Research in
ExperimentedMedicine
Res. Exp. Med. (Berl.) 174, 13--28 (1978)
.© Springer~Verlag1978
Plasma Amino Acid Response to Single Test Meals in Humans* II. Healthy Young Adults Given Synthetic Amino Acid Mixtures B. G. Ljungqvist 1, U. S.-O. Svanberg 1, and V. R. Young 2 1Department of Medical Biochemistry, University of GOteborg, Fack, S-40033 GOteborg 33, Sweden 2Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass., USA
Summary. The postprandial plasma amino acid response to test meals containing different amino acid mixtures was analysed in six young adults. The amount of amino acids given in each test meal was equivalent with 3.5 g of nitrogen. Each test meal also contained a large amount of carbohydrate (mainly as wheat starch). Five different amino acid mixtures were used, and the effects on the plasma amino acid response of different dietary levels of lysine, isoleucine and methionine were studied in detail. The plasma amino acid responses were evaluated both by a modified Longenecker and Hause procedure (PAA ratios) and by the procedure proposed by G r a h a m and Placko (percentage changes in the postprandial essential amino acid molar ratios). The plasma amino acid response of lysine was found to be less sensitive to reductions in the dietary supply than the isoleucine and methionine responses evaluated by these procedures. The influence of altered levels of dietary supply on the postprandial plasma concentrations of isoleucine and methionine seemed to be specifically great at the intake levels where these essential amino acids become limiting for protein utilization according to the amino acid score of the test mixtures. The nutritional significance of the postprandial pattern and level of essential amino acids is discussed on basis of the present results. Experimental conditions and evaluation models are examined with regard to the use of single-meal plasma amino acid responses to assess the protein quality of human diets. Key words: Amino acids - Nutrition - Diet - Protein quality - Plasma amino acid response * This study was supported by grants from the Swedish Medical Research Council (grant no. K75-03X-3139-04A and no. B76-03X-3139-05B) and from the Swedish Nutrition Foundation Offprint requests to: B.G. Ljungqvist, M.D. (address see above)
0300-9130/78/0174/0013/$3.20
14
B.G. Ljungqvist et al.
If p r e d i c t a b l e n u t r i t i o n a l relationships can be established for the effects o f the a m i n o acid c o m p o s i t i o n o f a m e a l on the p a t t e r n a n d levels o f free a m i n o acids in venous b l o o d p l a s m a , then it s h o u l d be possible to exploit a m i n o acid changes in p l a s m a following a single test m e a l to assess the q u a n t i f y a b l e nutritive value o f a f o o d p r o t e i n o r mixture o f p r o t e i n sources. In a p r e v i o u s p a p e r we e x a m i n e d the prerequisites o f such single meal tests in relation to the m e t a b o l i c processes k n o w n to affect the p l a s m a a m i n o acid levels in the a b s o r p t i v e state [1]. The p r e s e n t investigation was p e r f o r m e d in o r d e r to evaluate the a d e q u a c y o f the principles o f e x p e r i m e n t a l design that were p r o p o s e d on basis o f these considerations. Since the m a i n objective o f o u r study was to define further the r e l a t i o n s h i p between d i e t a r y a m i n o acid c o m p o s i t i o n a n d its influence on the acute changes in p l a s m a a m i n o acid levels, mixtures o f crystalline a m i n o acids were used in the e x p e r i m e n t a l model. These mixtures c o u l d be m o d i f i e d readily for their a m i n o acid c o n t e n t a n d p a t t e r n , facilitating e x p e r i m e n t a l design in the investigations. The p l a s m a a m i n o acid single-meal technique was chosen for further s t u d y because this a p p r o a c h m a y p r o v i d e a convenient a n d p r a c t i c a l a p p r o a c h for h u m a n biological e v a l u a t i o n o f p r o t e i n - r i c h vegetable mixtures for children. Therefore, the present e x p e r i m e n t s utilized diets supplying a m i n o acids, c a r b o h y d r a t e s a n d fat, in a p r o p o r t i o n c o m p a r a b l e to that in the vegetable p r o t e i n mixtures s t u d i e d p r e v i o u s l y [2--5].
Materials and Methods
Composition of Test Meals The composition of the test meals is shown in Table 1. All the ingredients were suspended in water and consumed in this form. The total amino acid content of the test mixtures studied are given in Table 2. The basic 0 mixture had the same amino acid composition as a "Reference" protein meal that was being used in a parallel experiment [3]. Test mixtures 1, 2, and 3 provided levels of methionine, isoleucine and lysine in amounts equal to either 40, 60 or 80% of those provided by the 0 mixture. The amounts of the other amino acids were held constant. 20% differences in intake levels were considered appropriate for evaluation because it was thought that it should be possible to detect the effect of such differences if the single-meal technique was to be of practical value. In addition, we used an amino acid mixture based on the egg protein pattern. The amino acid scores [5] of the various test meals are given in Table 3. The different amino acid patterns were offered to the subjects in a randomized order, except that all subjects received the amino acid mixture based on the egg patterns as the final test meal.
Table 1. The composition of the test meals (g) Wheat starch Sucrose NaCI Vegetable oil Amino acid mixture Water
65 8 2 2 21.9 350
Plasma Amino Acid Response
15
Subjects The volunteers were six healthy male MIT students, aged 24--32 years. All were judged to be in good health as determined by physical examination and medical history. The experimental protocol was approved by the MIT Committee on the use of humans as experimental subjects and the Policy and Excecutive Committees of the MIT Clinical Research Centre. The subjects were required to sign informed consent forms.
Experimental Procedure The test meals were consumed each week on Friday mornings, at 8 o'clock, following a 12h overnight fast. On the previous day each subject consumed as standard diet (tuna fish sallad sandwich for lunch and beef + baked potato for dinner). During the other days the subjects were requested to persue their usual habits, to eat regularly and to keep a diary of their diet and physical activities. Fasting blood samples were drawn into heparinized test tubes, from the antecubital vein at 8 a.m. just before consuming the test meal. Postprandial blood samples were drawn at 21/2 and 31/zh following the test meal. These postprandial sampling times were chosen in order to detect the maximal changes in the pattern of free essential amino acids in blood plasma caused by the diet [1]. The diet records for each subject did not reveal any notable features that were considered important in the evaluation of the results.
Analysis Immediately after blood samples were drawn the red cells were removed by centrifugation and the plasma deproteinized by addition of sulfosalicylic acid [7]. The samples were kept frozen at - 2 0 ° C until thawed for analysis with the aid of an automatic amino acid analyzer (JEOL 5AH). Tryptophan was analysed separately as described previously [8].
Calculations Evaluation models for single meal plasma amino acid responses have been discussed in a previous paper [1]. The present data were evaluated accoding to two techniques: First, the model proposed by Graham and Placko [9]; These workers calculated the molar ratios of the essential amino acids for fasting and postprandial blood samples. The limiting amino acids will show the largest percentage decrease in the postprandial molar ratio (AMR%). Second, a modification of the plasma amino acid ratio (PAA ratio).procedure as proposed by Longenecker and Hause [10, 11]; In their original study the PAA ratio = - f f @ - x 100, where A is the fasting and B is the postprandial plasma amino acid concentration and R is the relative requirement of each specific essential amino acid according to Longenecker and Hause. The lowest PAA ratio (it may be negative) should identify the limiting amino acid. A modification introduced by us concerns the estimation of the factor R, "relative amino acid requirement". Longenecker and Hause [11] based their estimates of R on the daily requirements for individual amino acids and on the essential amino acid content of ovalbumin. However, for the reasons discussed in a previous paper [1], we have suggested that a more useful or appropriate estimate of R would be provided by direct experimental measurements of the relative net transport rates of individual amino acids from the blood plasma compartment during the early postprandial period. Thus in the present experiment the relative transport rates were calculated from changes in the plasma amino acid concentrations between 21/2 and 3I/2h after the test meals. In these calculations valine was chosen as a reference and assigned an arbitrary value 10. The derived estimates of relative requirements, R, were then used in determination of PAA ratios, as described above. Furthermore, in order to facilitate comparisons between single test
16
B.G. Ljungqvist et al.
Table 2. The composition of the amino acid mixtures in the test meals (g) Test mixture"
Lys
Trp
Thr
Val
Cys
Met
Ile
Lev
Egg
1.36
0.35
1.05
1.58
0.50
0.67
1.41
1.87
0
1.82
0.28
0.92
1.21
0.41
0.47
0.98
1.66
1
1.46
0.28
0.92
1.21
0.41
0.19
0.59
1.66
2
1.09
0.28
0.92
1.21
0.41
0.37
0.39
1.66
3
0.73
0.28
0.92
1.21
0.41
0.28
0.78
1.66
Egg = amino acid pattern 0 = amino acid pattern 1 = 40% Met, 60% Ile 2 = 40% Ile, 60% Lys 3 = 40% Lys, 60% Met
of whole egg protein of a fish and wheat protein mixture and 80% Lys of 0 mixture + additional Ala and 80% Met of 0 mixture + additional Ala and 80% Ile of 0 mixture + additional Ala
Table 3. The amino acid score a of the test mixtures Test mixture
Lys
Trp
Thr
Val
Ile
Leu
Met + Cys
Phe + Tyr 163
Egg
113
160
121
144
161
122
152
0
151
127
105
110
112
108
114
128
1
120
127
105
110
66
108
77
128
2
90
127
105
110
44
108
102
128
3
60
127
105
110
89
108
89
128
a
F A O / W H O provisional amino acid scoring pattern, 1973 [6]
meals evaluated according to this modified Longenecker and Hause procedure, the PAA ratios for all the essential amino acids were expressed in relation to the PAA ratio of valine, taken to be zero for this purpose. Statistical
Methods
The results of the single-meal tests are presented as means + S.D. The limiting order of the essential amino acids in the plasma amino acid responses was statistically evaluated by a nonparametric approach, based on a signed rank test procedure [ 12]. The reasons for trying a nonparametric approach (rather than a parametric one like the t-test) are mainly the following: (a) the measurements of amino acid response may be characterized as ordinal measurements rather than interval-scaled measurements; (b) the "response level" may differ considerably between subjects, and between the test occasions for the individual subjects. A brief description of the underlying statistical model is given by the following: Let X i and Y/ denote the observed response values for the i : t h subject on the amino acids X and I1; let X / - Y/= Di. Model."
where
Di = a + ei (i = 1 . . . . . n) e i = random error component
a = response difference "effect"
Plasma Amino Acid Response
17
Tyr
Phe
His
Arg
Asp
Ser
Glu
Pro
Gly
Ala
0.92 0.58 0.58 0.58 0.58
1.23 1.11 1.11 1.11 1.11
0.56 0.69 0.69 0.69 0.69
1.42 1.27 1.27 1.27 1.27
1.49 1.95 1.95 1.95 .1.95
1.89 0.92 0.92 0.92 0.92
2.55 4.19 4.19 4.19 4.19
0.94 1.26 1.26 1.26 1.26
0.75 1.05 1.05 1.05 1.05
1.38 1.22 2.24 2.61 2.68
Assumptions: the e-components are mutually independent; each e-component is symmetrically distributed around zero Hypotheses: Hoa= 0 (no response difference "effect") Hla < 0 (significant response difference "effect") a > 0 (significant response difference "effect") Comment. For a detailed description of the test procedure the reader is refered to Wilcoxon [12] and Pratt [13]. The statistical properties (consistency and efficiency) of the test statistic used have been analysed by e.g. Pitman [14], van Eeden and Benard [15], Hodges and Lehmann [16]. The calculations are described in detail in Appendix 1.
Results Two blood samples were lost due to technical difficulties including one fasting blood sample for subject P.O. receiving test mixture 2. The fasting amino acid concentrations for this occasion were estimated from the mean values obtained with subject P.O. This was considered justified because of the small variation among the amino acid concentrations in the various fasting samples within individual subjects during the time course of the study (Table 4). The estimates of the R values to be used in the calculation of PAA ratios are given in Table 5. These are based on the decreases in the plasma concentration of the essential amino acids in venous blood plasma between 21/2 and 3~ h postprandially. As pointed out earlier [1], the best estimates of R should be found at the postprandial time coinciding with the most rapid flow of amino acids from the plasma. Therefore, data obtained with the test meals where the plasma valine levels decreased by more than 20pmoles/l between the 2~ to 3~ postprandial hour were used to estimate R. Table 5 gives the PAA ratios 21/2 and 3~2 h after the different test meals using the Longenecker and Hause equation [11], and the R values determined above. The PAA ratios for the egg and 0 mixtures represent plasma amino acid responses after test meals containing adequate supplies of all essential amino acids (cf. Table 3). The range of the means and the standard deviations of these PAA ratios thus indicate the range that can be expected after single-meal .tests with protein diets of high biological value.
18
B.G. Ljungqvist et al.
Table 4. Fasting plasma amino acid concentrations in the subjects before the consumption of the test meals. Means ± S.D. from five occasions. (P.O. four occasions) Amino acid
Lys Trp Thr Val Met Met+Cys Ile Leu Phe Phe+Tyr
Subjects D.P.
C.G.
F.F.
F.S.
P.O.
B.L.
254± 26 72± 6 140± 13 339+ 34 28± 3 136±11 83-+ 11 165± 24 60± 5 136± 15
264± 31 67± 9 168± 15 300± 22 36± 2 159± 12 78± 6 161± 11 66± 4 131± 6
205+ 20 68+ 8 102± 23 267_+ 58 27± 5 149± 4 77-+ 9 145+ 14 61± 4 109_+ 11
239± 24 68± 5 153± 32 322± 43 30± 3 161±20 89+ 13 163± 17 63± 4 123± 15
244± 12 68± 9 177± 23 285+ 9 31± 1 148± 7 80± 1 160± 3 65± 1 135± 6
251± 15 73± 11 169_+ 17 304± 13 29± 3 157± 3 79± 3 158± 6 60± 4 126± 9
Table 5. The postprandial decrease of the essential amino acid concentrations in blood plasma between 2~2 and 31/2h after the test meals. The values are calculated in comparison to Valine Amino acid Dietary mixtures of amino acids
Lys ~p Thr Val Met Met+Cys Ile Leu Phe Phe+Tyr
0 (n = 4)"
1 (n = 4)a
2 (n = 5)a
15.6±3.5 2.6±1.6 3.3±2.1 10 1.4±0.8 2.2±5.6 5.2±1.6 6.9±1.6 2.5±0.9 1.7±2.8
13.2±3.9 2.8±2.7 4.9±1.1 10 0.9±0.1 1.4±1.6 2.7±1.2 7.0±1.5 2.4±1.2 3.4±0.9
12.0±5.9 2.4±2.1 5.6±1.4 10 1.4±0.6 3.7±1.5 2.3±1.3 6.7±2.3 2.6±0.6 3.7±0.8
3 (n = 5)~
Egg (n = 5)a
8.0±1.3 2.0±1.1 5.2±1.7 10 1.0±0.2 3.4±1.4 3.4±1.2 6.0±I.0 2.5±0.4 3.8±1.0
9.3±3.1 2.2±1.0 5.0±1.6 10 1.8±0.4 2.7±1.6 6.1±1.9 7.0±2.0 2.3±0.4 3.0±1.7
Grand mean 11.4±4.5 2.3±1.6 4.8±1.7 10 1.4±0.6 2.8±2.5 4.2±1.6 6.7±1.6 2.4±0.6 3.1±1.6
a The single meal tests where the plasma Val decline was less than 20 I.tmol/l between 2~2 and 3~ h were excluded
The g r a d u a l omission of lysine, isoleucine a n d m e t h i o n i n e from the test meals (mixture 1, 2, a n d 3) results in decreased P A A ratios of these a m i n o acids c o m p a r e d to the 0 test mixture. This effect is m u c h more m a r k e d with regard to isoleucine a n d m e t h i o n i n e t h a n with regard to lysine. As a result, a limiting dietary supply of isoleucine a n d m e t h i o n i n e are more readily detected in the p l a s m a a m i n o acid response t h a n a limiting supply of lysine (see statistical evaluation).
Plasma Amino Acid Response
19
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I l l l l l
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ExperimentedMedicine
Res. Exp. Med. (Berl.) 174, 13--28 (1978)
.© Springer~Verlag1978
Plasma Amino Acid Response to Single Test Meals in Humans* II. Healthy Young Adults Given Synthetic Amino Acid Mixtures B. G. Ljungqvist 1, U. S.-O. Svanberg 1, and V. R. Young 2 1Department of Medical Biochemistry, University of GOteborg, Fack, S-40033 GOteborg 33, Sweden 2Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass., USA
Summary. The postprandial plasma amino acid response to test meals containing different amino acid mixtures was analysed in six young adults. The amount of amino acids given in each test meal was equivalent with 3.5 g of nitrogen. Each test meal also contained a large amount of carbohydrate (mainly as wheat starch). Five different amino acid mixtures were used, and the effects on the plasma amino acid response of different dietary levels of lysine, isoleucine and methionine were studied in detail. The plasma amino acid responses were evaluated both by a modified Longenecker and Hause procedure (PAA ratios) and by the procedure proposed by G r a h a m and Placko (percentage changes in the postprandial essential amino acid molar ratios). The plasma amino acid response of lysine was found to be less sensitive to reductions in the dietary supply than the isoleucine and methionine responses evaluated by these procedures. The influence of altered levels of dietary supply on the postprandial plasma concentrations of isoleucine and methionine seemed to be specifically great at the intake levels where these essential amino acids become limiting for protein utilization according to the amino acid score of the test mixtures. The nutritional significance of the postprandial pattern and level of essential amino acids is discussed on basis of the present results. Experimental conditions and evaluation models are examined with regard to the use of single-meal plasma amino acid responses to assess the protein quality of human diets. Key words: Amino acids - Nutrition - Diet - Protein quality - Plasma amino acid response * This study was supported by grants from the Swedish Medical Research Council (grant no. K75-03X-3139-04A and no. B76-03X-3139-05B) and from the Swedish Nutrition Foundation Offprint requests to: B.G. Ljungqvist, M.D. (address see above)
0300-9130/78/0174/0013/$3.20
14
B.G. Ljungqvist et al.
If p r e d i c t a b l e n u t r i t i o n a l relationships can be established for the effects o f the a m i n o acid c o m p o s i t i o n o f a m e a l on the p a t t e r n a n d levels o f free a m i n o acids in venous b l o o d p l a s m a , then it s h o u l d be possible to exploit a m i n o acid changes in p l a s m a following a single test m e a l to assess the q u a n t i f y a b l e nutritive value o f a f o o d p r o t e i n o r mixture o f p r o t e i n sources. In a p r e v i o u s p a p e r we e x a m i n e d the prerequisites o f such single meal tests in relation to the m e t a b o l i c processes k n o w n to affect the p l a s m a a m i n o acid levels in the a b s o r p t i v e state [1]. The p r e s e n t investigation was p e r f o r m e d in o r d e r to evaluate the a d e q u a c y o f the principles o f e x p e r i m e n t a l design that were p r o p o s e d on basis o f these considerations. Since the m a i n objective o f o u r study was to define further the r e l a t i o n s h i p between d i e t a r y a m i n o acid c o m p o s i t i o n a n d its influence on the acute changes in p l a s m a a m i n o acid levels, mixtures o f crystalline a m i n o acids were used in the e x p e r i m e n t a l model. These mixtures c o u l d be m o d i f i e d readily for their a m i n o acid c o n t e n t a n d p a t t e r n , facilitating e x p e r i m e n t a l design in the investigations. The p l a s m a a m i n o acid single-meal technique was chosen for further s t u d y because this a p p r o a c h m a y p r o v i d e a convenient a n d p r a c t i c a l a p p r o a c h for h u m a n biological e v a l u a t i o n o f p r o t e i n - r i c h vegetable mixtures for children. Therefore, the present e x p e r i m e n t s utilized diets supplying a m i n o acids, c a r b o h y d r a t e s a n d fat, in a p r o p o r t i o n c o m p a r a b l e to that in the vegetable p r o t e i n mixtures s t u d i e d p r e v i o u s l y [2--5].
Materials and Methods
Composition of Test Meals The composition of the test meals is shown in Table 1. All the ingredients were suspended in water and consumed in this form. The total amino acid content of the test mixtures studied are given in Table 2. The basic 0 mixture had the same amino acid composition as a "Reference" protein meal that was being used in a parallel experiment [3]. Test mixtures 1, 2, and 3 provided levels of methionine, isoleucine and lysine in amounts equal to either 40, 60 or 80% of those provided by the 0 mixture. The amounts of the other amino acids were held constant. 20% differences in intake levels were considered appropriate for evaluation because it was thought that it should be possible to detect the effect of such differences if the single-meal technique was to be of practical value. In addition, we used an amino acid mixture based on the egg protein pattern. The amino acid scores [5] of the various test meals are given in Table 3. The different amino acid patterns were offered to the subjects in a randomized order, except that all subjects received the amino acid mixture based on the egg patterns as the final test meal.
Table 1. The composition of the test meals (g) Wheat starch Sucrose NaCI Vegetable oil Amino acid mixture Water
65 8 2 2 21.9 350
Plasma Amino Acid Response
15
Subjects The volunteers were six healthy male MIT students, aged 24--32 years. All were judged to be in good health as determined by physical examination and medical history. The experimental protocol was approved by the MIT Committee on the use of humans as experimental subjects and the Policy and Excecutive Committees of the MIT Clinical Research Centre. The subjects were required to sign informed consent forms.
Experimental Procedure The test meals were consumed each week on Friday mornings, at 8 o'clock, following a 12h overnight fast. On the previous day each subject consumed as standard diet (tuna fish sallad sandwich for lunch and beef + baked potato for dinner). During the other days the subjects were requested to persue their usual habits, to eat regularly and to keep a diary of their diet and physical activities. Fasting blood samples were drawn into heparinized test tubes, from the antecubital vein at 8 a.m. just before consuming the test meal. Postprandial blood samples were drawn at 21/2 and 31/zh following the test meal. These postprandial sampling times were chosen in order to detect the maximal changes in the pattern of free essential amino acids in blood plasma caused by the diet [1]. The diet records for each subject did not reveal any notable features that were considered important in the evaluation of the results.
Analysis Immediately after blood samples were drawn the red cells were removed by centrifugation and the plasma deproteinized by addition of sulfosalicylic acid [7]. The samples were kept frozen at - 2 0 ° C until thawed for analysis with the aid of an automatic amino acid analyzer (JEOL 5AH). Tryptophan was analysed separately as described previously [8].
Calculations Evaluation models for single meal plasma amino acid responses have been discussed in a previous paper [1]. The present data were evaluated accoding to two techniques: First, the model proposed by Graham and Placko [9]; These workers calculated the molar ratios of the essential amino acids for fasting and postprandial blood samples. The limiting amino acids will show the largest percentage decrease in the postprandial molar ratio (AMR%). Second, a modification of the plasma amino acid ratio (PAA ratio).procedure as proposed by Longenecker and Hause [10, 11]; In their original study the PAA ratio = - f f @ - x 100, where A is the fasting and B is the postprandial plasma amino acid concentration and R is the relative requirement of each specific essential amino acid according to Longenecker and Hause. The lowest PAA ratio (it may be negative) should identify the limiting amino acid. A modification introduced by us concerns the estimation of the factor R, "relative amino acid requirement". Longenecker and Hause [11] based their estimates of R on the daily requirements for individual amino acids and on the essential amino acid content of ovalbumin. However, for the reasons discussed in a previous paper [1], we have suggested that a more useful or appropriate estimate of R would be provided by direct experimental measurements of the relative net transport rates of individual amino acids from the blood plasma compartment during the early postprandial period. Thus in the present experiment the relative transport rates were calculated from changes in the plasma amino acid concentrations between 21/2 and 3I/2h after the test meals. In these calculations valine was chosen as a reference and assigned an arbitrary value 10. The derived estimates of relative requirements, R, were then used in determination of PAA ratios, as described above. Furthermore, in order to facilitate comparisons between single test
16
B.G. Ljungqvist et al.
Table 2. The composition of the amino acid mixtures in the test meals (g) Test mixture"
Lys
Trp
Thr
Val
Cys
Met
Ile
Lev
Egg
1.36
0.35
1.05
1.58
0.50
0.67
1.41
1.87
0
1.82
0.28
0.92
1.21
0.41
0.47
0.98
1.66
1
1.46
0.28
0.92
1.21
0.41
0.19
0.59
1.66
2
1.09
0.28
0.92
1.21
0.41
0.37
0.39
1.66
3
0.73
0.28
0.92
1.21
0.41
0.28
0.78
1.66
Egg = amino acid pattern 0 = amino acid pattern 1 = 40% Met, 60% Ile 2 = 40% Ile, 60% Lys 3 = 40% Lys, 60% Met
of whole egg protein of a fish and wheat protein mixture and 80% Lys of 0 mixture + additional Ala and 80% Met of 0 mixture + additional Ala and 80% Ile of 0 mixture + additional Ala
Table 3. The amino acid score a of the test mixtures Test mixture
Lys
Trp
Thr
Val
Ile
Leu
Met + Cys
Phe + Tyr 163
Egg
113
160
121
144
161
122
152
0
151
127
105
110
112
108
114
128
1
120
127
105
110
66
108
77
128
2
90
127
105
110
44
108
102
128
3
60
127
105
110
89
108
89
128
a
F A O / W H O provisional amino acid scoring pattern, 1973 [6]
meals evaluated according to this modified Longenecker and Hause procedure, the PAA ratios for all the essential amino acids were expressed in relation to the PAA ratio of valine, taken to be zero for this purpose. Statistical
Methods
The results of the single-meal tests are presented as means + S.D. The limiting order of the essential amino acids in the plasma amino acid responses was statistically evaluated by a nonparametric approach, based on a signed rank test procedure [ 12]. The reasons for trying a nonparametric approach (rather than a parametric one like the t-test) are mainly the following: (a) the measurements of amino acid response may be characterized as ordinal measurements rather than interval-scaled measurements; (b) the "response level" may differ considerably between subjects, and between the test occasions for the individual subjects. A brief description of the underlying statistical model is given by the following: Let X i and Y/ denote the observed response values for the i : t h subject on the amino acids X and I1; let X / - Y/= Di. Model."
where
Di = a + ei (i = 1 . . . . . n) e i = random error component
a = response difference "effect"
Plasma Amino Acid Response
17
Tyr
Phe
His
Arg
Asp
Ser
Glu
Pro
Gly
Ala
0.92 0.58 0.58 0.58 0.58
1.23 1.11 1.11 1.11 1.11
0.56 0.69 0.69 0.69 0.69
1.42 1.27 1.27 1.27 1.27
1.49 1.95 1.95 1.95 .1.95
1.89 0.92 0.92 0.92 0.92
2.55 4.19 4.19 4.19 4.19
0.94 1.26 1.26 1.26 1.26
0.75 1.05 1.05 1.05 1.05
1.38 1.22 2.24 2.61 2.68
Assumptions: the e-components are mutually independent; each e-component is symmetrically distributed around zero Hypotheses: Hoa= 0 (no response difference "effect") Hla < 0 (significant response difference "effect") a > 0 (significant response difference "effect") Comment. For a detailed description of the test procedure the reader is refered to Wilcoxon [12] and Pratt [13]. The statistical properties (consistency and efficiency) of the test statistic used have been analysed by e.g. Pitman [14], van Eeden and Benard [15], Hodges and Lehmann [16]. The calculations are described in detail in Appendix 1.
Results Two blood samples were lost due to technical difficulties including one fasting blood sample for subject P.O. receiving test mixture 2. The fasting amino acid concentrations for this occasion were estimated from the mean values obtained with subject P.O. This was considered justified because of the small variation among the amino acid concentrations in the various fasting samples within individual subjects during the time course of the study (Table 4). The estimates of the R values to be used in the calculation of PAA ratios are given in Table 5. These are based on the decreases in the plasma concentration of the essential amino acids in venous blood plasma between 21/2 and 3~ h postprandially. As pointed out earlier [1], the best estimates of R should be found at the postprandial time coinciding with the most rapid flow of amino acids from the plasma. Therefore, data obtained with the test meals where the plasma valine levels decreased by more than 20pmoles/l between the 2~ to 3~ postprandial hour were used to estimate R. Table 5 gives the PAA ratios 21/2 and 3~2 h after the different test meals using the Longenecker and Hause equation [11], and the R values determined above. The PAA ratios for the egg and 0 mixtures represent plasma amino acid responses after test meals containing adequate supplies of all essential amino acids (cf. Table 3). The range of the means and the standard deviations of these PAA ratios thus indicate the range that can be expected after single-meal .tests with protein diets of high biological value.
18
B.G. Ljungqvist et al.
Table 4. Fasting plasma amino acid concentrations in the subjects before the consumption of the test meals. Means ± S.D. from five occasions. (P.O. four occasions) Amino acid
Lys Trp Thr Val Met Met+Cys Ile Leu Phe Phe+Tyr
Subjects D.P.
C.G.
F.F.
F.S.
P.O.
B.L.
254± 26 72± 6 140± 13 339+ 34 28± 3 136±11 83-+ 11 165± 24 60± 5 136± 15
264± 31 67± 9 168± 15 300± 22 36± 2 159± 12 78± 6 161± 11 66± 4 131± 6
205+ 20 68+ 8 102± 23 267_+ 58 27± 5 149± 4 77-+ 9 145+ 14 61± 4 109_+ 11
239± 24 68± 5 153± 32 322± 43 30± 3 161±20 89+ 13 163± 17 63± 4 123± 15
244± 12 68± 9 177± 23 285+ 9 31± 1 148± 7 80± 1 160± 3 65± 1 135± 6
251± 15 73± 11 169_+ 17 304± 13 29± 3 157± 3 79± 3 158± 6 60± 4 126± 9
Table 5. The postprandial decrease of the essential amino acid concentrations in blood plasma between 2~2 and 31/2h after the test meals. The values are calculated in comparison to Valine Amino acid Dietary mixtures of amino acids
Lys ~p Thr Val Met Met+Cys Ile Leu Phe Phe+Tyr
0 (n = 4)"
1 (n = 4)a
2 (n = 5)a
15.6±3.5 2.6±1.6 3.3±2.1 10 1.4±0.8 2.2±5.6 5.2±1.6 6.9±1.6 2.5±0.9 1.7±2.8
13.2±3.9 2.8±2.7 4.9±1.1 10 0.9±0.1 1.4±1.6 2.7±1.2 7.0±1.5 2.4±1.2 3.4±0.9
12.0±5.9 2.4±2.1 5.6±1.4 10 1.4±0.6 3.7±1.5 2.3±1.3 6.7±2.3 2.6±0.6 3.7±0.8
3 (n = 5)~
Egg (n = 5)a
8.0±1.3 2.0±1.1 5.2±1.7 10 1.0±0.2 3.4±1.4 3.4±1.2 6.0±I.0 2.5±0.4 3.8±1.0
9.3±3.1 2.2±1.0 5.0±1.6 10 1.8±0.4 2.7±1.6 6.1±1.9 7.0±2.0 2.3±0.4 3.0±1.7
Grand mean 11.4±4.5 2.3±1.6 4.8±1.7 10 1.4±0.6 2.8±2.5 4.2±1.6 6.7±1.6 2.4±0.6 3.1±1.6
a The single meal tests where the plasma Val decline was less than 20 I.tmol/l between 2~2 and 3~ h were excluded
The g r a d u a l omission of lysine, isoleucine a n d m e t h i o n i n e from the test meals (mixture 1, 2, a n d 3) results in decreased P A A ratios of these a m i n o acids c o m p a r e d to the 0 test mixture. This effect is m u c h more m a r k e d with regard to isoleucine a n d m e t h i o n i n e t h a n with regard to lysine. As a result, a limiting dietary supply of isoleucine a n d m e t h i o n i n e are more readily detected in the p l a s m a a m i n o acid response t h a n a limiting supply of lysine (see statistical evaluation).
Plasma Amino Acid Response
19
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