The kinetics of amino acid disappearance in the small intestine is related to the extent of amino acids absorbed in growing pigs Carlos A. Montoya1,2*, Michael van Bemmel2, Kevin Kreutz2, Suzanne M. Hodgkinson2, Natascha Stroebinger2 and Paul J. Moughan2 1Smart Foods & Bioproducts, AgResearch, Te Ohu Rangahau Kai Facility, Palmerston North, New Zealand 2Riddet Institute, Massey University, Te Ohu Rangahau Kai Facility, Palmerston North, 4474, New Zealand (Submitted 1 June 2023 – Final revision received 10 October 2023 – Accepted 23 October 2023 – First published online 26 October 2023) Abstract This study evaluated the importance of a correction for amino acids (AA) released into the hindgut on a measure of AA absorption kinetics and tested whether AA absorption kinetics are related to the extent of AA absorption using the growing pig as a model for humans. Thirty-six nine- week-old pigs (22·3 kg) received a diet containing whey protein as the sole protein source for 8 d. Pigs received their last meal containing the indigestiblemarker titanium dioxide before being euthanised at 1, 2, 3, 4, 6 and 12 h post-feeding. The entire content of each gastrointestinal tract (GIT) region was collected to determine AA released into the hindgut, and the kinetics and extent of AA absorption (uncorrected and corrected for AA entering the hindgut). Amounts of AA released into the hindgut increased over time (e.g. 33 and 180mg of Glu for 4 and 6 h post-feeding). The corrected apparent amount of each AA absorbed from the GIT lumen after 4 h post-feeding was generally lower (P≤ 0·05) than the uncorrected counterpart. Differences in both the kinetics and extent of AA absorption were observed across AA. For example, the time to reach half of the apparent AA absorption (T50) was 1·5 and 3·4 h for Met and Arg, respectively, whereas their extent of apparent absorptionwas 93 and 73 %. Negative correlations between parameters related to kinetics and the extent of apparent absorption were observed (e.g. for T50 r=−0·81; P< 0·001). The kinetics of AA absorption is related to the extent of AA absorption. Keywords: Amino acids: Kinetics of absorption: Transit time: Pigs The kinetics of amino acid (AA) absorption are important in relation to the effect of AA on whole-body protein metabo- lism(1,2). For example, AA need to be delivered at specific times and amounts to the site of protein synthesis(3,4). The kinetics of AA absorption are likely to be one of the contributing factors determining the extent of AA absorption in the small intestine, but this remains to be demonstrated. For example, the rate and extent of lysine absorption were lower than that for the methionine counterparts (8·4 %/h and 81 %, respectively, v. 9·7 %/h and 93 %) in rats fed a 15N labelled wheat/yeast diet(5). The kinetics of AA absorption are commonly studied by measuring plasma AA concentrations over time. This informa- tion could potentially be combinedwith that generated from ileal cannulated animals to understand the association between the rate of AA absorption and the extent of AA absorption. However, plasma concentrations are difficult to interpret as they are influenced by several factors such as splanchnic AAmetabolism. In addition, the ileal cannulation approach allows for determin- ing the extent of AA absorption to the end of the small intestine only. The ileal cannulation method does not allow determining the kinetics of AA absorption, as the test meal reaches the terminal ileum several hours post-feeding, which means that absorption values cannot be determined earlier. Furthermore, the digesta collected at the site of the cannula only represent material found at the terminal ileum, while other parts of the test meal are still transiting proximal gastrointestinal tract (GIT) regions. An alternative classical approach is to use serial slaughter studies with animals to directly determine AA disappearance (assumed to equate with absorption) from the GIT lumen, by measuring total amounts of AA remaining in the GIT at different time points after ingestion of a meal(6,7). Although this method is time-consuming, relatively expensive, requires the use of multiple animals and can only be applied with an animal model, it gives direct estimates of the kinetics of AA absorption in the same animal. In these types of studies, it is generally assumed that the absorption of an AA as such is complete by the end of the small intestine (ileum) and that AA that have disappeared from the GIT lumen have been absorbed. With this approach, AA released into the hindgut are not usually accounted for, despite * Corresponding author: Dr C. A. Montoya, fax þ64 6 351 8003, email carlos.montoya@agresearch.co.nz Abbreviations: AA, amino acid; DSI, distal small intestine; GIT, gastrointestinal tract; PSI, proximal small intestine. British Journal of Nutrition (2024), 131, 762–772 doi:10.1017/S0007114523002441 © The Author(s), 2023. Published by Cambridge University Press on behalf of The Nutrition Society. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. https://doi.org/10.1017/S0007114523002441 Published online by Cam bridge U niversity Press mailto:carlos.montoya@agresearch.co.nz https://doi.org/10.1017/S0007114523002441 http://creativecommons.org/licenses/by/4.0/ http://crossmark.crossref.org/dialog?doi=https://doi.org/10.1017/S0007114523002441&domain=pdf https://doi.org/10.1017/S0007114523002441 that over time, unabsorbed AA do enter the hindgut. We hypothesise that the amounts of AA released into the hindgut are significant and need to be considered when determining apparent AA absorption with the latter serial slaughter approach. We also hypothesise that the kinetics of AA absorption are related to the extent of AA absorption. This study aimed to demonstrate the effect of correcting for the AA released into the hindgut on measures of the kinetics of apparent AA absorption and to determine whether the kinetics and extent of apparent AA absorption are related. The study also allowed us to evaluate the use of a single dose of titanium dioxide (TiO2) as an indigestible marker under our experimental conditions. The growing pig was used as an animal model for adult humans(8). Materials and methods Animals and housing Ethics approval for the animal trial was obtained from the Animal Ethics Committee, Massey University, Palmerston North, New Zealand (application number 17/05). Entire male pigs (n 36, PIC Camborough 46 × PIC boar 356L, mean 22·3 (SE 0·32) kg bodyweight) were obtained from a commercial farm. Pigs were housed individually in metabolism crates in a room maintained on average at 22°C with a 12 h/12 h light/dark cycle. Cages and toys were washed daily. Toys were rotated daily. During the feeding and cleaning, pigs were monitored for general health, alertness, dietary intake and scouring using a scoring system to determine whether pigs remained in the study. The endpoints to exclude pigs from the study were food intake, lethargy and diarrhoea. Diets and experimental design Pigs were randomly allocated to each of the post-feeding time points, and some researchers and technical support staff were aware of this allocation. Pigs received a semi-synthetic diet for 8 d to ensure that they were adapted to the environment and to the diet. The adaptation diet contained wheat starch (521·2 g/kg DM diet), soyabean oil (140 g/kg DM), sucrose (100 g/kg DM), cellulose (40 g/kg DM), dicalcium phosphate (20·5 g/kg DM), vitamin and mineral premix (3 g/kg DM), salt (3 g/kg DM), the antioxidant Endox ® (0·3 g/kg DM) and whey protein isolate (172 g/kg DM; NZMPTM Whey Protein Isolate 8855, Fonterra Co-Operative Group Ltd) as the sole protein source. To improve palatability, the diet was mixed with 400 ml of tomato soup (20 g Maggi-rich tomato soup mix (14 g carbohydrates, 0·5 g protein, 0·3 g fat), Nestlé New Zealand Limited). The diet fulfilled the nutrient requirements of the growing pig(9). During the feeding period, pigs received two equal meals (2 % bodyweight/meal) at 08:00 and 17:00 h. However, on the final day of the feeding period, the last meal for each pig was provided at different times to ensure a 12 h fasting period on the following test day. On day eight, pigs were deprived of water for 2 h before receiving their final morning test meal. The test meal did not include the vitamin and mineral supplements or the antioxidant contained in the adaptation diet(10), as the test meal was also consumed by humans in a parallel but separate study. The indigestible marker TiO2 (1 g) was included in the test meal to allow the determination of its concentration in eachGIT location. The test meal was given to the pigs after a 12 h fasting period, apart from a group of pigs (n 6) that were euthanised directly. The latter group of pigs received TiO2 in the last meal on day seven, and for the calculations, they were considered as being 12 h post-feeding of the last meal. The remaining pigs were anaesthetised 15 min before being euthanised at 1, 2, 3, 4 or 6 h post-feeding (n 6 pigs/post-feeding time). Considering the sampling time required for each pig (∼25 min), pigs received their last meal at 30 min intervals to ensure that they were euthanised at the assigned post-feeding time. In addition, the same number of animals per post-feeding time was euthanised in a day. The anaesthetic cocktail (0·12 ml/kg bodyweight of Zoletil 100 (50 mg/ml), Ketamine (50 mg/ml) and Xylazine (50 mg/ml); Provet) was administered by an intramuscular injection. Pigs were euthanised by an intracardiac injection of sodium pentobarbi- tone (0·3 ml/kg bodyweight of Pentobarb 300; Provet). The body cavity was opened, and the stomach (at the oesophageal and pyloric sphincters), terminal ileum (at the ileocaecal valve) and the terminal rectum were isolated with clamps and the whole GIT was dissected out as described previously(10). The stomach and the terminal ileum (last 30 cm before the ileocaecal junction) were then removed, and the remaining small intestine was uncoiled and divided into two equal lengths (proximal and distal small intestine, PSI and DSI). To ensure that all chyme and digestawere collected, the stomach and each small intestinal region were gently flushed several times with a saline solution (0·9 g NaCl/l). The caecum was removed, and the colon (including the rectum) uncoiled. Faeces excreted post-feedingwere collected. The full caecum and colon were weighed, opened longitudinally, digesta were collected and caecal and colonic tissues were then washed, dried with paper towels and weighed again to determine total digesta on a wet basis. This was considered to be the most accurate way to determine total digesta in these GIT locations, as digesta adhere to these tissues. Caecal and colonic digesta, in contrast to small intestinal digesta, are less easily collected with flushing. Caecal and colonic (including faeces) digesta were thoroughly mixed before taking a representative weighed aliquot. Chyme and digesta (PSI, DSI, terminal ileal, caecal and colonic) were immediately frozen in dry ice, stored at –20°C, freeze-dried and ground. Chyme and small intestinal digesta samples (without terminal ileum) for each animal were pooled. The amounts of DM for the representative aliquots of caecal and colonic digesta were then used to calculate the total DM contents in the caecal and colonic digesta. Chemical analysis The test meal, stomach chyme, digesta (PSI, DSI, terminal ileal, caecal and colonic) and food refusals were analysed for DM and TiO2 (11). The test meal, food refusals, pooled digesta and terminal ileal digesta were analysed for standard AA (using HCl hydrolysis, o-phthalaldehyde pre-column derivatisation fol- lowed by reversed-phase HPLC)(12) and tryptophan (alkali hydrolysis)(13). The test meal was also analysed for starch (Kit Kinetics of amino acid absorption 763 https://doi.org/10.1017/S0007114523002441 Published online by Cam bridge U niversity Press https://doi.org/10.1017/S0007114523002441 AA/AMG, Megazyme), crude protein (nitrogen × 6·25; using an elemental analyser LECO), total fat (using a Soxhlet apparatus and petroleum ether extraction) and total dietary fibre(14). Calculations The amount of each AA (AAi) in the diet, food refusals, pooled digesta and terminal ileal digesta at each post-feeding time were calculated as shown below with terminal ileal digesta as an example. The same calculations were used to determine the TiO2 content (g DM). The TiO2 content of the stomach, PSI and DSI were summed to determine the TiO2 content of the pooled sample. AAi contentTerminal ileal digestaðmg inDMbasisÞ ¼ AAi concentrationTerminal ileal digestað%Þ �Total contentTerminal ileal digestaðgDMÞ=100 The relative amounts of DM and TiO2 exiting the stomach over time (timei as 1, 2, 3, 4, 6 and 12 h post-feeding) were determined as follows (DM as an example): RelativeDMexiting the stomach ðg inDMbasisÞ ¼ ðDMIntake �DMTime iÞ=DMIntake � 100 To ascertain the importance of AA escaping the small intestine into the large intestine, it was necessary to determine the amounts of AA released into the large intestine. Considering that the AA in large intestinal digesta do not represent the AA escaping the small intestine, as many are expected to be of microbial origin, the AA released into the large intestine at each post-feeding time were determined considering the sum of TiO2 content in the caecal and colonic digesta and the ratio of AA content/TiO2 content at the terminal ileal digesta (Direct method). Alternatively, the AA released into the large intestine can be calculated based on the TiO2 ingested in the meal and the TiO2 measured in the upper GIT (stomach to terminal ileum) (Indirect method). Both values were then used to determine the corrected apparent AA absorption in the GIT lumen as follows: Directmethod: AAi released into the large intestine (mg inDM basis) = (TiO2 contentCaecal digesta þ TiO2 contentColonic digesta) × AAi contentTerminal ileal digesta/TiO2 contentTerminal ileal digesta Indirect method: AAi released into the large intestine (mg in DM basis)= (TiO2 contentDiet – (TiO2 contentPooled digestaþ TiO2 contentTerminal ileal digesta)) × AAi contentTerminal ileal digesta/TiO2 contentTerminal ileal digesta Apparent AAi unabsorbed (mg inDMbasis)= AAi pooled digestaþ AAi terminal ileal digesta þ AAi released into the large intestine Apparent AAi absorbed (mg in DM basis) = AAi contentDiet − (Apparent AAi unabsorbed þ AA contentRefusal) Apparent AAi absorption (%) = (AAicontentDiet − (Apparent AAi unabsorbed þ AA contentrefusal)) / (AAicontentDiet− AA contentrefusal) × 100 Themean foodDM intake andDM in pooled digesta (stomachþ PSIþDSI), terminal ileum and the large intestine as well as the lysine and TiO2 concentrations are shown in the Supplemental Materials andMethods to demonstrate the calculations described above. Data related to the study are available upon request. One of the pigs at 4 h post-feeding did not have enough ileal digesta for both the AA and TiO2 analyses but enough contents in the remaining GIT locations. The TiO2 content of the remaining GIT locations was determined and 4 % of TiO2 reached the large intestine. To estimate the amount of AA released into the large intestine for this pig, a ratio between the average amount of AA released and the average amount of TiO2 in the large intestine for the same post-feeding time calculated over all the relevant pigs was calculated and multiplied by the amount of TiO2 in the large intestine for the specific animal. Statistical analysis For the study, a sample size of six replicates per time point was deemed satisfactory (>80 % at a two-tailed 5 % significance level), based on an effect size of 1·86 obtained from data reported for the small intestinal AA digestibility(15). Statistical analyses were performed using SAS (SAS/STAT version 9.4; SAS Institute Inc.). A paired-t-test was performed to compare the determined and predicted amounts of TiO2 released into the large intestine. A polynomial analysis was conducted for the amounts of AA released into the large intestine. The best polynomial model (up to third order) for each response variable was selected after comparing higher- v. reduced-order models using the log-likelihood ratio test. Probability values of P≤ 0·05 were considered of statistical difference, and 0·05< P< 0·10 were considered a trend. Non-linearmodelswere fitted to the data, and the PROCNLIN of SAS was used to estimate the parameters of different models. To determine the transit time of the diet in each GIT location (stomach, PSI, DSI, terminal ileum, caecum and colon), the power exponential model (TiO2 remainingTime = α0 exp – [κ × Time]β) was first used. α0 is the amount of TiO2 consumed (0·94 g), β is the index of the curve and κ is the slope of the curve. β and κ were then used to determine the 10 % cumulative transit time (CTT10 = [1/κ] × [log[1/0·9]][1/β]). The time difference between cumulative transit times of different GIT locations was used to calculate the transit time of specific GIT locations (e.g. TT10PSI, h= CTT10 stomach and PSI – CTT10 stomach). Based on the sigmoidal shape observed over time for the apparent relative AA absorption, different non-linear models (modified Weibull equation, Chapman–Richard equation, Logistic function and Gompertz function)(16), commonly used for sigmoidal parameters, were firstly fitted for Asp, Glu and Leu. For these AA, the Gompertz function better fitted the data (online Supplementary Table 1). However, with the Gompertz model the point of inflection is not symmetric (i.e. the half time, T50 of apparent AA absorption cannot be determined). Thus, the Logistic function (relative absorptionTime = α/[1 þ exp [β – (γTime)]]), which was the model with the second best fit (online Supplementary Table 1 and Supplementary Fig. 1), was fitted for all AA. The asymptote α represents the extent of apparent AA absorption or the apparent amount AA absorbed. The slope γ, or apparent absorption rate coefficient at α/2 (or inflection point), controls the shape of the curve and β shifts 764 C. A. Montoya et al. https://doi.org/10.1017/S0007114523002441 Published online by Cam bridge U niversity Press https://doi.org/10.1017/S0007114523002441 https://doi.org/10.1017/S0007114523002441 https://doi.org/10.1017/S0007114523002441 https://doi.org/10.1017/S0007114523002441 the curve along the X-axis. β and γ were then used to determine the point of inflection for α (Tα/2 AAi h = β/γ) and the time of 50 % for absorption of each AA (T50 AAi h = β/γ). The model diagnostics for each response variable were tested after combining the PROC UNIVARIATE and the ODS GRAPHICS procedures of SAS. A natural log or square root transformation of the AA released into the large intestine was required to fulfil the model assumptions of normality and homoscedasticity. The mean values reported are both trans- formed and back-transformed. Results The chemical composition (g/kg DM diet) of the last meal consisted of 298 g of starch, 116 g of total fat, 48 of total dietary fibre and 214 g of crude protein. In the last meal, all animals, except three, consumed the whole meal in around 5min. For the other animals, the refusals were removed after 15 min. Food refusals were generally low, and any remaining food from the last meal was weighed and food intakes were corrected. The mean intake of the test meal on the final experimental day was 203 (SE 6·0) g. The mean recovery of TiO2 in the entire GIT and considering faeces excreted after consuming the testmeal was 99·7 (SE 1·1)%. The TiO2 exiting the stomach followed the same pattern (r= 0·948, P< 0·001) as the DM exiting the stomach (Fig. 1). There was a correlation (r= 0·96, P< 0·001; Fig. 2) between the amount of TiO2 determined and that predicted (calculated by the indirect method) to be present in the large intestine (Table 1). Only the results based on the direct approach are pre- sented here. Apart from one pig, AA were not released into the large intestine during the first 3 h post-feeding (Table 2). Therefore, the uncorrected apparent AA absorbed from the GIT lumen for the first 3 h post-feeding were the same (P > 0·05) as the apparent corrected values (Table 3). As expected, the amounts of AA released into the large intestine increased over time (P ≤ 0·05) from 4 to 12 h post-feeding (Table 2). For example, after back-transformation of the natural logarithm values (Table 2), the amount of Asp increased from 25 mg at 3 h post-feeding to 213 mg at 12 h. The corrected apparent amounts of AA absorbed from the GIT lumen after 4 h post- feeding were (or tended to be) lower (P ≤ 0·05) than the uncorrected counterpart. For example, at 6 h post-feeding the corrected apparent amount of Asp absorbed was 102 mg/g protein intake instead of 105 mg. Corrected values only were used to determine kinetic parameters for the apparent AA absorbed (mg/g protein intake) and apparent AA absorp- tion (%). The extent of the apparent AA absorbed (α) ranged from 15 mg/g protein for His to 154 mg/g protein for Glu (Table 4), whereas the apparent rate of absorbed AA (γ) at the inflection point (Tα/2) ranged from 0·8 mg/g protein/h for Trp to 1·2 mg/g protein/h for Arg. The time to reach half of the total AA absorbed (T50) ranged from 1·6 to 3·6 h for Met and Arg, respectively, whereas Tα/2 ranged from 1·2 to 3·0 h for Met and His, respectively. The observed and parameterised extent of apparent absorption for all analysed AA ranged from 73 to 95% for Arg and Lys, respectively (Table 5). As expected, the parameters related to the rate of apparent absorption (e.g. γ, T50) were similar to the values for the apparent amounts of AA absorbed. For instance, the rate of apparent absorption ranged from 0·8 to 1·1 %/h for Trp and Arg, respectively, as reported above. 0 20 40 60 80 100 0 20 40 60 80 100 Oi T 2 % , hca m ots e ht g n itixe DM exiting the stomach, % Fig. 1. Correlation between the DM and titanium dioxide (TiO2) exiting the stomach over time for pigs fed a whey protein isolate containing test meal. The correlation value was 0·948 (P< 0·001, n 36). 0 0∙2 0∙4 0∙6 0∙8 0 0∙2 0∙4 0∙6 0∙8 Determined TiO2, g C al cu la te d Ti O 2, g Fig. 2. Correlation between the determined and calculated titanium dioxide (TiO2) content in the large intestine of pigs fed a whey protein isolate containing test meal. The correlation value was 0·96 (P< 0·001, n 36). Table 1. Determined and predicted amounts of titanium dioxide over time in the large intestine of pigs fed a whey protein isolate containing test meal* (Mean values with their standard errors) Time, h Determined Predicted Mean SE Mean SE g 1 0·013 0·006 0·006 0·085 2 0·025 0·008 0·013 0·062 3 0·013 0·005 0·011 0·036 4 0·037 0·026 0·038 0·068 6 0·313 0·130 0·352 0·123 12 0·452 0·145 0·473 0·117 * Values aremeans and standard errors of themean, n 6 growing pigs per post-feeding time. Based on the paired t test results, themean difference between determined and predicted titanium dioxide was not significantly different (P> 0·05). Kinetics of amino acid absorption 765 https://doi.org/10.1017/S0007114523002441 Published online by Cam bridge U niversity Press https://doi.org/10.1017/S0007114523002441 There were significant negative correlations between the apparent extent of AA absorption with both γ (r=−0·69, P< 0·01) and T50 (r =−0·81, P < 0·001; Fig. 3). The average T50 of the essential AA was shorter than the average T50 of the non-essential AA (2·19 v. 2·95 h, respectively; P < 0·01). Thus, essential AA tended (P = 0·06) to have a greater apparent extent of absorption than non-essential AA (90·3 v. 84·8 %, respectively). The amounts of TiO2 consumed and collected in each GIT location (stomach, PSI, DSI, terminal ileum, caecum and colon) were used to determine the 10% transit time in each GIT location (i.e. the time required for 10% of TiO2 to transit the given GIT location). The 10% transit time for the stomach was 0·3 h (Fig. 4). The 10% transit time of the diet from the mouth to the end of the small intestine (i.e., terminal ileum)was 2·9 h, while it was 4·0 and 24 h for the caecum and the mid-colon, respectively. In the small intestine, the 10 % transit time from the duodenum to the mid-PSI was 0·2 h, while from the end-PSI to the mid-DSI was 1·7 h. In the large intestine, the 10% transit time in the caecum was 1 h, while from the beginning of the colon to the mid-colon it was 20 h. Discussion One objective of this study was to evaluate the effect of the amount of AA released into the hindgut on measures of the kinetics and apparent extent of small intestinal AA absorption. A second objective was to test whether the kinetics of AA absorption are related to the extent of AA absorption. As hypothesised, there was a practically significant amount of AA released into the large intestine, which increased over time and needed to be considered to avoid overestimation of the kinetics of apparent AA absorption, when basing absorption on undigested material found in the upper digestive tract. Further, based on the correlation between several of the parameters related to the apparent kinetics of AA absorption and the apparent extent of AA absorption, the rate of AA absorption is strongly related to the extent of AA absorption. Amino acids released into the large intestine and corrected apparent absorption Other studies have used portal vein cannulated animals to determine the kinetics of AA absorption(17,18). However, as AA (e.g. Thr, Glu) are highly metabolised in the intestinal epithelium(19), the portal vein AA flux only represents the transported AA. To determine the absorption of AA in the small intestine by mass balance, we needed to measure the AA remaining in the stomach, small intestine and those released into the large intestine over time. Here, we used TiO2 to track the flow of the testmeal throughout theGIT and to determine AA released Table 2. Amounts of amino acids released over time into the large intestine of growing pigs fed a whey protein isolate containing test meal* (Mean values with their standard errors) Post-feeding time, h P 1–3 4 6 12 Mean SE Mean SE Mean SE Mean SE TiO2, %† 2·0 0·27 4·6 1·20 34 5·9 49 6·4 n‡ 1 5 6 6 mg His§ 1·59 0·36 4·92 1·85 0·31 6·34 2·35 0·24 10·5 3·87 0·40 47·9 0·002L‖ Ile§ 2·26 0·33 9·54 2·51 0·29 12·3 3·01 0·23 20·2 4·51 0·37 90·9 0·001L Leu§ 2·81 0·36 16·7 3·07 0·31 21·4 3·57 0·24 35·4 5·08 0·40 159 0·002L Lys§ 1·42 0·59 4·15 2·41 0·33 11·1 3·91 0·39 49·8 4·66 0·40 106 0·047Q‖ Met§ 1·06 0·45 2·88 1·32 0·39 3·75 1·85 0·30 6·33 3·42 0·50 30·5 0·006L Phe§ 2·26 0·34 9·54 2·51 0·29 12·3 3·02 0·23 20·5 4·55 0·38 94·7 0·001L Thr¶ 4·63 1·23 21·4 5·55 1·07 30·8 7·38 0·83 54·4 12·9 1·38 165 0·001L Trp§ 1·71 0·35 5·52 1·95 0·30 7·01 2·43 0·24 11·3 3·86 0·39 47·5 0·003L Val§ 2·56 0·33 13·1 2·82 0·29 16·8 3·32 0·22 27·6 4·82 0·37 123 0·001L Ala§ 2·84 0·33 17·1 3·06 0·28 21·4 3·51 0·22 33·4 4·85 0·36 127 0·002L Arg§ 3·01 0·35 20·2 3·24 0·30 25·5 3·70 0·24 40·4 5·08 0·39 161 0·003L Asp§ 3·21 0·34 24·8 3·45 0·30 31·5 3·93 0·23 50·8 5·36 0·38 213 0·002L Glu§ 2·36 0·54 10·5 3·50 0·31 33·2 5·19 0·36 180 5·47 0·37 238 0·006Q Gly¶ 5·51 1·21 30·4 6·34 1·04 40·2 8·00 0·81 64·1 13·0 1·35 169 0·002L Ser§ 2·87 0·33 17·7 3·10 0·28 22·1 3·54 0·22 34·6 4·89 0·37 132 0·002L Tyr§ 2·23 0·35 9·28 2·46 0·30 11·7 2·94 0·24 18·8 4·35 0·39 77·8 0·002L Total§,** 5·48 0·36 241 5·70 0·31 298 6·13 0·24 458 7·42 0·41 1,661 0·007L EAA§,** 4·69 0·3 109 4·92 0·30 137 5·37 0·24 215 6·73 0·40 833 0·004L NEAA§,** 4·94 0·36 139 5·16 0·32 174 5·60 0·25 270 6·92 0·40 112 0·005L * Values are mean values with their standard error of the mean. † TiO2, titanium dioxideLarge intestine/titanium dioxideIntake × 100. ‡ n, indicates the number of replicates. For post-feeding time 1–3 h, terminal ileal digesta of only one of the pigs at the 3 h post-feeding time had values higher than the limit of detection. Thus, for the animals with values lower than the limit of detection it was assumed that amino acids did not reach the terminal ileum and therefore they were not released into the large intestine. For post-feeding time 4 h, terminal ileal digesta collected were not sufficient to analyse titanium dioxide for one pig. § A natural logarithm transformation of the raw data was required to achieve the model assumptions of normality and homoscedasticity. The values (third column of the post-feeding time) represent the mean for each response variable after back-transformation. ‖ L or Q, linear or quadratic effect for the amounts of amino acids released into the large intestine over time. ¶ A square root transformation of the raw data was required to achieve the model assumptions of normality and homoscedasticity. The values (third column of the post-feeding time) represent the mean for each response variable after back-transformation. ** Total, EAA and NEAA, total, essential and non-essential amino acids. 766 C. A. Montoya et al. https://doi.org/10.1017/S0007114523002441 Published online by Cam bridge U niversity Press https://doi.org/10.1017/S0007114523002441 Table 3. Uncorrected and corrected amounts of amino acids disappearing from the small intestine over time for pigs fed a whey protein isolate containing test meal* (Mean values with their standard errors) Post-feeding time, h† 1 2 3 4 6 12 Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE mg/g protein His 2·58 1·63 5·38 0·37 10·4 0·6 10·4 0·6 12·2 1·0 12·1 1·1§ 15·1 0·5 14·5 0·4* 16·0 0·2 15 0·5* Ile 18·1 5·0 24·5 2·1 43·2 1·8 43·2 1·8 47·3 2·6 47·1 2·7* 54·3 1·3 53·2 1·2* 56·2 0·4 54·4 0·9* Leu 47·6 11·5 65·9 4·9 104 4·3 104 4·3 117 5·4 116 5·4* 128 6·3 126 6·1* 137 0·6 133 1·6§ Lys 54·0 9·6 71·7 3·0 99·3 3·3 99·3 3·3 110 4·2 110 4·2* 126 1·3 124 1·2§ 127 0·8 124 1·9§ Met 14·5 2·0 16·3 1·3 22·6 0·9 22·6 0·9 24·0 1·2 23·9 1·2* 27·0 0·5 26·4 0·5§ 27·8 0·1 26·9 0·4§ Phe 9·69 3·24 15·1 1·1 27·4 1·2 27·4 1·2 29·8 2·3 29·5 2·3* 34·1 2·1 33·0 2·0* 36·9 0·4 35·0 0·9* Thr 11·9 4·0 18·0 1·0 32·4 1·5 32·4 1·5 35·8 2·8 35·1 2·9* 43·5 1·0 41·8 0·8* 45·6 0·4 42·9 0·7* Trp 13·1 1·8 16·2 0·7 22·2 0·9 22·2 0·9 24·8 1·0 24·7 1·0§ 28·0 0·4 27·5 0·4§ 29·1 0·1 28·3 0·4§ Val 13·9 3·9 19·9 1·4 33·8 1·5 33·8 1·5 37·2 2·5 36·8 2·5* 43·9 55 42·4 1·1* 45·2 0·7 42·9 1·2* Ala 13·5 4·3 21·0 1·0 36·2 1·6 36·2 1·6 41·1 2·6 40·6 2·7§ 47·2 2·9 45·6 2·7* 51·2 0·3 48·7 0·8* Arg 4·1 1·69 6·73 0·67 14·6 0·8 14·5 0·8 16·1 1·9 15·3 2·1§ 20·8 1·0 18·9 0·9* 22·0 0·4 18·6 1·1* Asp 17·7 10·1 31·0 3·6 68·7 3·9 68·6 3·9 80·6 6·3 79·8 6·3* 105 2·0 102 1·6* 108 0·8 104 1·5* Glu 28·6 14·5 52·2 5·4 102 5·5 102 5·5 123 7·2 122 7·3* 149 7·9 144 7·7* 164 0·9 159 2·2* Gly −9·91 2·27 −6·03 2·23 1·71 0·78 1·61 0·84 −0·10 4·90 −1·1 5·22* 7·11 1·53 5·2 1·5* 11·1 1·0 8·3 1·1* Ser 4·18 3·44 10·0 1·0 21·5 1·5 21·4 1·5 24·0 3·2 23·4 3·3* 31·3 1·6 29·6 1·5* 33·7 0·4 31·3 0·8* Tyr 7·93 2·81 11·7 1·3 22·5 1·1 22·4 1·1 24·1 2·3 23·8 1·2* 28·9 0·4 27·9 1·3* 31·0 0·4 29·3 0·8§ Total‡ 247 65·1 379 22·7 663 29 662 30 746 47·9 739 49·0* 890 30·1 863 27·4* 942 7·38 902 16·1* EAA‡ 186 35·9 260 13·5 410 16·0 410 16·3 454 24·1 450 24·5* 521 13·7 508 12·1* 543 3·97 522 9·30* NEAA‡ 61·7 29·5 120 10·7 253 13·4 252 13·6 292 24·2 288 24·8* 368 16·5 354 15·4* 399 3·62 380 6·82* * Values are mean values with their standard error of the mean, n 6 growing pigs per post-feeding time. Values (second means for post-feeding time 3 to 12 h) were corrected by amino acids released into the large intestine. Means with one asterisk differ (P≤ 0·05). † Corrected values are not reported for 1 and 2 h post-feeding times, as TiO2 did not reach the large intestine. Thus, uncorrected and corrected values are the same. ‡ Total, EAA and NEAA, total, essential and non-essential amino acids. § Mean values tended to differ (0·05390 min of half transit time) has also been reported in humans fed pancakes containing 15 g bran(32). In pigs fed diets containing different starch sources, it has been shown a shorter mean retention time in the PSI compared with the DSI(33). Surprisingly, 30 % of the intake of TiO2 was present at the DSI after 12 h post-feeding (data not shown), which suggests that an important amount of the undigested diet remained at the DSI (20 g) for an extended period of time. Such accumulation has also been reported at the ileumof humans(31,34) and rats(6,35,36) but has not been quantified. Direct v. indirect determination of amino acids entering the large intestine To determine the amounts of AA released into the large intestine directly, total collection of caecal and colonic digesta is required to allow determination of the amount of TiO2 in the large intestine. However, unrolling the colon and collecting total caecal and colonic digesta are difficult and time-consuming. In this study, the amount of TiO2 released into the large intestine was calculated after unrolling the colon and with an almost total collection of caecal and colonic digesta (i.e., direct method) but also by subtracting the amount of TiO2 found in the stomach to the terminal ileum, from the amount of TiO2 ingested (i.e. indirect method). The statistically significant high correlation between TiO2 released into the large intestine using the direct and indirect methods suggests that the indirect method could be used to calculate the amounts of AA released into the small intestine. A limitation, especially with the indirect method, however, is that the terminal ileum does not always contain digesta, which affects the calculation of TiO2 released into the large intestine. The direct method allows to determine the exact amount of TiO2 in the large intestine, and this information can be used to calculate the AA released into the large intestine. Nevertheless, the indirect method appears to be a satisfactory alternative. TiO2 reached the large intestine during the first 3 h post- feeding in small amounts, but when determining the TiO2 concentration in the ileal digesta only one of the pigs at the 3 h post-feeding time had values higher than the limit of detection (0·083mg/ml TiO2). Thus, for all the pigs within the first 3 h post- feeding, except for the one pig, it was assumed that AA were not released into the large intestine (i.e. all the food AA were either absorbed or found within the pooled luminal contents). It is important tomention that AAweremeasured in the terminal ileal digesta of those animals, and these AA, which could be from both dietary (previous meals) and endogenous origin, are released into the large intestine butmay not have been part of the test meal. Conclusions There are quantitatively important amounts of AA released into the large intestine during digestion, which increase over time and need to be considered to avoid overestimating apparent AA absorption values with the serial slaughter total GIT content recovery method. The practical significance of the amounts of AA lost to the large intestine is expected to be greater for less digestible proteins. High negative correlations between the kinetics of AA absorption and the extent of AA absorption were observed when growing pigs were fed whey protein isolate as the sole protein source. This suggests that the kinetics of AA absorption modulates the extent of AA absorption. Based on the determined kinetics of AA absorption, essential AA are absorbed faster than non-essential AA. The absorption kinetics combined with transit time appear to modulate the overall extent of absorption of each AA. Acknowledgements The authors also acknowledge Fonterra (New Zealand) for donating whey protein isolate, Ms. Paloma Craig and Ms. Anneminke Buwalda for help analysing titanium dioxide and MateoMontoya for all the suggestions towrite the first draft of the manuscript. Data are available upon request to the correspond- ing author. The authors acknowledge the financial support provided by the Centre of Research Excellence Fund from the New Zealand Tertiary Education Commission and the New Zealand Ministry of Education, and additional funding from the AgResearch Strategic Science Investment Fund through contract A25773 (Systems Nutrition for Consumer Wellbeing). The funders did not contribute to the study design. C. A. M. and P. J. M. were responsible for planning the study. M. V. B. and C. A. M. were responsible for conducting the experiment. S. H., N. S., K. K., M. V. B. and C. A. M. were responsible for coordinating all the personnel involved in sample collection. C. A. M. did the statistical analysis and prepared the first draft of the manuscript that was revised by P. J. M., S. H. and N. S. All authors read and approved the final manuscript. The authors disclose no conflicts of interest. 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Montoya et al. https://doi.org/10.1017/S0007114523002441 Published online by Cam bridge U niversity Press https://doi.org/10.1017/S0007114523002441 The kinetics of amino acid disappearance in the small intestine is related to the extent of amino acids absorbed in growing pigs Materials and methods Animals and housing Diets and experimental design Chemical analysis Calculations Statistical analysis Results Discussion Amino acids released into the large intestine and corrected apparent absorption Rate and extent of apparent amino acid absorption Transit time of the diet throughout the gastrointestinal tract Direct v. indirect determination of amino acids entering the large intestine Conclusions Acknowledgements Supplementary material References