorbed, and the absorbed nitrogen a t zero body protein gain measured by extrapolation. The protein minima for nitrogen equilibrium were

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determined also in adult rats using the techniques of hlelnick and Cowgill (1937), described previously in this review. These minimum requirements expressed on the basis of body surface area were essentially the same for young and adult rats, providing a basis for the calculation of the amount of nitrogen utilized for maintenance during the 42-day growth period used in this study. The average amount of protein utilized for maintenance was calculated assuming that whole egg was 100% utilized for maintenance a t low levels of intake, an assumption that is supported by the work of Mitchell and Carman (1926), Murlin et al. (1938), and by the nitrogen balance studies in the dog. The per cent utilization of soybean protein for maintenance and growth is illustrated in Fig. 5 (Barnes et al., 1946). As protein consumption increases the fraction of absorbed proteins utilized for maintenance decreases, during which the fraction utilized for growth rises to a maximum and then falls. Thus there is a decrease in biological value as the food consumption increases. The biological value at 100% ’ maintenance

175

BIOLOGICAL EVALUATION O F P R O T E I N S

corresponds in magnitude to measurements made on adult animals in the region of negative or low-positive nitrogen balance. The relative participation of growth and maintenance in the establishment of the biological value is recorded in Table 111. Since the protein requirements TABLE 111 lielalive Participation of Growth and Maintenance i n Establishment of Biologicd Value with Diets of about 10% Protein”

1

Protein sourre

Whole egg Soyflour No. 1 Soyflour No. 2 Wheat gluten

Protein apparently absorbed

36 27 20 20

1

Biological value, %

Relative Relative participation participation of growth, of maintenance, yo %

99 67 64 35

77 65 59

26

23 35 41 74 _i__l_

*Barnes and Bosshardt (1946).

for growth and maintenance are different, these two factors should be measured independently and not in combination (Barnes et al., 1946).

2. Protein Eficiency Ratios The methods used most extensively for the determination of nutritive quality of proteins in growing animals are derived from the work of Osborne, Mendel, and Ferry (1919). These authors expressed the nutritive quality of a protein as a protein efficiency ratio (grams gain in body weight per gram protein eaten). The original method of Osborne el al. required that the maximum ratio be established by feeding varying amounts of protein, this maximum being taken a s the index to the nutritive value of the protein for growth. Few laboratories have followed this requirement to determine the maximum ratio but have fed a constant, usually a lO%-protein, diet. Barnes and Bosshardt (1946) have reexamined this method in rats, applying it also to mice. They concluded that “the common practice of employing a 10 per cent protein diet, regardless of the nature of the protein, will result in a considerable distortion of nutritive values, and the magnitude of the error will increase as the nutritive quality of the protein decreases.” Paired feeding is often used in place of ad libitum feeding to equalize the ingestion of the test proteins under comparative assay. These authors have pointed out, however, that if the restriction is severe in paired feeding some of the better protein may be wasted as fuel. Zucker et al. (1941) emphasized:

176

JAMES B . ALLISON

“the absolute daily intake of any factor fed a t a constant level in a diet supplied ad libitum increases steadily with growth, and the absolute requirement may increase more slowly than the food intake, or actually decrease.” These are variables which often have not been considered adequately in evaluating proteins in growing tests. Hartr, Travers, and Sarich (1947) have made a comparison of ( a ) litter mates us. randomly selected males, and ( b ) moderate restriction of food intake us. ad Zzbitum feeding in the determination of protein eficiencirs in rats. They concluded that preconditioning of the population of rats by feeding 10%casein ration for one week improves subsequent tests somewhat. Partial restriction of food intake (10 g. of casein ration daily) reduced the growth response on 10% casein by approximately 20% below the mean for ad libitum fed animals. The variance, however, for the animals restricted in quantity of diet was only about one-eighth to one-tenth that observed for animals fed ad Izbitum. Thus, Cood restriction improved the discriminatory capacity of a protein efficiency assay on rats. These authors, emphasized, however, the need to study further the physiological implication of partially restricting the food intake.

3. Protein Eficiency and Nitrogen Retention Mitchell (1942, 1944) has discussed thoroughly the shortcomings of the protein efficiency method for comparative assay of dietary proteins, shortcomings such :is those recorded above. He emphasized th a t the method assumes (1) that there is no requirement for maintenance and (2) that the protein content of the gains in body weight of growing animals is constant. Similarly, Albanese (1947), in a review of the amino acid requirements of man, pointed out: “ th a t composition of weight gain differs in quality, sometimes it may predominate in fluids and other times in fat or protoplasmic tissues. This discrepancy also raises questions as to the function and metabolic fatc of the retained nitrogen which fails t o appear as body tissue.” Probably one of the best examples of “the disturbing effect of a variable cornposition body weight gains in the interpretation of a protein nutrition experiment” (Mitchell, 1944) is illustrated by a n experiment reported from Mitchell’s laboratory (Be‘adles et al., 1933). These authors demonstrated that in paired-feeding tests, the protein of Limburger cheese is equal to th at of fresh milk curd in growth-promoting value, although it is less digestible and has a lower biological value. The explanation lay in the composition of the body weight gains, the gain achieved by the Limburger cheese ration being definitely lower in protein and higher in fat content than the gains produced on the milk curd ration. The following experiments on the growth of puppies illustrates also

177

BIOLOQICAL EVALUATION OF PROTEINS

the effect that the quality of protein can have on the type of body weight gain. Six 12-week-old beagle puppies were put on a protein-free diet (Allison and Anderson, 1945) to which 25% wheat gluten had been added. These puppies came from two litters, four from one and two from another. The daily caloric intake/kg. body weight over a period of 70 days are plotted in Fig. 6. The caloric intakes gradually decreased from a n initial value of approximately 225 cal./day/kg. to around 125 cal./day/kg. a t the end of 70 days of wheat gluten feeding. The dogs grew well during

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this period and appeared to be in good physical condition but a close examination demonstrated that they were soft and fat. Three of the litter mates were removed from the wheat gluten diet a t the end of 40 days and placed on an equivalent amount of defatted whole egg nitrogen. The caloric intake of the dogs fed whole egg protein immediately decreased below the intake of those on wheat gluten. The circles in the rectangle in Fig. 6 illustrate the caloric intakes of these dogs. The growth of the dogs receiving wheat gluten is illustrated in Fig. 7. All dogs received wheat gluten for 40 days, a t which time dogs 90 and 89 were placed on the diet containing an equivalent amount of whole egg nitrogen (indicated by circles). The loss in weight which accompanied the transfer to whole egg is illustrated by these data. This loss in weight, however, is not the result of a decrease in nitrogen. The data in Table I V demonstrate that the dogs fed whole egg were gaining nitrogen better than those fed wheat gluten. These data were obtained during two

178

JAMES B. ALLISON

TABLE IV Nitrogen Source, Intake, Excretion, and Gain during Two Weekly Periods of Growth in Dogs'

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Period I Dog No.

Nitrogen ~ource

__

86 87 89 90

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NitroNitrogen gen intake excreted _

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60.9 42.4 26 .O 32.0

-

53.7 29.0 11.3 10.7

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Nitrogen gained

Nitrogen intake

7.2 13.4 14.7 21.3

59.7 44.2 33.6 26.2

_

_

NitroBody nitrogen gen excreted gained ~

52.9 40.1 15.2 10.7

6.8 4.1 18.4 15.5

See Fig. 7. Valuefi given in g./week.

successive weekly collection periods, beginning on the forty-third day, 3 days after two of the four dogs had been changed to the whole egg diet. The average gain in nitrogen of the wheat gluten dogs during those collection periods was 7.9 g. The average gain in nitrogen in the dogs receiving whole egg over those same periods was 17.5 g.

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In a second experiment a group of three litter mates were fed the whole-egg diet. Two from this same litter and one beagle puppy of the same age as the others (12 weeks) were placed on the wheat gluten diet. The grams body weight gained per gram nitrogen intake was measured over a 3-week period for each puppy. These values recorded in the third column of Table V are essentially the same whether the dogs were fed wheat gluten or whole egg. The nitrogen intake, nitrogen excreted, and nitrogen gained the last 2 weeks of the experiment are also listed in Table V. These data demonstrated that the body nitrogen gained per

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179

BIOLOGICAL EVALUATION OF PROTEINS

TABLE V Nitrogen Intake, Excretion, and Gain by Dogs on dl-Day Diet of Wheat Gluten or Whole Egg

-

Nitrogen source 91 93 94 95 96 97

Wheat gluten Wheat gluten Wheat gluten Whole egg Whole egg Whole egg

-

g./week

Nitrogen excreted, g./week

Body nitrogen gained, G, g./week

G Z

62.8 61.3 50.2 66.9 45.3 48.1

51.1 47.8 43.6 31.8 22.4 25.1

11.7 13.5 6.6 35.1 22.9 23.0

0.19 0.22 0.13 0.52 0.51 0.48

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G . gain/g. intake, I ,

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nitrogen 9.6 8.0 8.6 10.6 8.3 8.0

gram nitrogen intake is much greater for whole egg than for wheat gluten, emphasizing the error that may be encountered in using growth curves to evaluate proteins in dogs. Hegsted and Worcester (1947), however, found a very high correlation between gain in weight and protein efficiency measurements in rats. They found too that protein efficiency is a function of weight gain rather than a characteristic of the dietary protein. They concluded that little additional information is gained in these growth methods by calculating protein efficiencies. In some studies on the utilization of proteins of white and whole-wheat flour, Chick, Copping, and Slack (1946) found that protein efficiency ratios reflected the retention of nitrogen in the body of animals as determined by cwcass analysis. Both good and poor correlation between growth and nitrogen retained are obtained, therefore, depending upon the nature of the growth of the animal. One of the fundamental questions which a growth method presents is: “What is normal growth?” This question has been answered, in part, by studies of Zucker et al. (1941, 1942) on the growth of the rat in relation to the diet. Their analysis emphasizes the fact that the shape of the growth curve has great significance in interpreting the response of the animal to different nutritional states. They found that the growth of rats on good stock diets is characterized by a “progressive decrease in gain for each successive time interval after weaning.” The following equation describes their data: log

w

=

-(k/t)

+ log c

(14)

where W is the weight of the rat and t is time. “Neither natural variation in size of the animal nor artificial stimulation of growth rate causes a deviation from the empirical formula of growth. While inherent size

180

JAMES B. ALLISON

of rats varies considerably, the slope of the plot (k of the formula) varies but little for each sex in albino rats. I n all cases where data for males and females are available, the ratio of k for males and k for females is constant within 3%.” Thus B norm is established for rats in which there are few if any restrictions to growth. This norm furnishes a basis for studies on deviations in animals fed diets suboptimal for growth. B y this knowledge (Zucker and Zucker, 1943, 1944; Zucker et al., 1941, discovered that there is a factor necessary for optimal growth associated with liver and certain proteins of good quality. Standard procedures are needed in growth methods so that results from different laboratories can be compared. Barnes, Rosshardt, and associates are developing such procedures applying them particularly to the mouse (Bosshardt et al., 1946). The mouse has advantages over the rat, such as smaller size, lower food consumption, and shorter test periods. These authors demonstrated the importance of a standard pretest treatment of the animals. They found that a t dietary protein levels giving maximum protein efficiency ratios, body weight gain is a true index of comparative body protein gains. They pointed out, however, that growth cannot be used as an absolute index of utilization of absorbed protein for body protein gain. They determined, therefore, the true dietary levels in the mouse for maximal utilization for growth in body protein, finding it to be approximately 6.5% of extracted whole egg, 8.0% of casein, and 25% of wheat gluten, corresponding to absorption of 3.5 g., 4.5 g., and 12 g., respectively. The calculation of absorbed proteins was made possible by the measurement of “metabolic fecal nitrogen” using the method described by Bosshardt and Barnes (1945).

IV. EVALUATION THROUGH TISSUE REGENERATION Animals do not possess adequate stores of free amino acids. For th a t reason, when they are placed on diets low in proteins there is a n immediate loss of body protein stores, a loss which reveals the dynamic equilibrium that exists between the proteins of the various tissues of the body. This concept of a dynamic equilibrium, developed by Whipple and associates a t Rochester, is described by them briefly in one of their recent papers as follows: “Food proteins yield the amino acids absorbed from the intestinal tract, and the amino acids are synthesized, in the liver cells (and elsewhere) into plasma proteins. These plasma proteins, (and amino acids) supply the protein requirements of the body cells. Normally, there is a considerable reserve of plasma protein-forming material (1 to 5 times the circulating mass), which reserve may be reduced by fasting, low protein diet, or plasma depletion. This depletion of protein reserves lowers the body resistance t o infection and intoxication. Thus, body

181

BIOLOGICAL EVALUATION OF PROTEINS

protein stores, protein production, and protein wear and tear are in a nicely balanced or steady state, a dynamic equilibrium.” 1. Depletion in Proteins

Depletion in proteins reduces the body protein stores, causes a shift in water balance, and impairs the functions of organs. Depletion in protein stores of the body is studied most often through changes which take place in the plasma proteins. Chemical fractionation of the plasma proteins from animals in the protein-depleted state proved that albumin was reduced markedly below normal while the globulins were reduced less or not at all (see Fig. 8). Zeldis et al. (1945) studied the effects of

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low-protein feeding on the plasma proteins as revealed by electrophoretis analysis. Their findings agree with those of chemical analysis that the albumin fraction is markedly reduced. They point out, however, that the degree of depletion of electrophoretic albumin is considerably greater than that of chemical albumin. Allison, Anderson, and Seeley (1946) presented data also to show that albumin :globulin ratios, determined by salt fractionation, are always greater than ratios determined through electropheresis. Chow, Allison, Cole, and Seeley (1945) found a decrease in electrophoretic plasma albumin and an increase in a-globulin in depleted dogs. Similar changes in plasma proteins were found to be associated with malnutrition, tuberculosis, or cancer in man (Chow, 1946). Chow et al. (1945, 1948) reported also that the yglobulin fraction is reduced below normal in depleted dogs,?a reduction accompanied by a n increased susceptibility to infection.

182

J A M E S B . ALLISON

The increase in the a-globulin fraction during depletion is largely the result of a fall in plasma volume and is not a real increase in total circulating globulin. The plasma volume decreases and the extracellular fluid increases as the plasma albumin falls in the depleted animals (Allison et al., 1947). As pointed out by Zeldis and coworkers, it is difficult to deplete the globulin fraction of the blood, the globulin being more like essential organ tissue protein and less like labile protein reserves. Indeed, when a dog is depleted so that the globulin fraction is markedly decreased, repletion is difficult, the animal often dying in the depleted state. Accompanying an increased lipide fraction in the blood of the depleted dog is impairment of liver function and the production of a fatty liver. I,i and Freeman (1946a, 1946b), for example, demonstrated the production of a fatty liver in dogs fed a 33%-fat, protein-deficient diet for 10 to 16 weeks. Protein depletion impaired hepatic dye clearance and caused an elevation of serum phosphate in dogs (Seeley, 1947). Kosterlitz (1946) demonstrated that liver cytoplasm is lost during fasting and in protein deficiency. The quantity of protein in the liver is rapidly reduced when rats are placed upon low-protein or protein-free diet (Addis, Pool and Lew, 1936a, 1936b; Kosterlitz and Campbell, 1945, 1945-1946, 1946; Harrison and Long, 1945; Brown and Allison, 1947). Other tissues are also effected by depletion in proteins. Li and Freeman (194Gc) found that the incidence of “peptic” ulcers was high in protein-deficient dogs. Armstrong and Haydee (1947) demonstrated that bone atrophy will develop in mature rats fed a protein-deficient diet. Corneal vascularization in rats resulted also from the absence of proteins or different amino acids in the diet (Hall et al., 1946; Niven et al., 1946; Berg et al., 1947; Sydenstricker et al., 1947). Thus corneal tissue like all other tissues of the body require a daily complement of essential amino acids to maintain their integrity. 2. Repletion in Plasma Proteins

The first experimental evidence that the repletion in plasma proteins was influenced by diet was presented by Kerr, Hurwitz, and Whipple (1918). These investigators found that depleted dogs regenerated plasma proteins more rapidly on a mixed protein diet than during fasting. Plasmapheresis was used to deplete the dogs. Their results were substantiated by Smith, Belt, and Whipple (1930), who concluded that part of the increase in plasma proteins, following plasmapheresis, was the result of replacement from a reserve supply of protein held in the body cells. The effect of diet, therefore, upon the plasma protein regeneration was superimposed upon this exchange. IIolman, Mahoney, and Whipple (1934) developed a more quantitative method for the study of the influ-

BIOLOQICAL EVALUATION OF PROTEINS

183

ence of plasma protein formation in dogs. They reduced the plasma protein concentration by plasmapheresis each day, t o from 3.6 to 3.9 grams per cent. The bleeding was Continued over a period of 4 to 6 weeks until a relatively small and constant amount of plasma was removed each day to decrease the plasma protein concentration to a constant low level, the small daily regeneration of protein being attributed to the 7% of vegetable protein in the diet. Test proteins were fed for 7 days to these depleted dogs and plasmapheresis continued daily to reduce the plasma protein concentration to the standard hypopr0,teinemic level. Repletion was expressed a s the “grams of new plasma protein resulting from the feeding of 100 grams of test protein.” Pommerenke, Slavin, Kariher, and Whipple (1935) improved the determination by introducing weekly plasma volume determinations so that the daily removal of plasma could be calculated more accurately. Weekly nitrogen balances were determined also. McNaught, Scott, Woods, and Whipple (193G) found that dogs tolerated a plasma protein concentration of 4.0 to 4.2 better than 3.6 to 3.9 grams per cent, and they maintained their hypoproteinemic dogs a t this level. This method has been developed into one of the best tools for evaluation of proteins in animals (Madden and Whipple, 1940; Madden et al., 1943). 3. Production Ratio In their recent studies the Rochester group have used the doubly depleted dog. Double depletion is produced by sustained bleeding of dogs fed a protein-free or low-protein diet with adequate iron. Thus the reserve stores of blood-protein-producing materials are depleted, levels of 6 to 8 grams per cent of hemoglobin, and 4 to 5 grams per cent of plasma being maintained. New hemoglobin in these dogs may be derived in part from plasma protein. Further studies by Whipple, Miller, and Itobscheit-Robbins (1947) have shown that doubly depleted dogs will continue to produce much plasma and hemoglobin for many weeks while being fed a low-protein or protein-free diet with abundant iron. Thus the blood proteins take priority over other tissue proteins, an example of the “ebb and flow” between tissue, organ, and blood proteins. Body weight was lost as tissue and organ proteins were transformed into blood proteins, the average dog tolerating this raiding of body tissue proteins for from 7 to 11 weeks. For every kilogram of weight loss there was 50 to 140 g. blood proteins formed, the weekly blood protein production ranging from 40 to 66 g. This heavy demand on body protein did not bring about a “premortal rise” in urinary nitrogen, however, the excretion remaining low, conserving body nitrogen. Whipple el al suggest that “ premortal rise ” in many experiments may be

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associated with a terminal infection leading to catabolism of tissue nitrogen. Whipple, Robscheit-Robbins, and Miller (1946) found that relatively incomplete protein like globin can contribute effectively to the protein stores of the doubly depleted dog and they suggest that “it must obviously be supplemented by the amino acids inadequately represented in globin, drawn from some reserve store, to produce cell proteins, or plasma proteins.” They pointed out that these depleted animals will use efficiently a variety Qf proteins, digests, and the growth mixture (Rose) of pure amino acids. The capacity of dietary nitrogen to replete in these experiments is measured in terms of a production ratio, which is the ratio between protein output and intake. Relative production of hemoglobin with respect to plasma protein is expressed as a ratio between plasma protein and hemoglobin. Hemoglobin (by vein) , dog hemoglobin (tryptic digest by veins), and horse hemoglobin (oral) contribute to the production of blood proteins in these depleted dogs. The utilization of dog hemoglobin is improved by methionine supplementation but not isoleucine (Robscheit-Robbins et al. , 1946a). The addition of isoleucine was found essential, on the other hand, to support normal hematopoiesis with human or beef globin in the rat (Orten, Bourque, Underhill, and Orten, 1945). Further observations by Miller and Alling (1947) demonstrated that when Dbisoleucine is “added to a fed supplement of methionine or methionine and cystine, the utilization of parenterally given hemoglobin nitrogen is even better than the sulfur-containing amino acids alone.” These data emphasize the difficulty of determining the “ideal” amino acid pattern by feeding experiments alone. These authors suggest that the ideal pattern be defined in terms of the total amino acid pattern of each animal. The response in blood protein output and urinary nitrogen excretion to mixtures of essential amino acids has been studied by RobscheitRobbins, Miller, and Whipple (1947). The average production ratio for the ten essential amino acids (Rose) was 19. When one of the essential amino acids was removed the ratio rose to 25. On a good dietary protein the ratio was 15. The authors suggest that good dietary proteins like egg and lactalbumin are utilized to replete protein stores in organs and tissues, this repletion being reflected in a moderate way in the blood proteins. The amino acid mixtures, on the other hand, with or without one of the ten essential acids, cause weight loss, and from this loss materials were derived which accelerated the formation of blood proteins more than when the animal was receiving a good dietary protein. They found that methionine, threonine, phenylalanine, and tryptophan, when eliminated singly from the growth mixtures of amino acids, resulted in a

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sharp rise in urinary nitrogen, a rise which was corrected when the amino acid was replaced in the mixture. “Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when those individual amino acids are deleted from the complete growth mixture of amino acids during 3 t o 4 week periods. Tryptophane and to a less extent, phenylalanine and threonine, when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output. Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output.” None of the amino acid mixtures, whether they contain the ten essential acids or not, will prevent weight loss in the doubly depleted dog. A good dietary protein such as casein, lactalbumin, whole egg, or liver protein in amounts of 150 to 250 g. protein/week will produce positive nitrogen balance, maintain body weight, and produce considerable amounts of hemoglobin and plasma proteins. Under comparable conditions, mixtures of essential amino acids will produce positive nitrogen balance and large amounts of hemoglobin and plasma proteins, but such mixtures will not maintain body weight. Miller, RobscheitRobbins, and Whipple (1947) suggest that some unidentified substance is present in the dietary proteins and absent in the mixture of amino acids which may be responsible for maintenance of weight in the doubly depleted dog. This unidentified factor may be similar to that reported by Womack and Rose (1946), who pointed out that protein may contain unidentified substances which, like arginine, are required for maximum increases in weight. 4. Potency Ratio Melnick, Cowgill, and Burack (1936) altered the plasmapheresis method of Holman, Mahoney, and Whipple (1934) in an attempt to increase the significance of regeneration of plasma proteins in proteindepleted dogs. They fed, for example, a protein-free basal diet instead of a low-protein diet so that the basal diet could not stimulate the formation of plasma protein nor act as a supplement t o any test protein added to it. They added the test proteins to the protein-free diet in “equal absolute amounts above the minimum each required t o establish nitrogen equilibrium.” They did this so that an equivalent amount of each test protein, over and above that needed to maintain nitrogen equilibrium, would be available for repletion of plasma and tissue proteins. Seeley (1945) and Allison, Seeley, Brown, and Anderson (1946) have demonstrated that the repletion of plasma proteins in proteindepleted dogs does not take place until the animal is in positive nitrogen

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balance and that the magnitude of repletion increases with the increase in positive balance. The potency ratio of a protein was expressed by Melnick el al. as the “ratio of (a) the amount of serum protein per week removed by bleeding, above that regenerated by the animal when eating the basal protein-free diet, to (b) the dietary protein increments.”

5. Plasma Albumin Regeneration Weech el al. (1935, 1938) developed a method for studying plasma protein regeneration in dogs, a method which did not involve plasmapheresis. They believed that the maintenance of hypoproteinemia in dogs by repeated plasmapheresis could invoke a repletion in plasma proteins which would be different than that associated with repletion from a lowered concentration toward normal. They depleted dogs mildly, therefore, for 3 weeks by feeding a low-protein diet, followed by 1 week of feeding of the basal diet plus the test protein. The total rise in plasma albumin was measured during the week of protein feeding and this value was used as a measure of the potency value of the protein. By following the increase in plasma albumin they measured the plasma protein, which reflects best the magnitude of protein stores of the animal. 6. Repletion Areas

None of these methods for the determination of repletion in plasma proteins lend themselves easily to a correlation between repletion of the various plasma proteins and the nitrogen balance produced. Seeley (1945) developed a technique so that: “the nitrogen excretion before, during, and after regeneration could be measured, utilization of the protein calculated, regeneration of plasma broteins determined and nitrogen balances established.” The dogs were depleted by feeding a proteinfree diet until the plasma protein concentration was between 4.0 and 4.5 g./100 ml. plasma, and essentially constant. After the plasma protein had been reduced, the dogs were fed sufficient protein to develop a positive nitrogen balance for 5 days, following which they were returned to the protein-free diet. Thus, the dogs were taken from a depleted state through a regeneration period, back to the depleted state. Since the plasma volume was not altered significantly the repletion in proteins could be expressed in terms of concentration changes. This was done by measuring the areas under curves during repletion. Seeley found that repletyon in plasma proteins in hypoproteinemic dogs increased with increasing positive nitrogen balance. Under the experimental conditions used, beef serum protein favored repletion of plasma albumin while casein favored the formation of both albumin and globulin. Similar results were reported using the plasmapheresis technique, by Holman

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et al. (1934) and by Madden and Whipple (1940). Thus, different patterns of amino acids produce different types of repletion in the hypoproteinemic animal. To study repletion of different types of plasma proteins more adequately and also to measure the total nitrogen utilized by the animal during the repletion process, the techniques of Seeley were altered to embrace a 30-day rather than a 5-day repletion period (Allison, Anderson, and Seeley, 1946). The effects of depletion and repletion on the plasma protein fractions are illustrated in Fig. 8. Using these techniques Chow et al. (1948) demonstrated that a casein hydrolyzate brought about an increase in total circulating albumin and globulins, the globulins being raised to above control values. Lactalbumin hydrolyzate, on the other hand, did not bring about an increase in any of the plasma globulins except the y fraction; most of the repletion being associated with the albumin fraction. The data recorded in Table VI demonstrate that repletion with whole egg, casein, and wheat gluten are correlated well with the nutritive value of these proteins. The gains in plasma protein nitrogen and in body nitrogen are approximately in the ratio of 1:30 previously reported by Elman (1947). AUo in the last column of this table is the difference between protein-free urinary nitrogen excretion in the repleted and depleted states. Since the urinary nitrogen excretion on a protein-free diet increases with an increase in certain labile protein stores of the body, a positive value for A U Omeans an increase in these stores. Values for AUo demonstrate that these labile stores were not increased until the body nitrogen gained was greater than approximately 80 g./m.* of body surface area. Wheat gluten did not replete these stores even after 30 days of feeding (see Fig. 2).

7. Repletion in Liver Protein Liver protein is lost rapidly during short periods of fasting or feeding of diets low in protein (Addis, Poo, and Lew, 1936a, 193613). Much of the background for studies on depletion and repletion in liver prdteins has been prepared by Kosterlitz (1944) and Kosterlitz and Campbell (1945, 1945-1946, 1946), who have presented data to show that the protein lost or gained represents actual liver cytoplasm, both particulate and interparticulate matter taking part. These authofs have called the fractions of liver cytoplasm which are easily lost “labile liver cytoplasm.” They found that the amount of labile cytoplasm present in the liver is directly proportional to the logarithm of the protein intake. They developed two methods for the assay of proteins. The first method measured the ability of the dietary protein t o maintain the labile liver cytoplasm when

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TABLE VI Data Obtained during 30 Days of Repletion i n Different D(

m

P

N intake

Fecal N

Urinary N

Body N gained

Plasma protein N gained

Avob

Whole Egg

174 175 189 192 336

25.7 26.1 27.7 25.7 38.6

55.3 62.0 60.8 55.3 161.3

93 .O 86.9 100.5 93 .O 136.1

2.9 2.1 3.1 2.9 5.2

+0.7 +0.3 +0.2 +O.l +0.6

75.8 96 .O 95.9 113.7

1.7 2.5 3.9 3 .O

+0.8 +0.6 +0.3

1.7 3.3 2.2 3.4

-0.4 -0.5 -0.2 -0.3

Casein

182 195 196 228

16.3 9.6 15.1 18.9

89.9 88.4 85.0 95.4

-

Wheat Gluten

136 298 333 352

5.1 12.0 21.2 16.9

98.5 215.3 249.1 256.4

32.4 70.7 62.7 78.7

-

Allison, Anderson, and White (1949). Values in g./m.* Difference between protein-free urinary N excretion in repleted and depleted states, g./day /m .a 6

rats were transferred from a stock diet to the test diet. The second method measured ability of the dietary protein to form labile liver cytoplasm after the rats had been on a protein-free diet for 4 days. Similarly, Harrison and Long (1945) developed a method for assay of dietary protein based on the regeneration of liver protein in the rat. The rats were standardized'for 1 week on a diet containing 20% casein and then fasted for 48 hours, after which they were fed for 4 days synthetic diets containing the'protein to be tested. Their data demonstrate that the regeneration of liver protein in the rat, following a fasting period, can be used as a method of assay. Gliadin was found to be superior to rein, both proteins promoting the regeneration of liver proteins better than gelatin. Casein and lactalbumin were essentially the same in ability to stimulate liver protein regeneration, but were both much

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superior to zein or gliadin. They found too, that the addition of methionine or cystine to a diet inadequate in casein increased the regeneration of liver protein. Recently Gurd, Vars, and Ravdin (1947) reported on another approach to the relation of nitrogen metabolism to the regeneration of liver protein. They achieved a large reduction in liver protein in rats by feeding a nonprotein diet for 14 days followed by hepatectomy. They determined nitrogen balances and new liver protein during a 14-day postoperative period while the animals were being fed the test proteins. They concluded that “ a close correlation appeared to exist between the amount of new liver protein formed and the amount of nitrogen saved or spared, irrespective of whether the rats were in positive or negative nitrogen balance. It is suggested that any factor, or summation of factors, which causes an over-all change in nitrogen metabolifim will affect the liver protein.” Vars and Gurd (1947) showed that “the rate of liver protein production was most rapid in the first two post-operative days, and more rapid in the protein-starved than in casein-fed rats during this period.” The degree of regeneration of liver protein, however, which occurred in protein-starved rats was greatly enhanced by feeding protein postoperatively for 14 days. This regeneration was proportional to the level and nutritive value of the protein fed. These studies on regeneration of liver protein emphasize the unique role of the liver in protein metabolism. The rapidity with which the so-called labile cytoplasm can be decreased or increased according to the demands of the body recalls the concept of a dynamic equilibrium between blood and tissue proteins which has been used so successfully by Whipple and associates. Indeed, liver protein may be increased while skeletal muscle protein is decreased. Brown and Allison (1947), for example, have shown that a large excess of methionine in a diet containing casein is associated with an increase in plasma globulin, and liver and kidney nitrogen, while there is a loss in skeletal-muscle nitrogen. Whipple, Robscheit-Robbins, and Miller (1946) found that relatively incomplete protein and amino acid mixtures have high production ratios for plasma proteins. They suggest that these incomplete amino acid mixtures are supplemented internally. Thus regeneration of any tissue protein of the body will be a function of shifts in the dynamic equilibrium in which they are a part, as well as a function of the pattern of amino acids introduced into the animal body. 8. Repletion in Body Weight Frazier, Wider, Steffee, Woolridge, and Cannon (1947) studied the dietary utilization of mixtures of purified amino acids in protein-depleted

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adult rats. The rats were placed on a low-protein depletion diet for approximately 3 months. Animals were chosen for repletion using uniformity in initial weights, percentages of weight loss (25-33%), and concentrations of serum proteins (4.064.85%) and hemoglobin (1 1.215.3%) as criteria of depletion (Wissler, Woolridge, Steffee, and Cannon, 1946). The nitrogen source fed t o these rats, other than traces in cornstarch and vitamins, consisted of either sixteen (ration A), ten (ration B), or nine (ration C) purified amino acids. The influence of these rations upon (a) weight and recovery, ( b ) food consumption, and ( c ) plasma and red-cell volumes and regeneration of serum proteins and erythrocytes were studied. The mixtures with sixteen amino acids were patterned after the amino acid composition of casein, the mixture with ten amino acids contained the essential amino acids for growth, and the mixture with nine amino acids lacked one of the essentials. These studies demonstrated that the protein-depleted rats gained weight rapidly and maintained good appetites when fed ration A, B, or the nine essential amino acids, histidine, lysine, tryptophan, phenylalanine, methionine, threonine, leucine, isoleucine, and valine. Arginine was not indispensable. Thus, the same nine amino acids essential for growth are also essential for gain in weight in these depleted rats. Omission of any one of the nine essential acids from the diet led to marked loss of weight and of appetite, the daily food consumption being reduced one-third to one-half. The effects of omission of tryptophan from ration A on weight and appetite are illustrated in Figs. 9 and 10. The weight loss in most cases was more marked than when the rats received a comparable low-protein level ration, a result which suggests that the unbalanced amino acid mixture has abnormal effects in nitrogen metabolism in the rat as in the dog. These authors believe that the omission of any one of the nine indispensable amino acids will produce metabolic disturbances altering the sense of well-being to such an extent that appetite fails. Good appetite and weight gains were maintained when one amino acid (lysine) was administered pnrenterally and others consumed orally. When, however, the lysine was replaced by a salt solution both the weights and appetites of the animals declined. There was no evidences in these studies for accessory polypeptide or other faqtors necessary for normal utilization of amino acids. Benditt, Humphreys, Straube, Wissler, and Steffee (1947) studied the effect of these diets on the synthesis of plasma and erythrocytes in protein-depleted rats. They found that the nine amino acids essential for appetite maintenance and weight gain are also indispensable in the hypoproteinemic rat for construction of serum protein and erythrocytes. A rat bioassay for proteins and protein digests has been developed by

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0

191

-“* \

5

10

DAYS

15

20

FIQ. 9. CAmparison of weight changes in protein-depleted rats fed ration A, containing sixteen amino acids, or ration A from which tryptophan had been omitted (Frazier, Wissler, Steffee, Woolridge, and Cannon, 1947).

DAYS

FIQ. 10. “Food consumption areas” in a rat fed ration A, containing sixteen amino acids, followed by ration A from which tryptophan had been omitted (Frazier, Wissler, Steffee, Woolridge, and Cannon, 1947).

Tomarelli and Bernhart (1947) which takes into consideration tissue regeneration after a short period of negative nitrogen balance. They compared the daily amount of casein nitrogen with t,he daily amount of other forms of dietary nitrogen required to maintain the weight of an adult rat for 1 week, after a preliminary period of 1 week on a protein-free

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diet. They conducted experiments to demonstrate that weight changes are valid criteria of comparative retention of nitrogen in the body of the rat.

V. EVALUATION THROUGH AMINO ACIDANALYSIS The preceding discussions on evaluation emphasize the importance of the presence of essential amino acids in the diet. Recently Cannon (1947, 1948) has emphasized the need to present the essential amino acids to the animal in the proper proportions and in the proper amounts, these proportions and amounts varying with the state of depletion of the various tissues in the body. To quote: ‘If it is true that essential amino acids are not stored individually in the tissues, even temporarily, to be utilized later for purposes of tissue synthesis, and if, moreover, the synthesizing mechanisms which fabricate complete tissue proteins must have available all necessary building stones which make up the particular protein to be synthesized, it would seem likely that the synthesizing mechanisms operate on a ‘perfectionistic’ or ‘all or none’ principle to the extent tha t if they cannot build a complete protein when it is required they will build none a t all. This would suggest also, t h a t the process of synthesis must be total rather than partial, and to be effective the synthesizing mechanism must have available and practically simultaneously all the essential amino acids in adequate proportions and amounts.”

He emphasized too, that this pattern of amino acids and its utilization may vary with the physiological state of animals. A protein-depleted rat, for example, will utilize two to five times the amounts of essential amino acids utilized by a control animal. Moreover the utilization of lysine and leucine are especially elevated, probably because of the greater need for these essential amino acids in the regeneration of skeletalmuscle tissue. 1. Correlation between Amino Acid Composition and Biological Tests The obvious relationship between retention of nitrogen of a dietary protein and the presence of these essential amino acids in the protein have given impetus to correlations between amino acid composition and nitrogen retention. Harte and Travers (1947), for example, in their analysis of human amino acid requirements for nitrogen balance, point out that the nutritive values of proteins are determined largely by the essential amino acid fraction. Mitchell and Block (1946) and Block and Mitchell (1946-1917) have made an extensive study of the correlation of the amino acid composition of proteins with their nutritive value. They showed that the amino acid composition of the protein reveals much concerning the nutritive value of a protein. A correlation between amino acid composition of proteins and biological tests was established by expressing as: “percentage deviations of the contents of the indis-

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pensable amino acids from the corresponding contents of a protein mixture, that of whole egg which has been shown to be almost completely utilized in the nutrition of the growing rat. With the results expressed in this way, it is possible to detect the limiting essential amino acid in each food protein, and even to assign to each food protein a chemical score that has a high degree of correlation with both the biological value and the protein efficiency ratio determined with growing rats.” This method of correlation gives promise in adult human nutrition but fails in poultry nutrition. 2. Some Causes of Disagreement

The causes of disagreement between any chemical and biological estimations are discussed by Block and Mitchell. It is obvious that, if amino acids are not made available to the organism through digestion or absorption, correlation between amino acid compositions of the protein and nutritive value must fail. Russell and coworkers (1946), for example, found that the availability of methionine in legumes changed with the variety and that the nutritive value of the protein could not be predicted from an analysis for this amino acid. In this connection, Melnick, Oser, and Weiss (1946) made an interesting study of the rate of enzymic digestion of proteins as a factor in nutrition. They suggested that: “for optimum utilization of food proteins all essential amino acids must not only be available for absorption but must also be liberated during digestion in vivo at rates permitting mutual supplementation.” They believe that differences in nutritive value of proteins can be caused by differences in the rate of release of individual amino acids in the intestinal tract. Their data indicate that, during digestion, methionine is liberated at a slower rate than leucine, or lysine in soy protein and that heat processing increases the liberation of methionine, improving the nutritive value of the protein. Recently, the effect of heat treatment of soybean oil meals on digestion and nutritive value of the proteins observed first by Osborne and Mendel (1917) has been investigated extensively. Evans and McGinnis (1946) and Evans, McGinnis, and St. John (1947) demonstrated that moderate heating of raw soybean oil meal increased its nutritive value for growing chicks while autoclaving a t 130°C. for 30 to 60 minutea decreased the value. They found that moderately heated soybean oil meals were more completely digested by the chick or by trypsin and erepsin in udtro than the raw or overheated meals. Similarly, Clandinin, Cravens, Elvehjem, and Halpin (1916) have shown that heating solventextracted soybean flakes in an autoclave a t 15-lb. pressure for 4 minutes resulted in a meal of high nutritive value in chicks. Continued heating

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for 4 hours reduced the nutritive value. Riesen, Clandinin, Elvehjem, and Cravens (1947) have studied the liberation of the essential amino acids by acid, alkaline, and pancreatic hydrolysis of raw, properly heated, and overheated soybean oil meal. Prolonged heat resulted in a decreased liberation of lysine, arginine, and tryptophan from acid hydrolysis. Proper heat treatment increased, while excessive heat treatment decreased the liberation of essential amino acids by pancreatic hydrolysis. Their data indicate that trypsin inhibitor is not the only factor involved in the improvement of the nutritive value of soybean meal by heat. They point out that: “the amino acid composition of proteins based upon acid hydrolysis does not necessarily indicate the extent of liberation of each amino acid from the protein by enzymatic digestion.’’ Mitchell and Block (1946) have reported a marked reduction in the digestibility and nutritive value of a cereal breakfast food submitted to puffing process. Eldred and Rodney (1946) found that heating casein could bring about changes which reduced the amount of free lysine released by enzymes although the total amount present in the acidhydrolyzed protein was the same in raw (7.5%) as in heated casein (7.7%). Block el al. (1946) found that baking and drying a protein food reduced the availability of its lysine to rats. Recognizing the need for proteins of high nutritive value in a palatable form to supply a highprotein diet to presurgical and postsurgical patients, they prepared a food in which proteins were mutually supplementary. This food consisted of white flour, sugar, egg white, lactalbumin, hydrogenated vegetable oil, dried yeast, molasses, and salt with a distribution of essential amino acids similar to whole-egg pr.,t?ins. The raw cake had a very high protein efficiency (3.3-3.8). Baking snd drying of the cake reduced the protein efficiency to values as low as 1.0. Toasting slices of the cake (100-130”) reduced the values to less than 0.7. Addition of lysine restored the nutritive value (3.2). It is suggested that heat may cause the free carboxyl groups of the dicarboxylic amino acids to react with c-amino groups of lysine to form a new peptide linkage which is resistant to enzymic digestion but not acid hydrolysis. An amino acid analysis of a dietary protein would fail to identify polypeptides or unknown substances other than amino acid combinations which may be essential for maximum utilization of the protein. Woolley (1941, 1946), for example, suggested that streptogenin may be involved in the nutrition of the mouse. Womack and Rose (1946) and Rose and Rice (1939) gave evidence for an unidentified substance in proteins which is required for maximum increases in weight in rats. Miller, RobscheitRobbins, and Whipple (1947) suggested that whole protein may contain

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something, not found in amino acid mixtures, which is responsible for the increased conservation of nitrogen and gain in weight in doubly depleted dogs. Correlations between amino acid composition and biological evaluations could fail, therefore, because of substances, other than amino acids, present in the protein source which would effect the retention of nitrogen by the animal.

VI. SUMMARY The following summary is not a complete survey of this review on the biological evaluation of proteins, but is an attempt to present briefly some of the more salient features. (1) The protein stores of an animal are the proteins of the tissues and body fluids; these stores are drawn upon to maintain the nitrogen integrity of tissues. ( 2 ) The protein stores can be maintained if the proper complement of amino acids is made available in the diet. Essential amino acids are those which cannot be synthesized a t sufficient rates to meet the needs of the body and must, therefore, be included in the daily diet. The amount of dietary protein needed to maintain the stores of the body is a function of the pattern of essential amino acids and also of the physiological state of the animal. The amount of nitrogen excreted from the protein stores decreases, for example, as the stores are depleted so that less dietary nitrogen is needed to maintain nitrogen equilibrium in a depleted than in a normal animal. (3) The relationship between nitrogen balance and either dietary nitrogen intake or absorbed nitrogen is linear in the region of negative nitrogen balance, becoming curvilinear in the region of positive balance. The slopes of the lines are functions of the rate a t which the protein stores are filled, being high when the nutritive value of the dietary protein is high and low when this value is low. These slopes have been called the nitrogen balance indexes of nitrogen intake and absorbed nitrogen, respectively. They are constant for any one protein in normal animals but they increase in value when the animal is depleted in proteins. ( 4 ) The linear relationship between nitrogen balance and nitrogen intake extends farther and farther into the region of positive balance as the animal is depleted in proteins showing a greater and greater capacity t o grow in nitrogen as the protein stores are reduced. Each protein, representing a different pattern of amino acids, produces a characteristic maximum positive nitrogen balance, the magnitude of which decreases as the nutritive quality decreases. (6) The nitrogen balance index for absorbed nitrogen can be equal to the fraction of dietary nitrogen retained in the body of the animal if the

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excretion of secalled body nitrogen is constant. The fraction of nitrogen retained in the body has been defined as the “biological value’’ of the dietary protein. Evidence is presented, however, to show that dietary nitrogen can alter the excretion of body nitrogen from the protein stores making the measurement of “biological value” a difficult task. (6) The relative proportion of dietary nitrogen entering into growth and maintenance of animals varies markedly depending upon the amount fed and nutritive quality of proteins. (7) The grams gained in body weight per gram of protein eaten (protein efficiency ratio) under constant experimental condition may be correlated with the gain in nitrogen in the body of the animal. Poor correlations occur, however, when the gain in weight is associated more or less with increases in fat, water, or tissue constituents other than protein. (8) Depletion in proteins results in the reduction of protein stores of the body such as the proteins of the gut, of the liver, plasma albumin, globulin, y-globulin, and proteins in other tissues. Thirty times as much nitrogen may be lost from body tissues as from plasma during depletion in proteins. The stores are not depleted uniformly so that a depleted animal needs a different pattern of essential amino acids in thc diet than the normal animal requires. (9) During repletion in proteins restoration of nitrogen is a function of the patterns of amino acids fed, different patterns producing various rates of repletion of the protein. An imbalance of amino acids can cause one type of tissue to be built up at the expense of others. (10) Lack of any one of the essential amino acids in the diet produces negative nitrogen balance, stops repletion, and causes the animal to lose appetite and become sick. (11) Correlations between the nutritive value of a protein and the amino acid composition may be excellent when the composition represents the pattern of amino acids which are absorbed into the animal. Digestive and other processes which prepare the dietary protein for absorption do not, however, always make available the pattern of amino acids revealed by chemical analysis. REFERENCES Addis, T., Poo, L. J., and Lew, W. (1936a). J . Biol. Chem. 116, 111. Addis, T.,Poo, L. J., and Lew,W. (1936b). J . Biol. Chem. 116, 117. Albanese, A. A. (1947). Advances in Protein Chem. 3, 227. Allison, J. B. (1948). Am. J . Med. 6, 419. Allison, J. B.,and Anderson, J. A. (1945). J . Nutrition 29, 413. Allison, J. B., Anderson, J. A,, and Seeley, R. D. (1946). Ann. N . Y . Acad. Sci. 47,245.

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