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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 15N from orally administered [15N2]urea or 15NH4Cl to plasma free and intestinal microbial amino acids. Design: Six men received an r.-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 [15N2]urea or, in random order, 15NH4C1 (3.45 mg 15-kg-1-d-1). On day 10, a 24-h tracer protocol (12 h fasted/12 h fed) was conducted with subjects receiving the 15N tracer hourly. In a similar experiment, 15NH4Cl (3.9 mg 15N-kg-1-d-1) was given to 7 ileostomates. 15N Enrichments of urinary urea and plasma free and fecal or deal microbial protein amino acids were analyzed. Results: 15N Retention was significantly higher with 15NH4Cl (47.7%; P < 0.01) than with [15N2]urea (29.6%). Plasma dispensable amino acids after the 15NH4Cl tracer were enriched up to 20 times (0.2-0.6 15N atom% excess) that achieved with [15N2]urea. The 15N-labeling pattern of plasma, deal, and fecal microbial amino acids (0.05-0.45 15N 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 15N from urea and NH4Cl 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. Copyright (c) 1999 American Society for Clinical Nutrition [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bioactive peptides either present in foods or released from food proteins during digestion have a wide range of physiological effects, including on gut function. Many of the bioactive peptides characterized to date that influence gut motility, secretion, and absorption are opioid agonists or antagonists. The authors review a body of experimental evidence that demonstrates an effect of peptides from food proteins on endogenous (nondietary) protein flow at the terminal ileum of simple-stomached mammals, including adult humans. At least some dietary peptides (1000-5000 Da) significantly enhance the loss of protein from the small intestine, causing an increased amount of protein to enter the colon. Food-derived peptides appear to either stimulate protein secretion into the gut lumen or inhibit amino acid reabsorption or influence both processes simultaneously. The effect of dietary peptides on small-intestine secretory-protein dynamics is discussed in the context of the major components of gut endogenous protein, sloughed cells, enzymatic secretions, mucin, and bacterial protein. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has profoundly disrupted household food purchasing and preparation, including elements identified as important drivers of household food waste. The two main aims of this study were (1) to examine changes in food waste behaviors since the start of the COVID-19 pandemic in the U.S. and Italy; and (2) to investigate potential predictors of food waste behavior, including avoidance of supermarkets, increased home cooking, and increased role of health concerns in food choices. A sample of n = 478 (79% female) individuals from the U.S., mean (SD) age = 30.51 (10.85), and n = 476 individuals from Italy, (78% female), mean (SD) age = 33.84 (12.86), completed an online survey between April 8th and April 28 th 2020. Just under half of respondents (49%) reported decreased food waste since the start of the pandemic. Rates were significantly higher among the U.S. sample (61.5%, n = 294) compared to the Italian sample (38%, n = 180). Controlling for the time since restrictions were introduced, age, gender, and perceived financial security, logistic regression revealed greater reduction in food waste since the beginning of the pandemic for U.S. individuals relative to participants from Italy (OR = 0.47, p < .001). In addition, increased importance of health concerns when making food choices (OR = 1.34, p < .005) as well as more frequent cooking (OR = 1.35, p < .001), and greater avoidance of supermarkets (OR = 1.15, p = .049) were associated with greater probability of less food waste. Scarcity and greater reliance on cooking may encourage individuals to reflect on food waste practices. Further research should explore how these factors may be targeted to reduce food waste beyond the pandemic., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

The precipitation of mucin using high concentrations of ethanol has been used by many researchers while others have questioned the validity of the technique. In this study, analysis of an ethanol precipitate, from the soluble fraction of ileal digesta from pigs was undertaken using molecular weight profiling and polyacrylamide gel electrophoresis. The precipitate contained 201 mg·g⁻¹ protein, 87% of which had a molecular weight >20 KDa. Polyacrylamide gel electrophoresis stained with Coomassie blue and periodic acid/Schiff, revealed that most glycoprotein had a molecular weight between 37-100 KDa. The molecular weight of glycoprotein in the precipitate was therefore lower than that of intact mucin. These observations indicated that the glycoprotein in the ethanol precipitate was significantly degraded. The large amount of protein and carbohydrate in the supernatant from ethanol precipitation indicated that the precipitation of glycoprotein was incomplete. As a method for determining the concentration of mucin in digesta, ethanol precipitation is unreliable.

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., (© 2012 Blackwell Verlag GmbH.)

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.

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.

Background: Although there are several published estimates of the endogenous amino acid composition of ileal digesta in humans, to our knowledge, there are no systematic studies of ileal digesta endogenous proteins., Objectives: We determined the nature and composition of endogenous nitrogen-containing substances lost from the upper digestive tract of humans., Design: Digesta were collected from the terminal ileum for a period of 8 h by using a nasoileal tube in 6 adult subjects fed a single meal that contained 22% of casein as the only source of nitrogen., Results: The total nitrogen that passed the terminal ileum was 39.3 mg/g native digesta dry matter. Of this amount, 86% was proteinaceous, ~60% was bacterial protein, ~7% was soluble-free protein, ~15% was mucin protein, and ~5% was protein from intact mucosal cells. For nonprotein nitrogen, ~5% of the total nitrogen was ammonia, and ~4% of the total nitrogen was urea. Bacterial and human mucosal cellular DNA nitrogen were collectively ~0.5% of the total nitrogen. Approximately 30% of the nonprotein nitrogen (4% of the total nitrogen) remained unidentified. This amount was assumed to include free amino acids, RNAs, amines, and the tetrapyrroles bilirubin and biliverdin. Bacterial nitrogen, combined with ammonia and urea nitrogen, represented >68% of total ileal nitrogenous losses., Conclusions: Findings are presented on the endogenous nitrogen-containing compounds that left the terminal ileum. Of particular significance is the observation that mucin was the most abundant truly endogenous component within the terminal ileal digesta. Bacterial protein, which was strictly nondietary rather than endogenous, contributed the highest proportion, by far, of nondietary protein, the result of which makes a significant contribution to published estimates of ileal endogenous amino acids and protein. The high concentration of bacterial protein and the presence of ammonia and urea nitrogen indicate potentially substantial microbial activity within the human distal small intestine.

The aim was to compare five techniques commonly used to quantify mucin concentrations in ileal digesta collected from three growing pigs that had been fed a diet in which the sole protein was casein. Ileal mucin output was estimated by the periodic acid-Schiff, ethanol precipitation, and phenol-sulfuric acid methods as 25.1, 19.3, and 20.7 g kg-1 of dry matter intake (DMI), respectively. The mucin concentration estimated from sialic acid was only 5.9 g kg-1 of DMI. On the basis of the concentrations of the hexosamines N-acetylglucosamine and N-acetylgalactosamine, mucin output was estimated as 44.9 g kg-1 pf DMI. Of the five assays studied, the ethanol precipitation, periodic acid-Schiff, phenol-sulfuric acid, and sialic acid methods may considerably underestimate mucin in the digesta, which calls into question the accuracy of all of these approaches. In contrast, the gas chromatography method for the determination of hexosamines gave more information on the type and state of the mucin present.

Although amino acids (AA) synthesized by enteric microbiota in the upper gut of nonruminants can be absorbed, they do not necessarily make a net contribution to the host's AA supply. That depends on whether protein or nonprotein nitrogen sources are used for microbial protein production. We determined the contributions of urea, endogenous protein (EP), and dietary protein (DP) to microbial valine (M.VAL) at the distal ileum of growing pigs, based on isotope dilutions after a 4-d continuous infusion of l-[1-(13)C]valine to label EP and of [(15)N(15)N]urea. Eight barrows were assigned to either a cornstarch and soybean meal-based diet with or without 12% added fermentable fiber from pectin. Dietary pectin did not affect (P > 0.10) the contributions of the endogenous and DP to M.VAL. More than 92% of valine in microbial protein in the upper gut was derived from preformed AA from endogenous and DP, suggesting that de novo synthesis makes only a small contribution to microbial AA.

To gain a clearer understanding of the nature and composition of endogenous nitrogen containing substances lost from the upper mammalian digestive tract, digesta were collected from the terminal ileum of six growing pigs that had been fed a casein-based diet with titanium dioxide as an indigestible marker. Total nitrogen lost at the terminal ileum was in excess of 63 mg.g(-1) digesta dry matter. Of this, nearly 73% was proteinaceous, with nearly 45% being bacterial protein, 13% from soluble free protein, and 11% from mucin. Of the nonprotein nitrogen, 11% was as ammonia and 5% as urea. Bacterial and porcine cellular DNA nitrogen were collectively 0.2% of the total nitrogen. Only 8.3% of the total nitrogen remained unidentified and was assumed to include free amino acids, RNAs, amines, and the tetrapyrroles bilirubin and biliverdin. Although mucin contributed just 10.4% of the nitrogen losses, it was the single most abundant truly endogenous component, comprising 13% of the total dry matter. Bacterial nitrogen, combined with ammonia and urea nitrogen, represented nearly 61% of the total nitrogenous losses: this suggests substantial microbial activity in the stomach and small intestine of the pig. Centrifugal separation of a bacterial fraction from the digesta produced a microbial amino acid profile that, when subtracted from the overall amino acid content, provided an amino acid profile more representative of true endogenous amino acid losses.

New estimates of the indispensable amino acid requirements of adult humans are much higher than previously thought and questions the adequacy of cereal-based diets of low protein quality. However, dietary amino acid requirements may be supplemented by contributions from the intestinal microbiota. This study measured the contribution of intestinal microbes to leucine input in healthy adult men. Fourteen adult men were studied during each of 2 11-d periods (before and after intestinal antimicrobial treatment), in which leucine was supplied at 1.25 times the estimated average requirement (EAR) (d 1-7) and at 2.5 times the EAR (d 8-11) providing an l-amino acid diet. We estimated fasting- and fed-state leucine oxidation on d 7 and d 11 using a (13)C-leucine tracer infusion. The microbial contribution to body leucine input was calculated from the relationship of leucine oxidation to leucine intake and the reduction in leucine oxidation after antimicrobial treatment. Antimicrobial treatment did not affect the slope of the relationship of leucine oxidation to leucine intake. Mean and fed-state leucine oxidation declined by approximately 13 and 20%, respectively (both P < 0.05) after antimicrobial treatment with the 1.25 EAR diet, but not with the 2.5 EAR diet. The contribution of the intestinal microbiota to body leucine input was estimated to be between 19 and 22% at the 1.25 EAR diet. The contribution of the intestinal microbiota to body amino acid homeostasis may be significant at maintenance intakes, but its long-term nutritional importance remains to be determined.

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.

Because the digestion of many dietary proteins is incomplete, and because there is a continuous (but variable) entry into the intestinal lumen of endogenous protein and amino acid nitrogen that is also subject to digestion, the fluxes of nitrogen, amino acids, and protein in the gut exhibit a rather complicated pattern. Methods to distinguish and quantitate the endogenous and dietary components of nitrogen and amino acids in ileal chyme or feces include the use of a protein-free diet, the enzyme-hydrolyzed protein method, different levels of protein intake, multiple regression methods, and stable-isotope labelling of endogenous or exogenous amino acids. Assessment of bioavailability can be made, with varying degrees of difficulty, in man directly but, for routine evaluation of foods, the use of model animals is attractive for several reasons, the main ones being cost and time. Various animals and birds have been proposed as models for man but, in determining their suitability as a model, their physiological, enzymological, and microbiological differences must be considered. Fecal or ileal digestibility measurements, as well as apparent and true nitrogen and amino acid digestibility measurements, have very different nutritional significance and can, thus, be used for different objectives. Measurements at the ileal level are critical for determining amino acid losses of both dietary and endogenous origin, whereas measurements at the fecal level are critical in assessing whole-body nitrogen losses. A complementary and still unresolved aspect is to take into account the recycling of intestinal nitrogen and bacterial amino acids to the body.

Inevitable catabolism contributes to the inefficiency of using dietary lysine intake for body protein deposition (PD). This study was conducted to determine the effects of true ileal digestible (TID) lysine intake, body weight (BW), and growth potential on lysine catabolism in growing pigs. Starting at 15 kg BW, 16 female Yorkshire pigs were offered a purified diet providing all nutrients in excess of requirements for maximum protein deposition (PDmax). At approximately 25 kg BW, the pigs' PDmax was determined using the N-balance method. Thereafter, 4 pigs were allocated to each of 4 diets, first-limiting in lysine, providing lysine intakes corresponding to 60, 70, 80, and 90% of estimated requirements for PDmax. The pigs were surgically fitted with catheters in the jugular and femoral veins. Lysine catabolism was determined at 2 BW (40-45 kg, low; 70-75 kg, high) either directly (oxidation) using a primed, constant infusion of l-[1-(14)C]-lysine or indirectly (disappearance) using the N-balance method. There was no effect of BW on the rate (g/d) or fraction of TID lysine intake catabolized. Lysine catabolism decreased with increasing growth potential. Lysine disappearance and lysine oxidation (% of TID lysine intake) were independent of lysine intake, except for the lowest lysine intake level, where they were lower. When lysine catabolism was independent of intake, lysine oxidation based on plasma free lysine specific radioactivity (SRA) was lower (9.9% of TID intake) than lysine disappearance (17.4% of TID intake) or lysine oxidation based on liver free lysine SRA (13.4% of TID intake).

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.

This study used a digesta transfer protocol to determine the site of absorption of lysine synthesized by the gastrointestinal microflora of pigs. Eight pigs were used, four with reentrant cannulas in the terminal ileum, two with simple T cannulas in the terminal ileum, and two intact. All pigs were given, for 5 days, the same low-protein diet that included fermentable carbohydrates. The diet of two pigs with reentrant cannulas (donor) and of the two intact (control) pigs was supplemented with (15)NH(4)Cl. The two other pigs with reentrant cannulas (acceptor pigs) and those with simple cannulas (used to supply unlabeled digesta) were given the same diet but unlabeled NH(4)Cl. Ileal digesta were collected continuously from all of the reentrant cannulas and kept on ice. All digesta from each donor pig were reheated and returned to the distal cannula of its companion acceptor, whose ileal digesta were discarded. Unlabeled ileal digesta from the pigs with simple cannulas were instilled into the distal cannulas of the donor pigs. At the end of the experiment, the average (15)N enrichment in the plasma free lysine of control pigs was 0.0407 atom % excess (APE); that of donor pigs was 0.0322 APE (79% of controls), whereas that of acceptor pigs was only 0.0096 APE (24% of controls). Due to nitrogen recycling, acceptor pigs had labeled lysine in the digesta of the stomach and small intestine, and donor pigs had labeled lysine in the digesta of the large intestine. If account is taken of the higher (15)N enrichment of microbial lysine in the large compared with the small intestine, it can be estimated that >90% of the absorption of microbial lysine took place in the small intestine.

The synthesis of essential amino acids by the gut microflora of pigs, and their absorption, were assessed from the incorporation of (15)N from dietary (15)NH(4)Cl and of (14)C from dietary (14)C-polyglucose into amino acids in the body tissues of four pigs. Both (15)N and (14)C were incorporated into essential amino acids in body protein. Because pig tissues cannot incorporate (15)N into lysine or (14)C into essential amino acids, the labeling of these amino acids in body protein indicated their microbial origin. The absorption of microbial amino acids was estimated by multiplying the total content of each amino acid in the body by the ratio of the isotopic enrichment of the amino acid in the body to that in microbial protein. Because the ratio of (14)C:(15)N in body lysine was closer to that in the microflora of the ileum than to that of the cecum, absorption was assumed to take place exclusively in the ileum. The estimates of microbial amino acid absorption from (14)C-labeling were as follows (g/d): valine 1.8, isoleucine 0.8, leucine 2.0, phenylalanine 0.3 and lysine 0.9, whereas for lysine, the estimate from (15)N-labeling was 1.3 g/d. We conclude that the gastrointestinal microflora contribute significantly to the amino acid requirements of pigs.

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.

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).

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.

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.

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.

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.

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 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.

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.

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.

Three imaging methods, ultrasound imaging (UI), computer-assisted axial tomography (CAT) and magnetic resonance imaging (MRI), are widely used in medicine. Their application to the assessment of body composition in nutrition research is still being explored and developed. Ultrasound imaging yields poor image quality but, because it is cheap and safe, deserves further exploration. Both CAT and MRI can produce images with good discrimination among bone, muscle and adipose tissue. Movement artifacts tend to be more serious in MRI than in CAT due to the longer imaging time. On the other hand, the X-ray exposure in CAT is likely to limit its use in human nutrition research. Repeated measurements of tissue volumes by CAT and MRI give similar CV. In both CAT and MRI, intra-abdominal adipose tissue presents greater problems of measurement than subcutaneous adipose tissue. Validation studies with 77-kg pigs of MRI, using 13 slices, predicted total body lipid with residual standard deviation of 1.9%. In validating any of these methods, account should be take of the extent to which the information they give can augment that given by more simple measures like age and weight.

The generation of 14CO2 from [1-14C]lysine by hepatic mitochondria through the saccharopine pathway is controlled by intramitochondrial concentrations of lysine, 2-oxoglutarate and NADPH. Mitochondria, isolated from rats pre-treated with glucagon, exhibited higher activities of L-lysine: 2-oxoglutarate reductase, saccharopine dehydrogenase and 2-aminoadipate aminotransferase. The flux through this pathway is stimulated in liver mitochondria after glucagon treatment. Multiple regulation of lysine oxidation in liver mitochondria confirms a complex mechanism of 'mitochondrial activation' by glucagon.

The mean response of body protein accretion in growing animals to their amino acid intake is sometimes described by a rectilinear ("broken-line") model and sometimes by a curvilinear model. The response of a population may be curvilinear as a result of averaging individual rectilinear responses or because individual responses are themselves curvilinear. This experiment was undertaken to distinguish these possibilities by examining the responses of individual animals. Eighteen pigs with a mean initial weight of 35 kg and a mean final weight of 73 kg were each fed, in a different sequence, six diets providing a daily nitrogen intake of 0.5, 1.5, 1.83, 2.17, 2.5 or 3.5 g/kg body wt0.75. Each diet was given for 10 d, with complete collections of feces over the last 6 d and of urine over the last 4 d. Rates of nitrogen retention (expressed per kg0.75 per day) were related to rates of nitrogen intake (in the same units) using a rectilinear (broken line), an exponential or a saturation kinetics model. Mean square errors were significantly larger for the rectilinear model than for either of the curvilinear models and were slightly but not significantly less for the saturation kinetics model than for the exponential model.

Saline, naloxone, domperidone or metaclopramide was injected into lactating rabbits immediately before suckling. Blood samples were taken prior to injection (0 minutes) and then at 15, 30, 45 and 60 minutes after the start of suckling, after which the samples were assayed for plasma prolactin and LH concentrations. In all the does there was a significant increase in prolactin concentration, which was highest 15 minutes after the start of suckling, and which declined exponentially thereafter to levels significantly higher than before suckling. The increase in prolactin concentration was similar in does given saline and naloxone, but it was significantly enhanced in does given metaclopramide; with domperidone the increase was intermediate and not significantly different from that following treatment with saline. In does given saline, domperidone, and metaclopramide plasma LH concentrations declined slowly during the hour after suckling but the concentration was increased significantly in does given naloxone. The inverse correlations between prolactin and LH were low weak and were not significant.

In four experiments growing pigs were given a cereal-based diet alone or supplemented with unmolassed sugar-beet pulp (SBP), used as a model substrate for fermentation. The rates of production of methane and gaseous hydrogen were measured and, together with the molar proportions of volatile fatty acids (VFA) in the digesta, used in stoichiometric calculations of fermentation. The resulting estimates were only one-sixth of the observed extent of digestion of SBP. Bacteriostatic levels of antibiotics reduced fermentation by more than half, as judged from the digestion of non-starch polysaccharides: allowing for the incomplete suppression of fermentation it was estimated that the production of methane and VFA could account completely for the digested SBP. The potential contribution of various routes of hydrogen disposal to the error of the stoichiometric calculations is discussed.

The suitability of L-[3-3H]valine for measuring valine oxidation was studied by comparing its oxidation rate with that of L-[1-14C]valine in rats and pigs. L-[3-3H]valine was synthesized by removal of the tritium on carbon-2 of L-[2,3-3H]valine by acetylation. The acetyl group was removed enzymatically using pig renal acylase 1 (EC 3.5.1.14) and the product was purified by ion-exchange and paper chromatography. For the first rat experiment L-[3-3H]valine was synthesized in our laboratory; for the subsequent experiments it was produced by Amersham International plc. In the first experiment in rats the two tracers were given by injection and 14CO2 was collected for 2 h. The oxidation of tritiated valine was significantly higher than that of L-[1-14C]valine. In a second experiment there was no difference. This was probably due to the higher purity of the labelled valine which, for the second experiment, was shown by nuclear magnetic resonance to contain only one tritium atom. In a study with pigs in which the two tracers were given by continuous infusion there was no significant difference between them in flux or oxidation. The results of this experiment were used to evaluate a model to estimate amino acid requirements. With pigs given a methionine-limiting diet a reduction in methionine intake, by reducing protein accretion, increased valine oxidation by the same proportion.

Primiparous crossbred does were remated on Day 1 (n = 15) or 14 (n = 25) post partum and killed on Day 10 post coitum to assess their fertility. Blood samples were taken during the pre- (0-12 h post coitum) and post- (1-10 days post coitum) ovulatory periods and plasma was assayed for luteinizing hormone (LH), prolactin, oestradiol-17 beta and progesterone. Ovulation response was significantly greater (P less than 0.01) and ovulation rate significantly lower (P less than 0.001) in does mated on Day 1 than in those mated on Day 14 post partum. Does failing to ovulate on Day 14 post partum exhibited no preovulatory LH surge and had significantly lower (P less than 0.05) premating concentrations of oestradiol-17 beta and prolactin than those ovulating at this time. No significant differences in hormone concentrations were observed during the preovulatory period between does ovulating on Days 1 and 14 post partum, with the exception of oestradiol-17 beta. Concentrations of this hormone were significantly lower (P less than 0.01) in does mated on Day 1, at 1 h post coitum. We conclude that (i) fertility was affected by the remating interval after parturition, (ii) ovulation failure was associated with an absence of the preovulatory LH surge and a reduction in premating concentrations of oestradiol-17 beta and prolactin and (iii) the lower ovulation rate in early lactation was apparently caused by a reduction in ovarian competence to respond to the gonadotrophic stimulus.

To investigate in vivo the interconversion between glycine (Gly) and its N-methyl product sarcosine (Sar), [1-13C]Gly and [1-14C]Sar were infused into hourly fed pigs receiving diets with low- and high-threonine levels. An open two-pool model was developed to calculate Sar demethylation (DM) and Gly methylation (GM). During [1-14C]Sar infusion, intracellular Gly specific radioactivities (SA) in the liver and kidney were higher than plasma Gly SA, suggesting that demethylation of Sar occurred in those tissues. DM estimated by using hippuric acid (HA) as the production pool had a mean value of 1.55 mumol.kg-1.h-1, similar to the Sar production rate (mean 1.85 mumol.kg-1.h-1). GM was undetectable (less than 0.5 mumol.kg-1.h-1). These results suggest that, in fed pigs, Sar is produced mainly from choline catabolism and is degraded only to Gly in liver and kidney. On the assumption that Sar degradation gave rise only to Gly, the production rate of Gly (Gly PR) was calculated from [1-13C]Gly and [1-14C]Sar infusions using either the primary pools (plasma Gly and HA, respectively) or the secondary pools (HA and plasma Gly, respectively). The results were explained by a liver-plasma Gly exchange model. The whole body Gly irreversible loss, i.e., direct loss from plasma and liver, was calculated from this model to be 832 +/- 58 mumol.kg-1.h-1, showing that the estimation of Gly PR with [1-13C]Gly infusion and plasma Gly enrichment (599 +/- 56 mumol.kg-1.h-1) was a significant underestimate of the true value.(ABSTRACT TRUNCATED AT 250 WORDS)

A basic assumption of the doubly labeled water (DLW) and triply labeled water (TLW) methods for measuring water flux (rH2O), CO2 production (rCO2), and fractionated water loss (X) is that the H of body water only leaves the body as water. Any loss of isotopes in other products will introduce an error into these techniques. The body fat represents the largest potential sink for water H. 2H sequestration into the carcass fatty acids was investigated in eight pigs labeled with 2H2O for 21 days. rCO2 was measured simultaneously in respiration chambers to allow an accurate assessment of the effect of 2H sequestration on the estimated rCO2. The fat content of the diet (1.63%), level of intake, and stage of maturity were all designed to give the widest possible range of sequestration effects. Four animals were restricted to their estimated maintenance requirement and four were allowed to feed ad libitum giving a range of weight gain from 100 to 650 g/day. This was reflected in the estimated error on rH2O (+0.42% in the restricted group and +2.52% in the fast-growing animals) and on rCO2 (-1.30 and -7.59%, respectively). The error on the calculation of X using TLW was +0.03 units in the restricted group and +0.20 units in the fast-growing animals. The error of +0.2 on X propagates through to an underestimate of rCO2 of approximately 4%, and since this is additive with the error on DLW the ultimate error on rCO2 using TLW would be approximately -12%.(ABSTRACT TRUNCATED AT 250 WORDS)

Body fat content of seven lean women (body mass index (BMI) 20.6 (SD 1.8) kg/m2) and seven overweight women (BMI 31.1 (SD 3.3) kg/m2) was estimated by six different methods: underwater weighing (UWW), body-water dilution (BWD), whole-body counting (40K), skinfold thickness (SFT), bioelectrical impedance (BEI) and magnetic resonance imaging (MRI). Using UWW as the reference method, the differences between percentage fat by each other method and the percentage fat by UWW were calculated for each subject. The mean difference was lowest for SFT and highest for BWD. MRI showed the lowest variability in individual results, and 40K the highest. 40K and BWD methods used in combination gave better agreement with UWW results than either 40K or BWD methods alone. There was a weak negative correlation between the difference from the UWW results and percentage fat in the SFT measurements, but not in the BWD, 40K, BEI or MRI measurements, suggesting that for these methods the assumptions involved produced no greater inaccuracy in the overweight women than in the lean women. In all subjects the BEI offered little improvement over the traditional SFT measurements. The agreement between MRI and UWW estimates in both lean and overweight women suggests that MRI may be a satisfactory substitute for the more established methods of body fat estimation in adult women.

Kinetic aspects of threonine (Thr) metabolism were examined in eight pigs fed hourly with a diet containing either 0.68% (LT group) or 0.81% (HT group) of Thr (wt/wt), corresponding to 10 and 30% Thr excess, respectively, compared with an "ideal" diet. Primary production (PR) and disposal (DR) rates were obtained for Thr, glycine (Gly), and 2-keto-butyrate (KB) after a 12-h continuous infusion of L-[U-14C]-Thr together with [1-13C]Gly and a 6-h continuous infusion of [1-14C]KB. Transfer of Thr into secondary pools was also monitored, and from these the rates of Thr oxidation through the catabolic pathways of L-Thr 3-dehydrogenase (DR(Thr-Gly)) and threonine dehydratase (DR(Thr-KB)) were estimated. For the LT group the results were (mumol.kg-1.h-1) PR(Thr) 314 +/- 3, PR(Gly) 551 +/- 24, PR(KB) 41 +/- 3, DR(Thr-Gly) 22 +/- 2, and DR(Thr-KB) 7 +/- 1. For the HT group they were PR(Thr) 301 +/- 23, PR(Gly) 598 +/- 55, PR(KB) 39 +/- 4, DR(Thr-Gly) 32 +/- 2, and DR(Thr-KB) 8 +/- 1. The increase in Thr intake (14 mumol.kg-1.h-1, P less than 0.01) induced a commensurate increase in the sum of DR(Thr-Gly) and DR(Thr-KB) (14 mumol.kg-1.h-1, P less than 0.001) when liver was used as the precursor pool. This was mainly due to the increased DR(Thr-Gly) (13 mumol.kg-1.h-1, P less than 0.01); the change in DR(Thr-KB) was not statistically significant. By comparison of intracellular-to-plasma ratios of specific activities (or enrichments) for different tissues with each type of infusion, liver was shown to be the major site of production of Gly and KB from Thr. These data suggest that in fed growing pigs a 30% excess of Thr in the diet does not alter the partition of Thr oxidation, since 80% of Thr oxidation occurs through the L-Thr 3-dehydrogenase pathway for both LT and HT groups.