Human Nutrition and Metabolism

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Human Nutrition and Metabolism

An Acute Ileal Amino Acid Digestibility Assay Is a Valid Procedure for Use in Human Ileostomates1 Paul J. Moughan, Christine A. Butts,* Henk van Wijk,† Angela M. Rowan,** and Gordon W. Reynolds† Riddet Centre, Massey University, Palmerston North, New Zealand; *New Zealand Institute for Crop & Food Research Limited, Palmerston North, New Zealand; †Institute of Food Nutrition and Human Health, Massey University, Palmerston North, New Zealand; and **Fonterra Research Centre, Palmerston North, New Zealand


amino acids



Measures of dietary protein quality are of fundamental importance in nutrition and are largely a reflection of the amino acid composition of a protein and the digestibility of its respective amino acids. Amino acid digestibility may be determined over the total digestive tract (fecal digestibility) or at the end of the small intestine (ileal digestibility). However, it is more appropriate to determine amino acid digestibility at the end of the small intestine rather than at the fecal level because the digestion of protein and subsequent absorption of amino acids occur mainly in the upper small intestine, and are effectively completed by the end of the ileum. Bacteria resident in the large intestine transform undigested dietary protein and amino acids into bacterial protein and other compounds that are absorbed but provide little nourishment. Amino acids, as such, do not appear to be absorbed from the large intestine in nutritionally relevant amounts. The determination of ileal amino acid digestibility in humans, however, requires that ileal digesta be sampled from conscious subjects. Two approaches have commonly been used, nasogastric intubation (1) and the collection of digesta from ileostomates (2). In both cases, it has been common




practice, and for pragmatic reasons, to give a single meal including the test protein, without prior dietary adaptation (i.e., acute feeding), and then to sample digesta pertaining to that single meal (3–5). The experimental diets used are often somewhat unpalatable and are acceptable to subjects only if given as a single meal. There is a tacit assumption that the determined protein digestibility coefficient will not be influenced by the less than complete adaptation of the digestive system to the test diet. However, a paucity of published data exist addressing this assumption. The first objective of the work reported here, therefore, was to assess, in a preliminary study, the accuracy of an acute feeding/digesta sampling regimen by comparison with results from a traditional 14-d metabolism study design. Ileostomized pigs were used in the study as a model for adult humans (6). In the main study reported here, an acute digestibility assay (validated in the preliminary study) was applied using adult human ileostomates. The objective was to determine ileal amino acid digestibility data for 4 refined food protein sources. Although there is considerable information available on fecal protein digestibility in humans, few data on ileal amino acid digestibility exist. Healthy adult ileostomates are a common experimental model for determining the ileal digestibility of nutrients in humans (2,7). In these subjects, the large intestine had been removed surgically because of some prior health

Funded by Fonterra Co-operative Group Ltd., Palmerston North, New Zea-

land. 2

To whom correspondence should be addressed. E-mail: [email protected]

0022-3166/05 $8.00 © 2005 American Society for Nutritional Sciences. Manuscript received 26 July 2004. Initial review completed 30 September 2004. Revision accepted 29 November 2004. 404

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ABSTRACT An acute (24-h) feeding/digesta sampling procedure was evaluated in a preliminary study using growing pigs. The validated acute procedure was then applied using human ileostomates to determine apparent and true ileal amino acid digestibilities of 4 dietary protein sources. The acute method involved feeding ileostomized pigs a single meal containing the test protein as part of a purified diet, with no previous dietary adaptation, followed by an 8-h collection of digesta. Apparent ileal N digestibility did not differ between the acute and conventional (14-d study) procedures. Eight adult human ileostomates each received a single meal of protein-free biscuits and a drink containing sodium caseinate, whey protein concentrate, soy protein isolate, or soy protein concentrate; this meal was followed by a 9-h total digesta collection period. Acid insoluble ash was used as an indigestible marker. True ileal amino acid digestibilities (means ⫾ SE) ranged from 90.5 ⫾ 2.74% for cysteine in soy protein concentrate to 105.3 ⫾ 5.66% for cysteine in sodium caseinate and were markedly higher than their apparent counterparts. True ileal digestibilities for total nitrogen were 101.9 ⫾ 0.70, 98.3 ⫾ 0.80, 99.5 ⫾ 0.80, and 98.5 ⫾ 1.20% for sodium caseinate, whey protein concentrate, soy protein isolate, and soy protein concentrate, respectively. The 4 protein sources were virtually completely digested in humans by the end of the small intestine. J. Nutr. 135: 404 – 409, 2005.


disorder, and the output of the upper gastrointestinal tract empties via a stoma into plastic stoma bags attached directly to the outer body wall. The food samples tested included sodium caseinate, whey protein concentrate, soy protein isolate, and soy protein concentrate. MATERIALS AND METHODS Preliminary study

TABLE 1 Ingredient composition of the skim milk powder-based experimental diet Ingredient Skim milk powder1 Margarine Corn flour Sucrose Golden syrup2 Cellulose (Avicel) Baking powder3 Vitamin/Mineral mix4 Ground ginger Chromic oxide Polyethylene glycol5

g/kg air-dried weight 392.0 254.0 111.0 118.5 50.0 27.0 9.5 22.0 10.0 3.0 3.0

1 Skim milk powder contained 38.2% protein, 0.8% fat, 47.9% carbohydrate, as well as Na, K, Ca, Zn, Se, vitamins A, D, B-6, B-12, riboflavin, thiamin, and niacin. 2 A corn sugar syrup. 3 Contained wheat flour, sodium bicarbonate, and calcium carbonate. 4 The vitamin/mineral mix contained 17 g dicalcium phosphate and 5 g vitamin/mineral premix. The premix supplied (mg/kg diet): vitamin A, 5.4; cholecalciferol, 56.3; vitamin E, 50; vitamin K, 2.5; thiamin, 1.5; riboflavin, 4.0; vitamin B-6, 3.0; pantothenic acid, 12.0; niacin, 22.0; choline, 150.0; vitamin C, 20.0; Co, 0.5; I 1.0; Cu, 125.0; Fe, 100; Mn, 45; Zn, 120; (␮g/kg diet): vitamin B-12, 30; folate, 500; biotin, 75; Se, 300. 5 Polyethylene glycol was included as a marker compound but was not used in the calculation of digestibility coefficients.

d 9 and 14 for 8 h (0830 –1630 h) each day. Ileal digesta samples were collected hourly and were immediately placed in a plastic container (with 10 mL of 4 mol/L HCl) and cooled. The digesta collected over the 8 h for each regimen were weighed, pooled, frozen (⫺20°C), and freeze-dried. The freeze-dried digesta samples, along with diet samples, were finely ground and stored at ⫺20°C before chemical analysis. The design gave rise to 2 observations/pig for each dietary regimen (12 observations/dietary regimen). Total nitrogen (N), dry matter, and chromium were determined in duplicate in samples of digesta and diet. Total N was determined using the Kjeldahl method on a Kjeltec 1030 analyzer (Tecator) using the method of Hiller et al. (9). Dry matter was determined by drying to a constant weight using a forced air oven at 95°C. Chromium was determined as described previously (10). Estimates of apparent ileal N digestibility were calculated from the dietary ratio of N to chromium relative to the corresponding ratio in the ileal digesta. The N:marker ratios, marker recoveries, and apparent ileal N digestibility values were tested for homogeneity of variance using Bartlett’s test (11). The data were subjected to an ANOVA for repeated measures using the generalized linear models procedure (12). Data are presented as means ⫾ SE and differences were deemed to be significant at P ⬍ 0.05.

Main study 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). Subjects and diets. The 8 men and women who participated in the study were recruited through the local (Manawatu) Ostomy Society. Each had undergone a total colectomy for ulcerative colitis, but not ⬎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 at least 8 wk before the study. No subject was pregnant, lactating, or taking oral contraceptives. The subjects ranged in age from 45 to 62 y and their body weights from 67 to 97 kg. The subjects participated in the study while residing in their own homes; they were visited regularly throughout the study. The test diet (Table 2) consisted of a proteincontaining drink and a protein-free biscuit, with the test protein as the sole source of protein. The test proteins were sodium caseinate (Alanate 180, New Zealand Dairy Board), whey protein concentrate (Alacen 472, New Zealand Dairy Board), soy protein isolate (Supro 590, Columbit, NZ Ltd.), and soy protein concentrate (Danpro 180, Columbit, NZ Ltd.). Celite (acid insoluble ash, AIA) was included in the biscuits as an indigestible marker compound (13) to allow correction for incomplete collection of digesta. The biscuits were baked in a conventional oven at 120°C for ⬃20 min. The determined amino acid compositions of the 4 protein sources and of the essentially protein-free biscuits are given in Table 3. Experimental procedure. Each subject received each of the protein-containing test diets consecutively (3 d apart) following two 4 ⫻ 4 Latin Squares, such that within a square each diet followed all other diets only once. Subjects were randomly assigned to the dietary sequences within and across the Latin Squares. After an overnight (14-h) fast and commencing at 0800 h, each subject attached a new plastic ostomy bag and then consumed the test diet which consisted of 160 g of the protein-free biscuits and 750 mL of the protein-containing drink. The meal was consumed within 30 min and subjects were permitted to freely consume sweetened tea, coffee, soft drinks, or water after the test meal. Ostomy bags were changed and emptied every 2–3 h over the 9-h (0800 –1700) digesta collection period. The ileostomy output was transferred to a container to which 10 mL of 4 mol/L HCl had been added to prevent bacterial activity. The collection containers were kept chilled during the collection period and the contents were then frozen (⫺20°C). The digesta samples collected for each subject consuming the same diet on the 2 separate days were thawed and pooled before being freeze-dried and ground for chemical analysis. There were thus 8 experimental

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Animals and diets. Approval to conduct the study was obtained from the Massey University Animal Ethics Committee. Landrace ⫻ (Landrace ⫻ Large White) female pigs [n ⫽ 6; 20 ⫾ 0.5 kg (mean ⫾ SE) liveweight] were surgically prepared with ileostomies using a method similar to a published procedure (8). The surgery and postoperative care were as described previously (2). The ingredient composition of a skim milk powder– based experimental diet is given in Table 1. The diet was baked in 2 ⫻ 1-kg quantities at 120°C for 15 min in a conventional oven and fed to the pigs in a biscuit form to match the experimental conditions that would be used in the subsequent study with humans. The pigs were fed their diet mixed with water 3 times daily (0800, 1200, and 1600 h) in equal portions, at a set daily intake of 0.10 ⫻ metabolic body weight (W0.75). The experiment was conducted as a crossover design with 6 pigs randomly allocated to 2 feeding regimens. Acute and adaptation feeding regimens were used and there were 3 pigs/regimen at any one time. In the adaptation feeding regimen, the pigs were fed the experimental diet for 14 d, and ileal digesta were collected on d 8, 9, 13, and 14 for 8 h (0830 –1630 h) each day. Ileal digesta for d 8 and 9 were pooled for each pig as were the digesta for d 13 and 14. In the acute feeding regimen, the pigs were fed a semisynthetic meat and bone meal– based preliminary diet on d 1– 8 and d 10 –13 of the 14-d experimental period and were fed the experimental diet on d 9 and 14. There was a 16-h period between the final meal on d 8 or 13 and the first meal on d 9 or 14, which was considered sufficient time to clear the upper digestive tract of the previous meal. Ileal digesta were collected from these pigs on




TABLE 2 Ingredient composition of the protein-free biscuits and protein-containing drinks consumed by the human ileostomates Protein-free biscuit

g/kg dry matter

Ingredient Purified corn flour Margarine Sucrose Cellulose1 Baking powder2 Celite3 Ground ginger

415 286 232 50 7 5 5

Apparent digestibility 共%兲 ⫽

Diet 1

Amino acid output 共 g/g dry matter兲

Diet 3

Diet 4


⫽ AA concentration in digesta ⫻ True digestibility 共%兲

33.1 28.3 80.0


%AIA in diet %AIA in digesta


38.9 80.0

1 Avicel, Asahi Chemical. 2 Contained wheat flour, sodium bicarbonate and calcium carbon-


AA intake ⫺ 共 AA output ⫺ endogenous AA兲 ⫻ 100 AA intake

Endogenous amino acid flows were determined based on digesta collected from human subjects fed a protein-free diet (Moughan, P. J. and Butts, C., unpublished data). Data are means ⫾ SE.

3 Double acid-washed Celite as a source of AIA (Sigma Chemical). 4 Each protein and flavor/sucrose combination was dissolved in

water. 5 The amount of protein concentrate varied depending upon its protein and dry matter content. 6 Orange flavored and colored sucrose:glucose mixture (Raro, Cerebos Greggs) with 5 g/80 g polyethylene glycol added.

observations for each of the 4 protein sources. Generally, subjects consumed test diets either 2 or 3 days apart (2 test meals/wk); between test meals, they consumed their normal daily diet. The total study lasted for 4 wk. Chemical analysis. Samples of finely ground, freeze-dried digesta along with representative finely ground, freeze-dried samples of the

RESULTS Preliminary study. The ileostomized pigs remained healthy postsurgery except for 2 pigs that were removed from the trial due to malfunctioning stoma. Values for dietary chromium recovery, the digesta N:chromium ratio, ileal dry matter flows (based on chromium recovery), and apparent ileal N digestibility (based on chromium recovery) are given in Table 4. The feeding regimen did not affect any of the variables, with similar apparent ileal N digestibilities (76 and 77%) for the adaptation and acute feeding regimens, respectively. The within-treatment SE also did not differ between the 2 feeding regimens (1.8 vs 2.7, adaptation vs acute).

TABLE 3 Amino acid composition of the 4 protein sources and the essentially protein-free biscuit Protein-free

Sodium caseinate

Whey protein concentrate

Soy protein isolate

Soy protein concentrate

127.4 71.0 46.1 187.6 42.2 46.4 44.7 80.9 59.7 60.4 51.0 33.1 26.7 39.8 13.3 14.3

86.8 49.0 30.6 126.5 29.0 33.2 29.9 54.8 40.0 43.4 34.4 22.9 19.0 29.1 11.3 10.2

mg/g air-dried weight Aspartate Arginine Alanine Glutamate Glycine Serine Isoleucine Leucine Valine Lysine Phenylalanine Tyrosine Histidine Threonine Cysteine Methionine

0.24 0.16 0.13 0.31 0.19 0.18 0.08 0.16 0.11 0.15 0.06 0.04 0.04 0.11 0.06 0.00

69.6 28.7 28.0 192.5 17.1 48.1 43.1 84.1 72.6 68.3 44.1 41.3 28.0 39.9 4.3 30.2

96.2 19.9 45.5 141.8 16.0 39.7 48.0 90.9 48.4 74.8 27.3 22.2 17.0 59.5 22.4 20.2

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Ingredient4,5 Sodium caseinate Whey protein concentrate Soy protein isolate Soy protein concentrate Flavour/sugar6

Diet 2

AA intake ⫺ AA output ⫻ 100 AA intake

AA ⫽ amino acid (g/g dry matter) where

g dry matter/750 mL Protein-containing drink

respective test diets were subjected to analysis for dry matter, total N, amino acids, and AIA. Analysis was as described for the preliminary study. AIA was determined using a gravimetric procedure (14). Amino acids were determined using detection by ion-exchange HPLC after hydrolysis of the samples in 6mol/L HCl for 24 h at 110°C in evacuated sealed tubes. Methionine and cysteine were determined as methionine sulfone and cysteic acid, respectively, after hydrolysis of samples that had been oxidized using performic acid (14). Tryptophan was not measured. Analysis of data. Apparent and true amino acid digestibilities were determined using the following equations:


TABLE 4 Effect of feeding regimen on dietary chromium recovery and apparent ileal N digestibility for ileostomized pigs fed a skim milk powder-based diet Feeding regimen1 Adaptation


Overall SE

3.0 59 243 75.7

2.3 60 217 76.6

0.33 5.4 25.6 1.72


and highly variable. The next highest apparent digestibility value for sodium caseinate was 69.6% for glycine. The true ileal N and amino acid digestibilities (corrected for endogenous loss; protein-free diet) for the 4 protein sources are given in Table 5. True ileal amino acid digestibility ranged from 89.2% for histidine in whey protein concentrate to 105% for cysteine in sodium caseinate. DISCUSSION

Digesta N:chromium Chromium recovery, % Digesta dry matter,2 mg/g DMI Apparent ileal N digestibility, %

1 Values are means, n ⫽ 12 for adaptation regimen (6 pigs, duplicate measures) and n ⫽ 9 for acute regimen (4 pigs with duplicate measures, 1 with a single measure). Means did not differ between feeding regimens. 2 DMI, dietary dry matter intake based on the chromium marker.

TABLE 5 True ileal amino acid and total nitrogen digestibilities in humans given milk protein and soy protein products1,2 Amino acid

Sodium caseinate

Whey protein concentrate

Soy protein isolate

Soy protein concentrate

97.2 ⫾ 0.44 97.8 ⫾ 0.91 97.9 ⫾ 0.76 99.1 ⫾ 0.29 97.8 ⫾ 0.62 95.3 ⫾ 2.43 96.9 ⫾ 0.53 97.3 ⫾ 1.57 98.4 ⫾ 0.44 97.2 ⫾ 0.69 97.1 ⫾ 0.46 97.2 ⫾ 0.49 98.8 ⫾ 0.73 97.9 ⫾ 0.51 99.3 ⫾ 0.88 99.1 ⫾ 0.51 99.4 ⫾ 0.33 99.5 ⫾ 0.80

96.6 ⫾ 0.79 96.7 ⫾ 1.88 97.0 ⫾ 1.35 97.9 ⫾ 0.35 96.4 ⫾ 1.17 96.2 ⫾ 1.47 96.6 ⫾ 0.86 90.5 ⫾ 2.74 96.1 ⫾ 0.70 96.5 ⫾ 0.94 97.2 ⫾ 0.66 96.7 ⫾ 0.64 98.8 ⫾ 0.86 97.1 ⫾ 0.52 96.9 ⫾ 1.90 98.2 ⫾ 0.61 99.7 ⫾ 0.86 98.5 ⫾ 1.20

% Aspartate Threonine Serine Glutamate Proline Glycine Alanine Cysteine Methionine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total N

98.1 ⫾ 0.70 99.5 ⫾ 0.77 98.8 ⫾ 0.63 98.6 ⫾ 0.29 99.2 ⫾ 0.28 102.4 ⫾ 2.06 99.7 ⫾ 0.98 105.3 ⫾ 5.66 99.5 ⫾ 0.26 99.6 ⫾ 0.54 99.5 ⫾ 0.47 100.1 ⫾ 0.46 100.2 ⫾ 0.59 99.7 ⫾ 0.52 99.0 ⫾ 0.92 99.8 ⫾ 0.47 100.2 ⫾ 0.70 101.9 ⫾ 0.70

97.7 ⫾ 0.43 92.7 ⫾ 0.58 93.0 ⫾ 0.69 97.6 ⫾ 0.28 95.1 ⫾ 0.43 97.8 ⫾ 2.36 97.3 ⫾ 0.53 99.3 ⫾ 1.92 98.5 ⫾ 0.48 98.3 ⫾ 0.57 98.5 ⫾ 0.32 99.2 ⫾ 0.30 98.7 ⫾ 0.72 98.8 ⫾ 0.65 89.2 ⫾ 1.91 96.7 ⫾ 0.37 98.9 ⫾ 0.76 98.3 ⫾ 0.80

1 Values are means ⫾ SE, n ⫽ 8. 2 Digestibility coefficients were calculated using AIA as an indigestible marker to correct for incomplete digesta collection.

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Main study. The subjects completed the study without complications and there was ready compliance with the experimental protocol. The recovery of AIA was high after the 9-h collection of digesta, ranging from 86.2 ⫾ 6.81% for the soy protein isolate diet to 92.7 ⫾ 9.99% for the soy protein concentrate diet. The overall recovery of AIA for the 4 diets was 90.6%. The apparent ileal N digestibilities (n ⫽ 8) were 89.8 ⫾ 0.70, 86.6 ⫾ 0.80, 88.2 ⫾ 0.80, and 86.4 ⫾1.20% for sodium caseinate, whey protein concentrate, soy protein isolate, and soy protein concentrate, respectively. The ranges in apparent ileal amino acid digestibility were 39.6 ⫾ 5.67% for cysteine to 97.3 ⫾ 0.26% for methionine; 69.0 ⫾ 2.36% for glycine to 95.7 ⫾ 0.48% for methionine; 76.9 ⫾ 1.57% for cysteine to 95.5 ⫾ 0.29% for glutamate; 72.3 ⫾ 2.74% for cysteine to 95.1 ⫾ 0.86% for arginine in sodium caseinate, whey protein concentrate, soy protein isolate and soy protein concentrate, respectively. The apparent ileal digestibility value for cysteine in the sodium caseinate was inordinately low

The nutritional value of a protein-containing food is determined largely by its amino acid composition, in relation to the requirements for the various dietary essential amino acids, and is modified by the extent to which the food can be digested in the gastrointestinal tract. True ileal amino acid digestibility is considered the best parameter to use in estimating protein digestibility in foods (15,16). However, there are few published data. An FAO/WHO Expert Consultation (17) recommended that improved methods be explored to obtain measures of true ileal protein digestibility. The work described here contributes to such a development. Determination of the true ileal or fecal digestibility of food proteins has traditionally been made using laboratory rats as the animal model (17). This model can be criticized, however, because of marked differences in amino acid requirements, food intake, and ingestive behavior between rats and humans. A study using growing pigs as the model animal for humans found that true ileal amino acid digestibilities for a mixed diet did not differ between the 2 species (2), and growing pigs may be a suitable model animal. Information obtained directly with humans, however, provides the most relevant and acceptable data for use in human nutrition. The primary aim of the present study was to apply a digestibility assay with human ileostomates to determine the apparent and true digestibility of amino acids in several refined sources of dietary protein. In humans, this generally requires the use of an acute assay. In animal-based digestibility assays, animals are normally fed a single test protein, as part of a



subjects may not have fully adapted to the protein-free state, this may be less of a concern (20). The acute feeding procedure used in this study incorporated an overnight fast of 14 h duration and a 9-h digesta collection period. The 14-h time period in which subjects consumed no food was considered sufficient to clear the upper tract of residues from previous food (21,22); based on the preliminary trial results, a single 9-h collection of digesta should have been sufficient to obtain a representative sample of digesta. High recoveries of the indigestible marker, AIA, were obtained (86 –93%), which were greater than a previously reported recovery value (40%) found in a similar study using human ileostomates with an 8-h collection of digesta (3) and were also higher than the recoveries of chromium reported for the ileostomized pigs in the preliminary study. The marker recovery values suggest a nearly complete recovery of the digesta associated with the test meals given. The apparent ileal digestibilities of N and amino acids were generally ⬃80 –90% for the milk and soy proteins. A particularly low value was found for the apparent ileal digestibility of cysteine in the sodium caseinate, although the true digestibility value exceeded 100%. Sodium caseinate has a relatively low cysteine content, meaning that the endogenous cysteine has a disproportionate effect on the determination of apparent digestibility. Relatively low apparent ileal digestibilities were also found for the amino acid glycine and for histidine in the whey protein concentrate. The low apparent but not true digestibilities found for some amino acids highlight that apparent amino acid values, which are affected by assay conditions, are potentially misleading (23). The true ileal digestibilities generally ranged from 95 to 100%. Overall, the data demonstrate that for refined dairy and soy protein sources, amino acids are absorbed almost completely, anterior to the end of the small intestine. This finding is consistent with other results from human digestibility studies (1,24) and from studies with other monogastric species (25). The quality of such proteins is not limited by amino acid digestibility. This is not expected to be the case for more processed animal proteins and less refined plant-based proteins, to which research attention should now be directed. In conclusion, experimental evidence is presented for the accuracy of an acute (24-h) ileal protein digestibility assay for application to humans. The study highlights the potentially misleading nature of apparent ileal amino acid digestibilities. Based on true ileal amino acid digestibilities, it appears that high-quality soy and milk proteins are virtually completely digested anterior to the end of the small intestine. LITERATURE CITED 1. 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. 2. Rowan, A., Moughan, M., Wilson, P. J., Maher, M. N., Tasman, K. & Jones, C. (1994) Comparison of the ileal and faecal digestibility of dietary amino acids in adult humans and evaluation of the pig as a model animal for digestion studies in man. Br. J. Nutr. 71: 29 – 42. 3. Rowan, A. M., Moughan, P. J. & Wilson, M. N. (1993) Endogenous amino acid flow at the terminal ileum of adult humans determined following the ingestion of a single protein-free meal. J. Sci. Food Agric. 61: 439 – 442. 4. Birkett, A. M., Mathers, J. C., Jones, G. P., Walker, K. Z., Roth, M. J. & Muir, J. G. (2000) Changes to the quantity and processing of starchy foods in a Western diet can increase polysaccharides escaping digestion and improve in vitro fermentation variables. Br. J. Nutr. 84: 63–72. 5. Mahe´, S., Roos, N., Benamouzig, R., Sick, H., Baglieri, A., Huneau, J.-F. & Tome´, D. (1994) True exogenous and endogenous nitrogen fractions in the human jejunum after ingestion of small amounts of 15N-labeled casein. J. Nutr. 124: 548 –555. 6. Moughan, P. J., Cranwell, P. D., Darragh, A. J. & Rowan, A. M. (1994) The domestic pig as a model for studying digestion in humans. In: Digestive

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semisynthetic experimental diet for 14 d, and ileal digesta are collected over the last 3– 4 d. Such a testing regimen, however, is generally unacceptable to human ileostomates, thus making it necessary to use a more acute regimen. A practical procedure is to request the subject (who has been eating a varied diet) to eat a single meal of the test diet and then to collect ileal digesta for a defined period of time within the working day. The application of such a procedure raises 2 issues. The first is whether the lack of adaptation to the test diet affects the digestibility result, and second, whether a single 8-h collection period is adequate. The preliminary study reported here was designed to address these 2 important considerations. Ileostomized pigs were used as a model for humans. The recovery of the indigestible marker, chromium, for the ileostomized pigs indicated that the amount of digesta collected was associated with approximately two-thirds of the intake of the test diet. This was considered to be satisfactory for ensuring a representative sample of digesta. There were no significant (P ⬎ 0.05) differences in the ileal digestibility of N between the acute and adaptation regimens. Importantly, the acute feeding regimen did not give rise to a greater within-treatment variance for apparent ileal total N digestibility. It thus appears that a 14-d adaptation period is not strictly necessary for ileal protein digestibility assays. Further, a collection of digesta within a single 24-h feeding/ digesta collection period gives an adequate and representative sample of ileal digesta. These findings suggest that an acute assay procedure can be successfully applied with human ileostomates, although it is recognized that the present results are restricted to one source of dietary protein (skim milk powder) and relate to the digestibility of total N rather than to that of the individual amino acids. The aim of the main study was to determine the apparent and true ileal digestibilities of amino acids and N in 4 refined protein sources for humans. Ileal digestibility was determined after collecting digesta from the terminal ileum of human volunteers with well-established ileostomies. The effect on protein digestion of microbial activity in the terminal ileum of ileostomates due to colonization postsurgery is unknown. However, in a study of gut endogenous amino acid flow in humans (18), antibiotic treatment to reduce ileal bacterial activity in ileostomates had little effect on the flow of amino acids at the terminal ileum. Ileostomates have been used in a variety of digestion studies and are widely accepted as a suitable model for studying digestion in the small intestine of humans (3,4,18,19). In determining true ileal amino acid digestibility, a correction is made for the presence of endogenous protein and amino acids in the digesta. Endogenous sources of protein and amino acids found at the end of the ileum derive mainly from the gastrointestinal tract and include digestive enzymes, mucoproteins, sloughed epithelial cells, plasma proteins, free amino acids, and peptides. Microbial protein is also included in the measure of endogenous protein, although it is not strictly endogenous material. Endogenous ileal protein and amino acid excretions have traditionally been determined after the administration of a protein-free diet, although this may lead to some degree of underestimation of gut endogenous protein loss (15). The protein-free method was recommended by the FAO/ WHO (18) for the routine evaluation of proteins using model animals. In the present study, the digesta protein and amino acid flows determined were corrected for the endogenous component using data obtained from adult ileostomates who had consumed a protein-free diet. The values of the protein-free true digestibilities may be conservative if endogenous protein loss is underestimated, although with the acute assay, in which

ILEAL AMINO ACID DIGESTIBILITY IN HUMANS Physiology in the Pig. Proceedings of the VIth International Symposium on Digestive Physiology in Pigs, Bad Doberan, Germany (Souffrant, W. B. & Hagemeister, H., eds.), pp. 389 –396. Forschungsinstitut fur die Biologie Landwirtschaftlicher Nutztiere (FBN), Germany. 7. Sandberg, A.-S., Andersson, H., Hallgren, B., Hasselblad, K., Isaksson, B. & Hulten, L. (1981) Experimental model for in vivo determination of dietary fibre and its effect on the absorption of nutrients in the small intestine. Br. J. Nutr. 45: 283–295. 8. Brooke, B. N. (1969) Ileostomy. In: Operative Surgery, Vol. 5 (Rob, C., Smith, R. & Morgan, C. N., eds.), pp. 515–521. Butterworth, London, UK. 9. Hiller, A., Plazin, J. & van Slyke, D. D. (1948) A study of conditions for Kjeldahl determination of nitrogen in proteins. Description of methods with mercury as a catalyst and titrimetric and gasometric measurements of the ammonia formed. J. Biol. Chem. 176: 1401–1420. 10. Costigan, P. & Ellis, K. J. (1987) Analysis of faecal chromium derived from controlled release marker devices. N.Z. J. Technol. 3: 89 –92. 11. Sokal, R. R. & Rohlf, F. J. (1981) Biometry. The Principles and Practice of Statistics in Biological Research. A.H. Freeman and Company, San Francisco, CA. 12. SAS Institute Inc. (2001) SAS version 8.2, SAS Institute, Cary, NC. 13. Rowan, A. M., Moughan, P. J. & Wilson, M. N. (1991) Acid-insoluble ash as a marker compound for use in digestibility studies with humans. J. Sci. Food Agric. 54: 269 –274. 14. Moore, S. (1963) The determination of cysteine as cysteic acid. J. Biol. Chem. 238: 235–237. 15. Moughan, P. J. (2003) Amino acid availability; aspects of chemical analysis and bioassay methodology. Nutr. Res. Rev. 16: 127–141. 16. Fuller, M. F. & Tome´, D. (2004) In vivo determination of amino acid


bioavailability in humans and model animals. J. Assoc. Off. Anal. Chem. Int. (in press). 17. FAO/WHO (1990) Report of the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation. FAO, Rome, Italy. 18. Fuller, M. F., Milne, A., Harris, C. I., Reid, T.M.S. & Keenan, R. (1994) Amino acid losses in ileostomy fluid on a protein-free diet. Am. J. Clin. Nutr. 59: 70 –73. 19. Sandstrom, B., Andersson, H., Kivisto, B., & Sandberg, A.-S. (1986) Apparent small intestinal absorption of nitrogen and minerals from soy and meat protein based diets. J. Nutr. 116: 2209 –2218. 20. Hodgkinson, S. M., Moughan, P. J. & Reynolds, G. W. (2000) Effect of the duration of feeding of a protein-free diet on endogenous ileal nitrogen and amino acid loss in the growing pig. J. Sci. Food Agric. 80: 1407–1412. 21. McMinn, R.M.H. & Hobdell, M. H. (1974) Functional Anatomy of the Digestive System, Pitman Medical, UK. 22. Magee, D. F. & Dalley, A. F. (1986) Digestion and the Structure and Function of the Gut. Karger, Basel, Switzerland. 23. Boisen, S. & Moughan, P. J. (1996) Different expressions of dietary protein and amino acid digestibility in pig feeds and their application in protein evaluation. A theoretical approach. Acta Agric. Scand. 46A: 165–172. 24. Gaudichon, C., Bos, C., Morens, C., Petzke, K. J., Mariotti, F., Everwand, J., Benamouzig, R., Dare´, S., Tome´, D. & Metges, C. C. (2002) Ileal losses of nitrogen and amino acids in humans and their importance to the assessment of amino acid requirements. Gastroenterology 123: 50 –59. 25. Rutherfurd, S. M. & Moughan, P. J. (1997) True digestible amino acid composition of several milk proteins: application of a new bioassay. J. Dairy Sci. 81: 909 –917.

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