Plasma amino acid response to single test meals in humans

Res. Exp. Med. (Bet1.) 174, 121--130 (1979)

Re@earth in

Experimental Medicine (~ Springer-Verlag 1979

Plasma Amino Acid Response to Single Test Meals in Humans*

V. Ethiopian Presch'ool Children Given Loweost Protein Supplements

B. G. Ljungqvist 3, K. B. Bj6rnesj61, M. Gebre-Medhin 1, D. Habte 2, G.W. Meeuwisse 1, O. Mellander 3, and U. S.-O. Svanberg 3

Ethiopian Nutrition Institute, Addis Abeba, Ethiopia 2 Ethio-Swedish Pediatric Clinic, Addis Abeba, Ethiopia 3 Department of Medical Biochemistry, University of G6teborg Fack, S-40033 G6teborg 33,

Sweden

Summary. The plasma amino acid response to single test meals was studied in preschool children, aged 9 months--5 years. The amount of protein given in each test meal wa s 1 g per kg body weight , which represented one-third of the daily intake of the children who were recovering from protein-energy malnutrition. The test meals given was gruels made from wheat mixed with a supplementary weaning food (Faffa or Superamin) or fish protein concentrate (FPC). The plasma amino acid responses were evaluated both as PAA ratios (a modification of the Longenecker and Hause method), and as AMR% (percentage change in the postprandial essential amino acid molar ratios according to Graham and Placko). Both evaluation models indicated that lysine, threonine and methionine were the limiting amino acids in the Faffa/ wheat diet, and that lysine and threonine were limiting in the Superamin/ wheat diet. All essential amino acids seemed to be supplied in adequate amounts in the FPC/wheat diet. These results were in close agreement with the amino acid score of the diets (based on chemical analysis).

Key words: Plasma amino acid - Protein quality - Weaning food - Preschool children

There is at present disagreement about the importance of protein quality in human nutrition [1,2, 3]. It is estimated that seldom in practical life does unsufficient supply of specific amino acids limit the utilization of dietary proteins. However, most experts tend to agree that infants and small children constitute an exception in this respect [1,4]. In these age-groups the requirements of both protein and energy, as well as essential amino acids, are much higher than for adults on a per kilo body weight basis [5,6]. Small children are thus vulnerable to a reduced intake of any of these nutrients.

* Supported by grants from'the Swedish Medical Research Council (No.K75-03X-3139-04A and No. B76-03X-3139-05B) Offprint requests to: B. G. Ljungqvist, M.D. (address see above)

0300-9130/79/0174/012l/$ 2.00

122 B.G. Ljungqvist et al.

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Plasma Amino Acid Response 123

Wi th regard to p ro te in qua l i t y , the s i tua t ion is p r o b a b l y most cri t ical at the age when the cons tan t ly well ba lanced supply of essential amino acids f rom the mother ' s mi lk [7] is d iscont inued, i.e. at weaning. The p rob lems in this age-group are usual ly specifical ly recognized in nu t r i t ion p rogrammes . One f requent ly used a p p r o a c h is to p roduce special ly designed supp lemen ta ry foods for chi ldren of weaning ages (examples: Incapa r ina , Supe ramin etc.). These foods should conta in a rich supply of p ro te ins in well ba lanced p ropor t i ons at a low cost [8]. Our in- s t i tu t ions have for a long t ime been engaged in evaluat ing var ious nut r i t iona l aspects of these weaning foods , especial ly the E th iop ian variety, "Fa f fa" [9, 10].

One of the ma in object ives of the present series of invest igat ions was to s tudy the use o f the s ingle-meal p l a sma amino acid responses as a tool in the evalua t ion of supp lemen ta ry weaning foods. This technique has the advan tage of being direct ly app l icab le in small chi ldren [11, 12]. In our previous invest igat ions hea l thy young adul ts were used as subjects [13, 14, 15] as a number of me thodo- logical aspects requi red fur ther study. The test meals used in those exper iments were, however , au thent ic weaning foods [15] or models of these [13, 14]. The present repor t describes the results f rom single-meal tests pe r fo rmed with children.

Material and Methods

Clinical Material

The clinical and anthropometric data of the children that took part in our study are presented in detail in Table 1. The age of the children ranged from 9 months to 5 years. They were all ad- mitted to the Ethio-Swedish Pediatric Clinic or the metabolic ward at the Ethiopian Nutrition Institute for rehabilitation from protein-energy malnutrition. Their energy and protein intake before and between the test meals were thus exceptionally high. Three of the children given a protein-free meal differed from the other children in that they were healthy and had just taken part in a nitrogen balance test on the FAO/WHO [5] "safe level" of protein intake.

Test Meals

Three different test meals were used. The composition of these is given in Table 2. The dietary proteins were derived from wheat +"Faffa", wheat +"Superamin" and wheat +fish protein concentrate (FPC), respectively. Faffa and Superamin are supplementary weaning foods pro- duced in Ethiopia and Egypt, and these are presented in more detail elsewhere [15]. The diet containing fish protein concentrate was included as a reference meal of high biological value [cf. 14]. In addition to these three diets, protein-free single meals based on corn-starch and sugar (Table 2) were given to six children.

The amino acid contents of the test meals are calculated in Table 3 on the basis of our previous chemical analyses [14, 15].

Experimental Procedures

The test meals were given at 8 a.m. in the morning following a 12-h fast. The test meals were consumed as gruels (Table 2). The protein-free test meal was consumed as "chechebsa', an Ethiopian spiced bread.

Except where otherwise stated (Table 1), the amount given at each test meal corresponded to 1 g of protein per kilo body weight. This represented one-third of the daily intake of these children, who were recovering from PEM.


Table 2. Composition of test meals. The amounts given in each test meal corresponded to 1 g of protein per kg body weight

g Protein g Energy, kcal

Faffa/wheatporridge

Faffa 96 20.9 326 Wheat flour 96 10.1 309 Sugar 24 96 Salt 1.2 Oil 30 269 Water 1078

1325 31.0 1000

Superamin/wheat porridge

Superamin 96 Wheat flour 96 Sugar 24 Salt 1.2 Oil 30 Water 599

20.2 326 10.1 309

96

269

846 30.3 1000

Reference meal a porridge

Wheat flour 75 7.9 249 FPC b 25 22.6 92 Corn starch 46 183 Sugar 53 213 Salt 2.4 Vanilla 1.5 Oil 30 268 Water 762

994.9 30.5 1000

Protein-free meal c

Corn starch 204 Oil 20.4 Spices 3.0 Salt 1.5 Water added - 700

2.5 816 184

928.9 2.5 1000

In some cases given as a bread a

b Fish protein concentrate, Astra Nutrition, M61ndal, Sweden c Served as a bread

Two blood samples were collected at each test: one just before the meal was given and the other 3 h after the test meal. At the single-meal tests with protein-free diet the postprandial blood sample was taken 2 h instead of 3 h after the meal, since it was expected that more marked plasma concentration decreases could be found at this time [cf. 14].

The heparinized blood specimens were immediately centrifuged in order to remove the red blood cells. The plasma samples were transported to Sweden in a frozen state, where the plasma proteins were precipitated with sulfosalicylic acid [13] before analysis of the free amino acid

Plasma Amino Acid Response

Table 3. Amino acid composition and score of the test meals

125

Amino Composit ion (mg/g N)

acid Faffa/ Superamin/ wheat wheat mixture mixture

Amino acid score (%)

FPC/ Scoring a Faffa/ Superamin/ F P C / wheat pattern wheat wheat wheat mixture mixture mixture mixture

Lys 268 306 494 340 79 90 145

Trp 74 70 92 60 123 117 154

Thr 204 197 289 250 82 79 116

Val 308 304 361 310 99 98 116

Met 85 120 196

Me t+Cys 188 220 276 220 86 100 126

Ile 254 243 291 250 102 97 116

Leu 463 447 503 440 105 102 114

Phe 306 310 285

Phe +Tyr 501 464 516 380 132 122 136

Provisional amino acid scoring pattern, FAO/WHO, 1973 [5]

Table 4. The postprandial decrease of the essential amino acid concentrations in blood plasma 2 h after a protein-free test meal. The values are calculated in comparison to Valine

Amino acid Children Adults a (n = 6) (n = 5)

Mean + S.D. Mean + S.D.

Lys 7.3±2.0 4.0±1.5

Trp - - 2 .1±0.6

Thr 5.4±2.5 4.0±0.8

Val 10 10

Met 1.7±0.4 1.5±0.3

M e t + C y s 3.7±1.4 3.1±1.1

Ile 4 .3±0.6 4.6±0.4

Leu 7.7±1.3 8.1±0.5

Phe 2.6±1.1 2.4±0.3

P h e + T y r 6.4±1.8 5.5±0.4

From [14]

concentrations (Jeol 5-AH automatic amino acid analyser). The tryptophan and cystine concentrations were to low to provide sufficiently accurate determinations.

Calculations The evaluation of the single-meal plasma amino acid responses was carried out using two separate calculation models, which have previously been described and examined [13, 16].

1. The model introduced by Graham and Placko [11], calculating the postprandial percentage changes in the essential amino acid molar ratios (A MR %). (The limiting amino acid(s) will show the largest AMR% decrease.)


2. A modification of the plasma amino acid ratio (PAA ratio) procedure introduced by B-A Longenecker and Hause [17, 18]. Their equation is: PAA ratio = -----if-- × 100, where A is the

fasting and B the postprandial concentration, and R is the relative requirement of each essential amino acid. The limiting amino acid(s) will yield the lowest PAA ratios (they may be negative). Our modification refers to the estimates of the relative requirements (R) where we use experimental evidences [13, 16] instead of the overall daily requirements [18].

In the present study the R values were derived from the net concentration decreases of the essential amino acids observed after the protein-free test meals, exactly as in a previous study [14]. These values (Table 4) were then used to calculate the PAA ratios according to the equation above. The PAA ratios of the essential amino acids from each test meal are expressed relative to valine (PAA ratio =0) [13].

Statist ical Methods

The results obtained from our single-meal plasma amino acid responses are presented as means and standard deviations for each test meal. The limiting order of the essential amino acids in the test meals are evaluated statistically by a non-parametric approach, based on a signed rank test procedure [19], which has been previously described [13].

Results

T h e resul ts o f the s ing le -mea l tests e v a l u a t e d as P A A ra t ios are g iven in T a b l e 5.

T h e resul ts agree ve ry well wi th the a m i n o ac id scores based on chemica l analysis

o f the diets (Tab le 3). T h e F a f f a / w h e a t p l a s m a a m i n o ac id responses thus

ind ica t ed lysine, t h r e o n i n e a n d m e t h i o n i n e to be l imi t ing , whi le lysine and

t h r e o n i n e were f o u n d to be l imi t ing in the S u p e r a m i n / w h e a t diet. T h e re fe rence

Table 5. The plasma amino acid response after different test meals. The data and limiting order are calculated according to a modified Longenecker and Hause procedure (PAA ratio)

Amino acid PAA ratio

Faffa/wheat (n = 17) Superamin/wheat (n = 7) FPC/wheat (n = 11)

Mean ± S.D. Limiting Mean± S.D. Limiting Mean ± S.D. Lim. order order order

Lys -3 .8±1 .6 i -2 .8±1 .8 1 1.8±2.4 5

Thr -2 .7±2.1 3 -1 .6±1 .6 2 0.2±1.8 2

Val 0 5 0 4 0 3

Met -3 .6±2 .1 2 0.7±2.8 6 1.6±2.8 7

Ile 0.4±1.8 6 0.2±2.3 5 0.1±1.6 4

Leu -1 .9±1 .7 4 -0 .6±1 .8 3 -1 .5±1 .3 1

Phe+Tyr 2.5±1.9 7 3.5±2.2 7 1.0±2.0 6

Significance 1--2, n.s. 1--2, n.s. 1--2, P < 0.01

level for pair- 1--3, P < 0.05 1--3, P < 0.01 2--3, n.s. wise differences in 2--3, P < 0.05 2--3, P < 0.01 limiting order [13]


Table 6. The plasma amino acid response after different test meals. The data and limiting order are calculated according to Graham and Placko (A MR %)

Amino acid A MR %

Faffa/wheat (n = 17) Superamin/wheat (n = 7) FPC/wheat (n = 11)

Mean _+ S.D. Limiting Mean _+ S.D. Limiting Mean _+ S.D. Lim. order order order

Lys -23.2_+ 9.6 1 -24.1_+11.7 1 20.6_+17.2 6

Thr - 1 3 . 8 + 12.0 2 -15.7_+ 12.2 2 -5.8_+ 13.6 3

Val 0.5+- 8.3 4 -4.5+- 4.2 3 -8.8_+ 6.2 1

Met -12.8_+ 12.3 3 10.4+-20.9 5 28.8_+27.7 7

Ile 17.5+-11.9 6 10.0+-11.2 6 7.0_+ 9.7 5

Leu 2.3+- 11.2 5 1.3_+ 10.8 4 -3.9+- 8.6 2

Phe+ Tyr 25.5_+ 16.5 7 28.0+ 19.9 7 2.4_+ 13.9 4

Significance 1--2, P < 0.01 1--2, n.s. 1--2, n.s.

level for pair- 2--3, n.s. 1--3, P<0 .01 1--3, n.s. wise differences in 2--3, P < 0.05 2--3, n.s. limiting order [13]

Table 7. PAA ratio of Faffa and Superamin meals evaluated as differences from PAA ratios in the reference meal

Amino acid PAA ratio

Faffa/wheat - reference Superamin/wheat (n = 11) - reference (n = 4)

Mean + S.D. Limiting Mean + S.D. Lira. order order

Lys -5.5_+ 1.8 1 -5.6+_ 1.0 1

Thr -2.5_+ 2.1 3 -1.6_+ 1.4 3

Val 0 5 0 4

Met -5.4_+ 3.5 2 -0.8_+ 3.8 2

Ile 0.2_+ 1.9 6 -0.3_+ 3.0 6

Leu -0.3_+ 1.9 4 -0.4_+ 2.3 5

Phe+ Tyr 1.7_+ 2.6 7 3.5_+ 2.6 7

Significance 1--2, n.s. 1--2, P < 0.06

level for pair- 1--3, P < 0.001 2--3, n.s. wise differences in 2--3, P < 0.01 limiting order [13]


meal seemed to be low in leucine, but the responses were all within the range previously found to be compatible with a well balanced plasma amino acid response [15].

The Graham and Placko evaluation model gave results very similar to those reported above with the modified Longenecker and Hause procedure (Table 6).

In Table 7 the PAA ratios of the single-meal tests with Faffa and Superamin were evaluated in comparison with the PAA ratios found after the reference meal containing fish protein concentrate. The evaluation was performed on an individual basis. No improvements with regard to the variability of the plasma amino acid responses were found, suggesting that the variability within the same subject was not smaller than that between the subjects. It is also obvious that the relative excess of lysine and methionine in the FPC/wheat mixture (Table 3) will greatly affect the results of the plasma amino acid response of the other test mixtures when a comparative evaluation is performed (see Tables 5 and 7).

Discussion

Our previous single-meal plasma amino acid response tests were performed in healthy young adults. These were all well nourished as judged by physical examination and dietary habits. Their intake of proteins was certainly well above "safe levels" [5]. To what extent, then, do the acute postprandial plasma amino acid changes in these healthy adults correspond to the responses of small, partly protein-malnourished children given the same test meals?

The requirements for specific essential amino acids have been found to be different in young, growing children and in adults [5, 6]. Adults need considerably less total essential amino acids per kilo body weight, but the relative needs for each essential amino acid also seem to differ from those in infants. The obvious explanation for this is that there may be different amino acid requirements for growth and maintenance [4]. According to the original assumptions of Longen- ecker and Hause [17], infants should thus have different relative requirements (R) for essential amino acids to be used in evaluations of plasma amino acid ratios according to their equation (see Methods). However, when the "relative requirements" were estimated from postprandial relative rates of decrease of the free essential amino acid concentrations in plasma in some of our present children, no significant differences were found compared to the relative rates measured in adults (Table 4). One exception is lysine, which showed a more marked plasma concentration decrease in the children than was found in the adults. The reason may be that the children did not have as large intracellular pools of free lysine as the adults [20].

The nutritional status of the children differed considerably at the time when the test meals were given (Table 1). However, the plasma amino acid responses, evaluated according to the two calculation models used, gave very consistent results, and the results also agreed very well with the amino acid scores based on chemical analysis of the test diets.

The plasma amino acid levels in the children at the postprandial sampling time (3 h) were significantly elevated compared to the fasting levels. If valine is


taken as an example the 3h levels were 56% (_+49) above the fasting levels (Mean +S.D. , of 35 observations). This is in sharp contrast to the single-meal tests in adults [13, 14, 15], where the 3 h levels were close to those found in the fasting state. The reason for the more elevated levels in the children is probably the larger protein intake per kilo body weight in the test meals. In spite of this difference in protein intake, the order of limiting amino acid and the magnitude of the PAA ratio and A MR % responses from the present single-meal tests were in good agreement with our previously reported single-meal tests with adults given similar diets [14, 15]. The amino acids lysine and threonine may be exceptions, since they were not found to be limiting in adults when the test meals given had a low content of these amino acids [15]. It should be further evaluated if this difference between adults and children will be found in adults given the same amount of protein per kilo body weight as the present children.

The similar plasma amino acid responses in healthy adults and in children recovering from protein-energy malnutrition indicate that the acute changes of the free amino acids in blood plasma after a protein meal, primarily reflect the addition to the body stores of physiologically available dietary amino acids. I f the utilization of amino acids by the organism had exerted a predominant influence, then more marked differences would be expected between healthy adults and protein-energy malnourished children. It is also possible that the difference between adults and children with regard to specific requirements for essential amino acids is not as great as is commonly thought [21]. An important implica- tion of this is that before extensive plasma amino acid response tests are carried out in children, it should be carefully considered whether they will provide any additional information compared to tests in adult subjects.

The test meals in our present study correspond to a situation where a staple (in our case wheat) is being supplemented with a protein-rich weaning food (Faffa and Superamin). It is noteworthy that in the case of the Faffa /wheat diet, wheat is actually "supplementing" Faffa with sulphur-containing amino acids to a small extent (Table 3). The mixture of wheat and Faffa has thus a slightly higher amino acid score than Faffa alone. In our single-meal tests with the wheat /Faffa mixture, methionine was not found to be solely limiting (Tables 5 and 6), which was the result when only Faffa was given, although in this case the tests were performed in adults [15].

The implications of our present findings with regard to the nutritional value of Faffa and Superamin given as supplements to children of weaning ages, will be discussed in two subsequent series of papers where the results from nitrogen balance studies and a long-term evaluation of Faffa and Superamin under normal living conditions will be reported.

References

1. Arroyave, G.: Amino acid requirements by age and sex. In: Nutrients in processed foods, P. L. White, D. C. Fletcher (eds.), p. 15. Acton, Mass.: Publ. Sciences Group, Inc. 1974

2. de Waard: Protein quality--for whom? Lancet 1976 II, 100 3. Porter, J. W. G., Rolls, B. H. (eds.): Proteins in Human Nutrition, pp. 349--368. London:

Academic Press 1973


4. el Lozy, M., Hegsted, P. M.: Calculation of the amino acid requirements of children at different ages by the factorial method. Amer. J. Clin. Nutr. 28, 1052 (1975)

5. Report of a Joint FAO/WHO Ad Hoc Expert Committee. Energy and protein requirements. World Health Organization Techn. Rep. Ser. No. 522 (1973)

6. Irwin, M. I., Hegsted, D. M.: A conspectus of research on amino acid requirements in man. J. Nutr. 101,385 (1971)

7. Svanberg, U. S.-O., Gebre-Medhin, M., Ljungqvist, B. G:, Olsson, M. E.: Breast milk composition in Ethiopian and Swedish mothers. III. Amino acids and other nitrogenous substances. Amer. J. Clin. Nutr. 30, 499 (1977)

8. PAG. Guideline on protein food mixtures for older infants and young children. PAG, United Nations, New York, N.Y., U.S.A. PAG Guideline No. 8 (1970)

9. Bj6rnesj6, K. B., Engsner, G., Gobezie, A., Jagenburg, R., Ljungqvist, B., Mellander, O.: Plasma amino acids in feeding children with a supplementary food mixture. Environ. Child Health 19, 129 (1973)

10. ~gren, G., Eklund, A., Stenram, U., Nordgren, H.: Biological and toxicological feeding studies on Faffa, an Ethiopian protein-rich food for children. Ethiop. Med. J. 11,102 (1973)

11. Graham, G. G., Placko, R. P.: Postprandialplasmafree methionine as anindicatorofdietary methionine adequacy in the human infant. J. Nutr. 103, 1347 (1973)

12. Graham, G. G., MacLean, W. C., Jr., Placko, R. P.: Plasma amino acids of infants con- suming soybean proteins with and without added methionine. J. Nutr. 106, 1307 (1976)

13. Ljungqvist, B.G., Svanberg, U. S.-O., Young, V. R.: Plasma amino acid response to single test meals in humans. II. Healthy young adults given synthetic amino acid mixtures. Res. Exp. Med. (Berl.) 174, 13 (1978)

14. Ljungqvist, B. G., Olsson, M. E., Svanberg, U. S.-O.: Plasma amino acid response to single test meals in humans. III. Supplementary effect of dried skim milk and methionine on a vegetable protein mixture. Res. Exp. Med, (Berl.) 174, 29 (1978)

15. Ljungqvist, B. G., Bostr6m, S. L., Sj6berg, L. B., Svanberg, U. S.-O.: Plasma amino acid response to single test meals in humans. IV. Four low-cost protein supplements evaluated by chemical analysis, rat bioassay and plasma amino acid response. Res. Exp. Med. (Berl.) 174, 111 (1979)

16. Ljungqvist, B. G.: Plasma amino acid response to single test meals in humans. I. A back- ground review. Res. Exp. Med. (Berl.) 174, 1 (1978)

17. Longenecker, J. B., Hause, N. L.: Relationship between plasma amino acids and composition of the ingested protein. Arch. Biochem. 84, 46 (1959)

18. Longenecker, J. B., Hause, N. L.: Relationship between plasma amino acids and composition of the ingested protein. II, A shortened procedure to determine plasma amino acid (PAA) ratios. Amer. J. Clin. Nutr. 9, 356 (1961)

19. Wilcoxon, F.: Individual comparisons by ranking methods. Biometrics 1, 80 (1945) 20. Young, V. R.: The role of the skeletal and cardiac muscles in the regulation of protein

metabolism. In: Mammalian protein metabolism, H. N. Munro (ed.), pp. 585--674. New York: Academic Press 1970

21. Young, V. R., Scrimshaw, N. S.: Nutritional evaluation of proteins and protein requirements, with particular reference to studies in man. (To be published)

22. Rutishauser, J. H. E., Waterlow, J. C.: Malnutrition in man. In: Early malnutrition and mental development, J. Cravioto, L. Hambraeus, B. Vahlqvist (eds.), p. 13. Sweden: Alm- qvist and Wiksell 1974

Received June 20, 1978 / Accepted September 13, 1978

Plasma amino acid response to single test meals in humans

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