Human Nutrition and Metabolism

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1 Human Nutrition and Metabolism Postprandial Kinetics of Dietary Amino Acids Are the Main Determinant of Their Metabolism after Soy or Milk Protein Ingestion in Humans 1 Cécile Bos, Cornelia C. Metges,* 2 Claire Gaudichon, 3 Klaus J. Petzke,* Maria E. Pueyo, Céline Morens, Julia Everwand,* Robert Benamouzig and Daniel Tomé Institut National de la Recherche Agronomique (INRA), Unité de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique Paris-Grignon (INA-PG), Paris Cedex 05, France; *Deutsches Institut für Ernährungsforschung-Potsdam (DIfE), Unit Protein Metabolism, Bergholz-Rehbrücke, Germany; and Service de Gastroentérologie, Hôpital Avicenne, Bobigny, France ABSTRACT Soy proteins have been shown to result in lower postprandial nitrogen retention than milk proteins, but the mechanisms underlying these differences have not been elucidated. To investigate this question, we measured the postprandial kinetics of the appearance of individual 15 N-amino acids in the serum of healthy adults after the ingestion of either 15 N-soy (n 8) or 15 N-milk proteins (n 8) in a mixed single meal (46 kj/kg). The kinetics of total and dietary amino acids (AA) in the peripheral circulation were characterized by an earlier and higher peak after soy protein ingestion. Dietary AA levels peaked at 2.5 h in the soy group vs. 3.9 h in the milk group (P 0.02). This time interval difference between groups was associated with a faster transfer of dietary N into urea in the soy group (peak at 3 vs h in the milk group, P 0.005) and a higher level of incorporation into the serum protein pool from 3 to 8 h after the soy meal. The dietary AA pattern in the peripheral blood closely reflected the dietary protein AA pattern. Postprandial glucose, insulin, and glucagon levels and profiles did not differ between groups. Soy AA were digested more rapidly and were directed toward both deamination pathways and liver protein synthesis more than milk AA. We conclude that differences in the metabolic postprandial fates of soy and milk proteins are due mainly to differences in digestion kinetics; however, the AA composition of dietary proteins may also play a role. J. Nutr. 133: , KEY WORDS: dietary protein amino acids postprandial metabolism GC-C-IRMS humans We and others have recently shown that the postprandial nitrogen metabolism of intact dietary proteins differs after the ingestion of milk and soy protein, mainly because of the greater deamination of soy amino acids than of milk amino acids (AA) 4 (1 4), but also because of their differing capacity to support protein synthesis (5). First, soy and milk differ in their amino acid composition, i.e., soy protein contains lower levels of methionine, branched-chain amino acids (BCAA), lysine and proline, and higher levels of aspartate, glycine, arginine and cystine than milk protein. In addition, soy proteins are less digestible than milk proteins on a nitrogen basis (1,6), and the digestibility of individual amino acids differs between these two proteins (7). Threonine, in particular, reaches 93% digestibility in milk protein but only 89% in soy protein, whereas the values for 1 Supported by grants of ARILAIT RECHERCHES and PROCOPE grants of the Deutsche Akademische Austauschdienst, Bonn, Germany, and the French Foreign Office, Paris, France. 2 Present address: Research Institute for the Biology of Farm Animals, Research Unit Nutritional Physiology Oskar Kellner, Wilhelm-Stahl-Allee 2, Dummerstorf, Germany. 3 To whom correspondence should be addressed. gaudicho@inapg.fr. 4 Abbreviations used: AA, amino acid; APE, atom % excess; AUC, area under the curve; BIA, bioelectrical impedance analysis; GC-C-IRMS, gas chromatography-combustion-isotope ratio mass spectrometry; TBW, total body water. valine are 96 and 92%, respectively. Moreover, individual amino acid availability for extraintestinal tissues is further reduced by amino acid uptake into portal-drained viscera (8,9). Consequently, it can be assumed that the pattern of amino acids reaching the liver is relatively more unbalanced with soy protein than with milk protein, which may result in higher postprandial deamination due to their limited utilization for protein synthesis (10,11). Second, the digestion and absorption kinetics of dietary proteins influence catabolic and anabolic activities at the whole-body level (12) and in the liver (13). Milk proteins contain caseins ( 80%), which clot in the stomach during the course of digestion, and whey proteins, which are soluble and rapidly emptied from the stomach (14,15); in contrast, soy proteins are composed mainly of a soluble protein fraction. Despite these differences, the gastric emptying rates for soy and milk proteins have been found to be identical when measured on either a nitrogen or dry matter basis (16 20). However, few data (obtained in both rats and humans) are available to suggest that soy protein exhibits a higher intestinal transit rate than milk protein (13,20). Finally, the AA pattern of dietary proteins may have different effects on the secretion of insulin (21); indeed, it has been shown that insulin secretion was positively correlated with plasma leucine, phenylalanine and arginine concentrations (22,23). Because the AA compositions of soy and milk /03 $ American Society for Nutritional Sciences. Manuscript received 29 November Initial review completed 8 January Revision accepted 15 February

2 POSTPRANDIAL METABOLISM OF DIETARY AMINO ACIDS 1309 TABLE 1 Subject characteristics and composition of test meals Milk group Soy group Subject characteristics1 Gender 5 women, 3 men 4 women, 4 men Age, y Body weight, kg Height, m Body mass index, kg/m Body fat, % Test meals Energy, kj/kg N-labeled protein, g/kg Maltodextrin, g/kg Sucrose, g/kg Sunflower oil, g/kg Values are means SD, n 8/group. proteins differ, variations in hormone secretions may occur with different protein sources, which, in turn, would be likely to influence the postprandial sparing of dietary amino acids (24). Such hormonal differences between protein sources have been observed by some authors (23,25) but not by others, including our previous works in which we could not demonstrate any difference in insulin profile as a function of the nature of the protein ingested in a mixed meal, or any stimulation of insulin by the ingestion of protein alone (1,3,26). To better clarify the reasons for the differing postprandial deamination of soy and milk proteins, we traced the pattern of amino acids of dietary origin reaching the periphery, as well as the hormonal response induced by a single meal. For this purpose, healthy volunteers ingested a mixed meal containing 15 N-labeled milk or soy proteins, after a 1-wk period of standardization to a protein intake of 1 g/(kg d). The appearance of each 15 N-AA was monitored in the serum using gas chromatography-combustion-isotope ratio mass spectrometry (GC- C-IRMS), and postprandial hormonal profiles were determined. SUBJECTS AND METHODS Protocol. The study protocol was approved by the Institutional Review Board for Saint Germain-en-Laye (France). After obtaining their written, informed consent, 16 healthy volunteers (7 men and 9 women) were included in this study. Their mean age and body mass index were 28 5 y and kg/m 2, respectively (Table 1). Total body water (TBW) and the percentage of body fat were determined using multiple frequency Bio-Impedance Analysis (BIA); the vendor-provided equations were used for the calculations (Analycor 5w, Spengler, Cachan, France). During the week before the study, the subjects were asked to follow a controlled diet adjusted to their body weight, providing 138 kj/(kg d), 1 g protein/(kg d), 4.5 g carbohydrates/(kg d) and 1.2 g fat/(kg d). The subjects were instructed to comply strictly with the specified meals and to weigh precisely the foods ingested. After this diet standardization period, they were asked to attend the Avicenne Hospital, Bobigny, France, on the morning of the study day, having fasted overnight, and were randomly assigned to one of the two experimental groups, i.e., soy or milk protein. The test meals represented one third of their daily energy intake, i.e., 46 kj/kg, and had a standard nutrient composition, i.e., 15, 55 and 30% of total energy as protein, carbohydrate and fat, respectively (Table 1). The protein constituent of the test meals was either milk or soy protein and was intrinsically 15 N-labeled, as previously described (1,6). The 15 N-labeling of milk protein was obtained after administration of 15 N-ammonium sulfate to a lactating cow and the total protein fraction was concentrated by diafiltration and dried (81.5% of protein in the isolate). Soy beans (var. Chandor) were 15 N-labeled by foliar spraying of K 15 NO 3. Soy protein isolate was obtained after acid precipitation of the protein fraction, centrifugation, neutralization, sterilization (140 C for 40 s) and lyophilization (final protein content: 72.3% of protein). Amino acids of both protein isolates were uniformly 15 N-labeled (Table 2). The amount of dietary protein ingested was 0.41 g/kg, representing an absolute amount of g in the milk group and g in the soy group. The difference between groups was due to the lower mean body weight of the subjects in the soy group. A small catheter for blood withdrawal was inserted in a vein of the nondominant forearm. Baseline samples of blood and urine were collected immediately before the start of the experiment. The volunteers ingested their experimental meal within 10 min and were not given other food until the end of the 8-h observation period. Drinking water (100 ml) was consumed hourly. Blood samples were collected every 30 min for 3 h and then hourly for the remaining 5 h. Aliquots of serum or plasma were collected and stored at 20 C until analysis. Urine was collected every 2 h for 8 h, measured for volume and then stored at 4 C before analysis. Analytical procedures. Total urea levels in the urine and serum, and creatinine levels in the urine were determined using an enzymatic method on a Dimension Automate (Dupont de Nemours, Les Ulis, France). Ammonia was measured in the urine by an enzymatic method using a clinical analyzer (Kone, Evry, France). Plasma glucose was measured by a glucose oxidase method (kit Glucose GOD-DP, Kone). Plasma hormones (insulin, glucagon) were measured by RIA (insulin: Bio-Rad, Marnes la Coquette, France; glucagon: Nichols, Paris, France). For the determination of individual serum free AA concentrations, the protein fraction of 800 L serum was precipitated with 40 L of a sulfosalycilic acid solution (1 g/ml). After1hofprecipitation at 4 C and centrifugation for 20 min at 3000 g, the supernatant was removed and dried. It was then resuspended in a lithium citrate buffer (ph 2.2), filtered on 0.2- m filters and injected into a HPLC system TABLE 2 15N-enrichments and intakes of dietary amino acids of the milk and soy protein isolates ingested as part of a mixed meal by healthy subjects 15N-enrichment Intake2 Milk protein Soy protein Milk group Soy group APE1 mol/kg body Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Alanine Aspartate asparagine Glutamate glutamine Glycine Proline Serine Arginine ND3 ND Tyrosine Atom % excess (APE) of each AA was determined using gas chromatography-combustion-isotope ratio mass spectrometry. 2 Intakes of dietary protein in the two groups (n 16) were adjusted to the body weight (0.41 g protein/kg body). 3 ND, not determined.

3 1310 BOS ET AL. (Bio-tek Instruments, St Quentin en Yvelines, France) combined with a postcolumn ninhydrin derivatization. Separation was performed on cation exchange resin. All AA except proline (440 nm) were detected at 540 nm. -Aminobutyric acid was used as an internal standard. The 15 N-enrichment of individual AA was measured using gas chromatography/combustion/isotope ratio mass spectrometry (GC-C- IRMS) analysis. Free AA from serum samples were purified as previously described (27), and derivatized to N-pivaloyl-i-propyl AA esters. Briefly, the AA were treated with 1 ml of a thionylchloride and i-propanol solution. The mixture was heated for 30 min at 110 C and then dried under a gentle stream of nitrogen at 60 C and redissolved in pyridine (100 L). After the addition of pivaloylchloride (100 L), each solution was acylated for 30 min at 60 C, cooled and then 2 ml of dichloromethane was added. The solution was then passed through a silica gel column and the eluate dried in a gentle nitrogen stream at room temperature. The vials were covered and the derivatives kept refrigerated until GC-C-IRMS analysis; just before this procedure, the derivatized AA were resuspended in 50 L ethyl acetate. The analysis was carried out on a Finnigan Delta S system (Thermoquest, Bremen, Germany) as previously described (28). A combustion interface allowed the production and purification of N gas from GC-separated compounds to enter the IRMS. An Ultra 2 capillary column (50 m, Hewlett-Packard, Waldbronn, Germany) was used to separate the AA. The carrier gas was He. A standard N 2 gas (known isotopic composition) was introduced for calibration. 15 N-enrichment in urinary and serum urea was measured by IRMS after extraction on cation-exchange resins, as previously described (1). Briefly, the serum urea was isolated after the precipitation of serum protein with sulfo-salicylic acid. The supernatant was buffered to ph 7 and urea was extracted on a cation-exchange resin (Biorad Dowex AG50-X8, mesh , Interchim, Montluçon, France) in the presence of urease. The resin containing urea-derived ammonia from serum was washed with distilled water and stored at 4 C. The pellet was dried, weighed and used for the determination of 15 N- enrichment in the serum protein fraction. Urine samples (7 ml) were put on resins to extract ammonia. The supernatant was transferred on a second resin and kept at 30 C for 2 h with urease for urea hydrolysis. Before isotope determination, the resins were eluted with KHSO 4 (2.5 mol/l). The 15 N-enrichment was measured by isotopic ratio mass spectrometry (Optima, Fisons Instruments, Manchester, UK) coupled to the elemental analyzer (NA 1500 series 2, Fisons Instruments). A calibrated nitrogen gas was used as the reference to derive the 15 N/ 14 N ratio. Calculations and statistics. The time course of dietary N incorporation (N diet, expressed as a percentage of the ingested amount) into the different monitored body N pools (serum free AA, serum protein, serum and urinary urea) was calculated using the following equation: N diet (t) N tot APE s (t)/ape m /N m 100 where N tot is the N content of the pool (mmol N), APE s (t) isthe 15 N-enrichment above the baseline enrichment (expressed as atom % excess, APE) in the N pool sampled at time t, APE m is the 15 N- enrichment of the meal (APE) and N m is the N content of the meal (mmol N). N tot in the serum protein pool was determined as the serum content of protein N, respectively, multiplied by the serum volume, estimated to be 5% of the body weight (29). Body urea pool size was calculated as the product of the urea concentration and TBW, measured by BIA, which has been recognized as an accurate method for total body water determination in young, healthy adults (30). A factor of 0.92 was used to take account of the plasma water content. For urinary urea, N tot was calculated as the product of urinary urea N concentration and the volume of urine excreted. The transfer of dietary N to the urea pool (or dietary urea production, UP diet ) was evaluated from the dietary N excreted in the urinary urea for each 2-h interval, corrected for the change in the dietary N content of the body urea pool over the same period, as follows: UP diet t t 2 UU diet t t 2 BU diet t 2 BU diet t where UU is urinary urea N from dietary origin and BU is body urea N from dietary origin. Dietary N retention, or net postprandial protein utilization, was derived from the amount of N ingested, minus the amount nonabsorbed at the intestinal level, i.e., 5 and 8.5% for milk protein and soy protein, respectively, as published previously (1,6), and minus the amount of urea produced for the 8-h period. The appearance in the serum of dietary N-containing AA (AA diet,in mol/l) was calculated for each AA as follows: AA diet (t) AA tot (t) APE s (t)/ape m where AA tot (t) is the concentration of individual AA in the serum sample at time t ( mol/l), APE s (t) and APE m the 15 N-enrichment excess of the individual sample and the meal, respectively. These values represented the quantity of individual AA carrying a nitrogen atom of dietary origin. For AA that do not transaminate (lysine and threonine), the amount of dietary N-containing AA directly reflected the amounts of dietary AA entering the systemic circulation. Areas under the curve (AUC) for serum 15 N-AA and hormones were computed. The results are expressed as mean SD. Group differences for single planned comparisons were tested using an unpaired t test. Comparisons of the time course of serum concentrations and of the incorporation of dietary N into the monitored N pools between the two groups were performed using a repeated-measures ANOVA (General Linear Model, SAS/STAT 6.03, SAS Institute, Cary, NC), in which the factors were the protein source, time and their interaction. When the protein source time interaction was significant, Tukey s post-hoc tests were applied. Differences were deemed significant when P RESULTS Plasma glucose and hormone concentrations. The postprandial plasma glucose profiles did not differ, and the AUC above baseline values were almost identical in both groups (Fig. 1). When normalized for baseline values, the insulin and glucagon postprandial profiles did not differ in the two groups. Total and dietary N-containing AA in different serum free AA pools. The temporal fate of both total AA and dietary N-containing AA in the different serum AA pools, including total (TAA), indispensable, dispensable, branched-chain (BCAA) and gluconeogenic (i.e., alanine, serine, glutamine, glutamate, glycine, threonine) AA, was monitored after the ingestion of a soy or milk protein meal. In each AA pool, AA concentrations rose significantly (time effect) and peaked 1 2 h after the ingestion of soy, whereas after the ingestion of milk, the rise was less pronounced and occurred later, as exemplified by the time course of total AA, BCAA and gluconeogenic AA (Fig. 2). The maximum total concentrations in the total AA pool occurred after 2 and 3 h, with values of and mol/l after the soy and milk protein meal, respectively. There was a significant time protein source interaction for both total and dietary N-containing AA in TAA, indispensable, dispensable, branched-chain and gluconeogenic AA pools (repeated-measures ANOVA), reflecting a global difference in the shape of the curves between groups. Moreover, for the different plasma AA pools, the postprandial rise in total AA concentrations could be explained mainly by the appearance of the dietary N-containing AA fraction, as illustrated by the specific course of leucine, proline or alanine (Fig. 3). For all AA (with the exception of proline), intake of the soy meal produced an earlier and higher peak value, usually 2 h after the meal, compared with the findings in the milk group. The protein source had a significant effect on serum AA concentrations for phenylalanine, arginine, glutamate and aspartate (higher global levels after soy than after milk protein ingestion). There was also a significant time protein source interaction for almost all AA, except for alanine, histidine, glutamate, glycine and aspartate (not shown).

4 POSTPRANDIAL METABOLISM OF DIETARY AMINO ACIDS 1311 linked (Fig. 4). For instance, methionine was twice as abundant in the milk group as in the soy group, and the accumulated dietary N-containing methionine levels were also twice the values in the milk group compared with the soy group. Transfer of dietary nitrogen to urea and serum protein. The production of total urea ( and mmol N/kg body after soy or milk protein ingestion, respectively) and the time course of serum protein concentrations ( and mmol N/kg after soy or milk FIGURE 1 Plasma glucose concentrations and variations in insulin and glucagon levels for the 8-h period after the ingestion of milk or soy 15 N-labeled protein in a single mixed meal in healthy subjects. Each data point represents the mean SD of n 8 in each group. Insert bar panel shows the mean area under the curve over the 8-h period. The groups did not differ. Kinetics and levels of appearance of individual dietary N-containing AA in the serum. There were few differences between the two groups in either the peak of dietary N- containing AA concentrations or the AUC in serum (Table 3). The only significant differences were observed for proline, which was found at double the amount in the milk group, and histidine, which reached a higher value after soy protein ingestion. There was a trend (0.05 P 0.1) toward lower dietary N-containing methionine and aspartate asparagine AUC values after soy protein ingestion. However, there were major differences for almost all of the dietary N-containing AA in terms of their kinetics, characterized by their systematically earlier appearance in the serum after soy protein ingestion than after the milk protein meal. This difference was significant for leucine, methionine, threonine, valine, aspartate asparagine, glutamate glutamine, glycine and serine. When expressed as ratios between group results (milk:soy), the different accumulated appearances of dietary N-containing AA and initial AA levels in the ingested proteins were closely FIGURE 2 Serum concentrations of total (TAA), BCAA and gluconeogenic (alanine, serine, glutamine, glutamate, glycine, threonine) AA and their dietary N-containing fraction for the 8-h period after the ingestion of milk or soy 15 N-labeled protein in a single mixed meal in healthy subjects. Each data point represents the mean SD, n 8/ group. Note that the scales of the y-axes are different. There was a significant time protein source interaction for the time courses of both total and dietary N-containing AA (repeated-measures ANOVA). *Significant difference between soy and milk groups at a specific time point (Tukey s test, P 0.05).

5 1312 BOS ET AL. FIGURE 3 Total level and appearance of the dietary nitrogencontaining fraction (leucine, proline and alanine) in selected serum amino acids after the ingestion of milk or soy proteins for the 8-h period after the ingestion of milk or soy 15 N-labeled protein in a single mixed meal in healthy subjects. Note that the scales of the y-axes are different. Values are means SD. *Significant difference between soy and milk groups at a specific time point (Tukey s test, P 0.05). protein, respectively) did not differ over the 8 h period (not shown). The peak of dietary N urea production (Fig. 5) was earlier in the soy group than in the milk group (3 1.1vs h, respectively, P 0.005). The protein source ingested had a significant effect on the time course of dietary N transfer to the urea produced (protein source time interaction: P 0.02, repeated measures ANOVA). Two hours after the meal, the levels of dietary N transferred to urea were higher after soy than after milk protein ingestion, reaching and % of the N ingested, respectively; P 0.02). The level of dietary N recovered in the urea pool over the 8-h period was and % of the ingested amount in the milk and soy groups, respectively (P 0.05) and dietary N retention reached and % of the ingested amount in the milk and soy groups, respectively (P 0.05). The incorporation of dietary N into serum protein (Fig. 5) was also higher in the soy group than in the milk group, and the protein type (P 0.02) and protein source time interaction (P 0.04) had significant global effects (repeated-measures ANOVA). DISCUSSION The aim of this study was to gain a better understanding of the differences already observed in postprandial protein metabolism after the ingestion of dietary protein of animal and plant origins, i.e., milk or soy protein. We utilized GC-C- IRMS combined with the uniform 15 N-labeling of protein to provide original data on the appearance of each 15 NAAinthe blood after the ingestion of specific dietary proteins. The results showed significant differences regarding both the metabolic fate of dietary AA in the serum and the transfer of dietary N to urea and serum proteins. The present study confirms a difference in dietary N metabolism after soy and milk protein ingestion, as described earlier (1,6), and provides evidence of major differences between the two proteins in terms of the rate of appearance of dietary AA in the peripheral blood, which is representative of the digestion/absorption kinetics. The measurement of 15 N- containing AA in the systemic circulation cannot exactly be considered to be dietary AA because of the transamination that occurs during the first-pass metabolism of AA, except in the case of threonine and lysine, which do not participate in transamination. Nevertheless, there was a close resemblance between the dietary protein and serum free dietary N-containing AA patterns, allowing us to draw conclusions concerning the differences between serum dietary N-containing AA in the two groups. Our first conclusion is that the differences observed between the two proteins had an important kinetic component related to the intestinal rate of AA absorption. A nearly equivalent half-time of gastric emptying has been reported for milk and soy proteins (16 20). However, this measure may not accurately account for the differential behaviors of milk protein fractions or differences in their kinetics, which are more likely to be seen at the intestinal level because the gastric emptying rate and intestinal transit time may be independently affected by dietary factors (31,32). Indeed, some differences between proteins seem to occur at the intestinal level, characterized by the more rapid transit of soy protein than that of casein, as has been shown in rats (20). In line with this, we recently found in humans that the intestinal half transit time of soy proteins was 50 min shorter than that of milk proteins in humans (13). A possible mechanism for such a difference may be the release from casein digestion of -casomorphin, which have been reported to modulate gastrointestinal motility (33,34), or a delay in the intestinal proteolysis of casein. The reason for assuming that this kinetic difference influences the metabolic utilization of the two dietary proteins is that digestion and absorption kinetics determine the directions of dietary AA through the various metabolic pathways in organs. This is consistent with studies in both humans and animals comparing postprandial protein metabolism after the ingestion of casein (slow rate of absorption) or free AA/ oligopeptides mimicking the composition of casein (fast rate of absorption) (35 37). The rapid absorption of AA is associated with both enhanced AA catabolism and transiently higher protein synthesis at the whole-body level (12,35,36). Our results provide new evidence for an important role of the liver in this transient anabolic effect of fast protein, as illustrated by the higher incorporation of dietary N into serum protein after the ingestion of soy protein. The time-course of dietary N appearance in serum free AA, urea and serum protein was consistently always more rapid after the ingestion of soy than after that of milk protein. We have already reported in rats that an acute increase in protein intake resulted in a higher transfer of dietary N into plasma protein (38). In agreement with others (24), we conclude that a high influx of AA enhances both liver protein synthesis and deamination to prevent hyperaminoacidemia. This is also in line with the data obtained in rats showing a lower incorporation of lysine in the

6 POSTPRANDIAL METABOLISM OF DIETARY AMINO ACIDS 1313 TABLE 3 Variables characterizing the postprandial appearance of dietary N-containing amino acids (AA) in the serum for the 8-h period after the ingestion of milk or soy 15N-labeled protein in a single mixed meal in healthy subjects1 Maximum concentration Peak time Accumulated amount2 Milk group Soy group Milk group Soy group Milk group Soy group mol/l h mol/(kg body 8h) Histidine * Isoleucine Leucine * Lysine Methionine * Phenylalanine Threonine * Valine * Alanine Aspartate asparagine * Glutamate glutamine * Glycine * Proline * * Serine * Tyrosine AA * Values are means SD, n 8/group. 2 Calculated as the area under the curve of the postprandial kinetics of dietary N-containing AA concentrations multiplied by the serum volume for the 8-h period and adjusted for body weight. * Significantly different from soy group (Student s t test, P 0.05). splanchnic zone after the ingestion of whole casein, compared with its corresponding crystalline AA mixture (37). Whether the moderate imbalance of indispensable AA from soy protein may also have contributed to their more marked deamination and incorporation into liver protein is difficult to predict from the present study and would require a study of soy protein supplemented with crystalline AA to reach the same level as total milk protein. Apart from intestinal dynamics, the differences between soy and milk protein may have arisen from the combined influence of a relatively unbalanced AA composition in the FIGURE 4 Relative contents of amino acids in dietary protein and relative amounts of dietary N-containing amino acids appearing in the serum for the 8-h period after the ingestion of milk or soy 15 N-labeled protein in a single mixed meal in healthy subjects. Values represent ratios between milk group values and soy group values. FIGURE 5 Transfer of dietary N to body and urinary urea N pools (urea production, see Methods for calculations) and serum protein pools for the 8-h period after the ingestion of milk or soy 15 N-labeled protein in a single mixed meal in healthy subjects. Each point is the mean SD, n 8/group. The protein source ingested had a significant effect upon the time course of dietary N transfer to the urea produced and serum protein pool (protein source time interaction: P 0.05, repeated measures ANOVA) and on the incorporation of dietary N into serum protein (higher in the soy group than in the milk group, P 0.02, repeated-measures ANOVA). *Significant difference between soy and milk groups at a specific time point (Tukey s test, P 0.05).

7 1314 BOS ET AL. soy protein and its lower digestibility, resulting in a less favorable pattern of AA reaching the periphery (7). Despite supposed differences in the pattern of absorbed AA entering the portal circulation after milk or soy protein ingestion, the absolute amounts of dietary AA appearing in the systemic circulation did not differ between groups, with the exception of a consistently higher level of dietary proline in the milk group because of its higher concentration in the meal. This lack of difference was due mainly to diminished power resulting from the large interindividual variability of circulating AA levels and the number of subjects in each group (n 8). We would have expected some differences in the splanchnic first pass to have exacerbated differences in the AA composition pattern between milk and soy. However, the fact that the subjects had previously been adapted to a moderate level of protein [1 g/(kg d)] was likely to attenuate differences in AA deamination/oxidation. At this level, the difference in dietary N retention between milk and soy protein was due mainly to differences in digestibility because AA catabolism was not activated, but at higher levels of protein habituation, the gap between the deamination of these proteins widens (unpublished data) (1,6). Nevertheless, despite the absence of absolute differences in AA amounts in plasma, the dietary protein pattern was directly reflected by the circulating dietary AA pattern when expressed as ratios between groups. In this case, a direct influence of the AA composition of dietary protein on the indispensable AA available to the peripheral tissues for metabolic utilization and protein synthesis can be hypothesized, which is of particular interest with respect to AA such as BCAA. Human studies have shown that the composition of ingested AA mixtures has a direct influence on whole-body protein synthesis (39,40). Using compartmental modeling in humans, we showed that postprandial N retention was significantly higher in the peripheral N pools after milk protein ingestion than after soy protein, which could be interpreted as a more favorable pattern of AA available for peripheral protein synthesis in the case of milk protein ingestion (13). It is also consistent with the lower protein retention values observed in pigs fed a soybean protein based diet compared with a casein-based diet (4). Dietary proteins, through their AA composition and their specific insulinotropic effects, may influence insulin secretion. Because we know the role played by insulin in AA sparing (41), it was important to test this hypothesis. Arginine and leucine have been suspected of having the most potent insulinotropic effects (42,43). We found very similar hormonal responses to the acute ingestion of both the soy and milk meals, which is consistent with the findings of Lang et al. (26). Similarly, the study by van Loon and colleagues (22) did not demonstrate any differences in plasma insulin levels after the ingestion of casein or pea protein hydrolysates. In contrast, wheat ingestion induced a higher insulin response, suggesting an insulinotropic effect of glutamine. During our study, the glutamine intake was nearly identical in the soy and milk meals. Finally, as recently shown in humans, the degree of protein fractionation likely plays a predominant role in the modulation of postprandial insulin/glucagon release, independently of the protein source (23). In conclusion, our study shows that the principal differences in postprandial metabolism between soy and milk protein are observed in terms of the time of appearance of dietary AA in the serum, presumably because of the different behavior of proteins in the intestine, with soy protein acting as a fast protein and milk protein as a slow protein. The massive influx of dietary AA into the liver appears to elicit a larger metabolic availability of AA in this organ, including both their incorporation into exported liver protein and their catabolism. In addition, we showed that the pattern of dietary AA reaching the periphery reflects mainly dietary protein composition and may participate in the lower level of protein retention. Elucidating whether these two important factors (rate of digestion and AA composition) interfere with each other under different nutritional conditions will constitute an interesting further step in the study of protein metabolism. LITERATURE CITED 1. 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