Chapter 6 Role of intestinal first-pass metabolism on whole-body amino acid requirements

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Role of intestinal first-pass metabolism on whole-body amino acid requirements R. F. P. Bertoloa, P. B. Pencharzc,d and R. O. Ballb,d a Department

of Biochemistry, Memorial University of Newfoundland, St. John’s, NewfoundLand, Canada A1B 3X9 b Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 c Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8 d The Research Institute, The Hospital for Sick Children, Toronto, Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada

The small intestine utilizes a different profile of amino acids compared to whole-body requirements. Quantifying the gut requirements for amino acids is critical to understand the limiting availability of these amino acids during periods of rapid growth in animals. Many methods have been employed to determine amino acid requirements in man and animals including growth assays, nitrogen balance and amino acid oxidation methods. The most versatile approach is the indicator amino acid oxidation technique which can be safely employed in many vulnerable populations. The amino acid requirements of the gut have been estimated using this technique in the parenterally fed piglet, which is a model of a gut-deficient animal, and comparing requirements to enterally fed controls. The gut’s requirement for threonine is proportionately greatest of the amino acids tested due to its role in mucin synthesis. The sulphur and branched-chain amino acids are also significantly utilized by the gut. Tryptophan, lysine, phenylalanine and tyrosine utilization by the gut is not significant. The availability of threonine and sulphur amino acids may be limiting for growth in situations of gut stress or disease due to the higher maintenance requirements during such gut challenges.

1. INTRODUCTION The small intestine has classically been regarded as a digestive organ responsible for the absorption of nutrients from foods. Only recently has the gastrointestinal tract, whose metabolism is dominated by the small intestine, been studied as a significant metabolic tissue with

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great impact on whole-body metabolism. Through co-ordinated inter-organ pathways, the gastrointestinal tract is involved in the synthesis, conversion and catabolism of amino acids to be used by other tissues in the body. In addition to this critical role in whole-body nutriture, the gut also requires vast amounts of particular amino acids for maintenance and growth. The profile of these amino acid requirements does not seem to parallel those for growth and maintenance of the rest of the body. Rather, because of the gut’s specific functions in digestion, absorption and immunity, the gut requires a different profile of amino acids. Quantifying these requirements has become an important goal in understanding the role of the gut in amino acid availability for the rest of the body. In particular, this availability becomes of paramount concern in situations of gut disease or stress where increased maintenance requirements can limit the availability of certain amino acids for whole-body growth and physiological functions.

2. METHODS TO MEASURE AMINO ACID REQUIREMENTS Many methods have been employed in humans and animals to determine amino acid requirements. The advantages and disadvantages of many of these techniques have been extensively reviewed by others (Lewis, 1992; Fuller and Garlick, 1994; Young and el-Khoury, 1995; Zello et al., 1995; Waterlow, 1999). Many of these methods were originally developed in animals and then modified for humans. However, it is important to note that most animal research on amino acid requirements has primarily focused on the growing phase for economic reasons, whereas human research has almost exclusively focused on the adult phase which comprises most of the lifespan. The basic strategy employed by almost all studies involved with the determination of amino acid requirements includes the feeding of graded levels of the test amino acid and the measurement of a specific biological response. The choice of biological response depends on many factors including species, age, health status, sample availability as well as ethical considerations, analytical equipment availability, financial constraints and practicality. In choosing the biological response, the most important aspect of amino acid metabolism to consider is the need for an amino acid for incorporation into protein. If all other essential nutrients, especially energy and other amino acids, are at or above requirement levels, then whole-body protein synthesis will occur at a level determined by the intake of the most limiting amino acid (i.e. the test amino acid). If the intake of this test amino acid is below its requirement, then protein synthesis will be reduced and the intake of all other essential amino acids will be in relative excess; because amino acids cannot be stored, this excess must be catabolized by the body and excreted as bicarbonate and ammonia via carbon dioxide and urea, respectively. Increasing the intake of the test amino acid will result in greater protein synthesis and the concomitant reduction in excess amino acid catabolism indicated by lower carbon dioxide and urea excretion. At intakes above its requirement, the test amino acid will no longer be the first limiting one and additional intakes will not result in greater protein synthesis. At these intakes, the test amino acid itself is in relative excess and must be catabolized to carbon dioxide and urea. This general scheme has been used to develop almost all techniques employed to determine amino acid requirements, including growth assays, serial slaughter, nitrogen balance, plasma amino acids, plasma urea, direct amino acid oxidation, amino acid balance, and indicator amino acid oxidation (fig. 1). It is also important to note that the chosen biological response should be amenable to statistical modelling techniques so that an objective estimate for an amino acid requirement can be determined, preferably with an estimate of population variance. Because the pattern of the biological response is rarely predictable over deficient to excess intakes of the test amino acid, several statistical models have been proposed and employed. The overall response is

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Fig. 1. Graphical representation of various response curves to increasing intakes of the test amino acid. The “breakpoint” requirement is usually determined using two-phase linear regression.

often argued, from a biological standpoint, to be a quadratic model; however, a two-phase linear regression breakpoint model sometimes fits better. In practice, when both models fit well, the breakpoint estimate is usually similar between techniques (Baker et al., 2002). Using either model, the requirement can be determined within individuals and then averaged for a requirement estimate with population variance. Or the model can be applied to a complete data set of many animals over many different intakes and the error of the fit could be used to predict the population variance. In any event, it is obvious that the more data are available for statistical manipulation, the more versatile the modelling can be. This issue of statistical manipulation is not a trivial one. Indeed, it has been recently demonstrated that re-analysis of the classic nitrogen balance studies of Rose and Jones yielded very different conclusions about the amino acid requirements in humans using the exact same data (Rand and Young, 1999; Di Buono et al., 2001a). These studies have clearly demonstrated that the importance of the chosen statistical model is almost as important as the data. 2.1. Growth assays Because the primary role of an amino acid is its incorporation into protein, the measurement of protein synthesis itself, during varying intakes of the limiting amino acid, can be considered to be the most direct of approaches. As such, growth assays, and more specifically the serial slaughter technique, have often been considered as the “gold standard” of techniques in the determination of amino acid requirements in animals. As the test amino acid intake is increased towards its requirement, then more protein is synthesized which leads to increased lean tissue deposition. In young animals with minimal fat deposition, growth is almost directly proportional to lean tissue deposition and the growth assay is appropriate. Obviously, as animals approach maturity, more fat is deposited and this proportional relationship between lean and body growth is not constant. To resolve this discrepancy, the serial slaughter technique employs body composition analysis to accurately determine lean tissue content. Using a reference group of animals analysed at the starting body weight, lean tissue accretion can be determined. However, the main drawback to this approach is the necessity of using

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different animals at different time points. Also, body tissue analysis for amino acids is not very accurate given the heterogeneity of ground body samples and the problems associated with protein-bound amino acid analysis. So although these techniques are simple and direct, they are limited to fast-growing lean animals with low and constant fat deposition or must involve large numbers of slaughtered growing animals of similar genetic background to minimize the inter-animal variation. These techniques are not useful in adult, non-growing animals or in animals with special conditions (i.e. gestation, lactation, egg production, disease, etc.).

2.2. Nitrogen balance When protein synthesis is limited, excess amino acids are catabolized to their metabolic endproducts which for all amino acids include bicarbonate and ammonia. The measurement of these excreted biological products provides an indirect and inverse measurement of changes in protein synthesis and thus can be used to determine amino acid requirements. Ammonia enters the nitrogen pool of the body and is excreted primarily as urea in mammals and as uric acid in birds. Because such excreta are relatively easy to collect and analyse for total nitrogen, the nitrogen balance method was one of the first to be developed for assessment of amino acid requirements in humans and animals. The amino acid requirement can be determined from either balance calculations or over a range of test amino acid intakes. The balance approach regresses nitrogen balance on test amino acid intake and defines the requirement as the intake level at which optimum balance is achieved (i.e. zero or positive balance in adults). A considerable drawback with balance calculations is the need to make an assumption of unmeasurable losses (sweat, skin, nails, hair, etc.). Indeed, some of the original landmark experiments by Rose and Jones to determine human amino acid requirements did not include such an assumption; when these data were later corrected for an estimate of these losses, amino acid requirement estimates more than doubled (Young and Marchini, 1990; Rand and Young, 1999). And because two large numbers are being subtracted (nitrogen intake and nitrogen output), the difference is relatively very small and this assumption becomes extremely important. An alternative approach not involving this correction is to measure the qualitative change in nitrogen balance over a range of test amino acid intakes. Nitrogen output is high when protein synthesis is limited by a single amino acid because the other amino acids are in relative excess and are catabolized. As the test amino acid is added incrementally to the diet, nitrogen output decreases until the requirement is met and further increments will not stimulate protein synthesis and nitrogen balance will remain constant (assuming isonitrogenous diets). The many technical advantages and disadvantages of the nitrogen balance technique have been extensively reviewed over the years (Waterlow, 1999; Tome and Bos, 2000) and will not be discussed here. However, several important points need to be mentioned in the present chapter. An important advantage of the nitrogen balance technique is that unlike growth assays, this method can be successfully applied to adult as well as young species. The major disadvantage of the technique is that because of the very large urea pool in most species, its response to dietary manipulation is rather slow and thus adaptations of a week or greater are generally required. If one is studying 6 or 7 test amino acid levels, this long adaptation results in a lengthy experimental period which is not useful in special physiological situations such as gestation, lactation or disease progression. In addition, this technique is also inappropriate in vulnerable populations where long-term feeding of deficient diets is not ethical. The nitrogen balance technique has been extensively applied for all indispensable amino acids in many species.

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2.3. Direct amino acid oxidation technique As opposed to the nitrogen balance technique, oxidation methods monitor the excretion of carbon dioxide, the other obligatory end-product of amino acid catabolism. However, the basic principle is similar in concept to other methods to determine amino acid requirements. At deficient test amino acid intakes, the test amino acid is efficiently utilized for protein synthesis and its oxidation is low and constant. At intakes above requirement, protein synthesis is maximized and excess test amino acid is preferentially oxidized to its end-products. Instead of measuring changes in nitrogen excretion as in the nitrogen balance technique, amino acid oxidation methods measure changes in carbon dioxide excretion in breath. The use of isotopically labelled amino acids allows for an extremely sensitive means of measuring small changes in amino acid oxidation in response to changes in intake. Although all carbons of amino acid skeletons are eventually oxidized, the most sensitive approach is to monitor the expiration of the cleaved carboxyl group at the 1-carbon position. Thus, oxidation of uniformly labelled amino acids results in distribution of the label among many metabolites which makes interpretation difficult and somewhat less sensitive. Alternatively, the oxidation of carboxyl-labelled amino acids is more direct and easier to interpret provided the decarboxylation step is irreversible. However, because one is measuring carbon dioxide in breath, the carboxyl group must also enter general bicarbonate pools which equilibrate readily with carbon dioxide expiration at the lungs. For example, experiments with labelled threonine and methionine have found that non-linear responses are typical with infusion of these amino acids over varying intakes as a result of their more complex degradative pathways (Chavez and Bayley, 1976; Zhao et al., 1986; Storch et al., 1988; Ballevre et al., 1990). Thus, as summarized by Zello et al. (1995), there are general criteria for choosing appropriate carboxyllabelled amino acids for oxidation studies: (1) the amino acid must be indispensable; (2) it must be primarily partitioned between oxidation to carbon dioxide and protein incorporation; and (3) the labelled carboxyl group must be irreversibly oxidized and sufficiently equilibrated with labelled carbon dioxide in breath. These restrictions adequately apply for some indispensable amino acids such as phenylalanine (provided excess dietary tyrosine is fed), lysine and the branched-chain amino acids; as expected, these amino acids have been used extensively in direct oxidation studies. Although the carboxyl group of methionine is irreversibly oxidized, methionine can equilibrate reversibly with homocysteine prior to the irreversible oxidative pathway, thus complicating interpretation of oxidation data. Brookes et al. (1972) were the first to use the oxidation of isotopically labelled amino acids to determine amino acid requirements in rats. Since then, the direct oxidation technique has been used to determine the requirements of several amino acids in growing rats (Kang-Lee and Harper, 1977, 1978; Harper and Benjamin, 1984), adult rats (Simon et al., 1978), young pigs (Kim et al., 1983a,b; Ball and Bayley, 1984, 1986; House et al., 1997a,b), infants (Roberts et al., 2001a) and adult humans (Meguid et al., 1986a,b; Meredith et al., 1986; Zhao et al., 1986; Zello et al., 1990). For animals, the direct oxidation technique has yielded similar or slightly lower amino acid requirements compared to “classical” techniques such as nitrogen balance and growth assays; this finding validates the approach to a certain extent. In contrast, direct oxidation studies in adult humans have yielded much higher (i.e. 2- to 3-fold) requirements for all tested amino acids compared to those proposed by the FAO/WHO/UNU (1985) based upon nitrogen balance studies. This latter discrepancy has been extensively debated and reviewed (Young and Borgonha, 2000). However, one cannot ignore the problems demonstrated in the interpretation of the small amount of original balance data (Rand and Young, 1999). Indeed, the human lysine requirement estimated from the re-analysed

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original nitrogen balance data (Rand and Young, 1999) actually agrees very well with the estimates derived from various techniques using direct oxidation (Meredith et al., 1986), indicator amino acid oxidation (Zello et al., 1993; Duncan et al., 1996), 24 h oxidation (el-Khoury et al., 2000) and indicator amino acid balance (Kurpad et al., 2001). In addition, animal studies employing nitrogen balance techniques, which are numerous and well controlled, result in more reliable and agreeable results compared to those of more recent kinetic techniques. In any event, the direct oxidation technique provides as biologically valid an approach as the nitrogen balance technique and generally agrees with the growth assays performed to date in animals. A very important advantage of the oxidation method compared to the nitrogen balance technique is the more rapid adaptation of the biological response to test amino acid intake changes. Initial studies in direct amino acid oxidation fed particular test amino acid levels for 7–10 days prior to oxidation measurement, analogous to nitrogen balance studies. However, more recently it has been demonstrated that prior adaptation to amino acid intake (Zello et al., 1990; Motil et al., 1994) does not affect the breakpoint estimate of its requirement using the direct oxidation approach. Therefore, only hours of adaptation to a deficient or excess level of test amino acid seems to be necessary to measure changes in oxidation, and thus to determine requirement. To our knowledge, there have been no studies in animals designed to address this adaptation issue using the direct oxidation technique. However, we have successfully employed the direct oxidation technique to measure phenylalanine requirement in parenterally fed piglets using only 16 h of adaptation prior to oxidation measurement (House et al., 1997a). The adaptation issue has been carefully addressed in indicator amino acid oxidation studies in animals and will be discussed below. It is important to note that this issue of sufficient adaptation is the subject of considerable debate and has been reviewed (Young and Marchini, 1990). With long-term adaptation to a deficient diet, the subject will “accommodate” to this situation and possibly become more efficient in its metabolism. The question is: does this accommodation come at the cost of other unmeasured metabolic functions? If such costs were incurred, then it violates Waterlow’s (1985) reasonable definition of adaptation: the process that permits the organism to respond to a dietary change without adverse consequences. We also need to consider the definition of amino acid requirement which has been proposed by Young and Borgonha (2000) as the minimal intake level needed to maintain a specific nutritional criterion such as growth, body composition, body amino acid balance, organ or system function, etc: the choice of nutritional criterion then becomes the subject of debate. In spite of this ongoing debate, the studies in humans have methodically shown that the direct oxidation method has the distinct advantage over the nitrogen balance technique of very short adaptation periods resulting in more time-efficient and cost-effective studies. Although the direct oxidation technique has provided a more sensitive approach to determination of amino acid requirements, its disadvantages have limited its widespread use in many species. Most importantly, as mentioned above, not all indispensable amino acids can be easily used for direct oxidation measurements, limiting its general application. More specifically, of the indispensable amino acids, only phenylalanine (with excess dietary tyrosine), methionine, lysine and the branched-chain amino acids undergo irreversible oxidation of the carboxyl carbon so that amino acid oxidation can be calculated from expired breath carbon dioxide. However, Young and colleagues have attempted to predict the requirements of the other amino acids using previously determined tracer techniques, composition of body proteins and assumed obligatory oxidative amino acid losses (Young and el-Khoury, 1995); these predictions were subsequently validated (Raguso et al., 1999). An additional criticism

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from a kinetic standpoint is that the feeding of deficient to excess amounts of test amino acid changes that amino acid’s pool size dramatically, thereby diluting the tracer being measured. This variable dilution of the tracer in the pool increases variability and reduces sensitivity of oxidation measurements and hence requirement estimates. 2.4. Indicator amino acid oxidation technique The indicator amino acid oxidation (IAAO) technique is an extrapolation of the earlier work with the direct oxidation technique. Both require the accurate measurement of amino acid oxidation by collecting isotopically labelled carbon dioxide in breath. As with the direct oxidation method, the IAAO method is based on the hypothesis that the partitioning of amino acid metabolism between incorporation into protein and catabolism via oxidation is determined by the most limiting amino acid in the diet. However, the difference between the techniques is that instead of measuring oxidation of the test amino acid, the IAAO method measures the oxidation of one of the other amino acids that is also responding to changes in protein synthesis. When the test amino acid is deficient, protein synthesis is limited and other amino acids are in excess. Indispensable amino acids in excess must be catabolized at a level inversely reflecting the rate of protein synthesis which is dictated by the test amino acid intake. By monitoring the oxidation of one of these “indicator” amino acids over a range of test amino acid intakes, one can estimate the test amino acid requirement for protein synthesis. As intake of the test amino acid increases towards requirement, protein synthesis increases which utilizes more of the indicator amino acid resulting in a smaller excess and lower oxidation. Once the test amino acid intake equals requirement, then greater intakes of this amino acid will not lead to greater protein synthesis and therefore indicator amino acid oxidation remains constant. The choice of indicator amino acid depends on its metabolic characteristics. Phenylalanine (see below), lysine (Ball and Bayley, 1984; Roberts et al., 2001b) and leucine (Kurpad et al., 2001) have been used with success to determine various amino acid requirements. Methionine (Brookes et al., 1972) has been employed unsuccessfully due to its complicated metabolic pathways, as mentioned previously. When the requirement of a test amino acid has been determined using more than one of the indicators, the requirement estimates were very similar (Ball and Bayley, 1984; Zello et al., 1993; Kurpad et al., 2001). Phenylalanine has been used most often as the indicator with successful determinations of the requirements for lysine (Kim et al., 1983a; House et al., 1998a), histidine (Kim et al., 1983b), threonine (Kim et al., 1983a; Bertolo et al., 1998), tryptophan (Cvitkovic et al., 2000), methionine and total sulphur amino acids (Kim and Bayley, 1983; Shoveller et al., 2001), branched-chain amino acids (Elango et al., 2002a), arginine (Ball et al., 1986), proline (Ball et al., 1986) and total protein (Ball and Bayley, 1986) in piglets, tryptophan in trout (Were, 1989), lysine in chickens (Coleman et al., 2002), lysine in growing pigs (Bertolo et al., 2001), and in humans, lysine (Zello et al., 1993; Duncan et al., 1996; Kriengsinyos et al., 2002), threonine (Wilson et al., 2000), tryptophan (Lazaris-Brunner et al., 1998), methionine (Di Buono et al., 2001b), total sulphur amino acids (Di Buono et al., 2001a), branched-chain amino acids (Mager et al., 2001, 2002; Riazi et al., 2001) and tyrosine (Bross et al., 2000; Roberts et al., 2001b). The IAAO technique to determine amino acid requirements was first developed in the neonatal piglet by Bayley and colleagues. Following observations that amino acid catabolism depends on the balance of other amino acids (Brookes et al., 1972; Newport et al., 1976), these researchers successfully demonstrated that the IAAO technique can be used to determine the amino acid requirements in piglets. In particular, Kim et al. (1983b) showed that the

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estimate for histidine requirement in piglets was similar using both the direct oxidation and IAAO techniques with phenylalanine as the indicator. In addition, Ball and Bayley (1984) found that either phenylalanine or lysine could be used as an indicator because the oxidation of both responded to varying tryptophan intakes similarly. To further validate the theoretical concept, Ball and Bayley (1984) also demonstrated that liver protein synthesis was inversely correlated with phenylalanine oxidation. In addition, this research group demonstrated that the amino acid requirements for piglets determined using the IAAO technique, namely histidine (Kim et al., 1983b), sulphur amino acids (Kim and Bayley, 1983), lysine and threonine (Kim et al., 1983a), tryptophan (Ball and Bayley, 1984), proline and arginine (Ball et al., 1986) and total protein (Ball and Bayley, 1986), agreed very closely with those determined by classical techniques (NRC, 1979, 1998). From the initial studies of Bayley and colleagues, the IAAO method has been subsequently refined and expanded from the original approach. All indispensable amino acids have been tested using this method; this aspect is the main advantage of the IAAO method over the direct oxidation technique. In addition, the aforementioned criticism of the direct oxidation technique regarding amino acid pool size does not apply to the IAAO approach. Indeed, because the indicator amino acid is fed at the same level over varying test amino acid intakes, its pool size is not permitted to change and therefore the tracer is not variably diluted. This unchanging pool size is probably the main reason why the IAAO approach tends to give requirement estimates with less variability compared to direct oxidation estimates. As with the direct oxidation technique, the adaptation period required to a particular test amino acid intake has been shown to be minimal. Indeed, in recent human studies, the lysine requirement was similar whether hours (Zello et al., 1993; Duncan et al., 1996; Kriengsinyos et al., 2002), 7 days or 21 days of adaptation (Kurpad et al., 2002b) were employed. This finding is profound in context with the aforementioned ongoing debate about adaptation versus accommodation. Furthermore, this adaptation seems to be relatively insensitive to body size or growth. We have also recently found that phenylalanine oxidation after 1.5 days of adaptation (the shortest adaptation tested) to a high or deficient lysine diet was not different up to 8 days of adaptation in both 25 kg growing pigs and 250 kg sows (Bertolo et al., 2001). This distinct advantage of short adaptation over the classical nitrogen balance or growth assays allows great versatility in the application of oxidation techniques, especially to vulnerable populations (Brunton et al., 1998). Indeed, we have recently determined the branched-chain amino acid requirement of children with the inherited genetic disorder, maple syrup urine disease (unpublished), as well as the tyrosine requirements of parenterally fed infants (Roberts et al., 2001a) and the phenylalanine (unpublished) and tyrosine (Bross et al., 2000) requirements of children with phenylketonuria. Because an estimate of population variance is critical to recommending amino acid requirements, the more data in the breakpoint model, the better. Because of the short adaptation time associated with the IAAO method, it is possible to measure amino acid requirements in individuals. To accurately determine a breakpoint in a two-phase linear regression model, at least six test amino acid intakes should be included (i.e. three oxidation measurements per regression). In the nitrogen balance technique, this would require at least 6 weeks of experimentation assuming 7 days of adaptation per diet. Because we have shown that only 1.5 days of adaptation were necessary in the IAAO method (Bertolo et al., 2001), we recently determined the lysine requirement of individual growing pigs in 2 weeks by changing diets and measuring indicator oxidation every other day (Moehn et al., 2001). Similarly, we have demonstrated that individual amino acid requirements of chickens can be determined in less than 3 weeks (Coleman et al., 2002). With the determination of enough individual amino acid

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requirements, an accurate population variance can be calculated; for animal species, this technique allows such a calculation for the first time. Indeed, more accurate diet formulation and animal performance can be achieved with knowledge of such an error within a genetic population of animals. In addition, long-term genetic improvement can also be achieved if animals with low amino acid requirements, or more efficient utilization of dietary amino acids, could be selected and subsequently bred. The obvious potential for such a technique to be exploited by various animal production groups is enormous. 2.5. 24 hour oxidation/indicator amino acid balance The balance technique is based on the principle that in non-growing adults, protein synthesis is balanced with protein breakdown and thus protein or nitrogen intake is balanced with nitrogen excretion. These two balances are connected through the free amino acid pool which is relatively tightly regulated and represents a minute proportion of total body nitrogen. This relationship between the two balances is often expressed by the steady-state flux equation proposed by Waterlow et al. (1978): flux (Q) = synthesis (S) + oxidation/excretion (O) = breakdown (B) + input (I). This equation is rearranged so that protein balance (S − B) = input/output balance (I − O). This latter equation is the basis of the nitrogen balance calculation technique as well as the amino acid balance technique. A recent adaptation of the oxidation techniques incorporates the balance concept described by Waterlow et al. (1978). Young and colleagues have developed a new method involving a 24 h infusion of amino acid tracer and the measurement of labelled carbon dioxide output in adult humans (el-Khoury et al., 1994a,b, 1995). These data are then used to calculate carbon balance at different levels of test amino acid intake. The requirement is taken as the minimal amino acid intake necessary to maintain balance. This amino acid balance is the difference between the intake of the test amino acid and whole-body oxidation of that amino acid. This approach was first employed to determine the leucine requirement in adult humans which compared very well with their previous direct oxidation experiments (Young et al., 1989). Subsequently, the technique was successfully used to verify direct oxidation determinations of aromatic amino acid (Basile-Filho et al., 1997, 1998; Sanchez et al., 1995, 1996) and lysine (el-Khoury et al., 1998, 2000) requirements. These experiments also demonstrated that when the test amino acids were fed at the FAO/WHO/UNU (1985) requirement levels, subjects were in significantly negative amino acid balance, indicating that the present accepted requirements are too low (Young and Borgonha, 2000). This approach was advanced by the same group by applying the IAAO technique. Using 24 h labelled leucine infusions, lysine (Kurpad et al., 2001, 2002b) and threonine (Kurpad et al., 2002a) requirements were determined by measuring leucine oxidation and balance over a range of test amino acid intakes. These new techniques are particularly suited for non-growing adults and account for amino acid metabolism in both the fasting and fed states. However, fasting-state amino acid kinetics are not relevant to young suckling animals (Bertolo et al., 2000a). Furthermore, this approach has only been used in humans and may not be widely applicable to fast-growing meat-producing animals that are in continuous positive nitrogen balance due to high rates of lean tissue deposition. As with the nitrogen balance calculation technique, the most important criticism of the amino acid balance techniques is the reliance on absolute calculations based on various assumptions. With amino acid kinetic calculations, the most important assumption is that the precursor amino acid enrichment in readily accessible body pools (i.e. plasma) is representative of the enrichment of the true intracellular precursors for protein synthesis (i.e. tRNA)

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and oxidation; this assumption is almost always untrue in constant infusion experiments (Wolfe, 1992). Intracellular exchange of labelled amino acids is incomplete and variable among tissues. Indeed, plasma free amino acid enrichments have been found to be 2 to 3 times greater than those for corresponding amino acyl-tRNA enrichments for leucine, lysine and phenylalanine (Caso et al., 2001). Because α-ketoisocaproate (KIC) is synthesized from intracellular leucine only and is released easily into plasma, KIC enrichment has often been proposed as a suitable representation of intracellular leucine enrichments. However, several studies have shown that KIC enrichment is not in equilibrium with the entire intracellular leucine pool (Cobelli et al., 1991; Chinkes et al., 1993) or leucine-tRNA in pigs (Baumann et al., 1994) or rats (Watt et al., 1991). Alternatively, others have used enrichments of amino acids that have been incorporated into apolipoprotein B-100 (Reeds et al., 1992; Cayol et al., 1996; Stoll et al., 1999), fibrinogen (Bennet and Haymond, 1991; Stoll et al., 1999) or albumin (Cayol et al., 1996). These very rapidly synthesized plasma proteins of hepatic origin can reflect isotopic steady state within hours. These enrichments are much lower than KIC enrichments but similar to tRNA measurements. Another issue is the possibility that precursors for oxidation may not equilibrate with tRNA pools, either intracellularly or between tissues, so that different enrichments may need to be measured to accurately calculate balance. The amino acid balance techniques will need to address this precursor enrichments issue. An advantage of the “relative” techniques comparing biological outcomes across dietary intakes is the avoidance of absolute assumptions. Indeed, in direct oxidation and IAAO analysis of amino acid requirements, the most reliable estimate of requirement with the lowest error is when percent dose oxidized is used as the biological outcome (House et al., 1997a,b, 1998a; Bertolo et al., 1998; Lazaris-Brunner et al., 1998; Bross et al., 2000; Roberts et al., 2001a,b). This outcome is in contrast to equivalent measurements of oxidation rate, which employ flux calculations that also use assumptions about the precursor pool. Ultimately, the “black box” approach of total labelled carbons in and total labelled carbons out provides the most reliable requirement estimates. Despite these methodological comparisons, it is important to reiterate that the amino acid balance studies to date have calculated amino acid requirements that are very close to those determined by the more qualitative direct oxidation and IAAO techniques. Therefore, the error associated with these kinetic assumptions and calculations may not be as significant as some have proposed.

3. RECENT DEVELOPMENTS IN THE INDICATOR AMINO ACID OXIDATION TECHNIQUE Considering that all of the techniques used to determine amino acid requirements generally agree in their final estimate, one must choose the most versatile method available for wider application. Given the short adaptation time, applicability to all indispensable amino acids, ease of biological sampling (i.e. breath) and applicability to most populations, the IAAO technique has been successfully adapted for use in a variety of situations and purposes. An important advance in the method was its adaptation for use in parenterally fed piglets as models for parenterally fed infants (Wykes et al., 1993; Bertolo et al., 1998; House et al., 1998a). The original work by Bayley in piglets employed one or two oral bolus feeds which included the isotope dose; the total labelled carbon dioxide excretion was not kinetically quantified, but rather relatively compared over different test amino acid intakes. With parenterally fed piglets, primed constant intravenous infusions of labelled indicator amino acid were introduced to acquire added information through kinetic calculations. With these studies, it was shown that the indicator (phenylalanine) flux rate was unchanging across test amino

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acid intakes (Bertolo et al., 1998; House et al., 1998a). In addition, it was demonstrated that, statistically, percent dose oxidized was the most reliable estimate in terms of variability (House et al., 1997a,b, 1998a; Bertolo et al., 1998); this finding has been confirmed in human studies (Lazaris-Brunner et al., 1998; Bross et al., 2000; Roberts et al., 2001a,b). Recently, we have also directly compared amino acid requirements using either intravenous or oral/gastric infusion of labelled phenylalanine as the indicator amino acid. The breakpoint estimates of the lysine requirement in adult humans were the same whether the indicator was delivered intravenously or orally (Kriengsinyos et al., 2002); similarly, the tryptophan requirements in gastrically fed piglets were similar with intravenous or intragastric infusion of the indicator (Cvitkovic et al., 2000). These subsequent methodological developments were critical in adapting the IAAO technique for vulnerable populations. In order to use the IAAO technique in infants and children, the dietary interventions must be short and the sample collections must be non-invasive. We have recently validated the use of oral isotope dosing and collection of urinary amino acids to measure enrichment as representative of plasma enrichment (Bross et al., 1998). Study diets were fed hourly for 4 h prior to dosing and subsequent half-hourly meals with isotope led to enrichment plateaux within 2 h. This simple non-invasive protocol can be used to study many different vulnerable populations, provided that dietary ingestion and breath collection are feasible. Similarly, this oral dosing protocol has been shown to be very effective in determining amino acid requirements in large pigs where the implantation and maintenance of catheters can be problematic (unpublished data). In addition, such a simplified protocol allows for a broader application of the technique to other experimental models. Such models include neonatal and adult animals, gestating or lactating animals, as well as compromised populations which include disease or surgical interventions. The IAAO technique can also be applied to research investigating other aspects of protein metabolism where the goal is not simply to determine amino acid requirements. Because indicator amino acid oxidation responds to protein synthesis, intracellular changes in test amino acid availability are reflected in changes in indicator oxidation. Recently we have exploited this principle by adapting the technique to determine true metabolic availability of lysine from feedstuffs in growing pigs (Ball et al., 2001). We designed a low-lysine diet with all other indispensable amino acids above requirements. With incremental additions of synthetic lysine, which is assumed to be 100% available, phenylalanine oxidation declined linearly until the requirement was met. Within this deficient range of lysine intakes, we used the linear response equation to predict true availability of lysine from added feedstuffs (fig. 2). When peas were added to the low (50% of requirement) lysine diet so that the total true available lysine content was 90% of lysine requirement, the phenylalanine oxidation corresponded to the availability predicted by ileal digestibility estimates (i.e. the true available amount according to NRC, 1998). When peas were heated to render some lysine unavailable via Maillard products, the phenylalanine oxidation increased and corresponded to an availability of 50% of unheated peas. When synthetic lysine was added to the heated peas, phenylalanine oxidation decreased below that determined with 90% of requirement, demonstrating that the increase in oxidation due to heating was due solely to loss of available lysine. This technique is a new, rapid approach to determining true metabolic availability of lysine that does not require a series of tenuous assumptions about endogenous losses, which are problematic to the true ileal digestibility method. This novel application of IAAO principles to improve estimates of amino acid availability has demonstrated the technique’s versatility. We have also used the basic qualitative principle of the IAAO technique to determine whether amino acid formulations are inadequate. Recently, by adding amino acids suspected

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Fig. 2. Representation of novel technique to determine metabolic amino acid availability by measuring indicator amino acid oxidation.

of being deficient to TPN solutions and monitoring lysine (the indicator) oxidation, we have systematically demonstrated that the amino acid profile of some commercial TPN solutions are inadequate (unpublished data). This approach can be further adapted to simulate the strategy pioneered by Baker and colleagues to determine amino acid requirements by dietary amino acid supplementation and deletion by employing the ideal ratios to lysine (Mavromichalis et al., 1998). The important advantage of the IAAO approach is that multiple modifications can be made to the diets of individual animals because short oxidation measurements are used, as opposed to the more time-consuming growth assays. Because of the relatively simple and direct biological response in the IAAO technique, many more extrapolations and adaptations of the original technique will probably be developed in the future.

4. INTESTINAL FIRST-PASS METABOLISM In addition to being responsible for the digestion and absorption of nutrients, the gut is also a major metabolic organ in the body, responsible for the synthesis, conversion and degradation of amino acids. The gut has a very high metabolic activity and extracts a significant proportion of the absorbed dietary and endogenous amino acids before transport to the portal circulation and the rest of the body. Indeed, although the portal-drained viscera (PDV) (intestines, pancreas, spleen, stomach) represents only ~5−7% of body mass, these tissues disproportionately account for 20–35% of whole-body energy expenditure and protein synthesis (Lobley et al., 1980; McNurlan and Garlick, 1980; Burrin et al., 1990). This significant extraction of dietary amino acids by splanchnic tissues has been demonstrated by comparing amino acid kinetics when isotopes are delivered intravenously or orally. The “splanchnic disappearance” of labelled amino acids when given orally has led many researchers to speculate on the fate of this irreversible loss of label in kinetics experiments. From these types of studies, several researchers have determined that in humans and animals, the splanchnic tissues metabolize between 20% and 50% of dietary essential amino acids (leucine, lysine, phenylalanine) on first-pass (Yu et al., 1990, 1995; Hoerr et al., 1991, 1993; Biolo et al., 1992; Matthews et al., 1993; van Goudoever et al., 2000). In addition, with low-protein diets, this extraction is as high as 70% of lysine intake (van Goudoever et al., 2000). Albeit the data are very impressive, one problem with this approach is that it is difficult to separate the metabolism of the liver from the PDV, although this can be overcome by measuring amino acid

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enrichments and flow in the portal vein (van Goudoever et al., 2000). An additional problem with the technique is that these types of studies vastly underestimate arterial extraction of recirculating enteral isotope and hence overestimate first-pass extraction. Indeed, the elegant study by van Goudoever et al. (2000) demonstrated that a 46% portal mass balance extraction corresponded to a 22% isotopic extraction by the PDV, which when corrected for arterial recirculation amounted to insignificant utilization of dietary lysine on first-pass. The relative importance of nutrient processing by the small intestine (the predominant PDV organ) versus the liver has only recently been elucidated. Indeed, we have demonstrated that the small intestine is more important than the liver in modifying nitrogen utilization when pigs are chronically fed by central vein, portal vein or stomach (Bertolo et al., 1999). In addition, other researchers have demonstrated that intestinal metabolism dominates splanchnic metabolism of phenylalanine (Stoll et al., 1997) and lysine (van Goudoever et al., 2000) in pigs and leucine in dogs (Yu et al., 1990). In addition to dietary extraction of amino acids, the gut also transports an enormous amount of amino acids from the arterial circulation, especially during the post-absorptive state. Isotopic data describing the incorporation of amino acids from both arterial and dietary sources have demonstrated that both sources of precursor amino acids are critical and that the partitioning between them is dependent on specific amino acid and dietary protein level (MacRae et al., 1997; Stoll et al., 1999; van Goudoever et al., 2000). Recent work by van Goudoever et al. (2000) has determined that with high-protein feeding, almost all of the lysine utilized by the PDV was of arterial origin, whereas with low-protein feeding, approximately half of the lysine utilized was from both arterial and dietary sources. It is important to note that level of protein feeding did not affect total lysine use by the PDV, demonstrating an enormous obligatory utilization of amino acids by the gut for normal function and growth. Because of this high obligatory protein turnover in the gut, it follows that with gut challenges (i.e. tissue damage, increased growth, pathogen exposure, dietary anti-nutritional factors, etc.) the amino acid requirements of the gut increase. Indeed, first-pass intestinal extraction of amino acids is proportional to mucosal mass. Stoll et al. (1997, 1999) demonstrated that phenylalanine splanchnic extraction was 50% higher in pigs raised outdoors, where rooting and pathogen challenges are greater, compared to pigs raised in a relatively clean indoor research facility; this higher extraction correlated with measured mucosal mass. Infestation of pathogens is known to affect growth rate as well as gut function. The stimulation of whole-body and gut immune systems must impart a protein synthetic cost to the infected animal. Indeed, sepsis in rats has been shown to stimulate intestinal protein synthesis (von Allmen et al., 1992; Higashiguchi et al., 1994b); in particular, sepsis stimulates the synthesis of endogenous and secretory proteins, including certain gut peptides, in small intestine mucosa (Higashiguchi et al., 1994a). More recently, Yu et al. (2000) have shown that subclinical nematode infection in sheep increased total gastrointestinal tract leucine sequestration by 24% and gastrointestinal tract oxidative losses of leucine by 22–41%. In another study in pigs, the infusion of endotoxin led to enhanced intestinal catabolism of amino acids (Bruins et al., 2000). These intriguing studies suggest a possible mechanism for the growth-stimulating effect of feed-grade antibiotics; increased protein synthesis during infection by the already metabolically demanding intestinal tissues limits the availability of amino acids for extraintestinal lean tissue growth. With the impending discontinuation of prophylactic feeding of antibiotics in animal production, the prevention of subclinical infections or challenges needs to be a priority in the development of alternative strategies in the near future.

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The primary fate of indispensable amino acids is presumably to protein synthesis; however, recent intriguing data have demonstrated that catabolism dominates the first-pass utilization of these amino acids by the gut (Stoll et al., 1998; Wu, 1998; van Goudoever et al., 2000). This seemingly wasteful oxidation of indispensable amino acids amounts to a small but significant proportion of dietary intake (Yu et al., 1992, 2000; Cappelli et al., 1997; van Goudoever et al., 2000), but a large proportion of whole-body amino acid oxidation (van Goudoever et al., 2000). Indeed, we have recently demonstrated that phenylalanine oxidation is significantly greater when labelled phenylalanine is delivered orally, as opposed to intravenously in the IAAO technique (Cvitkovic et al., 2000; Kriengsinyos et al., 2002). This increased oxidation during feeding of adequate diets demonstrates that first-pass catabolism of phenylalanine by the gut is significant. Whatever the fate of indispensable amino acids extracted by the gut, the evidence clearly suggests that this tissue plays a significant role in modulating the profile of amino acids delivered to the rest of the body (Stoll et al., 1998; Wu, 1998; Bertolo et al., 2000b). Much of this role results in a net loss of amino acids to extraintestinal tissues. Presumably, when gut metabolic activity is increased by growth or stress, so is the loss of dietary amino acids from whole-body functions. This aspect of whole-body amino acid requirements has not been fully explored. In addition to gut metabolic activity, the quality of dietary protein (Deutz et al., 1998; Gaudichon et al., 1999; Mariotti et al., 1999) and type of dietary carbohydrate (van der Meulen et al., 1997) also influence first-pass extraction of amino acids. So the actual availability of dietary amino acids for muscular protein synthesis is highly dependent on the metabolic activity of intestinal tissues. It is important to note that this concept is accommodated by our recent adaptation of the IAAO technique mentioned above for determination of true metabolic availability of dietary amino acids from heat-treated feedstuffs. Given the significant demand of the gut for dietary and arterial amino acids, it is obvious that the maintenance and growth of this organ already constitutes a significant proportion of whole-body amino acid requirements. Furthermore, it follows that in certain situations that increase the metabolic activity of the gut, this proportion will increase at the expense of whole-body growth. Indeed, this hypothesis is supported by the abovementioned studies in subclinical nematode infection and the reduced growth rate commonly observed in gastrointestinal disease. If the availability of an indispensable amino acid is already limiting in an animal’s diet, then an unobservable subclinical challenge to the gut could feasibly limit animal growth further. Although this concept seems intuitive, it is very difficult to demonstrate experimentally.

5. IMPACT OF INTESTINAL METABOLISM ON AMINO ACID REQUIREMENTS Many of the approaches recently employed to demonstrate changes in amino acid utilization by the gut or PDV tissues could be adapted to quantify amino acid requirements of these tissues relative to whole-body requirements. It has been well demonstrated that the proportion of dietary amino acids extracted by the gut changes with dietary composition; but a systematic approach to dietary ingredient requirements for the gut has yet to be published. By employing graded intakes of indispensable amino acids, it is possible to determine the minimum amount required by the gut for normal function and growth. To date, our body of literature regarding amino acid requirements during intravenous or intragastric feeding in piglets provides the only attempt, to the authors’ knowledge, to quantify requirements with and without first-pass splanchnic metabolism.

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5.1. The parenterally fed piglet as a model of splanchnic metabolism The emerging evidence that the gut utilizes a significant proportion of dietary amino acids led us to speculate that in situations of gut bypass or stress, the whole-body amino acid requirements must be different. We proposed that total parenteral nutrition (TPN), which bypasses first-pass metabolism by the small intestine and liver and can cause gut atrophy (Johnson et al., 1975; Goldstein et al., 1985; Alverdy, 1995; Bertolo et al., 1999; Burrin et al., 2000), would lead to changes in amino acid requirements that can be measured. Indeed, we (Duffy and Pencharz, 1986; Bertolo et al., 1999) and others (Sim et al., 1979; Lanza-Jacoby et al., 1982; Jeevanandam et al., 1987) have shown that TPN feeding alters whole-body nitrogen metabolism compared to oral feeding. The different nitrogen utilization as a consequence of parenteral feeding may be due to reduced gastrointestinal metabolism associated with gut atrophy and/or due to lack of hepatic first-pass metabolism. With the development of the TPNfed piglet model (Wykes et al., 1993), we subsequently demonstrated using three infusion routes that intestinal atrophy has a greater impact on nitrogen metabolism than liver bypass (Bertolo et al., 1999). Whatever the fate of indispensable amino acids in the gut, an atrophied gut will utilize fewer amino acids and thereby affect whole-body requirements. Therefore, we have proposed that the TPN-fed piglet is a “gut-deficient” model. So the amino acid requirements of TPN-fed piglets approximate the requirements for extraintestinal tissues. When compared to a piglet gastrically fed identical diets, the amino acid requirements for the intact gut could at least be estimated (table 1). However, TPN feeding is only one of many relevant clinical scenarios that lead to compromised gut metabolic capacity. Gut dysfunction can also be caused by malnutrition, diarrhoea, chemotherapy, gastrointestinal surgery and gastrointestinal diseases. Furthermore, with relevance to the animal industry, gut stress is often associated with weaning, especially

Table 1 Effects of gut atrophy and bypass on whole-body amino acid requirements as determined in parenterally and enterally fed piglets using the indicator amino acid oxidation technique

Amino acid Threonineb Methioninec Total sulphursc Branched-chaind Tryptophane Lysinef Phenylalanineg Tyrosineh a

Oral requirement (g/kg/d)

Parenteral requirement (g/kg/d)

0.42 0.25 0.42 2.64 0.13 and 0.11 0.85 0.50 0.30

0.19 0.18 0.29 1.53 0.14 0.79 0.45 0.35

Gut bypass effecta (%) 55 28 31 42 0 7 10 0

This effect includes atrophy from 7 d on TPN as well as bypass of first-pass gut metabolism. Bertolo et al. (1998). c Shoveller et al. (2001). Methionine requirement was determined with excess dietary cysteine; total sulphurs refer to the methionine requirement determined with no dietary cysteine. d Elango et al. (2002a). Leucine, isoleucine and valine dietary ratio (NRC, 1998) was maintained across intakes. e Cvitkovic et al. (2000). Tryptophan requirement using intravenous (0.13) or oral isotope (0.11). f House et al. (1998a). Oral requirement for lysine was estimated from NRC (1998). g House et al. (1997b). Parenteral phenylalanine requirement was determined using direct oxidation technique; oral phenylalanine requirement was estimated from NRC (1998). h House et al. (1997a). Oral tyrosine requirement was estimated from NRC (1998). b

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with the increasingly popular early weaning practice in swine production. Any clinical situation which diminishes gut capacity would affect the metabolism of many amino acids; for example, increased amino acid requirements discovered during TPN feeding should also be applied to the treatment of any of the above conditions. Thus, our research has relevance to many conditions in addition to the TPN-fed neonate. However, we chose to use the TPN-fed piglet as a model for diminished small intestinal capacity because of demonstrated gut atrophy, reproducibility and ease of development compared to gut disease models. With this clinically relevant model, we could extrapolate our results to any situation involving gut dysfunction. 5.2. Threonine Perhaps the most impressive effect of reduced gut metabolism on whole-body amino acid requirement was demonstrated for threonine. Using the TPN-fed versus gastrically fed piglet, we have demonstrated that the threonine requirement is reduced by 55% when the gut is bypassed and atrophied (Bertolo et al., 1998). These data are supported by Stoll et al. (1998), who also observed that the PDV tissues extract 60% of dietary threonine measured by both net portal balance and labelled threonine extraction. In addition, van Goudoever et al. (2000) showed that when pigs were fed the high-protein diet, 84% of threonine was retained by the gut, and on the low-protein diet, all of the threonine was retained. This enormous demand for threonine by the gut is probably reflected by its role in mucin synthesis for maintenance of the luminal mucus layer (Lamont, 1992). Intestinal mucins are continuously secreted by the intestines and are critical in the defence of the mucosa from mechanical and pathogenic insults. The core protein of mucins contains a disproportionate amount of threonine, proline and cysteine (Specian and Oliver, 1991). With parenteral feeding and gut atrophy, mucin synthesis is reduced and so is the gut’s requirement for threonine. Recently we have demonstrated that feeding threonine-deficient diets to gastrically fed piglets reduces gut growth and goblet cell numbers and alters the mucin profile of intestinal mucus; in addition, parenteral threonine cannot completely restore normal gut function and histology compared to enteral threonine (Ball et al., 1999; table 2). This profound impact of gut metabolism on whole-body threonine requirement must also be considered as a minimum effect. Because the parenteral threonine requirement (0.19 g/kg/d) was so much lower than NRC (1998) recommendations (0.53 g/kg/d on a true ileal digestible basis), we introduced the gastrically infused control group which received identical diets to verify NRC estimates for piglets. The requirement for these control pigs (0.42 g/kg/d) was lower than that recommended by NRC, but still substantially higher than the parenteral requirement. This discrepancy with NRC values is not surprising given that the requirements recommended by NRC (1998) include digestibility estimates for corn–soybean diets and adjust these values for endogenous loss estimates, whereas our diet was completely elemental and available. In subsequent IAAO experiments, we have demonstrated that these NRC estimates are proportionately closer to our gastrically fed estimates for tryptophan (0.15 vs 0.15 g/kg/d for NRC), methionine (0.25 vs 0.23 g/kg/d for NRC) and methionine plus cysteine (0.42 vs 0.48 g/kg/d for NRC). The discrepancy in threonine requirements between that recommended by NRC and our oral estimates could be due to the increased sensitivity of the threonine requirement to endogenous losses. Because the mucin protein core is resistant to digestion and is almost completely recovered in ileal digesta (Mantle and Allen, 1981), a major component of endogenous losses at the ileum is mucins which are rich in threonine, proline, serine and cysteine (Specian and Oliver, 1991). Mucin secretion and hence losses are known

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Table 2 Goblet cell parameters in piglets fed adequate oral threonine (IG-A), deficient oral threonine (IG-D) or deficient oral threonine with adequate parenteral threonine (IV-A)a PAS/AB 2.5b Cellsc Duodenum

Ileum

IG-A IG-D IV-A SDpooled IG-A IG-D IV-A SDpooled

18.8a 15.2b 14.9b 2.7 20.9 22.6 27.5 5.9

Staind 39 41 39 13 79 68 82 18

AB 2.5b

AB 1.0b

Cells

Stain

Cells

Stain

17.6a 5.5c 12.6b 4.4 30.1a 13.9b 19.9a,b 7.8

44a 6b 25a,b 12 63a 14b 31a,b 18

16.3a 7.1b 13.5a,b 4.4 24.6a 13.3b 20.7a,b 6.5

27 12 31 15 30 13 31 11

Ball et al. (1999). Data are means for n = 7 piglets. For data with letter superscripts within a row, those not sharing a letter are different (P < 0.05, LSD comparisons). b PAS/AB 2.5: staining is combination of Alcian blue/periodic acid (5 min)-Schiff base (15 min) (PAS) reaction allowing unsubstituted α-glycol-rich neutral mucins (pink) and acidic mucins (blue) to be differentiated; AB 2.5: 1% Alcian blue (AB, pH 2.5, 1 h) for the localization of carboxylated and/or sulphated acidic mucins; AB 1.0: 1% Alcian blue (AB, pH 1.0, 1 h) for the selective identification of sulphomucins. c Goblet cells in the mucosa stained with PAS/AB 2.5, AB 2.5 or AB 1.0 were counted in 10 well-oriented crypt-villus units ~25 μm in each animal. d Semi-quantitative staining intensities based upon a scale ranging from 0 (unreactive) to 3 (intensely stained) were multiplied by total number of goblet cells. a

to be sensitive to dietary composition as well as presence of fibre and anti-nutritional factors (More et al., 1987; Sharma and Schumacher, 1995). Indeed, in a very recent experiment, we have demonstrated that dietary supplementation of wheat bran, a stimulant of mucin synthesis, leads to increased ileal losses of threonine which may affect whole-body availability of dietary threonine (Myrie et al., 2002). Therefore, this is probably the reason why the threonine requirement of pigs fed a fibre-free elemental nutrition solution (i.e. our gastrically fed control pigs) was lower than the estimated requirement of pigs fed a corn–soybean meal diet (i.e. NRC recommendations). Indeed, as a percentage of the NRC recommendation, the threonine requirement in parenterally fed pigs was 36% (instead of 45%), which translates to a gut utilization of 64% of dietary threonine. Therefore, the requirement for threonine by the gut versus the whole body depends on dietary composition. 5.3. Sulphur amino acids Although methionine is an indispensable amino acid, cysteine is not because it can be synthesized from methionine. However, increased metabolism of methionine to meet cysteine needs could limit methionine availability for protein synthesis and growth. As a result, dietary cysteine has a “sparing effect” on the amount of methionine required. In a series of experiments, we have recently determined the methionine requirements of piglets fed orally and intravenously, with and without dietary cysteine, using the IAAO technique (Shoveller et al., 2001; table 1). With excess or without dietary cysteine, the methionine requirements in parenterally fed piglets were 72% or 69% of the respective requirements in orally fed piglets. In other words, approximately 30% of dietary methionine is utilized by the gut whether dietary cysteine is present or not. These data are supported by those of Stoll et al. (1998),

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who showed that approximately 30% of dietary methionine disappears in first-pass metabolism by the PDV. Furthermore, the sparing effect of cysteine was similar in pigs fed either intravenously or orally (i.e. excess cysteine reduced the respective methionine requirements by 40% regardless of feeding route) (Shoveller et al., 2001). In addition to demands for protein turnover, this relatively high demand by the gut for both sulphur amino acids can also be attributed to other metabolic functions specific to methionine and cysteine. It is possible that the high nucleic acid turnover of intestinal cells requires a significant amount of methionine, an important methyl donor. Furthermore, cysteine, as a product of methionine metabolism, is incorporated to a large extent into intestinal mucins and the tripeptide antioxidant glutathione; both of these products are critical for the maintenance of the mucosal tissue and protection against pathogens (Martensson et al., 1990). Indeed, the impact of a pathogenic challenge on the methionine requirement may therefore be significant, but has yet to be studied. 5.4. Branched-chain amino acids The aforementioned data regarding substantial first-pass splanchnic metabolism of leucine suggest that the splanchnic tissues extract a surprisingly large amount (i.e. 20−50%) of branched-chain amino acids (BCAA). Recently, using a diet with a fixed ratio of BCAA (1:1.8:1.2, isoleucine:leucine:valine), we have determined that the BCAA requirement in intravenously fed piglets was 56% of that in intragastrically fed piglets (Elango et al., 2002a). The apparent uptake of 44% of enterally fed BCAA by the splanchnic tissues is a significant finding because it is generally accepted that the BCAA are predominantly metabolized by the extrahepatic tissues due to the higher activity of branched-chain aminotransferase (BCAT), the first enzyme in the catabolic pathway of the BCAA, in skeletal muscle compared to the liver. In addition, the pattern of BCAA in the plasma of enterally fed piglets, when compared with parenterally fed piglets, clearly demonstrates that the gut has a high demand for leucine and a clear preference for leucine compared to isoleucine or valine. The observation, during enteral feeding, that plasma valine and isoleucine concentrations increased while leucine concentrations remained low indicates that leucine is being extracted by the gut and therefore may be limiting protein synthesis in the rest of the body. Valine and isoleucine do not appear to be utilized by the gut to the same extent and are therefore being passed to the systemic circulation, but because protein synthesis is limited by leucine, these two amino acids, as well as most of the other indispensable amino acids, increase in concentration in the plasma. The difference in BCAA requirements between routes of feeding is supported by the data in humans (Gelfand et al., 1988; Hoerr et al., 1991, 1993; Biolo et al., 1992; Matthews et al., 1993) and dogs (Yu et al., 1990, 1995), regarding first-pass splanchnic extraction of leucine measured by isotope infusions. In addition, Stoll et al. (1998) reported that the pig PDV extracted 43% of leucine, 39% of valine and 31% of isoleucine. Altogether, the first-pass extraction data compare well with the 44% lower BCAA requirement in parenterally fed piglets observed in our recent study. In a subsequent study, we adapted the IAAO technique to systematically determine which of the BCAA was most limiting (Elango et al., 2002b). In both orally and intravenously fed piglets, diets moderately deficient in BCAA (75% of respective requirement) were fed and indicator amino acid oxidation determined. Piglets were then randomly assigned to receive one of three test diets containing either isoleucine, leucine or valine to meet 100% of requirement, with the remaining two amino acids at 75%.

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Fig. 3. The change in phenylalanine oxidation in parenterally (IV) or enterally (IG) fed pigs after supplementation of individual amino acids (Elango et al., 2002b). Diets were formulated to be slightly deficient in all branched-chain amino acids and indicator oxidation was performed before and after isoleucine, leucine or valine supplementation. * indicates oxidation change was different than zero.

The difference in phenylalanine oxidation between unsupplemented and supplemented diets was used as an indicator of BCAA adequacy (fig. 3). In orally fed piglets, the difference in percent dose oxidized was not significant for any supplemented amino acid. However, in parenterally fed piglets, isoleucine and valine supplementation decreased phenylalanine oxidation; isoleucine had the greatest effect and was first limiting (i.e. oxidation decreased from approximately 22% to 10%) and valine was second limiting (22% to 15%). Leucine, which is the preferred amino acid by the gut according to our previous data, had no effect when supplemented to gut-atrophied, parenterally fed pigs. The optimal ratio of BCAA for orally fed pigs is adequately predicted by requirement estimates (NRC, 1998). However, because the gut does not utilize the BCAA in this proportion, the ideal ratio of BCAA for maintenance of the gut is not the same as that for the whole body and has not yet been determined. 5.5. Tryptophan and lysine The tryptophan requirements of parenterally and orally fed piglets were not different when identical diets were employed (Cvitkovic et al., 2000). This result suggests that the gut’s requirement of tryptophan for protein synthesis and/or for oxidation does not significantly impact whole-body requirements, possibly due to either efficient recycling by an atrophied gut or to this amino acid’s low proportion in protein. We have also determined the lysine requirement of parenterally fed piglets but did not employ a gastrically fed control group (House et al., 1998a). Because this was the first amino acid for which we determined the requirement during TPN feeding, we did not appreciate the large impact of the gut. Because NRC estimates were proportionately similar to gastrically fed control pigs for other amino acids, we therefore compared the parenteral lysine requirement of 0.79 g/kg/d to the NRC (1998) estimate of 0.85 g/kg/d, providing a 7% difference due to gut metabolism. It is important to note that this comparison may not be valid given the lack of empirical data for amino acid requirements in young piglets (NRC, 1998). The net lysine utilization by the gut may still be significant as in previous studies regarding the

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substantial splanchnic extraction of lysine (Hoerr et al., 1993; van Goudoever et al., 2000); however, it appears that in piglets, this utilization is not nearly as profound as that for threonine, the sulphur amino acids or the branched-chain amino acids. The impact of parenteral feeding (i.e. with gut atrophy and lack of first-pass metabolism) on whole-body lysine requirements is probably accounted for by the reduced general protein turnover in the atrophied gut; in other words, there seems to be no disproportionate requirement for lysine by the gut versus the whole body. 5.6. Phenylalanine and tyrosine By employing the direct oxidation approach, we found only moderate differences in the phenylalanine (House et al., 1997b) and tyrosine (House et al., 1997a) requirements of parenterally fed piglets compared to the NRC (1998) estimates of oral requirements. The parenteral tyrosine requirement was estimated at 0.35 g/kg/d; in addition, the parenteral phenylalanine requirement (with excess dietary tyrosine) was only 10% lower than NRC estimates (0.45 vs 0.50 g/kg/d). Combined, the phenylalanine plus tyrosine requirement of parenterally fed piglets (0.80 g/kg/d) was equal to that estimated by NRC (0.80 g/kg/d). Furthermore, we have recently been able to compare these results in piglets to the parenteral tyrosine requirement in parenterally fed human infants (Roberts et al., 2001a); the tyrosine requirement determined for parenterally fed infants was similar to the broad range recommended for orally fed neonates (Snyderman, 1971). Again, this minor effect of gut metabolism on whole-body requirements is in contrast to dual-isotope infusion studies (Biolo et al., 1992; Matthews et al., 1993) where 29–58% of dietary phenylalanine was extracted by splanchnic tissues in adult humans; however, arterial recirculation (van Goudoever et al., 2000) was not estimated in these studies. In addition, we have shown that when the indicator amino acid is delivered orally or intravenously, basal phenylalanine oxidation is significantly increased when first-pass splanchnic metabolism is maintained in adults (Kriengsinyos et al., 2002) or piglets (Cvitkovic et al., 2000). However, an increase in phenylalanine oxidation with first-pass metabolism by the gut does not necessarily translate to a substantial extraction of dietary phenylalanine, as demonstrated with lysine by van Goudoever et al. (2000). Indeed, although phenylalanine oxidation increased by 70% when infused orally versus intravenously in humans, the percent extraction determined by flux rates was only increased by 30% (Kriengsinyos et al., 2002). Furthermore, phenylalanine oxidation amounted to less than 15% of intake during either route of infusion. In piglets, when the indicator phenylalanine was fed at the requirement, oxidation rates were increased from 0.6% to 0.8% of dose, which translated to 0.9% and 1.5% of phenylalanine intake or requirement (Cvitkovic et al., 2000). These latter data also provide a reason why phenylalanine is a good choice for the indicator amino acid. Even if there are large differences between diets or individuals in the level of phenylalanine oxidation, the total amount oxidized is still rather insignificant relative to the quantity used for protein synthesis which is driving the whole-body requirement for the amino acid. As with lysine and tryptophan, it appears that dietary phenylalanine is primarily utilized for non-specific protein synthesis, which does not appear to disproportionately impact wholebody requirements. It has been suggested that the gut may hydroxylate a substantial amount of phenylalanine to tyrosine (Stoll et al., 1998); however, the impact of this capacity on whole-body hydroxylation or requirements has yet to be explored. Estimates of phenylalanine hydroxylation to determine tyrosine requirements have been shown to be unreliable (House et al., 1998b; Thorpe et al., 2000; Roberts et al., 2001a).

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5.7. Arginine and proline Although for most species studied arginine is considered dispensable, arginine has been found to be an indispensable amino acid in some species such as cats (Morris and Rogers, 1978), chicks (Tamir and Ratner, 1963) and ferrets (Deshmukh and Shope, 1983). Furthermore, arginine may be conditionally indispensable in young mammals including the dog (Visek, 1984), rat (Borman et al., 1946) and piglet (Mertz et al., 1952; Ball et al., 1986; Brunton et al., 1999), which means arginine can be synthesized de novo, but not at sufficient rates to maintain required functions (i.e. syntheses of protein, urea cycle intermediates, creatine, nitric oxide, etc.). The neonatal small intestine has been suggested to be the major site of arginine synthesis (Wu et al., 1994; Stoll et al., 1998) and the ontogeny of the necessary enzymes in enterocytes has been well described (Wu, 1998). We initially planned to use the IAAO technique to determine the arginine requirement of piglets during parenteral feeding. An initial pilot experiment was conducted whereby parenterally fed piglets were fed arginine-free diets so that growth and nitrogen balance data could be assessed. Both pigs experienced severe hyperammonemia after only ~16 h without dietary arginine; one pig died and the other was comatose. Because plasma ammonia concentration was found to be a sensitive indicator of arginine deficiency, we used this biological outcome to determine if the arginine synthesis rate of the piglet gut was sufficient to maintain the urea cycle and whether proline, the primary precursor of arginine synthesis in the gut, must be available to maintain synthesis rates. The subsequent study successfully demonstrated that parenterally fed piglets could not synthesize sufficient arginine to maintain the urea cycle, let alone to maintain growth, whether or not proline was present in the diet. This study also demonstrated that orally fed piglets could not synthesize arginine and proline at rates sufficient to maintain plasma concentrations or to prevent hyperammonemia. However, unlike the gut-atrophied parenterally fed piglets, the gut-intact orally fed piglets experienced less severe hyperammonemia when proline was provided in the diet. These data suggested that the conversion of proline to arginine occurs in the piglet, but only during oral feeding. We therefore hypothesized that this conversion occurs almost exclusively in the gut and that parenterally fed piglets could not use proline for arginine synthesis because of gut atrophy and/or gut bypass during feeding. This experiment convincingly demonstrated the essentiality of arginine and proline in continuously fed piglets; we predicted that with voluntary feeding, animals would refuse feed if severely deficient, especially if elevated plasma ammonia levels develop. Indeed, vomiting is a symptom of hyperammonemia in pigs, which functions to lessen the ammonia load by expelling potentially toxic amino acids. This clear demonstration of arginine indispensability in piglets was followed by a multiisotope, dual-route infusion study whereby labelled proline, ornithine and arginine were infused intragastrically or into the portal vein to isolate the in vivo effects of small intestinal first-pass metabolism. This experiment demonstrated that the conversion from proline to arginine is completely dependent on the small intestine, confirming the conclusions from our previous experiment assessing hyperammonemia. Thus, in situations where gut metabolism is bypassed or compromised, such as during TPN feeding or gut disease, arginine synthesis is diminished, increasing the overall requirement compared to normal oral feeding. In addition, this study demonstrated that proline synthesis from arginine is also dependent on gut metabolism and its requirement also would be higher when gut metabolism is bypassed. Although the cumulative evidence clearly indicates that arginine synthesis is dependent on the neonatal small intestine, the impact of this first-pass metabolism on whole-body arginine requirements can only be estimated.

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Most of these data support the hypothesis that the arginine requirement is much higher during parenteral feeding, for both maintenance and growth, due to lower synthetic capacity of an atrophied gut. Indeed, we speculate that for arginine, a separate maintenance requirement can be distinguishable from the growth requirement. Such a hypothesis has enormous implications in neonatal populations in which small intestinal first-pass metabolism is bypassed or compromised by gastrointestinal disease or stress. Indeed, these implications have been demonstrated in parenterally fed infants (Heird et al., 1972) and adult rats with small intestinal resection (Wakabayashi et al., 1995). This latter study is interesting considering that adult rats normally can synthesize adequate amounts of arginine in the kidney, but the citrulline precursor originates in the small intestine (Morris, 1992). So although net intestinal arginine synthesis declines during late suckling as renal synthesis increases (Wu, 1998), the importance of intestinal metabolism in the inter-organ synthesis of arginine is still potentially critical in adult species that normally do not require arginine. Proline has been suggested to be an indispensable amino acid for the piglet (Ball et al., 1986), but subsequent studies indicated that proline indispensability is dependent on availability of precursors such as glutamate (Murphy et al., 1996; Wu, 1998) and arginine (Brunton et al., 1999). The extensive gut metabolism of glutamate and glutamine (Windmueller and Spaeth, 1980; Stoll et al., 1999; Reeds et al., 2000) may limit arginine and proline synthesis in certain conditions. Furthermore, the importance of proline for collagen synthesis probably increases in situations of injury and stress. The obvious interdependence of arginine and proline requirement on gut health as well as availability of precursors makes the quantification of such requirements very complicated. However, there is enough evidence to date to suggest that the impact of gut first-pass metabolism on whole-body requirements must be significant and warrants future investigation.

6. FUTURE PERSPECTIVES Albeit the parenterally fed piglet model has proved useful in estimating the impact of firstpass gut metabolism on whole-body requirements, a more direct determination of gut requirements for amino acids has yet to be developed. With a more direct technique, researchers could then attempt to quantify the effects of gastrointestinal stress, injury, disease or dysfunction on whole-body requirements. In particular, the recent intriguing work on leucine extraction and nematode infection in sheep provides preliminary evidence for the importance of this type of investigation. In addition, Klasing and Calvert (1999) have provided an important advance in this area by estimating that the percent of lysine intake consumed by the chicken immune system increases from 1.2% to 6.7% with an injected immune challenge. This “cost” of an immune challenge must be even more profound with a gastrointestinal pathogenic challenge where the additional costs of gut secretion stimulation, gut tissue repair and compromised dietary absorption of lysine must be considered. Furthermore, this cost mostly relates to lysine requirements for non-specific protein synthetic processes. The costs for threonine, the branched-chain amino acids, methionine and cysteine would be much greater due to their more specialized roles in gut maintenance. In addition, arginine and proline requirements would also be increased due to reduced synthesis as well as increased utilization. The nutritional consequences of gastrointestinal disease and stress require further investigation considering that its importance in the treatment of such conditions cannot be underestimated. Because emerging evidence is demonstrating a substantial impact of gut first-pass metabolism on amino acid requirements, the accommodation of this metabolism will undoubtedly

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generate interest, especially among researchers in animal production and clinical treatment of gut diseases. The role of the native microbial population and subclinical gut infections has an important impact on whole-body requirements; indeed, the higher amino acid utilization efficiency with growth-promoting feed-grade antibiotics may partially be explained by the minimization of subclinical challenges. Considering that the small intestinal capacity to digest and absorb protein and amino acids is substantially greater than possible dietary inputs (Burrin et al., 1999), one is tempted to consider that much of this organ’s demand for amino acids for maintenance may be an unnecessary burden. Considering that the protein component, and especially synthetic amino acids, is the most expensive component of animals feeds, the cost of maintaining the surplus capacity of the gut becomes significant. In addition, recent evidence has demonstrated that a significant proportion of whole-body amino acid catabolism occurs in the gut, presumably for energy. Alternative fuels may be sought to replace this perhaps unnecessary, expensive source of energy. Another compelling solution would be to select animals for lower intestinal metabolism without compromising gut absorptive capacity or protective functions. These animals would have a substantially greater amino acid utilization efficiency. The IAAO technique has proven very useful and versatile in determining amino acid requirements of animals. Its adaptation for vulnerable populations is especially advantageous if amino acid requirements during pathogenic challenges or disease states are to be explored. Furthermore, the development of the technique to determine metabolic availability of amino acids is an important advance in the field of amino acid digestibility, which is of critical importance when providing dietary amino acids to meet requirements. Because the IAAO technique relies on relative differences, its dependence on questionable kinetics assumptions is minimal. This is particularly important given the accumulating evidence that luminal and arterial amino acids are channelled differently intracellularly; the amino acid pools for oxidation and protein synthesis are probably separated to some extent so that respective precursor enrichments may be very different, rendering kinetic equations irrelevant. Further adaptation of the IAAO technique to answer many of these questions is eagerly sought.

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