Download The Effect of Protein Loads on Plasma Amino Acid Levels

Clinical Science and Molecular Medicine (1973) 45,827-832.
THE EFFECT OF PROTEIN LOADS O N PLASMA
AMINO ACID LEVELS
T . P A L M E R , M A R Y A. ROSSITER, B. LEVIN(')
V. G. OBERHOLZER
AND
Biochemistry Department, Queen Elizabeth Hospital for Children, London
(Received 18 June 1973)
SUMMARY
1. After ingestion of up to 1.2 g of protein/kg body weight by adults, plasma concentrations of all amino acids, including glutamine and glutamic acid, rose to a maximum
within 5 h.
2. The increases in concentration depended on the amount of protein ingested.
3. The changes were not due to diurnal variation in plasma amino acid levels,
so, protein loading tests may be of value in the assessment of protein absorption.
Key words: amino acids, plasma, meat, circadian rhythm.
The amount of amino-nitrogen ingested in a single protein meal may be up to ten times that
normally present in the extracellular pool, but the rate of uptake or utilization by liver and
tissues is so rapid (Van Slyke & Meyer, 1913-14) that peripheral plasma amino acid levels are
maintained within a very narrow range (Scriver, 1968). Yearick & Nadeau (1967), using ionexchange chromatography, did show that in adults most plasma amino acid levels reached
maximum values 2-3 h after a meal and then began to fall, but the significance of this became
uncertain when a circadian periodicity in plasma amino acid levels was demonstrated (Feigin,
Klainer & Beisel, 1968; Feigin, Beisel & Wannemacher, 1971). Maximum levels were found to
occur between 12.00 and 20.00 hours and minimum between 04.00 and 08.00 hours.
We have investigated in adults the effects of meat meals on the plasma concentrations of
all amino acids, including glutamine and glutamic acid, and have done control studies to determine to what degree the changes after protein ingestion could be due to diurnal variation.
SUBJECTS A N D M E T H O D S
Nine healthy young adults, all volunteers, ate a weighed amount of meat giving 0.65485 g of
protein/kg body weight at about 09.00 hours following an overnight fast. The meat was lean
Present address: East Ham Memorial Hospital, London E7 8QR.
Correspondence:Dr T. Palmer, Biochemistry Department, Queen Elizabeth Hospital for Children, Hackney
Road, London E2 8PS.
(l)
827
T. Palmer et al.
828
steak (steamed or grilled) or lean boiled ham, and the protein content was calculated from the
tables of McCance & Widdowson (1946). Blood samples were taken fasting and 2 h after
the start of the meal (14 h in two cases). In order to investigate in more detail the rise and fall
of plasma amino acid concentrations, further blood specimens were taken from two of these
subjects at 3* and 5 h after the meal. Blood was also collected from an additional subject during
the first 2 h after a meat meal containing 1.2 g of protein/kg given at 09.30 hours and from
between 24 and 8 h after an identical meal given on a separate occasion at 08.00 hours. These
three subjects were males of very similar age and weight. As controls, six healthy young adults
fasted overnight until 11.30 hours, and blood was collected from these at 09.30 and 11.30 hours.
All blood specimens were collected from the forearm by venepuncture into lithium heparin.
The subjects included both males and females, and all pursued their normal activities during the
course of the tests.
Plasma specimens from the meat loading tests were deproteinized with picric acid (Stein &
Moore, 1954)and amino acids were determined using a Technicon single column (140 cm x 0.6
cm, Chromobeads 'A' resin) analyser with a modification (Palmer, 1968) of the standard techTABLE
1. Plasma amino acid concentrations bmol/l) in adults before and after a meat meal
(0.65485 g of protein/kg) and during a continued fast, with a statistical comparison of the
changes found. The probability P indicates the significance of the differences observed
between the individual changes after protein ingestion and those during continued fast (see
the text). ABA = a-amino-n-butyric acid.
Protein load (n = 9)
Fasting
Mean SD
Taurine
HYP
Thr
Ser
Gln
Glu
Pro
G~Y
Ala
ABA
Val
Met
Ile
Leu
Tyr
Phe
Orn
LYS
His
'4%
53
12
130
105
680
45
165
220
310
21
175
17
50
100
44
49
48
170
94
85
9
3
20
25
95
20
40
25
65
9
35
4
13
20
11
12
10
30
19
16
Continued fast (n = 6)
2 h after
09.30 hours
11.30 hours
Mean SD
Mean SD
Mean SD
90
18
205
150
830
62
235
265
390
27
325
37
130
240
78
76
76
330
135
160
34
32
41
14
P < 0405
130 30
130 35
715 75
70 30
145 35
245 100
315 60
7
15
240 45
5
14
54 14
110 15
51 10
5
48
61 15
175 35
85 16
70 37
115
125
15
35
80
13
65
80
65
3
P < 0.001
P <0.001
P < 0~001
60
5
30
30
85
10
75
45
70
9
25
9
30
40
22
15
12
60
20
30
645
61
155
235
300
16
235
18
51
105
50
46
56
185
87
83
0.05<P<0.10
0.10<P<0.25
P = 0.005
P<O.01
0.10<P<0.25
55
P<0.001
7
P<0.001
P < 04w1
12
30
P<0.001
8
P<0.001
8
P < 0~001
9
P<O~Ool
35
P<O.Ool
14
P<0.001
P<O001
37
Efect of protein loads on plasma amino acids
829
nique. The recommended Technicon sodium citrate buffer system and varigrad composition
(Technicon, 1967) were used, except that no methanol was added, and the column was kept
at 35°C for 44 h before raising the temperature to 60°C for the rest of the run.
The plasma specimens during prolonged fast were taken at a later date when a Locarte
'Automatic' Amino Acid Analyser, giving a much shorter analysis time, was available. The
samples, after deproteinization with an equal quantity of sulphosalicylic acid solution (30 g/l),
were analysed on this instrument (25 cmx0-9 cm column) using sodium citrate buffers of
standard composition. The timing programme was pH 3.25 for 80 min, pH 4.25 for 90 min,
pH 6.65 for 130 min, NaOH for 40 min and pH 3.25 for 140 min. The temperature was 28°C
for the first 75 min and then 55°C for the rest of the run.
The two instruments were calibrated independently with standard mixtures, and thus give
identical results.
RESULTS
In the series of subjects who ate between 0.65 and 0.85 g of protein/kg body weight, the mean
plasma concentration of each amino acid 2 h after protein ingestion was higher than the correTABLE2. Changes in plasma amino acid concentrations in individual adults after protein loads. Results at 2 h
and before, and those at 24 h and after, are from separate tests on the same individual (see the text). ABA =
a-amino-n-butyric acid.
Increment above fasting concentration (,anol/l)
(1.2 g protein/kg body weight)
Taurine
HYP
Thr
Ser
Gln
Glu
Pro
Cit
GlY
Ala
ABA
Val
Met
Ile
Leu
TYr
Phe
Orn
LYS
His
Art3
th"'
l h
4
5
15
10
-20
4
20
5
5
25
22
17
17
8
70
60
30
35
260 145
-9 -11
55
90
4
6
60
90
165 105
2
3
50 105
11
11
73
35
60 105
19
31
18
18
21
11
125 165
42
42
74
54
-5
5
-1
12
15
5
9
0
35
-3
8
1)h
(0.85 g/kg)
5h
64h
8h
lth
13
16
45
2
13
4
80 120 110
35
75
40
260 260 220
4 -20 -26
70
45 105
13
18
12
55 100
30
60
140 155
-2
6
14
140 185 285
22
39
43
88 103 148
125 145 205
53
58
33
43
46
34
42
35
23
170 225 215
57
37
42
64 105 100
11
11
-3
29
9
17
25
13
0
2 1 4
2
6
65
65
65
30
50
40
30
30
30
30
120
70 150 115
40
36
46
5 -20
40
40
50 100
60
50
5
15
4
22
17
50
0 -15
10
5
50 -10
160
25 -40
1
1
1
0
2
5
120 165 175
95 180
18
27
24
12
14
88 118 108
42
95
145 195 165
65 165
35
46
18
18
18
12
18
5
16
17
20
26
27
26
41
140 110
40 120 130
35
25
20
38
20
90
70
50
37
52
2h
24h
0
5
25
-15 -25
65
75
-44
-40
-44
-60
3
6
-55 -65
-85 -115
-3
13
80
120
1
14
17
38
20
45
19
0
10 -2
9
18
5 -20
-3
-6
-6
0
Time after meal.
34h
(0.65 g/kg)
5h
l g h 34h
5h
-1
0
0
0
65
-30
-20
11
-35
-85
1
110
0
42
65
7
1
27
5
13
-2
T. Palmer et al.
830
1
300
Lysine
Valine
A
200
100
0
I
4
.-c
I
I
I
I
I
I
I
I
0
2
4
6
8
200
0
0
S
0
v
100
200
0
I00
- 100
0
0
2
4
6
8
Time after load (h)
FIG.1. Changes in plasma levels of selected amino acids in individual subjects after meat meals.
A , 1.2 g of protein/kg body weight (results at 2 h and before, and those at 24 hand after, are from
separate tests on the same individual, and this is indicated by a break in the line); 0,0.85 g of
protein/kg; W, 0.65 g of protein/kg.
sponding fasting mean (Table 1). The sum of the mean concentrations of each individual amino
acid 2 h after the meal was found to be 1-5times the sum of the mean fasting concentrations.
Glutamine, the most abundant amino acid in plasma, and glycine showed the smallest changes,
their mean concentrations 2 h after the meal being 1.2 times the mean fasting concentrations,
while the greatest changes were found in the concentrations of the branched-chain amino acids,
the mean concentration of isoleucine after the meal being 2.6 times its fasting value.
In three subjects studied more fully, the largest protein load produced the most sustained
increase in plasma amino acid concentrations (Table 2 and Fig. 1). The observed rises and falls
in the concentrations of individual amino acids were similar to those reported by Yearick &
Nadeau (1967), maximum concentration occurring between 1 and 5 h after the load. Glutamine,
which was not estimated by Yearick & Nadeau (1967), responded similarly. Alanine was the
first to reach a maximum and the branched-chain amino acids the last. N o more than trace
amounts of aspartic acid could be detected at any time. The glutamic acid concentration
scarcely rose above its fasting level, to which it returned within 5 h, whereas Yearick & Nadeau
(1967) found its concentration rose throughout the 7 h after a protein load.
Most plasma amino acid concentrations in the subjects on a continued fast fell slightly
Efect of protein loads on plasma amino acids
83 1
between 09.30 and 11.30 hours, confirming the recent findings of Armstrong & Stave (1973).
The probability that the observed rises after protein ingestion could be due to diurnal variation
was tested for each amino acid by comparing the difference in the mean of the changes in the
individual subjects from 09.30 to 11.30 hours between the group having protein and the group
on prolonged fast. The differences were significant (P<O.Ol) for all except glutamic acid, proline and a-amino-n-butyric acid (Student's t-test, small-sample method), and even concentrations of these rose by a greater amount after protein ingestion (Table 1). The test was unpaired
since only three subjects were common to both groups.
DISCUSSION
Feigin et al. (1968, 1971) have clearly established that a circadian periodicity in plasma amino
acid concentrations does exist. However, the same investigators also found evidence to indicate
that although a large protein load ingested during the evening does not disrupt the natural
rise and fall in concentrations, a similar one ingested during the morning does so for a short
period of time. They were unable to detect the short-term effect of meals in their main study
because they took blood specimens only at 4-hourly intervals. In the present study, the different
responses to different amounts of ingested protein and the relative stability of amino acid
concentrations during a continued fast both confirm a connection between the ingestion of a
protein meal during the morning and subsequent changes in concentrations. The sum of the
mean concentrations of all the amino acids 2 h after a meal was 1.5 times the sum of the fasting
means, whereas Feigin et al. (1968) found that the sum of the means even at the time of maximum daily values (20.00 hours) was only 1.3 times that at the time of the minimum (08.00
hours). This underlines the differences between the short-term and long-term variations in
concentrations.
The major difference between the observed response of plasma amino acids to protein meals
and the findings of Yearick & Nadeau (1967) is in respect of glutamic acid. Their findings
could be explained by assuming that their samples were estimated consecutively, starting with
the fasting sample, so that the later samples suffered decay of glutamine on storage. This can
be quite rapid, even at - 15"C, glutamic acid being one of the products (Dickinson, Rosenblum
& Hamilton, 1965; Palmer, 1973).
The ingestion of protein stimulates the release of large amounts of endogenous protein into
the intestinal lumen, and hydrolysis of the mixture of exogenous and endogenous proteins has
been reported to yield an amino acid pool with relatively constant molar ratios regardless of
the meal ingested (Nasset, 1965). Thus little correlation would be expected between the amino
acid content of ingested protein and the individualamino acid response. Even so, the observation
that there was little or no increase in plasma aspartic acid and glutamic acid concentrations
after the meals is remarkable, since these are the major amino acid constituents of meat protein
(Spector, 1956). However, it is known that amino acids may undergo changes during absorption, and the main result of the ingestion of aspartic acid and glutamic acid is a rise in plasma
alanine (Wiseman, 1968).
Factors influencing the response to protein meals are complex, and in addition to the above
include rates of absorption from the intestine, rates of utilization or uptake by liver and tissues,
and rates of stimulated release from the tissues of individual amino acids (Wiseman, 1968;
Palmer, 1973). A prolonged absence of dietary glucose leads to increased utilization of amino
832
T. Palmer et al.
acids, especially alanine, by the liver for gluconeogenesis and decreased uptake by the tissues
(Felig, 1973). The branched-chain amino acids are deaminated more slowly than other amino
acids in the liver, and normally depend on the extrahepatic tissues for their metabolism (Miller,
1962; Ning, Lowenstein & Davidson, 1967; Felig, 1973). This explains the finding that they
show the most sustained plasma response when meat, which had a low carbohydrate content,
is given after an overnight fast. It cannot be explained by a slow intestinal absorption of the
branched-chain amino acids, since these are known to be absorbed fastest of all (Adibi, Gray
& Menden, 1967).
The present study has shown that the changes in plasma amino acid concentrations after
protein loads, although dependent on many factors, are meaningful. Their investigation may
be of clinical value, provided results are compared with control tests performed at the same
time of day.
ACKNOWLEDGMENTS
We are grateful to those of our colleagues who volunteered to take part in this study.
M.A.R. was supported by a grant from the Heinz Foundation.
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