Abstract:
239 ENDOGENOUS AMINO ACID LOSS IN SIMPLE-STOMACHED ANIMALS P J MOUGHAN* SUMMARY It is important to be able to accurately determine the flow of endogenous amino acids in different parts of the digestive tract. Endogenous loss has traditionally been determined after feeding animals a protein-free diet, but recent evidence indicates that this may lead to considerable underestimation. It appears that endogenous amino acid loss in animals given a protein-free diet over relatively short periods of time is not lowered due to a deranged body protein metabolism generally, but rather to the absence of the stimulatory action of gut peptides. Techniques need to be developed to allow the routine determination of gut endogenous amino acid losses for animals under conditions of normal protein alimentation. INTRODUCTION Endogenous amino acid loss from the entire digestive tract or regions of the gut of animals is a fundamental measure in Nutritional Science. Estimates of endogenous amino acid loss are used in the factorial calculation of amino acid requirements for growth and maintenance (Moughan 1989) and for correcting apparent digestibility coefficients to true values. There is some controversy concerning the application in practice of true as opposed to apparent coefficients of amino acid digestibility. True values, however, are an attribute of the feedstuff itself and are less affected by a number of methodological aspects of the digestibility assay. The argument as to whether apparent or true digestibility values are preferred for practical dietary formulation is also inextricably linked to the approaches adopted in estimating amino acid requirements for growth. Care must be taken whenformulating diets, and with respect to how amino acid requirements and dietary amino acid levels have been defined, that part of the maintenance requirement for amino acids is not allowed for twice. There is an increasing trend to define amino acid requirements at the tissue level using computerised simulation models and in this case true coefficients of amino acid digestibility are most meaningful for dietary formulation. An increased need for dietary amino acids by the animal to offset a heightened loss of amino acids from the gut due to components of a mixed diet such as anti-nutritional factors or complex non-starch polysaccharides can be accounted for when modelling the amino acid transactions. The traditional approach to determining endogenous nitrogen or amino acid loss, involves feeding animals diets which are almost entirely devoid of protein.The 'protein-free' method has been frequently criticised, however, as being unphysiological. When animals are deprived of dietary protein and enter negative body nitrogen balance, their rate of whole-body protein synthesis falls (Millward et al. 1976) and this may affect the entry of protein into the gut lumen.THE PROTEIN DEPLETE STATE Two studies have recently been conducted to test the hypothesis that state of body nitrogen balance per se affects endogenous amino acid loss. In the first study (Skilton et al. X988), growing rats were given either a protein-free diet or a similar diet but containing synthetic free amino acids as a source of dietary nitrogen. The latter diet was devoid of specific dietary non-essential *Department of Animal Science, Massey University Palmerston North, New Zealand 240 . amino acids. The animals receiving the amino acid based diets grew normally and were in positive nitrogen balance. The endogenous amino acid flows at the terminal ileum determined using diets devoid of certain amino acids are given in Table 1 along with values for the rats given the protein-free diet. TABLE 1 Mean (n=6) endogenous ileal amino acid flows determined by feeding rats synthetic diets devoid of specified dietary non-essential amino acids or a protein-free diet These results indicate that the negative body nitrogen balance caused by the protein-free feeding regime, which was presumably accompanied by a fall in the rate of body protein synthesis, did not lead to a lowered endogenous,loss of amino acids from the ileum. Nor does it appear that dietary free amino acids stimulate endogenous amino acid secretion. It is possible, though unlikely, that administration of the dietary synthetic amino acids could have masked an effect of positive nitrogen balance on endogenous loss. This does not appear to be the case. At the University of Alberta, de Lange et al. (1989) fed growing pigs a protein-free diet while intravenously infusing half the animals with physiological saline and the remainder with a balanced mixture of synthetic amino acids. There were no significant differences in ileal endogenous amino acid excretion between the pigs in positive nitrogen balance and those receiving the protein-free diet with intravenous infusion of saline, except that proline loss was significantly (P<O.O5) lower for animals given the amino acid infusion. It does not appear, therefore, that the protein-deplete state per se affects endogenous amino acid loss at the distal ileum of animals. It remains possible, however, that dietary peptides exert a direct stimulatory effect on gut endogenous protein secretion. THE EFFECT OF PEPTIDES In examining the effect of dietary peptides on endogenous amino acid losses from the gut methods have had to be devised to distinguish between amino acids of dietary and endogenous origin. The most common approach adopted, although it has several shortcomings, has been to label the animal's body protein using radioactive or stable isotopes. Using this technique with growing pigs, de Lange et al. (1990) sh owed that endogenous nitrogen flow at the ileum is considerably enhanced when natural protein-containing feeds are given as opposed to proteinfree diets. Two further methods, which allow more definitive study of the effect of peptides and protein on endogenous amino acid loss, have recently been developed. The first approach which involves feeding animals guanidinated proteins (lysine has beentransformedto homoarginine) allows determination of the endogenous loss of lysine (Hagemeister and Erbersdobler 1985) when dietary protein is present in the gut. Moughan and Rutherfurd (1990) completely guanidinated gelatin protein and in a study with growing rats demonstrated (Table 2) that when dietary 241 protein is present in the endogenous lysine loss from the terminal ileum is considerably enhanced above that found with protein-free alimentation. gut 7 Mean endogenous flows of lysine' (+ SE) at the terminal ileum of TABLE 2 growing rats given either a guanidinated gelatin based diet or a protein-free diet With the second method (Moughan et al. 1990), the ileal flows of all endogenous amino acids are able to be determined.. In this case the animal is fed a semi-synthetic diet containing enzymically hydrolysed casein (peptides < 5000 MW) as its sole nitrogen source. Ileal digesta are collected and the protein (X0,000 MW) is immediately separated physically by ultra-filtration. Any unabsorbed peptides or amino acids along with endogenous amino acids and small peptides which are found in the ultra-filtrate are discarded and the retentate fraction is analysed to give an estimate of endogenous amino acids. The degree of underestimation of endogenous amino acid flow consequent upon discarding any endogenous free amino acids and small peptides has been shown to be of only minor significance ( P. J. Moughan, unpublished). Butts et al. (1991) applied the latter technique with growing rats, and their results some of which are presented in Table 3 provide further evidence that dietary peptides have a stimulatory effect on endogenous amino acid secretion and loss from the smallintestine. A similar effect has been found with growing pigs (P.J. Moughan and G. Schuttert, unpublished). TABLE 3 Mean endogenous amino acid flowsa (+ SE) at the terminal ileum of rats given an hydrolysed casein based diet and with treatment of the digesta, or a protein-free diet In light of the above-documented evidence for a stimulatory effect of peptides on gut endogenous amino acid loss, it is interesting to note that bioactive peptides formed during the digestion of foods have been isolated and are thought to play a role in stimulating gut secretory processes (Schlimme et al. 1989). There is now little doubt that peptides either administered per OS or derived from the natural digestion of dietary protein have a considerable influence on the loss of endogenous amino acids from the digestive tract. 242 CONCLUDING REMARKS Although there is an inherent effect of feeding protein or peptides to the animal, the source of dietary protein appears to have little effect on endogenous amino acid excretion. However, other compounds often associated with protein in a feedstuff may have a marked effect upon endogenous amino acid loss. Among these are the so-called anti-nutritional factors (eglectin, tannins) andvarious fractions of the dietary fibre (Sauer and Ozimek, 1986). The effects that such factors may have an endogenous amino acid loss, with the result that endogenous loss is likely to vary among different feedstuffs, poses problems for the determination of endogenous loss in practice. The loss of amino acids from the small intestine and over the entire digestive tract of animals is quantitatively much more significant than has been recognised traditionally. It is now clear that the often-used protein-free technique gives unrealistic estimates of endogenous amino acid excretion and that several dietary factors may influence endogenous loss. These effects may be dependent upon the concentration of the given factor in the feedstuff and its interaction with other dietary factors. Consequently, the effect on endogenous amino acid excretion may differ from the case when a single feedstuff is given to an animal to that when the same feedstuff is one of several ingredients in a compound diet. This further highlights the potential dangers in adhering to a system of feed evaluation based on apparent estimates of amino acid digestibility. There is an urgent need to develop methods to allow accurate measurement of endogenous amino acid loss when specific feedstuffs are given singly to animals and to afford description of the effects of various factors present in mixed diets and the effects of level and method of feeding. REFERENCES BUTTS, C.A., MOUGHAN, P.J. and SMITH, W.C. Press. (1991). J. Sci. Food Agric. (In J. Anim. Sci. 67:755. de LANGE, C.F.M., SAUER, W.C. and SOUFFRANT, W. (1989). de LANGE, C.F.M., SOUFFRANT, W-B, and SAUER, W.C. (1990). J. Anim. Sci. u:409. HAGEMEISTER, H. and ERBERSDOBLER, H. (1985). Proc. Nutr. Soc. D 44:133A. 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