Amino Acid-Enriched Plasteins: A Source of Limiting Amino Acids for the Weanling Rat

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1 Amino Acid-Enriched Plasteins: A Source of Limiting Amino Acids for the Weanling Rat DAVID V. M. ASHLEY, ROMAN TEMLER, DENIS BARCLAY, CHARLES-ANDRE DORMONO AND ROLF JOST Nestlà Research Department, CH-1814 La Tour-de-Peilz, Switzerland ABSTRACT The biological availability to the weanling rat of two amino acidenriched plasteins, tryptophan and methionine plastein, was tested. In both experiments rats were fed diets containing graded levels of the amino acid either in the free form or as the amino acid-enriched plastein. The first experiment tested the utilization of tryptophan plastein. The results as indicated by weight gain, food intake and plasma levels of tryptophan showed that the tryptophan in the plastein was utilized to the same extent as the free amino acid. Brain tryptophan levels correlated better with plasma Trp:neutral amino acid ratios (r = 0.83) than with plasma tryptophan itself (r = 0.63) suggesting that the small changes observed in plasma amino acid profiles were respon sible for differences in the brain tryptophan concentrations among the L-tryptophan and tryptophan plastein groups. In the second experiment, utilization of methionine plastein was tested. Based on weight gain and food intake data, it was concluded that methionine plastein was also utilized to the same extent as free L-methionine. J. Nutr. 113: 21-27, INDEXING KEY WORDS â tryptophan Supplementing low biological value pro tein with the limiting amino acid is an ac cepted principle for improving the nutri tional value of a protein. In commercial prep arations, however, this process has limited applicability because the free amino acids produce objectionable odors and flavors dur ing processing and cooking (1). Free amino acids may also react during processing with other components and may be rendered un available (2). A chemically more stable form of an amino acid may be obtained by incorporat ing it into high-molecular-weight proteinlike substances called plasteins (3). Low biological value proteins could then be preferentially supplemented with amino acid-enriched plasteins rather than with free amino acids. An additional advantage offered by the plastein synthetic reaction is its stereospecificity for incorporation of the L-enantiomer when a racemic amino acid ester is used as sub strate (4). plasteins â biological availability â methionine Plasteins have been used to upgrade the essential amino acid profile of many proteins, among them proteins from algae, bacteria and leaves (3). Despite some earlier indica tions that the growth of rats fed soy protein diets was improved when methionine-enriched plastein was added to the diets (5), the bioavailability of amino acid-enriched plas teins has never been studied in any depth. In the present paper, we describe studies which show the extent to which two amino acid-enriched plasteins, L-tryptophan and L- methionine plasteins, are utilized by the weanling rat. MATERIALS AND METHODS Tryptophan- and methionine-enriched plasteins were prepared from a peptic digest of soy protein and the DL-amino acid ethyl  1983 American Institute of Nutrition Received for publication 28 June

2 22 ASHLEY ET AL. ester hydrochloride according to a procedure previously described (6). The tryptophan and methionine contents of the amino acid-en riched plasteins were 225 and 325 mg amino acid residue per gram plastein, respectively. Less than 1% of either amino acid was pres ent in the free form as shown by amino acid analysis of the plastein prior to acid hydro lysis. The amino acid composition of tryp tophan- and methionine-enriched plasteins is shown in table 1. Experiment 1 acidthreonineserineglutamic food intake recorded. After 10 days, total acidprolineglycinealaninecystinevalinemethionineisoleucineleucinetyrosinephenylalaninelysinedistillineargininetryptophanmethionine weight gain and food intake were calculated Blood samples were not taken in this exper iment. All diets were stored at 4Â,and rats were IK! housed under the same conditions as de scribed for experiment Statistical analysis nd the plastein-containing diets were calculated to supply eventually about 8% more trypto phan than did the diets containing the free amino acid. All rats were housed singly in hanging wire-mesh stainless-steel cages in a room lighted for 12 hours daily ( hours) and maintained at 23-25Â. Food and water were supplied ad libitum throughout the 2- week study period. On day 14, rats were weighed and food intake measured. At 0900 hours, the rats were placed under ether anes thesia and bled from the abdominal vena cava. Blood was collected in heparinized tubes and processed for amino acid analysis as previously described (9). Brains were Male, weanling Sprague-Dawley rats (Süd deutsche Versuchstierfarm GmbH, Tuttlingen, W. Germany), initial body weight 50- quickly removed and stored at â 20 until 60 g, were randomly assigned to 6 groups of 6 rats each. One group was fed an 18% casein analyzed for tryptophan (10). diet (absolute control), another fed a basal amino acid diet containing 18% by weight Experiment 2 of an amino acid mixture (7) devoid of tryp Twelve equally weighted groups of 10 tophan (table 2A). The other 4 groups were weanling rats, initial body weights g, fed diets in which either free tryptophan or were fed 10% amino acid-based diets (table tryptophan-enriched plastein supplied B) in which either free methionine or the and 0.176% tryptophan. As the calculations methionine-enriched plastein provided 0.07, for these dietary additions were based on the 0.09, 0.11, 0.13, 0.15 or 0.17% methionine in tryptophan residue content of the plastein, the diet. For the same reasons as experiment 1, the methionine-plastein-containing diets supplied â 8%more methionine in the diet TABLE 1 than did the diets containing free-methionine. In this experiment, all diets were in Amino acid composition of methionine and tryptophan-enriched soybean plastein corporated into an agar gel (Difco Labs, De troit, MI) and contained 35% water. The de acidaspartic Amino Tryptophan fined amino acid mix was designed to conform plasteinplasteinmg to the nutritional requirements of the grow acidresidue amino ing rat. plastein52.7 /g Small portions of the diets were weighed daily and given to the rats ad libitum. At the same time, the rats were weighed and their Data from both experiments were ana lyzed by one-way ANOVA in which initial body weight was taken as a covariate. Spe cific comparisons of two means was made by

3 AMINO ACID-ENRICHED PLASTEINS 23 TABLE 2 Composition of diets IngredientsA. Completeamino acidi*180.0 amino acid+amino plastein â Basal Trp 1Casein1Amino Experiment mixture2'3cornstarchl-tryptophan3troptophan acid â plasteincorn oilvitamin mixture4mineral mixture5ingredientsb. â Completeamino Experiment 2Amino mixture3-6sucrosecornstarchl-methioninel-cysteinemethionine acid plasteincorn oilvitamin mixture4mineral mixture5agar-agarcasein188.9â 651.1â â Basalamino acid+ amino acid â Basal acid â Basal Met plastein (0.17%) â Casein hammerstein high protein (95.3% protein as supplied). Technosa, S.A., importers in Switzerland of ICN Nutritional Biochemicals, Cleveland, OH 2 Composed of (grams/kilogram): arginine, 62.2; histidine (HC1), 24.3; isoleucine, 46.1; leucine, 60.7; lysine (HC1), 123.6; methionine, 45.0; phenylalanine, 64.0; threonine, 45.0; valine, 45.0; alanine, 19.1; aspartic acid, 19.1; glutamic acid, 191.7; glycine, 128.2; proline, 19.1; cysteine, 19.1; tyrosine, 19.1; asparagine, 33.7; serine Fluka A.G., CH-9470 Buchs, Switzerland. All amino acids were of the L- isomer. 4 Composed of (grams/kilogram): vitamin A (200,000 U/g), 4.5; vitamin D (400,000 U/g), 0.25; o- tocopherol, 5.0; inositol, 5.0; choline bitartrate, 13.6; menadione, 2.25; p-aminobenzoic acid, 5.0; niacin, 4.5; riboflavin, 1.0; pyridoxine â HC1, 1.0; thiamin-hcl, 1.0; Ca-pantothenate, 3.0; biotin, 0.02; folie acid, 0.09; vitamin B-12, Technosa, S.A., importers in Switzerland of ICN Nutritional Bioehemicals. 5 Bernhardt-Tomarelli mineral mixture (8) ICN Nutritional Biochemicals. 6 Composed of (grams/kilogram): arginine, 78.0; histidine (HC1), 34.0; isoleucine, 49.0; leucine, 77.0; lysine (HC1), 88.5; phenylalanine, 54.0; threonine, 52.0; valine, 48.0; alanine, 39.0; aspartic acid, 119.0; glutamic acid, 205.0; glycine, 128.2; proline, 53.0; serine, 55.1; tyrosine, 37.0; tryptophan, Students' i-test and the dependence of two variables tested by correlation analysis (11). Experiment 1 RESULTS AND DISCUSSION Weight gain and food intake are shown in table 3. There was no statistical difference in either weight gain or in food intake be tween the 18% casein (absolute control group) and the complete 0.176% tryptophan-supplemented amino acid diet, showing the nutri tional adequacy of the complete defined amino acid diet. Both growth and food intake were directly proportional to the level of tryptophan or tryptophan equivalent pro vided in the diet (r = 0.99). Equivalent in creases in weight gain and food intake were obtained with each supplement of either free tryptophan or tryptophan-enriched plastein. Plasma tryptophan levels (table 4) corre lated with dietary tryptophan (r = 0.96). The lowest plasma tryptophan levels were found in the group fed the basal amino acid diet, and highest levels in the group fed the 18%

4 24 ASHLEY ET AL. TABLE 3 Weight gain and food intake of rats fed diet supplemented with either L-tryptophan or tryptophan-enriched plasteinli2 Experiment 1 Group Weight gain Food intake Casein(growth control) AA0.088% Basal Trp 0.176% Trp 0.088% Trp plastein 0.176% Trp plastein50.4 g/14 days g/14 days ±4.2" b22.7 ± ±0.8C 58.9 ±2.1" 26.9 ±2.5C 55.8 ±1.3a85.2 ±6.0a 0.7b ± ±1.9 94.5 ±2.9a 69.3 ±3.0 91.0 ±1.7a 1Means ±SEM;(n = 6). 2 Means with different su perscripts are significantly different (P «0.05). casein diet. This was not unexpected as the tryptophan content of the casein diet was higher (0.26%) than in any of the other diets. As with growth, no differences were found in plasma trytophan levels among groups fed equivalent dietary levels of trytophan either as the free amino acid or as the enriched plastein. Thus it can be concluded that for the weanling rat the biological availability of free tryptophan and plastein-bound tryp tophan were equivalent. We also examined the profile of the re maining amino acids in the plasma (table 5) to determine the manner in which the plastein might have contributed to a change in the rats' amino acid status. The plasma amino acid concentrations of casein-fed rats were in general higher than rats fed the basal amino acid diet, although serine, aspartic acid and glycine concentrations were lower and glutamic acid and histidine were un changed. Increasing the dietary tryptophan level from 0 to 0.176%, either as free tryp tophan or as tryptophan plastein in the basal amino acid diet changed the plasma amino acid profiles. In many cases there was a direct relation to the amount of food eaten. Highly significant positive correlations with food in take were found for glycine (r = 0.97), tyrosine (r = 0.96) and ornithine (r = 0.98). Weaker positive correlations were found for proline (r = 0.83), valine (r = 0.81), methionine (r = 0.85), isoleucine (r = 0.86) and ly sine (r = 0.72) suggesting that whereas in creasing food intake influenced to a large extent an increase in plasma levels, food in take per se was not the sole determinant. Food intake did not correlate with plasma levels of leucine, phenylalanine and arginine and correlated negatively with plasma levels of histidine (r = â 0.72),glutamic acid (r = â 0.86),serine (r = â 0.81), threonine (r = -0.87) and aspartic acid (r = -0.80). Al though interpretation of these findings is con founded by different rates of protein synthe sis among different groups, these data suggest that concentrations of amino acids in the free plasma pool are not simply a function of food and protein intake. Levels of individual amino acids or groups of amino acids appear to be independently controlled and may or may not depend on previous total food or protein intake. The profile of plasma amino acids in rats fed 0.088% tryptophan plastein resembled TABLE 4 Plasma tryptophan, the sum of the neutral amino acids in plasma, ratios of plasma tryptophan to neutral amino acid and brain tryptophan in rats fed diets supplemented with tryptophan or tryptophan-enriched plastein1'2 DietCasein Trpnmol/liter71.5 NAA NAA TrpMg/g7.2 AA0.088% Basal Trp 0.176% Trp 0.088% Trp plastein 0.176% Trp plasteinplasma ±3.4a 6.2b ± ±S.l ±7.6ac 45.5 ±5.4^ 61.5 ±6.7a ±28.4" 11.1 ± ±25.5 ±24.4b ±21.5b ±30.3bTrp/Z ± ± ± ± ± ±0.014Brain ±0.4" 0.7b ± ±0.3" 7.6 ±0.6' 5.8 ±0.5ab 7.2 ±0.7" 1Means ±SEM;(n = 6). 2 Means with different superscripts are significantly different (P «0.05). 3[Val] + [Leu] + [Phe] + [Isl] + [Tyr].

5 AMINO ACID-ENRICHED PLASTEINS 25 TABLE 5 Plasma amino acid concentrations in rats fed diets supplemented with either tryptophan or tryptophan-enriched p/asfein1>2 Diet Amino acid Casein Basal AA 0.088% Trp 0.176% Trp 0.088% Trp plastein 0.176% Trp plastein timol/liter AlanineThreonineMethionineLysineHistidineArginineProlineSerineClutamic 30.4«662.9 ± 5b924.2 ± b792.2 ± 25.5'b778.7 ± 26.8"776.8 ± 51.3'b653.1 ± 66.7*115.2 ± 110.3b33.2 ± 46.6'b37.9 ± 51.2"b57.3 ± 37.3*b584 ± 30.3'64.0 ± 8.4"867.5 ± \.fr760.0 ± 2.3b742.8 ± 5.5C856.8 ± 11.1e854.0 ± 9.5C992.7 ± 40.8«88.4 ± 37.41«81.2 ± 40.8C73.4 ± 51.7'b74.5 ± 12.4ab81.1 ± 32.7d72.7 ± acidaspartic 3.1*96.6 ± 10.2*336.6 ± 32.7'373.1 ± 20.2"136.6 ± 10.5"23.7 ± 7.2«b79.2 ± 10.6'145.2 ± 15.7b729.3 ± 48.4b133.9 ± 10.2"38.0 ± 3.6b114.4 ± 15.6"b153.7 ± 7.9""618.0 ± 19.01«100.7 ± 7.6bc25.7 ± 3.3k95.8 ± 6.3a164.1 ± 14.9b643.8 ± Ob105.3 ± "28.5 ± 3.1'b141.2 ± 9.3b169.7 ± 8.0>>652.4 ± 16.3b112.1 ± 19.5«25.7 ± 4.3b105.2 ± 5.1*171.3 ± ILO ± 11«89.1 ± e23.3 ± acidglycinevalineleucineisoleucinephenylalaninetyrosine *213.8 ± 2.7b575.8 ± 1.3*771.3 ± 2.0" ± 1.9"864.1 ± 0.5" ± 13.3«249.9 ± 45.4b108.0 ± ±9.0b d134.9 ± 55.5C140.2 ± 87.7d139.7 ± 9.1«170.6 ± 7.5b93.8 ± 11*97.9 ±6. 9.5C106.8 ± 2.4C94.7 ± 6.4'113.3 ± 3.3«60.1 ± 4.5"147.8 ± 2.1b54.8 ± 2.3b47.4 ± IJ&*43.6 ± 7.9b57.1 ± 5.1b41.9 ± 2.4bc57.4 ± 3.7b75.9 ± 6.2e50.1 ± 2.0"1>C88.4 ± 6.6b73.8 ± 5.4^52.4 ± 3.3* 66.0 ± 5.5b77.5 ± 9.7C48.1 ± 2.4bc92.3 ± ±2.4«400.4 ±2.0b411.3 ±1.9"417.0 ±7.2e527.8 ±4.2*427.5 ±1.9e 1Means ±SEM(n = 6). 2 Means with different superscripts are significantly different (P < 0.05). more closely the profile in the 0.176% L-tryptophan and 0.176% tryptophan-plastein groups than in the 0.088% L-tryptophan group. This was particularly true with regard to the essential amino acids, lysine, methionine, arginine, valine and isoleucine and the nonessential amino acid alanine (table 5). Glutamic acid levels were lower in the 0.176% tryptophan-plastein than in the 0.176% tryptophan group. Examination of the amino acid profile of the plastein sug gested no evident reason for these changes. Neither were they accountable by the small differences in the dietary tryptophan content as supplied by free tryptophan or by the plastein. However, as plasma amino acid levels did not reach, for the most part, the same high levels as casein-fed rats and as there were no significant differences in growth and food intake among groups fed almost equiv alent levels of tryptophan, it is clear that the observed differences in the plasma amino acid profiles were not detrimental to the whole organism's body protein metabolism. Changes in the concentrations of plasma neutral amino acids (NAA) have been shown to affect brain tryptophan levels (12). The large NAA, in particular valine, leucine, iso leucine, phenylalanine and tyrosine, compete with plasma tryptophan for entry into the brain. Brain tryptophan is the precursor for synthesis of the neurotransmitter serotonin, which may play a role in the control of sev eral behaviors such as sleep (13), appetite (14) and mood (15). Competition between plasma tryptophan and large NAA at the blood brain barrier can be expressed in terms of the ratio of the plasma concentrations of tryptophan to the sum of the concentrations of the car rier-competitive NAA in the plasma: i.e., Trp/2 NAA ratio. Plasma 2 NAA concentrations (table 4) of the rats fed the amino acid-based diets sup plemented with 0.088% tryptophan plastein were higher than for the group fed the equiv alent amount of free tryptophan without any differences in plasma tryptophan concentra tion. This explains the lower brain trypto phan levels of the former group (table 4), and suggests that brain tryptophan levels corre late better with plasma Trp/NAA ratios than with plasma tryptophan. As predicted, brain tryptophan levels cor related relatively poorly with plasma tryp-

6 26 ASHLEY ET AL. tophan levels (r = 0.63), and rather better with plasma Trp/NAA levels (r = 0.83). These results therefore reaffirm the importance of NAA changes in influencing brain tryptophan levels (12). Experiment 2 Experiment 2 was designed to determine whether another amino acid, methionine, was also available when covalently bound in a plastein. As plasma amino acids were not to be measured in this second experiment, several low levels of methionine were sup plied in the diet either as free methionine or equivalent amounts in the form of methio nine plastein. Thus even a small decrease in biological availability from the plastein would be reflected in the growth profiles. As shown in table 6, there were no differences in the weight gain, food intake and protein effi ciency ratio when equivalent levels of me thionine were supplied either as the free amino acid or the enriched plastein. The dif ferences, always slightly higher but nonsig nificant, in weight gain and food intake of the plastein groups were due possibly to the TABLE 6 Effect of supplements of methionine-enriched plastein on weight gain, food intake and protein efficiency ratio (PER) of rats fed a basal amino acid diet1 Levelof methi oninedietinitialweightweightgainfoodintakeper Free methionine g/10 days g/10 dayt am ± ± Melhtonine plastein ± ± ± ± ± ± ± ± ± »7OX»Oil II ± ± ± ± ± ± ± ± ± ± ± Means - SEM; (n = 10). ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.11 8% more methionine supplied in the diets containing the plastein. It was nonetheless clear that the methionine in the enriched plastein was as nutritionally available as free methionine. Based on the growth data and on experiment 1, there was no reason to sug gest that blood methionine levels differed among groups fed equivalent levels of me thionine. Small changes in the dietary avail ability, particularly at levels above 0.1% of the diet, would have resulted in substantial growth differences. The results from both these studies show that tryptophan and methionine covalently bound into plasteins are biologically equiv alent to the free amino acid for the growing rat. These data extend the observation of Yamashita et al. (5) showing that supple mentation of soy protein with methionineenriched plastein increased the protein effi ciency ratio of soy protein. As methionine and tryptophan are often the most limiting amino acids in proteins, these observations open the possibility for the use of plasteins in improvement of the nutritional value of inadequate proteins and for exploring the use of unconventional proteins in commercial food preparations. In addition, plasteins may be useful in the creation of new tailor-made proteins for dietetic products designed for therapeutic use. ACKNOWLEDGMENTS The authors are indebted to E. Bise, J. C. Monti and D. Moennoz for skilled technical assistance, and to H. Rahim for statistical analysis. LITERATURE CITED 1. Ballance, P. E. (1961) Production of volatile com pounds related to the flavour of foods from the Streker degradation of DL-methionine. J. Sci. Food Agrie. 12, Hurrell, R. (1980) Interaction of food components during processing. In: Food and Health Science and Technology (Birch, G. G. and Parker, K. J., eds.), pp , Applied Science Publishers Ltd., Lon don, U.K. 3. Fujimaki, M., Arai, S. & Yamashita, M. (1977) Enzymatic protein degradation and resynthesis for protein improvement. In: Food Proteins: Improve ment through Chemical and Enzymatic Modifica tion (Feeney, R. & Whitaker, J. R., eds.), pp , American Chemical Society, Washington, DC. 4. Yamashita, M., Arai, S., Imaizumi, Y., Amano, Y. & Fujimaki, M. A. (1979) One-step process for incorporation of L-methionine into soy protein by

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