Endogenous proteins in the ileal digesta of adult humans given casein-, enzyme-hydrolyzed casein- or crystalline amino-acid-based diets in an acute feeding study.

BACKGROUND/OBJECTIVES: To ascertain if the form of dietary nitrogen (free amino acids (AA), small peptides, or intact protein) affects the endogenous nitrogen containing substances lost from the upper digestive tract of humans. SUBJECTS/METHODS: Digesta were collected via a naso-ileal tube from the terminal ileum of 16 adult humans in a single parallel study following an acute feeding regimen. Subjects were given an iso-nitrogenous and isocaloric test meal containing 150 g of casein (CAS) (n=6), enzyme-hydrolyzed casein (HCAS) (n=5) or crystalline AA (n=5) dissolved in 550 ml of water, as the sole sources of nitrogen. RESULTS: The mean concentrations and flows of total nitrogen, protein nitrogen, and soluble protein nitrogen passing the terminal ileum were significantly higher (P <0.01) for the CAS and HCAS test-meal groups compared to the AA meal group. Dietary CAS and HCAS had a considerable influence on digesta mucin concentrations and flows compared to free AA (+41%). Only 3-4% of the total nitrogen remained unidentified. CONCLUSIONS: The form of dietary nitrogen (protein, small peptides or free AA) had an acute effect upon the secretion or reabsorption of endogenous proteins in the small intestine of healthy humans, as evident from significant differences in both the quantity and composition of the proteins found in digesta at the end of the ileum.

Comparison of three markers for the determination of bacterial protein in terminal ileal digesta in the growing pig.

The aim of the study was to compare three methods commonly used to determine the concentrations of bacterial protein in digesta collected from the terminal ileum of growing pigs that had been fed a casein-based diet. The amounts of bacterial protein in terminal ileal digesta were determined using three different markers: 2.6-diaminopimelic acid (DAPA) and the d-amino acids, d-aspartic acid (d-Asp) and d-glutamic acid (d-Glu). The effectiveness of each marker was compared against a control based on physical fractionation by centrifugation. The total bacterial protein concentrations derived from the markers d-Asp and d-Glu were significantly different (p = 0.05) to those calculated from DAPA and the control, but there was no difference between DAPA and the control. The percentage of bacterial nitrogen ranged from 40% to 52% dependent on the marker used. Bacterial protein expressed as a percentage of the total protein, ranged from 48% to 62%, a substantial proportion of which (12-28%) was derived from lysed bacterial cells. Statistical correlations between the estimation methods were low. Such poor correlation between the markers may be the result of random errors such as variance in the epimerization of the two d-amino acids during protein hydrolysis. DAPA was accepted as a reliable marker for determining microbial protein in ileal digesta.

The impact of lower gut nitrogen supply on nitrogen balance and urea kinetics in growing pigs fed a valine-limiting diet.

An N-balance and isotope dilution study was performed to determine the effect of lower gut N supply on N retention and CO(NH(2))(2) kinetics in growing pigs. Nine cecally cannulated and jugular-catheterized barrows (initial BW 22.4 ± 1.2 kg) were randomly assigned to 1 of 3 cecal N infusion treatments: saline, casein, or CO(NH(2))(2); the latter 2 treatments were infused at a rate of 40% of daily N intake. All pigs were fed a Val-limiting corn (Zea mays) starch and soybean (Glycine max) meal-based diet. Cecal N infusions did not affect apparent total tract digestibility of N (P > 0.05). The efficiency of using N [% of apparent ileal digestible intake; 72.9 ± 1.9, 84.9 ± 1.9, and 85.6 ± 2.3% (P = 0.01) for saline, casein, and CO(NH(2))(2), respectively] and Val (76.9 ± 1.9, 86.5 ± 1.9, and 86.5 ± 2.4; P = 0.02) for whole body protein and Val retention increased for casein and CO(NH(2))(2). Urea flux and urinary N excretion increased (P < 0.05) similarly for both N infusions, but this increase did not fully account for lower gut N disappearance. Lower gut N disappearance is in the form of NPN, which can be used for microbial AA production in the upper gut and should be considered when determining N and AA supply and requirements.

Lower gut nitrogen supply does not affect apparent ileal digestibility of nitrogen or amino acids in growing pigs.

An implicit assumption in measures of ileal digestibility (ID) to estimate bioavailability of AA and N is that ID is not influenced by lower gut N metabolism. The absorption of nitrogenous compounds from the lower gut, derived from fermentative AA catabolism, may have an impact on N metabolism and microbial AA synthesis in the upper gut as a result of CO(NH(2))(2) recycling. The objective of this trial was to determine the apparent ID of N and AA in growing pigs fed a corn (Zea mays) starch and soybean (Glycine max) meal-based diet and receiving an infusion of N into the caecum at 40% of N intake. Eight pigs (initial BW of 23.3 ± 0.55 kg) were fitted with simple T-cannulas in the ileum and cecum and randomly assigned to 1 of 3 continuous cecal infusion treatments [saline, sodium caseinate, or CO(NH(2))(2)] according to a crossover design with 3 periods. Digesta samples were collected and pooled per pig for each 2-d period, freeze-dried, ground, and analyzed for DM, OM, total N, and AA. Lower gut N supply did not affect apparent ID of DM, OM, total N (85.4, 83.4, and 82.7 ± 1.74%; P = 0.31), or any AA (e.g., 90.1, 89.0, and 89.9 ± 1.08% for Lys; P = 0.70) for saline, casein, and CO(NH(2))(2) treatments, respectively. Apparent ID may be an insufficiently sensitive measure to determine effects of lower gut N metabolism on N absorption from the lower gut and subsequent recycling into the upper gut.

Invited review: Amino acid bioavailability and digestibility in pig feed ingredients: terminology and application.

In this review, the terminology that is used to describe the bioavailability and ileal digestibility of AA in pig feed ingredients is defined. Aspects of the methodology to establish bioavailability and ileal digestibility values also are discussed, and recommendations about the use of these values are provided. Two main factors can contribute to differences between bioavailability and ileal digestibility of AA. First, some AA, such as Lys, may be absorbed in chemical complexes that preclude their use for metabolism. Second, fermentation in the upper gut may result in a net loss or gain of AA to the animal. In addition, dietary effects on the efficiency of using bioavailable AA intake for tissue growth or milk production should be considered and may be attributed to endogenous AA losses in the hindgut and the metabolic costs associated with endogenous gut protein synthesis and losses. Ileal digestibility values may be expressed as apparent ileal digestibility (AID), standardized ileal digestibility (SID), or true ileal digestibility (TID). These terms are used to specify how ileal endogenous AA losses are reflected in digestibility values. Ileal endogenous AA losses may be separated into basal losses, which are not influenced by feed ingredient composition, and specific losses, which are induced by feed ingredient characteristics such as levels and types of fiber and antinutritional factors. Values for AID are established when total ileal outflow of AA (i.e., the sum of endogenous losses and nondigested dietary AA) is related to dietary AA intake. A concern with the use of AID values is that these are not additive in mixtures of feed ingredients. This concern may be overcome by correcting AID values for defined basal endogenous losses of AA, which yields SID values. Furthermore, if the AID values are corrected for basal and specific endogenous losses, then values for TID are calculated. However, reliable procedures to routinely measure specific endogenous losses are not yet available. It is recommended that basal ileal endogenous losses of AA should be measured in digestibility experiments using a defined protein-free diet and that these losses are reported with observed AID and SID values. It is suggested that SID values should be used for feed formulation, at least until more information on TID values becomes available.

Feeding frequency and type of isotope tracer do not affect direct estimates of lysine oxidation in growing pigs.

Oxidation contributes to the inefficiency of lysine utilization for protein deposition. The influences of feeding frequency and type of isotope tracer on estimated lysine oxidation were studied in growing pigs fed lysine-limiting diets. Yorkshire gilts (n = 11) weighing 40-45 kg were fitted with venous catheters. They were fed, in 3 or 8 equal meals daily, a purified diet based on casein and cornstarch. Lysine intake limited the pigs' protein deposition to 70% of their potential. After a 5-d N-balance period, lysine oxidation was estimated by a primed, constant 26-h infusion of [1-14C]L-lysine and [6-3H]L-lysine. Feeding frequency and type of tracer did not affect lysine oxidation (P > 0.1). Increasing feeding frequency from 3 to 8 times daily reduced the variance and fluctuation of lysine oxidation by 46 and 30%, respectively. The mean lysine oxidation, as a fraction of the true ileal digestible lysine intake, was 9.2% based on the free lysine specific radioactivity (SRA) in plasma, 20.1% based on free lysine SRA in liver and 21.8% calculated from N-balance data. On the basis of liver free lysine SRA, tracer dilution methods and N-balance data give similar quantitative estimates of lysine oxidation (P > 0.10). Isotope tracer studies that cover one or more complete feeding cycles, i.e., feeding-to-feeding periods, can be used to obtain valid daily lysine oxidation values.

Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on nitrogen retention and amino acid utilization.

An experiment was conducted to investigate the effects of branched-chain amino acid (BCAA) interactions on their utilization by growing pigs and the effects of excessive amounts of BCAA (leucine, isoleucine, valine) on the utilization of methionine. A semipurified diet containing 100 g crude protein/kg with a balanced amino acid pattern was prepared using casein supplemented with free amino acids. Three further diets were made by reducing the concentration of methionine + cyst(e)ine, valine or isoleucine by 20%. Each of these four diets was then supplemented with leucine (50% excess) or a mixture of BCAA (50% excess of each but excluding the limiting amino acid). All diets were isoenergetic and were made isonitrogenous by replacement of glutamic and aspartic acids. The twelve diets were given to twenty-four growing pigs (30-40 kg) in three periods according to a randomized block design. Each period lasted 8 d and N retention was measured during the last 5 d of each period. Reducing dietary methionine, valine or isoleucine reduced the utilization of N (N retained/N digested) by approximately 20% (P < 0.05). Adding leucine to the isoleucine-limiting diet decreased the utilization of N by 9% (P < 0.05). This was reversed by simultaneous addition of valine. Excess leucine in a valine-deficient diet did not significantly reduce N utilization. In methionine-limiting diets an excess of either leucine alone or of all three BCAA increased the utilization of N by 8% (P < 0.05).

Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on plasma amino- and keto-acid concentrations and branched-chain keto-acid dehydrogenase activity.

The present experiment was designed to elucidate the mechanism of the methionine-sparing effect of excess branched-chain amino acids (BCAA) reported in the previous paper (Langer & Fuller, 2000). Twelve growing gilts (30-35 kg) were prepared with arterial catheters. After recovery, they received for 7 d a semipurified diet with a balanced amino acid pattern. On the 7th day blood samples were taken before (16 h postabsorptive) and after the morning meal (4 h postprandial). The animals were then divided into three groups and received for a further 7 d a methionine-limiting diet (80% of requirement) (1) without any amino acid excess; (2) with excess leucine (50% over requirement); or (3) with excesses of all three BCAA (leucine, isoleucine, valine, each 50% over the requirement). On the 7th day blood samples were taken as in the first period, after which the animals were killed and liver and muscle samples taken. Plasma amino acid and branched-chain keto acid (BCKA) concentrations in the blood and branched-chain keto-acid dehydrogenase (BCKDH; EC 1.2.4.4) activity in liver and muscle homogenates were determined. Compared with those on the balanced diet, pigs fed on methionine-limiting diets had significantly lower (P < 0.05) plasma methionine concentrations in the postprandial but not in the postabsorptive state. There was no effect of either leucine or a mixture of all three BCAA fed in excess on plasma methionine concentrations. Excess dietary leucine reduced (P < 0.05) the plasma concentrations of isoleucine and valine in both the postprandial and postabsorptive states. Plasma concentrations of the BCKA reflected the changes in the corresponding amino acids. Basal BCKDH activity in the liver and total BCKDH activity in the biceps femoris muscle were significantly (P < 0.05) increased by excesses of leucine or all BCAA.

Incorporation of urea and ammonia nitrogen into ileal and fecal microbial proteins and plasma free amino acids in normal men and ileostomates.

BACKGROUND: The importance of urea nitrogen reutilization in the amino acid economy of the host remains to be clarified. OBJECTIVE: The objective was to explore the transfer of (15)N from orally administered [(15)N(2)]urea or (15)NH(4)Cl to plasma free and intestinal microbial amino acids. DESIGN: Six men received an L-amino acid diet (167 mg N*kg(-)(1)*d(-)(1); 186 kJ*kg(-)(1)*d(-)(1)) for 11 d each on 2 different occasions. For the last 6 d they ingested [(15)N(2)]urea or, in random order, (15)NH(4)Cl (3.45 mg (15)N*kg(-)(1)*d(-)(1)). On day 10, a 24-h tracer protocol (12 h fasted/12 h fed) was conducted with subjects receiving the (15)N tracer hourly. In a similar experiment, (15)NH(4)Cl (3.9 mg (15)N*kg(-)(1)*d(-)(1)) was given to 7 ileostomates. (15)N Enrichments of urinary urea and plasma free and fecal or ileal microbial protein amino acids were analyzed. RESULTS: (15)N Retention was significantly higher with (15)NH(4)Cl (47.7%; P < 0.01) than with [(15)N(2)]urea (29.6%). Plasma dispensable amino acids after the (15)NH(4)Cl tracer were enriched up to 20 times (0. 2-0.6 (15)N atom% excess) that achieved with [(15)N(2)]urea. The (15)N-labeling pattern of plasma, ileal, and fecal microbial amino acids (0.05-0.45 (15)N atom% excess) was similar. Appearance of microbial threonine in plasma was similar for normal subjects (0.14) and ileostomates (0.17). CONCLUSION: The fate of (15)N from urea and NH(4)Cl differs in terms of endogenous amino acid metabolism, but is similar in relation to microbial protein metabolism. Microbial threonine of normal and ileostomy subjects appears in the blood plasma but the net contribution to the body threonine economy cannot be estimated reliably from the present data.

Responses in the absorptive phase in muscle and liver protein synthesis rates of growing rats.

The effect of time after beginning of a meal (30, 60, 90 and 120 min) on liver and gastrocnemius muscle protein synthesis was tested in growing male rats using the large dose technique, based on a 10 min exposure to [15N]phenylalanine. The fractional synthesis rate was estimated from the ratio between the atom percent excess of tissue protein-bound and free labelled phenylalanine. The latter was measured by gas chromatography mass spectrometry using the tertiary-butyldimethylsilyl amino acid derivatives. The protein-bound phenylalanine of gastrocnemius muscle was separated from the other amino acids using preparative amino acid chromatography and then oxidised to N2 in an automated carbon-nitrogen Roboprep (CN) combustion module attached to a continuous flow isotope ratio mass spectrometer (IRMS), with m/z ions 28 and 29 monitored. The protein-bound phenylalanine from liver was separated by a gas chromatograph attached to a sample preparation module and an isotope ratio mass spectrometer (GC C-IRMS), with again m/z ions of 28 and 29 monitored. The following results were obtained: the daily fractional protein synthesis rates (ks) in gastrocnemius muscle and liver were 13.9% and 65.6% respectively, in 12 h fasted 145 g rats. These ks increased within 30 min after ingestion of meal to 14.9% and 91.8% for muscle and liver, respectively, and remained at these values for the next 90 min (14.6% and 87.4% at 60 min, and 14.3% and 88.6% at 120 min after the beginning of feeding). It was concluded that measurement of protein synthesis rates characteristics for the absorptive phase can be undertaken in a period from thirty minutes to two hours after start of a meal, without significant changes in the ks values.

Availability of intestinal microbial lysine for whole body lysine homeostasis in human subjects.

We have investigated whether there is a net contribution of lysine synthesized de novo by the gastrointestinal microflora to lysine homeostasis in six adults. On two separate occasions an adequate diet was given for a total of 11 days, and a 24-h (12-h fast, 12-h fed) tracer protocol was performed on the last day, in which lysine turnover, oxidation, and splanchnic uptake were measured on the basis of intravenous and oral administration of L-[1-(13)C]lysine and L-[6,6-(2)H(2)]lysine, respectively. [(15)N(2)]urea or (15)NH(4)Cl was ingested daily over the last 6 days to label microbial protein. In addition, seven ileostomates were studied with (15)NH(4)Cl. [(15)N]lysine enrichment in fecal and ileal microbial protein, as precursor for microbial lysine absorption, and in plasma free lysine was measured by gas chromatography-combustion-isotope ratio mass spectrometry. Differences in plasma [(13)C]- and [(2)H(2)]lysine enrichments during the 12-h fed period were observed between the two (15)N tracer studies, although the reason is unclear, and possibly unrelated to the tracer form per se. In the normal adults, after (15)NH(4)Cl and [(15)N(2)]urea intake, respectively, lysine derived from fecal microbial protein accounted for 5 and 9% of the appearance rate of plasma lysine. With ileal microbial lysine enrichment, the contribution of microbial lysine to plasma lysine appearance was 44%. This amounts to a gross microbial lysine contribution to whole body plasma lysine turnover of between 11 and 130 mg. kg(-1). day(-1), depending on the [(15)N]lysine precursor used. However, insofar as microbial amino acid synthesis is accompanied by microbial breakdown of endogenous amino acids or their oxidation by intestinal tissues, this may not reflect a net increase in lysine absorption. Thus we cannot reliably estimate the quantitative contribution of microbial lysine to host lysine homeostasis with the present paradigm. However, the results confirm the significant presence of lysine of microbial origin in the plasma free lysine pool.

Nitrogen cycling in the gut.

This review examines the involvement of the gastrointestinal tract in the utilization of nitrogen, the identities of the nitrogenous substances entering and leaving the gut, and the significance of this recycling in the overall nitrogen economy of the body. It is concerned with nonruminant mammals, including man.

Nutrient intake and protein metabolism: responses to feeding.

Lean tissue growth occurs when the rate of protein synthesis exceeds the rate of protein breakdown. Although absolute rates of protein synthesis and breakdown rise during growth from birth to maturity fractional rates fall. Both these processes are sensitive to nutrient intake but responses to feeding vary greatly amongst different tissues. Protein, carbohydrate and fat can all stimulate body protein accretion in immature animals and in children but the mechanisms by which they do so, and the energy expenditures involved, seem to be different.

Microbial amino acid synthesis and utilization in rats: incorporation of 15N from 15NH4Cl into lysine in the tissues of germ-free and conventional rats.

The absorption of lysine synthesised by the gastrointestinal microflora was estimated by comparing the 15N incorporated into body lysine in four germ-free (15N-GF) and four conventional (15N-CV) rats. They were fed for 10 d on a protein-free diet containing fermentable carbohydrates and 15NH4Cl; another four conventional rats (control), fed on the same diet but with unlabelled NH4Cl, were used to estimate the natural abundance of 15N. The eviscerated carcass of each rat was homogenized and a sample hydrolysed. Lysine was isolated by ion-exchange chromatography and its 15N enrichment was measured by isotoperatio mass spectrometry. The 15N-CV rats significantly incorporated 15N into their body lysine. The 15N-GF rats had a statistically significant, although small, incorporation of 15N into their body lysine, probably arising from a measurement artifact. It was concluded, therefore, that all [15N]lysine was of microbial origin. The total lysine content in the body and the 15N enrichment of lysine in the microbial fraction of the faeces of the 15N-CV rats were also determined. The amount of microbial lysine absorbed by the 15N-CV rats was estimated by dividing the total amount of [15N]lysine in the body by the enrichment of microbial lysine. It was estimated that the daily absorption of microbial lysine by the conventional rats was 21.3 (SE 2.04) mg/kg body weight0.75.

Microbial amino acid synthesis and utilization in rats: the role of coprophagy.

Four rats were housed in cages with mesh floors; another four rats were housed in tubular anticoprophagy cages, in which they could not turn round to reach their own faeces. Both groups were fed for 6 d on a low-protein diet containing fermentable carbohydrates and 15NH4Cl. At the end of the experiment the rats were killed and their carcasses were homogenized, lysine was isolated by ion-exchange chromatography and its 15N enrichment measured by isotope-ratio mass spectrometry. The 15N enrichment in the lysine of the microbial fraction of faeces and the total amount of lysine in the body were also determined in order to estimate the amount of microbial lysine absorbed. The 15N enrichment in body lysine of non-coprophagic rats was not different from that previously measured in rats given unlabelled NH4Cl, but in coprophagic rats it was significantly higher. The daily absorption of microbial lysine by the coprophagic rats accounted for 20.7 (SE 2.55) mg/kg body weight0-75, but was only 0.5 (SE 1.04) mg/kg body weight0-75 for the non-coprophagic rats. This value was not significantly different from zero. The utilization of microbial amino acids via coprophagy resulted in a higher weight gain (adjusted for intake) in the coprophagic group (15.5 g/6 d) than in the non-coprophagic rats (3.1 g/6 d). It was concluded that, in rats, the utilization of microbial lysine occurred exclusively via coprophagy.

The effects of excessive amounts of protein on lysine utilization in growing pigs.

Two experiments were conducted to investigate whether the utilization of lysine in growing pigs is affected by the level of excess protein in the diet. Nine lysine-deficient diets containing 100, 200 or 300 g crude protein/kg and between 1.2 and 6.8 g ileal digestible lysine/kg were prepared. In the first experiment the apparent ileal digestibility of lysine in three of the nine diets was determined using pigs with simple T-cannulas and Cr2O3 as an indigestible marker. Ileal digestibility of lysine in the other diets was calculated by interpolation. In the second experiment N retention, as a measure of lysine utilization, was determined in all nine diets using growing pigs over the weight range 30-50 kg. The effect of excess protein on lysine utilization was assessed by comparing the regression of N retention v. lysine (ileal digestible) intake at the three levels of protein. Increasing ileal digestible lysine in the diets resulted in a linear increase in N retention with all three protein levels and there was no significant difference amongst the three regressions, indicating that lysine utilization was not affected by the level of protein. Therefore, all data were pooled together to calculate a single regression for all treatments. An increase of 1.0 g ileal digestible lysine led to an increase of 1.43 g N or 8.96 g protein (N x 6.25) retained. Assuming a lysine concentration in the retained body protein of 65-72 mg/g, lysine was utilized with an efficiency of 0.58-0.65.

Lysine utilization by growing pigs: simultaneous measurement of protein accretion and lysine oxidation.

Nitrogen retention and lysine oxidation were measured in growing pigs given diets which supplied 0, 0.2 or 0.8 of the lysine requirement, with other amino acids in relative excess. Eight groups of three female littermate pigs were used: one of each group was given each of the three diets. In half the pigs (four groups) N retention was measured at body weights (W) of approximately 25, 35 and 45 kg. The other four littermate groups of three pigs were given the same three diets; when they reached 35 kg W they were given a continuous (6 h) primed infusion of L-[6-3H]lysine. Lysine oxidation was estimated from the production of tritiated water. Rates of both N retention and lysine oxidation increased significantly with lysine intake; mean values (g/kg (W)0.75 per d) for the three diets respectively were for N retention, 0.00, 0.32 and 1.22, and for lysine oxidation 0.051, 0.058 and 0.078. From the N balance results (assuming a constant lysine concentration in body protein) the efficiency of utilization of absorbed lysine was estimated to be 0.85; from the oxidation results (assuming lysine absorbed but not retained is oxidized) the estimate was 0.95.

Validation of the doubly labeled water method in growing pigs.

The CO2 production (rCO2) of eight growing pigs was determined by continuous collection of CO2 over 21 days and simultaneously estimated using the doubly labeled water (DLW) method. The aim was to assess the accuracy of the method before and after correction for known sources of error and to test for any residual discrepancy arising from as yet unidentified sources of error. Mass spectrometer accuracy was verified by analyzing serial dilutions of the dose material in the form of an artificial decay curve; no significant bias was detected. The physiological errors were linearly dependent on weight gain. DLW-derived rCO2 (corrected only for fractionated water loss) underestimated the true value by 0.270 l CO2/g wt gain or -8% in the restricted (group R) and -16% in the ad libitum-fed (group AL) groups. Known sources of error accounted for -0.006 (methane), -0.032 (fecal 2H losses), -0.108 (fat synthesis), and -0.146 (changing pool size) l CO2/g wt gain. After correction for these sources of error the DLW-derived rCO2 differed from the true value by -2 +/- 3% in group R and 0 +/- 3% in group AL. Thus there was no significant bias in the DLW method after correction for known sources of error, even during rapid weight gain or at weight stability with or without correction. The precision estimates include both dose and background errors and uncertainty in the correction factors used. Strategies for optimizing precision are presented.