Ileal Losses of Nitrogen and Amino Acids in Humans and Their Importance to the Assessment of Amino Acid Requirements

Transcription

1 GASTROENTEROLOGY 2002;123:50 59 Ileal Losses of Nitrogen and Amino Acids in Humans and Their Importance to the Assessment of Amino Acid Requirements CLAIRE GAUDICHON,* CÉCILE BOS,* CÉLINE MORENS,* KLAUS J. PETZKE, FRANÇOIS MARIOTTI,* JULIA EVERWAND, ROBERT BENAMOUZIG,* SOPHIE DARÉ,* DANIEL TOMÉ,* andcornelia C. METGES *Institut National de la Recherche Agronomique (INRA), Unité de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique de Paris-Grignon (INA PG), Paris, France; and Deutsches Institut für Ernährungsforschung-Potsdam (DIfE), Unit Protein Metabolism, Bergholz-Rehbrücke, Germany Background & Aims: Irreversible amino acid losses at the human ileum are not taken into account when tracer-derived amino acid requirements are calculated because the data available are scarce.we have investigated amino acid losses at the ileal level in humans after ingestion of a protein meal. Methods: Thirteen volunteers ingested a single meal of 15 N milk or soy proteins.the appearance of 15 N and 15 N amino acids in the ileal effluents collected using an ileal tube was monitored for 8 hours. Results: In the soy group, higher losses of endogenous nitrogen, especially originating from amino acids, were observed, as well as a higher flow rate of dietary non amino acid nitrogen.with soy protein, the digestibilities of valine, threonine, histidine, tyrosine, alanine, and proline were significantly lower than with milk.ileal losses of leucine, valine, and isoleucine amounted to 12, 10, and 7 mg kg 1 day 1, respectively.threonine ileal loss (9 12 mg kg 1 day 1 ) was particularly high compared with the current amino acid requirement. Conclusions: Amino acid losses at the human terminal ileum are substantial and depend on the type of dietary protein ingested.although it remains unclear whether intact amino acids are absorbed in the colon, we suggest that ileal losses should be considered an important component of amino acid requirements. Amino acid oxidation is the major component of irreversible losses of indispensable amino acids (IAAs) from the body. However, amino acid losses at the terminal ileum may also be of nutritional importance. Indeed, it has been shown that intact amino acids are not absorbed from the large intestine at nutritionally relevant amounts, although this question is not completely answered yet in view of new evidence on colonic amino acid and peptide transporters. 1 3 When IAA requirements are estimated using the nitrogen-balance technique, amino acid (nitrogen) losses are not an issue. 4 When determined by the direct tracer-balance technique, 5 IAA requirements represent only whole-body oxidation and do not account for losses at the terminal ileum, because these are thought to be small compared to oxidation. In contrast, the indirect amino acid oxidation method includes all of the obligatory losses. 6,7 Daily nitrogen losses from the small intestine in humans have been previously estimated at 2 5 g/day, with endogenous losses contributing to about 60% of the total Ileal endogenous nitrogen losses include proteins and amino acids as well as urea and ammonia derived from intestinal, biliary, and pancreatic secretions. Endogenous losses of amino acids in humans are not easy to measure and recently were characterized for the first time in ileostomized human volunteers receiving a protein-free diet. 8 In that study, IAA losses in ileostomy fluid accounted for 14% 61% (for threonine) of the current IAA requirements established by the Food and Agriculture Organization/World Health Organization (FAO/WHO) in This finding emphasized the need for a careful examination of IAA intestinal losses as a potential component of the IAA requirement. In addition, ileal IAA losses have never been established in healthy human subjects ingesting protein-containing diets. The presence of protein in the diet may influence endogenous nitrogen losses, as has been reported in pigs. 11 Several studies have suggested that the type of protein and the form of dietary amino acids ingested influence both endogenous and dietary losses. In pigs, Leterme et al. 12 found significantly more endogenous Abbreviations used in this paper: ANOVA, analysis of variance; AP, atom percent; APE, atom percent excess; FAO, Food and Agriculture Organization; GC-C-IRMS, gas chromatography-combustion-isotope ratio mass spectrometry; IAA, indispensable amino acids; WHO, World Health Organization by the American Gastroenterological Association /02/$35.00 doi: /gast

2 July 2002 ILEAL AMINO ACID LOSSES IN HUMANS 51 amino acids in the ileum of animals fed a wheat diet than in those fed a pea diet. We recently reported that dietary free amino acids show different appearances in the upper and lower small intestine than do whole proteins. 13 In a series of studies in humans, we found that the type of dietary protein could modulate the level of endogenous (1 2 g/day) and dietary nitrogen ( g/day) recovery in the gut. 9,14-17 Moreover, the contribution of dietary amino acids to nitrogen losses must be considered. Based on the digestibility values of pea protein obtained in pigs, dietary lysine loss in the small intestine gut reaches 8% 20 % of ingested lysine. 12,18 Taken together with endogenous lysine losses. 8 this could result in a total ileal lysine loss via the gut as high as 50% of the estimated requirement. We have previously shown that our ileal tube method can be used to determine dietary and endogenous nitrogen losses after the ingestion of various 15 N-labeled proteins in humans, 15,17,19 but no data on individual dietary amino acid loss at the ileum have been obtained so far. The present study aims to provide the first data on the fate of individual dietary amino acids in the ileum of healthy humans. Using 15 N-labeled milk and soy protein and an ileal tube method, we quantified the endogenous and dietary ileal losses of amino acids and non amino acids based on the analysis of 15 N enrichments of individual amino acids in the protein meal and the ileal effluent. These losses were compared with current IAA requirements 20 and the tentative set of IAA requirement values determined using the tracer balance technique. 21 Materials and Methods Experimental Meals We compared 2 different purified protein test meals, fed to the subjects on the morning of the experimental day: a milk protein meal (295 mmol N) and a soy protein meal (316 mmol N), providing 502 kj. The meals consisted of 30 g of milk or soy protein isolate stirred into water to obtain a final volume of 500 ml. Dietary protein purification and 15 N labeling of milk and soy were performed as described in our previous studies. 16,17,22 Briefly, milk was obtained from a lactating cow given orally ( 15 NH 4 ) 2 SO 4 for 11 days. 15 N- labeled milk was skimmed, and uniformly 15 N-labeled milk proteins were concentrated by diafiltration and lyophilized. Soy seeds were grown under controlled conditions using K 15 NO 3 foliar spraying. The soy protein isolate was purified from the intrinsically and uniformly 15 N-labeled soy seeds. The global 15 N isotopic enrichment of milk proteins was atom % (AP), whereas that of soy protein was 1.16 AP. The relative proportions of amino acids in milk and soy protein, together with the individual 15 N enrichments, are shown in Table 1. Table 1. Amino AcidComposition of Milk andsoy Proteins and 15 N Enrichment of Individual Amino Acids Amino acid composition a (%) 15 N amino acid enrichment b (AP) Milk Soy Milk Soy Aspartate asparagine Threonine Serine Glutamate glutamine Proline Glycine Alanine Valine Methionine ND ND Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine ND ND a Relative contribution to the 15 amino acids that were determined. b In atom %. Experimental Protocol The study protocol was approved by the Institutional Review Board of Saint Germain-en-Laye (France). After obtaining their written informed consent, 13 volunteers (age, 28 8 years; body mass index, kg/m 2 ) participated in this study. The subjects were healthy, as determined by a thorough medical examination and had no history of gastrointestinal disorder. They were randomly assigned to 1 of the 2 test meals containing 30 g of 15 N-labeled protein of milk (n 7) or soy (n 6) origin. The volunteers entered the Avicenne Hospital in Bobigny, France, the day before the experiment, and a double-lumen tube was introduced through the nose up to the digestive tract. One tube was used to perfuse a saline solution of phenol red as a nonabsorbable marker of intestinal effluents, and the other was used to aspirate ileal effluents. After overnight fasting, the perfusion site in the ileum was located using radioscopy. A saline solution containing 400 mg/l phenol red was perfused into the ileum at a flow rate of 1 ml/minute as described previously. 19 Phenol red was used as a marker to calculate the flow rate of intestinal effluents. After 30 minutes of ileal sample collection, the subjects ingested 1 of the 2 test meals. The ileal effluents were collected continuously on ice and pooled at 1-hour intervals for a total of 8 hours. The samples were treated immediately with the protease inhibitor di-isopropylfluorophosphate (1 mmol/l; Sigma, St.Quentin Fallavier, France), and were kept frozen at 20 C until analysis. Analytical Procedure Phenol red concentrations in the ileal effluents of human subjects were measured using a colorimetric method, as described previously. 19

3 52 GAUDICHON ET AL. GASTROENTEROLOGY Vol.123, No.1 Total nitrogen and 15 N enrichment. Measurements of global 15 N enrichments in the gastrointestinal effluents were performed using an isotope ratio mass spectrometer (Optima; Fisons Instruments, Manchester, UK) coupled with an elemental analyzer (NA 1500 Series 2; Fisons Instruments). Calibrated N 2 gas was used as the 15 N/ 14 N reference. Enrichments were expressed as atom percent (AP 15 N/( 14 N 15 N)) and atom percent excess (APE AP baseline 15 N abundance of the sample). Total nitrogen was measured using an elemental nitrogen analyzer (Carlo Erba Intruments, Fisons, Arcueil, France) with atropine as a standard, as described previously. 19 Amino acid concentrations. The gastrointestinal samples were dried and hydrolyzed with 6 mol/l HCl for 24 hours at 110 C. After drying, the samples were resuspended in lithium citrate buffer (ph 2.2), filtered over 0.2- m filters, and analyzed by high-pressure liquid chromatography using postcolumn ninhydrin derivatization (Biotek Instruments). Separation was achieved on a cation exchange resin (C.I.L., Sainte-Foy-la-Grande, France). All amino acids were detected at a wavelength of 540 nm, with the exception of proline (440 nm). 15 N amino acid enrichments. 15 N enrichments of individual amino acids were analyzed by gas chromatographycombustion-isotope ratio mass spectrometry (GC-C-IRMS) (Finnigan Delta S; ThermoFinnigan, Bremen, Germany) as described previously. 23 Amino acids from hydrolyzed gastrointestinal samples were purified as previously described in detail 23 and then derivatized to generate N-pivaloyl-i-propyl amino acid esters. Briefly, the amino acids were treated with a thionylchloride/i-propanol solution. The mixture was heated for 30 minutes at 110 C and then dried in a gentle stream of nitrogen at 60 C and redissolved in pyridine. After the addition of pivaloylchloride, samples were acylated for 30 minutes at 60 C; after cooling, 2 ml of dichloromethane was added. The resulting solution was passed through a silica gel column, and the eluate was dried. The vials were capped and the derivatives kept frozen ( 20 C) until GC-C-IRMS analysis was done. A 50-m capillary column (Ultra 2; Hewlett-Packard, Waldbronn, Germany) was used to separate the amino acids. The carrier gas was helium. Standard N 2 gas (with a known 15 N/ 14 N isotopic composition) was introduced into the GC-C-IRMS system at timed intervals to calibrate the amino acids. Values are expressed in APE as mentioned earlier. Calculations and Statistical Analysis Ileal flow rates of nitrogen and amino acids. The flow rate of ileal effluents (F) was calculated from phenol red concentrations for each 1-hour period, as described previously: 24 F F m C m /C s, where F m is the flow rate of the nonabsorbable marker phenol red and C m and C s are the phenol red concentrations in the perfused solution and the ileal sample, respectively. The total nitrogen (N tot ) and amino acid (AA tot ) flow rates were derived from nitrogen and amino acid concentrations and ileal flow rates, respectively : and N tot F N s / 100 AA tot F AA s /100, where N s is the nitrogen content of the ileal sample and AA s is the content of each amino acid in the ileal sample. Ileal losses of dietary nitrogen and amino acids. The level of dietary nitrogen present in gastrointestinal samples (N diet ) was determined from the dilution of the isotopic marker ( 15 N) in the samples, as follows: N diet N tot (APE s / APE m ), where N tot is the amount of total N in the sample and APE s and APE m are the 15 N enrichment excess (i.e., above the baseline value) of the sample and the meal, respectively. Similarly, the amount of dietary amino acid (AA diet ) recovered in gastrointestinal samples was calculated for each amino acid as follows: AA diet AA tot (APE s / APE m ), where AA tot is the amount of individual amino acid (i.e., free amino acid peptides and protein-bound amino acids) in the sample and APE s and APE m are the 15 N enrichment excess of the individual sample and the meal, respectively. Endogenous nitrogen and amino acid contributions were derived from the differences between total and dietary nitrogen and amino acids, respectively. Nitrogen and amino acid digestibility. Nitrogen and amino acid true digestibilities (TD) in humans were calculated from the cumulated amounts of nitrogen and amino acids recovered at the ileal level and thus not absorbed in the small intestine, using the equation TD (intake ileal content) * 100/intake where intake and ileal content are the cumulative amounts of nitrogen or amino acids ingested and recovered at the terminal ileum after 8 hours. Statistics The results are presented as means and their standard deviations. Comparisons between soy and milk proteins were performed using 1-way analysis of variance (ANOVA) or 1-way repeated-measures ANOVA (procedure GLM, SAS/ STAT 6.03; SAS Institute, Cary, NC). Significance was considered to be reached at P 0.05.

4 July 2002 ILEAL AMINO ACID LOSSES IN HUMANS 53 and at 2 hours in the soy group. Endogenous nitrogen losses under postabsorptive conditions, when calculated between 5 and 8 hours after the meal, reached mmol/h in the milk group and mmol/h in the soy group. Cumulative dietary nitrogen losses were significantly higher between 3 and 8 hours after the soy meal ( mmol at 8 hours) than after the milk meal ( mmol). Figure 1. (A) Endogenous nitrogen flux in human ileal effluents after ingestion of milk or soy protein. (B) Cumulative dietary nitrogen appearance in human ileal effluents after the ingestion of milk or soy protein. Values are mean SD. Asterisks indicate significant differences between groups. (Repeated-measures ANOVA, P 0.05.) Results Endogenous and Dietary Nitrogen in the Ileal Effluents The flow rate of endogenous nitrogen and the cumulative appearance of dietary nitrogen in the ileal effluents are shown in Figure 1. The endogenous flow rate was enhanced during the first 2 3 hours after the meal, with a peak occurring at 1 hour in the milk group Composition of Endogenous Nitrogen Losses Table 2 shows the contribution of amino acid and non amino acid nitrogen to the endogenous nitrogen flow rate in the ileum. Endogenous amino acid losses over 8 hours were significantly higher with soy than with milk protein (P 0.05). On average, amino acid nitrogen represented 31% of endogenous nitrogen in the milk group and 46% in the soy group throughout the experimental period. The composition of endogenous amino acid secretions present in the ileum was very stable throughout the 8 hours after the meal (Figure 2). The contribution of amino acids to endogenous nitrogen reached its maximum after 2 hours. There appeared to be a considerable flux of endogenous glycine with both dietary proteins. Among the endogenous amino acids measured in the ileal effluents, high concentrations were also found for proline, aspartate asparagine (specifically in the soy group), threonine, serine, and alanine. Of lesser importance was the endogenous inflow of histidine, phenylalanine, lysine, tyrosine, and isoleucine. The greater losses of endogenous amino acids observed in the soy group than in the milk group were due to higher losses of valine, leucine, lysine, alanine, and aspartate asparagine (repeated-measures ANOVA, P 0.05). Composition of Dietary Nitrogen Losses Table 3 shows the contribution of amino acid and non amino acid nitrogen compounds to cumulative dietary nitrogen recovery in the ileum. Most of the unabsorbed dietary nitrogen was of amino acid origin, espe- Table 2. Contribution of Amino Acids and Other Nitrogenous Compounds to the Endogenous Nitrogen Flux in the Ileum 8 Hours After the Ingestion of Milk or Soy Protein in Healthy Volunteers Protein 0 1 h 1 2 h 2 3 h 3 4 h 4 5 h 5 6 h 6 7 h 7 8 h 0 8 h Amino acidmilk (mmol N/h) Soy a a Non amino acid b Milk (mmol N/h) Soy a NOTE. Values are expressedas means SD. a Significantly different from milk for the same parameter. b Non amino acid Total nitrogen (free peptide protein) amino acidnitrogen.

5 54 GAUDICHON ET AL. GASTROENTEROLOGY Vol.123, No.1 Figure 2. Flux of endogenous (A) indispensable and (B) dispensable amino acids over time in the human ileal digesta after the ingestion of milk or soy protein. Values are means; standard deviations are omittedfor clarity. Note the different scaling of the y-axis in A and B. cially in the milk group, where 82% of dietary nitrogen was explained by the amino acid content. In the soy group, this value reached 69%. Dietary amino acid losses did not differ between groups, unlike non amino acid losses (repeated-measures ANOVA, P 0.05). The cumulative appearance of individual amino acid of dietary origin in the terminal ileum is depicted in Figure 3. Among IAA, the highest losses were for leucine in the soy group and for threonine in the milk group. There was a significantly higher ileal recovery of leucine with soy when compared with milk (repeated-measures ANOVA, Figure 3. Cumulative appearance of individual dietary amino acids in the human ileal digesta after the ingestion of milk or soy protein. Asterisks indicate significant differences between groups. (Repeatedmeasures ANOVA, P 0.05.) P 0.05). Among the dispensable amino acids, higher levels of alanine, glycine, and aspartate asparagine were recovered in the ileum after the ingestion of soy rather than milk. Digestibility of Individual Amino Acids and Nitrogen The lowest digestibility was observed for threonine in the soy group (89.0%) and the highest was for tyrosine in the milk group (99.3%) (Table 4). Signifi- Table 3. Contribution of Amino Acids and Other Nitrogenous Compounds to Cumulative Dietary Nitrogen Recovery in the Ileum 8 Hours After the Ingestion of Milk or Soy Protein in Healthy Volunteers Protein 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h Prot a Prot t b Amino acidmilk (mmol N) c Soy Non amino acidmilk (mmol N) Soy d d d d d d NOTE. Values are expressedas means SD. a P value for protein effect (repeated-measures ANOVA). b P value for interaction protein * time effect (repeated-measures ANOVA). c Non amino acid Total nitrogen (free peptide protein) amino acidnitrogen. d Significantly different from milk for the same parameter.

6 July 2002 ILEAL AMINO ACID LOSSES IN HUMANS 55 Table 4. True Digestibility Values of Dietary Nitrogen and Amino Acids After the Ingestion of Milk or Soy Protein in Healthy Human Volunteers Milk Soy Aspartate asparagine Serine Glutamate glutamine Proline a Glycine Alanine a Tyrosine a Threonine a Valine a Isoleucine Leucine Phenylalanine Lysine Histidine a Average amino aciddigestibility b Nitrogen digestibility a NOTE. Values are means SD, in % of ingestedamino acidor nitrogen, n 7 and6 for milk andsoy, respectively. a Significantly different from soy group (ANOVA, P 0.05). b Calculatedfrom amino aciddigestibilities weightedby the proportion of each amino acidin the dietary protein. current FAO/WHO requirements for comparison, 20 endogenous lysine and threonine losses in the milk group were equivalent to 17% and 63% of the daily requirements, respectively. When subjects ingested soy protein, we observed the lowest value for histidine (21% of the current requirement) and the highest value for threonine (75% of the current requirement). Dietary threonine losses corresponded to 40% of the daily requirement after milk protein ingestion and 52% of the daily requirement after soy protein ingestion the highest levels of all of the amino acids considered. Discussion The present study allowed further characterization of nitrogen compounds in ileal intestinal effluents in humans. To the best of our knowledge, this study also provides the first data on individual amino acid losses of both endogenous and dietary origin in healthy human subjects. We demonstrated that an IAA amount corresponding to 40% 100% of current IAA requirements 20 cantly lower digestibility was found for threonine, valine, histidine, tyrosine, alanine, and proline with soy protein intake as compared with milk protein. Nitrogen digestibility was significantly lower in the soy group than in the milk group. In contrast, when total nitrogen digestibility was calculated from individual amino acid digestibilities, the difference between milk and soy was not significant. Daily Amino Acid Losses and Contribution to Requirements We used our experimental data to estimate daily losses of nitrogen compounds from the small intestine (Table 5). For this purpose, we assumed that the amounts of nitrogen compounds in the ileal perfusate are irreversibly lost to the body, and that the experimental meal represented one third of the daily protein intake. We multiplied the 8-hour cumulative individual amino acid recovery of both endogenous and dietary origin by a factor of 3 to calculate the daily amino acid losses (in mg kg 1 day 1 ), taking into account the average weight of subjects in each group. The highest levels of endogenous amino acid loss were observed for aspartate asparagine (8 13 mg kg 1 day 1 ), proline (6 7 mg kg 1 day 1 ), and threonine (5 7 mg kg 1 day 1 ). Dietary amino acid losses were highest for aspartate asparagine and glutamate glutamine (5 9 mg kg 1 day 1 ). These losses were compared with estimated IAA requirements for adult humans. Using the Table 5. Ranges of Daily Ileal Endogenous and Dietary Amino AcidLosses in Humans Ingesting Milk or Soy Protein, andcomparison With Amino Acid Requirement Values Endogenous and dietary ileal losses (mg kg 1 d 1 ) Total ileal losses (% of requirement) c Endogenous Dietary FAO a MIT b Aspartate asparagine Alanine Glutamate glutamine Glycine Proline Serine Isoleucine Leucine Valine Lysine Phenylalanine tyrosine Histidine Threonine NOTE. The lower value was extrapolatedfrom subjects ingesting milk protein andthe upper value from subjects ingesting soy protein, except for proline, for which the dietary loss was lower in the soy group than in the milk group. a Percentage of amino acidrequirements measuredusing the nitrogen balance method. 20 b Percentage of amino acidrequirements determinedby the tracer balance method. 46 c During the publication of this work, new requirements close to the MIT pattern have been establishedby the joint committee FAO/OMS, April 9 16, 2002, Geneva.

7 56 GAUDICHON ET AL. GASTROENTEROLOGY Vol.123, No.1 is recovered at the terminal ileum and thus should be considered when estimating IAA requirements. Amino Acid and Non Amino Acid Nitrogenous Compounds at the Terminal Ileum In line with previous results, the total nitrogen flow at the terminal ileum ranged from 2 to 5 g/day, with endogenous and dietary nitrogen losses ranging from 0.7 to 4 g/day and 0.3 to 1 g/day, respectively. 8 10,17 Endogenous and dietary amino acid losses were g/day and g/day, respectively. The results clearly show that a significant proportion of the nitrogen flow (about 40% 50%) in the human ileum is of nonprotein origin, which is consistent with the previous findings. 25 Using the pig as a human model, it has been reported that urea is mainly secreted in the small intestine, presumably by the pancreatic juice and the bile. 26,27 It appears that the production of ammonia from urea breakdown in the small intestinal lumen is very low, 28 probably due to lower urease activity in the small intestine as compared with the large intestine. 29 Consequently, a potential loss of ammonia is unlikely to cause underestimation of total nitrogen measurements in the ileal effluents. Data on individual ileal amino acid losses of endogenous and dietary origin are not available in humans. Few data were obtained in pigs using the 15 N leucine dilution method. In humans, we directly measured the disappearance (presumably corresponding to the absorption) of dietary amino acids using 15 N-labeled protein, which contrasts with the more indirect 15 N leucine dilution method that requires an intravenous infusion for a significant number of hours, if not days. 12,18,30 Using the latter method, Hess et al. 18 reported in pigs higher individual amino acid digestibilities for pea protein than when a 15 N-labeled pea protein was used, 31 suggesting an overestimation caused by inadequate consideration of endogenous protein. Leterme et al. 12 argued that the transamination of leucine, as well as the choice of the trichloroacetic acid soluble fraction of plasma as the precursor pool, led to an overestimation of digestibility. The amino acid composition of endogenous losses reported here reveals that glycine is the most abundant amino acid in this protein fraction, followed by proline, aspartate asparagine, glutamate glutamine, and threonine, albeit to a lesser extent (Figure 2). This is in line with previous results obtained in the jejunum of fasting humans, where glycine represented about 20% of endogenous amino acids. 32 Adibi and Mercer 33 found that in the human ileum, the proportion of glycine was 16% in the free amino acid fraction and 20% in the peptide fraction. In pigs fed protein-free diets, proline was the most abundant amino acid, followed by glycine, glutamate glutamine, and aspartate asparagine, whereas threonine had the highest concentration among IAAs. 34,35 High concentrations of glycine, aspartate asparagine, and glutamate glutamine could be due to biliary secretions, mucins, and also microbial amino acids, which contribute to endogenous nitrogen. 36 Another important issue to consider is the differing digestibility of milk and soy protein in humans, as well as their respective effects on endogenous amino acid and non amino acid ileal losses. We observed that the ingestion of soy protein induced higher ileal nitrogen flow rates than did the ingestion of milk protein, due both to a higher contribution of amino acid losses to the endogenous nitrogen flow rate and a higher contribution of non amino acid losses to the dietary nitrogen flow rate. The digestibility values of proline, alanine, tyrosine, threonine, valine, and histidine were lower after soy ingestion. Threonine was the lowest digestible amino acid in soy, but not in milk protein. Because the absorbed fraction of threonine is also highly used by both gut and liver tissues, 37,38 a relative imbalance between threonine and other IAAs in the free amino acid pools may occur. This imbalance may limit the entry of amino acids into protein synthesis, which in turn would expose them to oxidation. 39 This hypothesis provides a possible explanation for the higher transfer of dietary nitrogen to urea observed in humans ingesting soy protein as compared with those ingesting milk protein. 17,40 Deutz et al. 41 also reported that soy induced higher urea production than milk protein. Because urea is present in all body fluids, it should also be recovered in the intestine. This may explain why in the present study, levels of non amino acid nitrogen of dietary origin recovered in the ileum were also somewhat higher with soy protein (Table 3), leading to underestimation of nitrogen digestibility in the soy group but not in the milk group when compared with the average amino acid digestibility. Contribution of Daily Amino Acid Losses in the Gut to IAA Requirements Our results show that the levels of endogenous IAA recovered at the terminal ileum ranged from 3 to about 7 mg kg 1 day 1 (Table 5). These levels are somewhat higher than, but of a similar magnitude to, those found earlier in ileostomized human volunteers consuming a protein-free diet. 42 In addition, ileal IAA losses of dietary origin varied from 1 to 6 mg kg 1 day 1. The methods used to determine human IAA requirement estimates differ inasmuch as nitrogen-balance based values take into account ileal amino acid

8 July 2002 ILEAL AMINO ACID LOSSES IN HUMANS 57 losses by measuring fecal nitrogen excretion. In contrast, tracer-balance derived values used to establish the Massachusetts Institute of Technology (MIT) pattern of IAA requirements do not include ileal amino acid losses, because they have been considered very small compared to oxidative losses. 43 Our findings strongly suggest that this is not the case in healthy human subjects. Indeed, if we assume that amino acids cannot be absorbed at the colonic level, then the contribution of ileal losses to the current IAA requirements would range from 26% for histidine to 103% for threonine in the milk group, and from 29% for histidine to 127% for threonine in the soy group (Table 5). As for other IAAs measured in this study, the contribution ranged from about 40% to 60%. There is a growing consensus in favor of revising adult IAA requirements even if the methodologic debate has still not been resolved completely, 6,7,21,42,44,45 and the present study provides additional evidence that this revision is necessary. Although the tracer-balance method is clearly not designed to account for intestinal amino acid losses, we used this method to compare our results to the IAA requirement values established by Young et al., 46 because this pattern is purported to represent the total IAA requirements. In doing so, the contribution of intestinal losses reached about 20% 40% of the requirement for all of the IAAs except threonine (44% 54%). A question then arises as to whether or not the ileal losses as determined in our study must be added to the IAA requirements derived from the MIT studies. In this case, the requirements would range from 27 mg/kg/day for isoleucine to 48 mg kg 1 day 1 for phenylalanine tyrosine. For lysine, the requirement would amount to 45 mg kg 1 day 1. This value is similar to that proposed by Duncan et al. 47 using the breakpoint method, the alternative tracer-balance method, which provides an estimate of total IAA losses including the intestinal route. As mentioned earlier, an important issue is whether the amounts of IAA recovered at the ileal level are indeed equivalent to irreversible amino acid losses. It was generally agreed that there is no nutritionally important absorption of free amino acid in the large intestine of mammals, because until very recently, no amino acid transporters were known. New findings show that the B 0 amino acid transporter is expressed on the apical side of colonic cells in the mouse. 2 This transporter mediates the epithelial uptake of neutral and cationic amino acids (e.g., glycine, alanine, and lysine). However, further investigation is required to determine whether this transporter is present in the human colonic epithelium and to measure the contribution of a potential amino acid colonic reabsorption to whole-body amino acid fluxes. In addition, there is some indication that the large intestine also harbors peptide transporters, which might play a role in the colonic uptake of amino acids. 3 It appears that in pigs and humans, nitrogen absorption does occur, although the studies performed to date have shed no light on the nature of these nitrogen compounds. 36,48 Therefore, it is very likely that ileal amino acid losses might be somewhat overestimated, because small amounts of IAA recovered at the ileal level in humans may be reabsorbed. However, this question can be resolved only when more information on the potential for amino acid and small peptide absorption by the large intestine becomes available. In conclusion, our study provides the first data on ileal losses of endogenous and dietary amino acids in healthy humans. We have demonstrated that potential ileal amino acid losses are not negligible. We tested 2 dietary proteins to identify the impact of the protein source on these losses and found that endogenous ileal recovery accounted for a high percentage of daily branched-chain amino acid and threonine requirements, especially in the case of soy protein. This clearly supports the notion that tracer-derived requirements should take ileal losses into account, and thus further indicates that current FAO/ WHO IAA requirements are far too low. The clinical significance of the ileal amino acid losses may be of particular importance in elderly and malnourished persons and in persons suffering from inflammatory bowel disease, an issue that has not been addressed to date. Our study highlights the need to investigate the ileal losses in such conditions. References 1. Metges CC. Contribution of microbial amino acids to amino acid homeostasis of the host. J Nutr 2000;130:1857S 1864S. 2. Ugawa S, Sunouchi Y, Ueda T, Takahashi E, Saishin Y, Shimada S. Characterization of a mouse colonic system B(0 ) amino acid transporter relatedto amino acidabsorption in colon. Am J Physiol Gastrointest Liver Physiol 2001;281:G365 G Doring F, Walter J, Will J, Focking M, Boll M, Amasheh S, Clauss W, Daniel H. Delta-aminolevulinic acidtransport by intestinal and renal peptide transporters andits physiological andclinical implications. J Clin Invest 1998;101: Rose WC. The amino acidrequirements of adult man. Nutr Abstr Rev 1957;27: El-Khoury AE, Fukagawa NK, Sanchez M, Tsay RH, Gleason RE, Chapman TE, Young VR. The 24-h pattern andrate of leucine oxidation, with particular reference to tracer estimates of leucine requirements in healthy adults. Am J Clin Nutr 1994;59: Bos C, Gaudichon C, Tome D. Isotopic studies of protein and amino acidrequirements. 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10 July 2002 ILEAL AMINO ACID LOSSES IN HUMANS Fuller MF, Garlick PJ. Human amino acidrequirements: can the controversy be resolved? Annu Rev Nutr 1994;14: Young VR, el-khoury AE. Can amino acidrequirements for nutritional maintenance in adult humans be approximated from the amino acidcomposition of body mixedproteins? Proc Natl Acad SciUSA1995;92: Millward DJ, Fereday A, Gibson NR, Pacy PJ. Human adult amino acidrequirements: [1-13 C]leucine balance evaluation of the efficiency of utilization andapparent requirements for wheat protein andlysine comparedwith those for milk protein in healthy adults. Am J Clin Nutr 2000;72: Reeds PJ. Dispensable and indispensable amino acids for humans. J Nutr 2000;130:1835S 1840S. 46. Young VR, Bier DM, Pellett PL. A theoretical basis for increasing current estimates of the amino acidrequirements in adult man, with experimental support. Am J Clin Nutr 1989;50: Duncan AM, Ball RO, Pencharz PB. Lysine requirement of adult males is not affected by decreasing dietary protein. Am J Clin Nutr 1996;64: Fuller MF, Reeds PJ. Nitrogen cycling in the gut. Annu Rev Nutr 1998;18: Received January 10, 2002.Accepted March 7, Address requests for reprints to: Claire Gaudichon, Ph.D., Institut National Agronomique Paris-Grignon (INA PG), Unité de Physiologie de la Nutrition et du Comportement Alimentaire, 16 Rue Claude Bernard, Paris, France. gaudicho@inapg.inra.fr; fax: (33) Supported by grants from ARILAIT Recherches (Paris, France), PRO- COPE grants from the Deutsche Akademische Austauschdienst (Bonn, Germany), and the French Ministry for Foreign Affairs (Paris, France). The authors thank ARILAIT Recherches for their comments on the manuscript and the staff of the DIfE Protein Metabolism Unit for the excellent technical assistance with 15 N gas chromatography-combustion-isotope ratio mass spectrometry measurements. Dr.Metges is currently affiliated with the Research Institute for the Biology of Farm Animals, Dummerstorf, Germany.