Dietary peptides increase endogenous amino acid losses from the gut in adults 1 3

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1 Dietary peptides increase endogenous amino acid losses from the gut in adults 1 3 Paul J Moughan, Christine A Butts, Angela M Rowan, and Amélie Deglaire ABSTRACT Background: Accurate estimates of endogenous ileal total nitrogen and amino acid flows are necessary to ascertain true dietary amino acid digestibility coefficients and for the factorial estimation of dietary amino acid requirements. Objective: The objective was to ascertain endogenous amino acid losses from the small bowel in human subjects consuming a proteinfree diet or a diet with enzyme-hydrolyzed casein (EHC; MW 5000) as the sole source of nitrogen. Design: The subjects were 8 men and women with terminal ileum ileostomies after ulcerative colitis who consumed the protein-free and EHC-based diets in a crossover design. Each subject received each test diet in single meals followed by 2 consecutive 9-h total collections of digesta. Digesta samples for the EHC treatment were centrifuged and ultrafiltered ( MW cutoff), with the precipitate-plus-retentate fraction ( MW) providing a measurement of endogenous ileal amino acids. Results: The mean endogenous flows for most of the amino acids and nitrogen were significantly (P 0.05) higher when determined with the EHC-based diet than with the protein-free diet. Mean (n 8) endogenous ileal nitrogen flows were 2061 and 4233 g/g dry matter intake for the protein-free and EHC-based diets, respectively. Conclusion: The traditional protein-free method underestimates endogenous ileal amino acid loss in adults. Am J Clin Nutr 2005;81: KEY WORDS Adults, endogenous, gut, amino acids, ileum, peptides, obligatory losses INTRODUCTION Endogenous protein is found throughout the lumen of the digestive tract and is protein of body origin rather than of dietary origin. The primary sources of endogenous protein are desquamated cells, digestive enzymes, and mucin (1, 2). Although not strictly endogenous, bacterial protein is commonly a component of the endogenous measurement. As digestion proceeds, both dietary and secreted endogenous proteins are broken down and partly absorbed. Both undigested dietary protein and endogenous material accumulate at the end of the small intestine. It is important to be able to measure the amount of endogenous amino acids (AAs) at the terminal ileum. It is used to correct the apparent ileal digestibility of protein to true digestibility, which is a better measurement of the absorption of AAs from the gut (3). In addition, ascertaining the endogenous loss of AAs from the gut is an important component in establishing the daily amino acid requirement (4). Several methods are available for measuring endogenous AA loss. Traditionally, the subject receives a protein-free (PF) diet, and AA flow measured at the terminal ileum represents the endogenous flow. Although this method is used routinely in dietary protein evaluation (5), it has been criticized as leading to a physiologically abnormal metabolism. A PF diet may result in a decrease in the secretion of pancreatic and gastric enzymes (2) and in a general decrease in the rate of protein synthesis in the body and in the gut (6). Alternative methods have been developed that allow the determination of endogenous AA losses under protein alimentation (7). One of these methods, the enzymehydrolyzed protein technique (8), involves measuring AAs in the ileal digesta of subjects consuming a diet in which the sole source of nitrogen is free AAs and small peptides (hydrolyzed casein; MW 5000). The fresh digesta samples are centrifuged and ultrafiltered ( MW cutoff), and any undigested dietary AAs are discarded. The precipitate-plus-retentate fraction ( MW) constitutes a measure of endogenous ileal AA loss, measured under conditions in which the gut is supplied with dietary AAs and peptides, which mimicks the breakdown products of natural digestion. The enzyme-hydrolyzed protein method may slightly underestimate ileal AA losses because endogenous free AAs and peptides are removed from digesta along with undigested dietary nitrogenous material. Several studies with rats (9 12) and pigs (13 15) showed that the endogenous loss of AAs from the small intestine is higher with a diet containing peptides than with PF alimentation. Such a comparison has not been undertaken with human subjects, and, although ileal AA losses have been determined in humans after administration of a PF diet (4, 16, 17), there is a dearth of endogenous loss data obtained after the ingestion of dietary protein. Only one study (4), using the stable nitrogen isotope 15 N, has provided information on endogenous ileal AA flows in humans given a proteincontaining diet. The aim of the current study was to compare, in humans, ileal AA and nitrogen losses after the administration of a PF diet and 1 From the Riddet Centre, Massey University, Palmerston North, New Zealand (PJM and AD), the Fonterra Research Centre, Palmerston North, New Zealand (AMR), and the NZ Institute for Crop & Food Research Ltd, Palmerston North, New Zealand (CAB). 2 Supported by Fonterra Cooperative Group Ltd. 3 Address reprint requests to PJ Moughan, Riddet Centre, Private Bag , Palmerston North, New Zealand. p.j.moughan@massey.ac.nz. Received October 31, Accepted for publication February 22, Am J Clin Nutr 2005;81: Printed in USA American Society for Clinical Nutrition 1359

2 1360 MOUGHAN ET AL TABLE 1 Ingredient compositions of the protein-free foods and enzyme-hydrolyzed casein (EHC) based foods Protein-free biscuit an enzyme-hydrolyzed casein (EHC) based diet. The contribution these endogenous losses make to current recommended AA requirement values (18) was assessed. We chose human ileostomates as the experimental model in which to measure endogenous ileal losses (17). SUBJECTS AND METHODS EHC-based diet Drink Protein-free diet g/kg dry matter g dry matter/750 ml solution 1 Purified corn flour 415 Margarine 286 Sucrose 232 Cellulose 2 50 Baking powder 3 7 Celite 4 5 Ground ginger 5 EHC Flavor or sugar Solids were dissolved in 750 ml water. 2 Avicel; Asahi Chemical Company Ltd, Tokyo, Japan. 3 Contains wheat flour, sodium bicarbonate, and calcium carbonate. 4 Sigma Chemical Company, St Louis, MO. Celite is a diatomaceous clay. 5 Casein peptone; NZ Pharmaceuticals Ltd, Palmerston North, New Zealand; peptide MW Orange-flavored and -colored sucrose and glucose mixture (Raro; Cerebos Greggs Ltd, Auckland, New Zealand) with 5 g/80 g polyethylene glycol added. Subjects The 8 male and female subjects who participated in the study were recruited through the local (Manawatu) ostomy society. Each subject had undergone a total colectomy for ulcerative colitis, but 10 cm of the terminal ileum had been removed. The ileostomies were well established and functioning normally. The subjects were in good health, and none had taken antibiotics or medication that might alter small intestinal function for 8 wk before the study. No subject was pregnant, lactating, or taking oral contraceptives. The subjects ages ranged from 45 to 62 y, and their body weights ranged from 67 to 97 kg. The subjects participated in the study while in their own homes, and they were visited regularly throughout the study. Written informed consent was obtained from all participants. Approval to conduct the study, which involved the cooperation of fully informed and consenting adult subjects with established ileostomies, was obtained from the Massey University Human Ethics Committee and the Manawatu-Wanganui Area Health Board Ethics Committee (Palmerston North, New Zealand). Test diets Two experimental diets were given (Table 1). The EHC-based diet consisted of a drink containing EHC and a virtually PF biscuit. The PF diet consisted of a PF drink and the PF biscuit. The AA compositions of the diets are shown in Table 2. The TABLE 2 The amino acid compositions of the protein-free diet and enzymehydrolyzed casein (EHC) based diet Amino acid Protein-free diet EHC diet g/kg dry matter Threonine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Tyrosine Lysine Histidine Serine Glutamic acid Alanine Proline Arginine Aspartic acid Glycine indigestible marker celite (acid-insoluble ash, AIA) was added to both of the diets (19, 20). Experimental procedures Each subject received the respective test diet as a single meal, without previous adaptation. After ingestion of the meal, a 9-h collection of ileal digesta was realized. A previous study (21) showed that such an acute feeding and sampling regimen is a valid procedure for digestibility studies in human ileostomates. Ileostomates were randomly assigned to the PF- and EHC-based diets so that there were 4 subjects per diet at any one time, and the study was repeated over time by using a crossover design to obtain 8 observations per dietary treatment. Each subject consumed each test diet twice, 2 or 3 d apart, and, between test meals, the subjects consumed their normal diet. Digesta samples collected on the days when subjects received the same experimental diet were pooled so that each observation comprised 2 separate collections of ileal digesta. The total study covered a 2-wk period. Starting at 0800, after an overnight (14-h) fast, each subject attached a new plastic ostomy bag and then consumed the test diet, which consisted of 160 g PF biscuits and 750 ml drink (either PF or EHC-based). The meal was consumed within 30 min, and subjects were permitted to freely consume water after the test meal. Ostomy bags were changed and emptied every 2 3 h over the 9-h ( ) digesta collection period. The ileostomy output was transferred to a container to which 10 ml of 4 mol HCl/L had been added to prevent bacterial activity. The collection containers were kept chilled during the collection period, and the contents were frozen ( 20 C). Chemical analysis Digesta samples collected on the 2 separate days from each subject consuming the same diet were pooled. Digesta samples for the EHC-based diet were adjusted to ph 3 with 6 mol HCl/L and then centrifuged at 1400 g for 30 min at 3 C. The precipitate was washed and recentrifuged, and the supernatant fluids were pooled. The latter were ultrafiltered (Centriprep; Amicon, Inc, Beverly, MA; cutoff: MW). The retentates (

3 ILEAL ENDOGENOUS AMINO ACIDS 1361 TABLE 3 Endogenous ileal flows of amino acids in humans fed a protein-free (PF) diet or an enzyme-hydrolyzed casein (EHC) based diet 1 Total ileal flows Ileal flows 2 Amino acid PF diet EHC diet PF diet EHC diet P 3 mg/9 h g/g DMI Aspartate Arginine Alanine Glutamate Glycine NS Proline Serine Isoleucine Leucine Valine Lysine Phenylalanine NS Tyrosine NS Histidine Threonine Cysteine NS Methionine DMI, dry matter intake. 2 Related (by correction for marker recovery) to the food DMI. 3 Statistical analysis was performed on the log 10 -transformed data (data shown are untransformed) and applies to both columns of data. 4 x SEM (all such values). MW) from the ultrafiltration step were washed, ultrafiltered again, and then added to the precipitate from the centrifugation step. These samples were then freeze-dried and finely ground. The pooled digesta samples for the PF diet were immediately freeze-dried and finely ground. The nonfractionated digesta samples from the PF diet and the digesta precipitate-plus-retentate fractions (MW ) from the EHC-based diet were analyzed for total nitrogen, AAs, AIA, and dry matter. Samples of the diets were analyzed for AAs, AIA, and dry matter. All chemical analyses were carried out in duplicate. Total nitrogen was measured by the Kjeldahl method with the use of a Kjeltec Auto 1030 Analyzer (Tecator, Höganäs, Sweden) (22). Dry matter was measured by drying to constant weight in a forced air oven at 95 C. AIA was measured by a gravimetric method (19). AAs were measured by using ion-exchange HPLC after hydrolysis of the samples in 6 mol HCl/L for 24 h at 110 C in evacuated sealed tubes. Methionine and cysteine were measured as methionine sulfone and cysteic acid, respectively, after hydrolysis of samples that had been oxidized by using performic acid (23). Tryptophan was not measured. Statistical analysis Endogenous nitrogen or AA flows at the terminal ileum were determined by using the following equation: Endogenous flow ( g/g DMI) concentration of endogenous N or AA in the digesta ( g/g DM) (% AIA in diet/% AIA in digesta) (1) where DMI is food dry matter intake and DM is dry matter. The data were tested for homogeneity of variance. For some of the AAs, the variances were not homogeneous, and so data were transformed. The log 10 -transformed data were fitted to a general linear model on MINITAB 14 software (Minitab Inc, State College, PA). Subject and period were included as random effects, and treatment was included as a fixed effect. Because the terms for subject and period were not significantly different (P 0.05), a model was fitted with treatment as the sole effect. RESULTS The subjects fully complied with the protocol and completed the study without complication, except that 2 subjects found the EHC-based diet unacceptably bitter and did not consume the test meal. The mean ( SEM) recovery of the AIA marker was % for the PF diet (n 8) and % for the EHC diet (n 6). The total endogenous AA flows per 9 h and the endogenous flows corrected for the marker recovery and expressed in g/g dry matter intake are given in Table 3. The endogenous ileal flows for most of the AAs were significantly (P 0.05) higher with the EHC-based diet than with the PF diet (broadly, 1.2- to 2.2-fold). Only the ileal losses for glycine, phenylalanine, tyrosine, and cysteine did not differ significantly (P 0.05) between the diets. The mean endogenous ileal total nitrogen flows were 2061 and 4233 g/g dry matter intake (overall SE 279, P 0.001) for the PF and the EHC-based diets, respectively. DISCUSSION The PF diet method has traditionally been used to measure gut endogenous AAs. However, several studies with animals (11, 13, 14, 24) and one study with humans (4) showed that the ingestion of protein or peptides significantly increases the endogenous flow of AAs at the terminal ileum. The aim of the current study was to ascertain whether dietary peptides (ie, EHC) influence endogenous ileal AA flow in humans. To this end, an EHC

4 1362 MOUGHAN ET AL ( 5000 MW)-based diet was consumed by volunteer ileostomates, and ileal digesta samples were collected and ultracentrifuged ( MW). With this method, any unabsorbed dietary peptides are discarded in the ultrafiltrate, which leads to an unambiguous measurement of endogenous ileal AA loss. The method, however, underestimates the endogenous loss, because any endogenous free AAs and peptides ( MW) are also removed in the ultrafiltrate. In the growing pig given a PF diet, Moughan and Schuttert (25) found that only 14% of the nitrogenous compounds in ileal digesta had MW , whereas Leterme et al (26) reported a proportion of 22% of the compounds. It is more difficult to accurately ascertain the amounts of small endogenous AAs and peptides in the ileal digesta of protein-fed animals than in that of animals fed a PF diet, although, according to the results from the studies in which PF diets have been used, the concentration is expected to be low. That the method probably somewhat underestimates gut endogenous losses serves to make the peptide-to-pf comparison conservative. A further criticism of the enzyme-hydrolyzed protein method (27, 28) is that the peptides may elicit specific stimulatory effects (29) not seen when an intact protein is fed and digested naturally. This is possible, although the tryptic hydrolysate is prepared to simulate the natural products of protein digestion (30). A major advantage of the peptide alimentation method over other methods is that it allows the endogenous flows of total nitrogen and all AAs to be measured directly. Endogenous flows were measured in the current work by collection of digesta samples from the terminal ileum of human volunteers with well-established ileostomies. It is known (31) that a distinct microflora establishes in the small intestine of ileostomates, but the effect this may have on the endogenous protein flow is unknown. In a study of gut endogenous AA flow in human subjects, antibiotic treatment to reduce the numbers and the activity of microorganisms in the terminal ileum of ileostomates did not greatly affect the flow of AAs (16). Ileostomates have been used in numerous digestion studies and are widely accepted as a suitable model in which to study digestion in the small intestine of humans (32 36). Another important aspect of the method of the current study is that an acute feeding and digesta sampling regimen was used. It has been shown in an accompanying study (21), using the growing ileostomized pig as a model for ileostomized humans, that such an acute procedure does not affect in vivo measures of protein digestion and should be a valid procedure, therefore, for measuring gut endogenous protein. The recovery of the marker AIA in the current study was high (78 79%), which indicated that a representative sample of digesta pertaining to the meal had been collected. The recovery of AIA was greater than that found in a similar study with human ileostomates (40%; 17) but close to that found in another study of 30-kg live weight ileostomized pigs from which digesta samples were collected for 8 h (75%; 37). AIA has been shown to be a valid indigestible marker for use in human digestion studies (19). The endogenous flows for most of the AAs were significantly (P 0.05) higher with the EHC-based diet than with the PF diet. Significantly higher ileal endogenous flows of AAs have been reported for an EHC-based diet than for a PF diet in other simplestomached species such as the rat and pig (11 14, 24, 38). In particular, the flows of aspartate, glutamate, serine, proline, methionine, and total nitrogen measured by using the EHC method were considerably higher than those measured by using the PF method. Endogenous flows of glutamate and methionine were 4 times higher, and flows of aspartate, serine, proline, and total nitrogen were between 2 and 3 times higher than the respective flows measured with the PF method. Other studies comparing the endogenous flows of AAs in the rat reported smaller differences between the 2 methods for flows of glutamate and aspartate (11, 12). In those studies, the relative differences between the 2 methods for the endogenous flows of most of the essential AAs were similar to those found in the present study. The SEMs of the endogenous flows were high (CVs ranged from 12% to 67%; average CV: 30%), which indicated much variability among subjects in overall gut metabolism. The endogenous losses of AAs and total nitrogen were compared with earlier published results in ileostomized humans who were consuming either PF diets (16, 17) or 15 N-labeled proteins (4) (Table 4). The endogenous losses measured with a PF diet were in the same range. Rowan et al (17) reported values somewhat higher than the present ones, especially those for methionine and cysteine losses (2 times higher). Endogenous AA losses measured by using the EHC or 15 N dilution methods were also similar, with a ratio ranging from 0.8 to 1.2 times (except for glutamic acid, which was exceptionally high in the current study). Globally, the endogenous losses determined with a PF diet were lower than those determined with a peptide- or proteinbased diet, which supports the general view that the PF diet underestimates endogenous losses. In a related study (PJ Moughan, CA Butts, unpublished observations, 2000), 4 intact proteins (sodium caseinate, whey protein concentrate, soya protein isolate, and soya protein concentrate) were given to adult ileostomates (n 8) as the sole sources of protein. Mean apparent ileal digestibility coefficients for the AA lysine of 94.4, 92.7, 93.3, and 92.2% were determined for the sodium caseinate, whey protein concentrate, soya protein isolate, and soya protein concentrate, respectively. When the currently reported EHC-based endogenous ileal lysine flows were used to correct these apparent digestibility values, true ileal digestibilities of lysine of 103.0, 99.2, 102.6, and 101.9% were found for sodium caseinate, whey protein concentrate, soya protein isolate, and soya protein concentrate, respectively. This indicates, on the basis of estimates of endogenous loss that are more valid physiologically than are PF-based flows, that such high-quality protein sources are almost completely digested and absorbed. Ileal digestibility coefficients close to unity for refined milk and soybased proteins concur with the result of Gaudichon et al (4). A best estimate of the daily endogenous loss of essential AAs in humans was calculated as a mean of the values obtained using the protein- or peptide-based diet (Table 5). When the latter endogenous AA losses are expressed as a proportion of estimates of the daily AA requirement (18), the gut amino acid losses are found to be a sizeable proportion of the requirement, ranging from 9% to 20% for most of the AA losses; valine and threonine accounted for a greater part of the requirement 24% and 25%, respectively. The major contribution of the endogenous AA losses to the requirement particularly the contribution of the threonine loss has been noted in earlier studies (4, 16, 17). The high contribution of the threonine loss to its requirement may be related to the high proportion of threonine in the gastrointestinal mucins (41), which, through their resistance to hydrolysis, constitute one of the major forms of gut AA loss. When the endogenous AA losses were expressed as a proportion of the daily

5 TABLE 4 Interstudy comparison of endogenous ileal amino acid losses measured in human ileostomates 1 ILEAL ENDOGENOUS AMINO ACIDS 1363 Protein- or peptide-based diet Protein-free diet EHC diet 15 N isotope Rowan et al (17) 2 Without antibiotics Fuller et al (16) 3 With antibiotics Current study 4 Current study 4 Gaudichon et al (4) 5 mg/d mg/d Threonine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Tyrosine Phenylalanine tyrosine Lysine Histidine Serine Glutamic acid Alanine Proline Arginine Aspartic acid Arginine aspartic acid Glycine Nitrogen EHC, enzyme-hydrolyzed casein. 2 In this study, the values were reported in mg/8 h. The experimental meal was assumed to represent one-third of the daily intake, and the endogenous losses of amino acids were assumed to be similar every 8-h period. The initial values were multiplied by 3 for expression in mg/d. 3 Values in this study were reported in mg/d, so no conversion was necessary. 4 Values in the current study are reported in mg/g dry matter intake. Values were multiplied by 410 g (estimated average daily food dry matter intake; 23, 39, 40). 5 Values, reported in mg kg 1 d 1, were multiplied by the mean body weight of the subjects (70 kg) for expression in mg/d. dietary AA requirements as published by the Food and Agriculture Organization and the World Health Organization (FAO/ WHO; 42), the endogenous ileal losses contributed to an even greater proportion of the daily essential AA requirement. Most of the AA losses represented 26% to 40% of the dietary essential TABLE 5 Comparison of a best estimate of the endogenous ileal loss of essential amino acids in human ileostomates with recommended daily dietary amino acid requirement values Endogenous ileal losses 1 Percentage of requirement 2 mg/d % Threonine Valine Isoleucine Leucine Phenylalanine tyrosine Methionine cysteine Lysine Histidine The best estimate is the mean of the values determined by using the enzyme-hydrolyzed casein based diet in the current study and the 15 N milk or soy proteins in the study of Gaudichon et al (4) (also, see Table 4). 2 Human dietary amino acid requirements for persons aged 19 y (70 kg body weight; 18). AA requirement. Threonine accounted for 80% of the requirement. According to several workers, however, the threonine requirement should be 2 (43), 3 (44) or even 4 (45) times the current recommendation of the FAO/WHO (41), and thus the estimated proportional contribution of ileal threonine loss, and also that for other AAs, may be overestimated when expressed as a proportion of the FAO/WHO values. An assumption is made that the ileal losses represent obligatory AA losses from the body and that no reabsorption of endogenous AAs occurs in the colon. Metges et al (46) suggest that some AA reabsorption does occur in the human large intestine. Studies have shown that AA transporters are expressed in the colonic cells of mice (47) and that peptide transporters may play a role in the colonic uptake of AAs (48). More information about the contribution of the colon to the potential uptake of endogenous AAs is needed. On the other hand, the ileal flows given in Tables 4 and 5 represent unabsorbed endogenous secretions from the upper digestive tract only. Endogenous proteins are also secreted into the large intestine and will be lost from the body. In addition, in the current study, a relatively highly purified diet was given. The total gut endogenous AA loss can be expected to be higher for practical diets that contain plant fiber and antinutritional factors (eg, tannins, lectins, and trypsin inhibitors). In conclusion, our study shows that dietary peptides exert a positive influence on endogenous ileal AA loss in humans and supports the general finding that the PF method underestimates the gut endogenous AA losses. The endogenous ileal losses of

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