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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. REFERENCES E. (1967) The kinetics of amino acid absorption and alteration of plasma ADIBI,S.A., GRAY,S.J. & MENDEN, composition of free amino acids after intestinal perfusion of amino acid mixtures. American Journal of Clinical Nutrition, 20, 24-33. ARMSTRONG, M.D. & STAVE,U. (1973) A study of plasma amino acid levels. Metabolism, 22,549-560. 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