Analysis of an ethanol precipitate from ileal digesta: evaluation of a method to determine mucin.

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.

Endogenous proteins in terminal ileal digesta of adult subjects fed a casein-based diet.

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.

Methods for mucin analysis: a comparative study.

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.

Dietary and endogenous amino acids are the main contributors to microbial protein in the upper gut of normally nourished pigs.

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.

Endogenous components of digesta protein from the terminal ileum of pigs fed a casein-based diet.

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.

Intestinal microbial contribution to metabolic leucine input in adult men.

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.

Food-derived bioactive peptides influence gut function.

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.

In vivo determination of amino acid bioavailability in humans and model animals.

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.

Growth potential, but not body weight or moderate limitation of lysine intake, affects inevitable lysine catabolism in growing pigs.

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).

Pigs' gastrointestinal microflora provide them with essential amino acids.

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.

Lysine synthesized by the gastrointestinal microflora of pigs is absorbed, mostly in the small intestine.

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.