Measurement of the Nutritional Availability of Amino Acids in Foods

12 Measurement of the Nutritional Availability of Amino Acids in Foods

Downloaded by TUFTS UNIV on June 22, 2017 | http://pubs.acs.org Publication Date: January 1, 1966 | doi: 10.1021/ba-1966-0057.ch012

A. B. MORRISON and M. N A R A Y A N A RAO Nutrition Division, Food and Drug Research Laboratories, Department of National Health and Welfare, Ottawa, Canada.

The amounts of amino acids available to the body after ingestion of food proteins may be much less than indicated by the amino acid composition of the dietary protein. Chemical score data, based on amino acid composition, may therefore provide a misleading estimate of the value of processed food in ameliorating protein deficiency. Although a variety of chemical and biological procedures have been developed to evaluate amino acid availability, there is a continuing need for improved rapid procedures. A rapid chemical method for determining availability of the sulfur amino acids is particularly needed, since these amino acids often are limiting factors in human diets.

T

he classical investigations of Osborne and Mendel (58) led to a realiza tion that the nutritional value of proteins depends upon their content of the amino acids required for essential metabolic functions. It is not surprising, therefore, that in general the nutritive value of proteins can be predicted with reasonable accuracy from knowledge of their amino acid composition. This fundamental relationship between nutritive value and amino acid composition has provided the basis for a number of methods of evaluating proteins in foods. F o r example, Block and Mitchell (3) developed a procedure for determining the chemical score of proteins, based on their amino acid composition relative to egg protein, which is almost completely utilized by human subjects or the growing rat. A good correlation was found between the chemical score and nutritive value determined i n animal studies. More recently, McLaughlan et al. (40) found a high degree of correlation between simplified chemical score and protein efficiency ratio ( P E R ) values for 43 foods deficient in either lysine or the sulfur amino acids (Figures 1 and 2). I n studies on Canadian meals, Morrison (51) observed that 159 Altschul; World Protein Resources Advances in Chemistry; American Chemical Society: Washington, DC, 1966.

160

WORLD PROTEIN RESOURCES

prediction of per cent net dietary protein calories from simplified chemical score data gave values which agreed closely with those determined ex perimentally, if a prediction equation was used which takes into account the nature of the relationship between net protein utilization ( N P U ) and per cent protein calories. These data, summarized in Table I, indicate that the amino acids in the meals tested were available to the body.

100


Id QC Ο Ο CO

< ο χ ο ο LU

-Ο

LYSINE

DEFICIENT

PROTEINS r» + 0.965

ο.

- ·

METHIONINE DEFICIENT

CO

(+ CYSTINE) PROTEINS

r« + 0.834

1.0

2.0 PROTEIN

3.0

4.0

EFFICIENCY

RATIO

Courtesy Canadian Journal Biochemistry

Figure 1. Correlation between SCS and PER for 48 foods Foods apparently deficient in lysine plotted separately from those apparently deficient in methionine (plus cystine).

Miller and Donoso (47) concluded that i n mixed human diets the sulfur-containing amino acids often are limiting factors. Miller and Naismith (48) showed that the net dietary protein value of various human diets could be estimated accurately from their sulfur content. In subsequent studies, Miller and Donoso (47) proposed a chemical method for measuring nutritive value, based on the sulfur and nitrogen content of foods. They concluded that for practical purposes the protein scores of mixed diets are equal to 1000 S / N and calculated net dietary protein values from the following equation : N D - p Cals % = protein score Χ Ρ (54 - Ρ) (54 - Pm)

Altschul; World Protein Resources Advances in Chemistry; American Chemical Society: Washington, DC, 1966.

12.

MORRISON AND RAO

Amino Acids in Foods

161

where N D - p Cals % = % net dietary protein calories Ρ = % protein calories


Ρm = 400/protein score The validity of M i l l e r and Donoso's prediction equation is based upon a number of assumptions, one of which is that the amino acids in foods are available to the body. That this assumption may not always be valid is indicated by the studies of Miller and Carpenter (45), who reported that for a series of seven fish, meat, and whale meals, there was no correlation between the sulfur content and net protein utilization for rats, although the quality of the protein was limited by the sulfur amino acids. Cystine and methionine accounted for less than two thirds of the sulfur found in five of the seven samples. The digestibility of sulfur ranged from 53 to 9 0 % and the product of sulfur amino acid content and sulfur digestibility correlated closely (r = 0.93) with values for N P U . The studies of Miller and Carpenter (45) illustrate a principle which is becoming increasingly apparent—that the nutritional value of processed proteins cannot be predicted accurately from data on amino acid composi-

0

1.0

2.0 3.0 4.0 PROTEIN EFFICIENCY RATIO, Courtesy Canadian Journal Biochemistry

Figure 2. Correlation between SCS and PER for 43 foods after adjustment of (methionine plus cystine) scores



162


tion. It now is well established that, during processing, the amino acids of foods may be made partially unavailable to the body, with the result that the amounts of amino acids available for metabolic functions may represent only a small percentage of those present in the food protein. Continued population pressure on conventional food resources indicates that man will be forced to make ever-increasing use of highly processed protein foods, many of which now are used largely for fertilizer or animal feeds. In preparing such foods, careful attention must be paid to factors which influence the availability of the amino acids. It is not surprising, therefore, that many investigators have devoted considerable time and effort to develop procedures for measuring amino acid availability. This report summarizes available information on this important subject. N o attempt has been made to provide an encyclopedic coverage of the literature. The scope of the paper has been restricted to a discussion of recent papers which illustrate important principles. Table I.

Meal 2l

Prediction of ND-p Cals % from Chemical Score Data for Canadian Meals Protein Cal,

% 1 2 3 4 5 6

16.0 16.1 21.4 18.7 18.1 22.6

Simplified Limiting Chemical Amino Score Acids Found 52 80 79 60 83 72

L M +C M + C M +C L Tr

5.8 8.4 9.0 6.9 8.3 8.4

ND-p Cals % Calculated M M a

5.2 8.1 9.1 7.2 8.8 8.8

&P

b

6.8 9.9 11.3 8.4 11.0 10.6

(49).

b

Evaluation of Amino Acid Availability Chemical Methods. Perhaps the most useful chemical method developed thus far to measure amino acid availability is the procedure of Carpenter and Ellinger (7), which employs Sanger's reagent (61). It is based on the reaction of dinitrofluorobenzene ( D N F B ) with free amino groups in proteins. The e-dinitrophenyllysine (e-DNP lysine) released after subsequent hydrolysis is measured spectrophotometrically. This simple procedure proved useful for estimating available lysine in a range of animal materials (6), but was affected significantly by inter ference from α-dinitrophenylarginine when applied to autolyzed materials and from dinitrophenol arising from the breakdown of excess D N F B if carbohydrates were present (10). Furthermore, vegetable foods may give



12.

MORRISON AND RAO

163


rise to colors which interfere with the procedure. This interference was avoided by a modification (4) in which methoxycarbonyl chloride was used, but treatment with this reagent led to the unexpected development of a colored histidine derivative (8). Conkerton and Frampton (10) took advantage of differences in the absorbance of dinitrophenol at 360 ηΐμ in acid and alkaline media to correct for the dinitrophenol present in protein hydrolyzates. Other yellow substances such as dinitroaniline also may occur in the hydrolyzates of some dinitrophenylated proteins and introduce errors in colorimetric estimation of available lysine (21). The water-soluble imidazole-DNP histidine and O-DNP-tyrosine, which are colorless but absorb slightly at 360 πΐμ, are also likely to be found in dinitrophenylated protein hydro lyzates. In addition, the brown human pigments that occur in acid hydrolyzates of proteins may contribute to the error in estimating available lysine by the D N F B procedure. Interference in the D N F B procedure from a colored histidine derivative was reduced by a modification of the method (6) which involved removal with diethyl ether of the lysine-methoxycarbonyl chloride reaction product, and use of the resultant ' 'blank' ' value to correct for interference due to histidine. Suitable corrections were applied to allow for losses of D N P lysine during the acid hydrolysis, by making use of the recovery of an internal standard of DNP-lysine added to the samples prior to hydrolysis. Handwerck et al. (21) observed that the conditions of the chemical pro cedure partly protected D N P - l y s i n e from the destructive effect of acid hydrolysis in the presence of sugars. The protection was less effective with starch, in the presence of which the recovery of added D N P - l y s i n e was only 74%, compared with a mean recovery of 9 2 % obtained with animal products essentially free of carbohydrates (6). In an attempt to overcome errors in the colorimetric estimation caused by interfering materials, Baliga et al. (2) resorted to a chromatographic separation of e-DNP-lysine, followed by colorimetric estimation. The e-DNP-lysine was separated from the other water-soluble D N P - a m i n o acids on filter paper, using butanol-acetic acid-water (4:1:1) for irrigation. The e-DNP-lysine, which separates as a heavy band with an R value of 0.6, was eluted with IN HC1, the color density determined at 363 ηΐμ, and the value read from a standard curve. The reaction with D N F B was carried out at 40°C. as recommended by Sanger (61). Attempts to use higher temperatures in hope of shortening the reaction time gave lower values, probably due to slow destruction of the D N P derivative. Raghavendar Rao et al. (60) successfully used ion-exchange resin chromatography for separating e-DNP-lysine quantitatively from other yellow and brown components in protein hydrolyzates. The column was developed with a mixture of methyl ethyl ketone and SN aqueous H C 1 . Calculations of available lysine were made from measurement of absorbance f



164


at 435 ηΐμ. The authors claim that the method lends itself to routine analysis, since one operator can carry out eight determinations simultaneously. Carpenter's procedure using Sanger's reagent has been widely used by various workers for estimating available lysine in vegetable and animal products. There is a need for further modifications of the procedure for use with foods rich in carbohydrates to reduce their destructive effect on DNP-lysine during acid hydrolysis. More recently, Mauron and Bujard (41) suggested guanidation as an alternative approach for determining available lysine in foods. Guanidation with o-methylisourea transforms lysine molecules with a free e-amino group into homoarginine, which is stable during acid hydrolysis. The sample containing the protein is made to react with o-methylisourea, and the guanidinated proteins are precipitated with trichloroacetic acid and hydrolyzed with 6N HC1. The homoarginine present then is estimated by column chromatography. I n lyophilized and spray-dried skim milk powder, about 9 5 % of total lysine was converted to homoarginine and was therefore considered "available." I n peanut flour, the homoarginine value was slightly lower than the corresponding €-DNP-lysine value, but i n soybean flour it was higher. F r o m the limited data reported by Mauron, it is difficult to appraise the method fully, except to indicate that it appears promising. M o r a n et al. (50) used the reaction of the dye Orange G with basic amino acids (16) to determine the availability of free amino, imidazole, and guanidyl groups of proteins. They noted that the dye-binding capacities of soybean meals heated for varying lengths of time were closely related to the growth of chicks fed the meals. Ascarelli and Gestetner (1) also found that the dye-binding capacity of soybean meal was a good measure of its biological value. I n other studies, however, we could find no correlation between the ability of Canadian meals to bind Orange G and the nutritional value of the protein contained therein, as determined in the growing rat (51). Suitable chemical procedures to evaluate the availability of sulfur amino acids have not been developed, although the need for them is obvious. It is possible that advantage may be taken of the reaction between C N B r and the free terminal methionine residues in a peptide chain (18). Cyanogen bromide can cleave methionine peptide bonds in proteins, producing cyanosulfonium derivatives of methionine, which under acidic conditions yield homoserine lactone. This substance can be deter mined colorimetrically. It is conceivable these reactions may serve as the basis for a rapid chemical procedure for determining available methionine. Enzymatic Methods. I n vitro enzymatic studies have demonstrated clearly that amino acid availability and amino acid content of foods may differ markedly. Comparative measurements of the enzymatic and



12.

MORRISON AND RAO


165

chemical liberation of a number of amino acids have been made by numerous investigators, including Evans and Butts (14) using soybean protein and Hankes et al. (22) using casein. Evans and Butts (14) demonstrated that autoclaving soybean protein with sucrose for 4 hours did not seriously affect the in vitro enzymatic release of phenylalanine, threonine, leucine, isoieucine, and valine. Under the same test conditions, however, 8 4 % of the lysine and 4 1 % of the methionine were not released by enzymatic digestion of the protein-sucrose mixture. Approximately half of the loss of lysine was attributed to destruction and half to the formation of bonds within the protein molecule which resisted attack by proteolytic enzymes. The deterioration of methionine appeared to be caused by the formation of enzyme-resistant linkages within the protein molecule. In the study of Hankes et al. (22), casein was autoclaved for 20 hours i n the absence of carbohydrates. The rate of release of lysine by enzymes was markedly reduced by the heat treatment, whereas other amino acids were not so severely affected. In vitro analytical procedures for determining amino acid availability based on enzymatic release of amino acids from proteins have been deveoped by Sheffner et al. (64) and Mauron et al. (43). Sheffner et al. (64) studied the relationship between the pattern of amino acids released by digestive enzymes and the biological value of food proteins. A n amino acid index was described which attempts to take into account the physiological availability of amino acids during digestion. The index combines the pattern of essential amino acids released by in vitro pepsin digestion with the amino acid pattern of the pepsin-resistant residue to produce an integrated index termed the pepsin digest residue ( P D R ) amino acid index. P D R index values were closely correlated with N P U values for a variety of proteins. Division of the P D R index by the digesti bility coefficient of the respective proteins yielded values which accurately predicted the biological value of the proteins studied, as determined by nitrogen balance techniques i n the rat. The conditions established by Sheffner et al. (64) for obtaining in vitro pepsin digests were determined from feeding experiments with rats, which indicated that approximately 3 0 % of ingested egg protein nitrogen is absorbed before the chyme has reached the area of the intestine where tryptic activity is significant. The quantity of pepsin used and the duration of incubation in the i n vitro procedure therefore were adjusted to produce approximately 3 0 % release of microbiologically available essential amino acids from egg protein. W i t h pepsin of the proper activity the conditions are such that small variations i n time, temperature, and quantity of enzyme did not appear to cause significant variation in the results. Although Sheffner's procedure appears to give results which agree with those found in the rat, the amount of work involved in the method renders it unacceptable for routine evaluation of available amino acids in foodstuffs.


166



The procedure of Mauron et al. (43) is concerned with measurement of only three essential amino acids—lysine, methionine, and tryptophan— the amino acids which are most likely to be limiting i n foodstuffs. Briefly, the method is as follows: The sample is first dialyzed against tap water to eliminate substances of low molecular weight and then digested in the dialysis bag with pepsin at 37° at p H 2. After 15 hours of pepsin digestion the sample is digested for 24 hours with pancreatin at 50° and p H 7 to 7.5. The dialyzed fractions containing the amino acids are siphoned every hour and analyzed by appropriate procedures. Tryptophan and methionine are determined colorimetrically, lysine with a specific decarboxylase, and amino nitrogen gasometrically. This method has been extensively used by Mauron et al. (42, 4$) for determining the availability of amino acids in processed foods of vegetable and animal origin and appears to provide useful information. In Vivo Methods. Although in vitro results are of value in providing information on the release of amino acids, they obviously cannot be used directly as an index of availability in the intact animal. As a result, many investigators have used in vivo procedures to measure the availability of amino acids i n foods. Schweigert and Guthneck (20, 62, 63) used the growth of protein-depleted rats and fecal excretion of amino acids to determine the availability of lysine and methionine i n foods. Purified rations containing oxidized casein or sesame meal plus diammonium citrate and amino acids were used as basal diets deficient in methionine or lysine, respectively. The weight gains of protein-depleted rats receiving the basal rations plus graded levels of pure lysine and methionine were compared with those of animals given equivalent amounts of these amino acids in foods to provide an estimate of the availability of the protein-bound amino acid. The reproducibility of results obtained with different levels of the test product indicated that this procedure could be used to study the utilization or availability of amino acids from foods. Since Schweigert's early studies, growth procedures using rats or chicks have been used by many investigators to determine the availability of amino acids in foods. Factors influencing availability values obtained from growth studies have been reviewed by Harper and de Muelenaere (23), and are therefore not described here in detail. Kuiken and L y m a n (27) estimated the availability of the ten essential amino acids in different foods by measuring the excretion of amino acids in the feces. The amounts of endogenous amino acids in the feces were calculated from control periods during which the animals received a low-protein diet containing small amount of egg protein. Using this technique, Kuiken (26) later demonstrated effects of processing on the


12.

MORRISON AND RAO


167


availability of essential amino acids in cottonseed meal. Watts et al. (67, 68, 69) evaluated amino acid availability in human subjects following a procedure similar to that of Kuiken and L y m a n (27). The interpretation of any study involving the amino acid content of feces is limited by the inability to measure the extent of degradation and synthesis of amino acids by intestinal bacteria. Intestinal microorganisms may alter the amino acid distribution in food residues as they pass through the intestine. Furthermore, endogenous losses of amino acids by subjects receiving a low-protein diet may be markedly different from those of the same subjects receiving an adequate diet. The problems of relating availability to fecal excretion of amino acids are even more difficult in coprophagic animals, such as the rat, where fecal amino acids may be recycled through the animal. The availability of an amino acid in foods also can be determined by measuring nitrogen balance during alternate periods of feeding a basal diet supplemented with equivalent amounts of the amino acid in free or proteinbound form (34). Using this technique, Linkswiler et al. (31, 32, 83) showed that valine, threonine, and isoleucine in corn are fully available to man. The nitrogen balance method is time-consuming and expensive since large quantities of pure essential amino acids are required. A t the same time, it suffers from the defects usually associated with a nitrogen balance study. Specifically, it may be questioned whether the nitrogen balance technique is sufficiently sensitive to detect relatively small changes in availability since a number of rat growth studies indicate isoleucine to be partially unavailable in corn (11). Plasma Amino Acid Methods. Early studies by Wheeler and Morgan (70) and Guggenheim et al. (19) indicated that the levels of free amino acids in the plasma after a test meal may provide information on the in vivo availability of the dietary amino acids. Investigations carried out in our laboratory (38) on adult humans showed that peak plasma lysine and methionine levels were much higher after a meal of alcohol-extracted fish solids, than after a meal of fish extracted with 1,2-dichloroethane. The latter sample was known from other studies to contain unavailable amino acids (52, 54). Smith and Scott (65,66) reported that chicks fed overheated fish meal had lower plasma concentrations of lysine and threonine than those receiving unheated fish meal. In other studies with rats, McLaughlan (39) observed a high degree of correlation between plasma levels of lysine, methionine, tryptophan, leucine, isoleucine, and phenylalanine and the amounts of these amino acids in a test meal. D a t a which illustrate those findings are summarized in Figure 3. F r o m the preliminary information available, therefore, it appears that measurement of plasma amino acid levels may provide a very useful index of the availability of amino acids in vivo.


W O R L D

168

100

LYSINE

EGG

α

MILK

à

FISH

*

CORN

•

CORN

RESOURCES

TRYPTOPHAN

METHIONINE •

PROTEIN

his

y

BLOOD

20 ο 2

Ο

ISOLEUCINE

3

4

05

10

15

PHENYLALANINE


Measurement of the Nutritional Availability of Amino Acids in Foods

Recommend Documents