Human Nutrition and Metabolism
Casein and Soy Protein Meals Differentially Affect Whole-Body and Splanchnic Protein Metabolism in Healthy Humans1,2 Yvette C. Luiking, Nicolaas E. P. Deutz,3 Martin Ja¨kel, and Peter B. Soeters Maastricht University, Department of Surgery, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), 6200 MD Maastricht, The Netherlands ABSTRACT Dietary protein quality is considered to be dependent on the degree and velocity with which protein is digested, absorbed as amino acids, and retained in the gut as newly synthesized protein. Metabolic animal studies suggest that the quality of soy protein is inferior to that of casein protein, but confirmatory studies in humans are lacking. The study objective was to assess the quality of casein and soy protein by comparing their metabolic effects in healthy human subjects. Whole-body protein kinetics, splanchnic leucine extraction, and urea production rates were measured in the postabsorptive state and during 8-h enteral intakes of isonitrogenous [0.42 g protein/(kg body weight 䡠 8 h)] protein-based test meals, which contained either casein (CAPM; n ⫽ 12) or soy protein (SOPM; n ⫽ 10) in 2 separate groups. Stable isotope techniques were used to study metabolic effects. With enteral food intake, protein metabolism changed from net protein breakdown to net protein synthesis. Net protein synthesis was greater in the CAPM group than in the SOPM group [52 ⫾ 14 and 17 ⫾ 14 nmol/(kg fat-free mass (FFM) 䡠 min), respectively; P ⬍ 0.02]. Urea synthesis rates decreased during consumption of both enteral meals, but the decrease tended to be greater in the subjects that consumed CAPM (P ⫽ 0.07). Absolute splanchnic extraction of leucine was higher in the subjects that consumed CAPM [306 ⫾ 31 nmol/(kg FFM 䡠 min)] vs. those that consumed SOPM [235 ⫾ 29 nmol/(kg FFM 䡠 min); P ⬍ 0.01]. In conclusion, a significantly larger portion of soy protein is degraded to urea, whereas casein protein likely contributes to splanchnic utilization (probably protein synthesis) to a greater extent. The biological value of soy protein must be considered inferior to that of casein protein in humans. J. Nutr. 135: 1080 –1087, 2005. KEY WORDS:
In response to food intake, metabolism changes from a catabolic to an anabolic state due to inhibition of protein breakdown and increased net protein synthesis (1). A large part of this protein gain accumulates in the gut and is referred to as the labile protein pool (2). This pool provides essential amino acids to the body’s free amino acid pool during the postabsorptive phase when protein in this pool is in turn broken down (3,4). Both the amount and the quality of protein in the diet are important in determining the magnitude of change in protein metabolism with food intake. With an increasing protein content in the diet and increased plasma amino acid levels, whole-body endogenous protein breakdown is more markedly inhibited and protein synthesis is stimulated, although the latter occurs to a lesser extent and is dependent on the amount of protein ingested (5– 8). Moreover, the nutritional value of dietary protein is related to both the bioavailability of ingested
nitrogen and amino acids and the efficiency of their metabolic utilization to meet nitrogen and amino acid requirements for growth and renewal of body proteins (9 –13). Protein digestibility and naturally occurring growth-depressing or antinutritional factors in proteins affect both the bioavailability of nitrogen and amino acids (14,15), and postprandial protein kinetics (16). Moreover, metabolic utilization depends on the composition of the meal with respect to the presence or absence of (in)dispensable essential amino acids and carbohydrates in the meal (1,17–19). For example, an isoleucinedeficient blood meal or a protein meal without carbohydrates reduces gut protein synthesis and increases liver urea production (17,18). Compared with casein, soy protein is deficient in the essential amino acids methionine and lysine and contains less BCAA. Moreover, the bioavailability of soy protein is further impaired by the presence of endogenous inhibitors of digestive enzymes (e.g., trypsin inhibitors), and the poor digestibility of raw soybean [see (20) for review]. An oro-ileal digestibility for soy protein isolate of 91% was reported in the literature (21); this is slightly lower than the 95% found for milk protein concentrate (22). Recently, we observed in pigs that liver urea production and net production of essential amino acids by the portal drained viscera were higher with soy than with casein protein.
1 Presented in part at the Experimental Biology ’02, April 23, 2002, New Orleans, LA [Luiking, Y. C., Breitfeld, S., Jaekel, M., Hulsewe, K., Soeters, P. & Deutz, N. (2002) The metabolic effects of casein and soy protein meals in healthy subjects. FASEB J. 16: A788 (abs.)]. 2 Supported by a grant from the European Dairy Association (Brussels, Belgium). 3 To whom correspondence should be addressed. E-mail: [email protected]
0022-3166/05 $8.00 © 2005 American Society for Nutritional Sciences. Manuscript received 30 August 2004. Initial review completed 21 October 2004. Revision accepted 2 March 2005. 1080
METABOLIC EFFECTS OF CASEIN AND SOY PROTEIN
TABLE 1 General characteristics of the study population1
n Gender Age, y BMI, kg/m2 Height, m Body weight, kg Fat-free mass, kg
12 6M/6F 22 ⫾1 21.3 ⫾ 0.5 1.78 ⫾ 0.03 68 ⫾2 49 ⫾3
10 5M/5F 23 ⫾1 22.3 ⫾ 0.7 1.76 ⫾ 0.04 69 ⫾2 49 ⫾3
1 Values are means ⫾ SEM.
To assess these production rates, stable isotopes of urea, phenylalanine, and valine were used in a multicatheterized pig model (23). The design of this pig study, however, is not applicable in humans, and a different methodology to study the splanchnic area is therefore required. In humans, wholebody retention of dietary nitrogen was lower with soy (24), which was associated with more rapid intestinal absorption of dietary nitrogen from soy, increased transfer to urea, and reduced uptake by the peripheral area. In addition, net postprandial protein utilization, calculated from the true ileal digestibility of nitrogen and the true percentage of ingested nitrogen retained in the organism, was reported to be 78% for soy protein and 85% for milk protein (21,22), with the latter containing casein (⬃80%) and whey proteins. The present study, which involved studying metabolic variables as measures of the nutritional value of dietary proteins in humans, was designed to test the hypothesis that the quality of the ingested protein affects whole-body and splanchnic protein metabolism. Stable isotope techniques were used to compare whole-body protein kinetics and urea production rates of casein and soy protein meals. The double-labeled infusion technique (25,26) was used to test differences between casein and soy on the splanchnic level with respect to leucine extraction and protein synthesis. MATERIALS AND METHODS Subjects. Healthy subjects (n ⫽ 22) participated in the study, which took place during the period October 1999 to January 2000 (Table 1). None of the subjects were taking medication that could affect protein metabolism, and none of the women were pregnant. The Ethics Committee of Maastricht University approved the experimental study protocol. Informed consent was obtained from each subject. Experimental protocol. A randomized, single-blind study design was used to test the effect of consumption of a casein protein meal (CAPM)4 or a soy protein meal (SOPM) on both whole-body protein and urea metabolism, and on splanchnic leucine extraction (Fig. 1). The protocol started at 0630 h, after an overnight fast from 0000 h, when subjects had voided their bladder and body weight (BW) and height had been recorded. After introduction of a 14-Fr nasogastric tube (Vygon) and insertion of a catheter in the right antecubital vein, the first blood sample was taken for baseline measurements. Immediately thereafter, a primed-constant i.v. infusion of stable isotopes (80 mL/h) and intragastric (i.g.) tracer (20 mL/h) was started (T ⫽ ⫺2 h) using calibrated pumps (IVAC) (stable isotopes are described in more detail below). Next, a femoral arterial catheter was placed in the right groin with the catheter tip pointing distally for 4 Abbreviations used: BW, body weight, CAPM, casein protein meal, FFM, fat-free mass; PB, protein breakdown; QPhe3 Tyr, hydroxylation of phenylalanine; Ra, rate of appearance; Rd, rate of disappearance; SOPM, soy protein meal; SPE, splanchnic extraction; TTR, tracer-tracee ratio.
FIGURE 1 Scheme of the study protocol according to a randomized design. Enteral nutrition consists of a casein protein meal (CAPM; n ⫽ 12) or a soy protein meal (SOPM; n ⫽ 10).
arterial blood sampling, using local anesthesia (1% Lidocaine subcutaneously; Astra). For sampling arterialized blood (sampled in addition to validate our sampling technique), a venous catheter was placed retrogradely in a dorsal vein of the left hand, using the heated box technique (27,28). After 2 h of stable isotope infusion to reach steady-state enrichments, enteral nutrition was started (T ⫽ 0 h) via the nasogastric tube at a rate of 2 mL/(kg BW 䡠 h) using a calibrated infusion pump (IVAC), for a total of 8 h. Total fluid intake by i.v. infused solutions was 1345 mL during the 10-h experiment. Blood samples were taken at hourly intervals during the study, with 1 additional blood sample at T ⫽ ⫺0.5 h. In total, 260 mL of blood was taken from the different sampling sites. At T ⫽ 4 h, the femoral catheter was removed, local pressure was applied for 15 min and a sandbag placed on the puncture site for 1 h. During the experiment, fat-free mass (FFM) was measured using Bioelectrical Impedance Analysis (BIA) (Xitron 4000B, Xitron Technologies) (29) to express protein metabolism data/kg FFM. At T ⫽ 10 h, the subjects left the hospital. Subjects remained in supine position during the entire protocol. Enteral protein meals. Two different test meals were used, containing either 5.9 g sodium caseinate (casein protein meal: CAPM) or 6.8 g soy protein (soy protein meal: SOPM) in isonitrogenous amounts (0.83 g N) and equal amounts of maltodextrin (DE 20, 13.7 g), dissolved in ultrapure water to 200 mL volume and heated at 60°C (Table 2). In total, ⬃1200 mL enteral nutrition (based on a 75-kg subject) was supplied during the study. The test meals were adjusted with mineral salts to balance the mineral differences in the protein sources. The test meals supplied ⬃8.3 mg N/(kg BW 䡠 h) and 140 mg maltodextrin/(kg BW 䡠 h), resulting in 2016 kJ (480 kcal)/(kg
TABLE 2 Nitrogen and mineral composition of the experimental casein (CAPM) and soy (SOPM) protein-based meals1 Substance Raw product, g/L Protein, % Nitrogen, % Protein, g/L Nitrogen, g/L Maltodextrin, g/L Sodium, mmol/L Potassium, mmol/L Calcium, mmol/L Phosphorus, mg/L Trypsin inhibitor, IU/100 g protein Isoflavones, mg/L
29.5 89.3 (N ⫻ 6.38) 14.0 26.5 4.0 68.5 18.0 11.2 2.7 49.5 ⬍43 Not present
34 76.9 (N ⫻ 6.25) 12.1 26.5 4.0 68.5 18.0 11.2 2.7 49.5 309 240
1 Protein meals were infused enterally at 2 mL/(kg BW 䡠 h). Meals are isonitrogenous and contained similar amount of carbohydrates and minerals.
LUIKING ET AL.
BW 䡠 8 h) and 0.42 g protein/(kg BW 䡠 8 h) (26 En% protein) for a 75-kg person. Protein content of the meals is based on the daily protein requirement of 0.83 g protein/kg BW (9), which we considered equivalent to 16-h continuous intake. The meal therefore represents continuous moderate nitrogen infusion. The infusion rates of glucose and amino acids with the test meals are listed in Table 3 (not all amino acids listed). The protein composition of CAPM comprised a 1:1:1 mixture of commercially available French, Dutch, and Danish sodium caseinates. The protein composition of SOPM comprised a mixture of soy protein concentrate (Danpro S) and soy protein isolate (Supro 590). All meals were prepared during the evening before the experimental day. To ensure a complete dissolution of the proteins and to prevent bacterial growth, the meals were kept at 4°C until use. Isotope infusion protocol. Primed and constant infusion of the stable isotopes L-[ring-2H5]-phenylalanine [2H5-phe; prime: 2.5 mol/kg BW, infusion: 2.5 mol/(kg BW 䡠 h)], L-[ring-2H2]-tyrosine [2H2-tyr; prime: 0.9 mol/kg BW, infusion: 0.9 mol/(kg BW 䡠 h)], 2 2 L-[ H3]-leucine [ H3-leu; prime: 2.4 mol/kg BW, infusion: 2.4 mol/ (kg BW 䡠 h)], and [15N2]-urea [15N2-urea; prime: 34.5 mol/kg BW, infusion: 6.3 mol/(kg BW 䡠 h)] was given via the antecubital vein catheter. Primed infusion of L-[ring-2H4]-tyrosine (2H4-Tyr; 0.3 mol/kg BW) and L-[1-13C]-leucine (13C-leu; 2.0 mol/kg BW) was given in addition via the same catheter. Constant infusion of L-[113 C]-leucine [4.7 mol/(kg BW 䡠 h)] was given via the nasogastric tube. Primed and constant infusions of 2-[13C]-glycine and paraaminohippuric acid (PAH) also were given; those results will be reported elsewhere. Stable isotopes were purchased from Mass Trace. 2 H5-phenylalanine and 2H2-tyrosine are used to calculate wholebody protein metabolism; 2H4-tyrosine is used to prime the 2H4tyrosine pool for measuring phenylalanine hydroxylation. 2H3leucine and 13C-leucine are used to calculate splanchnic leucine extraction and endogenous leucine production. 15N2-urea is used for calculation of whole-body urea production. Analysis of blood. Blood samples were collected in prechilled, heparinized tubes (Becton Dickinson Vacutainer system), immediately put on ice, and subsequently centrifuged at 4°C for 10 min at
TABLE 3 Infusion rates of glucose and amino acids with the experimental casein (CAPM) and soy (SOPM) protein-based enteral test meals1 Substance
mol/(kg BW 䡠 min) Glucose Glu Ser Gln Gly Thr Ala His Arg Tyr Val Met Ile Phe Trp Leu Lys BCAA ⌺ ␣-amino groups2
13.60 0.83 0.64 0.70 0.24 0.37 0.35 0.19 0.21 0.32 0.58 0.20 0.46 0.31 0.06 0.70 0.54 1.74 5.85
13.60 0.68 0.41 0.41 0.46 0.28 0.40 0.14 0.36 0.18 0.35 0.08 0.30 0.27 0.05 0.50 0.35 1.16 5.25
1 Only those amino acids that also were measured in plasma in this study are shown. The manufacturers provided the amino acid composition of the proteins. 2 The total of the ␣-amino groups is from the sum of the amino acids measured.
FIGURE 2 Plasma enrichments (TTR) for phenylalanine, leucine, and urea. [ring-2H5]-phenylalanine, [2H3]-leucine, and [15N2]-urea in subjects infused i.v. for 2 h during the postabsorptive state and for 8 h during enteral feeding of a casein (CAPM; n ⫽ 12) or soy protein meal (SOPM; n ⫽ 10). Values are means ⫾ SEM.
3120 ⫻ g to obtain plasma. For amino acid analysis, plasma was deproteinized with sulfosalicylic acid (5%). For analysis of urea, glucose, lactate, acetate, pyruvate, and glycerol, plasma was deproteinized with trichloroacetic acid (5%). All samples were stored at ⫺80°C until further analysis. Plasma concentrations of amino acids were analyzed using an HPLC technique, after precolumn derivatization with o-phthaldialdehyde (30). Amino acid and urea enrichments were calculated as tracer:tracee ratios (TTR) and were analyzed by using a fully automated LC-MS (Thermoquest LCQ) (31). For urea enrichment, a newly developed method using reversed-phase LC in combination with an ion-trap spectrometer was applied (van Eijk, unpublished). Plasma glucose, lactate, acetate, pyruvate, urea, and glycerol were analyzed using a fully automated HPLC system (Pharmacia) application note number 5411, Alltech catalog (column IOA-1000, 300 ⫻ 7.8 mm, mobile phase 0.01 mol/L H2SO4, flow 0.3 mL/min, 60°C, refractive index detection). Calculations. We used a single-pool model for calculation of protein turnover. The model parameters used in the protocol are based on the assumption that there is an isotopic, but not necessarily a physiologic steady state (32). These isotopic steady-state conditions were obtained from a 60-min tracer infusion in the postabsorptive state (2-way ANOVA) (Fig. 2). During enteral nutrition, tracer steady state was reached again from 60 min of food intake, which did not differ between the subjects consuming CAPM or SOPM (2-way ANOVA) (Fig. 2). For the calculations of whole-body rate of appearance (Ra)urea during the postabsorptive state and during enteral feeding, a one-pool model nonsteady-state equation of Steele, modified for use with stable isotopes (33) was applied. Protein turnover is defined as the total flux into or out of the active metabolic amino acid pool. Influx (whole-body Ra) is considered to result from protein breakdown and nutrition (in the prandial state), whereas efflux [whole-body rate of disappearance (Rd)] includes amino acids used for protein synthesis and for hydroxylation (or oxidation). All other metabolic pathways are considered minor. Therefore, in the postabsorptive and prandial state, whole-body Ra (⫽infusion rate/TTR2H5-Phe in plasma) ⫽ whole-body Rd (under steady-state) ⫽ whole-body protein synthesis ⫹ hydroxylation of Phe to Tyr (34,35). For calculations of whole-body protein turnover and urea synthesis in the postabsorptive state, we used the TTR at 2 h after the start of tracer infusion (T0) using equations according to reference (33). Splanchnic leucine extraction (SPELeu) represents the fraction (in %) of ingested leucine, taken up by the gut and liver during its first pass and metabolized via oxidation or protein synthesis (25,26), calculated as (1): SPE LEU ⫽ 关1 ⫺ 共Ra 2H3-Leu /Ra 13C-Leu 兲兴 ⴱ 100%
METABOLIC EFFECTS OF CASEIN AND SOY PROTEIN
where Ra2H3-Leu and Ra13C-Leu represent whole-body Ra of leucine calculated from i.v. 2H3-Leu and i.g. 13C-Leu isotopes, respectively. The absolute splanchnic extraction of Leu from the meal [in nmol/(kg FFM 䡠 min)] can be calculated by multiplying SPELeu by the infusion rate of Leu with the meal. Whole-body Ra of leucine, not coming from dietary leucine [endogenous leucine (Raend-Leu)] is calculated from (2) and (3), and indicates whole-body protein breakdown: Corrected Leu intake ⫽ dietary Leu intake ⫻ 关1 ⫺ 共SPELeu ⫻ 0.01兲兴 (2) Raend-Leu ⫽ Ra2H3-Leu ⫺ corrected Leu intake
Whole-body Ra of phenylalanine, not coming from dietary protein [endogenous phenylalanine (Raend-Phe)], is estimated accordingly, using an estimated SPEPhe of 58% based on previous observations in the fed state (36). Whole body protein breakdown 共WbPB兲 ⫽ Raend-Phe
netWbPS ⫽ WbPS ⫺ WbPB
Statistical analysis. Results are expressed as means ⫾ SEM. Postabsorptive baseline values of arterial and arterialized blood were compared using paired samples t test and were not significantly different. Because postabsorptive baseline arterialized data had more missing values, we used arterial values as baseline. Postabsorptive baseline values between the 2 enteral protein meal groups were compared using independent-samples t tests. The meal-induced change in whole-body protein metabolism, splanchnic metabolism, and urea synthesis was calculated by subtracting the baseline values. Repeated-measures ANOVA was subsequently used to test changes during enteral feeding. A 2-way ANOVA was performed to compare meal and time differences (baseline values and values during the first 4 h and last 4 h of enteral feeding). When a time effect was observed, a Bonferroni test was used for post hoc analysis. Missing values, due mainly to occlusion of catheters during the experiment, were replaced by the series mean at the specific time for the specific protein meal, to make paired analyses possible. Urea kinetics (Ra) were calculated for n ⫽ 8 in the CAPM group and n ⫽ 7 in the SOPM group. SPSS software was used (SPSS for Windows version 7.5, SPSS). Statistical significance of differences was defined as a 2-tailed P ⬍ 0.05.
RESULTS Plasma substrate concentrations. Glucose levels increased in both subject groups (P ⬍ 0.05 for time effect). The area under the curve over the 8-h feeding period did not differ (P ⫽ 0.13) between subjects that consumed SOPM (7.7 ⫾ 1.0 mmol/L) and CAPM (5.7 ⫾ 0.8 mmol/L), but plasma glucose concentrations were higher in subjects that consumed the former during the last 4 h of the feeding protocol (P ⬍ 0.05) (Fig. 3). The glycerol concentration decreased in subjects during intake of both SOPM and CAPM (P ⬍ 0.001 for time effect), from 0.20 ⫾ 0.03 and 0.40 ⫾ 0.06 mol/L in the postabsorptive state to 0.08 ⫾ 0.01 and 0.09 ⫾ 0.02 mol/L after 8 h of CAPM and SOPM intake, respectively. Pyruvate, acetate, and lactate did not change during the feeding period (data not shown). Plasma concentrations of almost all amino acids increased with food intake (Table 4). In contrast, plasma concentrations of taurine decreased (P ⬍ 0.05 for time effect), whereas glutamate and glutamine did not change. In the subjects consuming CAPM, increases in plasma concentrations of serine, citrulline, tyrosine, valine, methionine, isoleucine, leucine, lysine, citrulline, BCAA, and total amino acids were greater than in those that consumed SOPM (P ⬍ 0.05 for time ⫻ meal effect). Plasma concentrations of arginine and tryptophan were lower in subjects consuming CAPM (P ⬍ 0.05 for time ⫻ meal).
FIGURE 3 Arterial plasma glucose concentrations (mmol/L) in subjects during the postabsorptive state (T0) and during enteral intake of casein (CAPM; n ⫽ 12) or soy (SOPM; n ⫽ 10) protein meals. Values are means ⫾ SEM. Significant P-values (⬍0.05) from the repeated measures ANOVA for time (T), and diet (D) are shown.
Whole-body protein turnover. Whole-body rate of appearance of endogenous phenylalanine (Raend-Phe), as an estimation of protein breakdown, did not change significantly during feeding in either group, using the assumption that 58% of phenylalanine from the meal is extracted in the splanchnic area. Protein breakdown in the postabsorptive state was 911 ⫾ 33 and 968 ⫾ 33 nmol/(kg FFM 䡠 min) for CAPM and SOPM, respectively. Whole-body production of endogenous leucine (Raend-Leu) (Fig. 4A), which also represents whole-body protein breakdown, decreased during enteral food intake compared with postabsorptive values (P ⬍ 0.001). However, this decrease did not differ between subjects who consumed CAPM and SOPM. The whole-body rate of hydroxylation of phenylalanine (QPhe3 Tyr) increased significantly during enteral food intake, but the rates differed from postabsorptive values only during the last 4 h of enteral food intake (P ⬍ 0.01). The increase did not differ between the CAPM and SOPM groups (P ⫽ 0.13). The ratio, QPhe3 Tyr/Raphe, which represents the percentage of total phenylalanine that enters the route of hydroxylation, tended to be higher in subjects that consumed SOPM than in those that consumed CAPM (P ⫽ 0.06). The ratios tended to increase over time (P ⫽ 0.08), with ratios of 0.123 ⫾ 0.012 and 0.111 ⫾ 0.016 in the postabsorptive state, 0.120 ⫾ 0.010 and 0.127 ⫾ 0.010 during the first 4 h, and 0.136 ⫾ 0.010 and 0.147 ⫾ 0.005 during the last 4 h of CAPM and SOPM intake, respectively. Whole-body protein synthesis rate increased during enteral feeding (Fig. 4B), and the increase (difference from postabsorptive values) tended to be higher in the CAPM group than in the SOPM group during the last 4 h of the enteral feeding period (P ⫽ 0.09). Net protein breakdown occurred during the postabsorptive period, but this changed to net protein synthesis during enteral food intake (Fig. 4C; P ⬍ 0.001 vs. baseline). The increase was greater in the subjects that consumed CAPM compared with those that consumed SOPM (P ⬍ 0.02). Splanchnic leucine extraction. Splanchnic leucine extraction (SPELeu) was higher (P ⬍ 0.05) in the postabsorptive state (49 ⫾ 5 and 39 ⫾ 5% for CAPM and SOPM, respectively) than during enteral food intake (32 ⫾ 3 and 35 ⫾ 2% at T1– 4 h for CAPM and SOPM, respectively). Although the percentage SPELeu did not differ between subjects that consumed CAPM and those that consumed SOPM, the absolute
LUIKING ET AL.
TABLE 4 Plasma amino acid concentrations in subjects in the postabsorptive state and during enteral food intake of casein (CAPM) and soy (SOPM) protein-based enteral test meals1 CAPM (n ⫽ 12) T02
SOPM (n ⫽ 10) T8
mol/L Glu Ser Gln Gly Thr Cit Ala His Arg Tau Tyr Val Met Ile Phe Trp Leu Orn Lys BCAA ⌺AA4 1 2 3 4
86 ⫾ 11 115 ⫾ 6 557 ⫾ 21 172 ⫾ 28 158 ⫾ 15 32 ⫾ 2 283 ⫾ 51 98 ⫾ 13 69 ⫾ 5 50 ⫾ 7 45 ⫾ 3 205 ⫾ 10 19 ⫾ 1 61 ⫾ 4 68 ⫾ 4 83 ⫾ 13 111 ⫾ 6 45 ⫾ 2 143 ⫾ 7 378 ⫾ 19 2466 ⫾ 100
80 ⫾ 37 135 ⫾ 6 562 ⫾ 18 192 ⫾ 26 178 ⫾ 15 35 ⫾ 2 343 ⫾ 24 111 ⫾ 13 81 ⫾ 6 47 ⫾ 6 65 ⫾ 3 245 ⫾ 10 28 ⫾ 1 80 ⫾ 4 84 ⫾ 3 84 ⫾ 13 150 ⫾ 7 55 ⫾ 3 210 ⫾ 11 474 ⫾ 20 2843 ⫾ 80
9⫾ 5 138 ⫾ 7 586 ⫾ 22 195 ⫾ 31 177 ⫾ 17 39 ⫾ 2 351 ⫾ 31 125 ⫾ 14 84 ⫾ 6 42 ⫾ 5 73 ⫾ 4 268 ⫾ 12 30 ⫾ 1 88 ⫾ 6 91 ⫾ 6 75 ⫾ 12 166 ⫾ 10 55 ⫾ 3 212 ⫾ 10 522 ⫾ 27 2954 ⫾ 115
75 ⫾ 4 120 ⫾ 9 556 ⫾ 31 184 ⫾ 32 162 ⫾ 21 32 ⫾ 2 205 ⫾ 9 94 ⫾ 9 71 ⫾ 3 39 ⫾ 3 46 ⫾ 2 202 ⫾ 12 19 ⫾ 1 63 ⫾ 4 70 ⫾ 5 85 ⫾ 16 117 ⫾ 5 43 ⫾ 3 134 ⫾ 9 382 ⫾ 19 2380 ⫾ 93
71 ⫾ 3 119 ⫾ 6 535 ⫾ 25 180 ⫾ 26 176 ⫾ 24 31 ⫾ 2 270 ⫾ 17 99 ⫾ 7 87 ⫾ 5 35 ⫾ 3 50 ⫾ 2 194 ⫾ 10 17 ⫾ 1 64 ⫾ 4 83 ⫾ 5 86 ⫾ 14 119 ⫾ 6 52 ⫾ 5 158 ⫾ 11 377 ⫾ 18 2507 ⫾ 77
74 ⫾ 4 129 ⫾ 5 561 ⫾ 19 172 ⫾ 26 192 ⫾ 29 33 ⫾ 2 280 ⫾ 20 104 ⫾ 10 97 ⫾ 2 37 ⫾ 3 62 ⫾ 3 214 ⫾ 13 19 ⫾ 1 77 ⫾ 7 93 ⫾ 6 98 ⫾ 16 138 ⫾ 10 49 ⫾ 3 171 ⫾ 8 429 ⫾ 27 2685 ⫾ 84
T T T T T T T T T T T
T T ⫻ T T ⫻ T ⫻ ⫻ ⫻ ⫻ T ⫻ ⫻ T ⫻ ⫻ ⫻
D D D D D D D D D D D
Values are means ⫾ SEM. Postabsorptive values (T0) and values at T ⫽ 4 and 8 h of enteral food intake. Significant P-values (⬍0.05) from the repeated-measures ANOVA for time (T), diet (D), and their interaction (T ⫻ D) are shown. Total AA: sum of measurable amino acids.
values of SPELeu (Fig. 5) were significantly higher in the subjects that consumed CAPM than in those that consumed SOPM throughout the feeding period (P ⬍ 0.01). Urea kinetics. Arterial plasma urea concentrations at baseline did not differ between the CAPM and SOPM groups (P ⫽ 0.2), but showed a different time course during meal consumption in the 2 groups (P ⬍ 0.05 for time ⫻ meal effect), reaching higher levels during the first 4 h of SOPM consumption (Fig. 6A). Whole-body urea production (RaUrea) decreased during enteral feeding, and the change from the postabsorptive values of 11.4 ⫾ 1.4 and 11.4 ⫾ 3.5 nmol/(kg FFM 䡠 min) for CAPM and SOPM, respectively, tended to be greater during consumption of CAPM than SOPM (P ⫽ 0.07), resulting in less urea production in the subjects that consumed CAPM (P ⫽ 0.07) (Fig. 6B). Urine production during the 8-h study did not differ between the subjects that consumed CAPM (1234 ⫾ 95 mL) and SOPM (1152 ⫾ 123 mL). DISCUSSION This study shows that after consumption of a soy protein meal, subjects produce more urea than after consumption of a casein protein meal. Moreover, net protein synthesis was lower in those subjects that consumed soy protein. Even though the splanchnic leucine extraction percentage was equal for the 2 meals, the absolute splanchnic extraction of leucine was lower in those subjects that consumed soy. These findings suggest that, in healthy human subjects, splanchnic protein synthesis is probably less stimulated by soy than by casein protein, and
that soy protein is degraded to urea to a greater extent than casein protein. Normal metabolic responses to food intake (casein) Plasma concentrations and whole-body protein kinetics. For most amino acids, changes in the arterial plasma level are related to the amino acid composition of the meal, except for those amino acids that are substantially metabolized in the splanchnic area. As an example, glutamine, of which ⬃65% is extracted in the splanchnic area (37–39), is partially converted to citrulline (40), and this explains the rise in plasma citrulline. Endogenous whole-body protein breakdown was reduced by food intake, as indicated by the 20% reduction of endogenous leucine release into the circulation. Together with the increase in phenylalanine hydroxylation, this observation reflects the normal change from a catabolic to an anabolic state and is in agreement with previous observations (1). Whole-body urea kinetics. We observed a decrease in whole-body urea synthesis with food intake, in contrast to the increase that we observed previously in growing pigs (23). The discrepancy is probably due to less reduction of endogenous protein breakdown in growing animals. The urea tracer technique that we used (41) cannot discriminate between urea production from endogenous amino acids and from dietary amino acids, but measures the total rate of urea production in the body. Although urea production from endogenous amino acids probably is decreased due to the change from a catabolic to an anabolic state, food intake increases the portal amino acids flux to the liver and as such stimulates urea production,
METABOLIC EFFECTS OF CASEIN AND SOY PROTEIN
FIGURE 5 Absolute splanchnic extraction of leucine (SPELeu) in subjects during enteral intake of casein (CAPM; n ⫽ 12) or soy (SOPM; n ⫽ 10). Values are means ⫾ SEM. Means without a common letter differ, P ⬍ 0.01.
the increase in urea synthesis from the diet in combination with the reduction in body protein catabolism. Taken together, these factors explain the decrease in urea production that we observed in our study.
FIGURE 4 Whole-body protein breakdown, indicated by Ra of endogenous leucine (PB; A), whole-body protein synthesis (PS; B), net whole-body protein synthesis (net PS: C) in subjects during the postabsorptive state (baseline) and during enteral intake of casein (CAPM; n ⫽ 12) or soy (SOPM; n ⫽ 10) protein meals during the first and the last 4 h of the prandial period. Values are means ⫾ SEM. Means without a common letter differ, P ⬍ 0.05.
in line with increased urea synthesis from 15N-labeled protein meals (42). Because urea synthesis from dietary nitrogen depends on the nitrogen content of the meal, which in our study was a continuous moderate nitrogen infusion [8.3 mg N/(kg BW 䡠 h), equivalent to ⬃0.4 g protein/(kg BW 䡠 8h); the daily protein requirement is defined at 0.83 g protein/(kg BW 䡠 d) (9)], this relatively low nitrogen content may have minimized
FIGURE 6 Urea concentration (A) in subjects during the postabsorptive state (T0) and during enteral intake of casein (CAPM; conc: n ⫽ 12) or soy (SOPM; conc: n ⫽ 10) protein meals. Whole-body Raurea (B), indicated as the difference during meal intake (CAPM; n ⫽ 8, SOPM: n ⫽ 7) from baseline. Values are means ⫾ SEM.
LUIKING ET AL.
Splanchnic metabolism. Splanchnic extraction of leucine decreased from ⬃45% in the postabsorptive state to ⬃34% with food intake in our study, in line with previous studies (25,36,43,44). Whether the increased amount of extracted leucine with casein intake is used for splanchnic protein synthesis or whether it is transaminated or irreversibly lost (25) cannot be concluded directly from our data because this requires 13KIC and 13CO2 measurements. However, the depression of urea formation and the increase of net protein synthesis suggest a favorable effect on splanchnic protein metabolism. The importance of the splanchnic area in protein anabolism after consumption of a meal, the last probably on the order of 50% of total intake, was described by us (18,23) and by others (24,45,46) previously, and has been related to the high protein turnover in this area (17,47). Moreover, the splanchnic region modulates the availability of amino acids for extra-splanchnic amino acids (48). Although the double-labeled tracer technique cannot discriminate between intestinal and liver metabolism, the liver uses a major part of extracted amino acids for protein synthesis (44), quantitatively equal to mucosal protein synthesis (45). This so-called labile protein pool in the splanchnic area could play an important role in the postabsorptive phase when renewed breakdown of this labile pool provides essential amino acids to the body free amino acid pool (4). Differences between casein and soy protein Plasma concentrations and whole-bodyprotein kinetics. The changes in plasma amino acid levels in the subjects consuming casein and soy meals largely reflected the different amino acid composition of the meals. The change in arterial concentration was less pronounced than expected from the differences in meal composition only for glycine, in agreement with previous studies (23,49). The difference in plasma citrulline between subjects consuming CAPM and those consuming SOPM could be related to the higher glutamine content of CAPM and subsequent intestinal conversion of glutamine to citrulline (40) (see above). Protein breakdown did not differ between subjects consuming casein protein and those consuming soy protein. However, a larger part of phenylalanine was hydrolyzed during soy protein consumption, which may point to increased oxidation of soy protein, whereas net protein synthesis was lower in the subjects who consumed soy protein. Thus, a substantially larger degree of oxidation and less net protein synthesis with soy protein intake suggest that the biological value of soy protein is less than that of casein protein. Our data on whole-body protein metabolism of soy and casein agree with previous findings comparing a high vegetable protein diet with a high animal protein diet (50), or low and high milk protein diets. Amino acids, in particular, are main acute regulators in the mechanism of the postprandial changes in protein and amino acid metabolism and are main determinants of protein synthesis (1,6). Whole-body urea kinetics. During soy consumption, urea synthesis and plasma urea concentrations (first 4 h) were higher than during the same period after consumption of casein. This may indicate increased breakdown from dietary soy amino acids to urea because body protein breakdown was similar in subjects who ate both meals. This is consistent with observations reported after consumption of a bolus of soy and milk protein (24,51). A higher biological value of the meal (such as for casein compared with soy protein) may in general result in less urea production, suggested by comparison of milk protein alone and milk protein supplemented with sucrose
(higher biological value) (22). Although urea production decreases during enteral feeding, the pattern of changes in urea concentrations differs from urea production. This seeming discrepancy probably reflects the fact that concentration is not determined only by urea production. Splanchnic metabolism. The absolute splanchnic extraction of leucine from SOPM was lower than from CAPM. This is in line with previous observations in pigs (23), where CAPM resulted in increased gut protein synthesis. Whether increased splanchnic protein extraction with CAPM in our study also indicates increased protein synthesis was not measured directly, but may be indicated by the fact that on the whole-body level, net protein synthesis was higher with CAPM. In general, it appears that changes in whole-body protein metabolism in subjects who consumed soy protein are more transitory than changes induced by casein protein. In contrast, more long-term effects on protein metabolism were present in subjects who consumed CAPM. As a consequence, a rapid amino acid delivery to the liver from SOPM and a slow and more continuous supply of amino acids from CAPM may have occurred, with increased transfer to urea and subsequent reduced uptake by the peripheral area when SOPM was consumed (24,51). These differential effects are probably due to the different behaviors of the 2 proteins in the intestine and mirror the slow and fast protein concept (16,51), which makes soy a “fast” protein and casein a very “slow” protein. This could also explain why, during our continuous food intake protocol, most differences between the subjects groups where found during the first hours after the start of food intake, when the response reflects bolus meal intake. We as well as others have suggested that the degree to which protein in a meal is digested, absorbed, and immediately reutilized for protein synthesis in the gut, determines protein quality. This process “protects” meal protein– derived amino acids from immediate release into the portal vein and potential degradation in the liver to urea. The findings in this study, including a lower net protein synthesis and higher ureagenesis after a soy-containing meal, confirm earlier findings in pigs, and suggest that casein has a higher biological value than soy protein. The stable isotope techniques as used in this study provide a physiologic model that can be used to compare metabolic effects in subjects after consumption of diets of different protein composition or to study effects of protein diets in conditions of net protein catabolism, such as proteinenergy malnutrition, renal disease, sepsis, and traumatic injury. ACKNOWLEDGMENTS The authors thank H.M.H. van Eijk (Ph.D.), J.L.J.M. Scheijen (B.Sc.), and G.A.M. ten Have (B.Sc.) for expert analytical assistance. S. Breitfeld (M.Sc.) is kindly acknowledged for her great assistance in data collection.
LITERATURE CITED 1. Millward, D. J., Fereday, A., Gibson, N. R. & Pacy, P. J. (1996) Postprandial protein metabolism. Baillieres Clin. Endocrinol. Metab. 10: 533–549. 2. Quevedo, M. R., Price, G. M., Halliday, D., Pacy, P. J. & Millward, D. J. (1994) Nitrogen homoeostasis in man: diurnal changes in nitrogen excretion, leucine oxidation and whole body leucine kinetics during a reduction from a high to a moderate protein intake. Clin. Sci. (Lond.) 86: 185–193. 3. Waterlow, J. C. (1995) Whole-body protein turnover in humans—past, present, and future. Annu. Rev. Nutr. 15: 57–92. 4. Soeters, P. B., de Jong, C. H. & Deutz, N. E. (2001) The protein sparing function of the gut and the quality of food protein. Clin. Nutr. 20: 97–99. 5. Pacy, P. J., Price, G. M., Halliday, D., Quevedo, M. R. & Millward, D. J. (1994) Nitrogen homeostasis in man: the diurnal responses of protein synthesis and degradation and amino acid oxidation to diets with increasing protein intakes. Clin. Sci. (Lond.) 86: 103–116.
METABOLIC EFFECTS OF CASEIN AND SOY PROTEIN 6. Gibson, N. R., Fereday, A., Cox, M., Halliday, D., Pacy, P. J. & Millward, D. J. (1996) Influences of dietary energy and protein on leucine kinetics during feeding in healthy adults. Am. J. Physiol. 270: E282–E291. 7. Motil, K. J., Matthews, D. E., Bier, D. M., Burke, J. F., Munro, H. N. & Young, V. R. (1981) Whole-body leucine and lysine metabolism: response to dietary protein intake in young men. Am. J. Physiol. 240: E712–E721. 8. Giordano, M., Castellino, P. & DeFronzo, R. A. (1996) Differential responsiveness of protein synthesis and degradation to amino acid availability in humans. Diabetes 45: 393–399. 9. Pellet, P. L. (1990) Protein requirements in humans. Am. J. Clin. Nutr. 51: 723–737. 10. Fuller, M. F. & Garlick, P. J. (1994) Human amino acid requirements: can the controversy be resolved? Annu. Rev. Nutr. 14: 217–241. 11. Young, V. R. & Marchini, J. S. (1990) Mechanisms and nutritional significance of metabolic responses to altered intakes of protein and amino acids, with reference to nutritional adaptation in humans. Am. J. Clin. Nutr. 51: 270 –289. 12. Millward, J. (1994) Can we define indispensable amino acid requirements and assess protein quality in adults? J. Nutr. 124: 1509S–1516S. 13. Hiramatsu, T., Cortiella, J., Marchini, J. S., Chapman, T. E. & Young, V. R. (1994) Source and amount of dietary nonspecific nitrogen in relation to wholebody leucine, phenylalanine, and tyrosine kinetics in young men. Am. J. Clin. Nutr. 59: 1347–1355. 14. Pellett, P., Bressani, R., Devadas, R., Dillon, J. C., Eggum, B., Hussein, M. A., Miller, E. L., Oyeleke, O. A., Slump, P., Yoshida, A. & Young, V. R. (1990) Joint FAO/WHO Expert Consultation on Protein Quality Evaluation. FAO/WHO, Bethesda, MD. 15. Sarwar, G. (1997) The protein digestibility-corrected amino acid score method overestimates quality of proteins containing antinutritional factors and of poorly digestible proteins supplemented with limiting amino acids in rats. J. Nutr. 127: 758 –764. 16. Boirie, Y., Dangin, M., Gachon, P., Vasson, M. P., Maubois, J. L. & Beaufrere, B. (1997) Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc. Natl. Acad. Sci. U.S.A. 94: 14930 –14935. 17. Deutz, N.E.P., ten Have, G.A.M., Soeters, P. B. & Moughan, P. J. (1995) Increased intestinal amino-acid retention from the addition of carbohydrates to a meal. Clin. Nutr. 14: 354 –364. 18. Deutz, N. E., Reijven, P. L., Bost, M. C., van Berlo, C. L. & Soeters, P. B. (1991) Modification of the effects of blood on amino acid metabolism by intravenous isoleucine. Gastroenterology 101: 1613–1620. 19. Zello, G. A., Pencharz, P. B. & Ball, R. O. (1993) Dietary lysine requirement of young adult males determined by oxidation of L-[1-13C]phenylalanine. Am. J. Physiol. 264: E677–E685. 20. Friedman, M. & Brandon, D. L. (2001) Nutritional and health benefits of soy proteins. J. Agric. Food Chem. 49: 1069 –1086. 21. Mariotti, F., Mahe, S., Benamouzig, R., Luengo, C., Dare, S., Gaudichon, C. & Tome´, D. (1999) Nutritional value of [15N]-soy protein isolate assessed from ileal digestibility and postprandial protein utilization in humans. J. Nutr. 129: 1992–1997. 22. Gaudichon, C., Mahe, S., Benamouzig, R., Luengo, C., Fouillet, H., Dare, S., Van Oycke, M., Ferriere, F., Rautureau, J. & Tome´, D. (1999) Net postprandial utilization of [15N]-labeled milk protein nitrogen is influenced by diet composition in humans. J. Nutr. 129: 890 – 895. 23. Deutz, N. E., Bruins, M. J. & Soeters, P. B. (1998) Infusion of soy and casein protein meals affects interorgan amino acid metabolism and urea kinetics differently in pigs. J. Nutr. 128: 2435–2445. 24. Fouillet, H., Mariotti, F., Gaudichon, C., Bos, C. & Tome´, D. (2002) Peripheral and splanchnic metabolism of dietary nitrogen are differently affected by the protein source in humans as assessed by compartmental modeling. J. Nutr. 132: 125–133. 25. Hoerr, R. A., Matthews, D. E., Bier, D. M. & Young, V. R. (1991) Leucine kinetics from [2H3]- and [13C]leucine infused simultaneously by gut and vein. Am. J. Physiol. 260: E111–E117. 26. Matthews, D. E., Marano, M. A. & Campbell, R. G. (1993) Splanchnic bed utilization of leucine and phenylalanine in humans. Am. J. Physiol. 264: E109 –E118. 27. Abumrad, N. N., Rabin, D., Diamond, M. P. & Lacy, W. W. (1981) Use of a heated superficial hand vein as an alternative site for the measurement of amino acid concentrations and for the study of glucose and alanine kinetics in man. Metabolism 30: 936 –940. 28. Brooks, D. C., Black, P. R., Arcangeli, M. A., Aoki, T. T. & Wilmore, D. W. (1989) The heated dorsal hand vein: an alternative arterial sampling site. J. Parenter. Enteral Nutr. 13: 102–105. 29. De Lorenzo, A., Andreoli, A., Matthie, J. & Withers, P. (1997) Predicting body cell mass with bioimpedance by using theoretical methods: a technological review. J. Appl. Physiol. 82: 1542–1558.
30. van Eijk, H. M., Rooyakkers, D. R. & Deutz, N. E. (1993) Rapid routine determination of amino acids in plasma by high- performance liquid chromatography with a 2–3 microns Spherisorb ODS II column. J. Chromatogr. 620: 143– 148. 31. van Eijk, H. M., Rooyakkers, D. R., Soeters, P. B. & Deutz, N. E. (1999) Determination of amino acid isotope enrichment using liquid chromatographymass spectrometry. Anal. Biochem. 271: 8 –17. 32. Wolfe, R. R. (1992) Radioactive and stable isotope tracers in biomedicine. In: Principles and Practice of Kinetic Analysis, 1st ed. (Wolfe, R. R., ed.) Wiley-Liss, New York, NY. 33. Engelen, M. P., Deutz, N. E., Mostert, R., Wouters, E. F. & Schols, A. M. (2003) Response of whole-body protein and urea turnover to exercise differs between patients with chronic obstructive pulmonary disease with and without emphysema. Am. J. Clin. Nutr. 77: 868 – 874. 34. Thompson, G. N., Pacy, P. J., Merritt, H., Ford, G. C., Read, M. A., Cheng, K. N. & Halliday, D. (1989) Rapid measurement of whole body and forearm protein turnover using a [2H5]phenylalanine model. Am. J. Physiol. 256: E631– E639. 35. Clarke, J. T. & Bier, D. M. (1982) The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C] tyrosine in the postabsorptive state. Metabolism 31: 999 –1005. 36. Biolo, G., Tessari, P., Inchiostro, S., Bruttomesso, D., Fongher, C., Sabadin, L., Fratton, M. G., Valerio, A. & Tiengo, A. (1992) Leucine and phenylalanine kinetics during mixed meal ingestion: a multiple tracer approach. Am. J. Physiol. 262: E455–E463. 37. Boza, J. J., Dangin, M., Moennoz, D., Montigon, F., Vuichoud, J., Jarret, A., Pouteau, E., Gremaud, G., Oguey-Araymon, S., Courtois, D., Woupeyi, A., Finot, P. A. & Ballevre, O. (2001) Free and protein-bound glutamine have identical splanchnic extraction in healthy human volunteers. Am. J. Physiol. 281: G267–G274. 38. Hankard, R. G., Darmaun, D., Sager, B. K., D’Amore, D., Parsons, W. R. & Haymond, M. (1995) Response of glutamine metabolism to exogenous glutamine in humans. Am. J. Physiol. 269: E663–E670. 39. Matthews, D. E., Marano, M. A. & Campbell, R. G. (1993) Splanchnic bed utilization of glutamine and glutamic acid in humans. Am. J. Physiol. 264: E848 –E854. 40. Windmueller, H. G. & Spaeth, A. E. (1981) Source and fate of circulating citrulline. Am. J. Physiol. 241: E473–E480. 41. Jahoor, F. & Wolfe, R. R. (1987) Reassessment of primed constantinfusion tracer method to measure urea kinetics. Am. J. Physiol. 252: E557–E564. 42. Bos, C., Mahe, S., Gaudichon, C., Benamouzig, R., Gausseres, N., Luengo, C., Ferriere, F., Rautureau, J. & Tome´, D. (1999) Assessment of net postprandial protein utilization of 15N-labelled milk nitrogen in human subjects. Br. J. Nutr. 81: 221–226. 43. Metges, C. C., El-Khoury, A. E., Selvaraj, A. B., Tsay, R. H., Atkinson, A., Regan, M. M., Bequette, B. J. & Young, V. R. (2000) Kinetics of L-[1(13)C]leucine when ingested with free amino acids, unlabeled or intrinsically labeled casein. Am. J. Physiol. 278: E1000 –E1009. 44. Cayol, M., Boirie, Y., Rambourdin, F., Prugnaud, J., Gachon, P., Beaufrere, B. & Obled, C. (1997) Influence of protein intake on whole body and splanchnic leucine kinetics in humans. Am. J. Physiol. 272: E584 –E591. 45. Stoll, B., Burrin, D. G., Henry, J., Yu, H., Jahoor, F. & Reeds, P. J. (1998) Dietary amino acids are the preferential source of hepatic protein synthesis in piglets. J. Nutr. 128: 1517–1524. 46. Morens, C., Gaudichon, C., Metges, C. C., Fromentin, G., Baglieri, A., Even, P. C., Huneau, J. F. & Tome´, D. (2000) A high-protein meal exceeds anabolic and catabolic capacities in rats adapted to a normal protein diet. J. Nutr. 130: 2312–2321. 47. Scornik, O. A., Howell, S. K. & Botbol, V. (1997) Protein depletion and replenishment in mice: different roles of muscle and liver. Am. J. Physiol. 273: E1158 –E1167. 48. Mariotti, F., Huneau, J. F., Mahe, S. & Tome´, D. (2000) Protein metabolism and the gut. Curr. Opin. Clin. Nutr. Metab. Care 3: 45–50. 49. Hagemeister, H., Scholz-Ahrens, K. E., Schulte-Coerne, H. & Barth, C. A. (1990) Plasma amino acids and cholesterol following consumption of dietary casein or soy protein in minipigs. J. Nutr. 120: 1305–1311. 50. Pannemans, D. L., Wagenmakers, A. J., Westerterp, K. R., Schaafsma, G. & Halliday, D. (1998) Effect of protein source and quantity on protein metabolism in elderly women. Am. J. Clin. Nutr. 68: 1228 –1235. 51. Bos, C., Metges, C. C., Gaudichon, C., Petzke, K. J., Pueyo, M. E., Morens, C., Everwand, J., Benamouzig, R. & Tome´, D. (2003) Postprandial kinetics of dietary amino acids are the main determinant of their metabolism after soy or milk protein ingestion in humans. J. Nutr. 133: 1308 –1315.