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The Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland, UK
3To whom correspondence should be addressed at IRTA-Centre de Mas Bové, Apartat 415, 43280 Reus, Spain. E-mail: david.torrallardona{at}irta.es.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • amino acid requirements • amino acid synthesis • amino acid absorption • intestinal bacteria • swine
One of the functions of the indigenous gastrointestinal microflora of animals is the provision of essential nutrients, such as amino acids and certain vitamins; this is especially important in ruminants and in nonruminant herbivores such as lagomorphs and horses that eat poorly digestible low protein diets (1 ,2 ). For nonruminant omnivores and carnivores, however, the nutritional benefits of the gut microflora are less clear. Earlier, we demonstrated the absorption of lysine of microbial origin in rats by showing that 15N from dietary [15N]ammonium chloride can be incorporated into lysine in the body protein of conventional but not of germ-free rats (3 ). We observed that microbial lysine contributed substantially to the requirements of rats, although its absorption depended on the practice of coprophagy (3 ,4 ).
The incorporation of inorganic 15N into body lysine has been shown not only in rats (5 –8 ), but also in pigs (9 ,10 ) and humans (11 –15 ), suggesting that other species may also benefit from essential amino acids synthesized by the gastrointestinal microflora. Recent studies in adult humans given [15N]ammonium chloride or [15N]urea suggested that the absorption of microbial lysine might be comparable in magnitude to the daily requirement (14 ). These estimates were based on the enrichment of [15N]lysine extracted from either feces or ileostomy fluid, although neither of those can be considered properly representative of digesta at the site of amino acid absorption in normal subjects. The present study was undertaken to obtain digesta samples from various sites along the intestine of pigs, to identify the site of absorption of amino acids synthesized by the microflora and to estimate their quantitative contribution to the pig’s requirements.
The incorporation of 15N from 15NH4Cl into microbial and body lysine and 14C from 14C-polyglucose into microbial and body lysine, valine, isoleucine, leucine, phenylalanine and histidine was studied. The changes in isotopic enrichments of lysine in the microbial protein were documented, both with time (in feces) and in different sections of the gastrointestinal tract at the end of the experiment.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals and procedures.
Four female Cotswold crossbred pigs (20.1 ± 0.70 kg body weight) were used. They were housed in metabolism crates to prevent coprophagy. The pigs were adapted for 8 d to a low protein diet containing unlabeled ammonium chloride and polyglucose; they were then given the same diet for 10 d but with labeled [15N]ammonium chloride and [14C]polyglucose. The diet was given in two daily meals, 600 g at 0800 h and 300 g at 1400 h. For one of the pigs, the incorporation of both isotopes into fecal microbial lysine was determined from feces collected on d 2, 4, 6 and 10. The pigs were killed under halothane anesthesia 3 h after their last meal on d 10. The gastrointestinal tracts were removed and, for one of the pigs, the digesta in the stomach, four sections (of equal length) of the small intestine, cecum, ascending colon and descending colon were sampled, taking care not to remove mucosal tissue; these samples were frozen until required. For the remaining three pigs, only the digesta in the last quarter of the small intestine and the cecum were sampled. For each pig, the carcass, viscera and blood were weighed and homogenized. Samples of the homogenates were frozen until analyses of amino acid concentrations and isotopic enrichments were made. The empty gastrointestinal tracts were processed separately to analyze their amino acid contents to avoid contaminating the other tissues with microbial amino acids.
Diet.
The experimental diet contained (g/kg): cornstarch, 616; cellulose, 103; casein, 51; soybean oil, 31; dicalcium phosphate, 7.3; limestone, 7.3; polyglucose (either labeled with 14C or unlabeled), 174; ammonium chloride (either labeled with 10 atom % 15N or unlabeled), 7.9; and vitamin and mineral mixture, 2.5. The diet was estimated to provide (per kg) 2768 kcal metabolizable energy, 47 g protein and 3.6 g Lys. The diet was given unlabeled during the 8-d adaptation period. During the 10-d labeling period, it included 15NH4Cl (10 atom %) and 14C-polyglucose. The total 15N and 14C doses consumed over 10 d by the pigs were 1.9 g/pig and 185 MBq/pig, respectively.
Synthesis of polyglucose.
Polyglucose, a low digestibility glucose polymer (16 ), was synthesized by anhydrous melt polymerization by heating a mixture of glucose (or [U-14C]glucose), sorbitol and citric acid (ratio of 90:10:1) in a vacuum oven at 140°C for 24 h at a reduced pressure of <1 mmHg (17 ).
Isolation of microbial protein.
The microbial fraction of feces or digesta was obtained by filtration and differential centrifugation as described previously (3 ). Briefly, samples were homogenized in 0.01 mol/L PBS and centrifuged (250 x g at 4°C for 25 min). The supernatant was filtered and centrifuged again (14,500 x g at 4°C for 30 min). The pellet was resuspended in PBS and centrifuged again (14,500 x g at 4°C for 30 min).
Tissue homogenization.
The carcasses and gastrointestinal tracts of the pigs were homogenized separately by mincing in a meat grinder. Samples were freeze-dried and subsamples were then ground in a freezer mill (Spex Industries, Metuchen, NJ) using liquid nitrogen.
Amino acid isolation.
Tissue and microbial samples were hydrolyzed under reflux with 6 mol/L HCl at 137°C for 18 h. Valine, isoleucine, leucine, phenylalanine, histidine and lysine were isolated by preparative ion-exchange chromatography (18 ). Amino acid concentrations were measured in an ion-exchange chromatography system with postcolumn ninhydrin detection (The Locarte Company, London, UK).
Measurement of amino acid labeling.
After desalting, the amino acids were concentrated and the specific activity of each was determined by liquid scintillation counting (Packard 1900CA Tri-Carb liquid-scintillation analyzer; Packard Instrument Company, Downers Grove, IL).
To determine its 15N-enrichment, lysine was digested to ammonium sulfate by a micro-Kjeldahl method, the ammonia was distilled and its 15N-enrichment measured by gas isotope ratio mass spectrometry (SIRA 12; VG Isogas, Middlewich, UK), as described previously (3 ).
Samples of the urine of the pigs, taken while they were consuming the unlabeled diet, were analyzed. The 15N-enrichments of these samples (0.3673 ± 0.00018) atom 15N % were taken as the natural abundance and all enrichments were calculated accordingly.
Amino acid analysis.
Samples of the dried, milled homogenates of body tissues and gastrointestinal tract were hydrolyzed under reflux with constant-boiling 6 mol/L HCl at 137°C for 18 h and measured in an ion-exchange chromatography system (The Locarte Co., London, UK).
Calculation of results.
Unlike bacteria, mammals cannot incorporate inorganic nitrogen (e.g., 15NH4Cl) into lysine, which does not engage in transamination. We confirmed this previously by showing the absence of [15N]lysine in germ-free rats, in contrast to conventional rats (3 ). Similarly, mammals cannot incorporate carbon from a carbohydrate source into essential amino acids (except possibly the methyl group of methionine), but bacteria of the gastrointestinal tract can. Therefore, the incorporation of dietary 15N into body lysine, or of dietary 14C into essential amino acids in body protein, indicates the absorption of microbially synthesized amino acids. Ignoring any loss or degradation of the labeled amino acid after it is absorbed from the gut, the absorption of a microbially synthesized amino acid can be estimated from the total quantity of the amino acid in the body and the relative isotopic enrichments of the amino acid in body protein and microbial protein.
The rate of microbial amino acid absorption (A, mg/d) can be calculated by the equation:
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where T is the total quantity of the amino acid in the body, t is the time during which the label was given, Et is the isotopic enrichment (or specific radioactivity) of the amino acid in the body at the end of the labeling study and Em is the average isotopic enrichment (or specific radioactivity) of the amino acid in the microbial protein at the site of absorption.
Statistical analysis.
The change with time in the labeling of the microbial fraction of feces was analyzed by linear regression. The isotopic enrichment of amino acids in the body protein of the pigs was compared with their corresponding natural enrichment by Student’s t test. The labeling in amino acids in ileal and cecal microbes was compared by Student’s paired t test. The statistical package MINITAB (19 ) was used.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Both 15N- and 14C-labeling of lysine in the microbial fraction of feces in the pig studied increased linearly during the experiment (r2 = 0.996; P < 0.001 and r2 = 0.985; P < 0.001 for 15N and 14C, respectively; Fig. 1 ).
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The 14C and 15N were incorporated into the essential amino acids and into lysine of body protein, respectively (Table 2) , indicating the absorption of amino acids of microbial origin. The 14C-labelings in the amino acids of the body tissues were significantly different from zero (Table 2 ; P < 0.05). The 15N-enrichment in the carcass (tissue) lysine was also different (P < 0.01) from the natural abundance (Table 2) .
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The values for T, t and Et in the equation used to estimate microbial amino acid absorption can be estimated with little difficulty. The enrichment of the microbial amino acids being absorbed (Em) is more problematic because the isotopic enrichment of microbial amino acids varied with both time and site. It was therefore necessary to determine the average enrichment at the site of absorption.
Changes with time in the isotopic enrichment of microbial amino acids.
Although digesta samples were obtained only on the last day of the experiment, both the 14C and 15N labeling of amino acids in the microbial fraction of feces increased linearly with time (Fig. 1) . Therefore, we made the assumption that the labeling of the microbial amino acids in digesta increased linearly in a similar manner during the 10 d of the experiment and that the mean enrichment can therefore be estimated as one half of that measured at the end.
The site of microbial amino acid absorption.
Although there is some evidence that intact amino acids can be absorbed through the mucosa of the large intestine (20 –22 ), experiments in which protein or amino acids have been infused into the terminal ileum or cecum have shown that such infusions have little or no nutritional benefit, even in animals fed amino acid–deficient diets (23 –27 ). This suggests that microbial amino acids are absorbed primarily from the small intestine.
We have further confirmed the small intestine as the primary site of absorption of microbial amino acids from crossover studies in which the digesta from the terminal ileum of pigs given [15N]ammonium chloride were quantitatively transferred to the cecum of pigs given unlabeled ammonium chloride (28 ). The 15N- labeling of body lysine in the donor and recipient pigs was approximately three quarters and one quarter, respectively, of that in intact pigs given [15N]ammonium chloride. Taking into account that the isotopic enrichment of microbial lysine in the large intestine was several times higher than that in the small intestine, it was estimated that considerably more than three quarters of the microbial lysine in the body was absorbed in the small intestine.
The present experiment supports this view. We assumed that the ratio of 14C:15N in the microbial lysine at the site of absorption was the same as that of the absorbed microbial lysine within the body. The ratio between 14C and 15N in the lysine of the body protein was 1.7 Bq · mol-1 · ape-1. The ratio in the microbial fraction of digesta measured along the gastrointestinal tract in one of the pigs varied from 2.1 in the ileum to 9.3–11.8 in the large intestine (Table 1) . Clearly, the ratio of the isotopes in body tissue is much closer to that of microbes in the ileum than in the large intestine. The ratio of 14C:15N in the lysine of fecal microbes remained relatively stable (17.4, 13.8, 10.8 and 11.8 on d 2, 4, 6 and 10, respectively), suggesting that the ratios measured on d 10 are representative of the whole experimental period. The same conclusion can be drawn from the combined data of the four pigs, i.e., the 14C:15N ratio in the microbial fraction of digesta was 3.8 in the ileum and 7.3 in the large intestine.
However, the 14C:15N ratio in body tissue did not correspond exactly to that of microbial protein in any part of the intestine. Unfortunately, we were not able to obtain enough digesta from the proximal small intestine for 15N measurements, but it may be that the ratio there was closer to that in the body. The increase in 14C:15N ratio along the gut may occur because ammonium chloride, which is readily soluble, is likely to have been absorbed rapidly in the upper digestive tract, whereas polyglucose, although susceptible to attack by microbial enzymes, probably became available much more slowly so that the ratio would likely increase all along the gut.
Estimates of microbial amino acid absorption.
To calculate the quantities of microbially synthesized amino acids absorbed we have assumed that absorption took place exclusively in the ileum. We have further assumed that the average isotopic enrichment of amino acids in ileal digesta was, like that in feces, half of that observed on d 10.
The estimates of microbial lysine absorption, based on the 15N enrichments or 14C specific radioactivities of samples from the distal ileum, were 1.3 and 0.9 g/d, respectively. These, and the estimates for other amino acids, based on 14C labeling, are presented in Table 3 .
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In considering the extent to which amino acids synthesized by the gastrointestinal flora might contribute to meeting the essential amino acid requirements of their host, it is important to consider two questions. The first is simply what proportion of the dietary requirement is represented by the estimated microbial supply. Estimates of the daily lysine requirement of 20-kg pigs (29
–31
) range between 7 and 12 g. The estimates of microbial lysine absorbed (Table 3)
are on the order of 1 g (0.9–1.3) or 
10% of the pigs’ requirement. It is worth noting, however, that the estimated requirement is for young pigs during rapid growth. If the absorption of microbial lysine can be assumed to be at least as great in adult pigs, it would be similar to its dietary lysine needs for maintenance, estimated to be 1 g/d in 150-kg pigs (32
).
The second question is whether amino acids synthesized by the gastrointestinal microflora make a net addition to the dietary supply. Metges et al. (14 ) put the issue as follows: "To the extent that the growth of the microbes that give rise to the labeled lysine is supported by the degradation of endogenous protein, the microbially derived lysine in plasma can be seen as part of the normal mechanism by which endogenous nitrogen and indispensable amino acids are recycled rather than as a net source of amino acids additional to those supplied in the diet." In the experiments of Metges et al. (14 ), lysine was the only amino acid investigated and 15N was the only tracer given. There is evidence of very extensive recycling of nitrogen in the gut, from endogenous secretions into the gut and back into the body (33 ). It is clear that the gut microflora play a part in this, for example, through the hydrolysis of urea and the degradation of complex glycoconjugates secreted by mucosal cells, but the extent to which the de novo synthesis of amino acids by the microflora is accompanied by the catabolism of endogenous amino acids, perhaps by another part of the microbial population, is not known. If such intricate transactions take place within the complex microbial ecosystem of the gut, the data obtained with 15N in the present experiment shed no further light on them. However, the 14C found in essential amino acids in the body came undeniably from dietary carbohydrate, and this evidence lends support to the view that de novo amino acid synthesis by the gastrointestinal microflora represents a net contribution to the total amounts of amino acid absorbed.
This conclusion does not invalidate existing estimates of the amino acid requirements of pigs, made from dietary dose-response experiments; it simply means that the metabolic requirement of pigs for an amino acid is supplied in part by the diet and in part by microbial synthesis. However, it does imply that as for some vitamins, metabolic requirement can not be equated with dietary requirement.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by The Scottish Office Agriculture and Fisheries Department. D.T. was supported by a studentship from INIA, Spain.
4 The mixture consisted of 3.44 mg retinyl acetate, 50 µg cholecalciferol, 15 mg dl-
-tocopheryl acetate, 1.5 mg vitamin K, 4.5 mg riboflavin, 3 mg vitamin B-6, 0.015 mg vitamin B-12, 10 mg pantothenic acid, 20 mg nicotinic acid, 0.75 mg folic acid, 100 mg Fe, 100 mg Zn, 40 mg Mn, 175 mg Cu, 2 mg I, 1 mg Co, 0.25 mg Se and 363 mg Ca.
Manuscript received 28 October 2002. Initial review completed 3 December 2002. Revision accepted 10 January 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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